Next Article in Journal
Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum
Previous Article in Journal
External Quality Assessment Evaluating the Ability of Dutch Clinical Microbiological Laboratories to Identify Candida auris
 
 
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Conference Report

9th Trends in Medical Mycology Held on 11–14 October 2019, Nice, France, Organized under the Auspices of EORTC-IDG and ECMM

1
UMR_S 1085—Inserm, Institut de Recherche en Santé, Environnement et Travail, CHU de Rennes, Université de Rennes, 35000 Rennes, France
2
Necker - Pasteur Center for Infectious Diseases and Tropical Medicine, Necker-Enfants malades Hospital, AP-HP, Paris Descartes University, 75015 Paris, France
3
Institut Pasteur, Molecular Mycology Unit, National Reference Center for Invasive Mycoses and Antifungals, CNRS UMR 2000, 75015 Paris, France
4
Department I of Internal Medicine, Clinical Trials Centre Cologne, ZKS Köln, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany
5
Department of Hematology, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
J. Fungi 2019, 5(4), 95; https://doi.org/10.3390/jof5040095
Received: 24 September 2019 / Accepted: 27 September 2019 / Published: 8 October 2019

Introduction

Dear Friends and Colleagues,
It is a great honor and pleasure for us to invite you cordially to participate in the 9th Congress on Trends in Medical Mycology (TIMM-9), which will be held in 11–14 October 2019 at Nice-Acropolis Convention Center, Nice, France. TIMM-9 is the 9th in the series of TIMM mycological international meetings organized jointly by the European Confederation of Medical Mycology (ECMM) and the Infectious Diseases Group of the European Organisation for Research and Treatment of Cancer (EORTC-IDG).
TIMM has become an important and essential meeting in the field of fungal infections, a forum in which researchers and clinicians from all over the world present the most important advances and research findings in clinical mycology. TIMM-9 will cover all aspects of mycology, with special focus on evidence-based and personalized approach to medical mycology, as well as diagnostic–therapeutic integrative efforts in the quest to improve the present knowledge of epidemiology, diagnosis, clinical course, and pathophysiological mechanisms of fungal diseases. It would be the place to present recent innovations in medical mycology.
The meeting is designed for medical microbiologists, medical mycologists, hematologists, oncologists, transplant physicians, intensivists, immunologists, and all those with interest in medical mycology. We expect TIMM-9 to be at least as successful as previous TIMM Congresses, which brought together around 1000 international delegates from all over the world. Therefore, we would like to invite you to TIMM-9 in Nice to enjoy with us excellent science in a stimulating environment.
We look forward to greeting you in Nice!
  • Jean-Pierre Gangneux
  • Olivier Lortholary
  • Oliver A. Cornely
  • Livio Pagano
TIMM-9 Executive Committee
 
COMMITTEES
Executive Committee
  • Jean-Pierre Gangneux, ECMM, France
  • Olivier Lortholary, EORTC, France
  • Livio Pagano, EORTC, Italy
  • Oliver A. Cornely, ECMM, Germany
Local Scientific Committee
  • Hamdi Akan, EORTC, Turkey
  • Valentina Arsić Arsenijević, ECMM, Serbia
  • Sharon Chen, ECMM, Australia
  • Raoul Herbrecht, EORTC, France
  • Martin Hoenigl, ECMM, Austria
  • Nikolai Klimko, ECMM, Rusland
  • Katrien Lagrou, ECMM, Belgium
  • Johan Maertens, EORTC, Belgium
  • Jacques F. Meis, ECMM, the Netherlands
  • Tony Pagliuca, EORTC, UK
  • Zděnek Racil, EORTC, Czech Republic
  • Esther Segal, ECMM, Israel
  • Don Sheppard, ECMM, Canada
  • Paul Verweij, EORTC, the Netherlands
  • Thomas Walsh, ECMM, USA
International Scientific Committee
  • Jean-Pierre Gangneux, Rennes
  • Olivier Lortholary, Paris
  • Alexandre Alanio, Paris
  • Jean-Philippe Bouchara, Angers
  • Marie-Elizabeth Bougnoux, Paris
  • Stephane Bretagne, Paris
  • Christophe D’Enfert, Paris
  • Jean-Paul Latgé, Paris
  • Laurence Millon, Besançon
CONTENT
Welcome Address   1
Introduction to the Scientific Programme   5
Scientific Programme   6
Honorary Lectures   16
Plenary Sessions   17
Meet the Expert Sessions   29
Symposia   31
Poster Presentations   97
Introduction to the Scientific Programme
Plenary Sessions:
No session will be held in parallel to these sessions.
  • Plenary sessions are indicated by the prefix: PS
Symposia:
Each day, symposia will convene renowned speakers from several continents, who will cover a wide range of recent developments in their fields.
  • Symposia are indicated by the prefix: S
A part of the symposia includes a selected submitted abstract. These abstracts are marked with an asterisk (*).
Meet the Expert Sessions:
The audience will actively participate in these small sessions.
  • Meet the expert sessions are indicated by the prefix: M
Poster Sessions:
All poster boards are situated on the exhibition floor of the congress centre. The poster exhibition is open to all participants during the entire congress. The numbers on the poster boards correspond with the abstract numbers in this abstract supplement.
All authors of odd poster numbers must be present at their poster on Saturday 12 October, from 11:30 to 12:30. All authors of even poster numbers must be present at their poster on Sunday 13 October, from 11:30 to 12:30.
  • All posters are indicated by the prefix: P
SCIENTIFIC PROGRAMME                        
FRIDAY 11 OCTOBER 2019
09:00 ECMM Council Meeting
12:25 Welcome Address
12:45–13:45 Plenary Session 1—Tropical
Chairs: Jean-Pierre Gangneux, FECMM, France & Olivier Lortholary, France
12:45PS1.1Histoplasmosis
Matthieu Nacher, France
13:15PS 1.2Emergomycosis
Nelesh Govender, FECMM, South Africa
13:45–15:15 Parallel Symposia 1–4
Symposium 1 Water Quality (Mis-)Management—An Opportunity for Fungal Contamination
Chairs: João Brandão, Portugal & Esther Segal, FECMM, Israel
13:45S01.1 Fungal Contaminants in Drinking Water Regulation? A Tale of Ecology, Exposure, Purification, and Clinical Relevance
Monica Novak Babič, Slovenia
14:05S01.2 Hospital Environment: Water Supply and Containment of Aerosolised Fungal Particles
How Far Must We Go in Times of Antimicrobial Resistance?
Raquel Sabino, FECMM, Portugal
14:25S01.3 Potential Transmission Pathways of Clinically Relevant Fungi in Indoor Swimming Pool Facilities
Ciska Schets, the Netherlands
14:45S01.4* Study of Fungal Environmental Contamination in Nests of the Penguin Enclosure of a Large French Animal Zoo Park
Guillaume Desoubeaux, France
14:55 S01.5* Spectrum of Indoor Fungi Isolated from Indoor Environments in Busia—Kenya
Olga Mashedi, Kenya
Symposium 2 Pneumocystis jirovecii, an Airborne Transmissible and Human-Derived Ascomycete Showing Strong Pulmonary Tropism
Chairs: Philippe Hauser, Switzerland & Gilles Nevez, France
13:45S02.1 Pneumocystis jirovecii: An Obligate Parasite of Human Lungs with Unique Camouflage and Sex Strategies
Philippe Hauser, Switzerland
14:10S02.2 High-Throughput Methodologies in Molecular Epidemiology of Pneumocystis
Jirovecii Olga Matos, Portugal
14:35S02.3 Airborne Acquisition and Transmission of Pneumocystis jirovecii: An Update
Gilles Nevez, France
15:00S02.5* Does Pneumocystis jirovecii Infection Aggravate the Prognosis of Invasive Pulmonary Aspergillosis? Data from the RESSIF Network in France (2012–2016)
Florence Robert-Gangneux, France
Symposium 3 Mucormycosis
Chairs: Fanny Lanternier, FECMM, France & Arunaloke Chakrabarti, FECMM, India
13:45 S03.1 Hospital-Related Mucormycosis
Anna Skiada, Greece
14:00 S03.3 The New Treatment of Mucormycosis
Livio Pagano, FECMM, Italy
14:15 S03.4 Clinical Features Associated to Fungal Species
Dea Garcia Hermoso, France
14:30 S03.5 Molecular diagnosis
Laurence Millon, France
14:45 S03.6 Management of Mucormycosis in Low- and Middle-Income Countries
Arunaloke Chakrabarti, FECMM, India
15:00 Discussion
Symposium 4 MSG Symposium (Trial Design in Clinical Mycology: Innovative Approaches)
Chairs: Peter Pappas, USA & Sharon Chen, FECMM, Australia
13:45 S04.1 Candidiasis
Bart-Jan Kullberg, The Netherlands
14:05 S04.2 Endemic Mycoses
George Thompson, USA
14:25 S04.3 Cryptococcosis
David Boulware, USA
14:45 S04.4 Aspergillosis and other molds
Tom Patterson, USA
15:05 S04.5* The Lung Transplant Community Is Interested in a Clinical Trial to Determine the Optimal Strategy to Prevent Invasive Mold Disease
Ricardo La Hoz, USA
18:30EDLE. Drouhet Lecture (ECMM)
Chair: Martin Hoenigl, FECMM, Austria
How to Convince European institutions that Medical Mycology is a Major Science
Jean-Paul Latgé, France
SATURDAY 12 OCTOBER 2019
08:00–08:45 Meet the Expert Sessions
08:00M01 Hematology
Malgorzata Mikulska, FECMM, Italy & Alessandro Busca, Italy
08:00M02 Peadiatrics
Simone Cesaro, Italy & Adillia Warris, FECMM, UK
08:00M03 Candida in the ICU
Matteo Bassetti, Italy & Jean-François Timsit, France
08:00M04 Diagnostics/Laboratory
Alida Talento, Ireland & Michaela Lackner, FECMM, Austria
08:00M05 Tropical
Arunaloke Chakrabarti, FECMM, India & Rita Oladele, FECMM, Nigeria
09:00–10:00 Plenary Session 2—Highlights on Fungal Biology
Chairs: Vishukumar Aimanianda, France & Don Sheppard, FECMM, Canada
08:50 PS 2.1 Candida
Christophe D’Enfert, France
09:20PS 2.2 Aspergillus
Agostinho Carvalho, FECMM, Portugal
09:50PS 2.3* Lineage-Specific Behavioural Differences in Isolates of Candida auris
Andrew Borman, UK
10:30–11:30 Plenary Session 3—One World One Guideline ECMM MSG-ERC (EFISG) ISHAM Guidelines Initiative
Chairs: Martin Hoenigl, FECMM, Austria & John Perfect, USA
10:30 PS3.1 Mucormycosis
Oliver Cornely, FECMM, Germany
10:45 PS3.2 Endemic
George Thompson, USA
11:00 PS3.3 Rare Moulds
Martin Hoenigl, FECMM, Austria
11:15 PS3.4 Rare Yeasts
Sharon Chen, FECMM, Australia
11:30-12:30 Poster Session
14:15-15:45 Parallel Symposia 5–9
Symposium 5 Lung Transplantation
Chairs: Shahid Husain, FECMM, Canada & Paolo Grossi, Italy
14:15S05.1 Pretransplant Assessment
Blandine Rammaert, France
14:35 S05.2 Pathophysiology and Epidemiology of Invasive Aspergillosis in Lung Transplant Recipients
Claire Aguilar, France
14:55 S05.3 Current Guidelines
Shahid Husain, FECMM, Canada
15:15 S05.4 Prophylaxis
John Perfect, USA
15:35 S05.5* From the Lung to the Heart: Fatal Dissemination of Azole-Resistant Aspergillus Fumigatus in a Lung Transplant Patient
Rose-Anne Lavergne, France
Symposium 6 Prophylaxis during Hematology Malignancies
Chairs: Livio Pagano, FEMCC, Italy & Oliver Cornely, FECMM, Germany
14:15 S06.1 AML—in the Era of FLT3 Inhibitors
Russel Lewis, Italy
14:35 S06.2 ALL—There Is a Role for Prophylaxis
Daniel Teschner, Germany
14:55 S06.3 Personalized Medicine/Approach by Genetic Risk Factors
Pierre-Yves Bochud, Switzerland
15:15 S06.4 Baseline CT upon Diagnosis of Acute Leukemia
Stefan Schwartz, Germany
15:35 S06.5* Investigating the Impact of Posaconazole Prophylaxis on Systematic Fungal Screening Using Galactomannan Antigen, Aspergillus qPCR and Mucorales qPCR
Anne-Pauline Bellanger, France
Symposium 7 Paediatric Mycology (EPMyN)
Chairs: Emmanuel Roilides, FECMM, Greece & Roger Brüggemann, FECMM, The Netherlands
14:15S07.1 Fluconazole and Micafungin Dosing in Neonates
Roger Brüggemann, The Netherlands
14:35S07.2 Antifungal Susceptibility of Pediatric Candidemia
Zoi-Dorothea Pana, Greece
14:55S07.3 Primary Immunodeficiencies Characterized by Fungal Infections
Fanny Lanternier, FECMM, France
15:25S07.5* Isavuconazole use in pediatric hematoncologic patients: the Italian Association of Pediatric Hematology Oncology (AIEOP) experience
Nunzia Decembrino, Italy
Symposium 8 Immunologic Markers for Diagnosis and Treatment Stratification in Invasive Mold Infection
Chairs: Martin Hoenigl, FECMM, Austria & Agostinho Carvalho, FECMM, Portugal
14:15S08.1 Antimold Immune Response: the Impact of the Host, the Pathogen, and Translational Implications
Agostinho Carvalho, FECMM, Portugal
14:35S08.2 Immunologic Markers for Diagnosis of Invasive Aspergillosis and Other Invasive Mold Infections
Carol Garcia-Vidal, Spain
14:55S08.3 Immunologic Markers For Treatment Stratification in Invasive Mold Infection
Philipp Köhler, FECMM, Germany
15:15S08.4 Fungal Translocation: From Persistent Inflammation to Non-AIDS Events
Martin Hoenigl, FECMM, Austria
15:35S08.5* Immunological Characteristics of Broncho Alveolar Lavage in Neutropenic Patients with Invasive Aspergillosis
Claire Aguilar, France
Symposium 9 Chronic Pulmonary Aspergillosis
Chairs: David Denning, FECMM, United Kingdom & Aleksandra Barac, FECMM, Serbia
14:15S09.1 Effect of Patient Immunodeficiencies on the Diagnostic Performance of Serological Assays to Detect Aspergillus-Specific Antibodies in Chronic Pulmonary Aspergillosis
Elizabeth Hunter, UK
14:35S09.2 Diagnosis of CPA. Where Do We Stand?
Aleksandra Barac, FECMM, Serbia
14:55S09.3Current Treatment Options for CPA
David Denning, FECMM, UK
15:15S09.4 Future Directions
David Denning, FECMM, UK
15:25S09.5* Raised Amphotericin B MIC in Aspergillus fumigatus Isolates from Patients with Chronic Pulmonary Aspergillosis
Fiona Lynch, UK
16:15–17:45 Parallel Symposia 10–14
Symposium 10 Sensing the Host
Chairs: Muriel Cornet, France & Mihai Mares, FECMM, Romania
16:15S10.1 S10.1 Cell wall of Aspergillus fumigatus in murine lung tissue
Thierry Fontaine, France
16:45S10.2 Hybrid Histidine Kinases: Major Sensing Proteins in Pathogenic Fungi
Nicolas Papon, France
17:15S10.3 Adapting to the Host: How Candida Causes Bloodstream Infection
Oliver Kurzai, Germany
Symposium 11 The Antifungal Pipeline
Chairs: David Denning, FECMM, United Kingdom & Oliver Cornely, FECMM, Germany
16:15S11.1Olorofim—A Novel Mould-Active Antifungal (F2G)
John Rex, FECMM, UK
16:40S11.2 Fosmanogepix: A Novel, Broad Spectrum Antifungal Therapy in Clinical Development (Amplyx)
Michael Hodges, USA
17:05S11.3 Ibrexafungerp (formerly SCY-078) (Scynexis)
David Angulo, USA
Symposium 12 Environment and Fungal Outbreaks
Chairs: Jean-Pierre Gangneux, FECMM, France & Tom Chiller, USA
16:15S12.1 Frogs
Matthew Fisher, UK
16:35S12.2 Cryptococcus gattii
Ferry Hagen, FECMM, The Netherlands
16:55S12.3 Natural Disasters
Tom Chiller, US
17:15S12.4 Genomic Sequencing
Marie Desnos, France
17:35S12.5* Outbreak of Fluconazole-Resistant Candida Parapsilosis in a Hospital Ward: Arguments for Clonal Transmission and Environmental Persistence
Arnaud Fekkar, France
Symposium 13 Fungal Respiratory Infections in Cystic Fibrosis (the ECMM/ISHAM Working Group Fri-CF)
Chairs: Jean-Philippe Bouchara, France & Petr Hamal, Czech Republic
16:15S13.1 Rasamsonia Species and Other Emerging Fungi in CF
Solène Le Gal, France
16:35S13.2 Immunodiagnosis of Scedosporium/Lomentospora Infection—Lessons from Immunoproteomic Studies
Andoni Ramirez-Garcia, Spain
16:55S13.3 Bacterial/Fungal Interactions in the CF Mucus
Françoise Botterel, France
17:15S13.4* Morphology, Growth, and Biofilm Formation of the Black Yeast-Like Fungus Exophiala dermatitidis is Influenced by Pseudomonas aeruginosa under in Vitro Cystic Fibrosis Conditions
Lisa Kirchhoff, Germany
17:25S13.6* Study of Antigenic Markers for Serological Detection of Scedosporium spp. in Cystic Fibrosis Patients
Leire Martin-Souto, Spain
17:35 Discussion
Symposium 14 Teaching Medical Mycology: What, How, to Whom and When
Chairs: Patricia Muñoz, Spain & Esther Segal, FECMM, Israel
16:15S14.1 Mycology Teaching in Medical School—How Much, What to Choose, at What Level
Peter Rath, FECMM, Germany
16:35S14.2 Mycology Teaching in Medical School—Is Student Demography Important to What Should Be Taught?
Ester Segal, FECMM, Israel
16:55S14.3 Specific Courses Outside the European Continent—The Indian Example
Ruth Ashbee, United Kingdom
17:15S14.4 Teaching Clinical Laboratory Mycology—How and Who Is the Audience?
Sevtap Arikan-Akdagli, FECMM, Turkey
17:35S14.5 The African Example
Aude Sturny, France
SUNDAY 13 OCTOBER 2019
08:00–08:45 Meet the Expert Sessions 6–10
08:00M06 Hematology—hematopoietic stem cell transplantation
Nikolai Klimko, FECMM, Russia & Tony Pagliuca, UK
08:00M07 Molecular Diagnostics
Maurizio Sanguinetti, Italy & Dieter Buchheidt, FECMM, Germany
08:00M08 Surgery/Transplantation
Patricia Munoz, Spain & Paolo Grossi, Italy
08:00M09 Neonates
Thomas Walsh, FECMM, USA & Emmanuel Roilides, FECMM, Greece
08:00M10 Meet Mr. and Mrs. Fungus
Cornelia Lass-Flörl, FECMM, Austria & Neil Gow, FECMM, UK
08:50–09:50 Plenary Session 4—Top 10 papers in Mycology
Chairs: Maiken Arendrup, Denmark & Malcolm Richardson, FECMM, UK
08:50PS4.1 The Clinical and Translational Perspective
Don Sheppard, FECMM, Canada
09:20PS4.2The Microbiology Perspective
Katrien Lagrou, FECMM, Belgium
ECMM Academy and Excellence Centers: A Story of Success
Chair: Martin Hoenigl, FECMM, Austria
09:50 ECMM Academy
Katrien Lagrou, FECMM, Belgium
09:55 ECMM Excellence Centers
Cornelia Lass-Flörl, FECMM, Austria
10:30–11:30 Plenary Session 5—ICU—Candida Infections/Breaking News from Hematology and ICU
Chairs: Johan Maertens, FECMM, Belgium / Maricela Valerio, Spain
10:30 PS5.1 Abdominal Candidiasis in ICU Patients
Philipp Köhler, FECMM, Germany
10:45 PS5.2 Are the New Management Strategies Useful?
Arnaldo Colombo, FECMM, Brazil
11:00 PS5.3 CNS Infections
Anna Candoni, Italy
11:15 PS5.4 CMV and Aspergillosis in HSCT Patients
Johan Maertens, FECMM, Belgium
11:30–12:30 Poster Session 2
11:30 Video Session
11:30V01 Subcutaneous Nodule Caused by Phaeoacremonium fuscum in a Non-Immunocompromised Patient
Sofie Colman, Belgium
11:40V02 Trichosporon Diagnosis—The Right Path
Thayanidhi Premamalini, India
11:50V03 Disseminated Rhinosporidiosis with Different Morphological Lesions Involving Various Anatomical Sites
Jagdish Chander, India
14:15–15:45 Parallel Symposia 15–19
Symposium 15 Aspergillus in the ICU
Chairs: Katrien Lagrou, FECMM, Belgium & Alessandro Pasqualotto, FECMM, Brazil
14:15 S15.1 Influenza
Joost Wauters, Belgium
14:35S15.2 Invasive Aspergillosis in Patients with Underlying Liver Cirrhosis
Juergen Prattes, Austria
14:55 S15.3 Renal Failure
Riina Richardson Rautema, FECMM, UK
15:15 S15.4* Fungal Pneumonia in Critically Ill Cirrhotics: Spectrum, Outcomes, Comparison of Diagnostic Methods and Biomarkers
Pratibha Kale, India
15:25 S15.5* IPAFLU Survey: Invasive Aspergillosis among Patients with Severe Influenza in Intensive Care Units
Joost Wauters, Belgium
15:35 S15.6* Trend of Candidemia with Bloodstream Infection in Intensive Care Units from 2006 to 2017: Results from the Korean National Healthcare-Associated Infections Surveillance System
Young Hwa Choi, Korea
Symposium 16 Dermatology
Chairs: Valentina Arsic Arsenijevic, ECMM, Serbia & Sevtap Arıkan Akdagli, FECMM, Turkey
14:15S16.1 Cutaneous Aspergillosis, Is It So Rare?
Sevtap Arıkan Akdagli, FECMM, Turkey
14:35S16.2 Is Resistance a Problem for Dermatophytosis?
Pietro Nenoff, Germany
14:55S16.3 Phaeohyphomycosis
Teresa Martin, Spain
15:15S16.4 Blastomycosis
Ilan Schwarz, Canada
15:35S16.5* Galleria mellonella as a Novelty Model to Study Host—Pathogen Interaction in Malassezia furfur CBS 1878
Adriana Marcela Celis Ramírez, Colombia
Symposium 17 HIV-Associated Cryptococcal Meningitis
Chairs: John Perfect, USA & Olivier Lortholary, France
14:15S17.1Screening in Low-Resource Areas
Elvis Temfack, Cameroon
14:35S17.2 Current Therapeutic Strategies
Olivier Lortholary, France
14:55S17.3 Towards Having Antifungal Drugs in Low-Resource Areas
Ida Kolte, UK
15:15S17.4 Early versus Delayed Antiretroviral Treatment in HIV-Positive People with Cryptococcal Meningitis
Tihana Bicanic, FECMM, UK
15:35S17.5 Current and Future Clinical Trials on HIV-Associated Cryptococcal Meningitis
Tom Harrison, UK
Symposium 18 Antifungal Stewardship in the Era of Resistance
Chairs: Paul Verweij, FECMM, The Netherlands & Souha Kanj, FECMM Lebanon
14:15S18.1 Antifungal Stewardship: A Practical Experience in a Tertiary Care Institution
Maricela Valerio, Spain
14:35S18.2Stewardship and Azole-Resistant Aspergillosis: A Challenge for Farmer or Physician?
Paul Verweij, FECMM, The Netherlands
14:55S18.3 Rapid Diagnosis of Fungal Infections: Impact on Stewardship
Souha Kanj, FECMM, Lebanon
15:15S18.4 Mycology Laboratory Diagnostic Capabilities in Different Areas of the World
Alessandro Pasqualotto, FECMM, Brazil
15:35S18.5* Optimising Antifungal Stewardship: An Evaluation of Candidaemia Guideline Compliance and Clinical Outcome
Laura Cottom, UK
Symposium 19 Pneumocystis
Chairs: Enrique Calderon, Spain & Stephane Bretagne, FECMM, France
14:15S19.1 Diagnosis
Alexandre Alanio, FECMM, France
14:40S19.2 Pneumocystosis in HIV and Non-HIV Patients
Joseph Kovacs, USA
15:05S19.4 Pneumocystosis in Neonates
Enrique Calderon, Spain
15:30S19.5* Evaluation of a Novel Commercial Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Pneumocystis jirovecii
Dirk Schmidt, Germany
16:15–17:45 Parallel Symposia 20–24
Symposium 20 Candida auris
Chairs: Nelesh Govender, FECMM, South Africa & Anuradha Chowdhary, FECMM, India
16:15S20.1Schizophrenic Gram-Negative Yeast Conquering the World
Jacques Meis, FECMM, The Netherlands
16:35S20.2 In the US
Tom Chiller, USA
16:55S20.3 Outbreak Control
Alba Ruiz, Spain
17:15S20.4 In Resource-Limited Countries
Anuradha Chowdhary, FECMM, India
17:35S20.5* Understanding Echinocandin Activity towards Candida auris
Milena Kordalewska, USA
Symposium 21 Immunotherapy for Opportunistic Fungal Infections
Chairs: Carol Garcia-Vidal, Spain & Dimitrios Kontoyiannis, FECMM, USA
16:15S21.1 CAR-T Cells, NK Cells
Dimitrios Kontoyiannis, FECMM, USA
16:35S21.2 WBC Transfusions
Livio Pagano, FECMM, Italy
16:55S21.3 Cytokines
George Chamilos, Greece
17:15S21.4* Novel Chimeric Antigen Receptor T Cells for Invasive Aspergillosis Immunotherapy
Michelle Seif, Germany
17:25S21.5* Comparison of Circulating Lymphocyte Populations CD4+ and CD8+ T Cells, B and NK lymphocytes According to the Favorable or Worsening Evolution of Patients with Pneumocystosis
Eléna Charpentier, France
17:35S21.6* Glucosylceramides from Lomentospora prolificans Induce Cytokines Production and Increase the Microbicidal Activity of Macrophages
Mariana Ingrid Dutra Silva Xisto, Brazil
Symposium 22 NGS and Mycobiota
Chairs: Laurence Delhaes, France & Jean-Pierre Gangneux, FECMM, France
16:15S22.1 Bacterial–Fungal Interactions at the Airway Mucosa and Implications for Chronic Rhinosinusitis
Emily Cope, USA
16:35S22.2 Respiratory Mycobiome—A Clinical Perspective
Robert Krause, FECMM, Austria
16:55S22.3 Characterization of Indoor Dust Microbiota in Homes of Asthma and Non-Asthma Patients
Jean-Pierre Gangneux, FECMM, France
17:15S22.4 Understanding the Role of the Gut Mycobiome in Health and Disease
Maria Vehreschild, FECMM, Germany
17:35S22.5* An ECMM-ESCMID Survey on Goals and Practices for Mycobiota Characterization Using Next-Generation Sequencing in European Laboratories
Jean-Pierre Gangneux, FECMM, France
Symposium 23 The Funny Life of C. albicans in the Oral Tract
Chairs: Marie-Elisabeth Bougnoux, France & Anna Maria Tortorano, Italy
16:15S23.1 Within-Host Genomic Diversity of Candida albicans in Healthy Carriers
Marie-Elisabeth Bougnoux, France
16:35S23.2 Activation of Oral Immune Responses by C. albicans
Julian Naglik, UK
16:55S23.3 Modulation of the Fungal–Host Interaction by the Intraspecies Diversity of C. albicans
Dominique Sanglard, Switzerland
17:15S23.4 Intestinal Th17 Drives Airway Inflammation in ABPA
Petra Bacher, Germany
17:35S23.5* Persistence of Clonal Azole-Resistant Isolates of Candida albicans from a Patient with Chronic Mucocutaneous Candidiasis in Colombia
Andres Ceballos-Garzon, Colombia
Symposium 24 Fungal Neglected Tropical Diseases
Chairs: Flavio Queiroz-Telles, Brazil & Leila Lopes Bezerra, Brazil
16:15S24.1 Mycetoma
Wendy van de Sande, FECMM, The Netherlands
16:35S24.2 Chromoblastomycosis
Flavio de Queiroz-Telles, FECMM, Brazil
16:55S24.3 Sporotrichosis
Leila Lopes Bezerra, Brazil
17:15S24.4 Novel Diagnostic and Treatment Strategies for the Southeast Asian Fungus Talaromyces marneffei
Thuy Le, USA
17:25 Discussion
MONDAY 14 OCTOBER 2019
08:00–8:45 Meet the Expert Sessions 11–15
08:00M11 HIV
Blandine Denis, France & Nelesh Govender (FECMM), South Africa
08:00M12 Environment
Ester Segal, FECMM, Israel & Raquel Sabino, FECMM, Portugal
08:00M13 Diagnostics/Imaging
Frédéric Lamoth, FECMM, France & Christopher Thornton, UK
08:00M14 Resistance
Maiken Arendrup, Denmark & Lewis White, FECMM, UK
08:00M15 Registries
Danila Seidel, Germany & Oscar Marchetti, Switzerland
08:50–10:00 Plenary Session 6—Management of IFD in Pediatrics (EPMyN)
Chairs: Adilia Warris, FECMM, United Kingdom & Andreas Groll, FECMM, Germany
08:50PS6.1 Biomarker-Based Diagnostic Work-Up of IFD in Immunocompromised Pediatric Patients
Thomas Lehrnbecher, Germany
09:20PS6.2 Pre-Emptive Versus Empiric Antifungal Therapy in Children with Cancer
Maria Elena Santolaya, Chile
09:50PS6.3* Invasive Mucormycosis in Children with Hematological Malignancies and Solid Tumors: Report from the Infection Working Group of the Hellenic Society of Pediatric Hematology Oncology (2008–2017).
Emmanuel Roilides, Greece
10:30–11:15 Plenary Session 7—Superficial and Dermatology
Chairs: Arnaldo Colombo, FECMM, Brazil & Yee-Chun Chen, Taiwan, Province of China
10:30PS7.1 When the Skin Is the Portal of Invasive Infections
Yee-Chun Chen, Taiwan, Province of China
11:00PS7.2 When the Skin Talks for Systemic Infections
Fanny Lanternier, FECMM, France
11:15–12:30 Plenary Session 8—New (Antineoplastic) Drugs, New Risks
Chairs: Livio Pagano, FECMM, Italy & Johan Maertens, FECMM Belgium
11:30PS8.1 In Hematology
Alessandro Busca, Italy
12:00PS8.2 In Oncology
Lubos Drgona, FECMM, Slovakia
12:30 Closing TIMM-9

Honorary Lectures

E. Drouhet lecture EDL. How to convince European institutions that Medical Mycology is a major science

J.-P. Latgé
University of crete, Heraklion, Greece
How to convince Europe that Medical Mycology is a major science
Biochemistry; Molecular biology; Immunology
There are many reasons for flying the flag of Medical Mycology in medicine but also in science in Europe. First, morbidity and mortality due to fungi is much higher than the one due to tuberculosis and malaria in Europe. Second, essential advances in immunology such as TLR or C-type lectins, had their root in the analysis of the host response against fungal infections. Fungi continue to be models of choice for deciphering new immunological pathways. Cell wall which is a unique feature of these eukaryotic microorganisms, has been instrumental on this medical theme but also on the finding of new essential enzymes in glycobiology. Third, Mycobiota is a majority underrecognized and understudied component of the microbiota which is more and more recognized as an important constituent of human health. Fourth, analysis of fungal biochemical and molecular pathways can help understanding human diseases or metabolic dysregulations or stem cell dormancy. Finally, more than other microbes, fungi have to be loved for their natural beauty. All these cases will be illustrated with examples taken from the study of Aspergillus fumigatus which has been my “baby” for the last 30 years.

Plenary session 1—Tropical

PS1.2. Emergomycosis

N. Govender
National Institute for Communicable Diseases, Johannesburg, South Africa
Five species of thermally-dimorphic fungi within a new genus Emergomyces cause a disseminated mycosis among immunocompromised persons. Distinct from the closely-related Emmonsia and Blastomyces genera, Emergomyces strains have only been isolated from human infections and all species produce yeast cells (usually <5 µm diameter and with narrow-based budding) in the thermotolerant phase. The type species, Emergomyces pasteurianus was first described from a case in 1992 and has an apparently cosmopolitan distribution with cases diagnosed in Europe, Africa and Asia. The other four species were described to have emerged over the last decade, coinciding with increasing use of molecular diagnostic techniques in clinical and research laboratories, and may be geographically restricted. Overall, Emergomyces africanus has been implicated in the largest number of reported cases of emergomycosis. Restricted to southern Africa and first described by Kenyon et al in 2013, E. africanus causes a multi-system disease among persons living with advanced HIV disease. Systemic infection is presumed to occur following inhalation of air-borne conidia from a soil reservoir, with a subsequent temperature-mediated phase transition to a yeast form and dissemination through the reticuloendothelial system among immunocompromised individuals. Most cases are diagnosed by conventional culture of blood, tissues and fluids and/or histopathological tissue examination, both of which require technical expertise. Limited pulmonary disease is probably under-diagnosed in resource-limited settings; this has only been described to occur in the single case of Emergomyces europaeus infection. The full spectrum of clinical infection and prevalence in different populations could potentially be determined by better use of non-culture-based methods, including antigen and PCR assays, in clinical settings and for epidemiological surveillance. For instance, E. africanus is known to cross-react with a commercially-available Histoplasma galactomannan antigen assay and Emergomyces canadensis with a commercial DNA probe for Blastomyces dermatitidis. Although the attributable mortality has not been defined, the crude mortality in a South African case series was approximately 50%. Screening for emergomycosis among high-risk patients in endemic areas could detect active disease earlier and thus reduce mortality associated with late presentation. Treatment recommendations for emergomycosis are the same as for patients with disseminated histoplasmosis and are based only on observational data.

Plenary session 2—Highlights on fungal biology

PS2.1. Candida albicans genome diversity: mechanisms and consequences

C. D’Enfert
Fungal Biology and Pathogenicity, Institut Pasteur, INRA, Paris, France
The fungal pathogen Candida albicans shows significant diversity at the genetic and phenotypic levels. Here, I will review our current knowledge of the C. albicans diploid genome and its variability, the genetic structure of the C. albicans population and the mechanisms that are involved in C. albicans genome dynamics, with a focus on the parasexual cycle and loss-of-heterozygosity events. I will further explore the impact of genetic diversity and genome dynamics on C. albicans phenotypic diversity. Finally, I will discuss how our current knowledge of C. albicans genetic diversity could be leveraged in the future in order to get insights in the mechanisms underlying important biological attributes that are subject to variations across C. albicans isolates.

PS2.2. Metabolic regulation of innate immunity to Aspergillus fumigatus

S. Gonçalves, C. Cunha and A. Carvalho
Life and Health Sciences Research Institute (icvs), University of Minho, Braga, Portugal
The reprogramming of cellular metabolism was recently recognized as a fundamental mechanism through which innate immune cells meet the energetic and anabolic needs during host defense against invading pathogens. Sensing of microbial ligands by macrophages drives the upregulation of glycolysis, which delivers a rapid source of energy to support antimicrobial functions and the production of cytokines and other inflammatory mediators. The stimulation of immune cells with structural components of the fungal cell wall, such as β-1,3-glucan, has been demonstrated to promote the metabolic and functional reprogramming of immune cells during infection with the yeast Candida albicans. However, how the immune response to other fungi, such as Aspergillus fumigatus, is also regulated at the metabolic level and whether other cell wall constituents also participate in these signaling events remains unknow. Fungal melanin is a major virulence factor, endowing A. fumigatus with the ability to survive killing by phagocytes. By resorting to in vitro and in vivo models of infection and patients suffering from invasive pulmonary aspergillosis (IPA), and using different pharmacological and genetic tools to manipulate both the host and the pathogen, we reveal a novel mechanism whereby fungal melanin is perceived by the host to regulate adequate immunometabolic responses and susceptibility to infection. Specifically, we demonstrate that the host counters the immune inhibitory mechanisms deployed by fungal melanin, by “sensing” its removal during intracellular swelling inside the phagosome, and using these signals to rewire cellular metabolism towards enhanced glycolysis and promote antifungal immune responses. The contribution of glucose homeostasis to human antifungal immunity is further supported by genetic variation in glycolytic regulators that act as cytokine quantitative trait loci and predispose patients at-risk to IPA. Given the current limitations in diagnosis and therapy of fungal diseases as well as concerns over the emergence of antifungal resistance, these results may contribute towards the design of innovative therapeutic approaches or metabolic adjuncts to reorient host cells towards immune protection against IPA.

PS2.3. Lineage-specific behavioural differences in isolates of Candida auris

A. Borman 1, A. Szekely 1, L. Majoros 2, S. Lockhart 3, G.S. De Hoog 4, R. Ben-Ami 5 and E. Johnson 1
1
Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom
2
Department of Medical Microbiology, University of Debrecen, Debrecen, Hungary
3
Centres For Disease Control and Prevention, Atlanta, United States of America
4
Westerdijk Institute, Utrecht, Netherlands
5
Tel Aviv Sourasky Medical Centre, Tel Aviv, Israel
Objectives:Candida auris is a serious nosocomial health risk, with widespread outbreaks in hospitals worldwide. Sequence analyses of outbreak isolates revealed that C. auris has simultaneously emerged as multiple distinct, geographically restricted clonal lineages. We previously reported multiple independent introductions of C. auris isolates from at least three of these lineages (S. Asia, S. Africa and Japan/Korea) into hospitals across the UK, and that isolates circulating in the UK displayed two different cell phenotypes which correlated with differences in virulence in Galleria mellonella. The present study aimed to more fully characterise clade-specific differences in the behaviour of Candida auris isolates. Methods: Multiple independent Candida auris isolates corresponding to 4 of the known geographically-restricted clonal lineages (S. Asia, S. Africa, S. America/Israeli and Japanese/Korean) were subjected to extensive phenotypic characterisation (cellular morphology, actidione tolerance, virulence in insect [Galleria mellonella] and mammalian [mouse] infection models, antifungal susceptibility profiles, antifungal drug-mediated morphological changes) using well-established methodologies. Results: Significant clade-dependent differences in C. auris isolate behaviour were noted with respect to all of the biological features examined, including cellular morphology, resistance to actidione, virulence, antifungal susceptibility and morphological responses to antifungal drug exposure. Several of these differences may be correlated with previously described differences in cellular aggregation capacity, ERG11 resistance mutations and gene copy numbers, biofilm formation and efflux pump activity. Conclusion: The present data demonstrate that “one size does not fit all for Candida auris” and that the behaviour of individual isolates is at least in part dependent on the clonal lineages from which they originate. Further studies will aim to elucidate whether such differences have clinical significance, and to attempt to establish why isolates of three of the principal clonal lineages (S. Asian, S. African, S. American/Israeli) have been reported from large scale nosocomial outbreaks but none to date have been attributed to isolates from the Japanese/Korean clade.

Plenary session 3—One World One Guideline ECMM MSG-ERC (EFISG) ISHAM Guidelines initiative

PS3.1. Global guideline for the diagnosis and management of mucormycosis: An initiative of the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium

O.A. Cornely 1,2,3,4, A. Alastruey-Izquierdo 5, D. Arenz 1,3, S.C.-A. Chen 6, E. Dannaoui 7, B. Hochhegger 8,9, M. Hoenigl 10,11, H.E. Jensen 12, K. Lagrou 13, R. Lewis 14, S. Mellinghoff 1,3, M. Mer 15, Z. Pana 16, D. Seidel 1,3, D.C. Sheppard 17, R. Whaba 18, A. Chakrabarti 19, M. Akova 20, A. Alanio 21, A. Al-Hatmi 22, S. Arikan-Akdagli 23, H. Badali 24, R. Ben-Ami 25, A. Bonifaz 26, S. Bretagne 21, E. Castagnola 27, M. Chayakulkeeree 28, A. Colombo 29, D.E. Corzo-León 30, L. Drgona 31, A.H. Groll 32, J. Guinea 33, C.-P. Heussel 34, A.S. Ibrahim 35, S. Kanj 36, N. Klimko 37, M. Lackner 38, F. Lamoth 39, F. Lanternier 40, C. Lass-Flörl 38, D.-G. Lee 41, T. Lehrnbecher 42, B.E. Lmimouni 43, M. Mares 44, G. Maschmeyer 45, J. Meis 46, J. Meletiadis 47, O. Morrissey 48, M. Nucci 49, R. Oladele 50, L. Pagano 51, A. Pasqualotto 52, A. Patel 53, Z. Racil 54, M.D. Richardson 55, E. Roilides 16, M. Ruhnke 56, S. Seyedmousavi 24,57,58, N. Sidharthan 59, N. Singh 60, J. Sinko 61, A. Skiada 62, M. Slavin 63,64, R. Soman 65, B. Spellberg 66, W. Steinbach 67, B.H. Tan 68, A.J. Ullmann 69, J.-J. Vehreschild 1,2, M.J.G.T. Vehreschild 1,2,70, T. Walsh 71, P.L. White 72, N. Wiederhold 73 and T. Zaoutis 74
1
Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany
2
German Centre for Infection Research (DZIF) partner site Bonn-Cologne, Cologne, Germany
3
Cecad Cluster of Excellence, University of Cologne, Cologne, Germany
4
Clinical Trials Center Cologne, University Hospital of Cologne, Cologne, Germany
5
Mycology Reference Laboratory, National Centre For Microbiology, Instituto de Salud Carlos III, Madrid, Spain
6
Centre For Infectious Diseases and Microbiology Laboratory Services, New South Wales Health Pathology, and The Department of Infectious Diseases, Westmead Hospital, School of Medicine, University of Sydney, Sydney, Australia
7
Unité De Parasitologie-mycologie, Service De Microbiologie, Université Paris-Descartes, Faculté de Médecine, APHP, Hôpital Européen Georges Pompidou, Paris, France
8
Radiology, Hospital São Lucas da Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS), Porto Alegre, Brazil
9
Radiology, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, Brazil
10
Section of Infectious Diseases and Tropical Medicine and Division of Pulmonology, Medical University of Graz, Graz, Austria
11
Division of Infectious Diseases, Department of Medicine, University of California San Diego, San Diego, United States of America
12
Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
13
Department of Microbiology, Immunology and Transplantation, Clinical Department of Laboratory Medicine and National Reference Center For Mycosis, KU Leuven, University Hospitals Leuven, Leuven, Belgium
14
Infectious Diseases Clinic, Sant’Orsola-Malpighi Hospital, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy
15
Divisions of Critical Care and Pulmonology, Department of Medicine, Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg, South Africa
16
Infectious Diseases Unit, 3rd Department of Paediatrics, Faculty of Medicine, Aristotle University School of Health Sciences, Hippokration General Hospital, Thessaloniki, Greece
17
Division of Infectious Diseases, Department of Medicine, Microbiology and Immunology, McGill University, Montreal, Canada
18
Department of General, Visceral and Cancer Surgery, University Hospital of Cologne, Cologne, Germany
19
Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research, Chandigarh, India
20
Department of Infectious Diseases, Hacettepe University School of Medicine, Ankara, Turkey
21
Institut Pasteur, National Reference Center For Invasive Mycoses and Antifungals, Department of Mycology, Cnrs Umr2000, Parasitology-mycology Laboratory, Fernand Widal Hospitals, Assistance Publique-Hôpitaux de Paris (AP-HP), Université de Paris, Paris, France
22
Directorate General of Health Services, Ministry of Health, Ibri, Oman
23
Department of Medical Microbiology, Hacettepe University School of Medicine, Ankara, Turkey
24
Department of Medical Mycology/invasive Fungi Research Center (ifrc), School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
25
Infectious Diseases Unit, Tel Aviv Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
26
Dermatology Service & Mycology Department, Hospital General de México “Dr. Eduardo Liceaga”, Mexico City, Mexico
27
Infectious Diseases Unit, Istituto Giannina Gaslini Children’s Hospital, Genova, Italy
28
Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
29
Special Mycology Laboratory, Division of Infectious Diseases, Department of Medicine, Universidade Federal de São Paulo (UNIFESP), Sao Paulo, Brazil
30
Department of Epidemiology and Infectious Diseases, Hospital General Dr. Manuel Gea González, Mexico City, Mexico
31
Oncohematology Clinic, Faculty of Medicine, Comenius University and National Cancer Institute, Bratislava, Slovak Republic
32
Infectious Disease Research Program, Department of Paediatric Hematology/oncology and Center For Bone Marrow Transplantation, University Children’s Hospital Münster, Münster, Germany
33
Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Madrid, Spain
34
Diagnostic and Interventional Radiology, Thoracic Clinic, University Hospital Heidelberg, Heidelberg, Germany
35
Division of Infectious Diseases, Los Angeles Biomedical Research Institute, Harbor-University of California, Torrance, United States of America
36
Department of Internal Medicine, Division of Infectious Diseases, American University of Beirut Medical Center, Beirut, Lebanon
37
Department of Clinical Mycology, Allergology and Immunology, North Western State Medical University, St. Petersburg, Russian Federation
38
Division of Hygiene and Medical Microbiology, Department of Hygiene, Microbiology and Public Health, Medical University Innsbruck, Innsbruck, Austria
39
Infectious Diseases Service, Department of Medicine and Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerland
40
Institut Pasteur, National Reference Center for Invasive Mycoses and Antifungals, Department of Mycology, CNRS UMR2000, Paris Descartes University, Necker-Enfants Malades University Hospital, Department of Infectious Diseases and Tropical Medicine, Centre, Paris, France
41
Division of Infectious Diseases, Department of Internal Medicine, Catholic Hematology Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea, Democratic People’s Republic of
42
Division of Paediatric Haematology and Oncology, Hospital for Children and Adolescents, Johann Wolfgang Goethe-University, Frankfurt, Germany
43
School of Medicine and Pharmacy, University Mohammed the fifth, Rabat, Morocco
44
Laboratory of Antimicrobial Chemotherapy, Ion Ionescu de la Brad University, Iasi, Romania
45
Department of Hematology, Oncology and Palliative Care, Klinikum Ernst von Bergmann, Potsdam, Germany
46
Department of Medical Microbiology and Infectious Diseases, Centre of Expertise In Mycology, Radboudumc/Canisius Wilhelmina Hospital, Nijmegen, Netherlands
47
Clinical Microbiology Laboratory, Attikon University Hospital, National and Kapodistrian University of Athens, Department of Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Athens, Greece
48
Department of Infectious Diseases, Alfred Health & Monash University, Melbourne, Australia
49
Department of Internal Medicine, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
50
Department of Medical Microbiology & Parasitology, College of Medicine, University of Lagos, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, United Kingdom
51
Department of Hematology, Fondazione Policlinico Universitario A. Gemelli –irccs–, Universita Cattolica del Sacro Cuore, Rome, Italy
52
Federal University of Health Sciences of Porto Alegre, Hospital Dom Vicente Scherer, Porto Alegre, Brazil
53
Infectious Diseases Clinic, Vedanta Institute of Medical Sciences, Ahmeddabad, India
54
Department of Internal Medicine, Hematology and Oncology, Masaryk University and University Hospital Brno, Brno, Czech Republic
55
UK NHS Mycology Reference Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom
56
Department Haematology / Oncology, Paracelsus Klinik, Osnabrück, Germany
57
Center of Expertise in Microbiology, Infection Biology and Antimicrobial Pharmacology, Tehran, Iran
58
Molecular Microbiology Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, United States of America
59
Department of Hemato Oncology, Amrita Institute of Medical Sciences, Amrita Viswa Vidyapeetham University, Kochi, India
60
Division of Infecatious Diseases, University of Pittsburgh Medical Center and VA Pittsburgh Helthcare System, Infectious Diseases Section, University of Pittsburgh, Pittsburgh, United States of America
61
Infectious Diseases Unit, Szent Istvan and Szent Laszlo Hospital, Budapest, Hungary
62
Department of Infectious Diseases, Laiko General Hospital, National and Kapodistrian University of Athens, Athens, Greece
63
University of Melbourne, Melbourne, Australia
64
The National Centre for Infections in Cancer, Peter MacCallum Cancer Centre, Melbourne, Australia
65
Dept. of Medicine, P. D. Hinduja Hospital & Medical Research Centre, Mumbai, India
66
Los Angeles County + University of Southern California (LAC+USC) Medical Center, Los Angeles, United States of America
67
Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, United States of America
68
Department of Infectious Diseases, Singapore General Hospital, Singapore, Singapore
69
Department For Internal Medicine Ii, University Hospital Würzburg, Würzburg, Germany
70
Department of Internal Medicine, Infectious Diseases, Goethe University Frankfurt, Frankfurt, Germany
71
Departments of Medicine, Pediatrics, Microbiology & Immunology, Weill Cornell Medicine, and New York Presbyterian Hospital, New York, United States of America
72
Public Health Wales Microbiology Cardiff, UHW, Cardiff, United Kingdom
73
Fungus Testing Laboratory, The University of Texas Health Science Center at San Antonio, San Antonio, United States of America
74
Division of Infectious Diseases, The Children’s Hospital of Philadelphia, Philadelphia, United States of America

Abstract:

Background Mucormycosis is a difficult to diagnose rare disease with high morbidity and mortality. Diagnosis is often delayed, and disease tends to progress rapidly. Urgent surgical and medical intervention is lifesaving. Guidance on the complex multidisciplinary management has potential to improve prognosis, but approaches differ between health care settings. Methods from January 2018, authors from 33 countries in all United Nations regions analysed the published evidence on mucormycosis management and provided consensus recommendations addressing differences between the regions of the world as part of the “One World One Guideline” initiative of the European Confederation of Medical Mycology (ECMM). The author group based in 17 time zones, relied on electronic media including video tutorial on methodology, and central document repository with several daily updates. Results Signs and symptoms of mucormycosis depend on organ patterns and underlying conditions. Diagnostic management does not differ greatly between world regions. Upon suspicion of mucormycosis appropriate imaging is strongly recommended to document extent of disease and is followed by strongly recommended surgical intervention. First-line treatment with high-dose liposomal amphotericin B is strongly recommended, while intravenous isavuconazole and intravenous or delayed release tablet posaconazole are recommended with moderate strength. Both triazoles are strongly recommended salvage treatments. Amphotericin B deoxycholate is recommended against, because of substantial toxicity, but may be the only option in resource limited settings. Conclusion Management of mucormycosis depends on recognising disease patterns and on early diagnosis. Limited availability of contemporary treatments burdens patients in low and middle income settings. Areas of uncertainty were identified and future research directions specified.

PS3.3. Rare moulds: Clinical Practice Guideline of the ECMM and the MSG-ERC

M. Hoenigl 1,2
1
Division of Infectious Diseases, University of California, San Diego, United States of America
2
Section of Infectious Diseases and Tropical Medicine and Division of Pulmonology, Medical University of Graz, Graz, Austria

Abstract:

In the context of a growing population of immunocompromised patients at risk for opportunistic infections, prevalence of invasive mould infections, including moulds other than Aspergillus and Mucorales, are on the rise. While new diagnostic and therapeutic options are now available to tackle rare invasive mould infections, up to date guidance for the correct utilization in the clinical setting is urgently needed. On that background, ECMM together with MSG-ERC set out an unprecedented orphan diseases guidance initiative involving all disciplines involved in diagnosis and treatment of rare mould infections. Utilizing the global network of the ECMM Academy and the ECMM Excellence Centers, clinicians, microbiologists and other medical professionals from around the world will be invited by ECMM to contribute their expertise to the project "One World-One Guideline". The rare mould guideline covers diagnosis and treatment of infections caused by Fusarium, Lomentospora, Phaeohyphomycetes/dematiaceous fungi/black fungi, Rasamsonia, Scedosporium, Schizophyllum and other basidiomycetes, Scopulariopsis, Penicillium, and Talaromyces other than marneffei. The guideline will include taxonomy and epidemiology and give detailed recommendations regarding diagnosis and clinical management of these rare mould infections for adults and the pediatric population. The guideline follows the structure and definitions of the ESCMID/ECMM guidelines on invasive fungal infection, and the ECMM MSG-ERC guideline on mucormycosis, which are in accord with the GRADE and AGREE systems.

Plenary session 5—ICU—candida infections/Breaking news from Hematology and ICU

PS5.1. Abdominal Candidiasis in ICU patients

P. Köhler
Department I for Internal Medicine, European Excellence Center for Medical Mycology (ecmm), Germany and Cecad Cluster of Excellence, University of Cologne, Cologne, Germany

Abstract:

With increased admission rates to intensive care unit and high morbidity and mortality in the presence of severe sepsis or septic shock abdominal candidiasis is a frequent complication in surgical patients. In the majority of cases Candida species are part of polymicrobial infections after upper gastrointestinal tract perforation, anastomotic leaks after bowel surgery, acute necrotizing pancreatitis or peritoneal dialysis. Further risk factors comprise premorbid conditions, total parenteral nutrition, and previous antibiotic therapy among others. Blood cultures are often negative and the serum beta-d-glucan assay can support diagnosis in patients with suspected intraabdominal candidiasis. The diagnosis is achieved by aspiration of fluid under ultrasound or CT guidance or at the time of surgery. Current treatment strategies comprise both surgical intervention and antifungal therapy, preferably with an echinocandin. Therapy should continue for at least two weeks and often longer, until the abscess and all signs and symptoms of peritonitis are resolved.

PS5.2. Are the new management strategies useful?

A. Colombo
Medicine, Federal University of São Paulo, São Paulo, Brazil

Abstract:

Along the last decades, we faced a substantial progress in the clinical validation of new diagnostic and therapeutic tools for treating patients with invasive Candidiasis (IC). However, we still have several gaps in knowledge that may mitigate our results in the clinical management of complex patients with invasive candidiasis. We selected 3 main topics to be addressed along this presentation: (1) How Candida Biomarkers might be utilized in the clinics, (2) Should we continuous to order universal screening of eye infection and endocarditis for candidemic patients? (3) Concerns in the clinical management of neutropenic patients with candidemia.

PS5.3. Breaking news from Hematology and ICU: CNS infections

A. Candoni
University Hospital, Asuiud, Division of Hematology and Stem Cells Transplantation, Udine, Italy

Abstract:

Fungal Infections of the Central Nervous System (IFI-CNS) are rare life-threatening infections in hematologic patients and their management remains a challenge despite availability of new diagnostic techniques and novel antifungal agents (14). In addition, analyses of large cohort of patients focusing on these rare IFIs, are still lacking. The most important causative agent remains Aspergillus species and a concomitant extra-CNS involvement (mainly lung) is common. Recent reports highlight that the fungal biomarkers in CSF could be highly informative reinforcing that diagnostic lumbar puncture should be performed when IFI-CNS is suspected, enabling diagnosis confirmation and prompt initiation of targeted therapy (2). Surgical approach of this complications is feasible only in a minority of cases (those with focal, cortical-subcortical lesions and no severe thrombocytopenia), and, even with a broad spectrum of antifungal drugs, overall response rate remains poor (less than 40%) with a 12-months survival probability less than 30% (14). Availability of new drugs, with better CNS permeability and less interaction with immunosuppressive agents and chemotherapy (such as Isavuconazole), and prompt diagnostic work-up (high diagnostic value of CSF biomarkers) can guide a rapid, specific and aggressive therapy (including combination antifungal therapy ± surgery) to further improve outcomes of IFI-CNS in hematologic patients (14). (1) Pagano L, Caira M, Falcucci P, Fianchi L. Fungal CNS infections in patients with hematologic malignancy. Expert Rev Anti Infect Ther. 2005;3:775–85. (2) Candoni A, Klimko N, Busca A, et al. Fungal infections of the central nervous system and paranasal sinuses in onco-haematologic patients. Epidemiological study reporting the diagnostic-therapeutic approach and outcome in 89 cases. Mycoses. 2019;62(3):252–260. (3) Schwartz S, Kontoyiannis DP, Harrison T, Runke M. Advances in the diagnosis and treatment of fungal infections of the CNS. Lancet Neurology. 2018;17:362–372. (4) McCarthy M, Rosengart A, Schuetz AN, et al. Mold Infections of the Central Nervous System. N Engl J Med. 2014; 10(371-2): 150–160.

Plenary session 6—Management of IFD in paediatrics (EPMyN)

PS6.1. Biomarker based diagnostic work-up of IFD in immunocompromised paediatric patients

T. Lehrnbecher
Pediatric Hematology and Oncology, University of Frankfurt, Frankfurt, Germany

Abstract:

Immunocompromised pediatric patients such as children undergoing therapy for hematological malignancies or receiving allogeneic hematopoietic stem cell transplantation are at high risk for invasive fungal diseases, which are a major cause of morbidity and mortality in these patient populations. Unfortunately, clinical signs and symptoms of invasive fungal disease are mostly unspecific, which makes early diagnosis difficult. However, early diagnosis and prompt treatment of invasive fungal disease is associated with better outcome. Non-invasive antigen-based assays such as galactomannan (GM) or β-d-glucan and molecular biomarkers in peripheral blood have the potential to indicate invasive fungal disease prior to the development of clinical symptoms, and may therefore be helpful in the decision to institute and choose antifungal compounds and also to guide duration of therapy. When GM in blood is used as a diagnostic test in neutropenic children with prolonged febrile neutropenia unresponsive to broad-spectrum antibiotics or in children with a pulmonary infiltrate in the CT scan, sensitivity and specificity of the assay do not significantly differ between children and adults. However, the utility of the assay is clearly limited by the poor positive predictive value and the fact that despite the high negative predictive value of the test, a negative GM assay does not rule out non-Aspergillus molds. Data on β-d-glucan in immunocompromised children are too limited to give a final recommendation on its use, and similarly, the results of PCR testing in blood are variable and do not allow a firm conclusion on its utility. However, despite these limitations, biomarkers in blood may help to plan invasive diagnostic procedures such as broncho-alveolar lavage (BAL) or bioptic procedures. As compared to blood, both galactomannan and PCR testing in BAL lavage seem to be superior, but again, further studies in the pediatric population are warranted. Regarding the value of biomarker in CNS samples such as cerebrospinal fluid, little is known in both pediatric and adult patients. Further pediatric studies are warranted to evaluate whether the combination of diagnostic methods can improve sensitivity and specificity, but in addition, new diagnostic tools are needed that will improve the early and reliable diagnosis of invasive fungal disease in children.

PS6.2. Pre-emptive versus empiric antifungal therapy in children with cancer

M.E. Santolaya
Hospital de niños Dr. Luis Calvo Mackenna, Santiago, Chile

Abstract:

Invasive fungal disease (IFD) causes significant morbidity and mortality in paediatric cancer patients with high-risk febrile neutropenia (HRFN), along with high utilization of resources for prevention, diagnosis and treatment. Early diagnosis of IFD and prompt implementation of aggressive antifungal treatment have proven to be critical for patient survival. Nevertheless, early identification of the causal pathogen of an IFD continues to be difficult. The classic approach is currently based on clinical, imaging, microbiological (cultures from sterile sites) and histological studies. Major advances for early diagnosis of IFD have been made by the development of non-culture assays such as detection of galactomannan (GM) antigen, (1-3)-b-d-glucan antigen detection and nucleic acid detection, by PCR techniques. Despite these advances, IFD diagnosis continues to be a challenge and current recommendations propose to initiate empirical antifungal therapy in IFD high-risk paediatric patients with persistent (96 h) fever and neutropenia that are unresponsive to broad-spectrum antibacterial agents. The downside of this approach is the overtreatment of patients meeting the above criteria but who do not have an IFD, leading to an increase in adverse events, prolonged hospitalizations and elevated costs associated with the use of antifungal drugs. A more reasonable approach in cancer subjects would be to consider early identification of patients at high risk of IFD, application of a complete screening diagnosis strategy followed by a rational approach to antifungal therapy based on results of this early and extensive diagnostic workup, adopting a more selective preemptive treatment strategy in patients with persistent fever and neutropenia. We performed a prospective, multicenter study in children with cancer and persistent HRFN, aimed to determine the efficacy of pre-emptive treatment compared with current standard empirical antifungal treatment. In our study, pre-emptive antifungal therapy was as effective as empirical antifungal therapy, with a significant reduction in antifungal use. A reduction of antifungal use, based on stringent diagnostic criteria, could favor the adoption of evidence-based management strategies in this population. This approach requires optimal laboratory support with rapid turnaround response. Introduction of non-culture-based diagnostic techniques into clinical practice could contribute to better management of these patients, favoring the possibility of patient-based individualized therapy. Active monitoring and early diagnostic workup is a necessary step prior to proposing an evidence-based management strategy. In our experience, 58% of patients of the pre-emptive group did not receive antifungal therapy, with similar clinical outcome to the empirical group, with the aim of reserving antifungal therapy for the subset of patients who have early evidence of IFD by careful clinical, laboratory, imaging and microbiological assessment. The main findings of our study lead us to propose a step forward in the rational approach to treating children with cancer focusing on one yet-unresolved issue in the management of the patients: adoption of a more selective pre-emptive antifungal treatment strategy in children with prolonged fever and neutropenia.

PS6.3. Invasive mucormycosis in children with hematological malignancies and solid tumors: Report from the Infection Working Group of the Hellenic Society of Pediatric Hematology Oncology (2008-2017)

C. Antoniadi 1, I. Lialias 2, H. Tsipou 3, N. Kazakou 4, E. Iosifidis 2, E. Papakonstantinou 5, S. Polychronopoulou 1, A. Kattamis 3, E. Roilides 2 and A. Tragiannidis 4
1
Department of Pediatric Hematology-oncology, "Agia Sofia" Children’s Hospital, Athens, ATHENS, Greece
2
3rd Pediatric Department, Aristotle University of Thessaloniki, Hippokration Hospital, THESSALONIKI, Greece
3
Pediatric Hematology-oncology Unit, 1st Pediatric Department, National and Kapodistrian University of Athens, "Agia Sofia" Children’s Hospital, ATHENS, Greece
4
Haematology-oncology Unit, 2nd Pediatric Department, Aristotle University of Thessaloniki, AHEPA Hospital, Thessaloniki, Greece
5
Department of Pediatric Hematology-oncology, Hippokration Hospital, THESSALONIKI, Greece
Objectives: Mucormycosis is an invasive, life-threatening fungal infection that mainly affects immunocompromised hosts. Zygomycetes can invade virtually any tissue or organ, resulting in a variety of clinical presentations. The Infection Working Group (IWG) of the Hellenic Society of Pediatric Hematology-Oncology (HELSPHO) collected and analyzed data of pediatric mucormycosis cases from 7 Hematology-Oncology Departments within 2008–2017.
Methods: Six cases of proven invasive mucormycosis were reported retrospectively (female/male: 2/4, median age: 9.6 years, range: 2–15 years) during chemotherapy for hematologic malignancies and solid tumors within 2008–2017.
Results: Among the 6 children with invasive mucormycosis, three presented with acute lymphoblastic leukaemia (ALL), one with acute myeloid leukemia (AML), one with neuroblastoma and one child with brain tumor, as the primary underlying malignancy. In 4/6 children (66%), mucormycosis occurred within the first 20 days from the diagnosis of the underlying disease. One child with ALL developed mucormycosis 4 months since the initial diagnosis, with prior invasive aspergillosis two months earlier treated with voriconazole. Four out of 6 patients had received prolonged corticosteroid treatment and 3/6 had severe neutropenia with a neutrophil count <500 μL at the time of diagnosis. Four out of 6 patients (66%) had received intensive chemotherapy within the last 4 weeks and all patients had been hospitalized for at least 20 days. All patients presented with fever (100%), 4/6 with pain (66%), 3/6 with palpable mass (50%) (one at the left forearm and two at the orbit) and 3 patients presented with central nervous system symptoms (seizures, headache, and blur of vision) (50%). Primary infection sites were combined paranasal sinus with orbital involvement (2), central nervous system (1), occipital area (1), orbital involvement (1) and forearm (1). The diagnosis of mucormycosis was documented by histology and mycology (PCR or culture) in all patients (2 Rhizopus arrhizus,1 Mucor spp., 1 Absidia spp., 2 unidentified Mucorales). Dissemination was defined by clinical signs, imaging studies and histology in 4/6 (66%) patients. Four (4) out of 6 patients had received prophylactic treatment with antifungal agents (2/4 patients received micafungin and 2/4 l-Amphotericin B). Therapeutically, all patients received l-Amphotericin B (l-AmB) (median dose: 7.5mg/kg, range: 3-10 mg/kg). Two (2) patients received combined antifungal treatment with l-AmB and caspofungin, one (1) l-AmB and voriconazole, one (1) l-AmB and posaconazole and one (1) l-AmB, voriconazole and caspofungin. Three (3) patients received maintenance treatment with posaconazole. Surgical excision was performed in 4/6 cases (66%). Two out of 6 patients (33%) died, after 6 and 12 months, respectively.
Conclusion: Pediatric invasive mucormycosis mainly presents as paranasal sinus and orbital disease and is still related to high mortality rates. Our data demonstrated that the disease occurs early after initiation of intensive chemotherapy and febrile neutropenia and prolonged corticosteroids are the main predisposing factors. Early recognition and prompt intervention with combined prolonged antifungal and surgical treatment may rescue the patients and improve overall survival.

Plenary session 8—New (antineoplastic) drugs, new risks

PS8.1. New antineoplastic drugs, new risks in hematology

A. Busca
Division of Hematology, AOU Citta della Salute e della Scienza, Torino, Turin, Italy

Abstract:

Over the last 10 to 15 years, therapeutic options for patients with hematologic malignancies have largely increased with the growth of new immunotherapeutic agents. At least two issues regarding invasive fungal infections (IFI) and the new immunotherapeutic agents should be addressed. First, there are relevant pharmacokinetic interactions of the new targeted therapies with coadministered mold active azoles, namely posaconazole and voriconazole that are strong inhibitors of CYP3A4. Awareness of these interactions is of paramount importance for optimization of the treatment of patients, since we are asked to monitor for potential toxicity, or to modulate the dose of the drugs or rather to avoid the combination and consider alternative therapy. Second, is the risk of IFI increased with the use of the new agents? For many of these, for instance monoclonal antibodies (MoAb), bcl-2 inhibitors (i.e. venetoclax) and tyrosine kinase inhibitors (TKI) (i.e. sorafenib and midostaurin) when used for the treatment of patients with acute leukemia, it is hard to realize whether they have an additive risk to the underlying disease: indeed, there are very few information respect the incidence of IFI based on the clinical trials published so far. By contrast, several studies raised concern that the Bruton’s tyrosine kinase inhibitor Ibrutinib may increase the risk for IFI in patients with lymphomas. In this respect, different mechanisms by which Ibrunitib may favor the occurrence of IFI have been described, including alveolar macrophage, neutrophil, T-cell, and platelet function as well as alterations of cytokine environment. A recent study of Ibrutinib treatment for primary CNS lymphoma reported a 39% rate of invasive aspergillosis in patients concurrently receiving steroids even in absence of neutropenia, while in other reports the incidence appears to be much lower in the 3–4% range. Conversely, the risk of IFI with the use of Idelalisib, another B-cell receptor inhibitor, seems to be restricted to Pneumocystis jirovecii pneumonia only. Until more detailed epidemiological data will be available, anti-mold prophylaxis is not recommended for patients receiving Ibrutinib or Idelalisib. Chimeric antigen receptor (CAR) T cell therapy is an innovative strategy to harness the immune system to treat patients with diffuse large B cell lymphoma (DLBCL). CAR-T cells are T lymphocytes genetically engineered to express a tumor-targeting receptor, directing T cells to bind to a tumor-associated antigen, namely the CD19 in the case of DLBCL. A recent study evaluated infectious complications in 133 patients with ALL and lymphomas treated with CD19 CAR-T cells: IFI occurred in 5% of the patients during the first 28 days after CAR-T cell infusion, all of whom had severe cytokine release syndrome (CRS) or neurotoxicity requiring tocilizumab and/or corticosteroids. CRS severity was the only factor after CAR-T cell infusion associated with infection in a multivariable analysis. In summary, the risk of IFI in patients receiving targeted therapies needs to be defined by further experience in clinical practice and ongoing research trials. Preventive strategies of IFI in patients managed long term with immunotherapy remain at the present a challenge.

PS8.2. In oncology

L. Drgona
Department of Oncohematology, Comenius University and National Cancer Institute, Bratislava, Slovak Republic

Abstract:

The field of new antineoplastic therapeutical agents is increasing rapidly over the last decade. New drugs in oncology are basically considered as targeted ones due to their mechanism of action. These drugs directly target the cells or pathways involved in pathophysiology of neoplastic disease. Cell surface receptors, cytokines, immunoglobulines, intracellular enzymes are the most common targets involved in the therapy. Other drugs are focusing on the patient’s own immune system with the aim to boost the responsible cells to fight against the disease. The new drugs have different mechanisms of action resulted in less “classical“ toxicity known from chemotherapy agents. The adverse events after the targeted therapy are mainly caused by interaction with the immune system—targeted sites are often key elements of physiological processes like cell cycle control. This may have an influence on responses to acute infection or on control of latent/chronic infection. Theoretically, the potential of the new drugs to predispose to infection depends on their site of action. But this is simplification of the whole problem, because cancer patients are treated for months or years before the installation of the innovative therapy. Thus, an old, traditional risk factors for infectious complications should be added to the overall risk for the given patient. Invasive fungal diseases (IFDs) are associated with the treatment of cancer, patients with solid tumors have lower risk than majority of patients with hematological malignancies. The obvious risk factors for IFDs in patients with solid tumors are neutropenia, steroids, central venous catheter, progressive or refractory malignancy, gastrointestinal surgery with leak of anastomosis among others. Invasive candidiasis followed by invasive aspergillosis are the most common IFDs in this setting, other pathogens are less involved. With the advent of the new therapeutic approaches in oncology patients and physicians are challenged with the new spectrum of adverse events including infections. Generally, monoclonal antibodies, tyrosine kinase inhibitors, check point inhibitors and other targeted and biological agents are not associated with the increasing risk of IFDs in solid tumor patients. In special circumstancies patient undergoing such antineoplastic therapy may have an additional risk for IFDs. An example are the patients with melanoma or lung cancer treated with check point inhibitor developing an immune related adverse event. High dose steroids with or without anti -TNF blocker are recommended in the management of the serious toxicity; these patients are potentially at risk for IFD development due the supportive treatment of AE. Risk of IFD will be discussed in the patients with solid tumors reflecting the advances of antineoplastic treatment. Despite the relatively low risk for the development of IFDs and slowly growing experience some recommendations could be made. Anticipation, prediction and alertness are the pillars of action in this setting of cancer patients.

Meet the expert sessions

M04. Novelties in the molecular diagnostics of fungal infections

M. Lackner
Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria
The aim of this meet the expert session is to provide an overview on the most recent developed diagnostic kits in the field of medical mycology. We aim to present and compare novel diagnostic tools with established assay based on our personal experience and available literature. The presentation will be based on a literature review of the past 18 months and a web search for commercially available products. A mix of currently developed assays of research groups will be presented, as well as CE-IVD labelled commercial tests. The most promising innovative diagnostic assays will be presented and discussed. The audience is invited to share their experience with novel assays to enable a lively discussion and exchange of opinions.

M12. Environment

R. Sabino 1 and E. Segal 2
1
Reference Unit for Parasitic and Fungal Infections, Infectious Diseases, National Health Institute Dr. Ricardo Jorge, Lisbon, Portugal
2
Sackler School of Medicine Tel-Aviv University, Tel-Aviv, Israel

Abstract:

Exposure to environmental fungi, whether this occurs indoors or outdoors, in leisure activities, in the workplace or in the home, is an everyday occurrence. Different types of environment may contain variable levels of fungal particles and include viable and non-viable yeasts, conidia, hyphal fragments, as well as fragments derived from the fungal cell wall. Mycotoxins and other volatile organic compounds should also be considered as environmental potential hazards. Recognition of the importance of the environment as a source of human infection has come about, at least in part, as result of the emergence of an unprecedented number of ubiquitous environmental fungi as major causes of disease. Exposure to environmental fungi is associated with high number of hospital admissions, and asthma related ailments. Allergic bronchopulmonary mycosis, rhinosinositis, asthma with fungal sensitization and hypersensivity pneumonitis are among the diseases more frequently associated with fungal exposure. In addition, immunocompromised patients are at higher risk for the development of invasive infections. Changes in environmental factors, including changing land-use patterns, use of antifungals in agriculture, and climate changes have led to epidemiological shifts. Therefore, special attention should be paid in regard to the isolated fungal species. Environmental fungal species such as Aspergillus spp., Mucorales, Candida spp., dermatophytes and dimorphic fungi will be discussed during this session; emphasizing their importance as etiological agents of fungal infections.

M13. Diagnostics/Imaging

C. Thornton
Biosciences, University of Exeter and ISCA Diagnostics Ltd., Exeter, United Kingdom

Abstract:

Bench-to-Bedside Diagnosis of Invasive Pulmonary Aspergillosis. Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of haematological malignancy and bone marrow transplant patients caused by the air-borne mould Aspergillus. Current diagnostic tests for the disease lack sensitivity or specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. While Computed Tomography (CT) is a non-invasive diagnostic procedure, it has limited clinical utility for IPA diagnosis, but is nevertheless used as a trigger for commencing antifungal treatment in a number of centres. Innovative approaches to the diagnosis of IPA are needed to enable diagnostic-driven treatment with mould active drugs. In this talk, I will discuss the development of diagnostic technologies for IPA detection based on the Aspergillus-specific mouse monoclonal antibody JF5, including the CE-marked Aspergillus lateral-flow device (OLM Diagnostics), and CE-marked Aspergillus-ELISA (Euroimmun AG). In addition, I will describe the humanisation of mAb JF5 for PET/MR imaging of Aspergillus lung infections in vivo, and translation of the imaging technology to the clinical setting. I will finish by describing the recent development of a mAb, PD7, specific to A. fumigatus, and its use to detect a novel protein biomarker of IPA in urine.

M14. Resistance—Utilizing molecular methods

P. White
Microbiology, Public Health Wales Microbiology, Cardiff, United Kingdom

Abstract:

Until recently the identification of antifungal resistance has been based on the availability of a cultured organism to perform susceptibility testing to determine a MIC against a particular antifungal agent. The development of molecular techniques has provided an alternative option, by identifying genetic markers potentially associated with resistance or identifying species that may by inherently resistant, or have an increased likelihood of resistance to particular drugs. Indeed, molecular assays offer the only alternative to culture dependent procedures, that themselves can suffer from limited sensitivity and delayed time to result. However, molecular approaches are not without limitations. PCR based methods targeting specific mutations (for example: single nucleotide polymorphisms and/or tandem repeats), by design have a limited detection range, missing existing, likely less prevalent mutations, but also the presentation of novel genetic mechanisms. PCR methods that identify species that are usually resistant to certain antifungal drugs will be reliant on the knowledge of local epidemiology and the subsequent susceptibility testing of relevant cultures to guide antifungal prescribing. The use of PCR-sequencing, but more so next generation sequencing, will help identify a wider range of polymorphisms in genes encoding the proteins targeted by antifungal therapy and also in promoter regions that may enhance up-regulation, but the detection of novel mutations may lack the necessary phenotypic evidence confirming associated resistance. In addition, PCR-sequencing is associated with a significant processing time and has only had limited evaluation of direct specimen testing, where sensitivity may be lacking when targeting single copy genes. As of yet none of these methods can be confidently associated with a specific MIC range, and the presence of well defined polymorphisms have been associated with typically susceptible MIC values. This presentation will describe the benefits and limitations of using molecular methods for determining antifungal resistance, while providing data on the clinical performance of assays, including those that are commercially manufactured.

Symposium 1 Water quality (mis-)management—an opportunity for fungal contamination

S01.1. Fungal Contaminants in Drinking Water Regulation? A Tale of Ecology, Exposure, Purification and Clinical Relevance

M. Novak Babič 1, N. Gunde-Cimerman 1 and J. Brandão 2
1
Department of Biology, University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia
2
Department of Environmental Health, National Institute of Health Doutor Ricardo Jorge, Lisbon, Portugal
Safe drinking water is one of the fundamental human rights according to the Resolution 64/292 issued by the United Nations Human Rights Council [1]. Although the parameters for safe drinking water depend on the living conditions and vary between continents and countries, they strive for a common goal: drinking water is suitable for users when it does not contain dangerous chemical substances and microorganisms [2]. To achieve this, raw water from an underground or surface source is subject to various purification techniques before entering the distribution system. The processes usually include aeration, coagulation, flocculation, sedimentation, and ultrafiltration, depending on the location and quality of raw water [3]. Finally, water quality in Europe is controlled based on the parameters listed in Drinking Water Directive (98/83/CE). Water not reaching the required microbiological minimum is additionally subject to chemical treatment, mostly chlorination. Fungi are not listed in the current directive and are therefore not specifically monitored. Yet, their presence in fresh water is well documented and has been studied in at least 19 countries of Europe over the past 30 years [3]. More than 400 different species of fungi have been reported from water sources intended for human consumption, with prevailing presence of ascomycetous fungi of the genera Alternaria, Acremonium, Aspergillus, Aureobasidium, Candida, Cladosporium, Exophiala, Fusarium, Penicillium, Phoma, Sarocladium, Scopulariopsis, Sporothrix, Stachybotrys and Trichoderma, followed by Absidia, Mortierella, Mucor and Rhizopus from subphylum Mucoromycotina. Most commonly encountered yeasts were basidiomycetous genera Cystobasidium, Naganishia (formerly Cryptococcus) and Rhodotorula [3]. While water purification procedures remove 8–90% of fungal propagules, the remaining will ultimately form mixed biofilms inside the tap water distribution systems, later affecting the taste and odour of drinking water. Also, several water-related fungal species were recognised as opportunistic or emerging pathogens, although their transmission and effect on the health of drinking water consumers remains poorly investigated. Besides drinking, consumers use potable water also for daily activities such as cooking, washing and personal hygiene. During these activities they are in constant contact with fungi and their metabolites directly with the skin while washing, the digestive system while drinking, and through the respiratory system by inhaling aqueous aerosols [3,4]. With increasing transitory and serious immune alterations among patients as well as an increase of azole-resistant fungal strains in recent years also a need for monitoring of fungi increased, not only in drinking water but also in relation to materials in contact with drinking water.

References:

[1]
UN. Resolution adopted by the General Assembly on 28 July 2010 64/292. The human right to water and sanitation. United Nations: New York, USA, 2010; p. 3.
[2]
WHO. Guidelines for Drinking Water Quality, 4th ed.; World Health Organization: Geneva, Switzerland, 2011; p. 564.
[3]
Novak Babič, M., et al. Fungal Contaminants in DrinkingWater Regulation? A Tale of Ecology, Exposure, Purification and Clinical Relevance. Int. J. Environ. Res. Public Health 2017, 14, 636.
[4]
DEFRA. A Review of Fungi in Drinking Water and the Implications for Human Health, 1st ed.; BIO Intelligence Service: Paris, France, 2011; p. 107.

    S01.2. Hospital environment: water supply and containment of aerosolised fungal particles. How far must we go in times of antimicrobial resistance?

    R. Sabino
    Reference Unit for Parasitic and Fungal Infections, Infectious Diseases, National Health Institute Dr. Ricardo Jorge, Lisbon, Portugal
    Invasive fungal infections depend on the interplay between host susceptibility and environmental exposure. Therefore, hospital environment is one of the major concerns in the management of nosocomial fungal infections, especially in wards bearing immunocompromised patients. Particular attention should be paid to the environmental risks associated with water since fungi can be aerosolized at water taps and showers. This may lead to fungal exposure by inhaled and ingested droplets, or even by direct contact with mucosae. Studies report that filamentous fungi and yeasts are commonly found on water-pipe inner surfaces, even in the presence of free chlorine. Air levels of Fusarium and Aspergillus conidia were found to increase in hospital environments after running showers multiple times. Species of these two genera are described as the most frequently found in this setting due to their conidial dispersion mode, as well as their ability to form biofilms. Despite the intrinsic resistance found in some species of these two genera, fungal exposure to antifungal agents via medical or agricultural use of these compounds, appears to have a major impact on acquisition of resistance to azoles; namely in Aspergillus fumigatus. The isolation of this species in hospital water and water reservoirs is therefore an even bigger matter of concern. More recently, several reports on nosocomial outbreaks caused by Candida auris have been described. This species is resistant to several classes of antifungals and is associated with high mortality. Contamination of hospital environment or transient colonization of medical devices and equipment may display an important role in the transmission of this species. C. auris was already found in water samples and therefore this reservoir should not be excluded as possible source of infection. In conclusion, fungal counts and detection of potential pathogenic species in water were, until a few years ago, the major concern of clinical and scientific community towards the reduction of nosocomial fungal infections originating in water devices. The emergence of infections caused by fungal isolates with intrinsic or acquired antifungal resistance triggered new levels of alert in this field.

    S01.5. Spectrum of indoor fungi isolated from indoor environments in Busia-Kenya

    O. Mashedi 1, E. Amukoye 2, C. Bii 3, B. Kimani 3, A. Chepchirchir 4 and S. Okoth 5
    1
    Centre For Respiratory Dise Ases Research, Kenya Medical Research Institute, Nairobi, Kenya
    2
    Centre For Respiratory Diseases Research, Kenya Medical Research Institute, Nairobi, Kenya
    3
    Centre For Microbiology Research, Kenya Medical Research Institute, Nairobi, Kenya
    4
    School of Health Sciences, University of Nairobi, Nairobi, Kenya
    5
    School of Biological Sciences, University of Nairobi, Nairobi, Kenya
    Objectives: The unplanned growth, rapid evolution of industrialization has deteriorated the ambient air quality. Anthropogenic indoor air pollution continues to be seen as an environmental and public health problem. Its seriousness lies in exposure to fungi that can trigger allergic reactions, hypersensitivity pneumonitis, allergic rhinitis, and some types of asthma. Very few studies in Kenya have been conducted to determine the possible types of indoor fungi allergens in indoor environment and to evaluate the relationship between allergen exposure in residential environments and respiratory morbidity. The aim of this study is to determine the prevalence of fungal indoor pollution in residential environments and to establish allergens which are associated with fungal pathogens.
    Methods: This was a cross-sectional study carried out in 60 households in Busia County. Sedimentary and Volumetric sample collection method was used on water, air, swabs of dust and surfaces from homes. Mycological procedures in identification and analysis were used in fungal characterization. Comprehensive questionnaires were administered to determine the risk factors associated with indoor fungi infestation.
    Results: 60 study participants were interviewed from Busia urban setting with mean age of 35.2 ± 7.7 (SD) years where most 60.3% of them were aged between 40–49 years; while 19.0% were aged 20–29 years. 68.4% of the respondents were females. As regard to education background, 61.4% of the respondents had primary education as the highest education level whereas 3.5% had tertiary level. 43.1% of the households were occupied by more than 5 people. Filamentous and yeast fungi were both isolated. Candida albicans and Rhodotorula accounted for the 39% of the isolated yeast species. Among the filamentous fungi Fusarium had the highest prevalence of 79.7%, followed by Cladosporium spp, Alternaria spp, Acremonium Apergillus and Penicillium respectively. There was a significant association between Fusarium and Acremonium moulds and respiratory conditions. Acremonium species was significantly associated with itchy eyes allergies, p = 0.023.
    Conclusion: There is a need for the identification and monitoring of fungi in our home environment. Residential environment could potentially be considered an effective target for interventions aimed at reducing inciting fungi exposures to prevent upper respiratory allergies and infection in urban populations.

    Symposium 2 Pneumocystis jirovecii, an airborne transmissible and human derived ascomycete showing strong pulmonary tropism

    S02.1. Pneumocystis jirovecii: an obligate parasite of human lungs with unique camouflage and sex strategies

    P. Hauser
    Institute of Microbiology, CHUV Hospital, Lausanne, Switzerland

    Abstract:

    Pneumocystis jirovecii is a fungus colonizing the human lungs. Should the immune system weaken, it can turn into an opportunistic pathogen causing severe pneumonia that can be fatal if not treated. A method of in vitro long-term culture for this pathogen is not available yet. Nevertheless, the advent of the high throughput DNA sequencing methods allowed sequencing the P. jirovecii genome out of the lung microbiome. The analysis of this genome revealed the loss of several synthesis pathways, demonstrating that this fungus is an obligate parasite. The presence of a fusion of the minus and plus mating-type loci suggested that its mode of sexual reproduction is primary homothallism (self-fertility). The sexuality of P. jirovecii proved to be obligatory with human lungs, which is compatible with asci and/or ascospores being the particles responsible for the transmission between humans by the air route. Long reads sequencing permitted characterizing six hypervariable gene families encoding surface antigens within the subtelomeres of all chromosomes. These gene families are responsible for surface antigenic variation allowing presumably escape from the human immune system, as well as for adhesion to host cells. The most abundant family is subject to mutually exclusive expression of a single gene, whereas each gene of the other families possesses its own promoter, suggesting independent expression. Recombinations between members of each family is thought to generate gene mosaicism that contribute to antigenic variation. The mechanisms involved in P. jirovecii antigenic variation suggest that the infecting populations are antigenically heterogeneous, i.e., made of subpopulations displaying each a unique antigen of the most abundant family, as well as a minority of antigens of the other families.

    S02.2. High-throughput methodologies in molecular epidemiology of Pneumocystis jirovecii

    O. Matos
    Medical Parasitology Unit, Group of Opportunistic Protozoa/hiv and Other Protozoa, Global Health and Tropical Medicine, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal

    Abstract:

    The microscopic fungus Pneumocystis jirovecii is a pathogen that can cause fatal pneumonia (PcP) in immunocompromised patients (AIDS, transplant recipients, patients with haematologic and solid malignancies, patients with rheumatoid conditions and connective tissue disorders, patients receiving immuno-modulatory therapies or with pre-existing chronic lung conditions) worldwide. In the absence of an in vitro culture system to isolate and maintain live organisms, previous efforts to understand transmission patterns to develop methods of intervention and management for PcP relied on PCR-based approaches. Several genotyping methods have been used so far to address the genetic heterogeneity of P. jirovecii, PCR of multiple loci followed by direct DNA sequencing (Sanger), restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), short tandem repeat (STR) or single-base extension (SBE). These methodologies were followed by the introduction of next generation sequencing (NGS), which is considered a revolution in the field of DNA analysis. NGS has been applied in the assembly of P. jirovecii whole genome and in amplicon analysis, especially for outbreaks investigation. Here we will explore the advantages and disadvantages of these approaches applied in the expansion of knowledge of the epidemiology of P. jirovecii.

    S02.3. Airborne acquisition and transmission of Pneumocystis jirovecii: an update

    G. Nevez 1,2, P. Laurence 1,2 and S. Le Gal 1,2
    1
    University of Brest, Brest, France
    2
    Host-Pathogen Interaction Study Group, SFR ICAT 4208, Univ Angers, Univ Brest, Institute of Biology in Health, IRIS, University Hospital Center, Angers, France, Angers Cedex, France

    Abstract:

    Airborne transmission of Pneumocystis sp. from host to host has been demonstrated in rodent models and several observations suggest that interindividual transmission occurs in humans. Moreover, it is accepted that the Pneumocystis organisms infecting each mammalian species are host specific. Thus, the hypothesis of an animal reservoir for Pneumocystis jirovecii (P. jirovecii), the human-specific Pneumocystis species, can be excluded. An exosaprophytic form of the fungus cannot be strictly ruled out. Nonetheless, these data point out that: (i) the specific host may serve as its own reservoir (ii) Pneumocystis infection in humans is an anthroponosis with humans as a reservoir for P. jirovecii. This review highlights the main data on host-to-host transmission of Pneumocystis sp. in rodent models and in humans by the airborne route. Specifically, recent data on P. jirovecii exhalation by patients developing PCP or with pulmonary colonization in their surrounding environment are described. Taken together, these recent findings render it possible to provide a rationale for prevention of P. jirovecii acquisition and transmission within the hospitals.

    S02.5. Does Pneumocystis jiroveci infection aggravate the prognosis of invasive pulmonary aspergillosis? Data from the RESSIF network in France (2012–2016)

    F. Robert-Gangneux 1, G. Bouzille 2, F. Dromer 3, J.P. Gangneux 4 and T.H.E. French Mycoses Study Group 5
    1
    Univ Rennes, CHU Rennes, Inserm, EHESP, IRSET UMR S_U1085, RENNES, France
    2
    Univ Rennes, CHU Rennes, Inserm, LTSI – UMR 1099, RENNES, France
    3
    National Reference Center For Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, Paris, France
    4
    Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France
    5
    National Reference Center For Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, RENNES, France
    Objectives:Pneumocystis jiroveci pneumonia (PjP) is a frequent opportunistic infection in immunocompromised patients with various immune deficiency backgrounds, whereas invasive aspergillosis (IA) mostly occurs in patients with neutropenia or neutrophil impairment. Occasionally both fungi can be detected simultaneously. In that instance, Pj detection is sometimes considered as simple colonization, particularly when microscopic examination is negative and PCR is positive. In this study, we addressed the question of the presence of Pj as an aggravating factor during IA.
    Methods: We took advantage of the surveillance of invasive fungal infections (IFIs) implemented by the National Reference Center for Invasive Mycoses and Antifungals through the RESSIF network. All cases of IA and PjP recorded during a 5-year period (2012–2016) were analyzed. Multivariate analysis compared cases of IA and cases of IA+PjP co-infection, using Cox regression after multiple imputation of missing data. All cases with a follow-up of 90 days were taken into account, extra-pulmonary aspergillosis was excluded. The following variable were included for analysis: age, sex, clinical background (hematological malignancy, allogeneic stem cell transplantation (allo-HSCT), auto-HSCT, graft versus host (GVH) disease, solid organ transplantation (SOT), cancer), corticotherapy or biotherapy, delay of AI onset, AI classification (proven, probable), and Aspergillus species.
    Results: 730 patients were included, of whom 701 had IA and 29 had IA+PjP. The mean age of patients with IA and IA+PjP was 58.5 and 61.3, respectively (p = 0.08). There were no statistical differences in the sex ratio, clinical background, type of SOT, corticotherapy or biotherapy exposure, time from transplantation to diagnosis, time from hospitalization to IA diagnosis, time to death, nor classification of IA, between the two groups. The mortality rate was higher in the group IA+PjP than in IA alone (65% versus 39%, respectively, p = 0.009). Species from the Fumigati section accounted for 58.6% and 43.1% of cases in IA+PjP and IA, respectively (ns). In the multivariate analysis, the factos independently associated with death at 3 months were: age > 60 years (HR = 1.74 ; CI95% [1.4–2.2]), solid cancer (HR = 2.2 ; CI95% [1.1–4.6]), co-infection AI+PjP (HR = 1.9 ; CI95% [1.2–3.1]) and section Fumigati IA (HR = 1.65 ; CI95% [1.3–2.1]).
    Conclusion: Pj infection is an aggravating factor of IA, particularly in patients with solid cancer. No other associated risk factor was uncovered probably due to the limited number of co-infections recorded. We plan to extend the study period to increase the statistical power of the analysis.

    Symposium 3 Mucormycosis

    S03.1. Hospital related mucormycosis

    A. Skiada
    1st Dpt. of Medicine, Laiko Hospital, Athens University, Athens, Greece

    Abstract:

    Mucormycosis is a rare invasive fungal infection due to fungi of the order Mucorales. The usual underlying diseases are hematological malignancies and diabetes mellitus, but a significant proportion of patients are immunocompetent. The main clinical presentations are rhinocerebral, pulmonary, cutaneous and disseminated. The agents of mucormycosis are ubiquitous in nature, and they are transmitted by inhalation, direct inoculation or ingestion. There have been multiple reports of healthcare associated mucormycosis, either as isolated cases or as outbreaks. In a publication from India, during an eighteen month period, 75 cases of mucormycosis were reported, of which 9% were nosocomial. Healthcare associated mucormycosis has been attributed to various exposures in the hospital environment, including: (a) Use of non-sterile products. This is the most commonly suspected cause of infection. Bandages, adhesives, nitroglycerin patches, contaminated linen, wooden tongue depressors, ostomy bags, probiotics, have all been implicated. There has even been a report of an outbreak due to allopurinol tablets and pre-packaged food. (b) Various procedures and medical devices, such as catheters, insulin pumps and finger sticks, as well as insertion of tubes, tooth extractions and surgery. (c) Environmental factors may also be a source of infection. Molds may be found in the air, dust, water or any surfaces in the hospital. Construction works increase the risk of invasive fungal infections. Outbreaks have been linked to defective ventilation systems and water leakage. The clinical presentation varies, depending on the source of infection. Infections due to bandages, adhesives or contaminated wound dressings are mostly cutaneous. Percutaneous exposure in immunocompromised patients has led to disseminated disease. Inhalation leads to pulmonary and rhino-cerebral infection, while ingestion of tablets or food, as well as the use of tongue depressors, are responsible for gastrointestinal mucormycosis. Dialysis catheters have been linked to peritonitis. It is not easy to investigate a possible mucormycosis outbreak. The Centers of Disease Control of USA have recently published a guide to investigating suspected outbreaks due to mucormycosis. Due to the rarity of the disease, investigation is warranted even when it is unclear if there is a true outbreak. The first step is to verify the diagnosis and define a case. Histopathology and culture are the mainstay of diagnosis. In recent years, newer tools have been added to our diagnostic armamentarium. These include PCR, whole-genome sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The next step in the investigation of an outbreak is to describe the epidemiologic features of the cases, scrutinize patient care activities and determine environmental involvement. The management of outbreaks is multi-disciplinary and should include treating physicians, hospital administration, workers in the microbiology laboratory and health departments. Rapid identification of cases and initiation of treatment is important, in order to decrease mortality of existing cases and prevent the emergence of new ones.

    S03.3. The new treatment of mucormycosis

    L. Pagano
    Fondazione Policlinico Universitario A. Gemelli—IRCCS—Universita Cattolica del Sacro Cuore, Rome, Italy
    The antifungal drug that showed the best in vivo activity in the past has been deoxycolate Amphotericin B (d-AmB). Although this molecule, although extremely effective, has been characterized by an important series of side effects (i.e. renal insufficiency, hypokalemia) which have made its use disadvantageous. In more recent years, the introduction of new formulations of AmB in lipid formulations (l-AmB, ABCD, ABLC) have mitigated the unpleasant side effects without eliminating them completely but have made their use more manageable. Nowadays lipid formulations, and especially l-AmB, are considered by all the international guidelines as the first-choice drugs for the treatment of mucormycosis. In recent years new azole formulations, first and second-generation azoles (i.e. posaconazole, isavuconazole) have shown considerable efficacy in the treatment of these molds even in highly immunocompromised patients such as those suffering from acute leukemia or undergoing bone marrow transplantation, whereas voriconazole and itraconazole show no effect on mucorales. Posaconazole was registered as a drug to be used as an alternative to liposomal amphotericin and often the availability of the oral solution alone has considerably limited its use. The oral-only route of posaconazole is anyway a great limitation, especially in hematologic patients, often inable to take drug per os or undergoing chemotherapy with subsequent high incidence of vomiting. However the availability of posaconazole in intravenous formulation and in coated tablets has favored a greater absorbability and, consequently, greater efficacy. Isavuconazole in registrative trial has shown a good efficacy in the treatment of this fungal complication and is now recognized as an excellent alternative. We must also take into account the possibility, supported in recent years by multiple studies in real life, to approach the most aggressive and disseminated forms of mucormycosis with the combination of multiple drugs. l-Amb combined with caspofungin would appear to be preferable due to the lack of negative interactions between the drugs used, however there are multiple reports showing that l-Amb plus posaconazole may have a positive role.

    S03.4. Clinical features associated to fungal species

    D. Garcia-Hermoso 1, K. Sitbon 1, C. Gautier 1, F. Dromer 2 and T.H.E. French Mycoses Study Group 2
    1
    National Reference Center for Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, PARIS, France
    2
    National Reference Center for Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, Paris, France
    Background Fungi of the order of Mucorales are ubiquitous saprobes that can be responsible for life-threatening infections especially in immunocompromised patients but also in immunocompetent individuals. An increased incidence of mucormycosis has been reported mostly due to the rise of populations at risk and the use of better diagnostic tools. There are more than 20 pathogenic species involved in this type of infections, with some of them associated with specific body sites. The main localizations of mucormycosis are rhino-orbito-cerebral and pulmonary infections, depending on the underlying conditions of the patient. We present here a preliminary analysis aiming to dissect the association between clinical manifestations and Mucorales species using the active surveillance system (RESSIF) implemented in France at the NRCMA since 2012.
    Methods Three-hundred cases of probable and proven mucormycosis were so far collected through RESSIF. The isolates were characterized using phenotypic analysis by morphological methods, and antifungal susceptibility testing of 8 antifungal drugs done using EUCAST microdilution method. In addition, a multi-locus sequencing based on LSU, ITS, TEF-1 was performed.
    Results Among those 300 cases of invasive mucormycosis, lung involvement was the main presentation (52%) followed by rhinocerebral (18%) and cutaneous (15%) involvement. Deep infections occurred most often in patients with hematological malignancies. Six major species of Mucorales were identified with Rhizopus arrhizus being the most frequent fungus. Our current data expand previous findings showing that distribution of these 6 major species depended on the body site localizations and underlying condition of the patient.

    S03.5. Molecular diagnosis of mucormycosis

    L. Millon
    Parasitology—Mycology—Umr6249 Cnrs Chrono-environnement, University Hospital Besançon, Besançon, France
    Molecular techniques have provided a new understanding of the epidemiology of mucormycosis. Several factors may explain the growing incidence of mucormycosis, especially the increase in the number of susceptible people and the change in antifungal practices (which may have altered the relative frequency of mucormycosis and aspergillosis among patients at risk for both infection). In addition, the routine use of molecular techniques has helped to accurately identify Mucorales fungi in tissue samples when cultures are negative. As a result, the number of mucormycosis cases diagnosed in the last 10 years has increased. The use of molecular techniques has also improved the therapeutic management of mucormycosis. Indeed, mucormycosis and aspergillosis have similar underlying conditions, and similar radiological and clinical signs, but their antifungal treatments are different. For each of these infections, early diagnosis and early initiation of directed treatment are essential to improving patient outcome Molecular techniques may help to provide fast, effective treatment by accurately identifying the causative fungi. PCR amplification and sequencing were first applied to better identify isolates grown from cultures of biopsies or bronchalveolar lavage samples collected in patients with Mucorales infection. Molecular techniques have also been used to identify the fungus directly from the tissue samples when cultures were negative. The first technical approach for detection fungi in tissue samples consists of using panfungal primers, targeting ITS regions, followed by sequencing. The second possible approach consists of using Mucorales-specific primers. Diverse PCR techniques were tested and were successful, and several studies confirmed that PCR results were better in fresh/ frozen samples than in formalin-fixed paraffin-embedded samples. Molecular detection of fungi on hyphal positive biopsy samples is now strongly recommended (AII) by the European ESMID and ECMM joint clinical guidelines. Recent studies confirmed that Mucorales quantitative PCR (qPCR) applied on bronchoalveolar lavage fluid could provide additional arguments favoring earlier initiation of specific antifungal therapy, thus improving the outcome of pulmonary mucormycosis patients. However, these tools require invasive sampling (biopsy, bronchoalveolar lavage), which is not feasible in patients in poor condition in Hematology or Intensive Care units. QPCR-based procedures to detect Mucorales DNA in non-invasive samples such as plasma or serum have proved successful in diagnosing mucormycosis early (as early as 8 days before mycological diagnosis and 3 days before imaging in patients with hematological malignancies). The test can be performed in all patients, even those who cannot endure a biopsy or bronchoalveolar lavage. The Mucorales qPCR performed on serum or plasma is becoming essential to improve the management of at-risk patients. The main problem at the moment is a lack of standardization, because of the use of multiple in-house assays. Efforts from the Fungal PCR Initiative (FPCRI)/Mucorales Lab working group of the International Society for Human and animal Mycology (ISHAM) are currently ongoing to improve standardization and provide recommendations, as was previously done for Aspergillus PCR assays by the European Aspergillus PCR Initiative (EAPCRI) working group.

    S03.6. Management of mucormycosis in low and middle income countries

    A. Chakrabarti
    Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research, Chandigarh, India
    The epidemiology of mucormycosis is different in low and middle income countries (LMIC) from developed nations. In LMIC the number of mucormycosis cases is alarmingly high in diabetic patients, the spectrum of causative agents is wide with lack of knowledge on antifungal susceptibility of the new agents. Occasional unusual clinical presentation baffles the clinicians about diagnosis, and delay in seeking healthcare by patients leads to poor outcome of the disease. Additionally, the lack of awareness of the disease among clinicians, the lack of availability of appropriate diagnostic facilities, poor availability and affordability of antifungal agents pose serious challenge in the management of the disease. In a recent descriptive study covering 465 mucormycosis cases from 13 centres in India, it was noted that 24% patients could not complete or start antifungal therapy due to affordability issue and nearly 20% patients were treated with amphotericin B deoxycholate. In 4.2% patients the therapy was shifted from liposomal to deoxycholate preparation of amphotericin B after certain period due to financial constraints, and improper antifungal therapy was prescribed in 7% patients. Due to those challenges, 90-day mortality was 52.0%, though majority (78.2%) of the patients had rhino-cerebral or cutaneous mucormycosis, which were not difficult for early diagnosis. Deferasirox of label use was also noted in certain number of patients.

    Symposium 4 MSG symposium (Trial Design in Clinical Mycology: Innovative Approaches)

    S04.5. The Lung Transplant Community is Interested in a Clinical Trial to Determine the Optimal Strategy to Prevent Invasive Mold Disease

    R. La Hoz 1, C. Jones 2, G. Mcgwin 3, T. Patterson 4, P. Pappas 3, J. Baddley 3 and O. Morrissey 5
    1
    Internal Medicine, UT Southwestern Medical Center, Dallas, United States of America
    2
    The Ohio State University, Columbus, United States of America
    3
    University of Alabama at Birmingham, Birmingham, United States of America
    4
    University of Texas Health Science Center at San Antonio, San Antonio, United States of America
    5
    Monash University, Melbourne, Australia
    Objectives: The objectives of this survey were to: (1) describe the strategies used to prevent invasive mold disease in lung transplant recipients, (2) quantify the interest in participating in a clinical trial to determine the optimal prophylactic strategy for invasive mold disease in lung transplant recipients and (3) to describe the design types and factors that influence willingness to participate in the clinical trial.
    Methods: We invited transplant providers to participate in an anonymous cross-sectional web-based survey via email between December 2018 and February 2019. The 139-item survey was hosted on the University of Alabama at Birmingham Research Electronic Data Capture (REDCap) server. The contact list was generated from a variety of sources. The study was approved the University of Alabama at Birmingham Institutional Review Board.
    Results: of the 238 practitioners contacted, 50 (21.0%) responded to the survey. Responses were mainly from the United States 85.7% (36/42) and were transplant infectious diseases physicians 65.2% (30/43). The number of transplants performed at the responder’s centers during 2016 was less than 20 in 44.4% (16/36), 21 to 50 in 19.4% (7/36) and more than 50 in 36.1% (13/16). Most responders, 81.0% (30/37), had a mold prevention protocol at their program. Universal prophylaxis was the most common prevention strategy 75.7% (28/37), followed by pre-emptive prophylaxis 10.8% (4/37), and targeted prophylaxis 5.4% (2/37). A quarter of the responders (7/34) used prophylaxis for <3 months, 58.8% (20/34) for 3 to 6 months, 11.8% (4/34) for 6 to 12 months and 8.8% (3/27) for more than 12 months. The majority of the responders, 84.6% (22/26) were interested in participating in a clinical trial to assess the optimal prophylactic strategy for mold infections in lung transplant recipients. Almost a quarter of the participants were willing to participate of the clinical trial with a universal prophylaxis study design of new azole formulation to mold active azole (Figure 1). The study design arms and the anti-fungals selected for the study arms were the factors that had the most influence in study participation (Figure 2).
    Jof 05 00095 i001Jof 05 00095 i002
    Conclusion: Universal prophylaxis was the most common strategy to prevent invasive mold disease in lung transplant recipients. The majority of the responders were interested in a clinical trial to determine the optimal strategy to prevent mold disease in lung transplant recipients. The study design arms and the anti-fungals selected for the study arms were the factors that had the most influence in study participation.

    Symposium 5 Lung transplantation

    S05.1. Pre-transplant assessment

    B. Rammaert 1,2,3
    1
    Infectious Diseases, CHU Poitiers, Poitiers, France
    2
    U1070, INSERM, Poitiers, France
    3
    Faculté De Médecine Et Pharmacie, Université de Poitiers, Poitiers, France
    All the three main chronic pulmonary diseases leading to lung transplantation are at risk for fungal respiratory tract colonization: cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and interstitial lung diseases (ILD). Aspergillus and Scedosporium species are the two main molds found in the lower respiratory tract of lung transplant candidates. Colonization in CF patients could be explained by impairment of muco-ciliary clearance, and in COPD and ILD patients by use of inhaled or systemic corticosteroids that alter pulmonary immunity. Mortality due to invasive fungal diseases in post-transplantation can reach 60% depending on the causal species. Therefore, fungal colonization of the lower respiratory tract is on the list of relative contraindications for lung transplantation. Although passage from colonization to infection is not well understood, fungal spores could remain quiescent in the lungs, and secondarily reactivate once patients have received immunosuppressive drugs. Donor-derived fungal infection is one of the best examples of this reactivation. Pre-transplant colonization is not considered predictive for fungal invasive disease in post-transplantation. However, the timing and bronchoalveolar lavage protocols for fungal screening in respiratory tract before lung transplantation are heterogeneous between centers. In addition, culture of lower respiratory tract specimens has to be done on specific media such as Sabouraud glucose agar or yeast extract-peptone-dextrose-agar (YPDA), which could miss out on some fungi. Utilization of culture media supplemented with antibiotics has increased the number of CF patients culture-positive for fungi in their respiratory specimens from 18% to 78%. Scedosporium specific media such as Scesel+ increase the detection of Scedosporium spp. compared to Sabouraud glucose agar. Active screening for fungal colonization during the pre-transplant period is consequentley crucial not only in patients on a waiting list, but also in donors. There is an urgent need for bronchoalveolar lavage and fungal culture standardization in the different mycological laboratories dealing with lower respiratory tract specimens before focusing on pre-transplant fungal colonization.

    S05.5. From the lung to the heart: fatal dissemination of azole-resistant Aspergillus fumigatus in a lung transplant patient

    R.-A. Lavergne 1,2, T. Lepoivre 3, T. De Groot 4, F. Jeddi 2, D. Boutoille 5, I. Danner-Boucher 6, F. Morio 1,2, J. Meis 7 and P. Le Pape 1,2
    1
    Ea1155 Parasitology and Mycology Department, Nantes University, Nantes, France
    2
    Parasitology and Mycology Laboratory, Nantes University Hospital, Nantes, France
    3
    Thoracic Transplantation Intensive Care Unit, Nantes University Hospital, Nantes, France
    4
    Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, Netherlands
    5
    Tropical Infectious Diseases, University Hospital of Nantes, Nantes, France
    6
    Pneumology Unit, Nantes University Hospital, Nantes, France
    7
    Department of Medical Microbiology and Infectious Diseases, Excellence Center For Medical Mycology (ECMM), Canisius Wilhelmina Hospital, Nijmegen, Netherlands
    Azole resistance in Aspergillus fumigatus has emerged as a worrisome problem since the last ten years. Patients suffering from cystic fibrosis (CF) are at risk of being colonized with azole-resistant isolates. At our CF reference center hospital, prevalence of azole resistance in A. fumigatus reached 6.8% in 2015. Though resistance is increasingly described, its impact in CF patients is yet poorly known. Here, we describe the case of a CF patient having long-term colonization of the respiratory tract with a non-cyp51A mediated azole-resistant Aspergillus fumigatus isolate, prior to lung transplantation. Then, proven azole-resistant invasive aspergillosis and mucormycosis were diagnosed in the first days after transplantation and treated with liposomal amphotericin B. Two years later, azole-resistant Aspergillus fumigatus mitral-aortic endocarditis was diagnosed. Despite surgery and multiple antifungal regimens, the patient died with intracerebral hematoma. Objectives: The aim of the study was to investigate the genetic relationship between isolates collected at the time of transplantation and those from the native valve. Methods: Five isolates were retrospectively investigated for azole susceptibility to itraconazole (ITR), voriconazole (VOR), posaconazole (POS) and isavuconazole (ISA) (EUCAST method). Identification of A. fumigatus sensu stricto was confirmed using partial beta-tubulin sequencing. Molecular mechanisms leading to resistance (nucleotide sequencing of cyp51A gene and its promotor) and STRAf genotypes were determined. Results: Two isolates were collected at time of transplantation (bronchial secretion (n = 1) and postoperative wound (n = 1)) and three isolates were collected during endocarditis episode (respiratory tract (n = 2) and mitral valve (n = 1)). All isolates were identified as Aspergillus fumigatus and were pan-azole resistant (MIC ITR: > 8 mg/L, VOR: 4 to 16 mg/L, POS: 0.5 to 1 mg/L, ISA: 8 to >16 mg/L). Sequencing of the cyp51A gene and its promotor (4 isolates) showed wildtype cyp51A and promotor. All isolates shared the same STRAf genotype (M2: 26-20-8 M3: 35-9-7 M4: 9-10-20) confirming dissemination of this isolate from the lung tissue to the heart. Conclusion: This case report highlights the need for regular screening for azole resistance in CF patients especially for those awaiting lung transplantation. Physicians should keep in mind that azole-resistant colonizing isolates could be a possible source of life threatening invasive infection after lung transplantation. Yet, antifungal treatment of Aspergillus endocarditis is based on voriconazole or lipid formulation of amphotericin B but no data are available in case of azole-resistant isolate.

    Symposium 6 Prophylaxis during hematology malignancies

    S06.1. AML—in the era of FLT3 inhibitors

    R. Lewis 1,2
    1
    Infectious Diseases Clinic, Sant’Orsola-Malpighi Hospital, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy
    2
    Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy
    FLT3 mutations are among the most common genetic mutations in acute myeloid leukemia (AML) associated with increased risk of disease relapse and poorer survival. A number of small molecule tyrosine kinase inhibitors (TKI) of FLT3 signalling have shown promise for reducing relapse of FLT3+ AML. One TKI, midostaurin, received fast-track approval in 2017 for the treatment of FLT3 positive AML on the basis of data demonstrating, for the first time in over 45 years, improved survival compared to standard induction 7+3 (cytarabine/anthracycline) chemotherapy. The use of FLT3 inhibitors, however, is problematic in patients who require antifungal prophylaxis or treatment with triazole antifungals. In general FLT3 inhibitors are extensively metabolized by CYP3A4 to active metabolites with high potency and potential off-target effects. When coadministered with triazoles, FLT3 exposures may increase between 1.3 to 10 fold and alter production of active metabolites essential for FLT3 inhibition or increase off-target effects. Indeed, higher rates of pulmonary toxicity including fatal events, were observed among patients receiving midostarin with azole antifungals. Currently, the midostaurin SmPC does not recommend the co-administration of strong CYP3A4 inhibitors such as voriconazole or posaconazole; although this recommendation is not practical for many patients especially with active fungal disease. Unfortunately, second-generation FLT3 inhibitors currently in clinical studies (e.g., quizartinib) also appear to have similar risks as midostaurin as well as greater risk for dose-limiting myelotoxicity and QTC prolongation. It is clear that striking the balance between optimal antifungal prophylaxis vs. optimal FLT3 inhibition will become a major challenge in the management of AML in the coming decade. This presentation will discuss the pros and cons of possible strategies for managing drug-drug interactions with FLT3 inhibitors and antifungals in the absence of clearly-defined guidance.

    S06.4. Baseline CT upon diagnosis of acute leukemia

    S. Schwartz
    Charité Berlin, Berlin, Germany
    Computed tomography (CT) is a routine diagnostic procedure in immunocompromised patients (i.e. patients with long-lasting neutropenia) with suspected fungal pneumonia. Particular radiological appearances of lung infiltrates (e.g. halo-sign) have been associated with lung infections caused by molds and findings have been incorporated into the well acknowledged definitions of invasive fungal infections developed by the EORTC/MSG (1). A conventional chest X-ray is usually performed in immunocompromised patients with fever, but the value of a conventional chest X-ray has been questioned due to its poor diagnostic yield and most recommendations advocate CT (2). A baseline chest CT in asymptomatic patients scheduled for therapies resulting in a profound immunosuppression (e.g. induction chemotherapy for acute leukemia) appears meaningful, but published data about this are limited. In a prospective study, 198 adult patients scheduled for hematopoietic stem cell transplantation or intensive chemotherapy were evaluated with a baseline CT (3). Overall, 72 (36%) patients showed lung abnormalities. of these, 19 exhibited disease-defining lesions according to the EORTC/MSG definitions (nodules, halo sign, cavitation) and 53 patients showed other abnormalities (most frequently consolidations or ground-glass opacifications). Nine patients in each group were subsequently categorized as having proven or probable invasive mold infections. The risk of developing an invasive pulmonary mold infection was significantly higher among patients with disease-defining lesions according to the EORTC/MSG definitions or, less pronounced, with other abnormalities compared to patients with a normal baseline CT. In another study, 145 baseline CT scans from patients with acute leukemia scheduled for their first induction therapy were assessed retrospectively (4). Remarkably, the majority of baseline CT scans disclosed abnormalities (127; 88%) with nodules in 71 (49%) patients. Cavitations or lesions with a halo- or an air-crescent-sign were not present in this patient series. However, an independent radiological review and results from a comprehensive diagnostic evaluation were not available from this study. The high frequency of lung lesions at baseline, including disease-defining lesions associated with invasive mold infections, in patients scheduled for intensive chemotherapy or hematopoietic stem cell transplantation is striking. A baseline CT scan in these patients appears reasonable, but the overall benefit of this diagnostic approach in asymptomatic patients should be explored further in future studies. It would be desirable to evaluate in more detail other underlying, infectious/non-fungal and non-infectious, conditions (e.g. opacities associated with hyperleukocytosis) associated with lung lesions (5). Literature 1. de Pauw B, et al. Clin Infect Dis 2008;46(12): 1813–21. 2. Maschmeyer G. Curr Opin Oncol 2001;13: 229-35. 3. Ceesay MM, et al. Br J Haematol 2018;182: 723–27. 4. Vallipuram J, et al. Leuk Lymphoma 2017;58: 834–41. 5. Stefanski M, et al. Medicine 2016;95: e5285.

    S06.5. Investigating the impact of posaconazole prophylaxis on systematic fungal screening using galactomannan antigen, Aspergillus qPCR and Mucorales qPCR

    A.-P. Bellanger 1, N. Tatoyan 1, A. Berceanu 2, H. Gbaguidi-Haore 3, T. Monnot 4, D. Bichard 4 and L. Millon 1
    1
    Mycology, University Hospital Jean Minjoz, Besancon, France
    2
    Hematology, University Hospital Jean Minjoz, BESANCON, France
    3
    Infectious Risk Control, University Hospital Jean Minjoz, BESANCON, France
    4
    Pharmacy, University Hospital Jean Minjoz, BESANCON, France
    Objectives: At the University Hospital of Besancon, a systematic panel of fungal biomarkers is applied systematically to screen for invasive fungal infection (IFI) highly immunocompromised patients of the Hematology Department. This panel of biomarkers does include the galactomannan antigen (GM), Aspergillus qPCR and Mucorales qPCR. Most of the stem cell transplant (SCT) recipients receive now posaconazole prophylaxis. The pertinence of systematic fungal screening has been discussed in the literature for patients under active antifungal prophylaxis, especially the realization of the GM. In this context, our main objective was to investigate retrospectively if the posaconazole prophylaxis influenced the numeric value of the GM. Our secondary aims were to assess the contribution of Aspergillus qPCR and Mucorales qPCR as well as the prevalence of IFI under active antifungal prophylaxis.
    Methods: The results of systematic fungal screening (GM, Aspergillus and Mucorales qPCR) were collected for all the SCT recipients, treated with posaconazole as prophylaxis treatment for more than 8 days, from January 2015 to December 2016. Similarly, results of systematic fungal screening were collected for patients without posaconazole as prophylaxis treatment (induction chemotherapy-January 2010 to December 2015). The statistical analysis of data was performed using the Stata 2 software.
    Results: For the group of patients receiving posaconazole prophylaxis: 60 patients were included, with a mean period of prophylaxis of 107 days and a total 916 fungal screenings. The mean value of the GM was 0.17 (±0.32), the frequency of positive GM was 4.37%, the frequency of positive Aspergillus qPCR was 0.68% and the frequency of positive Mucorales qPCR was 3.66%. The frequency of IFI in this group was estimated at 5%. For the control group: 38 patients were included with a total of 392 fungal screenings. The mean value of the GM was 0.22 (±0.64), the frequency of positive GM was 5.61%, the frequency of positive Aspergillus qPCR was 12.35% and the frequency of positive Mucorales qPCR was 5.33%. The frequency of IFI in this group was estimated at 13%. The statistical analysis showed that posaconazole prophylaxis did not affect significantly neither the numeric value nor the frequency of positivity of the GM. A significant decrease of the frequency of positive Aspergillus qPCR was observed (0.68 vs 12.35, p = 0.015). A tendency of decreased frequency of IFI was also observed (5% vs 13%, p = 0.076).
    Conclusion: Our study did not demonstrate any negative effect of the posaconazole prophylaxis on the GM. This study highlights the high efficacy of the posaconazole prophylaxis, especially when using the tablet form, which was supported by a significant decreased frequency of positive Aspergillus qPCR and relatively lower frequency of IFI. The current screening strategy combining GM and fungal qPCRs is of high interest, even if the posaconazole prophylaxis is efficient, in a context of emerging infection with azole resistant strains of Aspergillus fumigatus and risk of A. fumigatus-Mucorales mixed infections.

    Symposium 7 Paediatric Mycology (EPMyN)

    S07.5. Isavuconazole use in pediatric hematoncologic patients: the Italian Association of Pediatric Hematology Oncology (AIEOP) experience

    N. Decembrino 1, K. Perruccio 2, A. Colombini 3, E. Calore 4, P. Muggeo 5, E. Soncini 6, A. Comelli 7, M. Molinaro 8, B.M. Goffredo 9, S. De Gregori 8, I. Giardini 8, S. Cairoli 9, L. Scudeller 10, M. Zecca 1 and S. Cesaro 11
    1
    Pediatric Hematology/oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Pavia, Italy
    2
    Division of Pediatric Hematology, University Hospital of Perugia, Perugia, Italy
    3
    Pediatric Hematology/oncology, Fondazione Monza e Brianza per il Bambino e la Mamma, Monza, Monza, Italy
    4
    Clinic of Pediatric Hemato- Oncology, Department of Women’s and Children’s Health, University Hospital of Padova, Padova, Italy, Padova, Japan
    5
    Department of Pediatric Oncology and Hematology, University Hospital of Policlinico, Bari, Italy, Bari, Italy
    6
    Pediatric Hematology/oncology, Spedali Civili, Brescia, Italy, Brescia, Italy
    7
    Department of Infectious and Tropical Diseases, Spedali Civili, Brescia, Italy, Brescia, Italy
    8
    Clinical and Experimental Pharmacokinetics Lab, Fondazione IRCCS Policlinico San Matteo, Pavia, Pavia, Italy
    9
    Metabolic Pathology Lab, Ospedale Pediatrico Bambino Gesù, Roma, Roma, Italy
    10
    Clinical Epidemiology and Biometric Unit, Scientific Direction, Fondazione IRCCS Policlinico San Matteo, Pavia, Pavia, Italy
    11
    Pediatric Hematology/oncology, Azienda Ospedaliera Universitaria Integrata, Verona, Italy, Verona, Italy
    Objectives: Isavuconazole (ISA) is a new triazole approved for invasive aspergillosis and mucormycoses treatment in adults only. Advantages are activity against both moulds and yeasts spp, excellent bioavailability after oral administration without relevant food or gastric pH effect, a water-soluble prodrug without nephrotoxic excipients, poor drug-drug interactions. We analyzed ISA use, safety and efficacy in immunocompromised children.
    Methods: Italian Association Paediatric Hematology Oncology (AIEOP) multicentric retrospective analysis of pediatric hematoncologic patients who received ISA asinvasive fungal disease (IFD) treatment or prophylaxis. Due to the lack of recommended dosing in children and of PK/PD target value, therapeutic monitoring (TDM) was applied by a validated liquid chromatography-tandem mass spectrometry (HLPC-MS/MS) assay technique. ISA trough plasma concentrations (C0) and 3 hours after drug intake (C3h) were measured.
    Results: Twenty-nine patients were included, (M/F 20/9); median age: 14.5 years (range 3–18), median bodyweight 47 kg (range 15–80). Ten patients only received chemotherapy, 19 patients received allogeneic hematopoietic stem cell transplantation (HSCT). Donors were haploidentical in 9 patients, matched-sibling in 4, allogenic-unrelated in 4 cases. ISA was used as prophylaxis in 5 patients, as IFD treatment in 24 cases: 20 for previous therapeutic failure (7 l-AMB, 4 Voriconazole, 5 combination therapy, 1 Posaconazole, 3 Caspofungin), 4 as first line therapy. According to EORTC criteria, IFD was proven in 5 patients (1 Aspergillus spp, 1 Aspergillus flavus, 1 Aspergillus fumigatus, 1 Penicillium spp. and 1 Mucor spp), probable in 9 and possible in 10 patients. Lungs were the main localization (18 cases), liver was involved in 2 cases, 3 patients had sinus involvement (2 with central nervous system localizations), 1 had a disseminated infection. Patients under 30 kg bodyweight received half ISA dose, the others received adult schedule (200 mg TID loading dose on days 1 and 2, 200 mg/die maintenance); only 13 patients received loading dose. Ten patients only received oral therapy, in the others IV route was switched to oral during treatment. ISA was administered for a median of 75.5 days (range 6–523 day), in combination with other antifungals in 7 patients (Caspofungin in 3 cases, l-AMB in 4). Overall response rate was 70.8%. IFD complete remission was achieved in 12 cases, partial remission in 5. Treatment failure was experienced by 5 patients, in 3 cases fungal lesions remained stable. No breakthrough infections were registered in prophylaxis group. TDM was applied to 17 patients including 1with severe veno-occlusive disease and 1 with renal failure secondary to thrombotic-microngiopathy. Overall median ISA Ctroughss concentration observed was 4.91 mg/L (2.15–8.54); Conc/Dose (kg) ratio was 1.13 (0.47–3.42). In 6 cases a 12h-PK profile was carried out and median AUC0–12h of 153,16 mgxh/L (range 86.31–169.45) was obtained. CTAE grade II-III toxicity was observed in 6 patients, with increased transaminase and/or creatinine levels which resolved after temporary ISA withdrawal. We did not observe drug-drug interactions in the patients receiving immunosuppressant as graft versus host disase prophylaxis (5 cyclosporine, 2 sirolimus, 1 tacrolimus).
    Conclusion: Isavuconazole may be useful and safe in the pediatric population with hematoncologic diseases, even in the HSCT setting. Prospective studies are warranted.

    Symposium 8 Immunologic markers for diagnosis and treatment stratification in invasive mold infection

    S08.3. Immunologic Markers for Treatment Stratification in Invasive Mold Infection

    P. Köhler
    Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany
    The two major entities, invasive aspergillosis and mucormycosis account for the majority of fungal mould infections in haematology and oncology. With increased hospital admission rates and admission to intensive care unit and high morbidity and mortality the need for prompt identification and outcome prediction is needed. Current in vitro diagnostic approaches comprise direct observation of fungi in biopsy or bronchoalveolar lavage samples, blood cultures and detection of diagnostic antigens such as galactomannan. New immunologic markers for diagnosis and treatment stratification have been recently published. Fungus-reactive CD4 T cells, interleukin profiling and inflammation parameters will be discussed with regard to treatment stratification depending of response of invasive mould infections. Possible developments could lead to algorithms incorporating measurement of these biomarkers at admission and throughout initial treatment for the purpose of identifying patients who warrant more aggressive treatment.

    S08.4. Fungal Translocation: from persistent inflammation to non-AIDS events

    M. Hoenigl 1,2
    1
    Division of Infectious Diseases, Department of Medicine, UCSD, San Diego, United States of America
    2
    Section of Infectious Diseases and Tropical Medicine and Division of Pulmonology, Medical University of Graz, Graz, Austria

    Abstract:

    1,3-β-d-glucan is a fungal cell wall polysaccharide produced by ascomycete fungi including Candida albicans and other yeast that colonize the GI tract. Recently we and others have shown that in the absence of an invasive fungal infection blood 1,3-β-d-glucan levels may serve as a marker of microbial translocation in patient populations with hypoperfusion of the gut and those with persistent inflammation, including patients with advanced liver cirrhosis, sepsis or HIV infection, where blood 1,3 β-d-glucan have been shown to be elevated compared to HIV negative controls and to correlate with other biomarkers of microbial translocation, with markers of immune activation and with lower Lactobacillales proportion (i.e. less protective) in the gut microbiome. In animal models 1,3-β-d-glucan has been shown to decrease Dectin-1 (innate pattern recognition receptor essential for the control of fungal infections), and also NKp30 (NK receptor mediates direct fungal recognition and killing) on monocytes and NK cells respectively, and induced pro-inflammatory cytokines secretion, indicating a potential role in pathogenesis of clinical events. Going one step further, studies have shown clear correlations between blood 1,3-β-d-glucan levels and non-AIDS events, including cardiopulmonary morbidity, and neurocognitive impairment. Only few studies to date have evaluated a potential benefit of antifungal treatment for tackling fungal translocation and reducing blood 1,3-β-d-glucan levels. In vitro model of mixed rat neuronal cultures that screened more than 2000 compounds, half of which were US Food and Drug Administration approved drugs for putative neuroprotective effects against oxidative stress-mediated neuronal injury not only paroxetine, a selective serotonin reuptake inhibitor, but also fluconazole, protected hippocampal neurons. In another study, the combination of paroxetine and fluconazole protected macaques from simian immunodeficiency virus–associated neurodegeneration. However, a phase 1/2, randomized, double-blind, placebo-controlled study for the treatment of HIV-associated neurocognitive impairment did not show an improvement of neurocognitive outcomes among the 9 participants randomized to fluconazole treatment alone, indicating that more research and larger studies are needed on how to prevent/treat fungal translocation.

    S08.5. Immunological characteristics of Broncho alveolar lavage in neutropenic patients with invasive aspergillosis

    C. Aguilar 1, S. Moshkelgosha 2, W. Gohir 1, K. Yee 3, A. Schimmer 3, M. Minden 3, A. Schuh 3, C. Ryan 4, S. Juvet 2 and S. Husain 1
    1
    Transplant Infectious Diseases, University Health Network, Toronto, Canada
    2
    Lung Transplant Program, University Health Network, Toronto, Canada
    3
    Hematology Department, University Health Network, Toronto, Canada
    4
    Respirology, University Health Network, Toronto, Canada
    Objectives: Invasive aspergillosis (IA) is a major complication in patients with acute leukemia receiving high dose chemotherapy. Diagnosis of IA classically relies on radiological criteria associated with direct and/or indirect microbiological tests, which have some limitations. some limitations. Antifungal immunity involves phagocytic cells including neutrophils and macrophages, as well a dendritic cells presenting antigen to T-CD4+ T cells. Antigen presentation leads to differentiation of CD4+ T cells into Th1 and Th17 cells, under the influence of cytokinic environment. Those cells secrete cytokines and amplify effector functions of phagocytic cells in order to kill the fungus. The triggering of those mechanisms relies on the recognition of fungal patterns by receptors of innate immunity, both soluble (such as PTX-3) and membrane bound (such as Dectin-1 and Dectin-2). In this study we aimed to assess the immunological parameters of broncho alveolar lavage (BAL) fluid of neutropenic patients with or without IA.
    Methods: We included acute myeloid leukemia patients who were neutropenic after receiving high dose chemotherapy and who underwent a bronchoscopy for diagnosis of pneumonia. Ten milliliters of BAL fluid were collected for the research study. Supernatant and cell pellet were separated and frozen until use. The level of 35 cytokines (EGF, Eotaxin, FGF basic, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL1-ra, IL-1α, IL1-β, IL-2, IL-2r, IL-3, IL-4, IL-5, IL-6, IL-7, IL-7, IL-8, IL-9, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17A, IL-17F, IL-22, IP-10, MCP-1/CCL2- MIG, MIP-α, RANTES, TNF-α, and VEGF) was measured in BAL supernatant using Luminex technology. PTX-3 level in BAL supernatant was measured by Enzyme-linked immunosorbent assy. The cell pellet was analyzed by flow cytometry for expression of CD45, CD206, CD163, Dectin-1 and Dectin-2.
    Results: Forty three patients were included in the study. Seven patients were diagnosed with probable IA and 20 with possible IA. One patient had mucormycosis diagnosed on lung biopsy and 1 patient had fusariosis. While PTX-3 level was not significantly different in patients with probable IA compared to patients without IA, IL-6, IL-8 land GM-CSF levels were higher in patients with probable IA compared to patients without invasive fungal infection. Flow cytometry could be performed on 31 BAL cell pellets. The proportion of alveolar macrophages (CD163+ CD206+) among CD45 positive cells was similar in all groups. Dectin-1 and Dectin-2 expression on alveolar macrophages (CD163+ CD206+ cells) was similar in patients with and without invasive fungal infection.
    Conclusion: Neutropenic patients with probable IA exhibit higher IL-8, IL-6 and GM-CSF levels in BAL supernatant compared to neutropenic patients without any fungal infection. The cellular expression of membranar receptors Dectin-1 and Dectin-2, as well as the level of soluble PTX-3 were not significantly modified in patients with IA. Further studies are needed to assess the role of immunological parameters, especially cytokines profile, in the panel of tests used for the diagnosis of IA in immunocompromised patients.

    Symposium 9 Chronic Pulmonary Aspergillosis

    S09.2. Diagnosis of CPA. Where do we stand?

    A. Barac
    Clinic for Infectious and Tropical Diseases, Clinical Center of Serbia, Belgrade, Serbia

    Abstract:

    Chronic pulmonary aspergillosis (CPA) is an uncommon, slowly destructive pulmonary disease characterized by progressive cavitation, fibrosis, and pleural thickening. CPA is usually seen in immunocompetent individuals with underlying respiratory disorders. Estimates suggest that up to 3 million people are affected worldwide causing high rates of morbidity and mortality. Diagnosis of CPA is often challenging and delayed and should be based upon a combination of characteristics. Patients are present with indolent, non-specific symptoms such as fatigue, weight loss, sweats, cough, dyspnea, or hemoptysis. The disease often remains undiagnosed for years, resulting in progression. A high index of suspicion is required in patients with predisposing factors such as previous tuberculosis (TB) with residual cavities, severe chronic obstructive pulmonary disease, previous treatment for lung cancer, or nontuberculous mycobacteria. The most important diagnostic finding is changes on chest imaging, usually CT scan. The most common presentation is one or more cavities, characteristically with an irregular or thick wall, but occasionally thin-walled, with or without intracavitary material or well-formed aspergilloma. Cavities tend to affect the upper lobes and often cause para-cavitary or adjacent pleural fibrosis. In early stages, nodules may be present, which may develop into cavitary disease over time. Differential diagnosis includes mycobacterial infection, bacterial abscess, cancer, or vasculitis. Expertise of the reporting radiologist is essential in highlighting the possibility of CPA. In addition to compatible radiological findings, microbiological and/or serological evidence of Aspergillus infection is required, in addition to ruling out alternative diagnoses. Direct microscopy for hyphae, fungal culture of respiratory secretions and histology are all recommended for diagnosis of CPA. Sensitivity of sputum or bronchoalveolar lavage (BAL) culture remains moderate at best, even when specific fungal media are used. Culturing a higher volume of sputum may lead to increased sensitivity. Sensitivity is significantly higher with the high-volume culture, which in addition detected azole-resistant isolates that could be missed with conventional culture. When microscopy and cultures are negative, molecular markers like Aspergillus polymerase chain reaction (PCR), as well as beta D-glucan, galactomannan (GM) testing in BAL samples can be used to increase sensitivity. In addition, using these tests in combination may result in better PPV. The usefulness of testing GM in sputum is not clear in a cohort of patients with CPA and ABPA. Therefore, sputum GM cannot be recommended for the diagnosis of CPA at present. Serological methods can contribute to the diagnosis of CPA. Aspergillus precipitins were used previously but have been supplanted by more sensitive serologic assays. Several assays detecting Aspergillus-specific immunoglobulin G (IgG) are commercially available. In summary, clinical and radiological findings, along with microbiological and serological tests are used in combination to diagnose CPA. There is no individual reference-standard test as all tests are characterized by suboptimal sensitivity and specificity. A high index of suspicion is needed, along with the need to rule out alternative diagnoses like TB or lung cancer.

    S09.5. Raised amphotericin B MIC in Aspergillus fumigatus isolates from patients with Chronic Pulmonary Aspergillosis

    F. Lynch 1,2, C. Moore 2,3 and R. Rautemaa-Richardson 1,2,4
    1
    Infectious Diseases, Manchester University NHS Foundation Trust, Manchester, United Kingdom
    2
    National Aspergillosis Centre, Manchester, United Kingdom
    3
    Mycology Reference Centre Manchester, Manchester University NHS Foundation Trust, Manchester, United Kingdom
    4
    Division of Infection, Immunity and Respiratory Medicine, University of Manchester, Manchester, United Kingdom
    Objectives: Chronic pulmonary aspergillosis (CPA) is a fungal infection of the lung caused by Aspergillus species, which usually affects patients with an underlying respiratory condition. The mainstay of treatment is prolonged antifungal therapy to improve symptoms and prevent disease progression. Aspergillus fumigatus with elevated minimum inhibitory concentrations (MIC) for amphotericin B (AMB) have been isolated. The aim of this study is to investigate the emergence and clinical relevance of elevated AMB MICs in A. fumigatus.
    Methods: Patients with CPA reported with AMB resistant (MIC > 4 mg/L) A. fumigatus were identified from the Mycology Reference Centre Manchester databases. All A. fumigatus isolates reported from these patients were included in the analyses. The date of sampling and resistance patterns of the isolates were analysed against the antifungal history of each patient. EUCAST antifungal breakpoints were used to determine sensitivity. Clinic letters and dispensing records were used to determine medical history, antifungal therapy and clinical outcomes.
    Results: Nine patients reported with one or more AMB resistant A. fumigatus isolates and a total of 148 isolates from these patients were identified. of the isolates, 34 (22%) were resistant to AMB; 13 (38%) had an MIC of 4 mg/L, five (15%) had an MIC of 8 mg/L and 16 (47%) had an MIC of >8 mg/L. In addition, 18 (12%) isolates were reported as intermediate to AMB (MIC = 2 mg/L). Five of the nine patients had prior exposure to AMB (IV AmBisome). Patients received 3-5 mg/kg once daily and different treatment regimens were used; one patient had one 3-week course, two patients had two 3-week courses and two patients received long term therapy three times weekly (for up to 3 years). Four patients had no exposure to AMB. of the 5 patients exposed to AMB, 4 developed resistance during therapy, and 1 patient first showed resistance 5 years after their 3-week course. All patients had prior azole exposure, and the duration of azole exposure prior to AMB resistance was from 8 months to 5 years. All AMB resistant isolates showed some level of azole resistance and 27 (82%) were pan-azole resistant. Example patient timeline.
    Jof 05 00095 i003
    Conclusion: All patients reported with A. fumigatus with elevated MIC for AMB had previously been treated with azole antifungals and developed resistance against one or more azoles. Many AMB resistant isolates were pan-azole resistant suggesting a link between AMB and azole resistance. Antifungal drug resistance can have significant implications on patient care due to limited treatment options. A better understanding of the development, genetic mechanism, and clinical relevance of AMB resistance is needed. Therefore, all isolates identified in this study will next be sequenced and typed.

    Symposium 10 Symposium 10 Sensing the host

    S10.2. Hybrid histidine kinases: major sensing proteins in pathogenic fungi

    N. Papon 1, S. Kabbara 1, A. Hérivaux 1, B. Bidon 1, J. Kilani 1, Y. Le Govic 1, A. Gastebois 1, T. Dugé De Bernonville 2, M. Clastre 2, V. Courdavault 2, S. Le Gal 1, G. Nevez 1 and J.-P. Bouchara 1
    1
    Host-Pathogen Interaction Study Group, SFR ICAT 4208, Univ Angers, Univ Brest, University Hospital Center, Angers, France
    2
    Ea2106 “biomolécules Et Biotechnologies Végétales”, Université de Tours, Tours, France
    Protein phosphorylation is one of the most extensively used modifications in signal transduction pathways in both prokaryotic and eukaryotic cells. Prominent families of enzymes that perform these protein phosphorylations encompass serine/threonine kinases, tyrosine kinases, and histidine kinases (HKs). While HKs dominate prokaryotic signaling pathways, they are less prevalent in eukaryotes, and most importantly, absent in animals. Historically, a small number of eukaryotic HKs have been studied in plants, yeasts, filamentous fungi, and slime molds. Recent studies have expanded characterization of HKs in other eukaryotic lineages and archaea, allowing a broader assessment of the types of signaling systems mediated by HKs, their phylogenetic distribution and evolution. HKs are central to regulatory systems that impact agriculture, the environment, and both beneficial and pathogenic interactions of microbes with humans and other animals. Their great diversity, versatility, and broad distribution make them attractive targets for therapeutics, biotechnological interventions, and also as building blocks for engineered biosensor systems. From a structural perspective, fungal HKs are commonly composed of three main regions. The first region referred to as the “sensing domain” corresponds to a highly variable N-terminal sequence that determines the stimulus perceived. The central “transmitter region” is made of two conserved domains: a dimerization histidine phosphotransfer (DHp) domain and a catalytic ATP-binding (CA) domain. The DHp domain includes an H-box, usually containing the phosphorylatable histidine residue, and an X-box. The CA subdomain includes four distinct sequence motifs: the N-, G1-, F-, and G2-boxes. In contrast to prokaryotic HKs, most eukaryotic HKs harbor an additional C-terminal receiver domain (REC) that includes a phosphorylatable aspartate residue. Thus, eukaryotic HKs are generally called “hybrid HKs” (HHKs). From a mechanistic point of view, HHKs constitute in fungal cells the initial sensing proteins of a four-step phosphorelay signaling pathway involving phosphorylation events of two downstream effectors, i.e. histidine phosphotransfer shuttle proteins (Hpts) and response regulators (RRs) (Figure 1). The perception of a stimulus induces the trans-autophosphorylation of a conserved His residue in the DHp domain by the CA catalytic part of the HHK. The phosphate of this histidine is then transferred to the conserved Asp residue in the REC domain prior to transfer to the Hpt domain, and finally to the conserved aspartate residue of the protein belonging to the RR family. The phosphorylation state of this RR orchestrates subsequent molecular events underlying the response to the input signal.
    Jof 05 00095 i004
    Here, we review the distinct genetic approaches carried out over the last fifteen years and that allowed the elucidation of the function of several HKs in prominent fungal pathogens. However, because their roles in regulating fundamental cellular processes are still largely obscure in fungi, we will discuss the major challenges that remain to improve our knowledge about the function of HKs in fungi and to design specific inhibitors for future therapeutic development.

    S10.5. The analysis on the genes related to the biofilm formation of Candida glabrata

    X. Chen 1, T.W. Widiyanto 1, H. Chibana 2 and S. Kajiwara 1
    1
    School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan
    2
    Medical Mycology Research Center, Chiba University, Chiba, Japan
    Objectives: Candida glabrata is capable of developing biofilm on the surface of host cells and medical materials. The biofilm formation is considered as one of pathogenic factors on infection to the host, which shows increase in the resistance to antifungals and host immune defense compared to the planktonic cells. Until now, the mechanism of biofilm formation of C. glabrata has not been studied well compared with C. albicans. Therefore, the aim of our study was to isolate and characterize some genes related with the biofilm formation of C. glabrata. Methods: The in vitro model of biofilm formation was constructed by using multi-well plates and the metabolic activity were detected by XTT assay. The susceptibility of biofilm formation was also detected towards different antifungals. Gene expression was analyzed by qPCR. Vacuolar morphology was observed by staining with FM4-64 fluorescent dye. The growth fitness was detected in buffered media with different pH and intracellular pH was measured with flow cytometry. Results: Upon the result of a genetic comprehensive screening by using C. glabrata mutant library, two gene mutants decreased metabolic activity of biofilms drastically and the transcriptional expressions of these two genes in biofilm formation showed more than two times higher than those of planktonic cell growth. One gene showed high homology with Saccharomyces cerevisiae SYN8 gene, then was named as CgSYN8. S. cerevisiae Syn8p was identified as a SNARE protein, acting in membrane fusion of vesicles. C. glabrata syn8Δ mutant was defective in both the metabolic activity and the biomass of the biofilm. Deletion of SYN8 also led to decrease in the adhesion ability during the biofilm formation, which may link to the repression of two adhesin genes, EPA10 and EPA22. Additionally, C. glabrata syn8Δ mutant revealed the abnormal vacuolar morphology. Therefore, it was suggested Syn8p is not only required for the normal vacuolar function but also influence the biofilm formation of C. glabrata. Another gene related to the biofilm formation of C. glabrata was named as CgQDR2. QDR (quinidine drug resistance) family genes in S. cerevisiae belong to MFS (Major Facilitator Superfamily) transporters, which mediate the ability of membrane transport to actively efflux the drug out of the cell. C. glabrata qdr2Δ mutant exhibited significant reduction in the biofilm formation and fluconazole drug susceptibility during biofilm formation. In addition, our results suggested QDR2 deletion caused an impaired ability of C. glabrata to maintain pH homeostasis, then might lead to the reduction of cell growth at neutral-basic pH conditions. These results may explain how the QDR2 gene affect the biofilm susceptibility and drug efflux towards antifungal drugs in C. glabrata. Conclusion: CgSYN8 and CgQDR2 were suggested to be involved in biofilm formation of C. glabrata. These findings provide more understanding on the biofilm formation of this fungus and more information for the development of clinical treatment in future.

    Symposium 12 Environment & fungal outbreaks

    S12.1. ‘Once is a misfortune, twice is carelessness…’: Lessons to be learnt from pandemic fungal infections of amphibians

    M. Fisher
    Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom

    Abstract:

    Discovering that chytrid fungi cause the disease chytridiomycosis in amphibians represented a paradigm shift in our understanding of how emerging infectious diseases contribute to global patterns of biodiversity loss. In this lecture I describe how the use of multidisciplinary biological approaches has been essential to pinpoint the origins of pathogenic chytrids, to time their emergence and pathways of spread and to identify the core mechanisms that underpin their pathogenicity. I will discuss the potential for using bioinformatics toolkits and experimental methods to allow a fuller understanding of these chytrids biology in order to leverage emerging technologies aimed at their control. The impacts of infectious diseases in wild populations are not restricted to wild animals and plants; they also threaten public health through the attrition of ecosystem services as well acting as reservoirs and amplifiers of disease inocula. I will argue that the lessons learnt from tracking amphibian chytrid fungi are also of relevance to understanding and tackling emerging patterns of fungal disease in humans. Paraphrasing Oscar Wilde, ‘to allow one fungal pandemic is misfortune, to allow two is carelessness, to allow three…’.

    S12.2. The Cryptococcus gattii species complex

    F. Hagen 1,2,3
    1
    Medical Mycology, Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands
    2
    Medical Microbiology, University Medical Center Utrecht, Utrecht, Netherlands
    3
    Department of Dermatology, Jining No. 1 People’s Hospital, Jining, China

    Abstract:

    The Cryptococcus gattii species complex placed itself in the spotlights of the medical mycology community with the unprecedented and ongoing outbreak that emerged in the temperate climate of Vancouver Island (British Columbia, Canada) during the late 1990s. A few years thereafter the outbreak was found to have spread to the Canadian mainland and the Pacific Northwest of the U.S.A. This pathogenic yeast, at that time named Cryptococcus neoformans variety gattii, was mostly known as being a tropical or subtropical pathogen that caused disease among apparently immunocompetent subjects. It was observed that the outbreak in Canada and the U.S.A. was due to an until then rare genotype named AFLP6/VGII, mostly known from Australia and the South American continents. Subsequent molecular typing showed that the outbreak was caused by a major (AFLP6A/VGIIa) and a minor (AFLP6B/VGIIb) genotype that had different virulence properties. In 2002 C. neoformans variety gattii was raised to species level as Cryptococcus gattii. Using several molecular techniques five lineages were recognized, nowadays known as molecular types (genotypes) VGI (AFLP4), VGII (AFLP6), VGIII (AFLP5) and VGIV (AFLP7), the fifth lineage is extremely rare and known as genotype AFLP10/VGIIIc. Although C. gattii was just proposed as being a separate species, the debate was already ongoing whether or not to further differentiate it into more species related to the molecular defined lineages. A further refinement for a taxonomic revision was supported by the availability of more molecular, phenotypic, antifungal sensitivity, geographic distribution and predilection for certain hosts. In 2015 a proposal was made to raise the five lineages to species level: C. gattii sensu stricto (AFLP4/VGI), C. bacillisporus (AFLP5/VGIII), C. deuterogattii (AFLP6/VGII), C. tetragattii (AFLP7/VGIV) and C. decagattii (AFLP10/VGIIIc). Due to the outbreak with C. deuterogattii in North America most of the focus was on this species, but what do we know about the other members of the C. gattii species complex in terms of environmental distribution and clinical relevance?

    S12.4. Whole genome sequencing: a valuable tool to study outbreaks due to uncommon yeast species

    M. Desnos-Ollivier
    National Reference Center For Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, Paris, France

    Abstract:

    Rapid genotyping tools such as Multi Locus Sequence Typing (MLST) and Variable Number Tandem Repeat (VNTR) are frequently used for common yeast species involved in invasive human infection (Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Pichia kudriavzevii, Cryptococcus neoformans/gattii complex). These methods are very useful to determine potential link between clinical and/or environmental isolates but implementation/validation requires a large number of unrelated isolates. Grouped-cases of infection or colonization due to uncommon or emerging pathogenic yeast species are usually considered to be due to a single strain. Since there is no genotyping method for these uncommon species and considering that origin of the infection can be endogenous and/or exogenous, this hypothesis should be verified. Whole genome sequencing (WGS) has become relatively simple, fast and inexpensive but analysis remains time-consuming and requires bioinformatic expertise. Since 2012 and the first French outbreak due to Saprochaete clavata, we started using WGS to study relationship between isolates involved in grouped cases and belonging to uncommon yeast species. Different approaches involving different parts of the genome can be considered. For S. clavata, alignment of whole genome allowed to identify different clades, one of which was responsible for the national epidemic with specific Single Nucleotide Polymorphism positions (SNP). Then, some SNPs were selected to design a real time PCR with allele-specific oligonucleotide (ASO) which allowed us to rapidly detect isolate belonging to the epidemic clade. Another approach was the use of WGS data of Yarrowia lipoytica to identify intergenic regions highly polymorphic and usable as molecular tool. Finally, the alignment of SNPs positions localized all over the genome was used to study genomic diversity of Trichosporon asahii and Magnusiomyces capitatus population. Although the presence of a rare species in several patients in the same hospital over a short period of time suggest a common source of infection, this is not always the case especially for species that are supposed to be part of the normal biota of skin and/or gastrointestinal tract. Our data confirm the importance of data and isolate centralization in a Reference Laboratory for molecular epidemiology. The routine WGS as a typing method for uncommon yeasts requires prior validation with varied sampling together with bioinformatics experts and mycologists to avoid misinterpretation.

    S12.5. Outbreak of fluconazole-resistant Candida parapsilosis in a hospital ward: arguments for clonal transmission and environmental persistence

    A. Fekkar 1,2, A. Bougle 3, A.C. Normand 1, M. Palous 1, R. Piarroux 1 and S. Imbert 1
    1
    Laboratoire De Parasitologie-mycologie, APHP La Pitié Salpétrière, PARIS, France
    2
    Sorbonne Université, Inserm, CNRS, Centre d’Immunologie et des Maladies Infectieuses, Cimi-Paris, F-75013, PARIS, France
    3
    Réanimation Chirurgie Cardiaque, La Pitie Salpetriere Hospital, Paris, France
    Objectives: The emergence of multidrug-resistant fungal agent is a threat for human health. In recent years, breakthroughs of infections due to fluconazole-resistant Candida parapsilosis isolates have been increasingly reported in different countries. We have recently faced with invasive infections due to resistant isolates and have decided to further investigate this phenomenon.
    Methods: We conducted a monocentric study at La Pitié-Salpêtrière Hospital, a tertiary care center in Paris, France. All Candida parapsilosis isolates collected between January 2012 and April 2019 for which sensitivity to antifungal agents had been determined were analyzed. Antifungal susceptibility testing was performed using Etest (Biomerieux) method and confirmed according the EUCAST broth microdilution reference method. Minimal Inhibitory Concentrations were determined for azoles drugs (i.e. fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole). For fluconazole resistant isolates and a random selection of susceptible isolates, the erg11 gene was sequenced and isolates were genotyped by a microsatellite method based on 6 markers.
    Results: 193 isolates (193 patients) sampled between 2012 and 2019 were analyzed. We observed an increase of fluconazole resistance rate among Candida parapsilosis isolates (MIC > = 8 µg/mL) between the periods 2012-2016 (3/97, 3.1%) and January 2017-April 2019 (10/96, 10.4%) (p<0.05 by Fisher’s Exact test): three fluconazole-resistant isolates were detected in 2012-2013, none in 2014-2016 and 10 between January 2017 and April 2019. All the resistant isolates presented the previously described A395T mutation in the erg11 gene that confers the amino acid change Y132F. This mutation was absent in azole-sensitive isolates. Genotyping showed 4 different microsatellite profiles for resistant isolates (R1, R2, R3 and R4) while susceptible isolates were not related each other’s and showed a greater diversity (Figure 1).
    Jof 05 00095 i005
    Two resistant clusters (R1 and R3) were related to grouped cases in a single intensive care unit (ICU). Isolates belonging to the R3 profile (n = 7) were detected since November 2017, were responsible of 7 deep infections and presented the highest MICs to fluconazole (> 64 mg/L) and a cross resistance with voriconazole (MIC > 0.5 mg/L). Epidemiological and chronological analyses furnish clues for a patient-to-patient transmission but also suggest an ability of resistant isolates to persist in the ward environment. Indeed, two isolates of the same genotype R1 were detected 4 years apart in the same ICU.
    Conclusion: Our work shows an increase of the incidence of fluconazole-resistant Candida parapsilosis isolates in intensive care units in relation to the clonal spread of few strains. Our results provide evidence of i. patient-to-patient transmission and ii. persistence of resistant clone over time in the same unit. Our study urges for a better investigation of fungal resistance and improved eradication measures to limit the spread of this resistant emerging pathogen.

    Symposium 13 Fungal respiratory infections in cystic fibrosis (the ECMM/ISHAM working Group Fri-CF)

    S13.1. Rasamsonia species and other emerging fungi in CF

    S. Le Gal 1,2, G. Nevez 1,2, L. Favennec 3,4 and J.-P. Bouchara 5,6
    1
    Laboratory of Parasitology-Mycology, University Hospital Center, Brest, France, Brest, France
    2
    Host-pathogen Interaction Study Group Ea3142, University of Brest - University of Angers, BREST, France
    3
    Laboratoire De Parasitologie-mycologie, CHU de Rouen, Rouen, France
    4
    Protozooses Transmises Par L’alimentation, University of Rouen - University of Reims, Rouen, France
    5
    Host-Pathogen Interaction Study Group, SFR ICAT 4208, Univ Angers, Univ Brest, Institute of Biology in Health, IRIS, University Hospital Center, Angers, France, Angers Cedex, France
    6
    Laboratory of Parasitology-Mycology, University Hospital Center, Angers, France, Angers, France

    Abstract:

    Cystic fibrosis (CF) is the most common genetic inherited disease in the European Caucasian population. The disease is caused by mutations in the gene CFTR (Cystic Fibrosis Transmembrane conductance Regulator) which encodes a membrane transporter involved in electrolytic exchanges across the apical membrane of epithelial cells. Several organs are therefore affected by these mutations, but the clinical picture is dominated by respiratory infections. Indeed, because of the resulting thickening of the bronchial mucus, the respiratory tract is often colonized by microorganisms, leading to respiratory infections which are the major cause of morbidity and mortality in patients with CF. Bacteria, mainly Staphylococcus aureus and Pseudomonas aeruginosa, are the major cause of these infections. However, several yeasts and filamentous fungi may also colonize the airways of patients with CF, sometimes leading to pulmonary infections and to severe disseminated infections after lung transplantation. Moreover, even in the absence of clinical signs, this fungal colonization could contribute to the inflammatory reaction and thus to the progressive deterioration of the lung function. If the epidemiology and pathophysiology of fungal infections still remain largely unknown, the frequency of these infections increases together with life expectancy of CF patients. Aspergillus fumigatus ranks first among the filamentous fungi colonizing the airways of patients with CF, followed by Scedosporium species, Exophiala dermatitidis, Lomentospora prolificans (formerly Scedosporium prolificans), and Aspergillus terreus. The prevalence of these fungi varies from one study to another because of: i) differences in patient recruitment (age, CFTR genotype); ii) lack of standardization of the procedures used for mycological examination of respiratory secretions; iii) geographical and climatic variations. In addition, other fungal species have been increasingly reported during the past decade, such as the yeasts Candida blankii, Trichomonascus ciferrii and Blastobotrys adeninivorans / Blastobotrys raffinosifermentans. These yeasts share the capability to use diamines, such as putrescine, which may explain that they multiply in the bronchial mucus during pulmonary exacerbations since the concentration of putrescine increases in this context. Likewise, some filamentous fungi including Arthrographis kalrae and species of the Rasamsonia argillacea complex (formerly Geosmithia argillacea, but first reported in CF as Penicillium emersonii) seem to be emerging in CF. Although it is usually well tolerated, the chronic colonization of the airways by these fungi should not be disregarded since it constitutes a risk factor for disseminated infections in the case of lung transplantation. The emergence of these fungi could be explained at least in part by improvements in diagnostic methods, or by selection pressure caused by repeated antifungal cures. Our knowledge of the ecology of these filamentous fungi still remains in infancy; nevertheless, regarding species of the Rasamsonia argillacea complex, some observations suggest that they could be xylophagous fungi that have spread in Europe since the end of the last century as a consequence of global warming and of the many storms that have crossed Europe since the end of the nineties.

    S13.2. Immunodiagnosis of Scedosporium/Lomentospora infection - lessons from immunoproteomic studies

    A. Ramirez-Garcia 1, L. Martin-Souto 1, L. Aparicio-Fernandez 1, I. Buldain 1, A. Antoran 1, J.-P. Bouchara 2, M.T. Martin-Gomez 3, A. Rementeria 1 and F.L. Hernando 1
    1
    Immunology, Microbiology and Parasitology, University of the Basque Country (UPV/EHU), Leioa, Spain
    2
    Laboratory of Parasitology-Mycology, University Hospital Center, Angers, France, Angers, France
    3
    Microbiology, Vall d’Hebron Universitary Hospital, Barcelona, Spain

    Abstract:

    The detection and diagnosis of Scedosporium/Lomentospora still relays on traditional methods and, in spite of being the second most common etiological agent among filamentous fungal infections, very few advances have been achieved in this field during the last decades. This fact is especially worrying in Cystic Fibrosis (CF) patients, whose characteristics favor the microbial presence. In these patients, the fungal detection does not mean that an infection is occurring, but it may increase the risk of suffering from it or other health problems. Therefore, it would be interesting to dispose an easy, rapid and non-invasive detection method for monitoring these patients and study the evolution of the fungus. With this purpose, some groups are focusing their efforts on finding new approaches to design a serologic system. In fact, techniques such as counterimmunoelectrophoresis or ELISA using recombinant proteins of some well-described virulence factors are being used by some laboratories, which design their own assays. Our research group addressed this problem using different immunoproteomics-based approaches. First, four different Scedosporium boydii protein extracts were studied by ELISA to select the most useful one for IgG detection in CF patients. The extracts used were a whole-cell extract, fungal cell-surface extract, conidia cell surface extract and secreted extract, all of them being able to discriminate acceptably the Scedosporium/Lomentospora-infected patients from Aspergillus-infected and non-infected patients. However, the best results were obtained with the whole-cell extract, which showed specificity, sensitivity, predicted positive and negative values comparable with commercial kits for other fungal diagnosis purposes. In spite of the excellent results obtained, to increase the specificity and avoid the problems derived from the use of a complex extract, the immunoreactivity of a collection of sera from CF patients infected with Scedosporium/Lomentopora against S. boydii was also studied in comparing with sera from CF patients infected with Aspergillus or without fungus. The analysis, made by 2-DE and WB, allowed the identification of antigens specifically detected by CF patients with Scedosporium/Lomentopora, two of them being identified because of their promising results. In addition, to shed light on the problematic of whether the fungal presence in CF patients is a colonization or an infection, we used sera from mice infected intravenously with different species of Scedosporium/Lomentospora to detect infection-related antigens of S. boydii. Interestingly, all the sera showed a very similar immunorecognition patterns, but different from the recognized by sera from Aspergillus-infected mice. Therefore, we purified six antigens and tested their interest for diagnosis analyzing their immunoreactivity by WB against sera from CF patients, two of them showing interesting results. Altogether, these results obtained by immunoproteomics-based techniques open up door in the looking for a serologic test for the detection of Scedosporium/Lomentospora in CF patients, as well as the monitoring of the fungal presence. Specifically, although the results have to be further studied and standardized, the ELISA test using whole-cell extract appears to be a very useful, and the antigens identified seems to be interesting candidates to be include in the diagnostic procedure.

    S13.4. Morphology, growth and biofilm formation of the black yeast-like fungus Exophiala dermatitidis is influenced by Pseudomonas aeruginosa under in vitro cystic fibrosis conditions.

    L. Kirchhoff 1, P.-M. Rath 1 and J. Steinmann 1,2
    1
    Institute of Medical Microbiology, University Hospital Essen, Essen, Germany
    2
    Institute of Clinical Hygiene, Medical Microbiology and Clinical Infectiology, Klinikum Nuernberg, Nuremberg, Germany
    Objectives: Exophiala dermatitidis, belonging to the melanized fungi, is frequently colonizing the respiratory tract of cystic fibrosis (CF) patients in rates up to 19 %. It was recently reported that E. dermatitidis is capable to form biofilms in a strain-dependent manner. However, little is known about E. dermatitidis behavior in co-culture with other CF-relevant pathogens, e.g. one of the most prevalent bacterial species, Pseudomonas aeruginosa. Growth, biofilm formation capabilities and morphology of E. dermatitidis in an artificial sputum medium (ASM), mimicking the CF sputum conditions, were assessed in mono- and in co-culture with P. aeruginosa.
    Methods: P. aeruginosa (PA07 & PA14, including PA14 ΔlasR and ΔrhlR) and E. dermatitidis (clinical isolate) were analyzed in growth experiments over a period of 48 hours at 36°. Growth was determined by colony forming unit (CFU) counts. Biofilm, formed on polystyrene surfaces under standard protocols, was determined after 24 and 48 hours of incubation at 36°C without agitation by stain with crystal violet, by CFU counts after biofilm detachment using 0.1% dithiothreitol, for species-specific cell counts and by confocal laser scan microscopy determining the thickness of extracellular matrix (ECM). Morphology of the dimorphic E. dermatitidis was monitored in light microscopy. Lengths of fungal hyphae were estimated and compared.
    Results: P. aeruginosa showed growth inhibiting effects on E. dermatitidis. Cell count of the fungus was decreasing in co-culture with P. aeruginosa. In contrast, E. dermatitidis biofilm formation was induced after 24 h and reduced after 48 h in co-culture with P. aeruginosa. A lack of P. aeruginosa quorum sensing (QS) systems lasR and rhlR resulted in induced fungal biofilm formation. At the same time, presence of lasR and rhlR lacking mutants caused an increased amount of E. dermatitidis hyphal structures whereas co-cultivation with the P. aeruginosa wildtype resulted in decreased hyphae amounts, compared to mono-culture.
    Conclusion: Interactions between P. aeruginosa and E. dermatitidis result in altered growth, biofilm formation capabilities and morphology of the fungus in an in vitro CF model. A role of P. aeruginosa QS systems in these interactions is suggested.

    S13.6. Study of antigenic markers for serological detection of Scedosporium spp. in Cystic Fibrosis patients

    L. Martin-Souto 1, M. Areitio 1, L. Aparicio-Fernandez 1, I. Buldain 1, A. Antoran 1, J.-P. Bouchara 2, M.T. Martin-Gomez 3, A. Rementeria 4, F.L. Hernando 4 and A. Ramirez-Garcia 4
    1
    Immunology, Microbiology and Parasitology, University of the Basque Country (UPV/EHU), Leioa, Spain
    2
    CHU d’Angers, Angers, France
    3
    Microbiology, Hospital Universitari Vall d’Hebron, Barcelona, Spain
    4
    Immunology, Microbiology and Parasitology, University of the Basque Country (UPV/EHU), Leioa, Spain
    Objectives: Cystic fibrosis (CF) is the major genetic disorder among the Caucasian population. CF patients produce a thick and sticky bronchial mucus in their respiratory tract, which facilitates the growth of airborne pathogens. In addition to bacteria, several fungi colonize the respiratory tracts of these patients. Species of Scedosporium and Lomentospora genera are emergent fungal pathogens ranking the second, only behind Aspergillus spp., among filamentous fungi causing a chronic colonization of the airways of CF patients. This may lead to chronic inflammation or even to life-threatening invasive disease in cases of severe immunosuppression. Detection of Scedosporium/Lomentospora relies upon low sensitivity and specificity non-standardized procedures. To contribute to the finding of new diagnostic methods, this work aims to characterize some molecular targets and to study their usefulness for serological detection of infections caused by Scedosporium spp. in CF patients.
    Methods: A total of 191 CF patients’ sera were used in this study. First, we classified these sera into 3 groups: patients with positive sputum cultures for Scedosporium (S+), for Aspergillus (A+), and patients with negative fungal cultures (Ctrl). A serological study of the sera was carried out to detect specific IgG response against Scedosporium boydii total protein extract by ELISA. In the search of diagnostic markers that allow the discrimination between colonization and infection, we performed an in vivo infection in a murine model to obtain sera that allow the detection of antigens associated with a disseminated infection by two dimensional western blot. These antigens were electroeluted from protein gels and blotted individually against CF patients’ sera to observe the immunological response.
    Results: The ELISA assay provided good specificity, and positive and negative predictive values to discriminate among S+, A+ and Ctrl patients. However, the sensitivity was influenced by differences in the humoral response of the S+ group. This suggests different levels of fungal presence, from colonization to infection, among S+ patients. To solve this problem, we looked for S. boydii antigens associated with a disseminated infection in a murine model selecting the six proteins with the highest immunoreactivity. Two of them were specifically recognized by most of S+ sera, especially by those that were more reactive in the ELISA assay.
    Conclusion: Two proteins of S. boydii resulted to be potential diagnostic markers for the detection of the infection caused by Scedosporium spp. in CF patients. The use of them in combination with an ELISA using S. boydii total protein extract might help in the detection of Scedosporium spp. in CF patients and the monitoring of the fungal presence.

    Symposium 14 Teaching Medical Mycology: what, how, to whom and when

    S14.2. Mycology teaching in Medical School—Is student demography important to what should be taught

    E. Segal
    Sackler School of Medicine, Clinical Microbiology and Immunology, Tel Aviv University, Tel Aviv, Israel

    Abstract:

    Mycology which is the discipline devoted to the study of fungi, is one of the four parts comprising Microbiology, the basis for understanding infectious diseases. Medical Mycology is the part of Mycology which focuses on those fungi known to cause or potentially can cause infections in human hosts. As such, study of Medical Mycology is an integral part of the education of physicians and is included in the curriculum of Medical Schools. One of the questions associated with the teaching of Medical Mycology is at which level should Medical Mycology be taught: at the basic sciences level, with other parts of Microbiology, or at a later stage in the frame of Infectious Diseases, as part of Internal Medicine. The answer to this dilemma may vary among different Medical Schools. Another question associated with teaching Medical Mycology in Medical Schools is as to the content of the subject: should it reflect the specific geographic–climatic conditions of the given Medical School or should a more general, broader approach be used. An additional dilemma regarding teaching of Medical Mycology in Medical Schools is whether the specific student demography should affect what is taught and to what extent. The latter is the subject of the following presentation. In the presentation different teaching possibilities will be demonstrated and discussed, as to pro- or contra- of the impact of student demography on what is taught. Examples of different approaches to the dilemma will be shown and discussed. Participation of the audience in the discussion will be encouraged.

    S14.3. Specific courses outside European Continent – The Indian Example

    R. Ashbee
    University of Leeds, Leeds, United Kingdom
    HR Ashbee, RD Sahni & JS Michael Teaching medical mycology in the Indian setting presents some unique challenges and opportunities. The diversity of mycoses seen is extensive and presentations can be unusual with disease sometimes occurring in patients not traditionally ‘at risk’. Whilst dermatophytosis and superficial candidosis is often neither diagnosed nor treated, fungal keratitis and sinusitis, subcutaneous and systemic mycoses represent a significant burden on the healthcare system. In combination with this, diagnostic facilities vary from virtually nil to fully equipped laboratories with well-trained staff. The Christian Medical College Hospital in Tamil Nadu, south India has had a mycology laboratory since the 1950’s, has highly skilled staff and a massive number of clinical samples – hence it is ideally suited to deliver comprehensive mycology training. The first course in 2007 was aimed at technical staff working in laboratories and junior faculty, primarily focusing on basic medical mycology and identification of fungal pathogens. The course was attended by 30 participants from all over India, some traveling for 30 hours by train to take part in the course. The need for training was apparent and further courses were planned The current course has been re-designed and is run in two parts. The first 3-day part is aimed at registrars/residents in microbiology and provides an integrated approach to teaching with clinicians presenting cases and the laboratory aspects taught in practical, hands-on sessions after the cases. The full range of diseases are covered, including the superficial mycoses, subcutaneous mycoses, candidosis, cryptococcosis, mucormycosis, aspergillosis and endemic mycoses. Hands-on practical sessions also cover setting up of antifungal susceptibility tests and their interpretation. The second part of the course held over 2 ½ days is for consultant microbiologists and this includes a full day of practical molecular mycology, including DNA extraction, setting up PCR and trouble-shooting molecular assays, use of commercially available assays, as well as molecular detection of antifungal resistance. Antifungal susceptibility testing is taught in another full day practical session with participants taken through the whole CLSI method, from making up the medium, preparing antifungal dilutions, making up plates and preparing the inoculum, to reading and interpreting results. Other methods such as E-test and disc diffusion are also taught. The practicals take place in small groups so every participant can get hands-on experience of each technique. Discussion sessions make up the remainder of the time with ample opportunity for participants to ask questions and clarify any problems. Participants are also encouraged to maintain contact after the workshop and help is provided with testing or fungal identification by CMC staff if required. The courses represent a significant investment of time and resources by the staff of CMC and overseas faculty but feedback has been extremely good and demonstrates that provision of high quality, comprehensive mycology training is both very much needed but also very well received.

    S14.4. Teaching Clinical Laboratory Mycology: How and who is the audience?

    S. Arikan-Akdagli
    Medical Microbiology, Mycology Laboratory, Hacettepe University Medical School, ANKARA, Turkey
    Expertise in Clinical Laboratory Mycology requires hands-on experience and in-depth basic knowledge. Challenges regarding teaching Clinical Laboratory Mycology are numerous. First, the number of invasive fungal infections is increasing with more infections due to rare and less-recognized species and the education for management of this growing problem is yet inadequate. Second, Mycology education was rather less emphasized until recent years. Teaching for Clinical Laboratory Mycology practices is usually limited during training of a Clinical Microbiologist and the numbers of well-equipped Clinical Mycology Laboratories and experts are also relatively limited worldwide. Third, the characteristics of Y (and Z) generation(s) are unique. The members of generation Y are primarily among the current audience and they are largely influenced by technology. Therefore, broader training and diverse teaching methods are required to meet all these challenges. Clinicial Laboratory Mycology training is a part of speciality/subspeciality, PhD or Master of Science training programs in Medical Microbiology/Mycology. Also, Infectious Diseases/Infectious Diseases and Clinical Microbiology Joint Training, and Pathology Training Programs include training in Mycology at required levels. For Medical Faculty students, on the other hand, basic Clinical Laboratory Mycology teaching is incorporated in the program at some institutions in order to integrate the previously taught theoretical knowledge with that on mycological diagnosis in the last year(s) of education. Laboratory technicians are also among the audience for teaching Clinical Laboratory Mycology at required and specifically technical level. Combining conventional teaching methods with digital technologies is needed for contemporary high quality Clinical Laboratory Mycology education. The common approach for teaching requires expert(s), a well-equipped Mycology Laboratory, and reliable basic information sources, including physical books or e-books. In lack of these facilities or for advancement for higher levels, in-person courses and workshops are very beneficial. However, they are costly and require qualified instructors and resources. E-learning courses, webinars, and educational websites are also very helpful in expanding the trained audience. Teaching Clinical Laboratory Mycology is a difficult task and requires dedicated Mycologists.

    S14.5. Capacity building for invasive fungal infections in Africa: concrete examples through mycology courses and the DREAMM implementation project

    A. Sturny Leclère 1, D. Garcia-Hermoso 2, T. Boyer-Chammard 1, I. Kolte 3, F. Dromer 4, A. Alanio 5, O. Lortholary 6 and A. Loyse 3
    1
    Molecular Mycology, IP Paris, Paris, France
    2
    National Reference Center For Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, PARIS, France
    3
    Institute of Infection and Immunity, St. George’s University of London, London, United Kingdom
    4
    National Reference Center For Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, Paris, France
    5
    Paris-diderot, Sorbonne Paris Cité University, Institut Pasteur, Molecular Mycology Unit, Cnrs Cmr2000, Parasitology-Mycology Laboratory, Lariboisière Saint-Louis Fernand Widal Hospitals, Assistance Publique-Hôpitaux de Paris, Paris, France
    6
    Infectious Diseases, APHP, Hopital Necker-Enfants malades, Paris, France
    Across the world, fungal pathogens are omnipresent in the environment, affect over a billion and kill more than 1.5 million people. Despite these figures, invasive fungal infections (IFIs) do not gather as much press coverage as tuberculosis or malaria. AIDS represents one of the major risk factors for IFIs together with other acquired immunodeficiencies. Invasive fungal infections should be a concern everywhere by their incidence, severity and high burden. Worldwide IFIs are not recognized due to lack of surveillance systems together with a lack of mandatory reporting, limited teaching in medical schools and therefore limited awareness and expertise of clinicians and biologists, lack of reliable diagnostic procedures for many IFIs, and limited number of commercialized antifungal drugs. In resource limited countries, the lack of basic laboratory equipment and poor access to essential medicines are additional major barriers. Outcomes of cryptococcal meningitis, a leading cause of HIV-related death, are still poor despite updated WHO guidelines. The DREAMM project aims to reduce the mortality of cryptoccocal meningitis in resource limited settingsby providing rapid diagnostic tests, essential medicines and reinforcing basic and mycology trainings for all staff members involved (technicians, nurses, physicians, biologists). Through this program, a better awareness of all IFIs is expected and results are already visible.

    Symposium 15 Aspergillus in the ICU

    S15.1. Influenza-associated aspergillosis in ICU: when the flu get mouldy

    J. Wauters
    Medical Intensive Care Unit, University Hospitals Leuven, Leuven, Belgium
    Worldwide, 3-5 million people develop severe influenza every year, leading to 50.000-100.000 deaths annually in the European Union and the USA. of the patients hospitalized, 5-10% needs ICU admission due to severe illness. Patients with severe illness initially present with typical influenza symptoms, but are rapidly evolving into respiratory deterioration due to bacterial superinfection but also influenza in itself can cause severe acute respiratory distress syndrome (ARDS), the latter being associated with a mortality of 14% to 41%. Although bacterial co-infection is already known for decades, influenza-associated aspergillosis (IAA) was recently found to be a frequent and severe complication of influenza pneumonia in critically ill patients. Classically, invasive pulmonary aspergillosis (IPA) typically occurs in a severely immunocompromised host. Influenza-associated aspergillosis (IAA) was occasionally described decades ago and several small case series were reported recently. 65% of the reported cases did not have classic host factors for IPA as defined by the EORTC/MSG. Recently, we published a large multicentre case-control study within the Dutch-Belgian Mycosis Study Group (7 Belgian and Dutch ICUs, 2009-2016) including 432 severe influenza patients (positive influenza polymerase chain reaction (PCR)), 315 of them being EORTC negative. As a control group, 315 influenza negative and EORTC negative patients, admitted to the ICU with severe community-acquired pneumonia (CAP), were selected. In the 432 severe influenza patients, IPA was diagnosed in 19% (83/432), a median of 3 (IQR 0-7) days after ICU admission. The IPA incidence in the 117 immunocompromised (EORTC positive) influenza patients was as high as 32% (38/117), while 14% (45/315) of the non-immunocompromised (EORTC negative) influenza patients developed IPA. In contrast, only 5% (16/315) of the non-immunocompromised influenza-negative controls developed IPA (p<0·0001). The 90-day mortality in influenza patients with and without IPA was 51% and 28%, respectively (p<0·0001). Moreover, in this retrospective cohort study, influenza was found to be independently associated with IPA (aOR 5·2, 95% CI 2·6-10·3, p<0·0001), besides a higher APACHE II score, male sex and use of corticosteroids. Though awareness of IAA is rising, it remains unclear why patients with severe influenza infection develop IPA. Influenza-induced necrosis of the bronchial tree might provide a gateway for Aspergillus infection. Further, severe influenza might alter innate and adaptive host facilitating fungal infection and genetic variation might also influence the susceptibility to IPA. Finally, corticosteroid (CS) therapy is a known independent risk factor for the development of IPA at the ICU. In summary, IAA is an early and frequent complication of influenza pneumonia and increases the probability of death with a factor 2 to 3 in critically ill patients with severe influenza. An aggressive diagnostic approach should be pursued, but early diagnosis is difficult due to unspecific clinical and radiological presentation. Future studies should evaluate whether a faster diagnosis and/or antifungal prophylaxis could improve outcome of influenza-associated aspergillosis. Reference: Schauwvlieghe AFAD, Rijnders BJA, Philips N et al. Dutch-Belgian Mycosis study group. Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study. Lancet Respir Med. 2018;6(10):782-792.

    S15.2. Invasive aspergillosis in patients with underlying liver cirrhosis

    J. Prattes
    Section of Infectious Diseases and Tropical Medicine, Medical University of Graz, Graz, Austria

    Abstract:

    Invasive aspergillosis (IA) has increasingly reported in critical ill patients without classical risk factors like hematological malignancy, neutropenia or solid organ transplantation. Liver cirrhosis represents the main underlying disease in some of these patients, highlighting the relevance of cirrhosis as a risk factor for IA development. Whereas the overall incidence of IA in patients with liver cirrhosis is very low (<1%), the risk for IA increases when liver function deteriorates and patients are in need for intensive care. Reasons for susceptibility of cirrhotic patients for developing IA are diverse. Cirrhosis-associated immune dysfunction summarizes both, systemic inflammation due to cirrhosis and cirrhosis associated immunodeficiency. The latter one may be due to systemic glucocorticoid treatment but also due to alterations of the innate as well as adaptive immune functions. Impaired expression of Fcγ –receptor on neutrophils, impaired neutrophil mobilization, phagocytic activity, chemotaxis, bacterial phagocytosis and killing are present in cirrhotic patients. However, the degree of immune dysfunction correlates with the stage of the liver disease putting patients with end stage liver disease at highest risk for IA development. Awareness for IA in cirrhotic patients has to be warranted as diagnosis may be challenging. Blood biomarkers, like galactomannan and beta-d-glucan, lack sensitivity and clinical presentation may be atypical. In case of IA suspicion early chest CT scan and bronchoscopy may be needed.

    S15.3. Aspergillus in the ICU – Management of IA in Renal Failure

    R. Rautemaa-Richardson 1,2,3,4
    1
    Division of Infection, Immunity and Respiratory Medicine, University of Manchester, Manchester, United Kingdom
    2
    Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom
    3
    National Aspergillosis Centre, Manchester, United Kingdom
    4
    Department of Infectious Diseases, Manchester University NHS Foundation Trust, Manchester, United Kingdom

    Abstract:

    Renal failure is a common complication in critically ill patients. In addition, angioinvasive aspergillosis is associated with renal lesions and renal failure. The challenge is that the main intravenous drugs used to manage invasive aspergillosis (IA), namely amphotericin B and voriconazole (with cyclodextrin) are nephrotoxic. Also, in critically ill patients with multi organ failure, the pharmacodynamics and pharmacokinetics are highly variable, which results in a significant risk for toxicity as well as suboptimal dosing and treatment failure. The impact of renal failure on the management of IA depends on whether the patient is on renal replacement therapy (RRT) or not. Once on RRT, the main consideration is the risk for suboptimal drug levels until the system is saturated with the drug. However, it is important to keep in mind that the initiation of RRT does not remove the risk for further or permanent drug-induced kidney injury. Once the patient starts to respond to the treatment, their haemodynamics stabilise and their kidney function improves, there is a risk for both toxicity and subtherapeutic levels. Therefore, in the case of azoles, critically ill patients on intensive care unit (ICU) require regular therapeutic drug monitoring (TDM) and repeated dose adjustments. This also applies to azoles not associated with kidney toxicity (isavuconazole). Whilst TDM is an important part of monitoring the safety and efficacy of antifungal treatment, microbiological diagnostics are another important tool in patient management. Sputum high-volume culture, PCR and galactomannan measurements can be used to monitor treatment response whereas blood galactomannan is less useful in non-neutropenic setting. However, it is not rare that these tests remain positive for prolonged periods despite good clinical response.

    S15.4. Fungal pneumonia in critically ill cirrhotics: Spectrum, Outcomes, Comparison of diagnostic methods and Biomarkers

    P. Kale 1, V. Khillan 1 and S.K. Sarin 2
    1
    Clinical Microbiology, Institute of Liver and Biliary Sciences, New Delhi, India
    2
    Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India
    Objectives: Liver cirrhosis causes immune dysregulation and increased susceptibility to fungal infections. Futher there are associated co-morbidities like diabetes mellitus, renall impairment and malignancy which predispose to fungal infections. Hence early diagnosis of fungal infections and initiation of appropriate empiric or pre-emptive antifungal is essential to reduce morbidity and mortality associated with it. We studied the epidemiology, spectrum, risk factors of fungal infections and antifungal susceptibility pattern for guiding empiric therapy. We also compared the rapid diagnostic methods and biomarkers for early diagnosis fungal pneumonia in critically ill cirrhotics.
    Methods: Single-center, prospective cohort study of 100 critically ill cirrhotics with fungal pneumonia between January to September 2018. All patients met the diagnostic criteria for liver cirrhosis by clinical, biochemical, and ultrasonography findings. Respiratory samples were processed for fungal culture and real time PCR. Antifungal susceptibility testing was done by Broth microdilution method in accordance with CLSI guidelines. Biomarkers; bronchoalveolar lavage (BAL) and serum Galactomannan, and serum procalcitonin measured on day 1, 3 7. Mortality within one month of diagnosis or discharge was analyzed.
    Results: Aspergillus flavus was the most common species (70/100,70%). Risk factors for fungal pneumonia included neutropenia (p 0.03), steroids prior to ICU admission (p 0.02), prolonged (>21 days) hospitalization (p<0.05). Culture positivity was 80%. Culture was not inferior to real time PCR for diagnosis of fungal pneumonia. Antifungal susceptibility testing revealed, all A. flavus and A. fumigatus isolates sensitive to azoles, amphotericin B and echinocandins. BAL galactomannan was early prognostic marker with median rise >3.5 over the index value. Median PCT level was higher from day 1in the fungal pneumonia non-survivor than survivor group (3.29 vs. 0.8ng/ml), with higher 30-day mortality (72%). Baseline PCT at admission to ICU was higher in non- survivors; levels on D3 and D7 were persistently higher. Higher PCT level was associated with bacterial co-infection (48%), antibiotic (74%) and antifungal therapy and renal failure and mortality.
    Conclusion: Cirrhotics who are neutropenic, have prolonged hospitalization and exposed to steroids have a high risk of fungal pneumonia. Aspergillus flavus predominate as in consensus with Asian epidemiology. Culture methods are reliable and combination of molecular test with BAL galactomannan is useful for rapid diagnosis. However the cutoff should be defined with epidemiological correlation. High serum procalcitonin level is an independent prognostic biomarker of mortality risk in fungal pneumonia which reaches nearly 70%. In our study the baseline PCT at admission to ICU was higher in non- survivor group, levels on D3 and D7 were persistantly higher. High index of suspicion and early detection is required in advanced cirrhosis

    S15.5. IPAFLU survey: Invasive Aspergillosis among Patients with Severe Influenza in Intensive Care Units

    M. Holtappels 1, C. Jacobs 1, T. Chiller 2, J. Fortenberry 3, B. Jackson 4, K. Lagrou 5, M. Toda 4, F. Van De Veerdonk 6, P.E. Verweij 7 and J. Wauters 8
    1
    Laboratory For Clinical Infectious and Inflammatory Disorders, KU Leuven, Leuven, Belgium
    2
    Mycotic Diseases Branch, Centre for Disease Control, Altanta, United States of America
    3
    Pediatric Critical Care, Children’s Healthcare of Atlanta, Atlanta, United States of America
    4
    Mycotic Diseases Branch, Centre for Diseases Control, Altanta, United States of America
    5
    Department of Microbiology, Immunology and Transplantation, KU Leuven, Leuven, Belgium
    6
    Infectieziekten, Afdeling Interne Geneeskunde, Nijmegen, Netherlands
    7
    Medical Microbiology, RadboudUMC, Nijmegen, Netherlands
    8
    Medical Icu, UZLeuven, Leuven, Belgium
    Objectives: Invasive aspergillosis usually occurs in people with weakened immune systems. However, several reports describe fatal Aspergillus pulmonary infections in previously healthy patients who were hospitalized with a severe influenza virus infection. Recently, a retrospective cohort study, conducted at seven intensive care units (ICUs) in Belgium and the Netherlands during 2009–2016, found that invasive pulmonary aspergillosis occurred in 19% of patients with severe influenza requiring admission to the ICU. We conducted an international survey to determine broader knowledge and awareness of influenza-associated aspergillosis (IAA), as well as the diagnostic use of galactomannan (GM) in bronchoalveolar lavage (BAL) and serum in critically ill patients with severe influenza.
    Methods: Members of the Extracorporeal Life Support Organization (ELSO), the Society of Critical Care Medicine (SCCM), and the European society of intensive medicine (ESICM) network (n = 20,093) were invited to participate in an online 11-question survey. Participants were asked about their roles in the ICU, ICU size, number of patients with influenza admitted to the ICU per influenza season, use of neuraminidase inhibitors (NAIs), frequency of obtaining lower respiratory specimens and GM testing on serum and BAL samples, and their knowledge on the occurrence of IAA.
    Results: A total of 468 (2.3%) members responded, with 225 (48%) residing in the United States, 151 (32%) in Europe, and 92 (20%) in other countries, including Australia and countries in South America, Asia and Africa. Most (89%) participants were critical care physicians. Large ICU departments were more common in the United States compared with Europe. The majority of participants reported <30 influenza cases per season, and NAIs were the preferred antiviral treatment (92%). European participants reported obtaining lower respiratory specimens and determining GM on BAL and serum more commonly than U.S. participants (p < 0.001) (Figure 1). Globally, 66% (n = 303) of respondents denied hearing of or seeing IAA cases in the past five years (Figure 2). However, 57% of European participants reported seeing ≥1 patient with IAA compared with only 17% of U.S. participants (p < 0.001) and 36% of participants in other countries (p = 0.007).
    Jof 05 00095 i006Jof 05 00095 i007
    Conclusion: More participants in Europe reported having seen IAA cases in compared with the United States and other countries. Although the observed differences in IAA cases could be explained by true variation in IAA prevalence, the condition might be underdiagnosed outside Europe. The results suggest that such underdiagnosis might partly explain the differences in IAA awareness in Europe compared with the United States and other countries.

    S15.6. Trend of Candidemia with Bloodstream infection in Intensive care units from 2006 to 2017: Results from the Korean National Healthcare-associated Infections Surveillance System

    Y.H. Choi 1, E.J. Kim 1, Y.G. Kwak 2, H.M. Yoo 3, J.Y. Choi 4, M.J. Shin 5, S.R. Kim 6, S.-Y. Yoo 7 and N.-H. Cho 8
    1
    Department of Infectious Diseases, Ajou University School of Medicine, Suwon-si, Gyeonggi-do, Republic of Korea
    2
    Department of Internal Medicine, Inje University Ilsan Paik Hospital, Goyang, Republic of Korea
    3
    Infection Control Office, Inje University Sanggye Paik Hospital, Seoul, Republic of Korea
    4
    Infection Control Unit, Chung-Ang University Healthcare System, Seoul, Republic of Korea
    5
    Infection Control Office, Seoul National University Bundang Hospital, Seongnam, Republic of Korea
    6
    Infection Control Office, Korea University Guro Hospital, Seoul, Republic of Korea
    7
    Adjunct Assistant Professor, College of Nursing, The Catholic University of Korea, Seoul, Republic of Korea
    8
    Department of Infection Control, Gangnam severance hospital, Yonsei university, Seoul, Republic of Korea
    Objectives: Candidemia is an important healthcare-associated infections (HAIs) in intensive care units (ICUs). The incidence trend and distribution of candidemia over a 12-year period through the Korean National Healthcare-associated Infections Surveillance System (KONIS) data were analyzed.
    Methods: The KONIS system was established in 2006, and has performed prospective surveillance for HAIs including bloodstream infection (BSI) and causative pathogen in ICU. We evaluated yearly trends of the frequencies of the causative pathogens and candidemia. All statistical analyses were 2-sided and performed the Cochran-Armitage test for trend and the Cochran–Mantel–Haenszel mean score test, by use of SAS software.
    Results: From 2006 until 2017, 2,248 candidemia cases occurred in 9,184,264 patients-days. For BSI, the proportion of candidemia gradually increased (for BSI from 15.2% in 2006 to 16.6% in 2017, P < 0.05) (Figure 1). The pooled mean incidence rate of candidemia was 0.24 per 1,000 patient days. Most frequent pathogen for BSI was Staphylococcus aureus from 2006 to 2012, however, candida species emerged as the most frequent pathogen since 2013. Candida albicans (39.9%) was the most frequent pathogen for candidemia, followed by C. tropicalis (20.2%) and C. parapsilosis (18.2%) (Figure 2). There was no significant change in the distribution of candida species by year (P = 0.29). Central-line associated BSI were the majority (92.5%). In subgroup analysis, there was a significant association with the increase in the proportion of candidemia by year in the presence of organ transplant wards (from 18.9% in 2006 to 21.1% in 2017), hospitals with less than 500 beds (from 2.7% in 2006 to 13.6% in 2017), or surgical ICUs (from 16.2% in 2006 to 21.7% in 2017) (P < 0.05).
    Jof 05 00095 i008Jof 05 00095 i009
    Conclusion: The proportion of candida as nosocomial pathogen for BSI has increased in Korea. Especially, the proportion in hospitals with less than 500 beds and surgical ICUs is increasing, so appropriate infection control program is needed. This work was supported by the Research Program funded (M2018A060000011) by the Korea Centers for Disease Control and Prevention.

    Symposium 16 Dermatology

    S16.1. Cutaneous Aspergillosis: Is it so rare?

    S. Arikan-Akdagli
    Medical Microbiology, Mycology Laboratory, Hacettepe University Medical School, ANKARA, Turkey

    Abstract:

    Invasion of skin by Aspergillus is an uncommonly encountered clinical picture. Primary cutaneous aspergillosis (PCA) indicates lesions due to direct inoculation of the fungus at the otherwise injured site. The lesions usually develop following trauma, surgery or burn wounds. One significant feature of PCA is that it may be observed in immunosuppressed as well as immunocompetent individuals. Premature neonates also constitute a unique group of predisposed hosts due to skin fragility. PCA may disseminate and be lethal particularly in immunocompromised patients. Secondary cutaneous aspergillosis (SCA), on the other hand, develops either due to hematogenous spread or direct invasion of the skin from adjacent infected structures, such as paranasal sinuses. Skin and visceral involvement or multiple skin lesions in two or more noncontiguous areas suggest disseminated Aspergillus infection. It is estimated that skin involvement in invasive aspergillosis occurs at rates of 1-5%. The skin lesions in CA appear to have variable presentations, including macule, papule, plaque, subcutaneous nodule, ulceration with necrosis, and hemorrhagic bulla. The exact prevalence of CA is unknown and there are limited number of dedicated series and case reports. Cases of CA are probably underdiagnosed and underreported due also to difficulties in diagnosing fungal infections in general. In addition, the affected patient population is heterogeneous and complex with multiple comorbidities and this further complicates the issue. For optimal management of patients with CA, skin biopsy should immediately be taken from suspected lesions particularly in predisposed individuals for histopathological and direct microscopic examinations and mycological culture.

    S16.2. Is resistance a problem for dermatophytosis?

    P. Nenoff 1, M. Monod 2, S.B. Verma 3, A. Burmester 4, A. Ebert 1, A. Singal 5, S. Gupta 6, C. Wiegand 4, U.-C. Hipler 7, F. Wittig 1, C. Krüger 1, D. Koch 1, R. Vasani 8, A. Saraswat 9, R. Madhu 10, S. Panda 11, A. Das 11, M. Kura 12, A. Jain 13, Y. Graeser 14 and S. Uhrlaβ 1
    1
    Partnership Dr. C. Krueger & Prof. P. Nenoff, Laboratory of medical microbiology, Roetha OT Moelbis, Germany
    2
    Dermatology Service, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
    3
    “Nirvan” and “In Skin” Clinics, Vadodara, India
    4
    Klinik Für Hautkrankheiten, Universitätsklinikum Jena, Jena, Germany
    5
    Department of Dermatology & Std, University College of Medical Sciences & GTB Hospital (University of Delhi), Delhi, India
    6
    Department of Dermatology and Venereolgy, Maharishi Markandeshwar Institute of Medical Sciences and Research, Mullana, India
    7
    Klinik Für Hautkrankheiten, Universitätsklinikum, Jena, Germany
    8
    Bhojani Clinic, Mumbai, India
    9
    Dermatology, Indushree Skin Clinic, Lucknow, India
    10
    Department of Dermatology (mycology), Madras Medical College, Chennai, India
    11
    Department of Dermatology, KPC medical college, Kolkata, India
    12
    Department of Dermatology, Grant Medical College, Mumbai & Sir JJ Group of Hospitals, Mumbai, India
    13
    12415, Doctor’s Nest, New Rajeev Gandhi Nagar, Kota, India
    14
    Institut Für Mikrobiologie Und Hygiene, Universitätsmedizin – Charité, Berlin, Germany
    Objectives: Resistance of dermatophytes against antifungal agents was not perceived as a problem for decades. Therapy failure in onychomycosis or tinea capitis was never related to a reduced in vitro susceptibility of the causative species of dermatophytes. The situation has changed dramatically. The starting point, was, most likely, India. An incredible increase in chronic recalcitrant dermatophytoses over the past few years has been noted in India. The main causative pathogen is the zoophilic dermatophyte Trichophyton (T.) mentagrophytes ITS genotype VIII.
    Patients and methods: Three epidemiologic exercises, for the purpose of speciation and also to look into the possibility of antifungal resistance were undertaken with cooperation from nine Indian centers with German and Swiss mycologists. A total of 291 isolates (278 T. mentagrophytes VIII, and 13 T. rubrum) were included in the terbinafine antifungal susceptibility testing and genetic point mutation analysis of the squalene epoxidase (SQLE) gene. Itraconazole and voriconazole minimal inhibitory concentrations (MICs) were determined.
    Results: High resistance rates of 66.7% (T. mentagrophytes VIII), and 27.3% (T. rubrum) to terbinafine were found in the first multicentric study from India. 76.0% T. mentagrophytes VIII and 57.1% T. rubrum strains of two further studies (New Delhi and Mullana) showed in vitro resistance to terbinafine. The T. mentagrophytes VIII strains collected from multiple geographic regions of India showed high frequency of single point mutations in the SQLE gene leading to terbinafine resistance. All resistant T. mentagrophytes strains (200) harboured missense mutations with subsequent amino acid substitutions, most frequently Phe397Leu (90.0%), either as a single substitution or in combination with Ala448Thr. On the other hand, terbinafine sensitive strains (78) showed almost exclusively a SQLE wild type (23.1%) or a single Ala448Thr substitution (73.1%). Substitutions less commonly encountered included Leu393Phe, Leu393Ser, Ser395Pro, Gln408Leu, His440Tyr and Ser443Pro. The number of terbinafine resistant dermatophytes in Northern India has increased significantly from 2017 to 2019 (69.0% in 2017; 72.0% in 2018; 78.0% in 2019). Additionally, we noted a rise in SQLE double mutants, which shows a selection advantage for this genotype combination.
    Conclusion: The dramatic increase in terbinafine resistant T. mentagrophytes ITS VIII from all over India within such a short period of time underscores the issue of development of resistance in patients with chronic dermatophytoses. A strong association was found between in vitro terbinafine resistance of T. mentagrophytes and the occurrence of single point mutations of the SQLE gene and distinct amino acid substitutions, respectively. Alterations at position Phe397Leu, Leu393Ser, Leu393Phe, Gln408Leu, His440Tyr, of the SQLE were associated with terbinafine resistance. Increasing number of patients in Europe (Germany, Finland, Estonia) and from United States have been seen to be presenting with chronic recalcitrant dermatophytosis due to terbinafine-resistant T. mentagrophytes VIII from India. Transmission of the Indian T. mentagrophytes VIII to other countries due to travel, migration and in general, globalization, appears to be a serious issue even from a public health perspective. Further studies are the need of the day to understand this phenomenon better and find answers to combat it.

    S16.4. Blastomycosis

    I.S. Schwartz
    University of Alberta, Edmonton, Canada

    Abstract:

    Among the main endemic mycoses of North America, blastomycosis is least understood. Until recently, blastomycosis was thought to be a single clinical disease caused by a single pathogen, Blastomyces dermatitidis. However, recent re-analyses of isolates from North American and global collections have revealed substantial genetic and phenotypic diversity in Blastomyces. Classical blastomycosis, now known to be caused by B. dermatitidis and the cryptic species B. gilchristii, is limited primarily to eastern and midwestern parts of North America. In addition, B. helicus, a species morphologically and physiologically distinct from B. dermatitidis species complex, has been described as the cause of atypical, disseminated blastomycosis in immunocompromised hosts from western and mountainous parts of the United States and Canada. Moreover, blastomycosis from Africa and the Middle East - long recognized to have clinical differences from the disease in North America - has been determined to be primarily caused by B. percursus and an additional novel species. Clinicians and microbiologists should be aware of these additional species, how they differ from B. dermatitidis, and the clinical and epidemiological spectrum of blastomycosis.

    S16.5. Galleria mellonella as a novelty model to study host – pathogen interaction in Malassezia furfur CBS 1878

    A.M. Celis Ramírez 1, C.M. Parra Giraldo 2 and E.N. Pinzón Pineda 1
    1
    Biological Sciences, Universidad de los Andes, Bogotá, Colombia
    2
    Microbiology, Pontificia Universidad Javeriana, Bogotá, Colombia
    Objectives: Malassezia furfur is a lipid-dependent yeast that is part of the animal and human skin mycobiota. This species can cause dermatological infections in the immunocompetent host until fungemia in immunosuppressed patients and neonates with parental lipid nutrition. However, aspects related to virulence traits and antifungal resistance is not entirely understood. Thus, the establishment of a model to study host-pathogen interaction has been a matter of attention in the last decade. The silkworm Galleria mellonella is a useful animal model characterized by its tolerance to a wide range of temperatures, including human temperature 37 °C; also, the immune response against pathogens is similar to the innate immunity in humans. Hence, this exciting model has been used for the large-scale screening of fungal pathogens such as Candida spp., Cryptococcus neoformans, and the activity of antifungal drugs in dermatophytes. This study aim to evaluate the feasibility of using G. mellonella larvae as an invertebrate model for infection with Malassezia furfur CBS 1878
    Methods: M. furfur CBS 1878 (Westerdijk institute, Utrecht, The Netherlands) was used for all experiments. An inoculum was adjusted to four different concentrations (1.5 × 109 UFC/mL to 1.5 × 106 UFC/mL) and 10 μl were injected in the last pro leg of G. mellonella. Survival analysis was performed for statistical processing using Prism software (GraphPad Software, Inc.). Infected larvae and controls were maintained at 33°C and checked daily. Hemolymph analysis was conducted to evaluate the capacity of phagocytosis of hemocytes. Experiments to corroborate the fungal load were also conducted. Each treatment was conducted in triplicate (technical replicates), and the whole experiment was repeated three times (biological replicates). Statistical analysis were performed using R package.
    Results: Survival test reveals that at least all silkworms injected with M. furfur CBS 1878 at 1.5 × 109 UFC/mL died within five days, whereas all silkworms injected with saline were alive. Fungal load data were in agreement with the inoculums tested. Besides, activation of the immune system of G. mellonella and the interaction between the yeast with different cells of the hemolymph was observed.
    Conclusion: This study is the first approach to the implementation of an invertebrate model Galleria mallonella to the future study of the host-pathogen interaction and the antifungal activity in M. furfur CBS1878. We demonstrated that injection of this yeast into silkworm hemolymph killed silkworms. We found the most effective inoculum was 1.5 × 109 UFC/mL with a short time of survival on average of five days. This model promise to be an efficient infection model to conduct easy and reliable approaches that perform to underpin our knowledge in Malassezia genus.

    Symposium 17 HIV associated cryptococcal meningitis

    S17.3. Towards having antifungal drugs in low-resource areas

    I. Kolte 1, A. Sturny Leclère 2, T. Boyer-Chammard 2, S. Lesikari Kivuyo 3, S. Mfinanga 3, C. Kanyama 4, C. Kouanfack 5, O. Lortholary 6, F. Dromer 7, T. Bicanic 1, S. Molloy 1, T. Harrison 1 and A. Loyse 1
    1
    Infection & Immunity, St Georges University of London, London, United Kingdom
    2
    Molecular Mycology, IP Paris, Paris, France
    3
    National Institute for Medical Research, Dar es Salaam, Tanzania
    4
    UNC Project-Malawi, Lilongwe, Malawi
    5
    Hôpital Central de Yaoundé, Yaoundé, Cameroon
    6
    Infectious Diseases, APHP, Hopital Necker-Enfants malades, Paris, France
    7
    National Reference Center For Invasive Mycoses & Antifungals, Molecular Mycology Unit, Cnrs Umr2000, Institut Pasteur, Paris, France

    Abstract:

    The results of the ACTA trial underpinned the development of new 2018 WHO guidance for the treatment of cryptococcal meningitis with short course amphotericin B deoxycholate (AmBd) + flucytosine (5FC) becoming the new gold standard for resource limited settings. The alternative recommended regimen is a two-week oral course of 5FC and fluconazole. However, access to key diagnostic tests such as the cryptococcal antigen lateral flow assay (CrAg LFA) and essential antifungal medicines including 5FC for the treatment of HIV-related cryptococcal meningitis is severely lacking in African low-and middle-income countries (LMICs) where disease burden and mortality is highest. This talk will highlight longstanding advocacy and implementation efforts to ensure access to these lifesaving tools and train frontline healthcare workers (HCWs) working on their optimal use so as to effectively reduce mortality in resource limited settings. We will focus in particular on illustrating the respective work of the cryptococcal meningitis action group (cryptoMAG), the DREAMM project, and outline the recently announced Unitaid/CHAI project on advanced HIV disease. The cryptoMAG group is an international group of stakeholders including St George’s University of London (SGUL), Centers for Disease Prevention & Control (CDC), WHO, Médecins sans Frontières (MSF) and Institut Pasteur amongst notable others that has been advocating for access to diagnostic tests and essential medicines for cryptococcal meningitis since 2013. DREAMM (Driving REduced AIDS-associated Meningo-encephalitis Mortality) is an implementation project currently ongoing in Tanzania, Cameroon and Malawi. It uses mixed methodology including co-designed training programs and local health system strengthening centred around African leadership to effectively and sustainably reduce mortality linked to HIV-related meningitis, a leading cause of advanced HIV disease. Lastly, in January 2019 Unitaid announced its program on AHD in partnership with the Clinton Health Access Initiative (CHAI). This program will enable access to the CrAg LFA and essential antifungal medicines including 5FC in 7 African LMICs, cryptococcal meningitis being a leading cause of death from advanced HIV disease.

    S17.4. Fluconazole resistance in cryptococcal meningitis

    T. Bicanic
    Infection & Immunity, St Georges University of London, London, United Kingdom
    In much of sub-saharan Africa, where Cryptococcus is the most common cause of community acquired meningitis, the gold standard treatment of Amphotericin B with flucytosine is not available, and fluconazole monotherapy is the norm for all phases of CM treatment: induction, consolidation and maintenance. Even at higher doses of 800-1200 mg/d, fluconazole is slow to clear Cryptococcus from CSF. Although primary resistance is rare in global surveys using MIC testing, the intrinsic mechanism of fluconazole resistance, reversible disomy of chromosome 1, is unstable: in the absence of drug pressure, it occurs in only a very small subpopulation of colonies and will be missed by conventional MIC testing. In a study in Tanzania in patients with HIV-associated cryptococcal meningitis, we performed serial lumbar punctures in 20 patients receiving fluconazole monotherapy versus fluconazole combined with flucytosine, plotting the rate of clearance of the total population and subpopulation able to grow on fluconazole, and saving colonies for subsequent whole genome sequencing. Fluconazole heteroresistance was detectable in all clinical Cryptococcal isolates.from CSF of all patients prior to initiation of therapy. The proportion of resistant colonies in CSF increased during the first two weeks of treatment in all patients receiving Fluconazole at 800-1200mg/d, but was suppressed in those receiving combination with flucytosine. Genomic analyses revealed high rates of aneuploidy in heteroresistant colonies, as well as in clinical isolates from patiets experiencing relapse. The predmonant mechanism in human infectionwas disomy of chromosome 1 (containing the drug target gene ERG11 and the efflux pump AFR1) and heteroresistance positively correlated with in vitro drug efflux pump activity. We also undertook fluconazole PK analyses in CSF for a subpopulation of patients: Monte Carlo simulations from the clinical PK/PD model predicted that only a minority of patients (13%) receiving doses of fluconazole at 1200 mg/d sterilise their CSF after 2 weeks, with 83% having a persistant subpopulation resistant to fluconazole. Our findings demonstrate that fluconazole when used alone is a sub-optimal agent for treating cryptococcal meningitis due to rapid selection of a resistant sub-population which comes becomes predominant within 2 weeks. Efforts are underway to make the combination oral therapy of cluconazole with flucytosine more widely available and implementable in Africa.

    S17.5. Current and future clinical trials on HIV-associated cryptococcal meningitis.

    T. Harrison
    Centre For Global Health, Institute of Infection and Immunity, St George’s University of London, London, United Kingdom
    The ACTA trial demonstrated that in centres in Sub-Saharan Africa, an induction regimen of 1 week amphotericin B (AmB) and flucytosine (5FC), followed by fluconazole (FLU) 1200 mg/d in the second week, was associated with reduced 10-week mortality compared with the then internationally recommended regimen of 2 weeks of AmB+5FC. 2 weeks of the oral combination of FLU plus 5FC was non-inferior to this standard; and 5FC was superior to FLU, as the partner drug given with AmB. The results have been further supported through 12-month outcome and economic analyses, and have driven both a change in WHO guidelines for the treatment of HIV-associated cryptococcal meningitis, and increasing access to 5FC through a Unitaid programme. The ongoing AMBITION-CM trial is testing single high dose (10mg/kg) liposomal amphotericin B (L-AmB), given with a 2-week oral FLU+5FC backbone, against the new standard of 1 week AmB+5FC. A randomized phase II study had shown non-inferior early fungicidal activity (EFA) with single high dose L-AmB vs daily L-AmB at 3 mg/kg/d for 14 days. The hope is that the L-AmB arm will be at least as effective and better tolerated and easier to deliver than one week of AmB deoxycholate. The trial includes a pre-planned health economic evaluation. Over 300 participants have been enrolled. Any further improvements in treatment may depend on introduction of new drugs into induction regimens, or novel host-directed or mechanical strategies. Viamet-1598, a fungal Cyp51 inhibitor, and a compound series from Amplyx Pharmaceuticals that target Gwt1, an enzyme required for fungal cell wall localization of GPI-anchored mannoproteins, are 2 promising candidates, and it is hoped both can advance to phase II EFA studies. Immunotherapeutic approaches should be further investigated but will need to be carefully targeted based on rapid assays of patient specific immunity. Investigators at Duke University are exploring the possibility of using neurapheresis to filter fungal cells from the CSF and rapidly reduce the organism load. Meanwhile efforts continue to prevent the development of clinical cryptococcal infection in patients with advanced HIV disease, and improve the results of the cryptococcal antigen (CrAg) screen and pre-emptive treatment strategy for patients with low CD4 cell counts. Despite pre-emptive fluconazole, mortality for those screening CrAg positive is significantly higher than for those who are CrAg negative; many of those with high CrAg titre have evidence of sub-clinical meningitis; and fluconazole failures have been increasingly well documented. A study looking at single high dose L-AmB to treat CrAg positive patients in screening will take place in Uganda, and a trial to test the oral FLU+5FC combination against FLU alone for CrAg positive patients identified at screening is planned in South Africa and Tanzania.

    Symposium 18 Antifungal stewardship in the era of resistance

    S18.1. Stewardship and azole resistant aspergillosis: a challenge for farmer or physician?

    P.E. Verweij
    Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen, the Netherlands
    Aims of antibiotic stewardship include treating infectious diseases with the appropriate drug, preventing overuse and minimizing resistance selection. These goals are frustrated by the emergence of azole resistance in Aspergillus fumigatus. Recent studies showed that voriconazole-treated patients with invasive aspergillosis have a 20% lower survival when the infection was caused by a voriconazole-resistant isolate compared with voriconazole-susceptible infection. Appropriate initial therapy is thus critical but diagnosis of resistance is slow when based on fungal culture and MIC-testing. To ensure appropriate initial antifungal therapy combination antifungal therapy is recommended if resistance frequencies exceed 10%. The Dutch guideline for management of invasive aspergillosis indeed recommends voriconazole/isavuconazole in combination with an echinocandin of liposomal amphotericin B is the azole-susceptibility is unknown as resistance surveillance indicated a resistance rate of 15%. Therefore, many patients are exposed to increased toxicity and drug interactions, while the majority still have an infection due to an azole-susceptible isolate. To improve antifungal stewardship programs accurate resistance surveillance is required as well as rapid and sensitive diagnostic resistance tests. As the main driver of resistance to medical azoles in A. fumigatus is azole fungicide use in the environment, interventions are needed to reduce the burden. Recent studies have identified specific conditions that facilitate resistance selection and the next step is to design and evaluate interventions that preclude resistance selection in the environment.
    The azoles represent an important class for both medical treatments and food production. Maintaining this class for both application is therefore critical and requires investment in rapid diagnostic tests and stewardship programs.

    S18.2. Rapid diagnosis of fungal infections: Impact on stewardship

    S. Kanj
    Department of Internal Medicine, Division of Infectious Diseases, American University of Beirut Medical Center, Beirut, Lebanon
    Invasive fungal infections (IFI) remain a challenge to the treating physician because of difficulty in diagnosis and high rates of morbidity and mortality associated with these infections, which occur predominantly in the immunosuppressed and critically ill patients. The incidence of invasive fungal infections, especially candidiasis, aspergillosis, and mucormycosis, continues to rise. With the excessive use of azole antifungal agents, we have witnessed a global shift of fungal population to more resistant species and genera. In addition, the increased consumption of antifungal agents in agriculture have selected for resistant Aspergillus species. There is a wide variation in the epidemiology of fungal infections worldwide. It is no longer acceptable to manage IFI without identifying the species of the infecting organism and its susceptibility profile to the various antifungal agents. Neighboring countries might have a different epidemiology depending on antifungal practices in humans and the environment. Whereas guidelines can be of great help in the management of patients, making the correct diagnosis is of paramount importance. Studies have shown that delay in the initiation of appropriate antifungal therapy is associated with a significant increase in mortality. Therefore, there is an urgent need to make a prompt diagnosis of IFI. In the past, histopathology, fungal cultures, and Candida scores in the right clinical setting guided the diagnosis and therapy of IFI. More recently, the availability of newer diagnostic tools such as Aspergillus galactomannan, β-D glucan, and polymerase chain reaction, has been of great help in speeding the diagnosis of IFI. These tools have also been used for screening and guiding pre-emptive therapy in high-risk patients. Studies have shown that combining these new tools increases the sensitivity and specificity with an impact on fungal infection-free survival in some patients and allows for early discontinuation. More recently, the development of new molecular tools such as Matrix-Assisted Laser Desorption/Ionization – Time of Flight, FilmArray, Light Cycler, T2Candida assay, and others, allows the rapid detection of fungal pathogens and determines resistance markers with a turnaround time of only a few hours. Investigational tools such as Proximity Ligation Assay, Breath Fungal Secondary Metabolite Signature, and Siderophore-based Molecular Infection Imaging are also promising in facilitating the diagnosis of IFI in the future. In addition, the development of point-of-care testing is helpful, especially in diagnosing cryptococcal meningitis in some developing countries where access to microbiology laboratory is limited, or in diagnosing invasive Aspergillosis using Lateral Flow Devices. All these new diagnostic tools have a tremendous impact on antifungal stewardship as they allow physicians to discontinue antifungal agents earlier when not needed or to de-escalate them to narrower-spectrum agents. These efforts will ultimately translate into decreasing antifungal resistance and reducing costs to healthcare, which are essential elements of stewardship.

    S18.5. Optimising antifungal stewardship: An evaluation of candidaemia guideline compliance and clinical outcome

    L. Cottom 1 and B. Jones 2
    1
    Department of Medical Microbiology, Glasgow Royal Infirmary, NHS Greater Glasgow & Clyde, Glasgow, United Kingdom
    2
    Department of Medical Microbiology, Glasgow Royal Infirmary, Glasgow, United Kingdom
    Objectives: Candidaemia remains an important cause of nosocomial infection, associated with significant morbidity and mortality. Over the last decade there has been a growing awareness that improvements to stewardship and robust surveillance is needed to ensure the judicious use of antifungal therapy. A guideline should never be construed or serve as a standard of care, rather recommendations should always be interpreted alongside individual patient findings and be guided by local epidemiology and antifungal susceptibility knowledge. The aim of this study was to evaluate compliance with the current 2016 Infectious Diseases Society of America (IDSA) guidelines and assess the impact on clinical outcome. Adherence to management recommendations was assessed using the EQUAL Candida Score of the European Confederation of Medical Mycology (ECMM). A secondary aim was to assess clinical outcome (30 day, 90 day and all-cause mortality) with an echinocandin compared to fluconazole as the initial treatment (primary therapy) for candidaemia.
    Methods: A retrospective analysis was performed over 18-months (January 2017 to June 2018). Data was analysed from five large university teaching hospitals within Greater Glasgow and Clyde; the largest NHS organisation in Scotland serving a population of 1.2 million. Patients were identified using our laboratory information management system and electronic clinical record systems. Only cases where a diagnosis of candidaemia had been confirmed and the patient had been actively treated were included for analysis. The source of infection and antifungal management was reviewed for each patient. The EQUAL candida score was calculated for each case. The 30-day, 90-day and all-cause mortality rate was determined for the study population.
    Results: A total of 131 patients were identified as having a candidaemia. The most common Candida species isolated was C.albicans (40%; 53/131) followed by C.glabrata (27%; 35/131). An echinocandin was initiated as primary therapy in 52% (68/131) of cases. In 62% of cases the patient had an indwelling line that was attributed as the direct source/nidus of infection. 21% of cases were related to a urinary tract source and/or urological instrumentation. The mean EQUAL Candida Score was found to be 13 (range 9-17), with the maximum score for guideline adherence being 16 for non-CVC patients and 19 for CVC carrier (score adjusted as initial blood culture volume was not known for study population). No statistically significant difference in clinical outcome (30 day mortality) was found when comparing an EQUAL Candida Score of ≤13 with > 13 (chi-square calculation; p-value 0.76). For the study population, a statistically significant difference in clinical outcome (30 day mortality) was found when comparing fluconazole to an echinocandin as initial treatment/primary therapy with a survival benefit being found in favour of fluconazole (chi-square calculation; p-value 0.039).
    Conclusion: Our study provides a valuable evaluation of compliance. Interesting, increased compliance was not found to improve clinical outcome. Furthermore, the results from our study suggest that fluconazole as initial therapy was non-inferior to an echinocandin for the treatment of candidaemia, with a survival benefit being observed. This study supports the importance of an effective program of surveillance to promote antifungal stewardship and the judicious use of antifungal agents.

    Symposium 19 Pneumocystis

    S19.1. Recent diagnostic strategies in Pneumocystis pneumonia

    A. Alanio
    Paris-diderot, Sorbonne Paris Cité University, Institut Pasteur, Molecular Mycology Unit, Cnrs Cmr2000, Parasitology-Mycology Laboratory, Lariboisière Saint-Louis Fernand Widal Hospitals, Assistance Publique-Hôpitaux de Paris, Paris, France
    Pneumocystis jirovecii pneumonia (PCP) is one of the most commonly diagnosed opportunistic infection in HIV-positive patients. In addition, the rising number of immunocompromised HIV-negative patients at risk of P. jirovecii infection (immunosuppressive therapies, allogeneic bone marrow or solid organ transplantation) is an emerging concern. PcP is difficult to diagnose in HIV-negative patients owing to the non-specific pulmonary symptoms and signs associated. Microscopic visualization of cysts or trophic forms in respiratory specimens based on immunofluorescence stainings is the most sensitive microscopic method and still considered as the gold-standard test to diagnose this infection. Bronchoalveolar lavage fluids are the most sensitive specimen type, since P. jirovecii is an organism associated to lung alveoli. PCR-based methods play an increasing role in the lab, initially developed to circumvent decreased fungal load in HIV-negative patients and detection in upper respiratory specimens. Real-time quantitative PCR (qPCR) is definitely the only format adapted to detect P. jirovecii since the risk of contamination is minimal and quantification is possible. A negative qPCR in BAL, but not in non-invasive respiratory specimens, allows to rule out PCP diagnosis. Quantitative results have been used for years to try to discriminate PCP (high fungal load) from carriage/colonization (low fungal load). However, these methodologies have limited use since intermediate fungal load are inconclusive. Recent advances in the comparison of the performances of the different qPCR assays showed that standardization is certainly one of the clues to move forward with this question. In parallel, the combination with (1-3)-b-D-Glucan (BG) detection in serum helps but do not completely resolve the problem. BG detection has a high sensitivity and a high negative predictive value for diagnosis of PcP in immunocompromised HIV-positive and -negative patients, but a positive result may also indicate the presence of other invasive fungal infections. The clinical utility of these diagnostic tests and the diagnostic strategies in HIV or non-HIV patients should be further assessed in prospective, randomized interventional studies.In the meantime, the status of the host should be better defined with regard to the evolution of P. jirovecii carriage. New screening and prophylaxis strategies to prevent transmission in hospital settings should be proposed and discussed

    S19.2. Management of Pneumocystis Pneumonia in HIV-infected Patients

    J. Kovacs
    Associate Clinical Professor of Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, United States of America
    Pneumocystis pneumonia (PCP) remains one of the most common life-threatening opportunistic infections in HIV-infected patients, despite the tremendous benefits seen with combination antiretroviral therapies. PCP continues to be seen in the current era primarily because at-risk patients are unaware of their HIV infection or are not in continuous medical care. Infection appears to represent recent acquisition in many cases rather than reactivation of latent infection. Primary clinical manifestations include fever, shortness of breath, and a non-productive cough. CXR will typically show diffuse infiltrates though it may be normal in 10-20% of cases; chest CT scans will almost invariably be abnormal, however. Diagnosis is based on detection of the organism in respiratory samples such as induced sputum or BAL. Because the organism cannot be cultured, organism detection relies on colorimetric or immunofluorescent staining, or PCR assays. The latter are highly sensitive but less specific than the former, in part because PCR can identify colonization or subclinical infection not requiring therapy. Assays to measure (1-3)-β-D-glucan levels have poor specificity and should not be relied on as the exclusive diagnostic modality. The preferred first-line drug for treatment is trimethoprim-sulfamethoxazole, which targets 2 steps in Pneumocystis folate metabolism. Alternatives for patients with mild to moderate disease (A-a O2 gradient <45 mm Hg) include clindamycin-primaquine, trimethoprim plus dapsone, and atovaquone. Alternatives for patients with more severe disease requiring parenteral therapy include clindamycin-primaquine (primaquine is available only for oral administration) and pentamidine. Caspofungin should not be used as a single agent since it affects only the cyst form and not the more numerous trophic form of the organism; further, there are no controlled studies evaluating its efficacy. Corticosteroids should be administered in patients with PaO2 <70 mm Hg or (A-a) O2 gradient >35 mm Hg, ideally within 3 days of starting therapy, and be continued using a tapering regimen while anti-Pneumocystis therapy is administered. Although there are no randomized trials, meta-analyses suggest that clindamycin-primaquine is more efficacious than pentamidine. For patients failing therapy, there are no randomized trials to address optimal management, and whether it is better to add drugs or to change regimens. PCP can be effectively prevented in at-risk patients (e.g. CD4 <200 cells/mm3) by using trimethoprim-sulfamethoxazole, or alternatively dapsone, dapsone-pyrimethamine, atovaquone, or aerosol pentamidine. In patients recovering from an episode of PCP, secondary prophylaxis should be initiated after completion of therapy. Prophylaxis can be discontinued after the immune system has reconstituted to CD4 >200 cells/mm3 for at least 3 months. In patients with poor immune reconstitution (CD4 >100 but <200 cells/mm3) but with HIV suppression to below detection limits of commercial assays (typically <20 copies/ml) for 3-6 months, prophylaxis can also be discontinued. Prophylaxis should be continued in patients with CD4 <100 cells/mm3 even with HIV suppression. Mutations have been identified in the dihydropteroate (DHPS) gene of Pneumocystis, the target of sulfamethoxazole and dapsone, primarily in patients receiving those drugs for prophylaxis. Although these mutations appear to confer at least low level resistance, their clinical significance is uncertain.

    S19.4. Pneumocystosis in neonates

    E. Calderon
    Instituto de Biomedicina de Sevilla and CIBER de Epidemiología y Salud Pública, Seville, Spain
    Pneumocystosis has been well recognized as a severe pulmonary disease and an important cause of morbidity and mortality in premature infants or debilitated neonates since the mid-twentieth century when Pneumocystis was acknowledged as the causing agent of interstitial plasma cell pneumonia. This form of pneumocystosis was observed before, during and after World War II in Europe, where it reached epidemic proportion with more than 500 cases confirmed by autopsy. Unexpectedly, this epidemic pneumocystosis disappeared in the same manner it had been emerged in Europe. Although data on pediatric Pneumocystis pneumonia (PcP) from developing countries are scarce mostly based on small series of cases, PcP in infants seem to remain as an important medical problem in those areas. Nowadays, the interest in Pneumocystis infection goes beyond PcP because a new spectrum of Pneumocystis-related disease seems to emerge in immunocompetent infants. Serological studies and use of molecular techniques to identified Pneumocystis jirovecii have shown that this microorganism probably is one of the more frequent infectious agents faced by humans in everyday life and that the first exposition to this pathogen happen in most children early in life, probably in the neonatal period. However, primary Pneumocystis infection currently goes unrecognized because it has been presumed to be an asymptomatic or mild nonspecific disease. However, recent reports indicate that it can present clinically as a self-limiting upper or lower acute respiratory tract infection. Pneumocystis infection was identified by specific PCR in 24% of infants with bronchiolitis from France and in 29.4% of Cuban infants and toddlers with whooping cough. In another study of 422 children hospitalized with acute respiratory tract infection, Pneumocystis was detected in 16% of infants. However, a marked difference occurred in the age distribution, as the prevalence was 48% in infants ages 50 to 112 days, 13% in infants ages 113 to 265 days, and 2% in in infants younger than 49 days. In a study carried out in Santiago de Chile, Pneumocystis DNA was detected in specimens from 51.7% of infants who died unexpectedly in the community, but only 15% had pneumonia. This study also showed that Pneumocystis infection is more frequent than viruses before the age of 6 months, and that it has a consistent peak between 2 and 5 months of age. The significance of these findings is unclear but shows the high prevalence of Pneumocystis infection in infants. Recently, in Spain, a high prevalence of Pneumocystis infection has been described in preterm neonates and this infection was associated to higher risk to develop neonatal respiratory distress syndrome. Increasing evidence suggests that the most common respiratory infection-affecting infants is the mild and sneaky, primary infection by Pneumocystis. This infection goes currently unrecognized and has been neglected as a subclinical irrelevant infection by contrast with the severe PcP affecting the immunocompromised patients. However, compelling new evidence suggests that this infection may be pathogenic to certain infant age groups and that microbiome host interactions in early life may condition the development of altered immune responses in older infants or adults.

    S19.5. Evaluation of a Novel Commercial Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Pneumocystis jirovecii

    D. Schmidt, U. Scharmann, J. Buer and P.-M. Rath
    Institute of Medical Microbiology, University Hospital Essen, Essen, Germany
    Objectives: Pneumocystis jirovecii pneumonia (PCP) is an increasing problem in immunocompromised patients. Detection of P. jirovecii in respiratory samples is usually based on microscopy or PCR. Both methods require experienced technical personnel and are either insensitive or time-consuming. In this study, a prototype of the eazyplex Pneumocystis jirovecii assay (Amplex Diagnostics, Germany) was established. The assay is based on LAMP technique (loop-mediated isothermal amplification). It requires minimal hands on time for sample manipulation (below 5 minutes) and performance of the assay itself (25 minutes) and could be a reliable technique to detect P. jirovecii.
    Methods: In total, 145 respiratory samples from 134 critically ill patients (65% male, median age = 64 years) were investigated, using the LAMP assay in comparison with the RealStar Pneumocystis jirovecii PCR 1.0 (altona Diagnostics, Germany). For the LAMP assay 25µl of sample were mixed with 125µl RALF buffer (resuspension and lysis fluid, Amplex) and incubated for 3 minutes at 99°C. 25µl of this suspension were added to the wells of the test strip afterwards. The LAMP assay was performed in a Genie II Mk2 device (Amplex). For qPCR, 300µl of sample were extracted using the Maxwell16 nucleic acid extraction platform (Promega, Germany). 10µl of DNA were used for qPCR according to the manufacturer´s recommendations. QPCR was performed on a RotorGeneQ cycler (Qiagen, Germany) in a final volume of 30µl. To determine the detection limit of the LAMP assay, compared to the qPCR, tenfold serial dilution of extracted P. jirovecii DNA and of a high positive P. jirovecii respiratory sample were investigated in both assays. Additionally, for each serial dilution time-to-positivity of the LAMP assay was plotted against cycle threshold values (ct values) of the qPCR. For quantification of the fungal load the quantification standards of the RealStar qPCR were used (1x104 -1x101 copies/µl).
    Results: The LAMP assay detected 17 of 19 qPCR positive samples and 117 of 117 qPCR negative samples. The assay was invalid in nine samples that were negative by qPCR. Time-to-positivity of the LAMP assay ranged from 11-22 minutes in patient samples. Detection limit of the assay was approximately 15-20 copies of genomic DNA/µl in patient samples and in extracted DNA which corresponded with ct values of 28-29 in the qPCR.
    Conclusion: The LAMP assay is an accurate and time saving tool for the detection of P. jirovecii in respiratory samples. Two samples that were positive by qPCR were not detected by the assay which is probably due to the significantly lower sample volume and the crude extraction method the assay uses. Nevertheless, the convenience of the performance of the assay and interpretation of the results allows laboratories with less experience and less technical equipment to perform molecular assays on a high standard.

    Symposium 20 Candida auris

    S20.1. Schizophrenic gram negative yeast conquering the world

    J. Meis
    Department of Medical Microbiology and Infectious Diseases, Excellence Center For Medical Mycology (ECMM), Canisius Wilhelmina Hospital, Nijmegen, Netherlands
    Candida auris has been named as the new “fungal superbug” which poses a significant threat to public health during outbreaks. C. auris was first named and described only 10 years ago. Follow-up genomic analysis has revealed that C. auris has emerged almost simultaneously in this short period on 3 different continents. At present 4 major clades have been identified and last month a fifth clade was discovered. In the beginning C. auris was often mistaken for other Candida or yeast species when using phenotypic assimilation/fermentation identification tests. Wide introduction of MALDI-TOF technology has significantly improved accurate identification in the past few years and is now the gold standard in addition to molecular sequencing. C. auris has also shown resistance to several different antifungal drugs and classes of drugs. While its resistance profile varies geographically, this new yeast pathogen has acquired resistant to fluconazole, a key drug in the azole class of antifungal drugs. C. auris spreads in healthcare facilities in a similar way as bacteria while the occurrence and niche in the environment is elusive. Infections have been reported in a range of countries across continents, but current estimates are probably inaccurate. Impervious to both antiseptics and the three major classes of anti-fungal medications, C. auris has been quietly transmitted within hospitals and nursing homes in at least 37 countries such as the UK, Spain, India, Pakistan, Middle East, South Africa, Latin America and parts of the USA. Patients can be colonised with C. auris without becoming sick, but the pathogen seems to prefer to colonise patients who are already sick or immunocompromised (eg, cancer or transplant patients), and the very young or very old among hospitalised inpatients. Among at-risk patients who have C. auris candidemia or invasive candidiasis, depending on the world region, high mortality rates (~ 30–60%) have been observed. But because infection typically occurs when a person is already very sick, it may be difficult to disentangle the attributable mortality. It is important to realize that C. auris is predominantly a healthcare-associated infection. Prior broad-spectrum antibiotics and antifungal use can influence the risk of infection. Finally an important question is still unanswered C. auris: Unde venis et quo tendis? Reference Meis JF, Chowdhary A. Candida auris: a global fungal public health threat. Lancet Infect Dis. 2018;18(12):1298-1299

    S20.3. Outbreak control: "Controlling a multidrug-resistant Candida auris outbreak"

    A. Ruiz-Gaitan 1,2
    1
    Severe Infection Research Group, Medical Research Institute La Fe, Valencia, Spain
    2
    Department of Clinical Microbiology, La Fe University and Polytechnic Hospital, Valencia, Spain
    Background: Candida auris is an emerging, multidrug-resistant yeast causing hospital outbreaks. The outbreaks by C. auris described in Spain as well as in other countries with large outbreaks, are characterized by an exponential increase in the number of cases in a short period, suggesting a high transmission rate. Objectives We report the first 24 months of the ongoing C. auris outbreak in a tertiary hospital in Spain. The epidemiological, clinical and microbiological characteristics of candidemia episodes and environmental samples by C. auris were also analyzed. Results: 228 patients were involved in the case–control study (114 colonized/candidemia and 114 controls). All candidemia episodes were observed in adult patients (21–81 years old) and 87.8% of them were admitted to SICU. The most common underlying condition observed in both colonized and candidemia patients was polytrauma (n = 13, 32%) followed by cardiovascular disease (n = 10, 25%) and cancer (n = 7, 17%). Indwelling CVC (odds ratio {OR}, 13.48), parenteral nutrition (OR, 3.49), and mechanical ventilation (OR, 2.43) were the more frequently invasive procedures observed in these two groups and remained significant predictors of C. auris colonization/candidemia. All C. auris isolates were resistant to fluconazole (MICs >64 mg/L) and had significantly reduced susceptibility to voriconazole (GM, 1.8 mg/ L). All isolates were susceptible to itraconazole, posaconazole, isavuconazole, and echinocandins. Environmental sampling showed presence of the C. auris on sphygmomanometer cuffs (25%) patient tables (10.2%), keyboards (10.2%), and infusion pumps (8.2%). Conclusions: - Predictor conditions to C. auris colonization/candidemia are similar to other Candida species. C. auris colonizes multiple patient’s environment surfaces. All isolates are resistant to fluconazole and had significant reduced susceptibility to voriconazole. - Due to its high transmissibility and survival in the hospital environment, C. auris can cause long duration outbreaks that are difficult to detect in early stages, and it makes it difficult to control and eradicate. - The implementation of early and strict surveillance and control measures is essential to preventing the spread of the outbreak representing a significant risk to critical patients. - Immediate notification of C. auris to clinical and infection control teams, as well as to health authorities and institutions, is essential to implementing infection control precautions at all levels in a timely way, to prevent transmission inside and outside the hospital and to prevent the development of infections in patients who are already colonized. - This report is intended to demonstrate not only the complexity of handling and containing a C. auris outbreak, but also how, with the use of a series of effective infection prevention measures, the spread of this pathogen can be successfully controlled.

    S20.4. Candida auris: in developing world

    Anuradha Chowdhary
    Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
    Candida auris a multidrug-resistant(MDR) yeast that exhibits resistance to fluconazole and markedly variable susceptibility to other azoles, amphotericin B, and echinocandins has globally emerged as a nosocomial pathogen that can cause invasive infections. Candida auris was first described in 2009 by Satoh et al. as a novel Candida species, in the Candida haemulonii complex(Metchnikowiaceae), from a patient in Japan after its isolation from the external ear canal. Subsequent to the first nosocomial outbreak in South Korea in 2011, several hospitals associated outbreaks have been reported in developing world especially in India, Pakistan and Africa. Alarmingly, in less than a decade this yeast, which is difficult to treat, has become widespread across several countries causing a broad range of healthcare associated invasive infections that display clonal inter- and intra-hospital transmission. C. auris in routine microbiology laboratories remains an unnoticed pathogen as 90% of the isolates characterized by commercial biochemical identification systems such as API 20C, Vitek 2 (bioMérieux), Phoenix (BD), and MicroScan (Beckman Coulter), misidentify as a range of other Candida species. Most commonly, these isolates have been misidentified as C. haemulonii, but also C. famata, C. sake, Rhodotorula glutinis, Rhodotorula mucilaginosa, and Saccharomyces species. Rarely, C. auris has been identified as C. catenulate, C. lusitaniae, C. guilliermondii, or C. parapsilosis. However, MALDI-TOF MS is considered a more rapid and robust diagnostic technique for C. auris identification. Mass spectra can be easily added to the MALDI-TOF MS database, leading to accurate identification of C. auris to the species level. Sequencing of genetic loci, including D1/D2, RPB1, RPB2, and internal transcribed spacer (ITS) domains of the rRNA, has proven useful in the identification of C. auris, but it is not routinely used.
    However, in the routine microbiology laboratories this yeast remains unidentified especially in developing world due to lack of fully equipped mycological diagnostic facilities. Further, the true burden of C. auris remains unexplored as effective surveillance of Candida spp is not available in several countries in the developing world. Antifungal susceptibility data for C. auris is of primary concern as C. auris exhibit consistently high fluconazole MICs and variable susceptibility to the other azoles, echinocandins and amphotericin B. No antifungal clinical breakpoints reported for C. auris. Studies examining the susceptibility of this organism to antifungals have used a variety of methods, including Clinical and Laboratory Standards Institute (CLSI) broth microdilution, Etest, and the Vitek 2 yeast susceptibility system. Regarding azole resistance mutations in Erg11 associated with the development of fluconazole resistance in C. albicans have also been detected in C. auris isolates. The fact that this yeast exhibit MDR clonal strains which are nosocomially transmitted is unusual in other Candida species. Therefore, the possible threat of its rapid spread in affected countries and its emergence in unaffected countries will not only challenge clinicians for its effective therapeutic management but will also bring high economic burden to countries especially in resource limited settings where modern identification facilities and access to antifungals other than FLU are limited.

    S20.5. Understanding Echinocandin Activity Towards Candida auris

    M. Kordalewska 1, A. Lee 1, R. Garcia-Rubio 1, S. Park 1, I. Berrio 2, A. Chowdhary 3, Y. Zhao 1 and D.S. Perlin 1
    1
    Center For Discovery and Innovation, Hackensack Meridian Health, Nutley, United States of America
    2
    Hospital General de Medellín “Luz Castro de Gutiérrez” ESE, Medellin, Colombia
    3
    Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
    Objectives: Candida auris is a recognized cause of invasive infections and health care associated outbreaks around the world. Development of echinocandin resistance has been documented in C. auris isolates recovered from patients treated with these drugs. Thus, a thorough understanding of C. auris echinocandin susceptibility profiles and resistance mechanisms is crucial for improved management. In this study, we performed a comprehensive analysis of C. auris echinocandin susceptibility and molecular resistance determinants.
    Methods: A total of 106 C. auris isolates (Colombia, India, AR Bank) were investigated in this study. Antifungal susceptibility testing (AFST) with echinocandins (anidulafungin, caspofungin, micafungin) was performed in accordance with CLSI document M27-A3. FKS1 gene, encoding the target enzyme for echinocandin class antifungal drugs, was amplified and sequenced. Mutation prevention concentration (MPC) determination assay was performed for echinocandin-susceptible (micafungin MIC range 0.03-0.5 mg/l), FKS1 wild-type C. auris, C. albicans and C. glabrata control strains. Isolates were cultured in RPMI-1640 containing 2-fold increasing concentrations (0.03 to 128 mg/l) of micafungin, then culture aliquots were plated onto YPD plates and number of surviving cells were calculated. Recovered colonies were screened for the presence of FKS1 mutations. Moreover, in vivo drug response of C. auris isolates to caspofungin was assessed in a murine model of invasive candidiasis.
    Results: Four Indian isolates from a total of 106 isolates (3.8%) exhibited highly elevated MICs ≥4 mg/l at 24 h and were considered presumptively resistant to all tested echinocandins. These isolates were found to harbor an S639F amino acid substitution in Fks1. The remaining 102 echinocandin-sensitive isolates presented wild-type genotype. Micafungin was the most potent echinocandin (MIC50 = 0.125 mg/l). We encountered a significant challenge obtaining an accurate MIC readout for caspofungin since all tested isolates exhibited an Eagle effect (paradoxical growth effect), with the intensities of the Eagle effect varying among the isolates. In MPC determination assay with micafungin, C. auris isolates did not present a classical bi-modal killing pattern characteristic for fungicidal drugs activity against other Candida species. Response of C. auris to micafungin differed between isolates, but micafungin did not reach the fungicidal endpoint for any of the isolates, even when high drug concentrations (128 mg/L) were tested. Since no FKS1 mutation was revealed by sequencing, we hypothesize the presence of extreme tolerance mechanism in C. auris. Echinocandins were effective in vivo in the treatment of invasive murine candidiasis caused by FKS1 wild-type C. auris isolates (significant (P < 0.05) ~1 log10 CFU/g kidney burden reduction upon caspofungin treatment relative to vehicle controls), despite the presence of the Eagle effect in vitro. Mice challenged with fks1 S639F mutants failed to respond to the drug and their kidney burdens were not much different between caspofungin treated and untreated groups.
    Conclusion: Without established breakpoints for C. auris, AFST results interpretation and conclusions regarding resistance, especially when testing caspofungin, should be made with caution. The high echinocandin tolerance of C. auris observed in vitro should be further explored. Presently, the only validated determinant impacting C. auris pharmacodynamic response to echinocandins is the FKS1 genotype.

    Symposium 21 Immunotherapy for opportunistic fungal infections

    S21.1. CAR T cell and NK cell therapy of invasive mycoses

    D. Kontoyiannis
    Ut Md Anderson Cancer Center, 1515 Holcombe, Houston, United States of America
    Invasive fungal infections remain a major threat to the survival of immunocompromised patients and no curative therapies are currently available to treat drug resistant opportunistic fungi. In this brief talk, I will provide a conceptual overview of recent developments to use specific chimeric antigen receptor (CAR) expressing T cells and NK cells as adoptive immunotherapy. CAR T cells have been successfully used to treat B-cell malignancies. To render CAR T cell therapy specific for fungi, we designed a CAR which combines the carbohydrate-binding domain of Dectin-1 with T cell signaling domains, designated as Dectin-1-CAR (D-CAR). The pattern recognition receptor Dectin-1 was selected based on its property to bind selectively to β-1,3-glucan present on the cell wall of life threatening fungal species. I will be discussing our efforts to manufacture clinical grade D-CAR+ T cells and to increase T cell persistence. However, allogeneic CAR T cells carry a significant risk of graft-versus-host disease, and logistical problems of rapid and robust production remain. As the role of natural killer (NK) cells in fungal immunity has been increasingly deciphered, I will be discussing the potential benefits of cord-blood derived NK cells as an attractive, allogeneic, off-the-self source for future, logistically feasible immunotherapeutic approaches to treat invasive mycoses.

    S21.2. WBC transfusions Granulocyte Transfusions As Adjunctive Treatment of Invasive Fungal Diseases In Neutropenic Patients

    L. Pagano
    Fondazione Policlinico Universitario A. Gemelli - IRCCS - Universita Cattolica del Sacro Cuore, Rome, Italy
    The degree and duration of neutropenia are crucial prognostic factors in hematological patients (pts) with invasive fungal infections (IFIs). Since the introduction of granulocyte colony stimulating factor (G-CSF), there has been a renewal of interest in granulocyte transfusions (GTX). Granulocyte transfusions (GTX) are seldom used as a life-saving therapy for neutropenic patients with severe infections. Despite several compelling evidences of GTX efficacy in retrospective and prospective case series, no study has been successful in demonstrating a definite advantage for recipients in controlled clinical trials. Some specific issues relevant to the efficacy of this therapeutic approach, such as the primary infection, the delivered doses and schedules, and the immunological effects of GTX, are still subject to discussion today. Importantly, the awareness of biological effects accompanying the transfusion of neutrophils might support their use at standardized doses and may definitely convey significant advantages to the recipient patients. At present, despite statistical evidences are lacking, GTX are still perceived as a lifesaving tool to support neutropenic patients with life threatening IFIs until their bone marrow recovery. Sharing procedures for donor identification and cell mobilization, pursuing common criteria to identify which patients will benefit of GTX during febrile neutropenia and define indications and therapeutic cell doses are absolutely urgent to pinpoint the true advantage of using GTX. On the other hand, adopting equal end points and outcomes to evaluate both clinical response to treatment and biological functions of neutrophils and chemokines during IFI, need to be clarified.

    S21.4. Novel chimeric antigen receptor T cells for invasive aspergillosis immunotherapy

    M. Seif 1, T. Nerreter 1, M. Machwirth 1, F. Ebel 2, H. Einsele 1, M. Hudecek 1 and J. Löffler 1
    1
    Medizinische Klinik Und Poliklinik Ii, Universitätsklinikum Würzburg, Würzburg, Germany
    2
    Institut Für Infektionsmedizin Und Zoonosen, Ludwig-Maximilians-Universität München, München, Germany
    Objectives: Immunocompromised patients are susceptible to invasive fungal infections mainly caused by Aspergillus fumigatus (Af). Adoptive transfer of Aspergillus-specific T cells has been shown to reduce the burden of invasive aspergillosis. Such T cells are hard to isolate and expand. An alternative option is the use of T cells modified to express a chimeric antigen receptor (CAR). CARs are recombinant receptor constructs composed of an extracellular targeting element linked to an intracellular signaling module. A recent study using Dectin 1 as targeting element proved the applicability for antifungal CAR T cells in vitro and in vivo. One main limitation of Dectin 1 is the differential expression of its target β-glucan on Af morphotypes cell wall. β-glucan is mainly exposed on the surface of swollen conidia and early germ tubes but less on hyphae. Thus, better specificity would be highly valuable. Here we propose a highly specific and effective way of redirecting T cells towards Af.
    Methods: For the construction of AF-specific CARs, we fused an scFv, derived from an antibody directed against the Af hyphal cell wall, to extracellular IgG4-Fc spacer domains of different lengths followed by CD28 and CD3-ζ signaling domains. We non-virally expressed the CARs on the surface of primary human CD4+ and CD8+ T cells using the Sleeping Beauty retrotransposon system and co-cultured CAR T cells with Af germ tubes to evaluate specific T cell activation and direct hyphal damage.
    Results: We could show that our CAR T cells are specific for Aspergillus fumigatus, as no cross-reactivity with other Aspergilli was observed. Activated CD8+ CAR T cells secreted mainly Th1-related cytokines (IFN-γ, TNFα, IL-8 and GM-CSF) and chemokines (mainly CCL3 and CCL4), and only one Th2 cytokine (IL-13). Similarly, CD4+ CAR T cells secreted mostly Th1 cytokines and chemokines, but also some Th2 cytokines (mainly IL-13 and IL-4). CARs equipped with a long extracellular spacer containing the full hinge-CH2-CH3-motif conferred superior T cell activation as compared to CARs having a short "hinge-only" spacer. Noteworthy, mainly CAR T cells containing a long spacer underwent proliferation upon activation. Upon binding to the target, the cytolytic machinery of CD8+ CAR T cells was activated, leading to the release of perforin and granzyme B and up to 45% hyphal damage. Furthermore, when cocultured with macrophages, both CD4+ or CD8+CAR T cells enhanced antifungal activity of macrophages.
    Conclusion: Taken together, our results show that we successfully redirected CD4+ and CD8+ CAR T cells against Af hyphae and that fine-tuning of CAR constructs might allow the control of fungal infections in high-risk patients.

    S21.5. Comparison of circulating lymphocyte populations CD4+ and CD8+ T cells, B and NK lymphocytes according to the favorable or worsening evolution of patients with pneumocystosis

    E. Charpentier 1,2, C. Marques 1, S. Menard 1, N. Blanchard 1, P. Chauvin 2, E. Guemas 1, S. Cassaing 2, J. Fillaux 2, A. Valentin 2, A. Berry 1,2 and X. Iriart 1,2
    1
    Center for Pathophysiology of Toulouse Purpan - INSERM UMR 1043, Toulouse, France
    2
    Parasitology and Mycology, CHU de Toulouse, Toulouse, France
    Objectives: Pneumocystosis is a severe opportunistic disease in which the host inflammatory response to the infection is decisive for the disease evolution. Although a reduced count of CD4+ T lymphocytes has been associated to a major risk of pneumocystosis, especially in HIV-positive subjects, little is known about other lymphocyte populations (CD8+ T cells, B and NK lymphocytes) or about CD4+ and CD8+ T cells subpopulations. This study aimed to compare circulating lymphocyte populations in patients suffering from pneumocystosis according to the patient evolution.
    Methods: Levels of circulating lymphocyte populations were evaluated in the blood of 24 patients diagnosed with a pneumocystosis at Toulouse Teaching Hospital between 2016 and 2018. Among them, five were solid organ transplant (SOT) recipients, seven had a haematological malignancy (HM), five had a solid cancer (SC), three had an inflammatory disease (ID) and four were HIV-positive. CD4+ and CD8+ T cells, B lymphocytes and NK cells were evaluated with flow cytometry as well as Th1, Th2, Th17, Treg and naive, effector, memory subpopulations. The lymphocyte populations were compared between patients with favorable evolution (n = 16) and deceased patients (n = 8; 2 SOT, 2 HM, 3 SC, 1 HIV).
    Results: Interestingly, the most significant results concerned CD8+ T cell subpopulations. Although there was no difference in global CD8+ T lymphocyte count, the proportions of memory CD8+ T lymphocytes (both central memory and effector memory) were lower in deceased subjects. At the opposite, the proportions of naive and effector CD8 lymphocytes were increased in this group. Effector memory CD8+ lymphocytes of deceased patients harboured less CD127 (IL7 receptor that induces lymphocyte survival and proliferation) whereas their CD8+ effector cells contained more CD127+ CD25+ (IL2 receptor and activation marker) cells in comparison to the CD8+ effector cells of surviving patients. These differences in CD8+ T cells were associated with reduced Th1 CD4 subpopulation and Th1/Th2 ratio in deceased patients. NK cells were also reduced in this group. There was no significant difference, either in CD4+ T cells count or in other CD4 subpopulations (Th2, Th17, Treg, naive, effector, memory) or in B cells count. Patients age, fungal load and CMV presence or absence were not significantly different in the two groups.
    Conclusion: The implication of CD8+ T cells in pneumocystosis is currently a controversial topic with a discussed role of these cells in the fungus clearance and a suspected association with alveolar lesions. According to this study, high CD8+ memory subpopulations would be associated with a better evolution of the disease, along with Th1 CD4+ T cells. However, high naive and effector CD8 populations would be associated to a worsening evolution of the patient. The disparity of CD8+ lymphocytes subpopulations might be related to a difference of immunity stimulation by Pneumocystis jirovecii antigens or it could be connected with a difference of CD8+ subpopulations homeostasis possibly affected by some therapeutics or living conditions. Despite the dogma that establishes CD4+ T lymphocytes as the center of pneumocystosis pathogenesis, CD8+ T cells may also have an important role in the disease pathogenesis.

    S21.6. Glucosylceramides from Lomentospora prolificans Induce Cytokines Production and Increase the Microbicidal Activity of Macrophages

    M.I.D.S. Xisto 1, J.E. Muñoz 2, L.S. Dias 3, G.M.P. Santos 2, R.O.R. Calixto 1, M.C. Bernardino 1, C.P. Taborda 4 and E. Barreto-Bergter 1
    1
    Instituto De Microbiologia Paulo De Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
    2
    Instituto Biomédico, Departamento De Microbiologia E Parasitologia, Universidade Federal Fluminense, Niterói, Brazil
    3
    Department of Pediatric, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, United States of America
    4
    Institute of Biomedical Sciences, department of Microbiology, University of São Paulo, São Paulo, Brazil
    Objectives: Lomentospora prolificans is an emerging opportunistic fungus with a high resistance to antifungal agents and it can cause localized infections in immunocompetent patients and disseminated infections with a high mortality rate in immunosuppressed patients. Glucosylceramides (GlcCer) are synthetized in the majority of known fungal pathogens. They are bioactive molecules presenting different functions such as involvement in fungal growth and morphological transitions in several fungi. In this study, we report the characterization of GlcCer species in L. prolificans and the role of these molecules in the activation of the innate immune response.
    Methods: GlcCer species were isolated from mycelium and conidia forms of L. prolificans and their chemical structures were elucidated by mass spectrometry (ESI-MS). The reactivity of L. prolificans GlcCer species to anti-GlcCer Mab was analyzed by ELISA. The GlcCer distribution on the surface of L. prolificans was analyzed by immunofluorescence using anti-GlcCer Mab. The cytokine analysis was performed by the GlcCer injection in BALB/c mice and the cytokines were quantified by ELISA according to the manufacturer’s instructions. Recruitment of Cells to the Peritoneum Cavity was analyzed FACS.
    Results: GlcCer purified from both forms presented a major species at m/z 750 that corresponds to N-2-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against GlcCer could recognize L. prolificans GlcCer species from mycelium and conidia, suggesting a conserved epitope in fungal GlcCer. In addition, in vivo assays showed that purified GlcCer species from both forms was able to induce a high secretion of pro-inflammatory cytokines by splenocytes. GlcCer species also promote the recruitment of polymorphonuclear, eosinophils, small peritoneal macrophage (SPM) and mononuclear cells to the peritoneal cavity. GlcCer species were also able to induce the oxidative burst by peritoneal macrophages with NO and superoxide radicals production, and to increase the killing of L. prolificans conidia by peritoneal macrophages.
    Conclusion: Our results indicate that GlcCer species from L. prolificans are potent immune response activators. These molecules induce a strong production of NO in peritoneal macrophages with a high killing activity. In vivo, GlcCer species induce an immune response composed by a Th1 and Th17 cytokine profile, with recruitment of inflammatory cells to the peritoneum cavity. In this way, we believed that these molecules are very important to the immune response against L. prolificans, and could be used to produce antibodies, vaccines or as an adjuvant.

    Symposium 22 NGS and mycobiota

    S22.1. Bacterial-Fungal Interactions at the Airway Mucosa and Implications for Chronic Rhinosinusitis

    E. Cope, O. Kask, S. Kyman, I. Zhang and K. Lee
    The Pathogen and Microbiome Institute, Northern Arizona University, Flagstaff, United States of America
    Emerging studies of the human microbiome (the trillions of bacteria, fungi, viruses, and archaea that inhabit the human body) have demonstrated the myriad of ways that host-associated microbial communities contribute to host health. In the airways, the composition and diversity of the mucosal microbial communities are related to health status. However, the relationship between airway microbiota and inflammation is not well understood. Chronic rhinosinusitis (CRS) is a significant healthcare issue; current estimates suggest that CRS is responsible for 5% of the total healthcare costs in the US and affects up to 16% of the population. In a prior study of the bacterial microbiota of CRS patients, we’ve shown that CRS patients have decreased sinonsasal bacterial diversity compared to healthy individuals. CRS patients were characterized by enrichment of one of four bacterial pathobionts, including Staphylococcaceae or Pseudomonadaceae. The objectives of this study were to determine whether bacterial and fungal microbiota interact in the airways to influence host immune response. We used ITS2 sequencing in a cohort of 100 CRS patients and healthy controls and found that, although bacterial community composition is variable across CRS patients, Malassezia is the predominant fungal genus in the upper airways of healthy individuals and CRS patients. We then hypothesized that species-specific bacterial-fungal interactions differentially influence host mucosal immune response. Thus, we investigated in vitro and in vivo interactions between Malassezia sympodialis, P. aeruginosa, and S. aureus. In vitro interactions were evaluated using the modified Kirby-Bauer Assay and Crystal Violet assay for biofilm quantification. A murine model of acute sinusitis was used to investigate relationships with the host immune response. S. aureus and P. aeruginosa were intranasally instilled in the presence or absence of M. sympodialis. 16S rRNA gene sequencing and FungiQuant were used to evaluate changes in microbiota composition and burden, and host immune response was measured using quantitative real-time PCR (qRT-PCR). In vitro, late stage planktonic and biofilm P. aeruginosa inhibited M. sympodialis growth. Co-infection of mice with M. sympodialis and P. aeruginosa or S. aureus differently influenced the immune response. In co-infected mice, expression of fungal sensing receptors (Dectin-1), allergic responses (IL-5 and IL-13) and inflammation (IL-10, and IL-17) in the murine sinonasal cavity depended on the bacterial species that was co-instilled with M. sympodialis (p<0.05, Mann Whitney). Mice infected with M. sympodialis alone did not have increased Th1, Th2, or Th17 gene expression, suggesting that this taxon can act as a commensal. However, mice that were co-infected with M. sympodialis and S. aureus had increased IL-5 gene expression (p = 0.01, Mann Whitney) compared to M. sympodialis or S. aureus alone. These results show that species-specific interactions in airway associated microbiota relate to distinct host immune response. These interactions may be implicated in some subsets of CRS disease. Our understanding of the role of bacterial-fungal interactions in CRS will contribute to development of novel therapies toward manipulation of the airway microbiota.

    S22.3. Characterization of indoor dust microbiota in homes of asthma and non asthma patients

    J.P. Gangneux 1, M. Sassi 1, P. Le Cann 2 and P. Lemire 2
    1
    Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France
    2
    Inserm Irset- Umr 1085, EHESP, Rennes, France
    Objective: The exposure of occupants of housing to indoor air pollutants has increased in recent decades. Among microbiological contaminants, bacterial and fungal aerosols remain poorly studied and the debate on the impact of these aerosols on respiratory health is still opened. This study aimed to assess the diversity of indoor microbial communities in relationship with the health of occupants.
    Methods: Measurements were taken from dwellings of 2 cohorts in Brittany (France), one with children without any pathology and the other with children and adults with asthma. Thirty dust samples were analyzed by next generation sequencing with a 16S and 18S targeted metagenomics approach. Analysis of sequencing data was performed using qiime 2, and univariate and multivariate statistical analysis using R software and phyloseq package. Dust samples were collected by vacuuming the floor in the child’s bedroom using a Dustream Collector (Indoor biotechnologies, United Kingdom) sampler-fitted vacuum cleaner (40 μm mesh nylon filter, domestic vacuum cleaner).
    Results: A total of 2,637 prokaryotic (589 at genus level) and 2,153 eucaryotic taxa were identified (856 fungal taxa (39%) and 573 metazoa (26%)). Among Fungi, only 136 taxa were identified at genus level. The four main bacterial phyla were identified: Proteobacteria (53%), Firmicutes (27%), Actinobacteria (11%), Bacteroidetes (8%). The three main fungal phyla identified were: Ascomycota (84%), Basidiomycota (12%) and Mucoromycota (3%). No bacterial nor fungal taxa were significantly associated with asthma versus control group.
    A trend of over representation in Asthma group versus control was observed for Corynebacterium (p-value = 0.013, adj. p-value = 0.09) and Streptococcus (p-value = 0.025, adj. pvalue = 0.112) genus. No correlations between the populations of fungi and the asthma conditions as well as the habits of the occupants/the characteristics of the dwellings or other environmental characteristic were observed.
    Conclusion: Our findings provide evidence that dust samples harbor a high diversity of human-associated bacteria and fungi. Molecular methods such as NGS are reliable tools for identifying and tracking the bacterial and fungal diversity in dust samples, a less easy strategy for the detection of eucaryots at least using18S metagenomics approach. This study showed that the detection of some bacteria may be associated to indoor air of asthmatic patients. Regarding fungi, a higher number of samples and sequencing with more depth could allow to reach significant signatures.

    S22.5. An ECMM-ESCMID survey on goals and practices for mycobiota characterization using Next Generation Sequencing in European laboratories

    J.P. Gangneux 1, H. Guegan 1, L.-E. Vandenborght 2, S. Buffet-Bataillon 1, R. Enaud 3, L. Delhaes 4 and The Esghami-Efisg (Escmid) and ecmm NGS Study Group 1
    1
    Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France
    2
    Genoscreen, Lille, France
    3
    University of Bordeaux, Centre de Recherche Cardio-Thoracique de Bordeaux, U1045, CHU de Bordeaux, CRCM Pédiatrique, CIC 1401, Bordeaux, France
    4
    Parasitology Mycology, Univeristy of Bordeaux, Centre de Recherche Cardio-Thoracique de Bordeaux, U1045, F-33000, Bordeaux, France, Bordeaux, France
    Objectives: Although substantial efforts have been made to investigate about the composition of the microbiota, fungi that constitute the mycobiota play a pivotal role in maintaining microbial communities and physiological processes in the body. Next generation sequencing (NGS) techniques have clearly renewed methodology to characterize host-associated microbiota in a more comprehensively way, and have consequently revealed a much more diverse microbiota than was previously thought to exist.
    Methods: We constituted a panel of experts from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID-ESGHAMI and -EFISG study groups) and the European Confederation of Medical Mycology (ECMM) to address an international-survey focusing on laboratory’s current procedures regarding their goals and practices of mycobiota characterization using NGS.
    Results: The laboratories reported their main reason of using NGS to study the mycobiota primary to understand the pathophysiology of a dysbiosis (n = 20), at a second level, to contribute to a diagnosis (n = 16) to implement a therapeutic strategy (n = 12), or to evaluate the exposome (environmental studies)(n = 10). Respiratory tract represented the most studied site, followed by the digestive tract, Sample originated in half of the laboratories from sputum (n = 14), or from stool (n = 12), broncho-alveolar lavage (n = 11), environmental (n = 6), oral wash (n = 4) and skin (n = 1). Target used for mycobiota analysis was equal among 18S rRNA (n = 11), ITS1 (n = 11) and ITS2 (n = 11), while some centers used the shotgun approach (n = 5). When asked about data analysis, database used for alignment were mostly UnitE (n = 8 labs), and both Genebank and CBS databases (n = 7 labs). Other details will be presented.
    Conclusion: We can therefore report a photography of mycobiota analysis in 28 laboratories from 19 countries, affiliated to working groups from ESCMID and ECMM and draw some conclusions of the diversity of approaches. NGS to study mycobiota is a great challenge for understanding pathophysiological questions in experimental studies as well as for driving diagnostic strategies. Techniques will rapidely develop, moving from targeted NGS to shotgun approaches and whole genome sequencing will also be very useful in the next years in the field of mycology, but also for an holistic reasoning.

    Symposium 23 The funny life of C. albicans in the oral tract

    S23.3. Modulation of the Fungal-Host Interaction by the Intra-Species Diversity of C. albicans.

    A. Kannan 1, G. Laval 2, M.-E. Bougnoux 3, C. DEnfert 4 and D. Sanglard 1
    1
    Microbiology Institute, University Hospital Lausanne, University of Lausanne, Lausanne, Switzerland
    2
    Center For Bioinformatics, Institut Pasteur, Paris, France
    3
    Laboratoire De Parasitologie Mycologie, APHP Hôpital Necker-Enfants-Malades, Paris, France
    4
    Fungal Biology and Pathogenicity, Institut Pasteur, INRA, Paris, France
    Candida albicans clinical isolates intrinsically display high diversity in their population genetic structure. Regulation of several host factors and development of C. albicans diseases can be influenced by diverse genetic backgrounds of the fungus. Using a collection of 96 genome-sequenced isolates, we aim here to understand by so-called Expression Quantitative Trait Loci (eQTLs), the influence of C. albicans genome diversity on fungal transcriptomic profiles in contact with human keratinocytes (TR146 cells). The impact of the fungal genomes on the host transcriptome can thus be probed. Pilot experiments using 5 distinct commensal clinical isolates (including the reference isolate SC5314) in contact with TR146 cells at different time points (0.3-, 1-,2- and 6h) revealed distinct transcriptional differences between the isolates. After 6h co-culture, only 194 C. albicans genes were found to be commonly differentially regulated (2-fold threshold) as compared to SC5314 in all four isolates. These data suggest a heterogeneous response between these different isolates, even with a small number of isolates. This heterogeneous response underpins the large genome diversity existing between the different C. albicans clades. Based on the overall summary of top 200 variable C. albicans genes among all the five isolates, we observed that the isolate CEC3665 most varied from SC5314 upon infection. This isolate differed from the others principally by genes involved in hyphal development and adherence. Consistently, CEC3665 exhibited low adherence to TR146 cells as compared to the others. The use of these 5 initial isolates allowed the optimization of several other experimental parameters (optimal exposure time to TR146 cells, variations within biological triplicates) that was used for probing the transcriptional profiles of the entire 96 genome-sequenced collection. The current state of the 96 genome-sequenced collection analysis will be presented.

    S23.4. Intestial Th17 drives airway inflammation in ABPA

    P. Bacher
    Institute of Clinical Molecular Biology & Institute of Immunology, Kiel, Germany
    Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses are poorly understood. In humans, Th17 responses are particularly important against Candida albicans, a mucocutaneous fungal pathobiont. In contrast, the role of Th17 cells for other pathogenic fungal species is unclear. We used antigen-reactive T cell enrichment (ARTE) for the ex vivo analysis of human T helper cell responses against 30 common members of the human mycobiome. We identified the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungal species are induced by T cell cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans- and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, such as asthma, COPD and cystic fibrosis and especially during acute allergic bronchopulmonary aspergillosis (ABPA), suggesting their specific contribution to lung pathology. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identified heterologous immunity to a single, ubiquitous member of the mycobiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a risk factor for pulmonary inflammatory diseases.

    S23.5. Persistence of clonal azole-resistant isolates of Candida albicans from a patient with Chronic Mucocutaneous Candidiasis in Colombia

    A. Ceballos-Garzon 1,2, L.M. Wintaco 3, C. Hernández Padilla 4, A. De La Hoz 4, S.L. Valderrama Beltrán 4, C. Alvarez Moreno 4, P. Le Pape 1, J. Ramirez 3, C.M. Parra-Giraldo 2 and N. Velez 2
    1
    Department of Parasitology and Medical Mycology of The University of Nantes., Universite de Nantes, Nantes, France
    2
    Unidad de Proteómica y Micosis Humanas, Grupo de Enfermedades Infecciosas Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá D.C, Colombia, Bogota, Colombia
    3
    Grupo De Investigaciones Microbiológicas-ur (gimur), Universidad del Rosario, bogota, Colombia
    4
    Grupo De Investigación En Enfermedades Infecciosas, Unidad De Infectología, Hospital Universitario San Ignacio (husi) Pontificia Universidad Javeriana, Hospital Universitario San Ignacio, bogota, Colombia
    Objectives: To evaluate and to characterize phenotypically and molecularly clinical Candida albicans isolates, obtained from a patient with Chronic Mucocutaneous Candidiasis.
    Methods: sixteen C. albicans were isolated from a 25-year-old male with several recurrent fungal infections and admitted to the San Ignacio Hospital in Bogota, Colombia. The isolates were recovered during four-year from a different anatomical sites. The isolates were typified by MultiLocus Sequence Typing, and evaluate for susceptibility to triazoles, Caspofungin and Amphotericin B by microdilution method (CLSI). Their pathogenic capacity was evaluated in the Galleria mellonella model.
    Results: Genotyping of all clinical isolates showed the persistence of the same Diploid Sequence Type (DST). Isolates changed their susceptibility profile over time acquiring resistance to azole drugs. In Contrast there were no significant statistical differences in pathogenicity during the period.
    Jof 05 00095 i010
    Figure 1. Characteristics of Candida spp isolates A. Antifungal treatment over time B. Antifungal susceptibility results.
    Jof 05 00095 i011
    Figure 2. Survival curve of average according to anatomical origin. Kaplan-Meier plots of G. mellonella survival after infection with C. albicans 3x105 UFC/ml. Inside the brackets the number of samples.

    Symposium 24 Fungal neglected tropical diseases

    S24.1. Updates on mycetoma

    W. Van De Sande
    Department of Microbiology and Infectious Diseases, Erasmus Medical Center, Rotterdam, Netherlands

    Abstract:

    Mycetoma is a subcutaneous inflammatory and infectious disease of mainly the foot. It is endemic in tropical and subtropical regions where it causes a high morbidity. It was the first fungal infection recognized as a Neglected Tropical Disease by the World Health Organization by resolution WHA69.21 in 2016. Within this resolution, endemic regions were encouraged to assess the burden of mycetoma and the scientific community was encouraged to develop tools for early diagnosis and to improve treatment of mycetoma. It is now more than 3 years since this resolution and within these three years new information became available on all these subjects. In terms of assessing the burden of infection, case reports and single center studies were published from different regions in the world. Studies from Brazil, China, India, Mexico, the Philippines, Senegal, Sudan and Togo. Also reports from non-endemic regions such as Italy, Spain, Switzerland and the United States of America demonstrated that immigrants and refugees can also take mycetoma to their new homelands where they often cause problems in diagnosis due to lack of awareness. Within these reports it became evident that there are still new mycetoma causative agents discovered every year. To identify the causative agents, morphological identification and/or histology often leaded to misidentification demonstrating the need for more reliable diagnostic tools. To speed up diagnosis and make it more reliable, techniques were developed to isolate DNA directly from mycetoma grains and novel PCR primers were developed to be able to identify more causative agents. Isothermal alternatives were also explored and for histology sections an in situ hybridization probe was developed. For identification on cultures, a Maldi-Tof database was generated. To optimize treatment, an open source drug discovery platform was founded (MycetOS) which identified the fenarimols as a new class of potent anti-mycetoma drugs and other classes such as the orotomides were tested in vitro for their activity against mycetoma causative agents. Based on the in vitro potency of ravuconazole, the first ever clinical trial on a new drug was started at the mycetoma research centre in Khartoum. It is obvious that since the recognition as a Neglected Tropical Disease the mycetoma field has moved forward and will continue as such in the upcoming future.

    S24.2. Chromoblastomycosis: A true neglected tropical disease

    F. Queiroz-Telles
    Public Health, Federal University of Parana, Curitiba, Brazil
    Several infectious diseases, including helminthic, protozoal, bacterial, and viral infections but not fungal diseases other than mycetoma and chromoblastomycosis, are currently defined as “neglected diseases” by the WHO. After sporotrichosis, chromoblastomycosis is the second most prevalent implantation mycosis in the world. Chromoblastomycosis (CBM) is defined as a chronic and indolent granulomatous fungal disease caused by the transcutaneous inoculation of propagules from several species of melanized fungi from the Herpotrichiellaceae family. The most prevalent species causing chromoblastomycosis are Fonsecaea pedrosoi, F. monophora, and Cladophialophora carrionii. Advances in molecular taxonomy have shown that the biodiversity of the CBM agents has increased without substantial correlation with specific type of lesions or severity of the disease and response to therapy. Because several CBM sibling etiologic agents are found in soil and plant microbiota, this disease is strongly associated with cutaneous macro or micro traumas during diverse outdoor activities, such as agro pastoralism, hunting, eco-tourism, etc. Chromoblastomycosis mainly occurs in tropical and subtropical zones in Latin America, Africa, Asia and Oceania. According to recent data, the the highest incidence density of CBM (number of reported cases /10 million), occurs in the Dominican Republic (50.1), Madagascar (26.4), Gabon (23.5), Congo and Costa Rica (11.8), respectively (Santos DW, PhD Thesis) Melanized cell wall, muriform cell architecture, hydrophobicity and cell adhesion are considered virulence factors leading to increased pathogenicity, a key point for disease development. After an uncertain period of incubation, the initial lesion starts at the site of inoculation. With time, these can evolve to polymorphic cutaneous lesions. The clinical forms of the disease might be associated with cytokine cellular-mediated response and in severe cases, the production of TNF α and interleukin 10 has been detected. To confirm diagnosis, the visualization of pathognomonic muriform (sclerotic) cells is the most simple, inexpensive, rapid and 100% sensitive method. In culture, the organisms are fastidious. Molecular methods are mandatory for species identification, but they remain expensive and are not important for therapeutic guidelines. Although serologic tests (immunodiffusion and ELISA) have been validated, they are not commercially available. The therapeutic options, for CBM, include physical methods and chemotherapy. Without a doubt, excisional surgery is the best method for the treatment of CBM but only for initial small and well-delimitated cutaneous lesions. According to a few open non-comparative clinical studies, itraconazole (ITZ) is currently the best therapeutic option for CBM, at the daily dose of 200 to 400 mg from 8 to 36 months. The success rate ranges from 15 to 80%. The duration and cure rates are related to the severity of the disease, ITZ gut absorption and the etiologic agent. Terbinafine (TBF) at the daily dose of 250 to 500 mg is the second most used drug. For refractory cases, posaconazole may be used as well as the combination of ITZ with TBF or 5-flucytosine. The only way to prevent CBM is to avoid trauma Individual under occupational risk should use protective equipment such as adequate clothes, shoes and gloves. Similarly, to other implantation mycoses, there are no available immunoprophilaxis for CBM.

    S24.3. Sporotrichosis: past and future

    L. Lopes Bezerra
    CIETEC/USP, São Paulo, Brazil
    The description of new cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. The Pathogenic Clade of the Sporothrix genus has four pathogenic species: S. schenckii, S. brasiliensis, S. globosa and S. lurei. The new emerging pathogen, S. brasiliensis, shows higher virulence and is mainly related to zoonotic transmission while S. schenckii and S. globosa are sapronotic agents. After 20 years of the zoonotic outbreak of sporotrichosis in Brazil the challenges to manage the disease caused by S. brasiliensis are enormous, including clinical-epidemiological aspects, treatment and diagnosis. The cat-transmitted disease has already spread from the South to the Northeast of Brazil with impressive number of cases in either humans or felines being reported every year. The increment of cases reported by Centers of Zoonotic Control account for over 500% between 2016 and 2018. For this reason, a national effort among scientists, clinicians and veterinaries was established to propose a guideline for the management of S. brasiliensis infection. This group of experts from several endemic areas is working since 2018. The data and conclusions achieved by the Feline and Human Brazilian Working Groups will be presented.

    S24.4. Novel diagnostic and treatment strategies for the Southeast Asian fungus Talaromyces marneffei

    T. Le
    Duke University School of Medicine, DURHAM, United States of America
    Novel diagnostic and treatment strategies for the Southeast Asian fungus talaromyces marneffei – Thuy Le, MD DPhil Talaromyces marneffei (Tm) is one of seven endemic human fungal pathogens that causes substantial global morbidity and mortality. Over just two decades, Tm has emerged as a leading cause of opportunistic infection and death in adults and children with advanced HIV disease in southern China and Southeast Asia. The mortality on antifungal therapy in both HIV and non-HIV infected patients is as high as 30%. The current diagnosis still relies on microscopy of skin lesions (which is a late manifestation and is absent in 40% of patients) and on cultures of the pathogen which takes up to 14 days. Late diagnosis is the most challenging clinical problem and drive mortality from 24% to 50%. The current treatment options are suboptimal. Intravenous amphotericin B deoxycholate is recommended for induction therapy but has serious side effects and is still not widely available. Itraconazole tablets are cheap and are widely available, but has low oral bioavailability, variable pharmacokinetics and is inferior to amphotericin B in efficacy. This talk presents new data on the comparative performance of real-time PCR assays versus antigen detection assays for rapid diagnosis of talaromycosis. Both assays have excellent specificity of >95%; however, the antigen tests are significantly more sensitive than the real-time PCR tests (>90% compared to 70-80%). Tm antigen can be detected up to 16 weeks prior culture positivity in symptomatic hospitalized AIDS patients, and Tm antigen independently predicts 12-month mortality in patients with a CD4 cell count <100 starting antiretroviral therapy. This talk will also present results of the IVAP trial, a multi-center clinical trial comparing amphotericin B and itraconazole for induction therapy in Vietnam, which demonstrated a 50% mortality reduction in the amphotericin B group. The trial showed that 98% of patients in the amphotericin B group cleared fungemia within 8 days of therapy, paving the road for a planned phase II clinical trial testing short courses of liposomal amphotericin B therapy to reduce toxicity. Efforts to test novel antifungal compounds are ongoing and should offer new and less toxic treatment options in the coming years.

    S24.5. Urban sporotrichosis: neglected micro-epidemia in the State of Minas Gerais, Brazil

    M.A. Resende Stoianoff, D.B. Silva Navarro, D.L. Silva and D. Assis Santos
    Dept. of Microbiology, Federal University of Minas Gerais, Belo Horizonte MG, Brazil
    Objectives: Sporotrichosis is a subcutaneous mycosis caused by dimorphic fungus of the Sporothrix complex that can affect both humans and other animals, being more common and severe in cats, and occurring more frequently in regions of tropical climate, as is the case in Brazil. Because it is not a compulsory notification disease in most Brazilian states, the true incidence of the disease is unknown. The aim of this study was to report the occurrence of cases of human sporotrichosis in the state of Minas Gerais, Brazil. Methods: The secretions of the ulcerated lesions were collected with the aid of sterile swab and the material was subjected to Gram staining, as well as to the culture at 28°C and 37°C. It was confirmed by the micromorphological examination that the samples belong to the genus Sporothrix. The molecular identification of the species is underway and the patients were referred for treatment. Results: It was reported the occurrence of 11 cases of human sporotrichosis between August 2018 and May 2019, nine of them from the city of Ribeirão das Neves and two from Belo Horizonte, state of Minas Gerais, Brazil. Among the patients, nine belonged to the female gender and two to the male. The patients’ ages ranged from two to 61 years and all of them reported contact with sick cats. The mean time between contact with the animals and the appearance of the lesions was 15 days. Nine patients presented lymphocutaneous form and two presented fixed cutaneous form. The patients presented lesions in the arms, forearms, fingers or in the heel. The Gram staining revealed presence of yeasts of elongated and/or rounded form, sometimes with single bud. The thermal dimorphism and the micromorphology confirmed the identification of Sporothrix spp. Conclusion: The results suggest that there may be a sporotrichosis microepidemia in the municipality of Ribeirão das Neves, with marked importance of the zoonotic transmission of the disease. According to official information, the conditions of infrastructure and sanitation are precarious in this region. The notification of this disease and studies that relate the socio-environmental and behavioral conditions that can determine the variation in sporotrichosis transmission are of great importance, in order to adopt the appropriate prevention and control measures. The contact of individuals with cats at home environment may be increasing in the last years, a fact that contributes to the increase of the possibility of infection of the population. The control of the source of infection is fundamental in this situation, with priority being given to the investigation of factors involved in the transmission dynamics of the disease, to assist in the surveillance and control measures necessary to contain its growth. Key words: Sporotrichosis, Sporothrix spp., Urban sporotrichosis Financial Support: CNPq, CAPES.

    Posters

    P001. induction of antifungal resistance exerted by the exposure of paclobutrazol to Aspergillus fumigatus

    E. Marques De Araujo 1,2, P. Le Pape 2, R. Gonçalves De Lima-Neto 1, T. Gonçalves 1 and N. Buarque De Gusmao 1
    1
    Biologia De Fungos, universidade federal de pernambuco, Recife, Brazil
    2
    Ea1155 Iicimed, Nantes University Hospital, NANTES, France
    Objectives: The resistance of Aspergillus fumigatus to azolic drugs occurs mainly through two routes: in vivo as a consequence of antifungal therapy, and in the enviroment as a consequence of the use of fungicides in agriculture. Placobutrazol is a plant growth retardant used in agriculture, which inhibits cellular growth and hormones composition. Since it is excessively applied and presents chemical stability, paclobutrazol accumulates in the soil and can affect the existing microbiota.The present study aimed to induce to the resistance and consequently the induction of CYP51 gene mutation on azoles-susceptible isolates of Aspergillus fumigatus after exposition against paclobrutazol.
    Methods: he A. fumigatus (2 from environmental origin and 1 ATCC) were submitted to antifungal susceptibility testing by the microdilution method (MIC-EUCAST) to prove that all strains were sensitive at concentrations of 32 μg/mL of paclobrutazol and 0.5 μg/mL of voriconazole and itraconazole. For the resistance induction test, these fungi were exposed to paclobrutazol for five weeks in RPMI culture medium at 37°C in initial concentration of 4 μg/mL, and increasing to a concentration of 64 μg/mL, it was also checked whether concentration progression influenced induction using the same method, without elevation in concentration of the molecule in the medium. Over the five weeks of the test, the MIC was verified through the micro dilution method established by EUCAST and the yeastone sensitrite commercial method in order to evidence the elevation of MIC after exposure.
    Results: The MICs obtained over the five weeks of the test showed a decreasing of sensitivity in all strains (100%) against antifungals used in the hospital routine after exposure to the paclobrutazol.
    Conclusion: Thus, is possible to affirm the exposure of the Aspergillus fumigatus to drugs used on agriculture may induce resistance against azoles usually prescribed. These finds demonstrating the need of a better and more rigid regulation in the use of this molecule in agriculture. The next steps in this research are to confirm that placobutazol exposure may induce a permanent mutation on resistance gene.

    P002. Oral Candidiasis in HIV Infected Patients and its Antifungal Susceptiblity Pattern by Disc Diffusion Method.

    P. Chaudhary
    Microbiology, Sagarmatha chaudhary eye hospital, lahan, siraha, Nepal
    Objectives: Oropharyngeal candidiasis (OPC) is an opportunistic fungal infection that is commonly found in (Human Immunodeficiency Virus) HIV infected patients. Acquired Immunodeficiency syndrome (AIDS), a disease of human immune system caused by HIV, has emerged as a global crisis since its discovery in summer of 1981 in United States. The low absolute CD4+ T-lymphocyte count has traditionally been cited as the greatest risk factor for the development of OPC and current guidelines suggest increased risk once CD4+T-lymphocyte counts fall below 200 cells/mm³.Hence, a cross-sectional study was conducted in Sukraraj Tropical and Infectious Disease Hospital (STIDH), Teku, Kathmandu over 3 months period with a primary objective, to isolate and identify Candida species among HIV infected patients and susceptibility pattern of isolates by disc diffusion method and secondary objective, to estimate the frequency of oral candidiasis according to age, sex, and other factors (CD4+ count, tobacco consumption, URTI, recent antibiotic consumption) in HIV infected patients.
    Methods: A total of 408 throat swab samples were collected from patients with and without active lesion visiting ART center of STIDH. Flow chart for sample processing and identification of isolates is shown below.
    Results: 114 isolates were obtained from total sample processed. Among them, 65 isolates were Candida species and remaining 49 was bacterial flora and other yeasts. Based on various phenotypic methods, Candida albicans, 53 (81.5%), was the most commonly isolated organism followed by Candida tropicalis 3 (4.6%), Candida krusei 2 (3.1%), Candida glabrata 1 (1.5%) and 6 (9.2%) were other Candida species. of the 65 yeast isolates from HIV seropositive individuals, 26 patients had CD4+ ≤ 200 cells/mm³ and 39 patients had CD4+ ˃200 cells/mm³. 14 patients exhibited severe immune suppression with CD4+ count less than 100 cells/mm³. There was a significant association between oral Candida carriage and CD4+ cell count ≤ 200 cells/mm³ (p<0.001). Antifungal susceptibility test of Candida species showed highest sensitivity with Amphotericin B and resistance with Fluconazole respectively. Table below depicts about the susceptibility pattern of the Candida spp.
    Conclusion: C. albicans was the predominant species for oral yeast colonization in our study. Different isolates presented different susceptibility to various antifungal agents. Among the antifungal discs tested, Fluconazole was found to show resistance in most of the isolates and Amphotericin B was the most sensitive drug which is an excellent antifungal to control Candida spp. since it showed the best result among all antifungal tested. Oral yeast colonization was associated with low CD4+ count (<200 cells/mm³). Thus, oral lesion serves as early marker for HIV infection. The increase incidence of oral candidiasis in HIV patients may be due to low CD4+ count, URTI and recent antibiotic consumption.

    P003. The validation of VIPcheck™ plates to screen Aspergillus fumigatus isolates for phenotypic resistance to triazole antifungal agents in St. James’s Hospital, Dublin.

    A. Griffin
    Microbiology, St. James’s Hospital, Dublin, Dublin, Ireland
    Objectives: Triazole resistance is an emerging problem in Aspergillus fumigatus (AF) resulting in failure of azole therapy. Triazole resistant AF is acquired through one of two routes - previous exposure to triazole therapy or an environmental source. In vitro antifungal susceptibility testing (AFST) on all AF strains isolated in a microbiology laboratory would be both labour intensive and impractical. A method to screen for triazole resistance would be more favourable. The objective was to validate VIPcheck™ plates (Mediaproducts BV, Groningen, Netherlands) in SJH to investigate whether they would be a suitable screening method for triazole resistance in AF clinically isolated from patients in a large tertiary hospital with a significant cohort of immunocompromised patients.
    Methods: A total of 18 retrospective isolates (clinical and environmental) of AF were tested using the VIPcheck™ plates (n=2 wild type, n=16 resistant to ≥ 1 triazole drug as previously determined by AFST and/or molecular methods). VIPcheck™ plates are a simple agar based screening method, each 4 well plate containing a growth control (GC) well and 3 wells containing itraconazole (4mg/L), voriconazole (2mg/L) and posaconazole (0.5 mg/L). Briefly, 25ul of a 0.5-2McF suspension AF is inoculated into each well and plates are read after 48hrs incubation at 37 °C. Any growth in a triazole containing well is suggestive of resistance prompting full AFST be carried out.
    Results: A total of 18 isolates (clinical and environmental) of AF were tested using the VIPcheck™ plates as described above. The wild type isolates showed growth only in the GC well while the resistant strains all showed growth in one or more of the triazole containing wells.
    Conclusion: VIPcheck™ plates are not intended to give an exact MIC; the preparation of the inoculum has a broad McFarland range of 0.5-2 and only one concentration of each azole drug is included in the plates. Rather, they are useful as a screening method to determine the need for further AFST and/or molecular testing and more importantly for earlier detection of triazole resistance in patients who will require treatment. Currently in SJH, AFST is carried out using gradient strips (Liofilchem™) and results are interpreted using EUCAST breakpoints We validated the VIPcheck™ plates with the intention to include this screening method as part of our AFST for AF isolated from clinical samples and our results suggest that they provide a reliable screening method for triazole resistance.

    P004. In vitro susceptibility to isavuconazole of Fusarium clinical isolates; comparison between eucast and etest methods

    A. Broutin 1, J. Bigot 1, A.C. Normand 2, Y. Senghor 3, A. Moreno 3, M. Hendrickx 4, J. Guitard 1 and C. Hennequin 3
    1
    Parasitology-mycology, APHP, hôpital St Antoine, Paris, France
    2
    Parasitology-mycology, APHP, Hôpital Pitie-Salpêtrière, Paris, France
    3
    Parasitology-mycology, APHP, Hôpital St Antoine, Paris, France
    4
    Mycology, Scientific Institute of Public Health, Brussels, Belgium
    Objectives: To evaluate the in vitro susceptibility to isavuconazole of a panel of precisely identified clinical Fusarium isolates and to assess the value of Etest as an alternative method in this indication.
    Methods: We randomly selected 75 clinical isolates previously identified as Fusarium sp. The strains were identified using MALDI-TOF technology (MSI System). They were then tested comparatively against isavuconazole (ISA) either using the broth microdilution method proposed by the Eucast consortium according to the recent document E.DEF 9.1 and the Etest technique as recommended by the manufacturer (Liofilchem).
    Results: All strains could be identified at the cryptic species-level revealing the existence of 14 different species in the panel (Table 1). F. solani (FSSC), F. oxypsorum (FOSC) and F. fujikuroi (FFSC) were the species complexes the most frequent, encompassing 22, 17 and 31 strains, respectively. Overall, ISA exhibited a limited activity against the Fusarium strains with MICs90 >16 µg/ml whatever was the species complex. Strains of the FOSC appeared a little bit more susceptible with a geometric mean at 9.41 µg/ml versus 14.02 µg/ml and 13.68 µg/ml for FSSC and FFSC, respectively. Considering the diversity of species, it was not possible to test a possible difference in susceptibility between the cryptic species within a given complex. Finally, we observed MIC between 2 to >16 µg/ml for strains of the single species of the FOSC, suggesting that precise identification may not be enough to predict the susceptibility to ISA. The concordance between Etest and Eucast results was calculated at 100% within an interval +/- 2 dilutions. The Intraclass Correlation Coefficient was calculated at 0.91, indicative of a strong concordance between the 2 methods.
    Jof 05 00095 i012
    Table 1: Isavuconazole MICs distribution against 75 clinical isolates of Fusarium.
    Conclusion: ISA exhibits a limited in vitro activity against clinical isolates of Fusarium, with few variations between the species complexes. In the case the drug should be used, Etest appears a valuable alternative to Eucast for evaluating the in vitro susceptibility of the infecting strain.

    P005. Species Distribution and Antifungal Susceptibility of Candida Ear Isolates from a Hospital in Korea

    J.H. Shin, Y.J. Kwon, S.A. Byun, M.J. Choi, E.J. Won and S.H. Kim
    Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Republic of Korea
    Objectives: Candida auris was first isolated from the ears of Japanese and Korean patients. However, the prevalence of Candida species from ear cultures and their antifungal susceptibility profiles in these nations are unclear.
    Methods: We assessed Candida isolates recovered from cultures of ear specimens from a university hospital in Korea over the 4-year period from January 2014 to December 2017. Species identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry and/or sequence analysis. Antifungal minimal inhibitory concentrations (MICs) were determined by the broth microdilution method (M27A) of the Clinical and Laboratory Standards Institute.
    Results: Candida species were isolated from the ears of 80 patients (75 adult and 5 pediatric patients). Most of these patients suffered from chronic otitis media (83.8%), and had a history of antibiotic use (73.8%) and ear surgery (60.0%). Among 80 non-duplicate isolates from ear cultures, Candida parapsilosis was the most frequently detected Candida species (35.0%), followed by C. auris (28.8%), Candida metapsilosis (10.0%), Candida orthopsilosis (8.8%), Candida albicans (7.5%), and other species (11.1%). The MICs of the isolates were 0.125 to > 64 µg/mL, ≤ 0.03 to 4 µg/mL, 0.25 to 1 µg/mL, 0.125 to 1 µg/mL, and ≤ 0.03 to 2 µg/mL for fluconazole, voriconazole, amphotericin B, caspofungin, and micafungin, respectively. of the 80 isolates, 43.8% (35/80) showed decreased susceptibility to fluconazole (MIC ≥ 4 µg/mL). of 23 C. auris isolates, 19 (82.6%) had a fluconazole MIC of ≥ 32 μg/mL. None of the isolates showed resistance to amphotericin B or echinocandins.
    Conclusion: C. parapsilosis complex and C. auris were the Candida species identified most frequently in ear cultures and exhibited a high rate of fluconazole non-susceptibility, particularly C. auris.

    P006. Epidemiological pattern of Malassezia, its phenotypic identification and anti-fungal susceptibility profile to azoles by broth micro-dilution method.

    N. Romald 1, A.J. Kindo 2 and M. Veeraragavan 3
    1
    Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, India
    2
    Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, India
    3
    Dermatology, Sri Ramachandra Medical College and Research Institute, Chennai, India
    Objectives: Analyse the epidemiological pattern of disease with regards to age, sex and site of predilection To Speciate Malassezia phenotypically. To study the anti-fungal susceptibility (AFST) pattern of Azoles to pathogenic Malassezia species by recording their MIC values (MIC50 & MIC90) using Broth Micro dilution method in accordance with CLSI M27-A3. To identify resistant drug/drugs to Malassezia species.
    Methods: Prospective study was done from August 2016-October2018 with sample size of 150 Sample collection-Hyper/hypo pigmented skin lesions by Skin scrapings and swabs Microscopic examination with 10%KOH Those showing meat ball and spaghetti appearances were included in the study. Culture Samples inoculated into Sabourauds Dextrose Agar (SDA), SDA with olive oil overlay (SDA-O) and Modified Dixon’s agar (MDA) containing chloramphenicol and incubated at 32o C upto 3 weeks. Phenotypic Identification Done by colony growth characteristics, gram staining, urease test, catalase test, bile esculin with tween 60 hydrolysis and tween assimilation AFST AFST was performed by the broth micro-dilution method, according to the Clinical and Laboratory Standard Institute (CLSI) guidelines M27-A3 (2008) Systemic anti-fungals used-Fluconazole, Itraconazole, VoriconazoleTopical Anti-fungals used-Clotrimazole, Miconazole, Luliconazole Since Malassezia are lipid dependant yeast RPMI 1640 was supplemented with glucose, peptone, ox bile, malt extract, glycerol, tween 80, tween 40 and Chloramphenicol Culture-Malassezia isolates grown on MDA for 72 h at 32°C± 2°C were used for inoculum preparation. Anti-fungal stock solutions of drugs were prepared and stored at - 700C Quality Control-Each microtitre plate included growth control well with inoculum and supplemented RPMI -1640 medium without the antifungal agent. Candida krusei ATCC 6258 was used as a quality control strain for antifungal susceptibility testing. Incubation and Reading Results-The microtitre plates were incubated at 32˚C.Results read after 24hrs for candida isolate and after 72-96 hours for Malassezia isolates. The minimum inhibitory concentration (MIC90) (reduction of 90% growth compared to growth control well) and MIC50 (50% reduction of growth compared to growth control well) were recorded.
    Results:
    Jof 05 00095 i013Jof 05 00095 i014
    Age distribution- The most common age affected was 20 to 30yrs (36%), followed by 30-40yrs (21%) and 10-20yrs (19%) extremes of age showed lower incidence. Sex distribution-The incidence among male and female was almost equal, showing little female predominance Site of infection - Among the total 150, the most common site was back (47 samples), followed by neck and shoulder (31 samples), and scalp (27 samples). Skin lesions-Both hypo and hyperpigmented skin scrapings were collected. But more of hypopigmented lesions were noted in our study ANTI-FUNGAL SUSCEPTIBILITY PATTERN-fig
    Conclusion: Among 150 samples only 95(62%) yielded growth on both SDA with olive oil and MDA. Growth on MDA was soon than SDA with olive oil Overlay. All organisms were urease and catalase positive. Among the 95 samples grown the predominant species was Malassezia sympodialis-29(33%) followed by M.globosa-26(29.5%), M.furfur-21(23%), M.obtusa-12(13%) by conventional method. The systemic anti-fungal with the least MIC values were Itraconazole and Voriconazole. Fluconazole shows resistant Pattern with high MIC values The most effective topical anti-fungal was Clotrimazole with low MIC values Malassezia sympodialis responded to anti-fungal better than other species isolated

    P007. Prevalence and genotype distribution of fluconazole-resistant Candida parapsilosis bloodstream isolates from a neonatal intensive care unit during a 14-year period

    J.H. Shin 1, I.J. Hwang 2, E.J. Won 1, M.J. Choi 1, S.-I. Jung 3, E.S. Song 2 and Y.Y. Choi 2
    1
    Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Republic of Korea
    2
    Paediatrics, Chonnam National University Medical School, Gwangju, Republic of Korea
    3
    Chonnam National University Medical School, Gwangju, Republic of Korea
    Objectives: Candida parapsilosis bloodstream infection (BSI) is a significant problem in neonatal intensive care units (NICUs). C. parapsilosis is usually susceptible to azole antifungals; however, some recent reports have indicated the emergence of BSIs caused by fluconazole-resistant (FR) C. parapsilosis isolates. We investigated the prevalence and genotype distribution of FR C. parapsilosis BSI isolates recovered from a NICU during a 14-year period.
    Methods: We assessed a total of 64 C. parapsilosis BSI isolates comprising 22 non-duplicate isolates from 22 neonates and 42 isolates from 21 neonates (two isolates from each neonate obtained on the first and the last days of C. parapsilosis-positive culture). All isolates were obtained at the same NICU between 2002 and 2015. In vitro antifungal susceptibility testing for fluconazole, amphotericin B, caspofungin, and micafungin was performed using the Clinical and Laboratory Standards Institute (CLSI) M27-A3 microdilution method. All isolates were genotyped via microsatellite typing using four specific microsatellite markers (CP1, CP4, CP6, and B). Clonal strains were defined as the isolation of two or more strains with identical genotype as determined by microsatellite typing.
    Results: of the 64 C. parapsilosis BSI isolates, 11 (17%) obtained from eight patients were FR (fluconazole minimal inhibitory concentration [MIC] ≥ 8 mg/L). All 64 isolates were susceptible to amphotericin B, caspofungin, and micafungin. Based on microsatellite typing, the 64 isolates yielded 19 different genotypes, including 12 genotypes unique to a single isolate and seven genotypes shared by 49 isolates (77%). All 11 FR isolates shared two genotypes (1 and II) that displayed closer genetic relatedness than other isolates examined in the microsatellite analysis, according to as an unweighted pair group method with arithmetic mean (UPGMA) tree. of these two genotypes, type I strains (three patient isolates) were isolated within a 6-month period, whereas type II strains (five patient isolates) were isolated over a 4-year period. Among 21 patients showing more than one C. parapsilosis isolate on 2 or more separate days, two sequential strains from each patient had identical types in 76% (16/21) of patients and different types in 24% (5/21). No difference in antifungal susceptibility was detected between two sequential isolates from the same patient. Among eight patients with FR C. parapsilosis isolates, 88% had no known prior antifungal exposure. We detected no significant difference in the overall 30-day mortality of C. parapsilosis BSI due to FR strains, or in that of BSI due to fluconazole-susceptible (FS) strains (25 vs. 20%).
    Conclusion: The results of the present study suggest that clonal transmission may occur frequently among both FS and FR blood isolates of C. parapsilosis in a NICU setting. Increased incidence of BSI due to the FR C. parapsilosis strain in this NICU may be associated with clonal transmission rather than prior antifungal exposure.

    P008. Bloodstream Isolates of Azole-Resistant Candida glabrata in Korean hospitals: Resistance Mechanisms and Clinical Features

    J.H. Shin 1, E.J. Won 1, S.-I. Jung 2, I.J. Hwang 3, M.J. Choi 1, S.A. Byun 2, S.H. Kim 1, Y.-J. Park 4 and M.-N. Kim 5
    1
    Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Republic of Korea
    2
    Chonnam National University Medical School, Gwangju, Republic of Korea
    3
    Paediatrics, Chonnam National University Medical School, Gwangju, Republic of Korea
    4
    The Catholic University of Korea College of Medicine, Seoul, Republic of Korea
    5
    University of Ulsan College of Medicine and Asan Medical Center, Seoul, Republic of Korea
    Objectives: Candida glabrata has recently become the most common non-albicans Candida species causing bloodstream infection (BSI) in South Korea, and the occurrence of azole-resistant C. glabrata isolates has been observed. We investigated the azole resistance mechanisms and clinical features of patients with azole-resistant C. glabrata BSI isolates recovered from multicenter surveillance in Korea.
    Methods: In total, 248 C. glabrata BSI isolates comprising 43 azole-resistant (fluconazole minimum inhibitory concentration [MIC], ≥ 64 μg/mL and voriconazole MIC, ≥ 1 μg/mL) and 205 non-azole-resistant (non-AR) isolates (fluconazole MIC, ≤32 μg/mL and voriconazole MIC, ≤ 0.5 μg/mL) were analyzed. The isolates were collected from 10 university hospitals over 8 years (2009–2017). The PDR1 genes of the isolates were sequenced to identify mutations. Expression levels of CDR1, CDR2, and ERG11 genes after fluconazole exposure were quantified by real-time PCR. The clinical features related to risk factors for BSI and the mortality rates were compared. Risk factors for BSI caused by AR isolates and survival rates in patients were assessed using multivariate logistic regression.
    Results: Pdr1 mutations were identified in 42 of 43 azole-resistant isolates, while no Pdr1 mutation was found in 201 of 205 non-AR isolates. Azole-resistant isolates of C. glabrata had higher mean expression levels of CDR1 (8.3-fold) gene than non-AR isolates (P <0.05). However, there were no significant differences in the mean expression levels of CDR2 and ERG11 between two groups. Previous azole exposure (odds ratio [OR], 6.369; 95% confidence interval [CI], 2.434-16.664; P < 0.001), central venous catheter use (OR, 11.406; 95% CI, 2.462-52.837; P = 0.00211), and total parenteral nutrition (OR, 4.042 95% CI, 1.381-11.828; P = 0.011) were the independent risk factors for BSI caused by AR C. glabrata. C. glabrata BSIs caused by AR isolates resulted in a higher 30-day mortality rate (55.8%, 24/43) than BSIs caused by non-AR strains (36.6%, 75/205) (P = 0.019). In multivariate analyses, severe sepsis (OR, 5.592; 95% CI, 2.689-11.630; P < 0.001), a high age-adjusted Charlson comorbidity index (ACCI) score (OR, 1.131; 95% CI, 1.012-1.263; P = 0.03), lack of antifungal therapy (OR, 5.213; 95% CI, 2.487-10.930; P < 0.001), and azole-resistance in C. glabrata BSI isolates (OR, 2.856; 95% CI, 1.161-7.025; P = 0.022) were independent predictors of the 30-day mortality among the 248 patients.
    Conclusion: This study provides that Pdr1 mutation with overexpression of CDR1 may be the main resistance mechanism of C. glabrata in Korea, and that azole-resistance in C. glabrata BSI isolates is independent predictor of 30-day mortality, and is associated with antifungal exposure, central venous catheter, and total parenteral nutrition use.

    P009. A case of urinary tract candidosis: a risky business

    R. Barton and K. Sethi
    Microbiology, Leeds Teaching Hospitals Trust, Leeds, United Kingdom
    Case Report: A 62 year old man with a history of psoriasis treated with methotrexate, poorly controlled type 2 diabetes, and chronic obstructive pulmonary disease had a laparoscopic cholecystectomy for cholecystitis and repair of a cholecystoduodenal fistula. He then presented with severe dysuria, polyuria and complained of passing cloudy urine ever since the surgery. Following a series of urine samples which were positive for yeasts which were not identified and a course of fluconazole (details not available), he was seen at a haematuria clinic with continued dysuria, frequency and urgency, and a flexible cystoscopy showed a "snowstorm" appearance within bladder, and an ultrasound revealed mobile echogenic debris. He was then put on a 14/7 course of 200mg oral fluconazole, pending a cystoscopy and bladder biopsy which revealed erosive cystitis. A subsequent urine sample was identified as Candida glabrata which was resistant to fluconazole (MIC >256mg/L) but sensitive to flucytosine (MIC 0.06mg/L). Despite a further 14 day course of 800mg fluconazole, signs and symptoms of Candida cystitis were not resolved. The patient was then referred to a urologist at our hospital and various treatment choices were discussed with oral flucytosine and IV Amphotericin deoxycholate being the main two options, associated with the risk of resistance and toxicity respectively. It was decided to treat with a 7 day course of 25mg/kg of oral flucytosine (levels were not monitored due to the short course) but follow up urine samples 9 days after completing the flucytosine course were still positive for C. glabrata resistant to both fluconazole (MIC >256mg/L) and flucytosine (MIC >64mg/L). Approximately 2 weeks after completing the course of flucytosine, the patient was admitted via Accident and Emergency with suspected sepsis relating to a biliary source, and a new ultrasound showed that the left kidney was moderately hydronephrotic, the bladder was filled with echogenic debris. A nephrostomy was performed and blood cultures taken which grew C. glabrata with identical sensitivities to that from the most recent urine sample. The patient was prescribed a 2 week course of Anidulafungin (200mg loading dose then 100mg 24-hourly IV) to combat the systemic candidosis, and a 7 day course of intravenous Amphotericin deoxycholate at 0.6mg/kg/day. During the Amphotericin B deoxycholarte treatment, renal function was not markedly affected, though increases in alkaline phosphatase levels led to the treatment being stopped at 6 days. Subsequent blood and urine cultures remained negative, CRP declined from 150 to 5.8 mg/L and symptoms resolved. This case is one of the examples of how the significance of the presence of yeasts in urine of patients is not clearly understood by the clinicians. Our patient presented here highlights the increasing prevalence of C.glabrata in funguria and non-albicans candidaemia in at risk settings, the challenges in its management, the risks and drug related factors which need to be assessed when treating fluconazole resistant urinary tract candidosis.

    P010. Effects of efflux pump modulators on the azole antifungal susceptibility of Microsporum canis

    C. Aneke, W. Rhimi, D. Otranto and C. Cafarchia
    Veterinary Medicine, University of Bari, Aldo Moro, Bari, Italy
    Objectives: This study investigated the effect of efflux pump modulators, EPMs (i.e. haloperidol-HAL and promethazine-PTZ) and their inhibiting activity on the minimum inhibitory concentrations of itraconazole (ITZ) and fluconazole (FLZ), against selected M. canis strains.
    Methods: M. canis strains with low (≤ 1mg/ml itraconazole and <64mg/ml fluconazole) and high (>1mg/ml itraconazole and ≥64 μg/ml fluconazole) azole MIC values were tested, and data analyzed using the model-fractional inhibitory concentration index (FICI) method.
    Results: The MIC values of ITZ and FLZ of M. canis decreased in the presence of sub-inhibitory concentrations of HAL and PTZ, the latter being more effective with a greater increased susceptibility. Synergism was observed in all strains with high azole MICs (FICI<0.5) and no synergism in the strains with low azole MICs.
    Conclusion: These results suggest that the drug efflux pumps are involved in the defense mechanisms to azole drugs in M. canis strains. The synergism might be related to an increased expression of efflux pump genes, eventually resulting in azole resistance phenomena. Additionally, the most effective double combinations were those of PTZ with either FLZ or ITZ as this had a greater increase in azole susceptibility. Complementary studies on M. canis resistance are advocated in order to investigate the molecular mechanisms of this phenomenon.

    P011. Antifungal resistance pattern of Candida species isolated from women of child-bearing age in Nsukka Nigeria suffering from different clinical conditions.

    A. Mgbeahuruike
    Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Nsukka, Enugu, Nigeria
    Objectives: To isolate and identify Candida species in child bearing women suffering from malaria, typhoid and diabetes To investigate the resistance partarn of the identified Candida species using conventional azoles and plant extracts To trace the evolutionary relatedness of the isolated fungi using phylogenetic analysis
    Methods: High vaginal swabs were analyzed for Candida presence in women of child bearing age suffering from malaria, typhoid and diabetes using Chromogenic agar. Evolutionary relatedness of the Candida species was inferred using phylogenetic reconstruction. Growth rate assay was done using Sabourand Dextrose medium supplemented with NaCl and glucose. The resistance pattern of the Candida species was tested using fluconazole and extracts (methanol and ethyl) from Byrsocarpus coccineus.
    Results: Out of the 79 fungal samples analyzed, 21 (26.6%) were C. tropicalis, 25 (31.6%) were C. krusei, 10 (12.7%) were C. parapsilosis and 8 (10.1%) were C. albicans. C. tropicalis was highest in the malaria patients, C. albicans dominated in the typhoid patients while C. krusei was more in the diabetic patients. The healthy women presented a very low percentage (8.9%) of Candida species. Phylogenetically, the sequenced Candida species was more closely related to C. tropicalis. High concentration of glucose (1g/ml) and NaCl (0.5M) decreased the growth of the fungus. From the study, fluconazole showed a higher activity than the two extracts (methanol and ethyl acetate).
    Conclusion: In conclusion, although fluconazole showed a higher activity than the two extracts (methanol and ethyl acetate), the plant extracts demonstrated very high activity and can be used as potential antifungal agents. C. tropicalis and C. krusei were the most abundant Candida species found in this study.

    P012. Colistin and isavuconazole interact synergistically in vitro against Aspergillus nidulans and Aspergillus niger

    P. Schwarz 1,2 and E. Dannaoui 3,4
    1
    Department of Internal Medicine - Respiratory and Critical Care Medicine, University Hospital Marburg, Marburg, Germany
    2
    Center For Invasive Mycoses and Antifungals, Philipps University Marburg, Marburg, Germany
    3
    Hôpital Européen Georges Pompidou, Laboratoire de Parasitologie-Mycologie, Paris, France
    4
    Working Group Dynamyc, Faculté de Médecine, Hôpital Henri Mondor, Créteil, France
    Objectives: Invasive aspergillosis is a devastating disease in immunocompromised patients. It mostly affects patients with hematological malignancies, especially those with severe and prolonged neutropenia. Colistin is a polypeptide antibiotic from the class of polymyxins. Polymyxins bind to lipopolysaccharides and phospholipids of the outer cell membrane of gram-negative bacteria. Binding leads to competitive displacement of divalent cations from the phosphate groups of the membrane lipids, leading to bactericidal action by disruption of the outer cell membrane and leakage of intracellular contents. Colistin has also shown in vitro activity against some fungi. The aim of the present study was to assess the in vitro interaction of the broad-spectrum azole isavuconazole with colistin against the most important Aspergillus species responsible for human disease.
    Methods: A panel of 31 Aspergillus isolates belonging to 5 species responsible for human invasive aspergillosis was used for the experiments (10 Aspergillus fumigatus, 5 Aspergillus flavus, 5 Aspergillus terreus, 6 Aspergillus nidulans, and 5 Aspergillus niger). Combinations of isavuconazole with colistin were tested by a broth microdilution checkerboard technique based on the EUCAST reference methodology. Plates were read spectrophotometrically, and ninety percent of inhibition was used as an endpoint for both, the drugs alone and in combination. Results were interpreted with the fractional inhibitory concentration index (FICI). Drug interactions were defined as synergistic (FICI≤0.5), indifferent (FICI>0.5≤4) or antagonistic (FICI>4).
    Results: Isavuconazole MICs ranges from 0.5 to 16 µg/ml. When tested alone, colistin exhibited no antifungal activity with MICs of 128 µg/ml for all isolates. Combination of isavuconazole with colistin was synergistic for 100 and 60% of the A. nidulans, and A. niger isolates, respectively (Table). All other interactions were indifferent. Antagonism was never seen.
    Jof 05 00095 i015
    Conclusion: Combination of colistin with isavuconazole showed strong synergy against all tested A. nidulans isolates and synergy for the majority of the tested A.niger isolates. In vitro interaction of colistin and isavuconazole will be further evaluated by checkerboard and gradient concentration strips against a larger panel of A. nidulans and A.niger isolates.

    P013. Update 2016-2018 of the nationwide Danish fungaemia surveillance study: epidemiologic changes in a 15-year perspective

    M. Risum 1, R. Datcu 1, H.K. Johansen 2, H.C. Schønheyder 3, F.S. Rosenvinge 4, I.J.D. Knudsen 5, B.L. Røder 6, V.S. Antsupova 7, J.B. Gertsen 8, L. Kristensen 8, J.K. Møller 9, R. Hare 1, K.M.T. Astvad 1, K.M. Jørgensen 1, E. Dzajic 10 and M.C. Arendrup 1,2,11
    1
    Mycology Unit, Statens Serum Institute, Copenhagen, Denmark
    2
    Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
    3
    Department of Clinical Microbiology, Aalborg University Hospital, Aalborg, Denmark
    4
    Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark
    5
    Department of Clinical Microbiology, Hvidovre Hospital, Hvidovre, Denmark
    6
    Department of Clinical Microbiology, Slagelse Sygehus, Slagelse, Denmark
    7
    Department of Clinical Microbiology, Herlev Hospital, Herlev, Denmark
    8
    Department of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark
    9
    Department of Clinical Microbiology, Vejle Sygehus, Vejle, Denmark
    10
    Department of Clinical Microbiology, Esbjerg Sygehus, Esbjerg, Denmark
    11
    University of Copenhagen, Dept. of Clinical Medicine, Copenhagen, Denmark
    Objectives: The objectives of this study was to provide nationwide contemporary data on the epidemiology of candidaemia, often a serious complication of hospital stay with a crude 30-day mortality rate of about 40% despite recent advantages in diagnosis and management. The motivation was that outcome depend on early appropriate therapy necessitating knowledge on susceptibility pattern of involved species. Moreover, epidemiological changes reflect current practises in prophylactic, empirical and pre-emptive use of antifungals and therefore are also informative regarding the need for adjustment of antifungal stewardship programmes or treatment guidelines.
    Methods: As part of an active national surveillance programme, fungal isolates from blood culture were referred to Statens Serum Institut (Copenhagen) for confirmatory species identification and EUCAST E.Def 7.3.1 susceptibility testing. A total of 1452 unique isolates (same patient and species isolates were regarded unique if more than 21 days apart or with different susceptibility) were included. 13 isolates (0.9%) were not referred. When MICs for echinocandins were above the clinical breakpoints, FKS sequencing of the isolates was performed. An isolate was deemed echinocandin resistant if the MIC was above anidulafungin breakpoint and/or if the isolate had a known FKS hotspot-mutation. Results were compared to previous data in a 15-year perspective.
    Results: The mean incidence rate during 2016-2018 was 8.13/100.000 inhabitants. Candidaemia was more frequent in males than females (60% vs 40%) as in accordance with previous years. In comparison to previous data, the fungaemia rate was stable (8.64, 9.03 and 8.38 per 100.000 inhabitants in 2004-7, 2008-11 and 2012-15 respectively). In the years 2016-2018 C. albicans accounted for 42.1% of all unique isolates with a decline in 2017 to below 40%. C. glabrata accounted for 32.1%. The proportion of candidaemia in 2018 involving C. albicans decreased by a third from 64% in 2004 (P<0.0001), and C. glabrata almost doubled from 17% in 2004 (P<0.0001) (Figure). We found a fluconazole susceptibility rate across all Candida species of 59% of isolates which has decreased since 2004-7 from 69% (P = 0.02). Echinocandin resistance was detected in 18/1294 isolates (1.4%) including 11/459 (2.4%) in C. glabrata specifically. No echinocandin resistance was detected in 2004-7. FKS-mutations were identified in the following species C. glabrata: two L630Q/S663F, four F659S, one L662W, three S663F and one S663P; C. krusei: four L701M, two S659F and one S659S/F; C. albicans one P1354P/S; C. dubliniensis one S645P; and for Candida lusitaniae one H657Y/L1243F/I283V. H657Y/L1243F/I283V, P1354P/S and L701M are outside the hot spot, and L701M is not associated as a molecular cause of echinocandin resistance.
    Jof 05 00095 i016
    Conclusion: We report a stable incidence rate of candidaemia, but the proportion of C. glabrata continues to rise at the expense of C. albicans which accounted for less than 40% in 2017. The consequent decrease in fluconazole susceptibility along with the presence of FKS-mutations particularly in C. glabrata raises concern for future perspectives in the treatment of candidaemia.

    P014. In vitro potpourri: Aspergillus terreus and the activity of amphotericin B

    R. Vahedi Shahandashti
    Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria
    Objectives: Invasive aspergillosis caused by intrinsically resistant non-fumigatus Aspergillus species displays a poor outcome in immunocompromised patients. Aspergillus terreus holds an exceptional position within the aspergilli due to its intrinsic resistance against the polyene amphotericin B (AmB). Till now, the exact mode of action in polyene resistance is not well understood. Micro-environmental factors at the infection site influence the growth of fungal pathogens and most likely also the efficacy of antifungal drugs. The purpose of this study was to evaluate the impact of hypoxia versus normoxia on the in vitro activity (fungistatic and fungicidal) of AmB against A. terreus.
    Methods: In total, 100 clinical strains of Aspergillus section terrei, collected from 21 various countries were analyzed in detail. Antifungal susceptibility testing was carried out according to EUCAST guideline, with normoxic (20% oxygen/5% carbon dioxide/75% nitrogen) and hypoxic (1% oxygen/5% carbon dioxide/94% nitrogen) conditions. Final drug concentrations used for AMB were 0.03–16 μg/ml. Minimum inhibitory concentration (MIC) was defined as the lowest drug concentration showing no fungal growth and minimum fungicidal concentrations (MFCs) was taken as the first well with ≥90% killing of fungi. MFC was determined by agar and broth-based (flash microbicide method) methods. MICs (μg/ml) and colony forming units were determined after 48h at 37°C. Testing was performed in duplicates.
    Results: AmB MICs of A. terreus section terrei ranged from 0.125-2 µg/ml for hypoxia, and from 0.5-8 µg/ml for normoxia. These data indicate that some strains were more susceptible to AmB in hypoxic conditions compared to normoxia (Figure 1). The majority of strains showed that AmB had no fungicidal effect against tested A. terreus under any methods and atmospheric conditions (Figure 2). A. terreus representing lower MICs (n = 41, ≤0.5 µg/ml) did not result in modest MFCs (≥90% killing in 1-2 µg/ml).
    Jof 05 00095 i017
    Figure 1. In vitro susceptibility to AMB of 100 A. terreus isolates in normoxia versus hypoxia, measured by EUCAST.
    Jof 05 00095 i018
    Figure 2. The effect of oxygen on MFC distributions of AMB, agar-based method measured by colony forming units.
    Conclusion: Simulating the host environment in in vitro susceptibility testing will contribute to a better understanding of how these conditions influence antifungal activity. Hypoxic conditions had a marginal influence on the in vitro antifungal susceptibility pattern of A. terreus species to AmB. A slight reduction of MICs was demonstrated, however, A. terreus did not turn to be classified as AmB susceptible (AmB MICs < 0.125 µg/ml) under hypoxic conditions. Remarkably, AmB lacked any fungicidal activity in the majority of examined strains; comparing with MICs, MFC seems to be related more with in vivo outcome and might partially explain the high failure rate of AmB therapy in vivo.

    P015. In vitro activity of active ingredients of disinfectants against drug-resistant fungi

    R. Stauf 1, D. Todt 2, E. Steinmann 2, P.-M. Rath 3, H. Gabriel 4, J. Steinmann 5 and F.H.H. Brill 4
    1
    Institute For Clinical Hygiene, Medical Microbiology and Infectiology, Paracelsus Medical University Nuremberg, Nuremberg, Germany
    2
    Department For Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
    3
    Institute of Medical Microbiology, University Hospital Essen, Essen, Germany
    4
    Institute For Hygiene and Microbiology, Dr. Brill + Partner GmbH, Hamburg, Germany
    5
    Institute of Clinical Hygiene, Medical Microbiology and Clinical Infectiology, Klinikum Nuernberg, Nuremberg, Germany
    Objectives: Prevention of immunosuppressed patients against fungal pathogens is essential in times of emergence of antifungal-resistant yeasts and moulds. Therefore, the use of fungicidal disinfectants is an important measure preventing infections. In this study, the fungicidal activity of common microbial agents of chemical disinfectants against antifungal-resistant and antifungal-susceptible yeasts and moulds was tested in comparison to the reference strains Candida albicans, Candida tropicalis, Aspergillus brasiliensis and Aspergillus niger.
    Methods: In total, nine clinical isolates from the fungal collection of the University Hospital Essen and four reference strains were included. Species identification was performed by morphological methods and sequencing of the internal transcribed spacer region (ITS). The antifungal susceptibility testing was performed via microdilution assay according to EUCAST standard 9.3 for moulds and 7.3.1 for yeasts. The sensitivity of clinical yeasts and moulds isolates in comparison to the reference isolates was tested in a quantitative suspension test against peracetic acid and ethanol as the common active ingredients of chemical disinfectants.
    Results: Ethanol was sufficiently active with a log10 reduction factor of ≥ 4.0 against all yeasts in a concentration of 50 % v/v within 1 min and against all moulds as 80 % solution within 1 min. Peracetic acid was active as 0.25 % solution in 5 min against all yeasts and as 0.5 % solution in 5 min against all moulds. Compared to the reference strains the clinical isolates showed similar or higher sensitivity to the active ingredients. In addition, antifungal-resistant strains exhibited equivalent sensitivity in vitro against ethanol and peracetic acid compared to the susceptible ones.
    Conclusion: Clinical fungal isolates including multidrug resistant ones did show a similar or higher sensitivity to active ingredients of disinfectants than the reference strains.

    P016. In vitro susceptibility of olorofim against 1,682 clinical Aspergillus isolates

    J. Buil 1, J. Oliver 2, M. Birch 2, D. Law 2, J. Rex 2, M. Tehupeiory-Kooreman 1, H. Van Der Lee 1, W. Melchers 1 and P.E. Verweij 1
    1
    Medical Microbiology, Radboudumc, Nijmegen, Netherlands
    2
    F2G Ltd, Manchester, United Kingdom
    Objectives: Olorofim (OLO) is a novel antifungal agent with potent activity against filamentous fungi such as Aspergillus spp., Scedosporium spp. and Lomentospora prolificans. We included OLO in our routine MIC-panel for filamentous fungi and analysed the in vitro susceptibility of OLO against clinical Aspergillus isolates received in our laboratory between May 2017 and March 2019.
    Methods: The in vitro activity of OLO was tested against 1,682 clinical Aspergillus isolates (987 azole susceptible A. fumigatus isolates, 414 azole-resistant A. fumigatus, 72 A. flavus species complex (SC), 32 A. nidulans SC, 110 A niger SC, 21 A. terreus SC, 7 A. versicolor SC, 39 other species). OLO susceptibility was determined using broth microdilution based on EUCAST guidelines. Isolates were identified to the SC level using microscopy and growth characteristics.
    Results: The olorofim MICs of all clinical A. fumigatus, A. flavus species complex, A. terreus species complex, A. niger species complex, A. versicolor species complex were found to be uniformly <1 mg/L. The MIC50 and MIC90 of species with >10 different isolates are displayed in the table. No significant differences were found in the OLO MICs of azole-susceptible A. fumigatus isolates compared to azole-resistant A. fumigatus isolates. Isolates of some cryptic species within the Aspergillus aspergillus species complex (A. hollandicus, n = 1 A. glaucus, n = 4 and A. chevalieri n = 1) were found to have elevated MIC’s: 1 isolate (A. glaucus) with a MIC of 1 mg/L and the remainder with a MIC of >2 g/L).
    Jof 05 00095 i019
    Conclusion: OLO showed potent in vitro activity against a large collection of clinical Aspergillus species, including 1401 A. fumigatus isolates, with many of these resistant to one or more azoles. Interestingly, OLO was less potent against a small number of isolates belonging to the Aspergillus aspergillus SC (formerly Eurotium).

    P017. Detection and Typing of Fluconazole Resistance on Candida albicans with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass

    M. Delavy, M. Corthesy, G. Greub, D. Sanglard and A.T. Coste
    Microbiology Institute, University Hospital Lausanne, University of Lausanne, Lausanne, Switzerland
    Objectives: Candida albicans causes life-threatening systemic infections in immunosuppressed patients. These infections are commonly treated with fluconazole, an antifungal agent targeting the ergosterol biosynthesis pathway. Current Antifungal Susceptibility Testing (AFST) methods take time and are often subjective. An alternative to the classical AFST methods could use Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry (MS). This tool, already used in clinical microbiology for microbial species identification, has already offered promising results to detect antifungal resistance and could be used to detect specific peaks associated with fluconazole resistance.
    Jof 05 00095 i020
    Methods: We analyzed MALDI-TOF MS spectra acquired, with the Bruker microflex device, from 21 C. albicans clinical strains isolated from 9 patients. Those strains were exposed for 3h to 3 fluconazole concentrations (256, 16, 0μg/mL). We optimized a protein extraction protocol that allowed the acquisition of high-quality spectra. Two steps of quality controls were implemented. First, the quality of the spectrum was assessed through its identification score. Second, the variability of the biological and technical replicates was evaluated, and outliers were removed. We then developed an R pipeline to identify MALDI-TOF MS signature peaks associated with fluconazole resistance or with TAC1 or ERG11 mutations known to affect C. albicans susceptibility level, and we designed score-based diagnostic tests with these signature peaks. Finally, we evaluated the effects of adding a protein standard (Bacterial Testing Standard, BTS, Bruker) to the proteins extracts to reduce the variability of the peaks position. and then we tested the effect of the tolerance inhibitor cyclosporin A on the susceptible strains’ spectra quality.
    Results: Up to now, in absence of cyclosporin A or BTS, we identified a 4480 m/z peak, whose intensity after exposition to 16 μg/mL fluconazole, was inversely proportional correlated with fluconazole resistance. The intensity of the peak seems to be more inversely proportional correlated with TAC1 gain-of-function mutation than with ERG11 mutations. Since only a single peak was identified, it is likely that the trailing effect of fluconazole might hide other interesting peaks. We thus decide to inhibit fluconazole tolerance using cyclosporin A in further experiments. In addition, the main challenge of this first analysis was to reproducibly align the spectra. To circumvent this issue, we decided to introduce a protein standard (BTS) to the fungal proteins extracts to be analyzed. Effects of BTS or cyclosporin A are currently under investigation.
    Conclusion: This study acts as a proof-of-concept for the use of MALDI-TOF MS for the typing of fluconazole resistance in C. albicans and currently offer promising results.

    P018. Azole resistance in Aspergillus. The first six months data from the Danish nationwide surveillance study

    M. Risum 1, R. Hare 1, F.S. Rosenvinge 2, J.B. Gertsen 3, L. Kristensen 3, J. Bangsborg 4, B.L. Røder 5, E.S. Marmolin 6, S. Sulim 7, K.M.T. Astvad 8, M. Pedersen 8, R. Datcu 1 and M.C. Arendrup 1,9,10
    1
    Mycology Unit, Statens Serum Institute, Copenhagen, Denmark
    2
    Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark
    3
    Department of Clinical Microbiology, Århus University Hospital, Århus, Denmark
    4
    Department of Clinical Microbiology, Herlev Hospital, Herlev, Denmark
    5
    Department of Clinical Microbiology, Slagelse Sygehus, Slagelse, Denmark
    6
    Department of Clinical Microbiology, Vejle Sygehus, Vejle, Denmark
    7
    Department of Clinical Microbiology, Ålborg University Hospital, Ålborg, Denmark
    8
    Department of Clinical Microbiology, Hvidovre Hospital, Hvidovre, Denmark
    9
    University of Copenhagen, Dept. of Clinical Medicine, Copenhagen, Denmark
    10
    Department Og Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
    Objectives: Azole resistance (azole-R) in Aspergillus has been increasingly reported worldwide. The use of azole fungicides has been proposed to contribute to the emergence of azole-R A. fumigatus, and isolates harbouring the resistance mechanisms TR34/L98H or TR46/Y121F/T289A have been dominant among azole-R A. fumigatus in azole naïve patients and environmental samples. These two resistance genotypes have been sporadically found in clinical and environmental samples in Denmark, but the size of the problem remains unknown. The purpose of this study was to establish a nationwide surveillance programme of azole-R Aspergillus in Denmark and to report the data from the first six month’s study period.
    Methods: Unique Aspergillus isolates were included from all Danish clinical microbiological departments in the period October-2018 to March-2019. Isolates from same patients were defined as unique if 1) found >30 days apart, 2) confirmation of another species or 3) different susceptibility pattern. Inclusion criteria were: a) Aspergillus isolates regarded clinically significant, or b) any Aspergillus isolate detected on a Monday regardless of clinical significance. Referral practices varied depending on local mycology skills. Most laboratories referred all moulds or all Aspergillus isolates which then underwent species ID and susceptibility testing at the reference lab (SSI). One laboratory (Aarhus University Hospital) reported the species identification for Aspergillus (n = 214) and only referred part of the isolates. Thus susceptibility was performed for 46/138 A. fumigatus isolates from AUH at the time of writing. Referred Aspergillus isolates were identified to the complex level except A. fumigatus which was identified sensu stricto using thermotolerance testing. A. fumigatus isolates underwent screening for azole-R following the EUCAST E.Def 10.1 method and using VIPcheck azole agar plates (Mediaproducts BV, Grönningen, NL). Screening positive A. fumigatus isolates and all non-fumigatus Aspergillus isolates underwent EUCAST E.Def 9.3.1 susceptibility testing. Isolates were deemed intermediate when classified as such for >1 azole. Isolates with azole MIC(s) above the EUCAST BP(s) underwent CYP51A sequencing. Duplicates within 30 days were excluded.
    Results: A total of 695 Aspergillus isolates were included of which 503 were A. fumigatus. Susceptibility testing was performed for 411 A. fumigatus isolates from 319 patients at time of writing (Table 1). For A. fumigatus, 92.7% isolates were azole susceptible, 1.7% intermediate and 5.6% azole-R. Nineteen out of 319 patients harboured an A. fumigatus which was azole-R (6%). Among these 18/19 (95%) harboured an isolate with a CYP51A mutation (Table 2), and 14/19 (73.7%) a CYP51A mutation derived from the environment. Furthermore, 2/9 patients harboured three azole resistant A. terreus isolates, each with a CYP51A mutation (Table 2).
    Jof 05 00095 i021Jof 05 00095 i022
    Conclusion: We report a nationwide azole-R rate of 6% in A. fumigatus at the patient level. The underlying resistance mechanisms was target gene mutations in all but one case and notably, the vast majority were of environmental origin linked to the use of azole fungicides. The fact that such isolates have increasingly been found in Denmark since 2009 is concerning and suggests that a one-health approach involving human and environmental azole management is necessary to limit further rise in azole-R A. fumigatus

    P019. Multicentre study to determine the Etest® epidemiological cut-off values of antifungal drugs in Candida spp. and Aspergillus fumigatus SC

    M. Salse 1, J.P. Gangneux 2, S. Cassaing 3, L. Delhaes 4, A. Fekkar 5, D. Dupont 6, F. Botterel 7, D. Costa 8, N. Bourgeois 9, B. Bouteille 10, S. Houzé 11, E. Dannaoui 12, H. Guegan 2, E. Charpentier 3, F. Persat 13, L. Favennec 8, L. Lachaud 9 and M. Sasso 1
    1
    Laboratoire De Microbiologie, CHU Nîmes, Nîmes, France
    2
    Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France
    3
    , CHU de Toulouse, Toulouse, France
    4
    Laboratoire De Parasitologie-mycologie, CHU de Bordeaux, Bordeaux, France
    5
    Laboratoire De Parasitologie-mycologie, APHP La Pitié Salpétrière, PARIS, France
    6
    Laboratoire De Parasitologie-mycologie, Hospices civils de Lyon, Lyon, France
    7
    Laboratoire De Parasitologie-mycologie, APHP Hôpital Henri Mondor, Créteil, France
    8
    Laboratoire De Parasitologie-mycologie, CHU de Rouen, Rouen, France
    9
    Laboratoire De Parasitologie-mycologie, CHU de Montpellier, Montpellier, France
    10
    Laboratoire De Parasitologie-mycologie, CHU de Limoges, Limoges, France
    11
    Laboratoire De Parasitologie-mycologie, APHP Hôpital Bichat, Paris, France
    12
    Laboratoire De Parasitologie-mycologie, APHP Hôpital Pompidou, Paris, France
    13
    Parasitologie Mycologie Médicale, Hospices Civils de Lyon, Lyon, France
    Objectives: To determine the specific Etest®-based epidemiological cut-off values (ECVs) for the main antifungal agents used in clinical practice, against the most frequent Candida spp. and Aspergillus fumigatus species complex (SC), in order to harmonise the interpretation of the Etest® minimum inhibitory concentrations (MICs) results in daily routine.
    Methods: For each antifungal agent, the Etest® MICs in Candida spp. and A. fumigatus SC isolated from routine specimen cultures were retrospectively collected from 2004 to 2018 from the laboratories of 12 French university hospitals. To ensure that robust and comparable data were included for the ECV estimation, basic requirements and criteria have been fulfilled during the selection of MIC data: aberrant individual distributions were excluded, MIC data were pooled only if there are generated by at least three independent laboratories, and a minimum of 100 MIC values were required after data pooling for each species-antifungal agent combinations. The ECVs were then calculated using the iterative statistical method with a 97.5% cut-off.
    Results: Forty-eight Etest® ECVs were determined for amphotericin B, caspofungin, micafungin, anidulafungin, fluconazole, voriconazole, posaconazole and itraconazole, after pooling and analysing the MICs of 9,654 Candida albicans, 2,939 Candida glabrata SC, 1,458 Candida parapsilosis SC, 1,148 Candida tropicalis, 575 Candida krusei, 518 Candida kefyr, 241 Candida lusitaniae, 131 Candida guilliermondii, and 1,526 A. fumigatus SC isolates. The ECV results were identical (17/32 ECVs) or within one two-fold dilution (15/32 ECVs) to the previously reported Etest®-based ECVs. They were comparable to the Clinical and Laboratory Standards Institute (CLSI) ECVs in 76.1% (similar or within one two-fold dilution: 35/46 ECVs) and strictly identical in 39.1% of cases (18/46 ECVs). They were comparable to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) ECVs in 81.2% of cases (similar or within one two-fold dilution: 26/32 ECVs) and strictly identical in 28% (9/32 ECVs).
    Conclusion: On the basis of these and other previous results, we recommend the determination of method-dependent ECVs. As Etest® is the most widely used commercial method in French laboratories, the goal of this study was to calculate specific ECVs for this method and harmonise the interpretation of the Etest® MICs results in daily routine. Etest® ECVs should not be used in place of breakpoints but may identify non-WT isolates with a potential resistance to antifungal agents and suggest that an isolate may not respond as expected to the standard treatment.

    P020. Human cell cultures exposed to antibiotic amphotericin B: toxicity and defence

    E. Grela 1, M. Piet 2, R. Luchowski 1, W. Grudzinski 1, R. Paduch 2 and W. Gruszecki 1
    1
    Biophysics, Maria Curie-Sklodowska University, Lublin, Poland
    2
    Virology and Immunology, Maria Curie-Sklodowska University, Lublin, Poland
    Objectives: Amphotericin B (AmB) is a polyene antifungal antibiotic, which is synthetized by the bacteria Streptomyces nodosus. AmB is a “gold standard” in treatment of systemic fungal infections, due to its broad spectrum of action, high effectiveness and rare pathogens resistant to this drug. Unfortunately due to the low selectivity of the action of this antibiotic, it is very toxic for patients. Although, the exact mechanism of action of AmB is still unknown, it is used as a lifesaving medicine. Understanding the mechanism of action of AmB would enable to minimize its toxic side effects, and possibly increase its therapeutic effects. The aim of this study was to get to know the toxicity mechanism of action of AmB.
    Methods: To understand molecular mechanism underlying the toxicity of AmB, human normal colon epithelial cells CCD 841 CoTr (ATCC No. CRL-1807) and human colon adenocarcinoma cells HT-29 (ATCC No HTB-38) were exposed to the drug and were imaged using Fluorescence Lifetime Imaging Microscopy (FLIM) and Raman scattering spectroscopy. Cultures were incubated for 24 h with AmB. Fluorescence lifetime imaging was carried out on MicroTime 200 (Picoquant GmbH, Germany) linked with Olympus IX71 inverted microscope. The cell samples were excited by 405 nm pulsed laser. Fluorescence lifetimes and fluorescence anisotropy values were analyzed. Raman spectral analysis and imaging on a microscale was carried out with inVia Reflex confocal Raman microscope (Renishaw, UK) with Cobolt o8-NLD 405 nm laser (power at a sample 0.2 mW). Obtained results were analyzed by DCLS spectral deconvolution.
    Results: The results confirmed high toxicity of antibiotic AmB for both human cell lines but lower for HT-29 cells. The results show that AmB incorporates to the lipid membrane containing cholesterol. The cell defense mechanism against the action of antibiotic was also observed, which is based on removing AmB from the membrane in the form of sterol-deficient exosomes.
    Conclusion: The lower toxicity of AmB observed for HT-29 cells, may be associated with high proliferation rate or different membrane composition of adenocarcinoma cells compared to CCD 841 CoTr cells. AmB binds to the bilayer in the form of small aggregates, which can cause uncontrolled ion-leakage and lead to the cell death.
    Jof 05 00095 i023

    P021. African ST173 Cryptococcus deuterogattii strains are commonly less susceptible to fluconazole: a tricky mechanism of resistance

    V. Bellet 1, F. Roger 1, T. Gouveia 1, P. Drakulovski 1, F. Kassi 2, H. Menan 2, D. Krasteva 1 and S. Bertout 1
    1
    Laboratoire De Parasitologie Et Mycologie Médicale Umi 233, University of Montpellier, Montpellier, France
    2
    CEDRES CHU de Treichville, Abidjan, Côte d’Ivoire
    Objectives: Fluconazole, alone or in association, is often administered during cryptococcosis treatment especially in Sub-Saharan Africa. Its intensive use leads to the emergence of resistant strains. The mechanisms underlying resistance are poorly documented for yeasts belonging to the C. gattii species complex. Literature suggests that resistance could be due to mutations and/or surexpression of ERG11 gene and overexpression of efflux pumps, like MDR or AFR.
    Methods: In Ivory Coast, we highlighted the presence of strains with a rare subtype in the molecular type VGII associated with high minimum inhibitory concentrations to FCZ compared to VGII strains originating from the Pacific Northwest. We investigated the mechanisms of fluconazole resistance of 18 ivorian clinical strains of C. deuterogattii with high MIC to FCZ compared to 10 strains with low MIC to FCZ.
    Results: We demonstrated that (i) these strains exhibited no mutation in the ERG11 gene, (ii) some strains had an increased in ERG11 and MDR1 mRNA expression while AFR1 and AFR2 were not overexpressed in high MIC to FCZ strains versus the low MIC to FCZ strains and (iii) exposure to FCZ in strains with high MIC to FCZ induced AFR1 mRNA overexpression.
    Conclusion: We demonstrated that the FCZ-resistant mechanism commonly described in Cryptococcus neoformans or in Cryptococcus gattii from the Pacific Northwest were not responsible for the resistance to FCZ in this rare sub-type strains.

    P022. Novel Technique to Measure Susceptibility to Lipid-Associated Antifungals

    M. Frenkel 1, Y. Yunik 1 and E. Segal 2
    1
    Tel Aviv University, Tel Aviv, Israel
    2
    Sackler School of Medicine, Clinical Microbiology and Immunology, Tel Aviv University, Tel Aviv, Israel
    Objectives: Lipid associated antifungals, such as polyenes, are extensively used for therapy in patients with invasive fungal infections. Thus, susceptibility of fungi to the lipid- associated antifungals is of importance. The most reliable method used in susceptibility testing is the broth micro-dilution technique. However, visual- or spectral- assessment of the antifungal activity is difficult, and possibly unreliable, in lipid-associated drugs, due to turbidity caused by the lipid. Hence, the objective of this study was to adapt a method for spectral measurement of susceptibility of fungi to lipid-associated antifungal drugs.
    Methods: The technique was adapted for Amphotericin B- Intralipid (AMB-IL) and Nystatin- Intralipid (NYT-IL) in comparison to AMB and NYT. Drug preparation: AMB-IL was prepared by adding to 5 mg/ml of AMB 20% Intralipid in DDW to a final concentration of 0.2 mg/ml AMB and shaken at 280 rpm for 18 h at 240 C. Intralipid in DDW was used as control. NYT-IL was prepared similarly, by adding to 20 mg/ml nystatin 20% Intralipid in DMSO to a final concentration of 0.8 mg/mL NYT and shaken at 280 rpm for 18 h at 240 C. Intralipid in DMSO was used as control. Susceptibility test: Susceptibility of the Candida albicans strains to AMB, NYT, AMB-IL, and NYT-IL was assessed by the micro-broth dilution assay using micro-titer plates. Susceptibility was evaluated using spectral readings in an EMax+ Microplate Reader at 560nm and 650nm, for the standard (AMB, NYT) and experimental Intralipid-associated polyene formulations (AMB-IL, NYT-IL), respectively. For AMB-IL or for NYT-IL the drug solutions and the relevant controls were diluted in RPMI. Each well contained the same concentration of RPMI, IL, DDW or DMSO, and serial dilutions of the antifungal drugs (AMB, NYT).
    Results: The validity of the test was evaluated by testing susceptibility of 40 C. albicans isolates: 20 from human mucosal infections (M strains) and 20 from candidemia patients (S strains) vs. a control strain C. albicans CBS-562, for which susceptibility data were available. We determined 50, 90 & 100% MIC values by spectral readings (see an example of tests with the control stain – CBS 562–Figure 1). The spectral readings were compared to visual readings of the micro-titer plates using an inverted microscope. In addition, minimal fungicidal concentration (MFC) for each strain was determined (Figure 2).
    Jof 05 00095 i024
    Figure 1: Spectral readings of Control strain –CBS 562 treated with AMB-IL & NYT-IL.
    Jof 05 00095 i025
    Figure 2: Spectral readings vs. Visual assessment of M and S Candida albicans strains.
    Conclusion: This study presents a spectral method for measuring susceptibility of yeasts to lipid associated polyenes which deals with the difficulties of visual assessment of susceptibility to lipid associated antifungals.

    P023. Antifungal activity of antimicrobial peptides against clinical and environmental Aspergillus fumigatus isolates.

    E. Ballard 1, R. Yucel 2, W. Melchers 3, A. Brown 1, P.E. Verweij 3 and A. Warris 1
    1
    Mrc Centre For Medical Mycology, University of Aberdeen, Aberdeen, United Kingdom
    2
    Ifcc, University of Aberdeen, Aberdeen, United Kingdom
    3
    Medical Microbiology, RadboudUMC, Nijmegen, Netherlands
    Objectives: Antimicrobial peptides (AMPs) are a key defence against invading microorganisms. The activity of AMPs against human fungal pathogen Aspergillus fumigatus remains poorly understood. We characterised the anti-Aspergillus activity of specific human AMPs and determined whether in-host adaption could lead to resistance to specific AMPs.
    Methods: The antifungal activity of lysozyme, histones, lactoferrin, β-defensin-1 and LL-37, was tested against 15 clinical isolates (from patients with cystic fibrosis, chronic and acute invasive aspergillosis), 5 environmental isolates and 13 isogenic A. fumigatus isolates obtained from a single patient over 2 years showing in-host acquired azole resistance. Hyphae grown from 5x104 conidia and incubated with individual AMPs at various concentrations for 2 h at 37oC, after which metabolic activity was determined by XTT. Aspergillus conidia (1x105 per well) were incubated for 10 h at 37oC with specific AMPs and fixed in 4% paraformaldehyde fixing solution. Analysis of hyphal length was performed using the Amnis ImageStreamX MK II Imaging Flow Cytometer (Luminex, USA). Data analysis was performed using IDEAS software.
    Results: Dose-dependent decreases in hyphal metabolic activity in all isolates were shown for lysozyme (20-80 µM; 31-65% and 5-25% decrease respectively; p<00001) and histones (50-100 ug/ml; 35-66% and 6-45% decrease respectively; p<0.0001). No effect was observed for lactoferrin (10-40 µM), β-defensin-1 (1-10 µM) and LL-37 (5-50 µM). Imaging flow cytometry revealed that incubation with histones (100 µg/ml), β-defensin-1 (10 µM) or lactoferrin (40 µM) resulted significantly fewer cells in the hyphae gate (p<0.0001, p = 0.04 and p = 0.003, respectively). No significant differences were observed with lysozyme (80 µM) or LL-37 (12.5 µM).
    Conclusion: Lysozyme and histones show dose dependent antifungal activity against A. fumigatus hyphae, irrespective of the isolate’s azole resistance profile. In addition, histones, lactoferrin and β-defensin-1 display inhibitory activity against germinating conidia. No differences were observed in the susceptibility to AMPs between the groups of A. fumigatus isolates used. We did not identify any trends indicating increased resistance to AMPs as a result of in-host adaptation.

    P024. Characterization of I1380T a new FKS1 polymorphism in Candida parapsilosis that confers high-level echinocandin resistance when associated to P660A

    I. Accoceberry 1, S. El-Kirat-Chatel 2, F. Quilès 2, N. Biteau 1 and T. Noël 1
    1
    Cnrs Umr5234, University of Bordeaux, Bordeaux, France
    2
    Cnrs Umr 7564, Université de Lorraine, Villers-lès-Nancy, France
    Objectives: The aim of this study was to determine the impact on echinocandin resistance of a new mutation identified in a FKS1 allele of a clinical isolate of C. parapsilosis.
    Methods: To assess the role of mutations potentially implicated in echinocandin resistance, a C. lusitaniae strain specially engineered for the expression of a single chromosomal copy of FKS1 was used for the expression of I1380T and P660A FKS1 alleles. The echinocandin susceptibility of the mutants, as well as their growth rates and phenotypes of interaction with murine macrophages, were evaluated in the absence or in the presence of caspofungin. Atomic force microscopy (AFM) was used to observe the morphological changes induced by caspofungin exposition. Finally, infrared spectroscopy in attenuated total reflection mode (IR-ATR) spectroscopy was used to detect and quantify the biochemical signatures of the cell wall before and after caspofungin treatment.
    Results: A caspofungin-resistant C. parapsilosis bloodstream isolate was recovered from a patient who underwent allogeneic bone marrow hematopoietic stem-cell transplantation (HSCT) for an idiopathic medullary aplasia with positive PNH clone. Full-length nucleotide sequencing of the FKS1 gene showed that the strain harboured the heterozygous mutation I1380T (isoleucine at position 1380 replaced by threonine) located 4 amino acids beyond the HS2 region (amino acids 1369 to 1376), compared to Fks1p of the C. parapsilosis reference strain CBS 604, in addition to the naturally occurring P660A polymorphism responsible for increased echinocandin MICS in this species. To characterize the impact of each mutation on caspofungin resistance, FKS1 alleles bearing I1380T associated or not to P660A were genetically engineered and expressed in Candida lusitaniae. When compared to a wild type allele bearing no mutation, I1380T conferred a 12-fold MIC increase, P660A a 6-fold MIC increase and the combination of both mutations a ≥ 256-fold MIC increase. Measuring the cellular interactions between C. lusitaniae FKS1 mutants and murine macrophages showed that, in the presence of caspofungin, I1380T enhanced yeast phagocytosis but had no effect on intramacrophagic yeast survival rates. AFM analysis of FKS1 mutants showed that caspofungin treatment impacts the cell surface topography in both susceptible and resistant strains, and that the single I1380T mutation leads to morphological changes even in the absence of caspofungin treatment. Infrared spectroscopy in attenuated total reflection mode (IR-ATR) showed that under high drug concentration exposure, the FKS1 I1380T mutants exhibited a massive β-glucan synthesis inhibition that was not proportionally compensated by chitin synthesis.
    Conclusion: We have identified I1380T, a new mutation located close to the HS2 region of the FKS1 gene of a clinical isolate of C. parapsilosis. We demonstrated that the high caspofungin MIC for this isolate was depending on the combination of I1380T with the naturally occurring P660A polymorphism. Further characterization at the cell wall level suggested a potential fitness cost caused by I1380T.

    P025. Azole resistant Aspergillus fumigatus: surveillance from flowered patios to departments with at-risk patients

    C. Godeau, E. Scherer, A. Laboissière, C. Léchenault-Bergerot, G. Reboux, L. Millon and S. Rocchi
    Mycology - Umr6249 Cnrs Chrono-environnement, University Hospital Besançon, Besançon, France
    Objectives: The University Hospital of Besançon is geographically located in a highly agricultural area (wood, vegetables, cereals, fruit and vines), and is very concerned by the emergence of azole resistance in Aspergillus fumigatus (ARAf), having an increasing number of cases especially amongst cystic fibrosis patients. For more than 20 years, the University Hospital of Besançon has been taking weekly samples (n = 25) to assess fungal air contamination in the 3 departments of hematology and corridors. Since 2015, the number of ARAf found in corridors has been increasing. This study aimed to determine whether plant, tree and flowerbeds within or near the hospital are sources of ARAf and to assess their spread into the hospital.
    Methods: Four soil sampling areas within the hospital and in the surrounding environment have been identified. Zone A corresponds to an outdoor relaxation area within the hospital, zone B consists of flowerbeds in front of the hospital entrance, zone C (inside) corresponds to pots containing trees below the hospital reception area and finally zone D corresponds to an outdoor area at level -1 below the zone A. From January 2019, external samples were taken using a homemade dust vane sensor equipped with 2 wipes, placed on a terrace at second floor, to capture the prevailing wind. Dust aspiration was also achieved using a filter placed at the end of a vacuum cleaner, towards the emergency door of the department of hematology, in the corridor along zone A, then in hematology consultation department. Soils and dust (wipes and aspiration) samples were seeded on medium containing itraconazole and voriconazole (2mg/L). The usual weekly air samples taken in the hematology departments and corridors from January 2019 were also analyzed. Resistance of A. fumigatus growing on azole media was tested using EUCAST method and cyp51A gene of ARAf were sequenced.
    Results: From the 85 soils samples, 137 colonies were isolated on azole medium from zones A, B and D, grew on azole media. Resistance of 90 A. fumigatus isolates was evaluated with EUCAST method. Other strains (zone B) were Neosartorya fisheri. Moreover, 71% (64/90) of A. fumigatus isolates were resistant to at least one azole. So far, 20 strains have been sequenced and all harbored the TR34/L98H mutation. 59 ARAf were found in pots containing tulips coming from Netherlands and 5 in soil pots containing trees. Three isolates were detected on azole media with dust vacuum and 2 (hematology department and corridor along zone A) were resistant with TR34/L98H mutation. No resistant strains were collected with the external sensor. Four ARAf with the TR34/L98H mutation were also isolated from air of corridors.
    Conclusion: Patios containing Dutch tulips in particular, had a significant number of ARAf. On the other hand, samples taken upstream of the esplanades, in relation to the prevailing wind, did not present any ARAf. Addionally, ARAf were also found in hospital corridors and to emergency exits, but not in hematology departments. Genotyping analyses will be carried out to confirm that tulips can be a source of ARAf found inside the hospital.

    P026. Triazole-resistant Aspergillus fumigatus: Evaluation of the Fungiplex® Aspergillus and Azole-R kits

    S. Rocchi, E. Scherer, A. Laboissière, C. Léchenault-Bergerot, C. Godeau, A.-P. Bellanger, G. Reboux and L. Millon
    Mycology - Umr6249 Cnrs Chrono-environnement, University Hospital Besançon, Besançon, France
    Objectives: Cases of Aspergillosis with azole resistant Aspergillus fumigatus are increasingly described. Some resistance mechanisms, with two major mutations on the cyp51A gene (TR34/L98H and TR46/Y121F/T289A), are linked to the unintended impact of triazole fungicides in agriculture. At the University Hospital of Besançon, 216 resistant strains have been isolated since 2012 and 142 sequenced for beta-tubulin: 52% from professional environments, 28% from clinical samples, 10% from hospital corridors, 7% from the inside garden of the Hospital and 3% from patients’ homes. Given the seriousness of resistant infections, it is important to detect cases of resistance as soon as possible. It is in this context that we proposed to test to Fungiplex ® (Aspergillus et Azole R) kits (Bruker).
    Methods: The Fungiplex® Aspergillus Azole-R kit was retrospectively evaluated on DNA extracts from a collection for which cyp51A gene sequencing and genotyping was previously performed. According to the instructions, the Azole-R kit is made for the identification of azole resistance markers in Aspergillus positive species directly from serum, plasma and bronchoalveolar lavage. We are evaluating the kits for the routine testing of cystic fibrosis patients working from isolated strains and directly on sputum. We also are testing the performance of the kits to detect 1) Aspergillus fumigatus strains 2) resistant strains directly in soil samples. To do this, we worked on inoculated soils with increasing concentrations of resistant strains (from 5 to 50 000 spores/mL with 200µL sprayed on 250 mg of soil) and directly on soils containing resistant strains.
    Results: 97 resistant strains previously characterized by sequencing techniques (beta-tubulin and cyp51A) were analyzed: 82 TR34/L98H, 2 TR34/L98H/S297T/F495I, 9 TR46/Y121F/T289A and 4 other resistant strains. Results showed that 99% of strains carrying one of the two mutations are detected. The first prospective results (22 resistant strains in Etest, analysed directly with the kits) made it possible to detect 22 A. fumigatus with the TR34/L98H mutation. Verification sequencing is in progress. Sputum analysis and tests with cryptic species are also in progress. Tests on inoculated soils show that resistant strains can be detected even for the smallest inoculated quantities (200 µL of solution concentrated at 5 spores/mL). Real soil tests provide a signal for Aspergillus but not for the TR34 and TR46 mutations. Internal reaction controls are also not detected, suggesting a problem with DNA extraction and inhibitors rather than poor kit performance. Different methods for extracting DNA directly from the soil are under evaluation.
    Conclusion: The kits show ease of use and very good reliability on retrospective analyses with 99% of the strains tested validated. The performance of the Aspergillus kit will also be evaluated on cryptic species of A. fumigatus (Neosartorya fisherii, A. oerlinghausenensis, Neosartorya hiratsukae, A. lentulus). If performance is equally good in ongoing prospective analyses, these kits will be promising to improve early diagnosis.

    P027. Relation between hanseniasis and superficial mycoses: prevalence, fungal identification and antifungal susceptibility.

    M.L. Scroferneker 1, D. Heidrich 2, R. Vettorato 2,3, L. Eidt 4, A. Carvalho Ribeiro 1, D. Pagani 5, A.H. Da Silva Hellwig 2 and T. Amaro 3
    1
    Department of Microbiology, Immunology and Parasitology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
    2
    Postgraduate Program In Medicine: Medical Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
    3
    Serviço de Dermatologia do complexo Hospitalar Santa Casa de Misericórdia de Porto Alegre, Porto Alegre, Brazil
    4
    Ambulatório de Dermatologia Sanitária de Porto Alegre, Porto Alegre, Brazil
    5
    Postgraduate Program In Agricultural and Environmental Microbiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
    Objectives: The aim of this study was to evaluate superficial mycoses in patients with hanseniasis in relation to the prevalence of fungal species causing mycoses and susceptibility to antifungals.
    Methods: A cross-sectional study was carried out with patients who were seen between May and October 2017 at the Hanseniasis Service of the Sanitary Dermatology Ambulatory in the city of Porto Alegre, Brazil. The samples collected were submitted to direct (DME) and cultural (CME) mycological examinations at the G post of the Santa Clara Hospital in Porto Alegre, were identified by region sequencing specified for each genus of fungus and traced sensitivity profile to clinical antifungals using protocols M38-A2 and M27-A3 from the Clinical and Laboratory Standards Institute.
    Results: A total of 91 hanseniasis patients were evaluated and 37 presented suspicion mycosis. of these, 23 had DME positive and 14 CME cultures were identified, with eight dermatophytes (seven Trichophyton interdigitale and one Epidermophyton floccosum), two isolates of Fusarium keratoplasticum, two Acremonium sp., one Candida albicans and one Arthrinium arundinis. Three patients had claws, caused by hanseniasis, at the mycosis site, indicating a relation of the two diseases (3/5 patients with claw). Terbinafine presented the lowest minimum inhibitory concentrations (MICs) for dermatophyte isolates (0.0078-0.06 μg/ml), while fluconazole MICs were the highest (4-> 64μg/ml). Fusarium keratoplasticum and Acremonium sp. have higher MICs than all antifungal agents than dermatophytes.
    Conclusion: This is the second onychomycosis caused by Arthrinium arundinis from the literature and showed low sensitivity to antifungals. Itraconazole had higher MICs for dermatophytes isolated from patients with hanseniasis (0.25-1μg/ml) than without the disease cited in the literature, indicating a relation of susceptibility to antifungals between mycosis and hanseniasis.

    P028. Prevalence of azole-resistant Aspergillus fumigatus isolates from respiratory specimens of patients from Lyon and exploration of resistance mechanisms

    L. Simon 1, R. Kramer 2, D. Dupont 3, T. Déméautis 4, H. Garnier 5, B. Lina 6, M. Rabodonirina 7, M. Wallon 3, F. Persat 8 and J. Menotti 4
    1
    Parasitology and Medical Mycology, Hospices Civils de Lyon / CHU de Nice, Nice, France
    2
    Parasitology and Medical Mycology, and Virology, Hospices Civils de Lyon / EUPHEM, Lyon, France
    3
    Parasitology and Medical Mycology / Umr 5292, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France
    4
    Parasitology and Medical Mycology / Ea7426 Inflammation and Immunity of The Respiratory Epithelium, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France
    5
    Parasitology and Medical Mycology, Hospices Civils de Lyon, Lyon, France
    6
    Virology / U1111, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France
    7
    Parasitology and Medical Mycology / U1111, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France
    8
    Parasitology and Medical Mycology, Université Lyon 1 / Hospices Civils de Lyon, Lyon, France
    Objectives: Resistance of Aspergillus fumigatus strains to triazole antifungals is increasingly reported in Europe. The main mechanisms of resistance described are mutations in the cyp51A gene encoding a lanosterol 14-α-demethylase or its promoter. As few data are available in Southern France, our objective was to assess the burden of Aspergillus isolates with azole resistance from clinical specimens in Lyon University Hospitals
    Methods: In this retrospective cross-sectional study, 203 consecutive A. fumigatus isolates were identified during a seven-month period from February to September 2017 from respiratory specimens from 182 patients attending the inpatient and outpatient wards of the Pulmonary Medicine Departments of Lyon University Hospitals. Morphological identification was confirmed by sequence analysis of the β-tubulin gene. Minimum inhibitory concentrations were determined using E-test reagent strips for itraconazole, voriconazole, posaconazole, and isavuconazole. Resistance was defined according to the 2018 EUCAST clinical breakpoints. The molecular resistance mechanisms were searched for by sequence analysis of the cyp51A gene and its promoter region, as well as by gene expression analysis of the cyp51 genes and genes encoding several efflux transporters.
    Results: PCR and sequence analysis of the β-tubulin gene confirmed the identification of Aspergillus fumigatus for the 203 isolates. Four isolates presented with azole resistance: two isolates against itraconazole/posaconazole/isavuconazole and another two against all four triazoles. Out of these four strains, three presented silent polymorphisms in an intronic part of cyp51A and one presented simultaneously the F46Y, M172V and E427K mutations. No mutation was identified in the cyp51A promoter, but significant inductions of cyp51A and cyp51B gene expression were observed for all four isolates and three isolates, respectively. Significant inductions of atrF and crd1B gene expression were observed for two and three isolates, respectively. No significant induction of MDR1-4, MFS56 and M85 gene expression was observed.
    Conclusion: The prevalence of azole resistance in our study population was 2.2% (95% CI 0.9-5.6). Only polymorphisms were found in the cyp51A gene and no mutation was found in its promoter. Nevertheless, significant inductions of the expression of the cyp51 genes and two genes encoding efflux transporters were evidenced, underlying the diversity of resistance mechanisms to be explored.

    P029. Antifungal activity of a mastoparan analog peptide: mode of action and in vivo evaluation in a Galleria mellonella model

    J. Singulani 1, M.C. Galeane 1, M. Dorisse Ramos 1, P.C. Gomes 1, C. Santos 1, B. Monson De Souza 2, M.S. Palma 2, A.M. Fusco Almeida 1 and M.J. Soares Mendes Giannini 1
    1
    Clinical Analysis, UNESP, Araraquara, Brazil
    2
    Biology, UNESP (Sao Paulo State University), Rio Claro, Brazil
    Objectives: The purpose of this work was to evaluate the antifungal activity and toxicity of a peptide analog of mastoparan class from wasps (MK58911) under both in vitro and in vivo conditions.
    Methods: Firstly, MK58911 was tested against Cryptococcus neoformans ATCC 90112 using the microdilution susceptibility test according to the CLSI M27-A3 (2008). In addition, the mechanism of action on fungal membrane, death cell events (apoptosis and necrosis) and reactive oxygen species (ROS) was evaluated by flow cytometry. Secondly, the peptide toxicity tests on lung fibroblasts (MRC5) and glioblastoma cells (U87) were performed. Finally, the efficacy and toxicity of the peptide were evaluated in vivo using a Galleria mellonella model.
    Results: The results showed a promising activity of the peptide with minimal concentration inhibitory (MIC) of 31.2 μg/mL and low toxicity in MRC and U87 cells (IC50 > 500 μg/mL). Taken together, these results demonstrated that the peptide has a high selectivity by fungal cells compared to mammal cells (selectivity index - SI > 16). The peptide presented a mechanism of action on plasma membrane, leading to death of the fungus mainly by the necrosis process and without production of reactive oxygen species. Finally, the peptide showed no toxic effects on G. mellonella and significantly enhanced the survival rates and decreased the fungal burden of larvae infected with C. neoformans. These effects were independent of an immunomodulatory activity.
    Conclusion: The results showed the peptide as a potential new antifungal drug against cryptococcosis. "This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP grants no. 2017/06658-9 and 2016/16212-5)."

    P031. Screening for Triazole resistance in clinically significant Aspergillus species from Pakistan

    A. Zafar, S. Moin, K. Jabeen, J. Farooqi and S. Laiq
    Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan
    Objectives: To determine the frequency of triazole (itraconazole, voriconazole, posaconazole) resistance in clinically significant Aspergillus species isolated at a tertiary care centre, Karachi, Pakistan.
    Methods: A descriptive cross-sectional study was conducted in the Department of Pathology and Laboratory Medicine, Microbiology Section of the Aga Khan University Clinical Laboratories, Karachi, Pakistan from September 2016 to May 2019. One hundred and fourteen, clinically significant Aspergillus isolates [A.fumigatus (38; 33.3%), A.flavus (64; 56.1%), A.niger (9; 7.9%) and A.terreus (3; 2.6%)] were included in the study. They were assessed for their clinical significance. The clinical spectrum ranged from Invasive Aspergillosis (IA) (n = 25; 21.9%), further divided into proven invasive extrapulmonary aspergillosis (n = 8; 7%), proven invasive pulmonary aspergillosis (IPA) (n = 6; 5.3%), putative/probable IPA (n = 11; 9.6%) according to the AspICU and 2008 EORTC criteria, to Chronic Pulmonary Aspergillosis (CPA) (n = 58; 50.9%), Allergic Bronchopulmonary Aspergillosis (ABPA) (n = 4; 3.5%), Severe Asthma with Fungal Sensitization (SAFS) (n = 4; 3.5%) and saprophytic tracheobronchial aspergillosis (n = 23; 20.2%). Screening for triazole resistance was performed by antifungal agar screening method as described by Mortenssen et al. The minimum inhibitory concentration (MIC) of 41 representative isolates [A.flavus (n = 15; 13.2%), A.fumigatus (n = 15; 13.2%), A.niger (n = 8; 7%), A.terreus (n = 3; 2.6%)] representing a clinical spectrum of extrapulmonary IA (n = 7; 6.1%), IPA (n = 4;3.5%) putative/probable IPA (5; 4.4%), CPA (n = 18;15.8%), ABPA (n = 3; 2.6%), SAFS (n = 1; 0.9%), and saprophytic tracheobronchial aspergillosis (n = 3; 2.6%) were tested according to the CLSI broth microdilution method.
    Results: All the isolates were categorized as triazole-susceptible based on the triazole antifungal agar screening. The MICs of the three azole antifungals for 41 representative isolates tested were found to be ≤1 ml/L and hence according to CLSI breakpoints, all the isolates tested were found to be triazole-susceptible. The MIC 90 of itraconazole, voriconazole and posaconazole of the representative aspergillus isolates was 1mg/L, 1mg/L and 0.5mg/L respectively.
    Conclusion: This study may set precedence for future periodic antifungal resistance surveillance studies in our region on Aspergillus isolates causing invasive disease, as well as other syndromes requiring long term antifungal therapy.

    P032. Antifungal susceptibility profile of invasive Candida glabrata isolates (2009-2019) from a tertiary care hospital laboratory in Pakistan

    J. Farooqi 1, S. Memon 2, S. Laiq 1, F. Naqvi 1, A. Zafar 1 and K. Jabeen 3
    1
    Pathology and Laboratory Medicine, Aga Khan University, Karachi-KHI, Pakistan
    2
    Microbiology, Karachi University, Karachi, Pakistan
    3
    Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan, Pakistan
    Objectives: Candida glabrata invasive infections are increasingly associated with antifungal resistance and poor clinical outcomes. The objective of this study was to evaluate antifungal resistance and distribution of minimum inhibitory concentrations (MICs) against invasive C. glabrata isolates (200-2019) at Aga Khan University (AKU) at Karachi, Pakistan. This laboratory, through its network of satellite collection centers receives specimens from more than 100 major cities and towns of the country.
    Methods: 162 Candida glabrata strains were isolated from blood (101), body fluids (26), pus and wounds (21) tissue (4) and others (10). Isolates were identified by conventional method using API 20C AUX, gross morphology on chromogenic and microscopic morphology on corn meal agar. MICs were determined using colorimetric broth microdilution (YeastOne Sensititre, Trek diagnostics). Susceptibilities were interpreted according to Clinical Laboratory standard Institute breakpoints mentioned in “Performance Standard for Antifungal Susceptibility Testing of Yeasts M60-ED1:2017”.
    Results: Out of 1917 archived invasive candida, 162 (8.4%) were C.glabrata 76% of these isolates were from patients admitted at AKU. Male to female ratio was 1.4 and 62% of the isolates were from ages 18-64 year. MIC90 of these strains against fluconazole was 64 µg/ml, voriconazole 2µg/ml, itraconazole 1µg/ml and posaconazole 2µg/ml. Among echinocandins, MIC90 was 0.12µg/ml for caspofungin, 0.06µg/ml for anidulafungin and 0.03µg/ml for micafungin. MIC90 for amphotericin B was 0.5µg/ml.
    Conclusion: Antifungal sensitivity testing for invasive candidiasis is essential in the face of emerging resistance as empiric therapy may not have reliable outcomes. Surveillance data of antifungal resistance among common Candida species with potential for developing antifungal resistance, like C. glabrata, should be monitored closely for identifying resistant strains in circulation.

    P033. Clinical implications of azole-resistant vs. azole-susceptible invasive aspergillosis in hematological malignancy (CLARITY) – a multicenter study

    D. Seidel 1, O.A. Cornely 2, P. Köhler 1,3, J. Meis 4, M. Zarrouk 3, J. Salmanton-Garcia 3, D. Arenz 5, O. Blennow 6, D. Buchheidt 7, J.-J. Vehreschild 3, N. Alakel 8, A. Bergeron 9, G. Desoubeaux 10, I. Falces-Romero 11, N. Klimko 12, K. Lagrou 13, C. Lass-Flörl 14, Y. Le Govic 15, J. Maertens 16, A. Ostojic 17, J. Prattes 18, Z. Racil 19, A. Reséndiz Sharpe 16, E. Schalk 20, M. Stanzani 21, J. Steinmann 22, W. Melchers 23, M.J.G.T. Vehreschild 24 and P.E. Verweij 23
    1
    Department I of Internal Medicine, Ecmm Excellence Centre of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), University Hospital Cologne, Cologne, Germany
    2
    Department I of Internal Medicine, Ecmm Excellence Centre of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), German Centre For Infection Research, Partner Site Bonn-cologne, Clinical Trials C, University Hospital Cologne, Cologne, Germany
    3
    Department I of Internal Medicine, University Hospital of Cologne, Cologne, Germany
    4
    Department of Medical Microbiology and Infectious Diseases, Excellence Center For Medical Mycology (ecmm), Canisius Wilhelmina Hospital, Nijmegen, Netherlands
    5
    Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), University of Cologne, Cologne, Germany
    6
    Karolinska University Hospital, Stockholm, Sweden
    7
    Mannheim University Hospital, Heidelberg University, Mannheim, Germany
    8
    University Hospital Dresden, Dresden, Germany
    9
    Université Paris Diderot, APHP Saint-Louis Hospital, Paris, France
    10
    Cepr Inserm U1100, Université de Tours, Tours, France
    11
    Hospital Universitario La Paz, Madrid, Spain
    12
    Department of Clinical Mycology, Allergology and Immunology, North-Western State Medical University n.a. I.I. Mechnikov, Saint-Petersburg, Russian Federation
    13
    Clinical Bacteriology and Mycology, Katholieke Universiteit Leuven, Leuven, Belgium
    14
    Div Hygiene & Med. Microbiology, Med. Univ. Innsbruck, Innsbruck, Austria
    15
    Groupe d’Etude des Interactions Hôte Pathogène (GEIHP), Université d’Angers, Angers Cedex, France
    16
    KU Leuven, Leuven, Belgium
    17
    University Hospital Centre Zagreb, Zagreb, Croatia
    18
    Section of Infectious Diseases and Tropical Medicine, Medical University of Graz, Graz, Austria
    19
    Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic
    20
    Otto-von-Guericke University, Magdeburg, Germany
    21
    “Lorenzo e Ariosto Seràgnoli” S’Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy
    22
    Institute of Clinical Hygiene, Medical Microbiology and Clinical Infectiology, Klinikum Nuernberg, Nuremberg, Germany
    23
    Medical Microbiology, RadboudUMC, Nijmegen, Netherlands
    24
    Uniklinikum Frankfurt, Med. Klinik II, Infektiologie, Frankfurt, Germany
    Objectives: In recent years, survival of patients with invasive aspergillosis (IA) has improved mainly due to availability of extended spectrum triazoles. These advances are jeopardized by the emergence of azole resistance in Aspergillus fumigatus, the most common causative pathogen of IA. Despite several studies suggesting high probability of azole treatment failure in patients with azole-resistant isolates, the clinical implications of azole-resistant IA compared to azole-susceptible IA remain unclear. Thus, we seek to describe the epidemiology and determine the efficacy of antifungal therapy in patients with documented azole-resistant IA compared to azole-sensitive IA in patients with hematological malignancy.
    Methods: In patients with hematological malignancies, cases of proven or probable IA (EORTC/MSG 2008) caused by A. fumigatus are registered. Retrospective data are documented, comprising demographics, diagnosis, treatment, response and outcome. Participating sites provided susceptibility results or isolates. Provided isolates were analyzed in a central laboratory.
    Results: Since January 2018, 51 sites in 15 countries worldwide enrolled 154 cases diagnosed with IA between 2010 and 2019, of which 23 (14.9%) had azole-resistant IA. of 44 cases, the respective clinical fungal isolate was analyzed in the central laboratory. A mixed fungal infection was reported for 34 patients (22.1%), 1 (2.9%) in the azole-resistant group; most were related to non-fumigatus Aspergillus species (n = 12, 35.3%) and non-Aspergillus molds (n = 10, 29.4). Most patients were male (n = 98, 63.6%); 19 (82.6%) in the azole-resistant group, 79 (60.3%) in the azole-susceptible group. Age was documented in categories instead of the exact age. Median age group was 50-69 years in both groups (ranging from 7-11 to 70-89 years for azole-resistant cases, 1-12 months to 70-89 years for azole-susceptible cases). Underlying disease and survival are shown in the table.
    Jof 05 00095 i026
    Conclusion: A worldwide network of investigators contributes to the CLARITY registry study. Completion of recruitment and subsequent data analysis are planned for 2019. Further sites may be added if azole resistant cases are encountered.

    P034. Sensitivity of isolated dermatophyte strains to antifungal drugs in the Russian Federation

    M. Manoyan, V. Sokolov, A. Gursheva, N. Gabuzyan and A. Panin
    Veterinary Mycology, The All-Russian State Center for Quality of Animal Medicines and Feeds, Moscow, Russian Federation
    Objectives: The spread of dermatophytosis is a veterinary problem; the spread of dermatophytosis pathogens in the human population is a social problem. In the Russian Federation immunobiological preparations, as well as external agents in the form of suspensions or shampoos are used for a treatment of animals dermatophytosis. As a rule, such agents contain azoles (clotrimazole, miconazole, enilconazole) or terbinafine as an active substance. They are often used unsystematically and can trigger the emergence of resistant strains. Recently, there have been increasing reports about dermatophytoses strains, resistant to antifungal drugs: M. canis strains resistant to terbinafine and azoles, and T. rubrum strains resistant to terbinafine.
    Methods: For comparison, the strains of dermatophytes - M. canis and T. mentagrophytes, earlier collected and freshly isolated from cats and dogs, were chosen. Collection strains, stored in a lyophilized form, were isolated from animals between 1970 and 2000. For the study there were selected ten strains of M. canis and T. mentagrophytes. Freshly isolated strains were collected from animals between 2015 and 2018, they also were stored in the laboratory’s collection in a lyophilized form; we also selected ten strains of M. canis and T. mentagrophytes. The choice of antifungal drugs spectrum, for which MIC was determined, was dictated by the data on the use of antifungal drugs in the Russian Federation: for terbinafine there was evidence of resistant strains; ketoconazole and enilconazole were used for animal fungal infections treatment and prevention. Cultures were isolated on Saburo medium with chloramphenicol and cycloheximide, pure cultures for determining MIC were cultivated on Saburo medium without additives for 7 days at +28°C. A method based on EUCAST E.def 9.3.1 was used to determine the MIC (inoculum density was 1.0E+5 CFU/ml, the incubation time was 4days.
    Results: MIC studies of terbinafine, ketoconazole and enilconazole for strains isolated between 1970 and 2000 showed following. One of ten M. canis strains was resistant to the action of terbinafine (MIC > 32 μg/ml), there was no detected any strain resistant to ketoconazole and enilconazole. None of the ten T. mentagrophytes strains was resistant to the action of terbinafine, ketoconazole or enilconazole. MIC studies of terbinafine, ketoconazole and enilconazole for strains isolated between 2015 and 2018 showed following. Four of the ten M. canis strains were resistant to the action of terbinafine (MIC > 32 μg/ml); one strain was resistant to the action of enilconazole (MIC > 16 μg/ml); and we did not find a ketoconazole resistant strain. Four of the ten T. mentagrophytes strains were resistant to the action of terbinafine (MIC > 32 μg/ml); one strain was resistant to ketoconazole (MIC > 16 µg/ml); 2 strains were resistant to the action of enilconazole (MIC > 16 µg/ml). We did not find strains of M. canis or T. mentagrophytes, resistant to two or three drugs.
    Conclusion: The number of antifungal resistant dermatophyte strains was higher among those isolated for a later period. This may indicate the emergence of resistant dermatophytosis pathogens strains in the Russian Federation.

    P035. Evaluation of the in vitro susceptibility testing of dermatophytic isolates: preliminary comparison of four methods

    A.-M. Markantonatou, K. Samaras, E. Zachrou and T.-A. Vyzantiadis
    First Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece
    Objectives: Superficial infections caused by dermatophytes affect a high percentage of the population. Antifungal susceptibility testing (AST) of these pathogens may offer information about their susceptibility profiles, documentation of the appropriate treatment and reduction of the cost. However, there are two factors that could delay and affect the performance of the AST: the slow growth rate of these fungi and their poor sporulation. The proposed methods by the CLSI or the EUCAST are both laborious for the everyday routine. Though, there are alternative applications that propose the use of an inoculum consisting of a conidia-mycelium mixture or even from plain mycelia, as well as the use of resazurin in order to facilitate the reading. The aim of this study was to compare these approaches to the EUCAST method in order to evaluate their performance.
    Methods: Three alternative methods of dermatophytic AST were compared to the EUCAST proposed methodology for conidia forming moulds. The methods under evaluation were a) a fragmented mycelia method, b) the EUCAST method with the addition of resazurin sodium salt solution and c) the fragmented mycelia method with the addition of resazurin sodium salt solution. The susceptibility of twenty dermatophytic isolates (8 Trichophyton interdigitale, 6 T. rubrum and 6 M. canis) was tested against the antifungal agents of griseofulvin, terbinafine, fluconazole and itraconazole.
    Results: After the measurement of the MICs by all four different methods, the essential agreement between them was calculated in percentages. Data analysis revealed sufficient overall essential agreement of the methods with the addition of resazurin to the initial “uncoloured” ones (98.5% and 97.2% for the EUCAST and the fragmented mycelia method respectively). The fragmented mycelia method exhibited a relatively sufficient overall essential agreement to the EUCAST method (88.9%) but not a satisfactory correlation between the MIC values. The mean MICs (by the EUCAST method, in μg/mL) for the twenty isolates were 1.78 for griseofulvin, 0.034 for terbinafine, 25.2 for fluconazole and 0.57 for itraconazole.
    Conclusion: The addition of resazurin sodium salt solution facilitates the reading and provides a more objective evaluation. The fragmented mycelia method could serve as an alternative in cases of poor or no sporulating dermatophytes.

    P037. Distribution of azole minimal inhibitory concentration (MIC) values and Erg11 amino acid substitutions among Candida auris isolates from different geographic clades

    M. Kordalewska 1, K.R. Healey 2, C. Jimenez-Ortigosa 1, A. Singh 3, B. Williamson 2, A. Wilk 2, I. Berrio 4, A. Chowdhary 3 and D.S. Perlin 1
    1
    Center For Discovery and Innovation, Hackensack Meridian Health, Nutley, United States of America
    2
    Department of Biology, William Paterson University, Wayne, United States of America
    3
    Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
    4
    Hospital General de Medellín “Luz Castro de Gutiérrez” ESE, Medellin, Colombia
    Objectives: Candida auris isolates are often characterized with reduced susceptibility to fluconazole (FLC) and other azole class drugs: voriconazole (VRC), posaconazole (POS), itraconazole (ITC), and isavuconazole (IVC). Such a situation is extremely worrying, since azoles are a mainstay in the treatment of Candida infections and antifungals other than fluconazole might be unavailable in resource-limited countries. Moreover, no antifungal clinical breakpoints are available for C. auris. CDC provides guidance for C. auris MIC interpretation, based on information gathered for related Candida species and expert opinion. Tentative epidemiological cut off values (ECVs) were proposed only for C. auris isolates from India. The aim of this study was to analyze distribution of azole minimal inhibitory concentration (MIC) values for isolates belonging to different geographic clades together with the data regarding molecular resistance determinants.
    Methods: A total of 108 C. auris isolates representing four geographic clades (I – South Asian; II – East Asian; III – South African; IV – South American) were investigated in this study. Antifungal susceptibility testing (AFST) with azoles (FLC, ITC, IVC, POS, VRC) was performed in accordance with CLSI document M27-A3. The ERG11 gene, encoding the target for azole antifungal drugs, was amplified and sequenced. All identified ERG11 alleles were cloned onto a low-copy vector (pRS416) and expressed within a haploid strain of Saccharomyces cerevisiae (BY4741). Azole susceptibilities were then determined for the transformed strains of S. cerevisiae.
    Results: The results of C. auris AFST and ERG11 sequencing are presented in Table 1. Expression of ERG11 alleles in S. cerevisiae revealed that triazole resistance was induced only upon expression of pCauErg11-V125A/F126L, pCauErg11-Y132F, and pCauErg11-K143R, but not pCauErg11-WT, pCauErg11-K177R/N335S/E343D, pCauErg11-I466M, or pCauErg11-Y501H.
    Jof 05 00095 i027
    Conclusion: Differences in the distribution of MIC values and resistance-conferring ERG11 mutations between isolates from different geographic clades underscore the need for an extreme caution when categorizing isolates as sensitive/resistant on a basis on AFST results. Only V125A/F126L, Y132F and K143R Erg11 amino acid substitutions identified in C. auris isolates directly mediate resistance and may be used as diagnostic markers for C. auris azole resistance. Mechanisms other than ERG11 mutations may also contribute to reduced azole susceptibility in C. auris, although this remains to be determined.

    P038. To study the profile of conventional and newer antifungal agents against dermatophytes in a tertiary care hospital in South India

    A.J. Kindo 1, H. Veena 1, A. Subramanian 2 and A. Krishnan 2
    1
    Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, India
    2
    Dermatology, Sri Ramachandra Medical College and Research Institute, SRIHER, Chennai, India
    Objectives: 1.Speciation of dermatophytes from the clinical isolates 2.To study the antimicrobial pattern of frequently used antifungal agents 3.To compare the antifungal profile with the newer antifungal agents
    Methods: Clinically diagnosed dermatophytic patients were sampled for microscopy and culture, grown dermatophytes were subjected to phenotypic identification by conventional method. Antifungal susceptibility testing was done by broth microdilution method according to CLSI M38-A2 document guidelines. Trichophyton mentagrophytes ATCC 4439 was included in each run of susceptibility testing as a quality control. Final drug concentration in the microdilution plates ranged from 64 to 0.25 µg/ml for fluconazole and Amphotericin B and from 16 to 0.06 µg/ml for Itraconazole, Voriconazole, Griesofulvin, Terbinafine, Sertaconazole, Luliconazole, Fenticonazole tested.
    Results:
    Jof 05 00095 i028
    Out of the 100 clinical isolates tested, 58 of them were identified as Trichophyton mentagrophytes, 40 were Trichophyton rubrum. and the remaining two isolates were identified as Epidermophyton Floccosum and Microsoprum nanum Antifungal susceptibility testing was carried out for 100 isolates of Trichophyton species against 9 antifungal agents. All isolates showed detectable growth after 4-5days of incubation. MICS for Trichophyton mentagrophytes ATCC 4439 were within the established range.
    Conclusion: In this study Trichophyton mentagrophytes was the most common cause of dermatophytosis. The most challenging task is not the diagnosis but the treatment, compliance and recurrence. Since it requires a long term treatment, the management with appropriate and responsive drug is the need of the hour. Currently this study suggests that the overall activity of Luliconazole, Fenticonazole, Sertaconazole, as topical agent and Itraconazole, Voriconazole (systemic) are significantly effective against dermatophytes. Current solution to this could be to maintain skin hygiene, prudent use of antifungals appropriate dosing and duration, performing antifungal susceptibility testing, usage of conventional drugs or topical keratolytic agents in combination with newer drugs in appropriate dosages and combination therapy.

    P039. Terbinafine resistant Trichophyton mentagrophytes genotype VIII, Indian type, isolated in Finland

    H. Järv 1, S. Uhrlass 2, T. Simkin 1, P. Nenoff 2, E. Alvarado Ramirez 3, E. Chryssanthou 3 and M. Monod 4
    1
    SYNLAB Estonia, Tallinn, Estonia
    2
    Laboratory For Medical Microbiology, Mölbis, Mölbis, Germany
    3
    Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden
    4
    Department of Dermatology, Lausanne University Hospital, Chexbres, Switzerland
    Objectives: Objective We are reporting first four isolates of Trichophyton mentagrophytes representing novel Indian genotype VIII in Finland potentially resistant to terbinafine. Our study points out the need for antifungal susceptibility testing of dermatophytes as a new issue in microbiology laboratories. The roles of classical mycological culture vs DNA-based diagnostics for laboratory diagnostics of dermatophytosis will be discussed.
    Methods: Primary fungal cultures were made on Sabouraud dextrose and 2% malt extract agar. Identification of fungal isolates was made by micromorphological appearance in SYNLAB laboratory in Estonia and by sequencing the ITS region of rDNA and TEF1-alpha gene in clinical microbiology laboratory in Mölbis, Germany. The genotyping of squalene oxidase gene was made in dermatology department of Vaudoise, Lausanne, Switzerland. The minimal inhibitory concentration (MIC) to terbinafine was determined by broth microdilution method in microbiology laboratory of Karolinska Hospital, Sweden. MIC was detected spectrophotometrically after 5 days incubation at 28 °C.
    Results: We describe four cases of extensive tinea cutis glabrae caused by T. mentagropytes. All cases were diagnosed from January to April of 2019 and occurred in geographically distinct regions of Finland. Three of four of T. mentagrophytes sub-type VIII (Indian sybtype) strains have high MIC values of terbinafine from 4 mg/L to >8 mg/L.
    Conclusion: Dermatophytosis has taken epidemic dimensions in India during the last 5 to 6 years. Unusual clinical presentation of dermatophytosis and corticosteroid modified lesions are making diagnosis challenging. Indian experts panel ECTODERM India has published new treatment guidelines in 2018. Nenoff et al have demonstrated in a recently published study from different geographical locations from India that 93.25% of the dermatophytes isolated from patients suffering from dermatophytosis correspond to a special genotype (VIII) of T. mentagrophytes. Singh with co-workers have published data about alarming terbinafine resistance of T. interdigitale strains in India. Trichophyton spp isolates with Indian origin have demonstrated high MICs to terbinafine, up to 32 mg/L, and also to azoles. Terbinafine is considered to be the most effective drug for treatment of Trichophyton spp infections. In general, we know that pathogens and resistant strains follow the human migration routes. The lack of data about presence of this novel genotype in European countries is because most clinical laboratories do not use multilocus sequencing for routine identification of dermatophytes. SYNLAB Estonia uses classical culture-based diagnostics and DNA-based diagnostics (in house PCR) for dermatophytes, handling approx. 10000 sample per year. We are not able to identify 1/5 of dermatophytes on species level. Most of commercial diagnostic DNA panels for dermatophytes do not discriminate T. mentagrophytes and T. interdigitale. In the light of new data diagnostic laboratories need to put more effort to identify all dermatophytes on species level by molecular methods. It will help to map the changing epidemiological situation of dermatophytosis in Europe.

    P040. An emerging clone of Candida tropicalis bloodstream isolates with ERG11 gene mutations and overexpression conferring reduced activity of isavuconazole

    P.-Y. Chen, Y.-C. Chuang, U.-I. Wu, H.-Y. Sun, J.-T. Wang, W.-H. Sheng, Y.-C. Chen and S.-C. Chang
    National Taiwan University Hospital, Taipei, Taiwan
    Objectives: In Asia, in vitro activity of isavuconazole (ISA) against clinical Candida isolates are seldomly reported, especially a rising concern that azole-resistant Candida tropicalis is common (≥10%) in this region. Also, few studies characterized molecular mechanisms of reduced isavuconazole activity against C. tropicalis.
    Methods: Sixty-four non-duplicated C. tropicalis bloodstream isolates were prospectively collected in National Taiwan University Hospital in 2017. In vitro susceptibility to antifungal agents was performed by using the microdilution colorimetric Sensititre YeastOne panel and the results were interpreted by the Clinical and Laboratory Standards Institute. ISA MICs were determined using the EUCAST E.def 7.3.1 method. Wild-type upper limit (wtUL) was defined as two two-fold dilutions above the MIC50. Isolates with reduced ISA activity (RIA) were defined as their ISA MICs above wtUL. The ERG11 gene was sequenced for all isolates, and the results were compared to the ERG11 wild-type sequence from C. tropicalis reference strain ATCC 750 (Genbank accession M23673). Quantitative realtime reverse transcription-polymerase chain reaction (ERG11, CDR1, and MDR1) were performed. Multilocus sequencing types were analyzed by the eBURST to determine putative relationships between isolates.
    Results: Among sixty-four C. tropicalis bloodstream isolates, the resistant/ non-wild type rates of fluconazole, voriconazole, posaconazole or itraconazole were 21.5%, 30.7%, 64.6%, and 13.9%, respectively. The proportions of RIA was 18.8% (n = 12). ISA MICs correlated with other azoles MICs by calculating with nonlinear regression fits. The R2 values were in the following orders: posaconazole (0.74) < itraconazole (0.82) < voriconazole (0.86) < fluconazole (0.88). The mean percentage of ISA trailing was 27.4%. A total of 8 nucleotide mutations were detected in ERG11 gene of all isolates. Only 2 (A395T/W and C461T/Y) belonged to missense mutations resulting in amino acid substitutions (Y132F and S154F). There were fourteen isolates with one or two these mutations all resistant to fluconazole. Twelve of them had RIA, all of which belonging to the same clone. Gene relative expression levels among three groups (isolates with low and high ISA trailing, and those with RIA) are shown Figure. Only ERG11 expression levels demonstrated significant difference (P < 0.001), and neither did CDR1 nor MDR1 (P > 0.05). Those with RIA showed the highest mean ERG11 relative expression level (3.51 ± 0.59 [S.D.]) with statistically significance, while the mean ERG11 levels between high and low ISA trailing group were not statistically different (1.08 ± 0.69 and 0.93 ± 0.66, respectively).
    Figure
    Jof 05 00095 i029
    Conclusion: C. tropicalis bloodstream isolates in our report showed an emerging clone with pan-azole resistance, including RIA. Fluconazole MICs best correlated to ISA MICs. Combined ERG11 gene missense mutations and ERG11 overexpression conferred RIA.

    P041. Comparative genomics of Aspergillus fumigatus and the influence of agriculture on ecology and azole resistance

    A.E. Barber 1,2,3, J. Born 4, T. Sae-Ong 2, I. Schliebner 4, K. Kang 2, G. Walther 2,3, G. Panagiotou 2, H.B. Deising 4 and O. Kurzai 1
    1
    University of Wuerzburg, Wuerzburg, Germany
    2
    Leibniz HKI Jena, Jena, Germany
    3
    National Reference Center for Invasive Fungal Infections, Jena, Germany
    4
    Martin Luther University Haale-Wittenberg, Halle (Saale), Germany
    Objectives: Aspergillus fumigatus is an environmentally ubiquitous saprophyte capable of causing life-threatening invasive infections and allergic bronchopulmonary disease and current management strategies rely on the azoles. Unfortunately, over the last decade there has been a global emergence in resistance to the azoles in A. fumigatus and the dominant resistance mechanism is of environmental origin, suggesting that resistance is emerging through a selective pressure applied by the widespread usage of azoles in agriculture.
    Methods: To examine the link between the use of azoles in agriculture and the emergence of clinical resistance, systematic soil sampling was performed over a three year period on seven conventional agricultural sites in Germany utilizing azole fungicides and on six organic sites withholding these compounds. Conventional sites were evaluated both before and after azole application. We also performed WGS on 250 environmental and 50 clinical isolates.
    Results: In total, 2875 soil samples were analyzed between 2016 and 2018. We observed a high degree of variation in the abundance of A. fumigatus across sites, however, fields with high A. fumigatus density tended to be consistently so from year to year. Strikingly, we observed a significant reduction in the abundance of A. fumigatus on conventional fields following azole treatment – a finding that was not repeated on an organic agriculture control field – indicating that the application of azoles is imposing a bottleneck on A. fumigatus. We detected a low overall resistance frequency amongst agricultural isolates, with only 1-5% of isolates from 2016-2018 showing medical azole resistance – a rate lower than the 15-20% resistance frequency observed by the German National Reference Center for Invasive Fungal Infections during the same time period. Susceptibility to commonly-applied agricultural azoles was also assessed and isolates resistant to medical azoles almost always had elevated MICs to these agricultural azoles, suggesting cross resistance. Importantly, we observed an increased tolerance to both agricultural and medical azoles in the samples taken after the growing season and application of azoles. At the genome level, isolates from different regions and types of agriculture did not cluster separately, indicating a lack of population structure in the fungus. Comparison of environmental isolates with clinical isolates revealed several subgroups present in the environment that were not represented among clinical samples. We also observed differences in the distribution of resistance mutations between environmental and clinical isolates. Resistant environmental isolates were exclusively either wild type at the cyp51a loci or carried the environmentally-derived TR34/L98H mutation. Clinical isolates, in contrast, showed a much wider range mutations, including substitutions at positions F219, M220, S297, F495, and G448.Ongoing work is focused on defining fungal determinants critical for human infection as well as genetic mechanisms associated with cyp51a-independent azole resistance.
    Conclusion: We observed a marked reduction in A. fumigatus fields following azole application and increased azole tolerance after the growing season and azole exposure. No population structure was observed among environmental isolates, but whole genome phylogeny identified genetic backgrounds enriched for among clinical isolates.

    P042. MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and thirty-five uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method

    A. Borman, J. Muller, J. Walsh-Quantick, A. Szekely, Z. Patterson, M. Palmer, M. Fraser and E. Johnson
    Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom
    Objectives: Epidemiological cut-off values and clinical interpretive breakpoints have been developed for a number of antifungal agents with the most common Candida species which account for the majority of infections due to pathogenic yeasts species. However, less common species for which susceptibility data are limited are increasingly reported in high risk patients and breakthrough infections.
    Methods: The UK National Mycology Reference Laboratory performs routine antifungal susceptibility testing of clinical isolates of pathogenic yeast submitted from across the United Kingdom. Between 2002 and 2016, in excess of 32 000 isolates representing 94 different yeast species were referred to the laboratory. Here we present antifungal susceptibility profiles generated over this 15 year period for amphotericin B, fluconazole, voriconazole, itraconazole, anidulafungin and flucytosine against 35 species of uncommon yeast. Minimum inhibitory concentrations (MICs) were obtained using CLSI broth microdilution methodologies, and MIC data was interpreted against epidemiological cut-off values and clinical breakpoints developed with Candida albicans, in order to identify species with unusually skewed MIC distributions that potentially indicate resistance.
    Results: Potential resistance to at least one antifungal agent (>10% of isolates with MICs greater than the epidemiological cut-off or clinical breakpoint) was evidenced for 29/35 species examined here. Four species exhibited elevated MICs with all of the triazole antifungal drugs against which they were tested, and 21 species exhibited antifungal resistance to agents from at least 2 different classes of antifungal agent.
    Conclusion: The current study highlights the importance of rapid and accurate yeast identification, and provides data to aid clinicians in deciding which antifungal regimens are appropriate when confronted with infections with rarer yeasts.

    P043. Case-series of Candida albicans and glabrata echinocandin-resistant candidemia in Switzerland

    A.T. Coste 1, J. Li 1, N. Khanna 2, D. Goldenberger 3, C. Garzoni 4, Z. Zhender 5, K. Boggian 6, D. Neofytos 7, A. Riat 8, D. Bachmann 1, D. Sanglard 1 and F. Lamoth 9,10
    1
    Microbiology Institute, University Hospital Lausanne, University of Lausanne, Lausanne, Switzerland
    2
    Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Basel, Basel, Switzerland
    3
    University Hospital Basel, Clinical Microbiology Laboratory Medicine, Basel, Switzerland
    4
    Riva Caccia 1b, Lugano, Switzerland
    5
    SYNLAB Suisse SA, Bioggio, Switzerland
    6
    Department of Infectious Diseases, Cantonal Hospital of Saint Gallen, Saint Gallen, Switzerland
    7
    Infectious Diseases, University Hospital of Geneva, Geneva, Switzerland
    8
    Geneva University Hospitals, Service of Laboratory Medicine, Geneva, Switzerland
    9
    Microbiology Institute, University Hospital Lausanne, Lausanne, Switzerland
    10
    Infectious Diseases Service, CHUV, Lausanne, Switzerland
    Objectives: Echinocandin drugs (anidulafungin, caspofungin, micafungin) represent first-line therapy of candidemia. Echinocandin resistance, due to hotspot mutations in the target FKS gene, has emerged among C. glabrata and C. albicans, which is associated with poor outcome. Therefore, national surveillance programs of echinocandin resistance are mandatory. The aims of this study were: i) to assess the epidemiology of echinocandin resistance among C. albicans/glabrata in Switzerland, ii) to describe the characteristics of candidemic episodes due to echinocandin-resistant isolates, and iii) to assess the efficacy of high-dose echinocandin against echinocandin-resistant C. albicans in an invertebrate model of infection.
    Methods: Cases of candidemia attributed to C. albicans/glabrata resistant to echinocandins (CLSI criteria) were identified by: i) screening of a collection of Candida bloodstream isolates from the Fungal Infection Network of Switzerland (FUNGINOS) including all candidemic episodes from 26 hospitals in Switzerland from 2004 to 2013, and ii) screening of the microbiology databases of the most important university hospitals from 2014 to 2018. Echinocandin-resistant Candida isolates were sent to the reference laboratory for antifungal susceptibility testing (CLSI protocol) and sequencing of FKS hotspots. Clinical data of candidemia were collected. In order to assess a possible efficacy of high-dose echinocandins against these isolates, we tested the efficacy of escalating anidulafungin doses in a Galleria mellonella model of invasive candidiasis with a C. albicans FKS mutant (S645P) compared to the wild-type SC5314 strain.
    Results: Seven cases of candidemia due to echinocandin-resistant C. albicans/glabrata were identified: 2 from the 2004-2013 cohort (incidence 0.1%) and 5 from the microbiological database screening from 2014 to 2018. Four infections were due to C. albicans and 3 to C. glabrata. Candidemia originated from urine (2 cases), intravascular catheter (2), gastro-intestinal tract (1) and primary bloodstream infection (1). All patients received previous echinocandin therapy with a median duration of 18 days (range 11-60). Overall mortality was 43%. One patient (C. glabrata infection) was cured by caspofungin therapy despite in vitro resistance. Minimal inhibitory concentrations (MIC) ranges for anidulafungin, caspofungin and micafungin were: 2-4, 1->16 and 1-4 µg/mL, respectively. The following hotspot mutations were identified in C. albicans: S645P (n = 3) and R1361G (n = 1). Among C. glabrata, 2 isolates displayed the S663P mutation in FKS2, while no hotspot FKS mutation was recovered from one pan-echinocandin resistant isolate. Because anidulafungin displayed some in vitro activity against C. albicans S645P mutant (MIC 2 µg/mL), we assessed its in vivo activity in a Galleria mellonella model of infection (Figure 1). While a dose-dependent response was observed in C. albicans wild-type strain-infected larvae, no effect was observed against the C. albicans FKS mutant despite high anidulafungin doses (12 mg/kg).
    Jof 05 00095 i030
    Conclusion: Our analysis suggests an increase in echinocandin resistance among C. albicans/glabrata in Switzerland with 5 cases identified within the last 5 years, while only two cases were reported in a cohort including all candidemias from 2004 to 2013. Previous echinocandin exposure was observed in all cases. Our invertebrate model suggests that high-dose echinocandins are ineffective against C. albicans FKS mutants despite some level of in vitro activity.

    P044. Aspergillus isolates from patients with chronic pulmonary aspergillosis mycologically and clinically resistant to azole antifungals are sensitive to Ibrexafungerp (SCY-078)

    R. Rautemaa-Richardson 1,2, C. Moore 1,2, K. Rawson 2, L. Novak-Frazer 1,2, D. Angulo 3, S. Barat 3 and M.D. Richardson 1,2
    1
    Division of Infection, Immunity and Respiratory Medicine, University of Manchester, Manchester, United Kingdom
    2
    Mycology Reference Centre, Excellence Center For Medical Mycology (ECMM), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom
    3
    Scynexis Inc, New Jersey, United States of America
    Objectives: Ibrexafungerp (formerly MK-3118, SCY-078) is a novel and structurally distinct triterpenoid glucan synthase inhibitor whose oral availability and efficacy has already been demonstrated against a wide spectrum of Candida species. Currently the drug is being evaluated for efficacy in invasive and chronic pulmonary aspergillosis. Ibrexafungerp has been shown to be effective in vitro and in vivo against a broad range of Aspergillus species, including drug-resistant strains. In vitro activity is an important efficacy indicator of therapeutic success or failure. The objective of this study was to determine the activity of Ibrexafungerp against a panel of Aspergillus strains isolated from the respiratory tract of patients unresponsive to or failing azole therapy, and which were previously shown to be resistant to one or more azole antifungals (itraconazole, voriconazole, posaconazole and isavuconazole) using the EUCAST E.Def 10.1 standard.
    Methods: Patients failing azole therapy for chronic pulmonary aspergillosis were identified in the weekly departmental multi-disciplinary team meetings and their isolates selected for Ibrexafungerp sensitivity testing. Twenty-two Aspergillus fumigatus complex, three A. flavus complex and one A. niger complex strains with varying degrees of resistance to one or more azole antifungals were tested by measuring the minimum effective concentration (MEC) for Ibrexafungerp following the EUCAST E.Def 10.1 standard. Where required, isolates were sequenced (using primers to the internal transcribed spacers (ITS), beta-tubulin (bt) and calmodulin (cal) genes) to identify to the species level.
    Results: The Ibrexafungerp MEC range for the 26 Aspergillus spp. isolates was 0.008 to 0.25 mg/l. Isolates that were deemed to be resistant to all azoles had an Ibrexafungerp MEC of 0.015 to 0.25 mg/l. One isolate that was resistant to all azoles and amphotericin B was sensitive to Ibrexafungerp (MEC 0.125 mg/l).
    Conclusion: The exquisite in vitro activity of Ibrexafungerp against Aspergillus fumigatus, A. flavus and A. niger isolated from patients with chronic pulmonary aspergillosis suggests that this new compound has potential for treating patients with this condition and similar manifestations of pulmonary aspergillosis.

    P045. Analysis of the susceptibility pattern among Aspergillus isolates against the commonly used Antifungal agents in a tertiary care centre in South India.

    U. Almas Fathima 1, A.J. Kindo 1, S. Prasanna Kumar 2, M. Thirunarayan 3 and V. Lakshmi Sree 4
    1
    Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, India
    2
    Ent, Sri Ramachandra Institute of Higher Education and Research, Chennai, India
    3
    Microbiology, Apollo Hospitals, Chennai, India
    4
    Microbiology, Apollo Speciality Hospital, Chennai, India
    Objectives: To do in vitro susceptibility testing and analyse the difference in the Antifungal susceptibility profile of Aspergillus isolates by Broth Microdilution method.
    Methods: Aspergillus grown from various clinical samples (Ear swab, Bronchial wash, Endotracheal Aspiration, Paranasal sinus, BAL, Sputum) was subcultured on Sabouraud’s Dextrose Agar/Oat meal Agar. Tease mount/Slide culture was done to study the morphological features of the hyphae, size, shape and arrangement of the conidia. Antifungal susceptibility testing(AFST) was done according to CLSI guidelines M38-A2 2008 documented reference method for Broth Microdilution Antifungal susceptibility of Filamentous fungi. Antifungal stock solutions of AmphotericinB, Voriconazole, Posaconazole, Itraconazole and Caspofungin was prepared in di-methyl sulfoxide and stored at -700 C. RPMI medium was prepared, adjusted to pH 7 and filter sterilized. Sterility was checked and stored at 40 C. Dilution of the stock solution was done as per the guidelines. Inoculum was prepared by adjusting conidial suspension in sterile distilled water to 0.5 McFarland Turbidity standard; to 100 µl of drug solution, 100 µl of inoculum in RPMI 1640 medium was added. Test micro-titre plate was incubated for 48hrs at 350 C. Growth and sterility control wells were included in each test. AFST-READING RESULT: For AmphotericinB, Voriconazole, Posaconazole, Itraconazole - MIC is the first well without visible growth. For Echinocandins, eg. Caspofungin- MEC is the point (lowest value) where a visible change in growth as compared to the positive growth is noted.
    Results: A total of 30 Aspergillus isolates were collected-Aspergillus niger(14), Aspergillus flavus(9), Aspergillus fumigatus(6) & Aspergillus terreus(1). The result was compiled based on mean MIC values of 21 isolates are as follows.
    Jof 05 00095 i031
    The MIC values of other isolates and Amphotericin B is yet to be completed.
    Conclusion: The common Aspergillus species was Aspergillus niger which was obtained from Ear swab. The susceptibility profile showed high MIC for Itraconazole in case of Aspergillus niger and high MIC for Itraconazole, Voriconazole and Posaconazole in case of Aspergillus flavus. For Aspergillus fumigatus no such high MIC was recorded. In case of Aspergillus terreus, being resistant in nature the low MIC is recorded only with Caspofungin. Due to less susceptibility of Aspergillus to the common Antifungal agents, it is important to perform antifungal susceptibility testing to reduce the morbidity and mortality in patients especially with Invasive Aspergillosis.

    P046. CRISPR-Cas9 as a tool box to investigate antifungal resistance

    F. Morio 1, L. Lombardi 2, U. Binder 3, C. Loge 1, E. Robert 1, C. Lass-Flörl 3, G. Butler 2 and P. Le Pape 1
    1
    Ea1155 Iicimed, Nantes University Hospital, NANTES, France
    2
    School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland
    3
    Department of Hygiene, Microbiology and Public Health, Division of Hygiene and Medical Microbiology, Medical University Innsbruck, Innsbruck, Austria
    Objectives: Antifungal resistance in human pathogenic fungi is increasingly reported and a public health issue. Deciphering the mechanisms underlying drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi. Over the last decade, the groundbreaking discovery of Clustered Regularly Interspaced Short Palindromic Repeats and the associate protein Cas9 (CRISPR-Cas9) revolutionized genome editing. We evaluated the potential of a CRISPR-Cas9 method, as a tool to perform precise genome editing of the ERG11 gene in the context of azole resistance.
    Methods: A new plasmid-based and marker-less CRISPR-Cas9 genome-editing method, recently developed for Candida parapsilosis (1), was used to investigate the potential contribution to azole resistance of two different single nucleotide polymorphisms (G1126A and G1372A), leading to amino acid substitutions (L376I and G458S, respectively) in the ERG11 gene, identified in an azole-resistant clinical isolate of Candida orthopsilosis. These mutations were introduced by homology directed recombination (HDR) using a donor template into a fluconazole-susceptible C. orthopsilosis background isolate. In vitro susceptibility of the transformants displaying the expected mutations (two clones for each engineered mutation) to azoles was then performed.
    Results: This approach allowed us to engineer both mutations in the fluconazole-susceptible background isolate. Though engineering L376I was straightforward, editing G458S required the addition of silent mutations within the core of the Cas9 recognition site to prevent Cas9 from recutting again. Eventually, in vitro susceptibility testing of the corresponding transformants demonstrated that G458S, but not L376I, confers resistance to fluconazole (MIC increased by 8-fold by introducing the homozygous mutation) and also affected voriconazole susceptibility. Importantly, after 3 passages on YPD at 30°C, transformants were not resistant to nourseothricin (selection marker) anymore, showing the rapid loss of the plasmid containing all the CRISPR-Cas9 components, thus proving that it did not integrate in the genome.
    Conclusion: Our work supports the contribution of G458S but not L376I in fluconazole and voriconazole resistance in C. orthopsilosis (2). Besides these findings, this study highlights CRISPR-Cas9 genome editing as a powerful tool to streamline the study of antifungal resistance in human pathogenic fungi. Though critical points should be considered (e.g. HDR drops with cut-to-mutation distance), this approach paves the way for an easier screening approach of any SNP, occurring in any gene suspected to be involved in antifungal resistance. 1-Lombardi et al., Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species. mSphere. 2019 Mar 13;4(2). pii: e00125-19. doi: 10.1128/mSphere.00125-19. 2-Morio et al., Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis. J Antimicrob Chemother. 2019 May 18. pii: dkz204. doi: 10.1093/jac/dkz204.

    P047. Changes in the lipid microenvironment of β-(1,3)-D-glucan synthase of Aspergillus fumigatus is a new clinically important echinocandin resistance mechanism

    S. Satish 1, C. Jimenez-Ortigosa 1, Y. Zhao 1, M.H. Lee 1, E. Dolgov 1, T. Kruger 2, S. Park 1, D. Denning 3, O. Kniemeyer 2, A. Brakhage 2 and D.S. Perlin 1
    1
    Center For Discovery and Innovation, Hackensack Meridian Health, Nutley, United States of America
    2
    Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany
    3
    University of Manchester, Manchester, United Kingdom
    Objectives: Aspergillus fumigatus is a leading cause of opportunistic invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high Minimum Effective Concentration (MEC) A. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance in Candida is known to arise from amino acid substitutions in β-(1,3)-D-glucan synthase encoded by the fks1 gene. Yet, these clinical isolates did not contain mutations in fks1, indicating an undefined resistance mechanism. The objective of this study was to explore a novel mechanism of echinocandin resistance independent of fks1 mutations.
    Methods: To explore this new mechanism, we used a lab-derived strain, RG101, which does not contain fks1 mutations but is resistant to caspofungin (CAS). Inhibition of glucan synthase enzyme isolated from RG101 grown in the absence and presence of CAS was determined in vitro using radioactive substrate and IC50 (drug concentration required for 50% reduction of activity) values were measured. Post-translational modifications (PTMs) potentially leading to resistance was explored by evaluating enzyme derived from RG101 grown in the absence and presence of CAS (1 µg/mL) using nano LC-MS/MS. A comprehensive lipidomics analysis was also performed to compare lipid profiles of glucan synthase containing fractions derived from RG101 grown in the absence and presence of CAS (1 µg/mL) using LC-MS/MS. To measure Reactive Oxygen Species (ROS) levels induced by different echinocandins, a 2’,7’-dichlorofluorescin diacetate (DCFDA)-based fluorescence assay was used, and ROS levels measured after one hour of drug exposure.
    Results: Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet, exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log-orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole enzyme post-translational modifications, but found none linked to resistance. However, analysis of the lipid microenvironment of CAS-induced resistant enzyme revealed a prominent increase in abundance of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrial-derived ROS, and dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance.
    Conclusion: We conclude that CAS-induces cellular stress, promoting formation of ROS, triggering an alteration in the composition of plasma membrane lipids within the local environment of glucan synthase, rendering it insensitive to echinocandins. We have discovered a new mechanism of resistance in A. fumigatus independent of the well-characterized FKS mutation mechanism observed in Candida. This study also identifies an off-target effect of CAS, ROS production in A. fumigatus, and integrates oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.

    P048. In vitro activity of fluconazole in combination with magnolol against Candida auris

    I. De-La-Fuente 1, A. Matilla-Gutiérrez 1, A. Guridi 1, E. Sevillano 1, J. Pemán 2, E. Eraso 1 and G. Quindós 1
    1
    Immunology, Microbiology and Parasitology, University of the Basque Country (UPV/EHU), Bilbao, Spain
    2
    Servicio De Microbiología, Hospital Universitario y Politécnico La Fe, Valencia, Spain
    Objectives: Candida auris is an emerging pathogen that causes outbreaks worldwide. This fungus is responsible for severe infections such as invasive candidiasis that present a high overall mortality. Treatment of these infections suppose a clinical challenge due to the high levels of multidrug-resistance in C. auris, which makes the finding of new therapeutic alternatives an important issue. Combination of fluconazole with different natural compounds has shown to be effective against fluconazole-resistant Candida isolates. For this reason, the aim of this study was to assess the in vitro interaction between fluconazole and magnolol, a polyphenol extracted from Magnolia officinalis, or epigallocatechin gallate (EGCG), a polyphenol obtained from Camellia sinensis, against clinical isolates of C. auris.
    Methods: We studied 21 C. auris isolates from different clinical specimens (seven from blood, seven from oropharynx and six from urine). Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality controls. Different fluconazole combinations with magnolol or EGCG were studied. Fluconazole concentrations were selected according to the minimum inhibitory concentration (MIC) determined by the European Committee on Antibiotic Susceptibility Testing (EUCAST) EDef 7.3.1 document. MIC was defined as the lowest drug concentration causing 50% growth reduction after 24 h of incubation. Magnolol and EGCG concentrations ranged from 0.125 to 64 μg/mL. The interaction between fluconazole and magnolol or EGCG were assessed by a checkerboard microdilution method. In vitro interactions of drug combinations were interpreted in terms of fractional inhibitory concentration index (FICI) as follows: FICI ≤ 0.5, synergistic; 0.5 < FICI ≤ 1, additive; 1 < FICI ≤ 4, indifferent and FICI > 4 antagonistic.
    Results: Fluconazole MIC range against C. auris was 64 to > 64 μg/mL and magnolol MIC range 16 to > 64 μg/mL; however, combination of both drugs reduced the concentrations necessary for inducing 50% reduction in the growth of the isolates. The effect of the combination of fluconazole with magnolol against seven isolates (33.3%) was interpreted as additive. The effect against the other 14 isolates was interpreted as indifferent, although the fluconazole MICs decreased to 1-16 μg/mL. The combination of fluconazole with EGCG was indifferent against all isolates and did not have any influence in the MICs of fluconazole.
    Conclusion: The combination of fluconazole with magnolol significantly reduced the fluconazole MICs against all of the C. auris isolates, showing additive effect in seven of them. The combination of fluconazole with EGCG showed indifferent effect. Funding: This work was co-supported by the Consejería de Educación, Universidades e Investigación (GIC15/78 IT-990-16) of Gobierno Vasco-Eusko Jaurlaritza.

    P049. Resistance to medical importance antifungals in environmental yeasts

    M.L. Scroferneker 1, D. Pagani 2, C. C. Tavares De Souza 2, A. M. Vieira 3, A.H. Da Silva Hellwig 4, I. Da S. Rios 3, D. Heidrich 4, A. M. Fuentefria 2 and P. Valente Da Silva 2
    1
    Department of Microbiology, Immunology and Parasitology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
    2
    Postgraduate Program In Agricultural and Environmental Microbiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
    3
    Federal University of Rio Grande do Sul, Porto Alegre, Brazil
    4
    Postgraduate Program In Medicine: Medical Sciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
    Objectives: The aim of the study was to investigate the presence of potentially pathogenic yeasts from a lagoon in South America.
    Methods: The lagoon was sampled in the summer of 2019. The water samples were inoculated in acidified YM agar medium (1% glucose, 0.5 % peptone, 0.3 % malt extract, 0.3 % yeast extract, 2 % agar) and incubated at 25 ºC for 30 days. Representative yeast colonies were selected and stored in YM medium with 30 % of glycerol at -20 ºC and in GYMP agar medium (2 % glucose, 2 % malt extract, 0.5 % yeast extract, 0.2 % monobasic sodium phosphate, 2 % agar) agar slants coated with mineral oil, maintained at 4 °C. We tested 8 environmental yeast strains. The antifungal susceptibility test was performed according to CLSI protocol, to select yeasts with possible resistance to clinical importance antifungals. The yeasts were tested in the antifungal concentrations established for Candida species against fluconazole, caspofungin and amphotericin B.
    Results: Four isolates had MIC ≥ 64 μg/mL to fluconazole, none of them were susceptible to caspofungin, with MIC ≥ 2 μg/mL. To amphotericin B, six strains had MIC ≥ 1μg/mL. Three isolates were considered resistant to all tested antifungals, following the guidelines for Candida species.
    Conclusion: The preliminary findings feature this lagoon environment as a possible resistance genes reservoir and fungal infection source. Further studies are needed to have a better understanding of this environment and the fungal resistance.

    P051. A natural substitution in FksB Hot Spot 2 of Saprochaete capitata probably related to reduced susceptibility to echinocandins

    P. Menéndez-Manjón 1, I. Arrieta-Aguirre 1, M.S. Cuétara 2, L. López-Soria 3, J.C. García-Ruiz 4 and M.D. Moragues 1
    1
    Enfermería I, Universidad del País Vasco UPV/EHU, Leioa, Spain
    2
    Microbiologia, Hospital Severo Ochoa, Leganés, Madrid, Spain
    3
    Microbiologia, Hospital Universitario Cruces, Barakaldo, Spain
    4
    Biocruces Health Research Institute, Hospital Universitario Cruces, Barakaldo, Spain
    Objectives: Saprochaete capitata is an emerging opportunistic pathogen reported in patients with haematological malignancies. Antifungal clinical breakpoints are not established and therapy remains empirical. S. capitata shows reduced susceptibility to echinocandins, which inhibit the enzyme β-1,3-D-glucan synthase (Fks) involved in the cell wall synthesis. Resistance to echinocandins is linked to mutations in two highly conserved regions of Fks, Hot Spot 1 (HS1) and Hot Spot 2 (HS2). We already reported a natural substitution F-to-L in Fks-HS1 that might be linked to this fungus low susceptibility to echinocandins (Arrieta-Aguirre et al., 2018). Mutations in HS2 have also been related to reduced susceptibility to echinocandins. Therefore, we resolved to sequence HS2 of S. capitata and search for mutations that could be related to echinocandin resistance.
    Methods: DNA of 11 strains of S. capitata was extracted by mechanical cell disruption and purified with the DNeasy Plant Mini Kit (Qiagen). Since HS1 and HS2 are around 2500 bp apart, we applied a primer walking strategy of smaller fragments sequencing to locate them. With a specific primer from the HS1 sequence and a degenerate primer designed for the HS2 region, we obtained an amplicon for one of the strains that allowed us to design downstream specific primers that led us to HS2. Finally, we designed specific primers to sequence HS2 in the rest of the strains and the sequences were compared with other fungal species.
    Results: The alignment of Fks-HS2 of S. capitata and other species revealed a natural substitution R-to-Q in the fourth position of HS2. The recently published genome of S. capitata reveals two Fks, FksA and B. Our sequence corresponds to FksB, which is more similar to Fks2 than Fks1 of other species. In C. glabrata, when R in the fourth position of Fks2-HS2 is modified it results in a deep decrease in the susceptibility to echinocandins (Jensen, 2016). Therefore, the natural substitution R-to-Q in FksB-HS2 of S. capitata could be related to resistance.
    Conclusion: S. capitata has at least two natural substitutions (F-to-L in Fks-HS1 and R-to-Q in Fks-HS2) that may be involved in its reduced susceptibility to echinocandins. However, the effect of both substitutions still needs to be verified empirically. This work contributes to understanding the molecular basis of antifungal resistance of this fungus and may help to improve its treatment. Financial support: Grupos de Investigación Consolidados of the Basque Government (GIC15/103; IT913-16) and Grant FPI of the Basque Government to PM-M.

    P052. Fluconazole resistance in Candida tropicalis blood stream isolates collected from 16 Brazilian Medical Centers

    A. Melo 1, D. Da Matta 1, L. Favarello 1, C. Oliveira 2, T. Sukiennik 3, F. Telles 4, M. Nucci 5 and A. Colombo 1
    1
    Medicine, Federal University of São Paulo, São Paulo, Brazil
    2
    Medicine, Hospital Universitário do Oeste do Paraná, Cascavel, PR, Cascavel, Brazil
    3
    Medicine, Santa Casa de Porto Alegre, Porto Alegre, RS, Porto Alegre, Brazil
    4
    Medicine, Hospital de Clínicas da Universidade Federal do Paraná UFPR, Curitiba PR, Curitiba, Brazil
    5
    Medicine, Hospital Universitário da Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ, Rio de Janeiro, Brazil
    Objectives: C. tropicalis is highly prevalent among episodes of candidemia in Latin America. The progressive use of fluconazole and other triazoles in regimens of prophylaxis and empirical therapy in high-risk patients has led to selective pressure and emergence of secondary resistance among Candida spp. The aim of this study was to check for temporal trends in the rates of azole resistance among C. tropicalis blood stream isolates collected from patients assisted at 16 Brazilian medical centers at any time within 2007 and 2018.
    Methods: We selected isolates of C. tropicalis stored at the culture collection of Laboratório Especial de Micologia (LEMI) – Universidade Federal de São Paulo, Brazil, that were obtained from candidemic patients admitted in 16 medical centers along the period of study. The isolates were identified at species level by Matrix-Assisted Laser Desorption Ionization-Time of Fligh Mass Spectrometry (MALDI TOF MS). In vitro antifungal susceptibility tests to fluconazole and voriconazole were evaluated by the Clinical & Laboratory Standards Institute (CLSI ) microbroth method (documents M27-A3 and M27-S4). For quality control of in vitro assays we used the reference strains C. parapsilosis ATCC 22019, C. krusei ATCC 6258 and C. tropicalis ATCC 750.
    Results: In order to check for historical trends of azole resistance among C. tropicalis isolates, we compared the MIC values generated with fluconazole and voriconazole against 200 blood stream isolates collect at any time within two different periods: P1 (2007-2012) versus P2 (2013-2018). Statistical differences were checked by student T Test. After testing all 200 isolates, 3.5% of C. tropicalis strains were considered to be non-susceptible to azoles. As illustrated bellow, we found resistance rates of C. tropicalis against fluconazole of 1.9% and 3.2% in P1 and P2, respectively (p>0.05). In regard to voriconazole, resistance rates of 0 and 1% were documented in P1 and P2, respectively (p>0.05).
    Conclusion: After testing 200 blood stream isolates of C. tropicalis we identified a rate of 3.5% of isolates exhibiting limited susceptibility (SDD/R) to fluconazole and/or voriconazole. Otherwise, we were not able to demonstrate any historical trend in terms of increasing rates of azole resistance among C. tropicalis isolates collected along the 2 periods of study.

    P053. Evaluation of spectrophotometric reading of EUCAST antifungal susceptibility testing of Aspergillus fumigatus

    G. Jan 1, L. Kristensen 2, I. Bach Bentsen 2 and M. Nielsen 2
    1
    Department of Clinical Microbiology, Aarhus University Hospital, Aarhus N, Denmark
    2
    Department of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark
    Objectives: The rise in azole resistance in Aspergillus fumigatus1 highlights the importance of antifungal susceptibility testing. While azole containing agar plates have been shown to have excellent sensitivity and specificity in detecting azole resistance2, access to MIC testing is still important. As commercially available methods have serious limitations reference broth microdilution is the only feasible option at present. The EUCAST method for antifungal susceptibility testing of moulds relies on visual reading of MICs which is laborious and time-consuming. A recent study3 suggests that spectrophotometric determination of MICs of azoles may improve objectivity and allow automatisation of the EUCAST E.Def 9.3 method. The scope of this study is to evaluate the spectrophotometric metod in our center using seven antifungal drugs.
    Methods: 22 clinical isolates from A. fumigatus species complex were selected for the study. 9 isolates were resistant to at least one azole while 13 were wild type. All isolates were tested against itraconazole, voriconazole, isavuconazole, posaconazole, amphotericin B, natamycin and terbinafine using EUCAST E.Def 9.3. Visually determined MICs were read at complete inhibition of growth.
    Spectrophotometric reading was performed using a Thermo Multiskan FC microplate reader with a 492 nm filter. The OD values of the negative control wells were subtracted from all wells and MICs were determined as the lowest concentration corresponding to either 5% growth compared to the drug free control or as 0.02 OD values above background.
    Spectrophotometrically determined MICs were compared to visually determined MICs. For all drugs the quantitative agreement within 0, 1 and 2 two-fold dilutions was calculated. For itraconazole, voriconazole, isavuconazole, posaconazole and amphotericin B categorical agreement was calculated using EUCAST Clinical breakpoints for fungi v. 9.0.
    Results:
    Jof 05 00095 i032
    Results are shown in Table 1. When using the 5% growth endpoint we found a high level of essential agreement (95-100%) for all drugs and a high level of categorical agreement (86-95%) for itraconazole, voriconazole, isavuconazole, posaconazole and amphotericin B. of importance no major or very major errors was observed. When using the 0.02 OD values above background endpoint however we found only 64% essential agreement for terbinafine and a categorical agreement of 68% for posaconazole including 5% very major errors.
    Conclusion: Spectrophotometric determination of MICs using the EUCAST E-Def 9.3 for A. fumigatus species complex appears to be a promising method for all tested drugs. Highest agreement between visual and spectrophotometric MICs is obtained using the 5% growth endpoint. References 1. van der Linden J et al. Prospective Multicenter International Surveillance of Azole Resistance in Aspergillus fumigatus. Emerg Infect Dis. 2015;21(6):1041-1044. 2. Arendrup MC et al; Multicentre validation of 4-well azole agar plates as a screening method for detection of clinically relevant azole-resistant Aspergillus fumigatus, Journal of Antimicrobial Chemotherapy, Volume 72, Issue 12,Pages 3325–3333 3. Meletiadis, J. et al., Spectrophotometric reading of EUCAST antifungal susceptibility testing of Aspergillus fumigatus, Clinical Microbiology and Infection, Volume 23, Issue 2, 98 - 103

    P054. Molecular mechanism and frequency of olorofim resistance in Aspergillus fumigatus

    J. Buil 1, J. Oliver 2, D. Law 2, M. Tehupeiory-Kooreman 1, J. Rex 2, M. Hokken 1, W. Melchers 1, M. Birch 2 and P.E. Verweij 1
    1
    Medical Microbiology, Radboudumc, Nijmegen, Netherlands
    2
    F2G Ltd, Manchester, United Kingdom
    Objectives: Olorofim (OLO) is a new antifungal agent with a novel mechanism of action, targeting dihydroorotate dehydrogenase (DHODH) in the de novo pyrimidine biosynthesis pathway. It is active against both azole-susceptible and azole-resistant strains of Aspergillus fumigatus. Thus, OLO may be an important treatment option in patients with Aspergillus disease, including azole-resistant cases. Azole resistance selection occurs in >10% of patients with chronic pulmonary aspergillosis during itraconazole (ITZ) therapy. In this study, we analysed the frequency of resistance mutation induction of OLO in A. fumigatus in three different strains using two different methods. The underlying OLO resistance mechanism was also investigated.
    Methods: Method 1: From two A. fumigatus isolates, AZN 8196 and V139-36, 10 single colonies were separately inoculated onto Sabouraud agar and incubated at 37°C for 96h. From each culture 1 x 108 conidia were applied to 6 RPMI agar plates containing either 0.5 mg/L OLO or 8 mg/L ITZ.. Plates were incubated at 37°C for up to 7 days. Colonies growing on drug-containing plates were subcultured on RPMI agar containing 0.5 mg/L OLO or 8 mg/L ITZ to confirm resistance. Method 2: Spore stocks of A. fumigatus strain Af293 were prepared and inoculated onto yeast nitrogen base with glucose agar (YNBG) containing 0.25 mg/L OLO. A total of 8 x 109 spores were inoculated into 12 x 100 ml YNBG-OLO agar plates that were subsequently incubated for 5 days at 35°C. Colonies growing on drug-containing plates were subcultured on YNBG-OLO to confirm resistance. The mean rate of resistance was calculated for each isolate. The pyrE gene which codes for DHODH was sequenced for initial OLO-susceptible strains and -resistant progeny isolates and analysed for mutations.
    Results: Method 1: The resistance rate of OLO was 1 in 5 x 107 (frequency of 2 x 10-8) for AZN 8196 compared to 1 in 5 x 106 (2 x 10-7) for ITZ. For V139-36 the resistance rate of OLO was 1 in 9 x 107 (1.1 x 10-8) while the rate for ITZ was 1 in 8 x 106 (1.3 x 10-7) Method 2: Resistance to OLO in Af293 occurred at a rate of 1 in 6 x 108 (frequency of 1.7 x 10-9). Sequencing of pyrE revealed a hotspot for resistance mutations in the pyrE gene for OLO resistance in A. fumigatus at locus Gly119. Four amino acid substitutions were found; G119V, G119C, G119S and G119A. A single OLO-resistant mutant was obtained that carried the wild type DHODH sequence, but this mutant was slow growing compared with parent.
    Conclusion: We demonstrate that OLO resistance can be selected in A. fumigatus and can be mediated by mutations in the pyrE gene at locus G119. The frequency of resistance varied from approximately 2 x 10-8 to 1.7 x 10-9 for spontaneous mutations. The resistance rate of OLO was 5 to 10-fold lower compared to ITZ. The low resistance rate found in these experiments indicates that resistance selection may be less frequent in patients with Aspergillus diseases treated with OLO.

    P055. Comparison of killing activity of rezafungin, anidulafungin, caspofungin and micafungin against Candida auris in the presence and absence of serum

    Z. Tóth 1, L. Forgács 1, J.B. Locke 2, G. Kardos 1, F. Nagy 1, R. Kovács 1, A. Borman 3 and L. Majoros 1
    1
    Department of Medical Microbiology, University of Debrecen, Debrecen, Hungary
    2
    Cidara Therapeutics, San Diego, United States of America
    3
    Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom
    Objectives: Candida auris is an emerging, difficult-to-treat multiresistant pathogen against which echinocandins are the recommended standard-of-care treatment. Rezafungin is a next-generation echinocandin with similar in vitro activity to existing echinocandins yet it attains much higher in vivo concentrations and exposures due to its extended half-life and front-loaded dosing paradigm. Because in vitro killing data with existing echinocandins are limited against C. auris and are lacking for rezafungin, we compared rezafungin to anidulafungin, caspofungin, and micafungin in time-kill assays against C. auris isolates in standard RPMI-1640 medium and also investigated the impact of serum on in vitro killing trends.
    Methods: Two C. auris clinical isolates from each clade (Japanese/Korean, South Asian/Indian and South African, obtained from the National Mycology Reference Laboratory, UK) were tested. Both South African isolates were autoaggregative. MICs in RPMI-1640 +/-50% human serum were determined using the standard broth macrodilution method (CLSI M27 Ed4). Time-kill studies with the four echinocandins were performed from 0.25 to 32 mg/L in both media, and killing rates were compared. Positive k values indicate killing; negative values indicate growth.
    Results: MIC values were higher in 50% serum than in RPMI-1640 alone (Table 1). In RPMI-1640, at 1xMIC or higher concentrations all four echinocandins showed only fungistatic effect. The highest k value with rezafungin (1.34 1/h) was at 32 mg/L against isolate 209. However, none of the echinocandins produced any CFU decrease against aggregating isolates (k values in cases of isolates 2 and 204 were always negative). Similar results were found in cases of caspofungin and micafungin against isolates 12, 15 and 27, and isolate 15, respectively. Re-growth was frequently observed for all four echinocandins. In 50% serum, growth rates were significantly lower. At 1xMIC or higher concentrations, echinocandins showed concentration-dependent killing activity, however, this CFU reduction never reached the fungicidal threshold. The highest k values were 0.34, 0.33, 0.34 and 0.29 1/h for rezafungin, anidulafungin, caspofungin and micafungin, respectively. However, k values were always negative at 1-32 mg/L, in the case of isolate 2 (an autoaggregative isolate) for all echinocandins and in cases of isolates 27 and 204 for caspofungin. Table 1. MIC values for rezafungin and echinocandin comparators against C. auris strains in the presence and absence of 50% serum.
    Jof 05 00095 i033
    Conclusion: Killing activity in RPMI-1640 alone was less consistently positive than in 50% serum and only fungistatic activity was detected. Aggregating isolates were less susceptible to echinocandins than non-aggregating isolates. Rezafungin showed the same or better activity than anidulafungin and micafungin at clinically attainable concentrations. The trend towards stronger killing activity in the presence of serum helps to account for the disconnect between the modest time-kill activity of echinocandins in vitro versus their strong efficacy against C. auris in vivo, as rezafungin has previously demonstrated in animal models.

    P056. Frequency of paradoxical and trailing effects with rezafungin, anidulafungin, caspofungin and micafungin against Candida species

    L. Forgács 1, Z. Tóth 1, J.B. Locke 2, F. Nagy 1, B. Balázs 1, E. Prépost 1, G. Kardos 1, R. Kovács 1, A. Szekely 3, A. Borman 3 and L. Majoros 1
    1
    Department of Medical Microbiology, University of Debrecen, Debrecen, Hungary
    2
    Cidara Therapeutics, San Diego, United States of America
    3
    Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom
    Objectives: Currently, echinocandins are the first-line agents for treatment of invasive Candida infections. Echinocandins demonstrate excellent efficacy and fungicidal activity against Candida species in vivo. However, echinocandins can exhibit some disconnected growth phenomena in vitro, such as paradoxical effect (PE) where activity decreases at higher drug concentrations and the trailing effect (TE) where complete growth inhibition is never achieved or occurs at dilutions well above the 50% endpoint used to determine MIC values. PE and TE can significantly alter the determination/interpretation of MIC values and impact other in vitro assays. Rezafungin is a next-generation echinocandin capable of attaining high concentrations and exposures in vivo, exceeding those measured for the three approved echinocandins, due to its long half-life and front-loaded dosing regimen. Given the relevance of evaluating higher drug concentrations for rezafungin and that rezafungin PE/TE trends have not yet been characterized, herein we compared the occurrence of PE and TE for rezafungin with data generated in parallel for caspofungin, micafungin and anidulafungin against clinically important Candida species.
    Methods: We tested 365 non-duplicate Hungarian clinical isolates belonging to 13 species (see Table). Isolates were collected between 2005 and 2018 and were identified with MALDI Biotyper (Bruker, Bremen, Germany). C. auris were obtained from National Mycology Reference Laboratory, UK. MICs were determined by BMD method according to CLSI (M27 Ed4) in RPMI-1640. Concentration ranges of antifungals were 0.06-32 mg/L. PE was defined as visible growth occurring at higher but not at lower supra-MIC concentrations. TE was defined when yeasts show reduced but observable growth in supra-MIC concentrations.
    Results: The percentages of isolates showing PE or TE after 48 hours are shown in the Table. Cumulative percentage of PE plus TE was the highest for caspofungin (47.7%), followed by anidulafungin (35.9%), micafungin (25.8%) and rezafungin (17.7%). PE was observed most frequently for caspofungin (31.4%) but was uncommon for rezafungin (3.1%). PE with caspofungin, micafungin or anidulafungin occurred most frequently in cases of C.tropicalis, C. albicans, C. orthopsilosis and C. inconspicua but was never found incase of C. krusei. The majority of C. auris and C. dubliniensis isolates showed TE for all four echinocandins. Table. Percentages of Candida spp. isolates demonstrating paradoxical effect (PE) or trailing effect (TE).
    Jof 05 00095 i034
    Conclusion: PE or TE was widely observed among Candida species and were echinocandin- and species-dependent. PE was frequently found with caspofungin, micafungin and anidulafungin but not with rezafungin. This study is the first to characterize PE and TE trends for rezafungin and helps to inform future in vitro work with this novel echinocandin.

    P057. In vitro antifungal susceptibility testing of cryptic species of Aspergillus: a multi-centre study

    S. Imbert 1, A.C. Normand 2, F. Gabriel 3, C. Bonnal 4, L. Lachaud 5, D. Costa 6, S. Cassaing 7, L. Hasseine 8, L. Kristensen 9, C. Schuttler 10, H. Raberin 11, S. Brun 12, R. Piarroux 1 and A. Fekkar 1
    1
    Laboratoire De Parasitologie-mycologie, APHP La Pitié Salpétrière, PARIS, France
    2
    Ap-hp, Groupe Hospitalier Pitié-salpêtrière, Service de Parasitologie Mycologie, Paris, France
    3
    Parasitology Mycology, CHU de Bordeaux, Bordeaux, France
    4
    Laboratoire De Parasitologie Mycologie, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, PARIS, France
    5
    Laboratoire De Parasitologie-mycologie, CHU de Montpellier, Montpellier, France
    6
    Laboratoire De Parasitologie-mycologie, CHU de Rouen, Rouen, France
    7
    CHU de Toulouse, Toulouse, France
    8
    Laboratoire De Parasitologie Mycologie, CHU DE NICE, NICE, France
    9
    Department of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark
    10
    Laboratoire BioParisOuest, Levallois Peret, France
    11
    CHU Saint Etienne, Saint Etienne, France
    12
    Parasitology-mycology, Avicenne Hospital, Bobigny, France
    Objectives: In recent years, Aspergillus species taxonomy has been revolutionized with the introduction of the concepts of cryptic species and species complex. However, these “new” species remain usually rare and their characteristics, including antifungal susceptibility, are little known. Thus, the aim of this study was to collect a large number of isolates per cryptic species and to assess their antifungal susceptibility.
    Methods: From September 2017, users of the MSI application, a free online MALDI-TOF mass-spectrometry database, were asked to send to our University Hospital, isolates they identified as cryptic species of Aspergillus. Identification at the species level was confirmed by sequencing a part of beta-tubulin and calmodulin genes. Antifungal susceptibility for azole drugs (itraconazole, voriconazole, posaconazole and isavuconazole) and amphotericin B was assessed by EUCAST broth microdilution reference method.
    Results: During the study period, 192 isolates were collected from 11 centers in France and Denmark and were assessed for antifungal susceptibility. According to sequence-based identification, isolates corresponded to 43 species, belonging to 6 species complexes. Nidulantes species complex was the most represented (53 isolates, 12 species), followed by the species complexes Circumdati (43 isolates, 10 species) and Fumigati (40 isolates, 8 species). The last species complexes represented were Usti (23 isolates, 4 species), Flavi (19 isolates, 5 species) and Terrei (14 isolates, 4 species). Regarding the 3 main species complexes, diversity in antifungal susceptibility between cryptic species was observed. Indeed, inside the Fumigati complex A. thermomutatus, A. lentulus, A. udagawae, A. fischeri and (15, 7, 3 and 3 isolates respectively) showed pan-azole high minimal inhibitory concentrations (MICs), whereas A. hiratsukae (8 isolates) showed low MICs. All these species had low MICs for amphotericin B, except A. lentulus with MICs ³4 mg/L. Regarding the Circumdati species complex, A. westerdijkiae (12 isolates) showed the highest MICs for amphotericin B (> 16 mg/L), whereas A. sclerotiorum (17 isolates) showed lower MICs (MIC50 = 4 mg/L). In contrast A. sclerotiorum showed pan-azole high MICs, whereas azole MICs where much lower for A. westerdijkiae. Finally, inside the Nidulantes complex, all species showed low MICs for all drugs, except A. creber and A. unguis (6 and 2 isolates respectively) which showed high MICs (> 8 mg/L) for itraconazole only. MICs regarding the 3 other species complexes studied are shown in Table 1.
    Jof 05 00095 i035
    Conclusion: This study brings evidence on the diversity of antifungal susceptibility pattern inside the same species complex. However, including more isolates is needed to confirm these preliminary results. Nevertheless, this underline the importance of an accurate and easy-to-perform identification of Aspergillus at the species level for the management of Aspergillus diseases and the conduct of epidemiological studies.

    P058. Species identification, in vitro antifungal susceptibility testing and human pathogenicity of Fusarium species: a European multicenter study

    S. Imbert 1, A.C. Normand 1, S. Brun 2, S. Ranque 3, L. Hasseine 4, D. Costa 5, S. Cassaing 6, E. Chryssanthou 7, C. Bonnal 8, L. Delhaes 9, E. Rubio 10, N. Bourgeois 11, C. Schuttler 12, J. Guitard 13, M. Sautour 14, G. Jost 15, M. Brandenberger 16, L. Kristensen 17, B. Sendid 18, C. Gronfors Seeth 19, E. De Laere 20, M. Hendrickx 21, V. Sainte Rose 1, R. Piarroux 1 and A. Fekkar 1
    1
    Laboratoire De Parasitologie-mycologie, APHP La Pitié Salpétrière, PARIS, France
    2
    Parasitology-mycology, Avicenne Hospital, Bobigny, France
    3
    IHU-Méditerranée Infection, Marseille, France
    4
    Laboratoire De Parasitologie Mycologie, CHU DE NICE, NICE, France
    5
    Laboratoire De Parasitologie-mycologie, CHU de Rouen, Rouen, France
    6
    CHU de Toulouse, Toulouse, France
    7
    Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden
    8
    Laboratoire De Parasitologie Mycologie, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, PARIS, France
    9
    Laboratoire De Parasitologie-mycologie, CHU de Bordeaux, Bordeaux, France
    10
    Barcelona hospital, Barcelona, Spain
    11
    Laboratoire De Parasitologie-mycologie, CHU de Montpellier, Montpellier, France
    12
    Laboratoire BioParisOuest, Levallois Peret, France
    13
    Parasitology-mycology, APHP, hôpital St Antoine, Paris, France
    14
    Laboratoire Parasitology Mycology, CHU Dijon, Dijon, France
    15
    Dianalabs, Geneve, Switzerland
    16
    synlab, Lucerne, Switzerland
    17
    Department of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark
    18
    Laboratory of Mycology and Parasitology, University Hospital of Lille, Lille Cedex, France
    19
    Clinical Microbiology, Karolinska University Hospital, karolinska Institutet, Stockholm, Sweden
    20
    AZ Roeselare, Roeselare, Belgium
    21
    Mycology, Scientific Institute of Public Health, Brussels, Belgium
    Objectives: Fungi belonging to the genus Fusarium are responsible of a large number of infections that may be difficult to treat because of a low susceptibility to antifungal drugs available. However, in recent years, fungal taxonomy has been revolutionized with the introduction of the concepts of cryptic species and species complex. This is particularly true for the genus Fusarium, which taxonomy remains partially unclear. In consequence, pathogenicity and antifungal susceptibility specific to each Fusarium species is largely unknown. Thus, the aim of this study was to collect a large number of isolates per species, to collect clinical data on their pathogenicity and to assess their antifungal susceptibility.
    Methods: During 1 year (January-December 2018), users of the MSI application, a free online MALDI-TOF mass-spectrometry database, were asked when they identified a Fusarium sp. to send prospectively to our University Hospital laboratory the isolates and to collect clinical data. Identification at the species level was confirmed by sequencing a part of TEF1α gene. Antifungal susceptibility for azole drugs (voriconazole, posaconazole and isavuconazole), terbinafin and amphotericin B was assessed by measuring minimal inhibitory concentrations (MICs) by EUCAST broth microdilution reference method.
    Results: During the study period, 145 isolates from 20 centers in 6 European countries were collected. According to sequence-based identification, 23 species were identified and were distributed in 6 species complexes (SC) whose main ones are Fusarium oxysporum SC (FOSC, 54 isolates), Fusarium solani SC (FSSC, 48 isolates) and Fusarium fujikoroi SC (FFSC, 36 isolates). Regarding these 3 main species complexes, minimal inhibitory concentrations which inhibited 50% of our isolates (MIC50) were high for all azole drugs, FSSC showing the highest MICs for voriconazole. For amphotericin B, MIC50 were not high (1 mg/l) and no differences were observed between complexes. Finally, terbinafine susceptibility seemed to be related to the species complex. Indeed, FOSC and FSSC showed the highest MICs (MIC50 = 16 mg/l) whereas FFSC showed lower MICs (MIC50 = 2 mg/l). MICs results are shown in Table 1.
    Jof 05 00095 i036
    Among isolates collected, clinical data were available for 117 isolates. They were responsible for 11 invasive/disseminated infections, 19 keratitis and 40 superficial infections (34 onychomycosis and 6 intertrigo). Sixteen isolates were considered as pulmonary airways colonization, whereas 31 were considered as culture contaminants. The three main species complexes identified in the study were represented in each category.
    Conclusion: This study brings evidence on the diversity of Fusarium species implicated in human diseases and their differences in terms of in vitro antifungal susceptibility, mainly for terbinafin. This underlines the importance of an accurate and easy identification of Fusarium species, at least at the complex level, to improve therapeutic management of Fusarium-related diseases.

    P059. Tacrolimus and not cyclosporin A or sirolimus interacts synergistically in vitro with isavuconazole against Aspergillus species

    P. Schwarz 1,2 and E. Dannaoui 3,4
    1
    Department of Internal Medicine - Respiratory and Critical Care Medicine, University Hospital Marburg, Marburg, Germany
    2
    Center For Invasive Mycoses and Antifungals, Philipps University Marburg, Marburg, Germany
    3
    Hôpital Européen Georges Pompidou, Laboratoire de Parasitologie-Mycologie, Paris, France
    4
    Working Group Dynamyc, Faculté de Médecine, Hôpital Henri Mondor, Créteil, France
    Objectives: Invasive aspergillosis is a devastating disease in immunocompromised patients. It mostly affects patients with hematological malignancies, especially those with severe and prolonged neutropenia, but is also encountered in solid organ transplant recipients. Immunosuppressive agents are used as anti-rejection drugs in organ transplant patients, but besides their immunosuppressive activity, these drugs also possess intrinsic antifungal activity. The aim of the present study was to assess the in vitro interaction of the broad-spectrum azole isavuconazole with the calcineurin inhibitors (e.g. tacrolimus or cyclosporin A) or the mTOR pathway inhibitor (e.g. sirolimus) against the most important Aspergillus species responsible for human disease.
    Methods: A panel of 30 Aspergillus isolates belonging to 5 species responsible for human invasive aspergillosis was used for the experiments (10 Aspergillus fumigatus, 5 Aspergillus flavus, 5 Aspergillus terreus, 5 Aspergillus nidulans, and 5 Aspergillus niger). Combinations of isavuconazole with cyclosporine A, tacrolimus or sirolimus were tested by a broth microdilution checkerboard technique based on the EUCAST reference methodology. Plates were read spectrophotometrically, and 90% of inhibition was used as an endpoint for both, the drugs alone and in combination. Results were interpreted with the fractional inhibitory concentration index (FICI). Drug interactions were defined as synergistic (FICI≤0.5), indifferent (FICI>0.5≤4) or antagonistic (FICI>4).
    Results: Isavuconazole MICs ranged from 0.25 to 16 µg/ml. When tested alone, immunosuppressive drugs showed poor activity with MICs < 16 µg/ml for only 2, and 3 isolates for tacrolimus and cyclosporin A, respectively. Combinations of isavuconazole with tacrolimus, cyclosporine A or sirolimus were synergistic for 53, 20, and 10% of the tested isolates, respectively. There were some differences between species (Table). Antagonism was never seen.
    Jof 05 00095 i037
    Conclusion: Calcineurin is a key enzyme in the calcium-dependent signal transduction pathways of eukaryotes. Antifungal drug resistance and virulence of several pathogenic fungi, including Aspergillus species, is regulated by this pathway. Results of the present study showed synergistic interactions between isavuconazole and tacrolimus and underline the role of the calcineurin pathway as a target for the development of new antifungal drugs.

    P060. Synergistic antifungal effect of fluconazole combined with pure culture extract of Candida albicans against Trichophyton mentagrophytes

    T. Lemes 1, B. De Almeida 2, M. Caetano 3, C.R. Polaquini 4, L.O. Regasini 5, T. Maschio-Lima 3, N. Brizzotti 1,6, E. Castilho 6, J.P. Siqueira 7, M. Marques 6 and M. Almeida 8
    1
    Microbiologia, Unesp, São José do Rio Preto, Brazil
    2
    Unesp, São José do Rio Preto, Brazil
    3
    UNESP, São José do Rio Preto, Brazil
    4
    São Paulo State University - Institute of Biosciences, Humanities and Exact Sciences (UNESP - IBILCE), São José do Rio Preto, Brazil
    5
    Chemistry and Environmental Sciences, UNESP (Sao Paulo State University), São José do Rio Preto, Brazil
    6
    FAMERP, São José do Rio Preto, Brazil
    7
    Infeciosas Disease, FAMERP, São José do Rio Preto, Brazil
    8
    Infectious Disease, Medical School, sao jose do rio preto, Brazil
    Objectives: The present study evaluated the synergistic interaction between a pure culture extract of Candida albicans and fluconazole against Trichophyton mentagrophytes.
    Methods: This study used clinical strains of C. albicans from the Laboratory of Microbiology of the Medical School in São José do Rio Preto (FAMERP), Brazil. A 500-mL Inoculum prepared in Sabouraud Dextrose Broth was filtered through a 0.2 μm millipore membrane and separated using ethyl acetate as a counter-phase. The ethyl acetate phase was completely dried using a rotary evaporator and subsequently solubilized in sterile distilled water with 10% dimethyl sulfoxide (DMSO). Minimal Inhibitory Concentration (MIC) tests were performed for T. mentagrophytes strains following the Clinical and Laboratory Standards Institute (CLSI) M38-A2 guidelines. After obtaining the extract’s MIC, a checkerboard trial with fluconazole was performed to evaluate the synergistic interaction with the extract based on the calculation of the fractional inhibitory concentration index (ICIF) = (MIC fluconazole in the mix / MIC fluconazole alone) + MIC extract in the mix / MIC extract isolated). The synergistic interaction was classified using the method described by Kumar et al., where values ≤ 0.5 indicate significant interactions.
    Results: The results obtained for the C. albicans extract’s MIC against the T. mentagrophytes strain and fluconazole in isolation were 1000 μg/mL and 16 μg/mL, respectively. However, when the extract was used in combination with fluconazole, the MIC value was 0,001 μg/mL and of fluconazole it was 2 μg/mL with an ICIF value of 0,01.
    Conclusion: The extract of C. albicans in isolation shows antifungal activity against T. mentagrophytes, which may influence the laboratory diagnosis of mixed infections. Furthermore, the action of this extract is greater in association with an azole derivative, thus proving synergy. In the future, the isolation and identification of extract compounds may allow new therapeutic approaches in the control of fungal infections.

    P061. Screening of antifungal susceptibility of Malassezia pachydermatis isolates from dogs using disk diffusion test

    S. Hadina 1, I. Benvin 2, Z. Štritof 1, J. Habuš 1, V. Stevanović 1, M. Perharić 1, K. Martinković 1, V. Mojčec 1, L. Barbić 1, Z. Milas 1, V. Starešina 1, N. Turk 1 and L. Pinter 1
    1
    Department of Microbiology and Infectious Diseases With Clinic, Faculty of Veterinary Medicine, The University of Zagreb, Zagreb, Croatia
    2
    Faculty of Veterinary Medicine, The University of Zagreb, Zagreb, Croatia
    Objectives: Malassezia pachydermatis is lipophilic yeast commonly seen in skin and ear diseases of dogs. Lately, different studies are describing the appearance of reduced susceptibility of M. pachydermatis strains. In addition, reports about its zoonotic potential and emerged number of Malassezia yeast resistance in human medicine, highlight the need for study of its antifungal susceptibility. The problem of surveillance of potential resistance is lying in no established reference method for antifungal susceptibility testing and no interpretative criteria for this yeast. Various studies used different methodologies and interpreted obtained results based on different recommendations. Modified microdilution method is considered the most accurate, but at the same time more expensive and technically demanding to perform in clinical laboratories. Recent study demonstrated good correlation of results obtained by modified CLSI M44-A2 disk diffusion and broth microdilution method, indicating its suitability for in vitro susceptibility testing of M. pachydermatis in epidemiological studies (Pasquetti et al., 2015.). The aim of this study was to screen antifungal susceptibility of M. pachydermatis for miconazole (MCZ) and clotrimazole (CTZ) using disk diffusion method.
    Methods: A total of 96 isolates of M. pachydermatis were recovered from animals with clinical signs of otitis. Identification was based on their ability to grow on Sabouraud dextrose agar and typical macroscopic and microscopic appearance. Two antifungal tablets containing most commonly azole derivatives used for topical therapy were tested: MCZ and CTZ (10 µg, Neo-Sensitabs, Rosco, Denmark). Disk diffusion test was performed using Mueller-Hinton agar supplemented with 2% glucose and 0.5 μg/l methylene blue. Inoculum of M. pachydermatis was prepared in sterile saline from 3 days old cultures, with addition of the lipid source, containing approximately 1-5x107 CFU/mL. Readings were performed 72 hours after incubation at 37°C, and interpreted according to guidelines provided by manufacturer.
    Results: Out of 96 tested, all isolates demonstrated good susceptibility to CTZ with variations of inhibition zones from 34 to 73 mm (mean ± SD: 56 ± 8,06 mm). Susceptibility testing against MCZ resulted with the range of inhibition zones from 14 to 65 mm (mean ± SD: 41 ± 6,77 mm) and good sensitivity of 95 strains, while one isolate demonstrated intermediate sensitivity. Comparison of the size of the zones showed that CTZ had larger zones of inhibition than MCZ indicating its higher antifungal activity.
    Conclusion: Screening of antifungal susceptibility of M. pachydermatis using disk diffusion method resulted with identification of one strain with reduced susceptibility to MCZ. However, taking into consideration that there are no standardized interpretative criteria for Malassezia yeast, follow up study using broth microdilution assay is needed, in order to check if this isolate could be categorized as potentially resistant. Reference: Pasquetti, M., E. Chiavassa, P. Tizzani, P. Danesi, A. Peano (2015): Agar diffusion procedures for susceptibility testing of Malassezia pachydermatis: Evaluation of Mueller-Hinton agar plus 2% glucose and 0.5 µg/mL methylene blue as the test medium. Mycopathologia 180, 153-158.

    P062. Susceptibility to voriconazole of Aspergillus spp. strains, isolated from patients in Russia

    N. Vasilyeva 1, I. Vybornova 2 and T. Bogomolova 1
    1
    Kaschkin Research Institute of Medical Mycology. Department of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation
    2
    Kaschkin Research Institute of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation
    Objectives: Azole resistance of aspergillosis etiologic agents is an emerging problem in Europe and other parts of the world. The aim of the study: to determine susceptibility to voriconazole of Aspergillus spp. strains isolated from patients with aspergillosis in Saint Petersburg, Russia.
    Methods: Fungal species identification was done using morphological and molecular methods. A total of 230 strains of Aspergillus spp. isolated from biological samples of patients with different clinical forms of aspergillosis (invasive, chronic and allergic) in Saint Petersburg during 2014 – 2019 yrs. have been tested by standard disk diffusion method according to CLSI M51A Document. For 34 strains MICs of voriconazole have been determined according to EUCAST Document E Def 9.3.1.
    Results: Species distribution among Aspergillus spp. isolates was as follows: A. fumigatus – 68%, A. niger – 16%, A. flavus – 13%, rare species – 3% (A. terreus – 4 strains, A. calidoustus – 2, A. ustus – 1, A. sydowii – 1). By disk diffusion method no voriconazole resistant isolates were found among common species: A. fumigatus, A. niger, A. flavus. Only 4 resistant strains were revealed among rare species: A. terreus – 1, A. calidoustus – 2, A. ustus – 1 (inhibition zone diameter < 17 mm). Voriconazole MICs for A. fumigatus, A. flavus, A. niger strains ranged from 0,125 to 0,5 mg/L. Only A. calidoustus showed high MIC – 4 mg/L.
    Conclusion: Resistance to voriconazole among etiologic agents of aspergillosis in Saint Petersburg, Russia was found only among rare fungal species (A. calidoustus, A. ustus, A. terreus). Future surveillance is necessary to monitor antifungal resistance.

    P064. Antifungal susceptibility testing of Malassezia furfur

    T. Bogomolova 1, T. Bogdanova 2, N. Vasilyeva 1, A. Alexeyev 1 and V. Spiridonova 3
    1
    Kaschkin Research Institute of Medical Mycology. Department of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation
    2
    Department of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation
    3
    Kaschkin Research Institute of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation
    Objectives: Malassezia spp. may be etiologic agents of superficial and invasive mycoses. There are no standard protocols for evaluation of antifungal susceptibility of these yeasts. The aim of the study was to determine Malassezia furfur isolates susceptibility to antifungal agents by two different methods.
    Methods: Disk diffusion method CLSI M44A2 was modified by addition of lipids to the Muller-Hinton agar. Commercial test panels Sensititre Yeast One were also supplemented by fatty acid RPMI 1640.
    Results: We tested 11 strains of M. furfur isolated from blood of pediatric patients. Both methods showed resistance to fluconazole and voriconazole. By Sensititre Yeast One panels the following MICs were obtained: amphoterecin B – 1 mg/L, fluconazole > 64 mg/L, caspofungin >8 mg/L, voriconazole – 1-8 mg/L, anidulafungin >8 mg/L, micafungin >8 mg/L, 5-fluorocytozne - > 256 mg/L, itraconazole - < 0,015, mg/L, pozaconazole – 0,03-0,06 mg/L.
    Conclusion: Disk diffusion method may be used for screening antifungal resistance in Malassezia spp. isolates. Itraconazole and pozaconazole demonstrated highest activity against M. furfur.

    P065. Evaluation of the efficacy of antifungals against invasive aspergillosis in an invertebrate animal model

    J. Sana 1, É. Melloul 1, J. Guillot 1, F. Botterel 1,2 and E. Dannaoui 1,3
    1
    8 Rue Du General Sarrail, DYNAMYC, Creteil, France
    2
    Laboratoire De Parasitologie-mycologie, APHP Hôpital Henri Mondor, Créteil, France
    3
    Hôpital Européen Georges Pompidou, Laboratoire de Parasitologie-Mycologie, Paris, France
    Objectives: Evaluation of new therapeutic strategies for the treatment of invasive aspergillosis is needed due to emergence of azole-resistance in Aspergillus fumigatus. Animal models in rodents are generally used but are costly and their use is limited by ethical considerations. For these reasons, evaluation of antifungal efficacy in mini-host models such as Galleria mellonella is a promising alternative. The aim of our study was to develop an invasive Aspergillus infection in larvae of Galleria mellonella to evaluate the efficacy of antifungals.
    Methods: Three clinical strains of Aspergillus fumigatus (HEGP064, HEGP4017 and HEGP2666) have been used. Minimum inhibitory concentration for voriconazole (VRZ) was 0.5, 0.5 and 8µg/mL for HEGP064, HEGP4017 and HEGP2666 respectively. Inoculum finding experiments (with spore suspensions at 105 to 108 conidia/mL) have been performed to determine the concentration that gave 90% mortality at seven days post-infection (LD90). The presence of an invasive infection has been demonstrated by direct examination of tissues stained with Gomori-Grocott (GG) and by histopathology (H&S staining). In a first set of experiments, groups of 10 larvae were infected with the LD90 and treated with monotherapy by voriconazole (VRZ, [Vfend®] at 0.5, 1, 2, 4, and 8 μg/larva), amphotericin B (AMB, [Fungizone®], or caspofungin (CSP, [Cancidas®] at 0.2, 0.4 and 0.8 μg/larva). Subsequently, HEGP4017 or HEGP2666 infected larvae were treated after two hours by VRZ at 4µg/ml alone and in combination with CSP at 4, 2 or 1µg/larvae. The mortality was estimated daily for 7 days.
    Results: After infection, an invasive aspergillosis was obtained as shown by the presence of hyphae in tissues. The LD90 values for the HEGP064, HEGP4017 and HEGP2666 strains were 4.38×107,9.59×107 and 1×108 CFU/ml respectively. In infected and untreated groups, mortality was at least 90% on day 7 post-infection. The mortality decreased with increasing doses of amphotericin B. At 4 µg/larvae, the mortality was on average 20% for the 3 strains. In animals treated with VRZ, the mortality rates on day 7 post-infection were 35%, 40% and 60% for HEGP064 and were 55%, 65% and 95% for HEGP4017 and 85%, 95% and 95% for 8, 4 and 2 μg/larva, respectively (p = 0.0001 compared to control). VRZ at 8, 4 and 2 μg/larva was statistically more effective for the treatment of larvae infected with HEGP064 or HEGP4017 compared to those infected by HEGP2666. Administration of VRZ 4 did not decreased, significantly, the mortality of HEGP2666 infected larvae (p = 0.304) in contrast to those infected by HEGP4017 (p = 0.0082). The bitherapy VRZ 4µg/larvae and CSP 4µg/larvae lead to a more significant decrease in mortality compared to monotherapy (VRZ 4µg/larvae) and control group infected by HEGP4017(p = 0.026) and HEGP2666 (p = 0.005) strain.
    Conclusion: G. mellonella is a reliable model for inducing invasive aspergillosis. There was a clear dose-response when animals were treated with VRZ and AMB. This model could be used for screening and evaluation of different therapeutic strategies against Aspergillus spp.

    P066. Analysis of the efflux-pump activity against resistant Sporothrix schenckii complex isolates

    M.L. Scroferneker 1, T.R. Pinheiro 2, G. Seibert 2, A.L.R. Poletto 3, J.V. Prade 3 and C.D.O. Stopiglia 3
    1
    Department of Microbiology, Immunology and Parasitology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
    2
    Universidade Federal do Pampa, Uruguaiana, Brazil
    3
    Universidade Federal do Pampa, Uruguaiana, RS, Brazil
    Objectives: The present study aims to assess the efflux-pump activity in the resistant Sporothrix schenkii complex strains.
    Methods: The minimum inhibitory concentration (MIC) of itraconazole (0.03125-16 µg/mL) was determined through broth microdilution method, as described by protocol M38-A2 of the Clinical and Laboratory Standards Institute (CLSI, 2008). Ten clinical isolates of Sporothrix schenckii complex were prepared from 7-day-old cultures grown on potato dextrose agar at 28 °C. The inocula were adjusted to a final concentration of 0.5–2.5×103 cells/mL. The microdilution plates were incubated at 35 °C for 72h. The MIC of itraconazole was defined as the lowest drug concentration capable of inhibiting total of growth. For the analysis of the efflux-pump activity, the resistant Sporothrix schenckii complex strains (04/10) were tested against two efflux-pump inhibitors, promethazine and verapamil by broth microdilution method. Posteriorly, susceptibility test was performed with itraconazole and sub-inhibitory concentrations of promethazine (MIC/8 = 24 µg/mL) and verapamil (MIC/8 = 100µMol).
    Results: There were no reduction of MIC for itraconazole in the presence of the verapamil and prometazine inhibitors.
    Conclusion: Thus, the present study suggests that the mechanism of resistance of the isolated evaluated isn’t the presence of efflux pumps.

    P067. Terbinafine resistant Trichophyton mentagrophytes ITS genotype VIII from India in Germany

    P. Nenoff 1, A. Süβ 2, A. Ludes 3, A. Schuller 4, E. Fischer 5, S. Dessoi 6, W. Hofmann 6, S. Schmidt 7, S.B. Verma 8, M. Monod 9, K. Salamin 9, C. Krueger 1, A. Ebert 1 and S. Uhrlass 1
    1
    Partnership Dr. C. Krueger & Prof. P. Nenoff, Laboratory of medical microbiology, Roetha OT Moelbis, Germany
    2
    Hautärztin, Gemeinschaftspraxis Allgemeinmedizin und Dermatologie, Wittlich, Germany
    3
    Allgemeinmedizinische Praxis Dr. med. Alfons Ludes und Thorsten Koech, Leiwen, Germany
    4
    Hautarztpraxis, Halle/Saale, Germany
    5
    Hautarztpraxis Dr. med. Gunhild Kratzsch, Leipzig, Germany
    6
    Hautzentrum Nordwest, Frankfurt am Main, Germany
    7
    Hautarztpraxis im MVZ Friedrichstadt, Dresden, Germany
    8
    “Nirvan” and “In Skin” Clinics, Vadodara, India
    9
    Dermatology Service, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
    Case Report: Objectives: In India, a dramatic increase of chronic recalcitrant dermatophytoses due to Trichophyton (T.) mentagrophytes genotype VIII is on the rise. Due to travelling and migration, globalization of the mostly terbinafine resistant strains of the causative agent from India to other parts of the world is assumed. First strains of T. mentagrophytes genotype VIII have reached Germany, now. Patients: 1) A 6 months-old-female infant from Bahrain visiting Germany with her family for a holiday was seen for extensive dermatophytosis of the back, buttocks, chest and groins. Topical treatment by terbinafine for over 2 months was not successful. 2) A 28 years old male from Libya, living for 3 years in Germany, suffered from tinea cruris and tinea faciei involving the left upper and lower eyelids. Treatment by oral fluconazole and by terbinafine failed. His German girlfriend was affected by the dermatomycosis, too. Her child was free of signs of a dermatophytosis. The patient had no contact to India and Indians or Arabs, he did not visit Libya in the last years. The man, however, regularly goes to the gym. 3) After a trip to Saudi Arabia, a pregnant German woman suffered from tinea cruris and tinea corporis (trunk). Her husband was affected, too. 4) An Indian male, living in Germany, was treated because of a dermatomycosis of the buttocks and groins by oral terbinafine, without improvement. 5) A couple from Iraq, living for long time in Germany, suffered from chronic recalcitrant dermatophytosis of the groins for at least 1.5 years. Repeated topical treatments by „Combo Creams“ failed. Antifungal topical preparations (ciclopirox olamine, sertaconazole) for 5-6 weeks acted very slowly, progression of the dermatomycoses lead to therapy break-off. Oral itraconazole 200 mg daily was started. Results: Mycological examination of skin scrapings from all 5 patients revealed the detection of T. mentagrophytes. Four out of the five dermatophyte isolates were subject to sequencing of the “internal transcribed spacer” (ITS) region of the fungal rDNA. All these 4 strains belonged to the newly described genotype VIII of T. mentagrophytes. The until now investigated point mutations revealed for patient 1) TTC/TTA mutation of the squalene epoxidase (SQLE) gene which is closely associated with Phe397Leu amino acid substitution of the enzyme. T. mentagrophytes isolates of patient 2) and 3) were in vitro sensitive to terbinafine. The T. mentagrophytes strain of patient 3) showed GCT/ACT mutation (Ala448Thr). The above mentioned Phe397Leu and Ala448Thr substitutions are indicative of in vitro resistance of the dermatophyte against terbinafine. Conclusion: Transmission of the here found Indian ITS genotype VIII of T. mentagrophytes to other countries due to globalization is a serious issue to be considered. Moreover, a significant percentage of these Indian T. mentagrophytes strains are resistant to terbinafine both in vitro and due to point mutations in the SQLE gene. Some strains are also found to be partially resistant against itraconazole and voriconazole. The here described patients represent the first reports on an infection due to terbinafine-resistant T. mentagrophytes of the ITS genotype VIII from India in Germany.

    P068. Comparison of two antifungal susceptibility tests for Malassezia yeasts

    A. Abdillah 1, A. Packeu 2, S. Khelaifia 3, M. Hendrickx 4 and S. Ranque 1
    1
    Vitrome, Aix Marseille Univ, IRD, AP-HM, SSA, Marseille, France
    2
    Mycology & Aerobiology, Sciensano, Brussels, Belgium
    3
    Culturomics, IHU Méditerranée Infection, Marseille, France
    4
    Service of Mycology and Aerobiology, Bccm/ihem Fungal Collection, Sciensano, Brussels, Belgium
    Objectives: The lipid-dependent Malassezia spp. yeast are members of the normal human skin microbiota that are occasionally involved in various skin diseases or in blood stream infections in immunocompromised and neonates patients. A better knowledge of the antifungal susceptibility testing (AFST) profiles of these yeast is pivotal to enhance antifungal treatment strategies. This study aimed to compare two broth microdilution assays for the in vitro AFST of Malassezia spp.
    Methods: A total of 19 strains of the BCCM /IHEM collection, including 4 Malassezia species: (n = 6) M. furfur, (n = 4) M. pachydermatis, (n = 5) M. sympodialis and (n = 4) M. slooffiae were tested against ketoconazole, voriconazole, terbinafine and fluconazole using the broth microdilution method (CLSI - M27-A2). Two different media were used as diluent, modified Dixon broth (mDIXB) and FastFung broth, a new medium derived from the Schaedler agar. MIC values were visually read after 48 h of incubation at 30°C. The essential agreement (EA) (±1 or 2 dilution) of the MIC values between both media was estimated. The inter-laboratory reproducibility between Marseille and Brussels was tested.
    Results: Ketoconazole, voriconazole and terbinafine MICs (mg/l) were low, with MIC90 values of 0.06 to 0.5, 0.03 to 0.125, and 0.125, in mDIXB, and of 0.03 to 0.25, 0.03 to 0.125, and 0.06 to 1, in FastFung broth, respectively. Fluconazole displayed the highest MIC values. The EAs varied from 84.2 to 100% for ±1 dilution while EAs of 100% were observed for ±2 dilution. The inter-laboratory EAs (±2 dilutions) ranged from 73.8 to 100% and 94.7 to 100% in mDIXB or FastFung broth, respectively.
    Conclusion: Our findings showed that both media performed similarly, with relatively higher EAs (>60%), except for terbinafine. The FastFung broth can be used as alternative medium for Malassezia in vitro AFTS.

    P075. The effect of farnesol on Candida auris biofilms, virulence and azole susceptibility

    F. Nagy 1, Z. Tóth 1, L. Forgács 1, A. Borman 2, L. Majoros 1, B. Balázs 1 and R. Kovács 1
    1
    Department of Medical Microbiology, University of Debrecen, Debrecen, Hungary
    2
    Public Health England UK National Mycology Reference Laboratory, Bristol, United Kingdom
    Objectives: Farnesol is a fungal quorum-sensing molecule, which has a pivotal role in regulation of fungal morphogenesis. The aim of our study was to examine the effect of farnesol on biofilms, azole susceptibility (fluconazole, voriconazole, itraconazole, posaconazole, isavuconazole) against Candida auris clinical isolates as well as in vivo tissue fungal burden in a murine bloodstream infection model.
    Methods: The effect of farnesol on C. auris cells was tested in three experiments: i) biofilm forming ability of cells pre-treated with farnesol for 24-hours prior to biofilm formation, ii) biofilm forming ability of cells continuously exposed to farnesol for 24-hours during biofilm formation, iii) effect of farnesol on one-day-old biofilms. Farnesol concentrations tested were 10, 50, 100, 300 μM in all experiments. Metabolic activity of sessile cells was determined at 0, 2, 4, 6, 8, 10, 12 and 24 hours using XTT-assay. One-way ANOVA with Dunnett’s post-testing was used to analyze the metabolic activity change exerted by different farnesol concentrations compared to untreated control. Interactions between farnesol and azoles were examined against one-day-old biofilms by broth microdilution checkerboard method. The tested concentrations were 1.17-300 μM and 0.125-512 mg/L for farnesol and azoles, respectively. Fractional inhibitory concentration index (FICI) was used to assess the nature of interactions. Synergy was specified if the FICI was ≤0.5, between >0.5 and 4 was indifferent and as antagonistic if the FICI was >4. MICs were determined as at least 50% reduction of metabolic activity compared to control in case of individual drugs and isoeffective combinations. Farnesol pre-exposed (75 μM) cells were inoculated intravenously into immuncompromised female BALB/c mice to examine the day six fungal burden in kidneys, heart and liver.
    Results: The one-day-long farnesol pre-exposure (50-300μM) led to formation of biofilms with significant metabolic activity inhibition after 24 hours (p<0.01). Biofilms formed under farnesol exposure showed significantly decreased metabolic activity in first the 12, 12, 10 and 8 hours for 300, 100, 50 and 10 μM farnesol, respectively (p<0.05-0.001). In case of one-day old biofilms treated with farnesol after formation, only the highest farnesol concentration caused significant inhibition at 24 hours (p<0.001).The median MIC values of azoles and farnesol against sessile C. auris clinical isolates are showed in Table 1. Based on FICI values, farnesol was synergistic with all tested azoles (FICIs ranged from 0.061 to 0.375) (Table 1). Fungal tissue burdens caused by farnesol pre-exposed C. auris were significantly higher in all examined organs compared to organs infected by farnesol-unexposed fungal cells at sixth day postinoculation.
    Jof 05 00095 i038
    Conclusion: Farnesol inhibited the biofilm forming ability of C. auris and significantly reduced the metabolic activity of C. auris biofilms in a concentration-dependent manner; in addition, it showed a remarkable synergistic effect with azoles. On the other hand, farnesol pre-exposure led to increased tissue fungal burdens. In summary, though its systemic usage may be hindered by a potential enhancement of fungal virulence, farnesol may be a useful addition to therapy where biofilm formation is important, e.g. in catheter-lock therapy against C. auris.

    P076. Transcriptional and physiological effect exerted by tyrosol on Candida parapsilosis cells

    R. Kovács 1, Á. Jakab 2, Z. Tóth 1, F. Nagy 1, T. Emri 2, I. Pócsi 2 and L. Majoros 1
    1
    Department of Medical Microbiology, University of Debrecen, Debrecen, Hungary
    2
    Department of Molecular Biotechnology and Microbiology, University of Debrecen, Debrecen, Hungary
    Objectives: Tyrosol has a potent antifungal activity against Candida species; however, the background of its antifungal mechanism is still poorly understood. We examined the effect of tyrosol at supraphysiological concentration on growth, biofilm formation, redox homeostasis, virulence as well as on fluconazole susceptibility to reveal the background of its antifungal effect. To gain further insights into the physiological consequences of tyrosol treatment we examined the caused genome wide gene expression changes using RNA-Seq.
    Methods: CLIB 214 Candida parapsilosis (sensu stricto) strain was used and 15 mM tyrosol concentration was applied to examine physiological and transcriptional changes. Following 4 hours incubation of C. parapsilosis precultures, we added 15 mM tyrosol to the cultures then growth was examined at 1-hour intervals by determination of cell density both by means of measuring of the absorbance (at 640 nm) and by counting the living cells. Reactive species were measured in the presence or absence of tyrosol by a technique that converts 2’,7’dichlorofluorescin-diacetate to 2’,7’–dichlorofluorescein. One-day-old biofilm formation ability in the presence of tyrosol was determined by quantitative CFU determination of adhered cells. Interaction between fluconazole and tyrosol was evaluated by two-dimensional broth microdilution chequerboard assay. Afterwards, the nature of interaction was analyzed using Fractional Inhibitory Concentration Index determination. Tyrosol induced effect on virulence was examined in immuncompromised systemic BALB/c mouse model.To obtain global transcriptome data regarding tyrosol treatment, high-throughput mRNA sequencing was performed on Illumina NextSeq sequencing platform.
    Results: Fifteen mM tyrosol caused significant growth inhibition within two hours of the addition of tyrosol (p<0.01). Tyrosol increased the production of reactive oxygen species (p<0.001) and it was associated with elevated superoxide dismutase, glutathione peroxidase and catalase activities (p<0.05). Biofilm formation ability was comparable in the presence or absence of 15 mM tyrosol in terms of living fungal cells.The interaction between fluconazole and tyrosol was antagonistic (FICI 4.5). Fifteen mM daily treatment decreased the fungal tissue burden in the kidneys compare with untreated control (p<0.01).Tyrosol exposure resulted in 1,462 differentially expressed genes. Out of them, 261 and 181 genes with at least 1.5-fold increase or decrease in expression, respectively, were selected for further investigations (Figure 1). Genes involved in ribosome biogenesis showed down-regulation, while genes related to later biofilm events, oxidative stress response and ethanol fermentation were up-regulated. In addition, tyrosol treatment up-regulated the expression of certain fluconazole efflux pump genes including MDR1 and CDR1 and down-regulated the FAD2 and FAD3 virulence genes belonging to desaturated fatty acid formation.
    Jof 05 00095 i039
    Figure 1 Up-regulated (red) and down-regulated (dark blue) genes were defined as differentially expressed genes (p<0.05) where log2(FC)>0.585 or log2(FC)<–0.585, respectively, and FC stands for fold change FPKM (fragments per kilobase per million mapped fragments) value.
    Conclusion: Tyrosol exposure enhanced oxidative stress response and efflux pumps, while inhibited growth and ribosome biogenesis as well as virulence. Metabolism was modulated towards glycolysis and ethanol fermentation. Primer adherence was inhibited, and rather later biofilm events was enhanced. Our findings suggest that tyrosol may be a potential locally active and/or adjuvant agent in the development of alternative treatments targeting quorum-sensing in the future.

    P077. Nonyl 3,4-dihydroxybenzoate as a potent anti-biofilm compound for the treatment of dermatophytosis

    C. Orlandi 1, R.H. Pires 2, N.M. Bila 1, A. Serafim-Pinto 1, J.L. Bonatti 1, L. Scorzoni 1, J. Singulani 1, C. Santos 1, C.R. Andrade 3, P. Silva 4, M. Chorilli 4, L.O. Regasini 5, A.M. Fusco Almeida 1 and M.J. Soares Mendes Giannini 1
    1
    Clinical Analysis, UNESP (Sao Paulo State University), Araraquara, Brazil
    2
    Health Promotion, UNIFRAN (University of Franca)), Araraquara, Brazil
    3
    Physiology and Pathology, UNESP (Sao Paulo State University), ARARAQUARA, Brazil
    4
    Drugs and Pharmaceuticals, UNESP (Sao Paulo State University), Araraquara, Brazil
    5
    Chemistry and Environmental Sciences, UNESP (Sao Paulo State University), São José do Rio Preto, Brazil
    Objectives: This study aimed to verify the in vitro susceptibility of nonyl 3,4-dihydroxybenzoate against planktonic cells and biofilms of dermatophytes. In addition, efficacy and toxicity were tested in a murine model of dermatophytosis. The compound was incorporated into nanostructured lipid systems (NLS) in order to prove whether the incorporation was able to maintain its antifungal activity as well as, reduce toxicity in human cell lines and alternative models. In addition, the probable mechanism of action was evaluated.
    Methods: The preformed biofilms and planktonic cells of Trichophyton rubrum and T. mentagrophytes were treated with different concentrations of synthetic antifungal drugs and nonyl 3,4-dihydroxybenzoate. The metabolic activities were determined using the XTT reduction assay. The effects of the treatments on biofilms structures were visualized by scanning electron microscopy (SEM). The efficacy of the compound was evaluated in wild type (WT) C57BL/6 mice by determining the fungal burden and histopathological analysis. Hepatic and renal toxicity were assessed by blood analysis in the equipment Vitros 250 chemistry analyzer. After incorporation into NLS, susceptibility tests were conducted according the document M38-A2, proposed by the CLSI (2008). The toxicity of incorporation was evaluated in HaCaT cell lines by the sulforhodamine B method and in Caenorhabditis elegans model. Finally, the damage of cell ultrastructure was evaluated by transmission electron microscopy (TEM).
    Results: Both biofilms and planktonic cells were susceptible to nonyl compound in concentrations ranging from 125 – 250 mg/L. T. rubrum and T. mentagrophytes biofilms were significantly more resistant to fluconazole, griseofulvin and terbinafine than planktonic cells (p<0.05). Fluconazole, griseofulvin, terbinafine and nonyl were able to prevent biofilm formation in all reference strains and clinical isolates tested (p<0.05). SEM results revealed that the antifungal drugs caused minor or no damage to the structure of the biofilms, and in some cases, stimulated the development of resistance structures. In contrast, the biofilms treated with the nonyl compound were seriously damaged. The fungal burden was significantly reduced after treatment and histopathological analysis showed complete tissue regeneration. In addition, there was no systemic toxicity after treatment in mice. Nonyl incorporated to NSL formed by the surfactant Brij®98 Epikuron® (98-I) presented the best results of minimum inhibitory concentration (MIC), with values between 1.98 and 3.9 mg/L. Cytotoxicity tests indicated that the incorporation resulted in cell viability greater than 80% at all tested concentrations. These results corroborated with in vivo testing in C. elegans model, which indicated that the incorporation of nonyl significantly increased the survival of larvae. Finally, TEM results indicated that the compound might have more than one mechanism of action in the dermatophytes, causing enlargement of the vacuoles, besides derangement and formation of pores in the plasma membrane, similar to amphotericin B.
    Conclusion: These findings show that nonyl 3,4-dihydroxybenzoate may be a promising antifungal. Funding: CNPq, Capes and FAPESP Process # 2018/02785-9; 2017/18388-6.

    P078. Anti-dermatophyte biofilm activity of photodynamic therapy using 2-chalcone as photosensitizer

    N.M. Bila 1,2, C. Orlandi 1, L.C. Gonçalves 1, C.O. Vaso 1, C. Santos 1, P. Silva 3, M. Chorilli 3, L.O. Regasini 4, F.L. Primo 5, C.R. Fontana 1, A.M. Fusco Almeida 1 and M.J. Soares Mendes Giannini 1
    1
    Clinical Analysis, UNESP (Sao Paulo State University), Araraquara, Brazil
    2
    Para-clinics, UEM (Eduardo Mondlane University), Maputo, Mozambique
    3
    Drugs and Pharmaceuticals, UNESP (Sao Paulo State University), Araraquara, Brazil
    4
    Chemistry and Environmental Sciences, UNESP (Sao Paulo State University), São José do Rio Preto, Brazil
    5
    Bioprocess and Biotechnology, UNESP (Sao Paulo State University), Araraquara, Brazil
    Objectives: This study aimed to evaluate the anti-dermatophytic and anti-biofilm activity of 2-chalcone alone and combined with photodynamic therapy (PDT).
    Methods: Strains of Trichophyton rubrum, T. mentagrophytes and one clinical isolate of Microsporum canis were used. Susceptibility tests were conducted according to the M38-A2 document proposed by CSLI. Planktonic cells, biofilms with 24 h (initial stage) and 72- 96 h of growth (mature) were formed in RPMI-1640 medium and placed in contact with different concentrations of 2-chalcone (1 - 500 mg/L), fluconazole (1,25 - 512 mg/L) and terbinafine (0.002 – 32 mg/L). The metabolic activities after the treatments were quantified using the tetrazolium salt (XTT) reduction method. The topographies of initial and mature biofilms with or without treatment were visualized by scanning electron microscopy (SEM). For the PDT assay, 2-chalcone was used a photosensitizer and a blue led as a source of light with a dose of 150 J.cm-2.
    Results: All the strains, in the planktonic form, were inhibited by treatment with 2-chalcone (MIC = 3.9–7.8 mg/L), terbinafine (MIC = 0.008–0.03 mg/L) and fluconazole (1–64 mg/L). The substance 2- chalcona eradicated the 24 hour biofilm of all strains tested at the concentration of 15.6 mg/L, terbinafine ranging from 0.06 to 16 mg/L, and fluconazole inhibited only the formation of T. mentagrophytes biofilm in the concentration of 32 mg/L. Regarding mature biofilms, only 2-chalcone was able to reduce the metabolic activities in the concentration of 31.2 mg/L. Mature biofilms were resistant to both antifungal drugs tested. When planktonic and biofilms (initial and mature) were treated with PDT using 2-chalcone as photosynthesizer, the inhibitory concentrations were reduced by 4 times (2–8 mg/L). The SEM of biofilms treated with 2-chalcone showed a total collapse of the cell walls, resulting from a probable extravasation of cytoplasmic content.
    Conclusion: The substance 2-chalcone is a promising antifungal drug. Funding: CNPq, IBE process 150/2017, Capes and FAPESP Process # 2018/02785-9; 2017/18388-6.

    P079. Antifungal activity of voriconazole and micafungin against the biofilms and planktonic cells of Candida albicans and Aspergillus fumigatus in vitro

    D. Shi 1 and W. Liu 2
    1
    Jining No. 1 People’s Hospital, Jining, China
    2
    Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing, China
    Objectives: Candida albicans and Aspergillus fumigatus are among the most common invasive opportunistic pathogens known to have high morbidity and mortality. Micafungin (MCF) and voriconazole (VOR) are the most commonly used first-line therapies for these fungi. However, to our knowledge, there are few data comparing the efficacy of MCF and VOR on biofilm and planktonic cells on both fungi.
    Methods: We have carried out an in vitro study to evaluate the efficacy of VOR and MCF against biofilm development and inhibition of fungal viability within biofilm formed by these two pathogenic fungi.
    Results: We find that, when compared with planktonic cells, the corresponding fungal biofilm magnificently decreases drug sensitivities. To inhibit more than 50% of cell viability within a mature biofilm, the concentration of each drug must increase by at least 5 to 10 MIC – depending on the specific drug and fungus combination. With specific regard to biofilm formation itself, we find that MCF is more effective than VOR against developing biofilm in C. albicans, whereas VOR is more effective at high concentration against developing biofilm in A. fumigatus. We also find that the earlier the therapeutic intervention, the greater the biofilm inhibition that will be achieved. The critical time window for biofilm inhibition is 12h for A. fumigatus and 8h for C. albicans; beyond those points in time safe and effective drug intervention is almost impossible.
    Conclusion: Our results provide a few clinical hints that may help in choosing antifungal agents when treating common fungal infections.

    P080. Contribution of Galleria mellonella model to understand interactions between Aspergillus fumigatus and Stenotrophomonas maltophilia: study of human, animal and environmental strains.

    M.-F. Durieux 1,2, É. Melloul 2, L. Roisin 2, P.-L. Woerther 2,3, V. Risco 2,4, J. Guillot 2,4, M.-L. Dardé 1, E. Dannaoui 5, J.-W. Decousser 2,3 and F. Botterel 2,6
    1
    Hopital Dupuytren, Laboratoire de Parasitologie-Mycologie, Limoges, France
    2
    Dynamyc, Créteil, France
    3
    Microbiology, Hôpital Henri-Mondor, Créteil, France
    4
    École Nationale Vétérinaire D’alfort, Unité de Parasitologie-Mycologie, Maisons-Alfort, France
    5
    Hôpital Européen Georges Pompidou, Laboratoire de Parasitologie-Mycologie, Paris, France
    6
    Laboratoire De Parasitologie-mycologie, APHP Hôpital Henri Mondor, Créteil, France
    Objectives: Clinical and environmental strains of Aspergillus fumigatus (Af) and Stenotrophomonas maltophilia (Sm) can colonize the respiratory tract of patients with chronic lung diseases such as cystic fibrosis, specially in biofilm form. The interactions between these two pathogens are not well known (1, 2). For a decade, insect model Galleria mellonella (Gm) can be used to screen microorganism pathogenicity. The aim of this study was to compare several associations of Af – Sm strains of different origin in a Gm model to determine the virulence of these associations.
    Methods: An in vivo infection model with human, animal and environmental strains of Af and Sm, alone and associated, was developed using larvae of Gm. Several couples were tested using different strains of Sm and Af, especially human reference clinical strains (Af13073 and Sm13637). Virulence was analysed by survival curves at 7 days and histology method.
    Results: Mortality rate with Af alone showed no significant difference between human, animal and environmental strains. For Sm, mortality rates showed variations depending on the strains. The association between reference clinical strains Sm13637 and Af13073 showed a synergistic effect in Gm (60% of mortality at 7 days) versus Sm 13637 alone (10% mortality) and Af13073 alone (30% mortality). This effect was not observed with the other co-infections. Gomori-Grocott coloration of histological sections showed fungal nodules disseminated in the larvae and a Gram coloration demonstrated the presence of the bacteria, validating our infection model (3).
    Conclusion: For the first time, we described a coinfection of Af and Sm in a Gm model with an interesting synergistic effect between human clinical strains. This effect did not exist for environmental and animal strains showing a particular fitness between the human strains. These preliminary results provide complementary data to the in vitro data already obtained. The Gm model has demonstrated its relevance for the study of mixed infections, which remains to be improved (2). Indeed, the extracellular matrix could not be identified in Gm, which could validate the presence of mixed biofilm. (1, 2) Melloul et al. 2016 and 2018 (3) Société Française de Mycologie Médicale price of the best oral communication, 2019.

    P081. Antifungal susceptibility of Lictheimia corymbifera biofilms to Amphotericin B formulations and Posaconazole

    A. Vikelouda 1, M. Simitsopoulou 1, C. Antachopoulos 1, L. Skoura 2, A. Chatzimoschou 1, I. Stamouli 1 and E. Roilides 1
    1
    3rd Department of Pediatrics, Aristotle University of Thessaloniki, Thessaloniki, Greece
    2
    Department of Microbiology, Aristotle University, Thessaloniki, Greece
    Objectives: Mucormycosis is an emerging life-threatening opportunistic fungal infection that affects patients with underlying immunosuppressive conditions, such as hematological malignancies, trauma, hematopoietic stem cell transplantation and diabetes mellitus. The ability of Mucorales to form biofilms, structures with reduced susceptibility to antifungal agents, has recently been demonstrated., Lictheimia Corymbifera is the second most common clinically significant Mucorales species and is related mostly to pulmonary and trauma infections. We investigated the antifungal activity of deoxycholate amphotericin B (D-AMB), liposomal amphotericin B (L-AMB), posaconazole (POS) against planktonic cells and mature biofilms of Lichteimia corymbifera. Additionally, we investigated the combinational activity of L-AMB with POS against mature biofilms.
    Methods: Three L. corymbifera clinical strains, were isolated from bronchial secretions and incubated at 10^5 cfu/ml in 96-well microtiter plates at 37οC for 48h. Biofilm formation was assessed by 1% safranin staining and measured spectrophotometrically at 490 nm. In order to determine the MIC, two-fold dilutions of D-AMB, L-AMB and POS (0.007-256 mg/l) were incubated with biofilms or planktonic cells (2x10^5 cfu/ml) for 24h (n = 9). XTT metabolic reduction assay was used to assess biofilm damage compared to controls. MIC50 was determined as ≥50% biofilm damage. The activity of the combination of L-AMB (0.5-32 mg/l) with POS (0.125-64 mg/l) against biofilms was determined using a checkerboard microdilution method (n = 10). Drug interactions were analyzed using Bliss independence model. The combination effect was defined as synergistic, antagonistic or indifferent when the observed biofilm damage was significantly higher, lower or equal to the expected damage, respectively.
    Results: All L. corymbifera strains exhibited strong biofilm formation. Biofilm MIC50 of D-AMB, L-AMB and POS were 2, 4 and >256 mg/l, respectively, as compared to 0.007, 0.03 and 0.03 mg/l for planktonic cells. Synergistic effects were observed at 1-2 mg/l of L-AMB combined with 8-16 mg/l of POS (mean ΔE value of significant interactions:19% [range: 17% to 21%]; mean SE:1% [range: 1% to 3%]). None of the combinations exhibited antagonism.
    Conclusion: L. corymbifera can produce strong biofilms that possess comparable susceptibility profiles to D-AMB and L-AMB, but are resistant to POS. The combination of L-AMB with POS at super-therapeutic concentration ranges exhibits synergistic activity against mature L. corymbifera biofilms.

    P082. Biofilm formation by Histoplasma capsulatum in different culture mediums and oxygen atmospheres

    L.C. Gonçalves 1, C. Orlandi 1, N.M. Bila 1,2, C.O. Vaso 1, R.A. Moraes Da Silva 1, M.L. Taylor 3, M.J. Soares Mendes Giannini 1 and A.M. Fusco Almeida 1
    1
    Clinical Analysis, UNESP (Sao Paulo State University), Araraquara, Brazil
    2
    Para-clinics, UEM (Eduardo Mondlane University), Maputo, Mozambique
    3
    Microbiology and Parasitology, UNAM (Autonomous University of Mexico), México, D.F., Mexico
    Objectives: This work aimed to study Histoplasma capsulatum biofilms, by checking the influence of different culture media and oxygen atmospheres in the development of these communities.
    Methods: The biofilm formation by two strains (ATCC 26029 – G186A and EH315) was characterized in different nutrient conditions [Brain Heart Infusion (BHI), Roswell Park Memorial Institute (RPMI) with 2% of glucose, Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and nutrient medium HAM-F12 (HAM-F12) supplemented with glucose (18.2 g/L), glutamic acid (1 g/L), HEPES (6 g/L), and L-cysteine (8.4 mg/L)] and atmospheres of oxygen (aerobic and microaerophilic). Optical microscopy techniques, XTT reduction assay, as well as crystal violet and safranin staining, and scanning electron microscopy (SEM) were performed.
    Results: The results indicated that, although all the culture media stimulated the maturation of the communities, the nutrient-rich culture media, such as HAM-F12, provided a better biomass and extracellular matrix development when compared to the others. In addition, microaerophilic conditions were the most favorable than aerobic. The topographies observed in SEM showed yeasts surrounded at several points by an exuberant amount of extracellular matrix. However, the biofilms formed in BHI, RPMI and DMEM presented a reversal yeast for significant filamentation, which needs better investigation.
    Conclusion: The results obtained so far represent significant advances for the field of biofilms and open new possibilities for the study of virulence and Histoplasma-host interaction. Funding: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo Process # 2016/11836-0); CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico Process # 130018/2018-0) and Capes (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

    P085. Combined treatment of experimental paracoccidiodomycosis with Itraconazole and low-level LASER therapy

    E. Burger 1, J.C. Grisolia 1, L.A. Santos 1, N.A. Dias 1, L.C.C. Malaquias 1, A.M. Rodrigues 2 and Z.P.D. Camargo 2
    1
    Department of Microbiology and Immunology, Federal University of Alfenas, Alfenas, Brazil
    2
    Department of Microbiology, Immunology and Parasitology, Federal University of Sao Paulo, Alfenas, Brazil
    Objectives: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), requiring long treatment, justifying studies that amplify the therapeutic options. Itraconazole (Itra) is effective for PCM and requires shorter therapy than other drugs. We propose to study a shorter treatment model with fewer side effects through the activation of neutrophils by the simultaneous administration of the antifungal drug Itraconazole and adjuvant therapy with Low Level LASER Therapy (LLLT) to provide better control in the early stages of the infection, leading to a more efficient and protective immune response, resulting in the control or cure of this disease.
    Methods: We used a highly purified PMNs preparation by inoculating Pb in subcutaneous air pouches of mice. We evaluated the effect of Itraconazole (Itra) therapy and/or LLLT therapy on their size, cellular composition, mitochondrial activity, protein metabolism, production of reactive oxygen (ROS) and nitrogen (NO) species, production of IL-10, INF-y and TNF-α cytokines, and on tissue architecture and number of viable fungi.
    Results: The viability of air pouch cells was always greater than 70%, therefore, none of the treatments had adverse effects, and about 80% of the cells were PMN. Mitochondrial activity and cell counts differed significantly between LLLT-treated and controls and between mice treated with different Itra concentrations, with or without LLLT. Cells from 10mg/mL Itra-treated mice produced the highest levels of ROS followed by those of 3mg/mL. When Itra was associated with LLLT treatment, we found an increase in NO production in the 3mg/mL group. Total protein levels were higher when Itra was used, compared to the untreated group or to the group treated with LLLT alone, in which LLLT increased protein production in the 3mg/mL group and decreased in the 10mg/mL group. LLLT increased the production of cytokines IL-10, INF-y and TNF-α, and the use of antifungal decreased their production at all doses, both using only Itra treatments and in combination with LLLT. The highest dose of Itra (50mg/mL) resulted in a significant reduction of air pouch size, volume and weight. The intensity of the inflammatory process and the number of vessels and neovas decreased when the concentration of Itra in combination with LLLT increased. The numbers of viable Pb were significantly reduced when Itra was administered in a dose-response way and more markedly when used in combination with LLLT.
    Conclusion: Pb numbers decreased significantly when Itra was given at all concentrations, and this effect increased when used in combination with LLLT. Itra at 50 mg/mL caused a significant reduction in air pouch, indicating a dose-response control. Our results strongly suggest that the efficiency of Itra is enhanced by LLLT. Therefore, we could detect a possible additive or synergistic effect of PMNs, the antifungal drug Itra and LASER therapy, suggesting a new, more effective treatment for PCM. Grants: CNPq 305216/2016-3 and FAPEMIG APQ 012941-16. J.C.Grisolia and L.A. Santos are recipients of CAPES scholarships.

    P086. Clinical outcome of chronic pulmonary aspergillosis patients managed surgically

    F. Setianingrum 1,2, R. Rautemaa-Richardson 1,3,4, R. Shah 4,5 and D. Denning 1,3,4
    1
    Division of Infection, Immunity and Respiratory Medicine, University of Manchester, Manchester, United Kingdom
    2
    Department of Parasitology, Universitas Indonesia, Jakarta, Indonesia
    3
    National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom
    4
    Manchester Academic Health Science Centre, Manchester, United Kingdom
    5
    Department of Cardiology and Cardiothoracic Surgery, Manchester University NHS Foundation Trust, Wythenshawe Hospital, Manchester, United Kingdom
    Objectives: Surgery is one of the treatment options for chronic pulmonary aspergillosis (CPA), especially for single aspergilloma, for those with recurrent haemoptysis despite bronchial artery embolisation or with the unilateral azole-resistant disease. This study aims to evaluate the outcomes of patients managed surgically and the correlation between Aspergillus Immunoglobulin G (IgG) levels and response to treatment.
    Methods: This was a retrospective cohort study evaluating CPA patients who were treated at National Aspergillosis Centre, Manchester, UK over an 11-year period (2007-2018). Clinical variables including antifungal therapy and Aspergillus-IgG (Thermo Fisher/Phadia) were recorded and correlated with surgical success or relapse. Aspergillus-IgG were analysed based on 37 patients with complete serology database.
    Results:
    Jof 05 00095 i040
    61 patients with CPA underwent surgery with a mean duration of follow-up of 36 months. The mean age was 52.8 (± 14.3) years. The most common presenting symptoms pre-operatively were cough (n = 41, 67%), haemoptysis (n = 24, 39%), dyspnea (n = 23, 38%) and 5 patients (8%) were asymptomatic. Failed medical therapy (n = 31, 51%) and presumed malignancy (n = 23, 38%) were the most common indications for surgery. The most common sign of failed medical therapy was recurrent haemoptysis (n = 19, 31%). Twenty-six patients (43%) had single aspergilloma, 23 patients (38%) showed chronic cavitary pulmonary aspergillosis (CCPA) while 19 patients (31%) had one or more Aspergillus nodule. Main underlying diseases were chronic obstructive pulmonary disease (n = 19, 31%), asthma (n = 18, 30%), bronchiectasis (n = 14, 23%) and previous pneumothorax (n = 13, 21%). The procedures included lobectomy (n = 39, 64%), wedge resection (n = 17, 28%), segmentectomy (n = 3, 5%), pneumonectomy (n = 3, 5%) and decortication (n = 2, 3%). Main surgical complications were pleural infection (n = 7, 12%), prolonged air leak (n = 3, 5%) and persistent pneumothorax (n = 3, 5%). Assessed 6-8 months after surgery, 21 patients were asymptomatic (34%), 23 experienced cough (38%), 6 patients (10%) had haemoptysis, and the frequency of these symptoms reduced significantly after surgery (p< 0.05). Relapse occurred in 25 patients (41%) while the rest of the patients (n = 36, 59%) showed no sign of relapse. Haemoptysis, cough and dyspnea decreased significantly in the non-relapse group, whereas only haemoptysis was initially less frequent in relapse group (p<0.05). In this relapse group, 6 (24%) patients received antifungal before surgery compared to 23 (64%) in the non-relapse group (p = 0.002). Perioperative antifungal therapy was administered to 5 (20%) patients who relapsed and 19 (53%) who did not (p = 0.01). The median Aspergillus-IgG in the non-relapse group (n = 12, 32.4%) before surgery was 75 mg/L decreasing to 52 mg/L in the end of follow-up (p = 0.003). The median survival time (free-relapse) was 88 months in patients with antifungal therapy before surgery and 31 months in patients without antifungal therapy before surgery (p = 0.002).
    Conclusion: Surgery in these selected CPA patients resulted in favourable outcomes with a decrease in clinical symptoms and the Aspergillus-IgG level of the non-relapse group of CPA. Pre and perioperative antifungal therapy reduced the likelihood of CPA relapse, but confounding factors may contribute to this. The diagnosis of CPA before surgery is important to optimise pre and perioperative antifungal therapy in patients to improve prognosis.

    P087. The all new, updated antifungal drug interaction database at www.aspergillus.org.uk The all new, updated antifungal drug interaction database at www.aspergillus.org.uk The all new, updated antifungal drug interaction database at www.aspergillus.org.uk

    S. Niazi-Ali 1, D. Denning 2,3, G. Atherton 2,3 and M. Walczak 3,4
    1
    Fungal Interaction Trust, Macclesfield, United Kingdom
    2
    University of Manchester, Manchester, United Kingdom
    3
    National Aspergillosis Centre, Manchester University NHS Foundation Trust, Manchester, United Kingdom
    4
    Manchester Academic Health Science Centre, Manchester University NHS Foundation Trust, Wythenshawe, United Kingdom
    Objectives: A drug-drug interaction describes the influence of one drug upon another or the change in a drug’s effect on the body when the drug is taken together with a second drug. A drug-drug interaction can delay, decrease, or enhance absorption or metabolism of either drug. The antifungal drug interaction database was first launched in 2012. It is available as web (www.aspergillus.org.uk/content/antifungal-drug-interactions) and app versions to allow information on potential drug interactions with antifungals with a version for patients and another for health professionals. New drugs are constantly being launched and approved. In this era, with the requirement of speed and accurate information due to pressures on all healthcare professionals, a new and updated database with apps (on Apple and Android platforms) has been created. This allows all of the above with the added benefit of real time updates in a more time efficient manner.
    Methods: Horizon scanning documents were reviewed from 2017, 2018 and 2019 to date. Drugs that were not in the current database were identified and interactions determined using the Summary of Product Characteristics together with further exploration, as needed, with review of papers. CYP P450 and p-glycoprotein are the major interaction pathways where antifungal drugs are involved. However other pathways like OATP1B1 and BCRP were also explored, as there is new and emerging information on the effect of these pathways with drug-drug interactions.
    Results:
    Jof 05 00095 i041
    134 new preparations were identified to April 2019 leading to updated information. 16 classed as red (severe interactions with some antifungals), 29 as amber (moderate interactions where co-administration is possible and guidance on any management required) and 89 as green (interaction unlikely). These were added to the database together with references to ensure the user is able to, refer and, find references with ease to validate their decisions. of 16,324 possible interactions for drugs licensed by the end of 2018, 507 are potentially severe (3.1%), 1080 (6.6%) moderate and 809 mild interactions (4.9%). The antifungals with the least interactions are anidulafungin (n = 3 mild) and micafungin (n = 4 moderate & 8 mild) which contrasts with voriconazole with 459 interactions (140 severe) and itraconazole with 404 interactions (134 severe).
    Conclusion: The new and revised version of the web and smartphone app-based database of these drug interactions with antifungal agents is now available. This will allow clinicians to effectively evaluate the use of antifungals with all the new drugs on the market and allow the best outcome for their patients. It will also enable them to make the most effective and evidence-based decisions for those drugs that are co-administered and require a little more clinical management to provide the most successful treatment for the wellbeing of their patients.

    P088. Posaconazole Serum Drug Levels Associated with Hypertension and Hypokalemia: The Syndrome of Pseudohyperaldosteronism

    M.-V. Nguyen 1, M. Davis 1, R. Wittenberg 1, I. Mchardy 1, J. Baddley 1, B. Young 1, A. Odermatt 2 and G. Thompson 1
    1
    Infectious Disease, University of California at Davis, Davis, United States of America
    2
    Toxicology, University of Basel, Basel, Switzerland
    Objectives: Posaconazole tablets are well tolerated and efficacious in the prophylaxis and treatment of aspergillosis, mucormycosis, and other invasive fungal infections There have been case reports of posaconazole-induced pseudohyperaldosteronism, however, its occurrence and association with serum posaconazole drug levels has not previously been investigated.
    Methods: In this single-center retrospective observational study, we examined the occurrence of posaconazole-induced pseudohyperaldosteronism (PIPH) in outpatients newly starting posaconazole and evaluated differences in serum posaconazole concentrations and clinical characteristics between those with and without this syndrome.
    Results: Sixty-nine patients receiving posaconazole were included, of whom 16 (23.2%) met the definition of PIPH. Patients with PIPH were significantly older (61.1 vs 44.7 years, P = 0.007) and more frequently had hypertension prior to starting posaconazole (68.8% vs 32.1%, P = 0.009). Patients with PIPH had a significantly higher median serum posaconazole level than those without PIPH (3.0 vs 1.2 µg/mL, P < = 0.0001). There was a positive correlation between serum posaconazole levels and changes in systolic blood pressure (r = 0.37, P = 0.01), a negative correlation between serum posaconazole levels and changes in serum potassium (r = -0.39, P = 0.006), and a positive correlation between serum posaconazole levels and serum 11-deoxycortisol (r = 0.69, P < 0.0001).
    Conclusion: Posaconazole is associated with secondary hypertension and hypokalemia, consistent with pseudohyperaldosteronism, and development is associated with higher serum posaconazole concentrations, older age, and baseline hypertension.

    P089. A case of primary joint infection caused by Candida pelliculosa

    S.-H. Kim 1, K.Y. Song 2 and J.-H. Choi 1
    1
    Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea
    2
    Department of Orthopaedic Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea, Seoul, Republic of Korea
    Case Report: Candida joint infection is most often due to hematogenous seeding during an episode of candidemia in immunocompromised patients. Exogenous inoculation after trauma or surgical procedures also can lead to infection, but primary joint infection caused by Candida species is extremely rare. Until now, some Candida joint infections by Candida albicans, Candida krusei, Candida lusitaniae, Candida parapsilosis, and Candida tropicalis have been reported. Herein, we report the first case of primary knee joint infection caused by Candida pelliculosa without predisposing factors. A 75-year-old woman came to our hospital with a 3-year history of pain and swelling of the left knee, with the pain particularly aggravated for the past 1 month. The patient had undergone a left total knee arthroplasty (TKA) for incapacitating knee pain at a local hospital 32 months ago. At that time, C. pelliculosa was isolated from the tissue taken by intraoperative biopsy, but no antifungal treatment was performed. One year after TKA, C. pelliculosa was repeatedly isolated from left knee synovial fluid and the patient was treated with intravenous amphotericin B deoxycholate (0.5 mg/kg/day) for 4 weeks, then intravenous fluconazole (6 mg/kg/day) for 3 weeks, followed by oral fluconazole (4 mg/kg/day) for 2 weeks. However, progressive bone loss around prosthetic components was seen on follow-up radiographs and the patient was referred to tertiary hospital. The patient had no other significant medical history. The patient was admitted to our hospital and arthrocentesis of the left knee was performed. Results of synovial fluid analysis were as follows: leukocyte, 608 cells/μL (neutrophil, 54%); protein, 5.9 g/dL; glucose, 7 mg/dL. Magnetic resonance imaging showed a large abscess around the antero-lateral aspect of the left knee. Fungal culture of synovial fluid reported as C. peilliculosa susceptible to amphotericin B (minimum inhibitory concentration [MIC], ≤0.25 mg/L), caspofungin (MIC, ≤0.25 mg/L), fluconazole (MIC, 2 mg/L), micafungin (MIC, ≤0.06 mg/L), and voriconazole (MIC, ≤0.12 mg/L) by Vitek 2 yeast identification system. The isolate was confirmed as C. pelliculosa using Internal transcribed spacer 1 and 4 region PCR and sequencing. At 2 weeks of micafungin (100 mg/day) therapy, the patient underwent prosthesis resection with debridement of soft tissue and bone, total synovectomy, and placement of an antibiotic-impregnated spacer. Operative findings showed infected granulation tissue in whole joint space and bony fistula extended to the anterolateral aspect of the lateral tibial component. Postoperatively, the patient was given micafungin for 4 weeks, followed by oral fluconazole (6 mg/kg/day). There were no signs of infection around the left knee and the patient was discharged from the hospital. Oral fluconazole will be given for at least 6 months and implantation of new prosthesis is planned at 3 months after the stage 1 procedure. Rare Candida species could be a causative organism of primary joint infection. Even a single colony of Candida on synovial fluid or bone biopsy should be considered as pathogenic, and the patient should be treated with antifungal agents. A high index of suspicion is required to establish the diagnosis.

    P090. Transdiaphragmatic Mucormycosis

    P. Köhler 1, R. Reimer 2, R. Whaba 3, B. Schömig-Markiefka 4 and O.A. Cornely 1
    1
    Department I For Internal Medicine, European Excellence Center For Medical Mycology (ecmm), Germany and Cecad Cluster of Excellence, University of Cologne, Cologne, Germany
    2
    Department of Diagnostic and Interventional Radiology, University Hospital Cologne, Cologne, Germany
    3
    Department of General, Visceral and Cancer Surgery, University Hospital of Cologne, University of Cologne, Cologne, Germany
    4
    Institute of Pathology, University Hospital Cologne, Cologne, Germany
    Objectives: Introduction Mucormycosis is a life-threatening infection in immunocompromised patients and in haematological malignancy patients in particular. Objectives To report the phenomenon of transdiaphragmatic mucormycosis and to raise concerns with regard to the extent of imaging studies in patients with mucormycosis.
    Methods: Retrospective chart review was performed on patients with Mucormycosis treated at the University Hospital of Cologne between January 2011 and January 2019.
    Results: In a series of three patients we observed a previously unreported contiguous disease pattern, where the pulmonary type of disease contiguously spread through the diaphragm into abdominal organs, mostly liver or spleen. Patient 1, a 53-year-old women with history of B-NHL, ASCT and chronic renal disease developed mucormycosis of the lungs with continuous growth through the diaphragm into the liver. Histology showed invasive growth of fungal hyphae of the Mucorales order in liver and diaphragm. Patient 2, a 35-year-old man with history of AML and no further underlying conditions developed mucormycosis of the lungs with continuous growth through the diaphragm into the spleen. Splenectomy was performed and histology showed invasive growth of fungal hyphae. Fungal cultures of the surgical sample remained negative, but panfungal PCR resulted in Rhizomucor spp. Patient 3, a 42-year-old man with history of poorly controlled IDDM and injection drug use developed mucormycosis of the lungs with continuous growth through the diaphragm into the liver. Fungal culture of BAL fluid showed growth and PCR positivity of Rhizopus microsporus.
    Conclusion: This is the first report of the clinical observation of transdiaphragmatic mucormycosis in 3 patients. We observed this phenomenon in 11% (3/27) of patients, raising concerns with regard to the extent of imaging studies in patients with mucormycosis. Clinicians should become aware of this new-described entity.

    P091. Baseline predictors influencing the prognosis of invasive aspergillosis in adults

    P. Köhler 1, J. Salmanton-Garcia 2, S. Gräfe 3, F. Köhler 3, S. Mellinghoff 4, D. Seidel 1, A. Steinbach 5 and O.A. Cornely 6
    1
    Department I of Internal Medicine, Ecmm Excellence Centre of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), University Hospital Cologne, Cologne, Germany
    2
    Department I For Internal Medicine, European Excellence Center For Medical Mycology (ecmm), Germany and Cecad Cluster of Excellence, University of Cologne, Cologne, Germany
    3
    Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany
    4
    Department I of Internal Medicine, Ecmm Excellence Centre of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), German Centre For Infection Research, Partner Site Bonn-cologne, University Hospital Cologne, Cologne, Germany
    5
    Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), German Centre for Infection Research, Partner Site Bonn-Cologne, Cologne, Germany
    6
    Department I of Internal Medicine, Ecmm Excellence Centre of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), German Centre For Infection Research, Partner Site Bonn-cologne, Clinical Trials C, University Hospital Cologne, Cologne, Germany
    Introduction: Invasive aspergillosis (IA) is a serious hazard to hematological and critical care patients.
    Objectives: To understand prognostic variables we analyzed original articles identifying baseline factors that predict treatment outcome in patients with IA.
    Methods: PubMed database was searched for publications since database inception until May 2018. Inclusion criteria were published baseline prognostic factors present at the diagnosis of IA.
    Results: In total, 58 studies from 267 centers reported 7,320 patients with IA, and 40 different predictors. Unfavorable predictors in medical history were kidney (7.4%, 10/136) and liver failure (3.7%, 5/136), ICU admission (3.7%, 5/136), and uncontrolled underlying disease (3.7%, 5/136). Regarding state of immunosuppression, negative outcome predictors were prolonged neutropenia (12.5%, 17/136), corticosteroid treatment (8.1%, 11/136), and graft-versus-host disease (3.7%, 5/136). On the pathogen side, relevant predictors were galactomannan positivity (8.1%, 11/136), Aspergillus terreus infection (2.2%, 3/136), and lack of amphotericin B susceptibility (1.5%, 2/136). IA-specific predictors were disseminated disease (5.1%, 7/136) and CNS involvement (2.9%, 4/136). Imaging results associated with negative outcome were multiple consolidations (2.9%, 4/136), bipulmonary lesions (2.2%, 3/136), and pleural effusion (2.2%, 3/136).
    Conclusion: At diagnosis of IA, most frequently identified predictors of outcome were neutropenia, corticosteroid use, elevated galactomannan, renal failure, and disseminated disease.

    P092. EQUAL Aspergillosis Score 2018: An ECMM score derived from current guidelines to measure QUALity of the clinical management of invasive pulmonary aspergillosis

    O.A. Cornely 1, P. Köhler 2, D. Arenz 3 and S. Mellinghoff 2
    1
    Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), Cecad Cluster of Excellence, Clinical Trials Centre Cologne (zks Köln), University of Cologne, Cologne, Germany
    2
    Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), Cecad Cluster of Excellence, University of Cologne, Cologne, Germany
    3
    Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), University of Cologne, Cologne, Germany
    Introduction: Invasive pulmonary aspergillosis is a serious threat to immunocompromised and critical care patients. Recent detailed guidelines and treatment algorithms lead microbiologists and clinicians in diagnosis and treatment of invasive aspergillosis.
    Objectives: Currently, there is no tool available that allows to measure guideline adherence and quality of managing invasive pulmonary aspergillosis.
    Methods: To develop such a tool, we reviewed current guidelines provided by 5 scientific societies: European Society for Clinical Microbiology and Infectious Diseases, European Confederation of Medical Mycology, the European Respiratory Society, Infectious Diseases Society of America (IDSA), and Infectious Diseases Working Party of the German Society for Hematology and Medical Oncology. We selected the strongest recommendations for management as key components for our scoring tool, broke down guidelines, and grouped recommendations into 3 groups: diagnosis, treatment, follow-up. First, we selected “AI” and “AII” recommendations. Then, we added aspects that are part of a complete work-up, but are either recommended with a lower grade of evidence or not specifically recommended in current guidelines. In a last step, we allocated score points according to levels of evidence and clinical importance.
    Results: We developed the EQUAL Aspergillosis Score 2018 to measure the quality of clinical management of invasive pulmonary aspergillosis. We integrated diagnostic measures (chest computed tomography, bronchoalveolar lavage with galactomannan, fungal culture, fungal polymerase chain reaction analysis, species identification, and susceptibility testing; histology with silver stain, Periodic acid–Schiff staining, and molecular diagnostics), treatment (antifungal choice and therapeutic drug monitoring), and follow-up computed tomography. For ease of use, we have summarized the EQUAL scores on laminate pocket cards, which will be translated in 15 different languages. The EQUAL Aspergillosis Score 2018 can be used to evaluate guideline adherence in daily clinical practice and thus aims to support antifungal stewardship.
    Conclusion: The EQUAL Aspergillosis Score 2018 aggregates and weighs the components recommended in the ideal management of invasive pulmonary aspergillosis and provides a tool supporting antifungal stewardship and quantifying guideline adherence.

    P093. EQUAL Mucormycosis Score 2018: An ECMM Score Derived From Current Guidelines to Measure QUALity of Mucormycosis Management in Hematology

    P. Köhler 1, S. Mellinghoff 1, A. Alanio 2, D. Arenz 3, M. Hoenigl 4, K. Lagrou 5, C. Lass-Flörl 6, J. Meis 7, M.D. Richardson 8 and O.A. Cornely 9
    1
    Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), Cecad Cluster of Excellence, University of Cologne, Cologne, Germany
    2
    Paris-diderot, Sorbonne Paris Cité University, Institut Pasteur, Molecular Mycology Unit, Cnrs Cmr2000, Parasitology-Mycology Laboratory, Lariboisière Saint-Louis Fernand Widal Hospitals, Assistance Publique-Hôpitaux de Paris, Paris, France
    3
    Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), University of Cologne, Cologne, Germany
    4
    Division of Infectious Diseases, Department of Medicine, UCSD, San Diego, United States of America
    5
    Laboratory of Clinical Bacteriology and Mycology, Department of Microbiology and Immunology, Excellence Center For Medical Mycology (ecmm), KU Leuven, Leuven, Belgium
    6
    Division of Hygiene and Medical Microbiology, Excellence Center For Medical Mycology (ecmm), Med. Univ. Innsbruck, Innsbruck, Austria
    7
    Department of Medical Microbiology and Infectious Diseases, Excellence Center For Medical Mycology (ecmm), Canisius Wilhelmina Hospital, Nijmegen, Netherlands
    8
    Mycology Reference Centre, Excellence Center For Medical Mycology (ecmm), Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom
    9
    Department I For Internal Medicine, Excellence Center For Medical Mycology (ecmm), Cecad Cluster of Excellence, Clinical Trials Centre Cologne (zks Köln), University of Cologne, Cologne, Germany
    Introduction: Mucormycosis is a serious infectious threat to immunocompromised patients in general and in hematological malignancy in particular. Detailed standards and treatment algorithms guide microbiologists and clinicians in the diagnosis and treatment of mucormycosis.
    Objectives: Currently, there is no tool available that allows the measurement of guideline adherence and the quality of managing mucormycosis.
    Methods: To develop such a tool, we reviewed current guidelines provided by four scientific societies: the European Society for Clinical Microbiology and Infectious Diseases (ESCMID), the European Confederation of Medical Mycology (ECMM), the European Conference on Infections in Leukemia (ECIL), and the Infectious Diseases Working Party of the German Society for Hematology and Medical Oncology. We selected the strongest recommendations for management as key components for our scoring tool, broke down guidelines, and grouped recommendations into three groups: diagnosis, treatment, follow-up. First, we selected “AI” and “AII” recommendations. Then we added aspects that are part of a complete work-up, but are either recommended with a lower grade of evidence or not specifically recommended in current guidelines. In a last step, we allocated score points to the various levels of evidence and clinical relevance of diagnostic and therapeutic procedures.
    Results: We developed the EQUAL Mucormycosis Score 2018 to measure the quality of mucormycosis management. We integrated diagnostic measures (chest computed tomography, bronchoalveolar lavage with fungal culture, fungal polymerase chain reaction analysis, species identification, susceptibility testing, histology with silver stain, Periodic acid–Schiff staining, and molecular diagnostics), treatment (antifungal choice, surgical debridement and therapeutic drug monitoring), and follow-up computed tomography. For ease of use, we have summarized the EQUAL scores on laminate pocket cards, which will be translated in 15 different languages. The EQUAL Mucormycosis Score 2018 can be used to evaluate guideline adherence in daily clinical practice and thus aims to support antifungal stewardship.
    Conclusion: The EQUAL Mucormycosis Score 2018 aggregates and weighs recommendations for management of mucormycosis and provides a tool supporting antifungal stewardship and quantifying guideline adherence.

    P094. Compliance with recurrent vulvovaginal candidiasis guidelines – An audit of RVVC referrals

    L. Brown 1 and R. Rautemaa-Richardson 1
    1
    Faculty of Biology, Medicine and Health, University of Manchester, pl, United Kingdom
    2
    Division of Infection, Immunity and Respiratory Medicine, University of Manchester, Manchester, United Kingdom
    Objectives: Recurrent vulvovaginal candidiasis (RVVC) is a debilitating, chronic condition which affects over 138 million (6%) women of reproductive age annually. It is defined as four or more episodes in a 12-month period. Symptoms include vulval itch, soreness and discharge. Diagnosis is often difficult, as it is based on a combination of these non-specific clinical symptoms and detection of Candida by high vaginal swab (HVS) microscopy, which is part of the normal vaginal flora in many women. A number of other infectious and non-infectious conditions can present similarly. Treatment of RVVC comprises of vulval skin care with the use of emollients for personal hygiene and antifungal treatment combined with long-term antifungal suppression for 6 months or longer. The British Association for Sexual Health and HIV (BASHH) has published guidelines on the management of RVVC. The aim of our study is to evaluate the compliance of referring doctors to the current guidelines.
    Methods: This retrospective audit was undertaken in the Infectious Diseases (ID) department of Wythenshawe Hospital, Manchester from October 2017 to May 2019. All patients referred to the tertiary clinic with suspected RVVC were included in the analyses. Electronic patient records were analysed to determine whether appropriate investigations and treatments had been offered prior to referral. The type and pattern of recorded symptoms were assessed against the typical clinical picture of RVVC and whether alternative differentials should have taken precedence.
    Results: Patient records from a total of 47 RVVC referrals were analysed. Patient age at the time of referral ranged from 17-66 years with a mean age of 37.7 years. Symptom duration prior to referral ranged from 4 months to 24 years, with an average duration of 6.7 years. “Dry”, “sensitive” skin or “eczema” were mentioned in 36% of the cases. RVVC was the final diagnosis in 80% of cases. 19% of referrals were deemed inappropriate due to repeatedly negative HVS for Candida and lack of response to antifungal therapy, or reported symptoms not fitting with RVVC. 57% of patients had a secondary diagnosis, the most common being vulvodynia and vulval dermatitis. 94% of the patients were appropriately investigated by high vaginal swab (HVS) microscopy or culture prior to referral. Appropriate antifungal regimens, including long term suppression, were trialled in 53% of patients prior to referral. Only 11% patients were practicing appropriate vulval skin care before attending clinic. Excessive or otherwise inappropriate personal hygiene likely to irritate vulval skin, including the use of soap and shower gel, was mentioned in 21% of cases. MBL deficiency was diagnosed in 28% of the patients.
    Conclusion: RVVC is often complicated by coexisting vulval pathologies and vulval symptoms labelled as RVVC are often due to alternative diagnoses. Education on vulval skin care is the mainstay of RVVC management and prevention but healthcare professionals are failing to deliver this. Also, there is lack of awareness of the efficacy and safety of long term antifungal suppression. The morbidity related to this debilitating condition could be easily reduced by following the principles summarised in the BASHH guidelines.

    P095. Validation of a clinical decision rule for selecting empiric treatment for invasive aspergillosis in a setting with high triazole resistance.

    R. Van De Peppel, R. Van Grootveld, B. Hendriks, J. Van Paassen, J. Koopmans, P. Von Dem Borne, M. Van Der Beek and M. De Boer
    Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands
    Objectives: World-wide, increasing triazole resistance complicates treatment of invasive aspergillosis (IA). In case of detected triazole resistance, the first choice treatment is replaced by lipid formulations of Amphotericin B (LAmB). In settings with substantial (>10%) prevalence of triazole resistance, empiric combination therapy with both a triazole and LAmB has been suggested. However, the expected benefits of this strategy need to be weighed against a higher rate of serious adverse events associated with combination therapy, as well as higher costs. To evade unnecessary toxicity while optimizing outcome, a clinical decision rule guiding to monotherapy with either voriconazole or LAmB was designed and validated.
    Methods: In 2015, all medical specialties involved in the diagnosis and treatment of patients with IA participated in constructing a treatment algorithm that provided rationale for empiric monotherapy with either voriconazole or LAmB (Figure 1). The designed algorithm was approved by the institution’s antimicrobial steering committee. Protocolized CT-scanning and bronchoalveolar lavage (BAL) were performed upon suspicion of IA. BAL samples were examined by direct microscopy, culture, galactomannan assay (cut off at 0.5 OD) and from 2017 onwards also by PCR. Triazole resistance was routinely tested by four well agar plate. All adult patients diagnosed with IA and treated for this condition in the Leiden University Medical Center from 2015 to 2019 were included. Data about underlying diseases, age, gender, results of diagnostic tests and outcomes, were extracted from the hospital’s electronic patient files and anonymized. Primary outcomes were 30- and 100-day crude- and attributable mortality rates.
    Jof 05 00095 i042
    Results: At first evaluation, 102 patients had been treated for IA with monotherapy according to the designed algorithm and 3 patients received off-protocol treatment regimens (Figure 2). In the period of study, prevalence of voriconazole resistance in all Aspergillus isolates varied per year between 16.3% and 24%. of included patients, 65 (64%) were male; the median age was 64 (range 18-82), and 80 (78%) patients were treated for an underlying hematological malignancy. Fifty-nine patients (58%) were started on voriconazole and 43 (42%) were started on LAmB. Cultures were positive in 16 (15.7%) patients and phenotypical voriconazole resistance was detected in 7/16 (44%). Upon clinical evaluation of empiric therapy and/or resistance data if available, a switch was made to LAmB in 23/59 (39%) of patients started on voriconazole. Crude mortality rates per treatment stratum are listed in Figure 2. Later adaptation of antifungal therapy to LAmB was not associated with a higher crude mortality rate (p = 0.73). Definite therapy consisted of voriconazole in 41 (40%) and of LAmB in 61 (60%) of cases.
    Jof 05 00095 i043
    Conclusion: By applying a comprehensive clinical decision algorithm in our area with high triazole-resistance rates, 58% of patients were empirically treated with monotherapy voriconazole, without excess crude mortality even if a later switch to LAmB was needed. In 40% of patients therapy with LAmB could be safely avoided during the entire course of treatment. As treatment allocation was not randomized, and confounding by indication plays a role, it remains unclear whether patients directed to initial LAmB monotherapy would have benefited from the addition of a triazole.

    P096. Multicenter study on antifungal prophylaxis in high risk hematology patients - The SAPHIR Study

    J.P. Gangneux 1, C. Padoin 2, M. Michallet 3, E. Saillio 4, A. Kumichel 5, R. Peffault De La Tour 6, P. Ceballos 7, T. Gastinne 8 and A. Pigneux 9
    1
    Laboratoire De Parasitologie-mycologie, CHU de Rennes, Rennes, France
    2
    Pharmacy Department, CHU de Martinique, Fort-de-France, France
    3
    Clinical Haematology Department, CH Lyon-Sud, Pierre-Bénite, lyon, France
    4
    MSD France, courbevoie, France
    5
    ClinSearch, Malakoff, France
    6
    Saint-Louis Hospital, APHP, Paris, France
    7
    CHRU Lapeyronie de Montpellier, Montpellier, France
    8
    CHU de Nantes, Nantes, France
    9
    Hospital group Haut Leveque de Pessac, Pessac, France
    Objectives: The main objective of the SAPHIR study was to describe the diagnostic and therapeutic management of hematology patients at high risk of Invasive Fungal Disease (IFD) under antifungal prophylaxis (AFP) in France. The secondary objectives were the description of (1) parameters and diagnostic procedures concomitant to AF therapy management and (2) preventive procedures adopted in French hematology services.
    Methods: SAPHIR was an observational, prospective, multicenter, non-comparative French study. The included patients were ≥18 years, in a context of myelosuppressive chemotherapy for acute myeloid leukemia (AML) and initiated an AF prophylactic treatment.
    Results: The 23 participating hematology services included 404 patients, whose mean age was 56.4±14.0 years and of whom 51.2% were male. Duration between AML diagnosis and inclusion was 65.4±178 days with 65.6% being newly diagnosed. A previous chemotherapy had been undergone by 26.7%. The index chemotherapy, was started 1.06±4.49 days before inclusion and its nature was either induction 79.0%, consolidation 12.6% or relapse 8.4%. Among the 404 patients, 91.6% had experienced a profound neutropenia period with 1.08±0.51 periods per patient and a duration of 23.3±16.6 days. For 373 patients (92.4%), the initially prescribed AFP was posaconazole tablets (90.6%) or suspension (1.73%). Considering all patients, 8.17% changed AFP between 1 and 3 times, with the first change 17.3±23.9 days after inclusion mostly due to absorption issues (65.0%). The mean AFP period was 24.2±32.1 days after which 267 patients (66.8%) stopped because they were at the end of the high-risk period and 126 (31.2%) switched to a non-prophylactic AF treatment (2/3 empirical, 1/3 preemptive/curative), mainly because of fever (67.1%). A total of 18/404 patients (4.46%) have received a curative treatment based on positive mycological exams, imaging, antifungal sensitivity tests and/or blood cultures. Among them, 9/404 (2.23%) were true probable IFDs: 5 probable invasive aspergillosis, 2 pulmonary pneumocystosis, 1 mucormycose and 1 fungemia due to Saccharomyces sp. At 7-15 days after AFP discontinuation, 365/387 patients (94.3%) showed no signs and symptoms of infection. In the course of the study, 20 patients (5.0%) had deceased within 57.6±50.3 days after inclusion. of those, 7 patients (35.0%) showed signs and symptoms of infection and the death of 3 patients was directly linked to IFD infections (1 candidiasis and 2 aspergillosis). In order to prevent IFD, additionally to AFP, French haematology services provide the patient with sterile/protected food (94.3%), digestive decontamination (35.8%), and accommodation in a sterile/isolated area (93.5%). To gain a better understanding of the patient characteristics possibly indicating the necessity for a consecutive empirical/preemptive/curative AF treatment, data of patients with and without such a consecutive treatment were compared. Several parameters proved to be significantly different, for instance: cytogenetics and molecular biology prognosis, AML onset, previous chemotherapy treatment but also duration between chemotherapy initiation and inclusion and the type of current chemotherapy cycle used.
    Conclusion: AF prophylactic treatment is frequently prescribed in French hematology services. Only few prophylactic treatment changes are necessary which reflects treatment tolerance and effectiveness regarding decreased numbers of intercurrent infections and mortality.

    P097. EQUAL Cryptococcus Score 2018: A European Confederation of Medical Mycology Score Derived From Current Guidelines to Measure QUALity of Clinical Cryptococcosis Management

    A. Spec 1, C. Mejia-Chew 1, W. Powderly 1, P. Köhler 2,3 and O.A. Cornely 2,4
    1
    Internal Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO, United States of America
    2
    Department I For Internal Medicine, European Excellence Center For Medical Mycology (ecmm), Germany and Cecad Cluster of Excellence, University of Cologne, Cologne, Germany
    3
    Department I of Internal Medicine, Ecmm Excellence Centre of Medical Mycology, Cologne Excellence Cluster On Cellular Stress Responses In Aging-associated Diseases (cecad), University Hospital Cologne, Cologne, Germany
    4
    German Centre For Infection Research, Partner Site Bonn-cologne, Cologne, Germany and Clinical Trials Centre Cologne (zks Köln), University of Cologne, Cologne, Germany
    Objectives: Cryptococcocis is an opportunistic fungal infection with high morbidity and mortality. Guidelines to aid clinicians regarding diagnosis, management, and treatment can be extensive and challenging for clinicians. Currently, there is no tool available that allows the measurement of guideline adherence and the quality of cryptococcosis management.
    Methods: We reviewed current guidelines from the Infectious Diseases Society of America, the World Health Organization, the American Society of Transplantation and recent significant publications to select the strongest recommendations as vital components of our scoring tool.
    Results: Items included diagnostic tests (blood, tissue, and cerebrospinal fluid cultures, Cryptococcus antigen, India ink, histopathology with special fungal stains, central nervous system imaging), pharmacological (amphotericin B, flucytosine, azoles) and nonpharmacological treatments (intracranial pressure management, immunomodulation, infectious disease consultation), and follow-up of central nervous system complications. For ease of use, we have summarized the EQUAL scores on laminate pocket cards, which will be translated into 15 different languages.
    Conclusion: The EQUAL Cryptococcus Score 2018 weighs and aggregates the recommendations for the optimal management of cryptococcosis, providing a tool that could measure guideline adherence or facilitate clinical decision-making.

    P099. An ongoing malady, mucormycosis from a tertiary care centre at Chandigarh, India.

    J. Chander 1, A. Bhagat 1, N. Gulati 1, N. Singla 1, R.P.S. Punia 2, D. Agarwal 3, A.K. Attri 4 and A. Dass 5
    1
    Microbiology, Government Medical College Hospital, Chandigarh, India
    2
    Pathology, Government Medical College Hospital, Chandigarh, India
    3
    Pulmonary Medicine, Government Medical College Hospitaln, Chandigarh, India
    4
    General Surgery, Government Medical College Hospital, Chandigarh, India
    5
    Ent, Government Medical College Hospital, Chandigarh, India
    Objectives: Mucormycosis is an emerging necrotizing angioinvasive infection caused by commonly encountered filamentous fungi of the class Mucormycetes. In our institution, as the awareness is gradually increasing, newer and newer species causing mucormycosis are being recognized. In this very reference a study was undertaken to further enhance our knowledge on risk factors, species distribution and their antifungal susceptibility testing.
    Methods: The study was conducted on forty-four cases of mucormycosis over a period of eighteen months from January 1, 2017 to June 30, 2018. The samples were subjected to microbiological examination like KOH/CFW wet mount and fungal culture and histopathology. The isolates obtained were identified phenotypically and confirmed by DNA sequencing of internal transcribed spacer region. The antifungal susceptibility testingwas done for amphotericin B, posaconazole, itraconazole and terbinafine by microbroth dilution as per CLSI Reference Method.Study performed by Almyroudis, et al, was referred to define susceptibility for some of the breakpoints for Mucorales.
    Results: The mean age of patients of mucormycosis was 48.89 ± 17.1 years. The proportion of female patients (54.5%) was slightly more than male patients (45.5%). Rhino-orbito-cerebral mucormycosis (ROCM) was the commonest presentation (61.4%, 27) followed by pulmonary (20.4%, 9) and cutaneous (15.9%, 7). One case of mucormycosis of the middle ear was also seen. The most common risk factor associated with mucormycosis was diabetes mellitus (77.3%, 34/44), which was found to be statistically significant (P = 0.003). Tooth extraction was seen as a risk factor in 18.5% (5/27) of ROCM cases. The commonest isolate was Rhizopus arrhizus (32.3%, 10), followed by Rhizopus microsporus (29%, 9) and Apophysomyces variabilis (12.9%, 4). Rhizopus homothallicus was isolated from two cases, one of middle ear and other from pulmonary mucormycosis. A case of cutaneous mucormycosis caused by Thamnostylum piriforme, which is the second case of this genus and first of this species, is being reported. Amphotericin B was the most effective drug with an MIC range of 0.0313-4 µg/ml giving 94.7 % isolates as sensitive. The only isolate resistant was Rhizopus homothallicus with an MIC of 4 µg/ml. Posaconazole gave an MIC range of 0.0625-4 µg/ml with 68.4% isolates as sensitive. Dual management with surgery and antifungals proved to be significantly better (P < 0.001) than amphotericin B alone. Treatment with liposomal amphotericin B (LAmB) was associated with a higher survival rate (81%), compared to 28.6% survival when patients were treated with conventional amphotericin B deoxycholate. Twelve (27.3%) patients either left against medical advice or were referred. A total of 12 patients expired giving a mortality rate of 27.3% and 37.5% among the treated patients.
    Conclusion: India has second largest diabetic population globally with nearly 70% being found with uncontrolled diabetes leading to high incidence of mucormycosis. Due to an ever-growing population at risk and the fatal outcome, need for more prospective studies in this field are emphasized. Increased awareness among clinicians, timely diagnosis and prompt treatment are the key to the successful management.

    P100. The Protective Role of Cinnamaldehyde against Tenuazonic acid-induced Mycotoxicity in Murine model

    A. Kumari 1 and K. Singh 2
    1
    Animal Mycology Laboratory, Zoology, Mmv, Banaras Hindu University, Varanasi, India
    2
    Animal Mycology Laboratory, Zoology, Mmv, Banaras Hindu University, VARANASI, India
    Objectives: Cinnamaldehyde is a natural product obtained from cinnamon and has been reported to have insecticidal, antimicrobial, anti-inflammatory, immunomodulatory, anticancer, anti-angiogenic and potential anti-fungal effects. The present study investigated the possible protective effect of cinnamaldehyde against tenuazonic acid-induced mycotoxicity in murine model. Tenuazonic acid (TeA), a toxin produced by Alternaria alternata is a common contaminant in tomato and tomato based products.
    Methods: The in-vivo experimental plan was as follows.
    Jof 05 00095 i044
    All the experimental groups were administered with TeA for 8 weeks. After 2 weeks, the prophylactic group was administered with cinnamaldehyde along with TeA for the next 6 weeks. Haematological (differential leukocyte count), histopathological (haematoxylin and eosin), biochemical (superoxide dismutase, catalase, malondialdehyde, alanine aminotransferase, aspartate aminotransferase) and cell apoptotic factor (caspase-3) analyses of the experimental and control mice were performed.
    Results: Behavioral observations- Unlike mycotoxicosis induced groups, no behavioural abnormalities were observed in the animals who received cinnamaldehyde as prophylactic agent. Weights of the mice of the prophylactic group showed reduced body weight gain as compared to that of the mycotoxicosis induced groups. Morphological observations- Hair loss indicating mycotoxicosis was not observed in the prophylactic groups. Anatomical observations- Spleenomegaly, hepatomegaly and gross lesions on liver and kidney were noted on autopsy in the mycotoxicosis induced groups. On the other hand, the prophylactic groups showed normal weights of liver and spleen. The body:organ weight indices of all the organs remained at par to control group in the prophylactic groups except for the lungs and brain where the weights of the organs were significantly high. Haematological analysis- Neutrophilia was observed in IP (mycotoxicosis induced) while neutropenia was observed in the oral (mycotoxicosis induced) group while the differential leukocyte count of the prophylactic groups were found to lie in normal range. Biochemical analyses- Acute intoxication of mice with TeA showed elevated malondialdehyde (MDA), reduced catalase (CAT) and superoxide dismutase (SOD) production; abnormal levels of aspartate transaminase (AST) and alanine transaminase (ALT). Treatment with cinnamaldehyde reversed TeA-induced alterations of antioxidant defense enzyme activities. Histological observations- Histopathological changes characterised by non alcoholic fatty liver, nuclear pyknosis and infiltration of immune cells were observed in the mycotoxicosis induced groups while cinnamaldehyde significantly prevented the TeA-induced organ damage. Cell apoptosis factor- Reduced activity of caspase-3 enzyme was noted in the mycotoxicosis induced groups while significantly high activity of the enzyme was observed in the prophylactic groups.
    Conclusion: Thus, from the present study we concluded that, cinnamaldehyde showed therapeutic effects and toxicity reduction in TeA induced mycotoxicosis.
    Jof 05 00095 i045

    P101. Candi-SoL: A multi-site retrospective audit and analysis of Candidaemia across three South London Teaching Hospitals

    C. Logan 1, A. Fife 2, A. Goodman 3, C. Ward 2 and T. Bicanic 1
    1
    Infection & Immunity, St Georges university, London, London, United Kingdom
    2
    Department of Microbiology, Kings College Hospital, London, United Kingdom
    3
    Department of Infection, Guy’s & St Thomas’ Hospital, London, United Kingdom
    Objectives: Candidaemia is associated with high mortality, particularly in critical care and immunocompromised patients. Emergence of multi-resistant candida species, notably C. glabrata and C. auris, pose further challenges in treatment. This retrospective surveillance and audit study reviews both adult and paediatric candidaemia cases between January 2012- December 2018 across three Tertiary South London Trusts; St George’s Hospital in London, Kings College hospital London and Guys & St Thomas’ Hospital. Collectively these trusts have over 3500 beds, including over 220 ICU beds (adult surgical, neurological, cardiac and general ICUs and paediatric and neonatal ICU) and onsite specialist surgical, cardiothoracic and medical services, including liver, bone marrow and renal transplant. The aims of this study were to assess trends in candidaemia epidemiology across the South London region, categorise at risk groups, antifungal therapy used and review clinical outcomes.
    Methods: Blood cultures for adults and children isolating Candida species between January 2012-December 2018 were retrospectively identified from the Microbiology database. Species and antifungal susceptibility were reviewed. Persistent candidaemia (identical Candida sp. isolated ≤ 30days after the initial blood culture) was analysed as a single episode. Demographics (age, sex); location; risk factors; focus of candidaemia; antifungal treatment received and duration; ECHO performed and outcome; ophthalmology performed and outcome; line removal and culture result; length of stay; 30-day and in-hospital mortality was collected from electronic patient records. Analyses were conducted in Microsoft Excel 2013 and GraphPad Prism V7.
    Results: Between January 2012 – December 2018, 509 candidaemia episodes were identified across the three Trusts. In total 42% were C. albicans, 30% C. glabrata, 12% C. parapsilosis and 1% of cases were due to C. auris following an outbreak in 2016. There was no significant trend towards an increasing proportion of non-albicans species over the 7-year period (Chi-squared test for trend, p = 0.22). Regarding age at the time of candidaemia, 4% were neonates (< 1 month), 9% were children (aged 1 month - <18 years), and 86% were adults (> 18 years). Patients on an intensive care unit (adult, paediatric or neonatal) accounted for 43% of cases. Clinical data regarding the candidaemia foci, the ECHO and ophthalmology findings, antifungal therapy and duration, B-D-glucan result if available, length of stay and mortality will also be presented. Susceptibility profile of isolates will be interrogated and presented to assess if there has been a change over time across the three trusts.
    Conclusion: This retrospective audit of candidaemia across three large South London Trusts illustrates the changing regional candida epidemiology, with non-albicans species accounting for over half of all isolates and highlights the emergence of multi-drug resistant species such as C. auris in the London. The remaining analyses of this large dataset will highlight which are the dominant causes of candidaemia across the South London Trusts and examine clinical outcomes with candidaemia over a diverse group of patients.

    P102. Antifungal Efficacy of Isavuconazole and Liposomal Amphotericin B for Treatment of Experimental Exserohilum rostratum Meningitis and Therapeutic Monitoring with CSF (1→3)-β-D-glucan: A Preclinical Paradigm for Treatment of CNS Phaeohyphomycosis

    V. Petraitis, R. Petraitiene, E. Naing, B.B.W. Maung, A. Garcia, P. Kavaliauskas and T.J. Walsh
    Infectious Diseases/medicine, Weill Cornell Medicine of Cornell University, New York, United States of America
    Objectives: Phaeohyphomycosis of the central nervous system (CNS) is a life-threatening infection with serious morbidity and mortality. Treatment options for CNS phaeohyphomycosis are limited. This severity of CNS phaeohyphomycosis was tragically illustrated in the contamination of a corticosteroid injection preparation that resulted in epidemic Exserohilum rostratum meningitis (ERM), resulting in meningoradiculitis, brain abscess, neurologic deficits, and death despite antifungal therapy with voriconazole with or without liposomal amphotericin B (LAMB). We hypothesized that isavuconazole may be an alternative approach for patients with ERM and other causes of CNS phaeohyphomycosis, particularly those with severe infection or intolerance to initially high dosages of voriconazole therapy. We therefore investigated the in vitro antifungal activity of isavuconazole with or without LAMB followed by treatment in a new model of experimental ERM.
    Methods: Clinical isolates of E. rostratum recovered from patients suffering from ERM were used in all experiments. Broth microdilution methodology was used to prepare an inoculum for each of five clinical isolates of E. rostratum. Combination checkerboard plates were used to test the activity of isavuconazole and LAMB, either alone or in combination. Minimum inhibitory concentrations (MICs), minimal lethal concentrations (MLCs) and fractional inhibitory concentration (FIC) indices were determined. For in vivo therapeutic studies, E. rostratum were inoculated intracisternally with 1.0x106 microconidia to anesthetized rabbits. Immunosuppression was produced with cytarabine-induced profound persistent neutropenia and methylprednisolone. Study groups consisted of isavuconazole, 60 mg/kg/d, LAMB at 5.0, 7.5, and 10 mg/kg/day and untreated controls (UC).
    Results: As there were no in vitro additive, synergistic, or antagonistic interactions for combinations of isavuconazole plus LAMB against the E. rostratum isolates, in vivo studies in the rabbit model of ERM were conducted with isavuconazole and LAMB as monotherapies. Isavuconazole 60 mg/kg/d and LAMB at 5.0, 7.5, and 10 mg/kg/day demonstrated significant reductions of the residual fungal burden of E. rostratum in cerebrum, cerebellum, spinal cord, and paravertebral muscle (p<0.01) in comparison to those of UC. Reduction of the residual fungal burden correlated with significant reduction of CSF (1→3)-β-D-glucan levels in comparison to those of UC (P<0.05).
    Conclusion: Isavuconazole alone or LAMB alone significantly reduced residual fungal burden in parallel with reduction of CSF (1→3)-β-D-glucan levels in treatment of experimental E. rostratum meningitis and may serve as alternative regimens for treatment of CNS phaeohyphomycosis.

    P103. Pulmonary mucormycosis caused by Lichtheimia ornata (experimental model)

    N. Vasilyeva 1, I. Bosak 2, T. Bogomolova 1, I. Vybornova 2, A. Stepanova 2, Y. Avdeenko 2, G. Chilina 2, O. Aak 2 and G. Solovyeva 1
    1
    Kaschkin Research Institute of Medical Mycology. Department of Medical Microbiology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation
    2
    Kaschkin Research Institute of Medical Mycology, North-Western State Medical University named after I.I. Mechnikov, Saint-Petersburg, Russian Federation
    Objectives: Lichtheimia ornata (A.K. Sarbhoy) Alastr. – Izq. & Walter due to the prevalence and virulence is one of three most common species causing mucormycosis. This species was revealed among the agents of invasive mucormycosis in Russia. The purpose of the present research was to develop a murine experimental model of pulmonary mucormycosis caused by L. ornata.
    Methods: For the infection of mice we used the strain RCPF-1507 of L. ornata, isolated from the tissue of accessory sinuses of nose from a patient with lymphatic leukemia. For modeling the pulmonary mucormycosis we used outbred mice, males, with body weight 18-20 g. For inducing the condition of neutropenia we intraperitoneally injected cyclophosphamide at the dose 150 mg/kg four times (-3, 0, 4 and 8 days). From fungal culture grown on Sabouraud glucose agar for 3 days at 37° C we made the spore suspension in sterile 0.85% NaCl solution at the concentration 1·107 CFU/ml. Infection of animals was carried out by intranasal injection of 50 μl of fungal spores suspension to a narcotized mouse.
    Results: Data on mice lethality and results of cultural and histological investiga-tions of murine lungs with invasive mucormycosis were obtained. Death of animals was observed from 8 to 18 days after infection. No mice survived. Culturing of lung tissue from all dead mice revealed growth of L. ornata. During the histological investigation of murine lungs taken after eight days from the beginning of infection the different area of localization of hyphal cells were found. Hyphae were wide (10-12 µm), with rare branching at right angle. Hyphal cells in the lung parenchyma generally were located densely and in parallel relatively each other that is characteristic of tissue forms of mucormycetes. In lungs vessels massive aggregation of densely and chaotically oriented fungal hyphae were revealed. Also we observed the hyphal aggregation in gleams of bronchial tubes and an interstitium. The inflammatory process due to a mycotic infection was poorly expressed, provided by poor neutrophilic and lymphocytic infiltration, alteration processes prevailed.
    Conclusion: The scheme of immunosuppression used in this work (intra-peritoneal introduction of cyclophosphamide) showed efficiency for producing infection in mice by L. ornata. Data on survival of the infected animals and also results of cultural and histological investigations confirm existence of mycotic damage of lungs at the infected animals. The developed model of invasive pulmonary mucormycosis can be used for future scientific research, testing of new antifungal drugs and diagnostic methods.

    P107. Case of successful treatment of chronic pulmonary aspergillosis in a patient with polycystic disease

    O. Shadrivova 1, V. Kuznetsov 2, E. Desyatik 2, Y. Borzova 2, S. Ignatyeva 2, T. Bogomolova 2, N. Vasilyeva 2 and N. Klimko 1
    1
    Department of Clinical Mycology, Allergy and Immunology, North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation
    2
    Kashkin Research Institute of Medical Mycology, North-Western State Medical University named after I.I.Mechnikov, St. Petersburg, Russian Federation
    Case Report: Background. Publications onchronic pulmonary aspergillosis (CPA) in patients with polycystic diseaseare limited.
    Objection: We present a case of successful treatment of CPA in a patient with polycystic disease.
    Methods: Diagnosis of CPA was made in accordance with the criteria ESCMID/ECMM/ERS, 2016.
    Result: Patient V., 70 years old, was admitted to the mycological clinic with complaints of productive cough and dyspnea in September 2018. It is known that in 2015 lesions in left lung lower lobe were detected on chest CT scan. The CPA diagnosis was made on the basis of chest CT scan (multiple aspergillomas in left lung S6), elevated serum Aspergillus IgG level, and A. fumigatus and A. niger in BAL culture. The patient was treated with itraconazole 400 mg/day for 2 weeks. In October 2018, the patient underwent surgery removal