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Molecular Tissue Diagnosis of Fungal Infections

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A special issue of Journal of Fungi (ISSN 2309-608X). This special issue belongs to the section "Fungal Pathogenesis and Disease Control".

Deadline for manuscript submissions: closed (1 February 2022) | Viewed by 30143

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Special Issue Editor

Dr. Volker Rickerts

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Guest Editor

Robert Koch Institut, 13353 Berlin, Germany
Interests: identification of fungi in tissue samples; epidemiology of cryptococcosis

Special Issue Information

Dear Colleagues,

Despite progress in fungal diagnostics from respiratory specimens such as BAL and from easily obtained samples such as blood, the demonstration of fungal elements in host tissue is one of the pillars in the diagnosis of invasive fungal infections. The demonstration of fungal elements together with a tissue response is paramount for establishing the diagnosis of an invasive fungal disease.

The identification of causative agents by cultivation or morphological characteristics is considered to be important to guide patient management. However, as many fungi do not show discriminative morphologic features and cultures may fail to grow an organism even from samples with positive histopathology, molecular tools have been studied as an adjunct to classic diagnostic techniques.

Molecular tools may help identify the causative fungal species, providing information on intrinsic resistance patterns leading to optimized patient therapy. The use of specific antibodies or hybridization with oligonucleotides may localize one or more infecting agents in the disease process, establishing causality. Furthermore, strain typing or the detection of resistance determinants directly from tissue may clarify additional epidemiological issues.

While the potential of molecular tools to generate important insights leading to the improved management of individual patients and gaining insights into the epidemiology of invasive fungal infections is widely appreciated, a lack of standardization makes generalizations on their impact difficult.

This Special Issue welcomes reviews on best practices and novel approaches, systematic studies, case series or instructive case reports, as well as reports on quality control issues or standardization involving tissue for the diagnosis of invasive fungal infections using conventional and molecular techniques.

Dr. Volker Rickerts
Guest Editor

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Keywords

histopathology
epidemiology of invasive fungal infections
PCR
FISH
NGS
FFPE

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Published Papers (9 papers)

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Research

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15 pages, 17103 KiB

Open AccessArticle

Animal Histoplasmosis in Europe: Review of the Literature and Molecular Typing of the Etiological Agents

by Dunja Wilmes, Ursula Mayer, Peter Wohlsein, Michael Suntz, Jasmin Gerkrath, Christoph Schulze, Ina Holst, Wolf von Bomhard and Volker Rickerts

J. Fungi 2022, 8(8), 833; https://doi.org/10.3390/jof8080833 - 9 Aug 2022

Cited by 6 | Viewed by 2524

Abstract

Histoplasmosis has been previously diagnosed in animals from Europe. The aim of this study is to review the literature on these reports, to analyze cases diagnosed at our laboratory (2000–2022) and to improve molecular typing of Histoplasma capsulatum directly from tissue to study the molecular epidemiology of Histoplasma capsulatum causing animal infections in Europe. Including 15 cases studied in our laboratory, we identified 39 cases of animal histoplasmosis between 1968 and 2022. They were diagnosed mostly in superficial tissue biopsies from cats and badgers from Central Europe. Using phylogenetic analyses of six partial genes, we were able to classify eight of the etiological agents as belonging to a highly supported lineage within the Eurasian clade. This study confirms the occurrence of autochthonous histoplasmosis in animals in Central Europe and proposes the addition of new loci to the MLST scheme to study the molecular epidemiology of histoplasmosis using either formalin-fixed paraffin-embedded tissue and fresh or cadaveric biopsies. Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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15 pages, 4713 KiB

Open AccessArticle

Comparison Approach for Identifying Missed Invasive Fungal Infections in Formalin-Fixed, Paraffin-Embedded Autopsy Specimens

by Sota Sadamoto, Yurika Mitsui, Yasuhiro Nihonyanagi, Kazuki Amemiya, Minoru Shinozaki, Somay Yamagata Murayama, Masahiro Abe, Takashi Umeyama, Naobumi Tochigi, Yoshitsugu Miyazaki and Kazutoshi Shibuya

J. Fungi 2022, 8(4), 337; https://doi.org/10.3390/jof8040337 - 24 Mar 2022

Cited by 7 | Viewed by 3021

Abstract

Invasive fungal infection (IFI) has a high mortality rate in patients who undergo hematopoietic stem cell transplantation, and it is often confirmed by postmortem dissection. When IFI is initially confirmed after an autopsy, the tissue culture and frozen section are challenging to secure, and in many cases, formalin-fixed, paraffin-embedded (FFPE) samples represent the only modality for identifying fungi. Histopathological diagnosis is a useful method in combination with molecular biological methods that can achieve more precise identification with reproducibility. Meanwhile, polymerase chain reaction (PCR) using fungal-specific primers helps identify fungi from FFPE tissues. Autopsy FFPE specimens have a disadvantage regarding the quality of DNA extracted compared with that of specimens obtained via biopsy or surgery. In the case of mucormycosis diagnosed postmortem histologically, we examined currently available molecular biological methods such as PCR, immunohistochemistry (IHC), and in situ hybridization (ISH) to identify fungi. It is reasonable that PCR with some modification is valuable for identifying fungi in autopsy FFPE specimens. However, PCR does not always correctly identify fungi in autopsy FFPE tissues, and other approaches such as ISH or IHC are worth considering for clarifying the broad classification (such as the genus- or species-level classification). Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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10 pages, 1452 KiB

Open AccessArticle

Identification of Mucormycosis by Fluorescence In Situ Hybridization Targeting Ribosomal RNA in Tissue Samples

by Jill Jasmine Dalimot, Ilka Mc Cormick Smith, Jasmin Gerkrath, Sylvia Hartmann, Oliver A. Cornely, Soo Chan Lee, Joseph Heitman and Volker Rickerts

J. Fungi 2022, 8(3), 289; https://doi.org/10.3390/jof8030289 - 11 Mar 2022

Cited by 3 | Viewed by 3532

Abstract

Mucormycosis is an invasive fungal infection associated with high mortality, partly due to delayed diagnosis and inadequate empiric therapy. As fungal cultures often fail to grow Mucorales, identification of respective hyphae in tissue is frequently needed for diagnosis but may be challenging. We studied fluorescence in situ hybridization (FISH) targeting specific regions of the fungal ribosomal RNA (rRNA) of Mucorales to improve diagnosis of mucormycosis from tissue samples. We generated a probe combination specifically targeting Mucorales. Probe specificity was verified in silico and using cultivated fungi. Mucorales hyphae in tissue of a mouse model demonstrated a bright cytoplasmatic hybridization signal. In tissue samples of patients with mucormycosis, a positive signal was seen in 7 of 12 (58.3%) samples. However, autofluorescence in 3 of 7 (42.9%) samples impaired the diagnostic yield. Subsequent experiments suggested that availability of nutrients and antifungal therapy may impact on the FISH signal obtained with Mucorales hyphae. Diagnosis of mucormycosis from tissue might be improved by rRNA FISH in a limited number of cases only. FISH signals may reflect different physiological states of fungi in tissue. Further studies are needed to define the value of FISH to diagnose mucormycosis from other clinical samples. Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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12 pages, 978 KiB

Open AccessArticle

Diagnosing Fungal Keratitis and Simultaneously Identifying Fusarium and Aspergillus Keratitis with a Dot Hybridization Array

by Ming-Tse Kuo, Shiuh-Liang Hsu, Huey-Ling You, Shu-Fang Kuo, Po-Chiung Fang, Hun-Ju Yu, Alexander Chen, Chia-Yi Tseng, Yu-Hsuan Lai and Jiunn-Liang Chen

J. Fungi 2022, 8(1), 64; https://doi.org/10.3390/jof8010064 - 7 Jan 2022

Cited by 3 | Viewed by 1851

Abstract

Fungal keratitis (FK) is one of the most common microbial keratitis, which often leads to poor prognosis as a result of delayed diagnosis. Several studies implied that early differentiation of the two major FK, Fusarium and Aspergillus keratitis, could be helpful in selecting effective anti-fungal regimens. Therefore, a novel dot hybridization array (DHA) was developed to diagnose FK and differentiate Fusarium and Aspergillus keratitis in this study. One hundred forty-six corneal scrapes obtained from one hundred forty-six subjects impressed with clinically suspected FK were used to evaluate the performance of the DHA. Among these patients, 107 (73.3%) patients had actual FK confirmed by culture and DNA sequencing. We found that the DHA had 93.5% sensitivity and 97.4% specificity in diagnosing FK. In addition, this array had 93.2% sensitivity and 93.8% specificity in diagnosing Fusarium keratitis, as well as 83.3% sensitivity and 100% specificity in diagnosing Aspergillus keratitis. Furthermore, it had 83.9% sensitivity and 100% specificity in identifying Fusarium solani keratitis. Thus, this newly developed DHA will be beneficial to earlier diagnosis, more precise treatment, and improve prognosis of FK, by minimizing medical refractory events and surgical needs. Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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12 pages, 3494 KiB

Open AccessCommunication

Rapid Diagnosis of Central Nervous System Scedosporiosis by Specific Quantitative Polymerase Chain Reaction Applied to Formalin-Fixed, Paraffin-Embedded Tissue

by Robert J. Lauerer, Emely Rosenow, Rudi Beschorner, Johann-Martin Hempel, Georgios Naros, Anna Hofmann, Katharina Berger, Jennifer Sartor-Pfeiffer, Annerose Mengel, Ulf Ziemann, Volker Rickerts and Katharina Feil

J. Fungi 2022, 8(1), 19; https://doi.org/10.3390/jof8010019 - 27 Dec 2021

Cited by 3 | Viewed by 2733

Abstract

Scedosporium (S.) apiospermum is a typical mold causing cerebral abscesses, often after near-drowning. Infections are associated with high morbidity and mortality due to diagnostic challenges including the need for prolonged incubation of cultures. In addition, histopathological differentiation from other filamentous fungi, including Aspergillus fumigatus, may not be possible, excluding early specific diagnosis and targeted therapy. Polymerase chain reaction (PCR) on tissue samples can rapidly identify fungi, leading to an earlier adequate treatment. Due to an extensive spectrum of causative fungi, broad-range PCRs with amplicon sequencing have been endorsed as the best DNA amplification strategy. We herein describe a case with brain abscesses due to S. apiospermum in a 66-year-old immunocompromised female patient. While broad-range PCR failed to identify a fungal pathogen from a cerebral biopsy demonstrating hyaline mold hyphae, specific quantitative PCR (qPCR) identified Scedosporium and ruled out Aspergillus, the most prevalent agent of central nervous system mold infection. A panel of specific qPCR assays, guided by the morphology of fungal elements in tissue or as a multiplex assay, may be a successful molecular approach to identify fungal agents of brain abscesses. This also applies in the presence of negative broad-range fungal PCR, therefore providing diagnostic and therapeutic potential for early specific management and improvement of patient clinical outcome. Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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13 pages, 997 KiB

Open AccessArticle

Identification of Pneumocystis jirovecii with Fluorescence In-Situ Hybridization (FISH) in Patient Samples—A Proof-of-Principle

by Débora Raysa Teixeira de Sousa, João Ricardo da Silva Neto, Roberto Moreira da Silva, Jr., Kátia Santana Cruz, Sven Poppert, Hagen Frickmann and João Vicente Braga Souza

J. Fungi 2022, 8(1), 13; https://doi.org/10.3390/jof8010013 - 25 Dec 2021

Viewed by 2810

Abstract

In resource-limited settings, where pneumocystosis in immunocompromised patients is infrequently observed, cost-efficient, reliable, and sensitive approaches for the diagnostic identification of Pneumocystis jirovecii in human tissue samples are desirable. Here, an in-house fluorescence in situ hybridization assay was comparatively evaluated against Grocott’s staining as a reference standard with 30 paraffin-embedded tissue samples as well as against in-house real-time PCR with 30 respiratory secretions from immunocompromised patients with clinical suspicion of pneumocystosis. All pneumocystosis patients included in the study suffered from HIV/AIDS. Compared with Grocott’s staining as the reference standard, sensitivity of the FISH assay was 100% (13/13), specificity was 41% (7/17), and the overall concordance was 66.7% with tissue samples. With respiratory specimens, sensitivity was 83.3% (10/12), specificity was 100% (18/18), and the overall concordance was 93.3% as compared with real-time PCR. It remained unresolved to which proportions sensitivity limitations of Grocott’s staining or autofluorescence phenomena affecting the FISH assay accounted for the recorded reduced specificity with the tissue samples. The assessment confirmed Pneumocystis FISH in lung tissue as a highly sensitive screening approach; however, dissatisfying specificity in paraffin-embedded biopsies calls for confirmatory testing with other techniques in case of positive FISH screening results. In respiratory secretions, acceptable sensitivity and excellent specificity were demonstrated for the diagnostic application of the P. jirovecii-specific FISH assay. Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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Review

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15 pages, 941 KiB

Open AccessReview

Rapid Genomic Diagnosis of Fungal Infections in the Age of Next-Generation Sequencing

by Chi-Ching Tsang, Jade L. L. Teng, Susanna K. P. Lau and Patrick C. Y. Woo

J. Fungi 2021, 7(8), 636; https://doi.org/10.3390/jof7080636 - 5 Aug 2021

Cited by 43 | Viewed by 7916

Abstract

Next-generation sequencing (NGS) technologies have recently developed beyond the research realm and started to mature into clinical applications. Here, we review the current use of NGS for laboratory diagnosis of fungal infections. Since the first reported case in 2014, >300 cases of fungal infections diagnosed by NGS were described. Pneumocystis jirovecii is the predominant fungus reported, constituting ~25% of the fungi detected. In ~12.5% of the cases, more than one fungus was detected by NGS. For P. jirovecii infections diagnosed by NGS, all 91 patients suffered from pneumonia and only 1 was HIV-positive. This is very different from the general epidemiology of P. jirovecii infections, of which HIV infection is the most important risk factor. The epidemiology of Talaromyces marneffei infection diagnosed by NGS is also different from its general epidemiology, in that only 3/11 patients were HIV-positive. The major advantage of using NGS for laboratory diagnosis is that it can pick up all pathogens, particularly when initial microbiological investigations are unfruitful. When the cost of NGS is further reduced, expertise more widely available and other obstacles overcome, NGS would be a useful tool for laboratory diagnosis of fungal infections, particularly for difficult-to-grow fungi and cases with low fungal loads. Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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Other

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8 pages, 1896 KiB

Open AccessCase Report

Exclusion of Mucorales Co-Infection in a Patient with Aspergillus flavus Sinusitis by Fluorescence In Situ Hybridization (FISH)

by Johanna Kessel, Michael Hogardt, Lukas Aspacher, Thomas A. Wichelhaus, Jasmin Gerkrath, Emely Rosenow, Jan Springer and Volker Rickerts

J. Fungi 2022, 8(3), 306; https://doi.org/10.3390/jof8030306 - 16 Mar 2022

Viewed by 2250

Abstract

Invasive fungal infections are associated with increased mortality in hematological patients. Despite considerable advances in antifungal therapy, the evaluation of suspected treatment failure is a common clinical challenge requiring extensive diagnostic testing to rule out potential causes, such as mixed infections. We present a 64-year-old patient with secondary AML, diabetes mellitus, febrile neutropenia, and sinusitis. While cultures from nasal tissue grew Aspergillus flavus, a microscopic examination of the tissue was suggestive of concomitant mucormycosis. However, fluorescence in situ hybridization (FISH) using specific probes targeting Aspergillus and Mucorales species ruled out mixed infection. This was confirmed by specific qPCR assays amplifying the DNA of Aspergillus, but not of Mucorales. These results provided a rational basis for step-down targeted therapy, i.e., the patient received posaconazole after seven days of calculated dual therapy with liposomal amphotericin B and posaconazole. Despite clinical response to the antifungal therapy, he died due to the progression of the underlying disease within two weeks after diagnosis of fungal infection. Molecular diagnostics applied to tissue blocks may reveal useful information on the etiology of invasive fungal infections, including challenging situations, such as with mixed infections. A thorough understanding of fungal etiology facilitates targeted therapy that may improve therapeutic success while limiting side effects. Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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7 pages, 47490 KiB

Open AccessCase Report

Disseminated Fungal Infection and Fungemia Caused by Trichosporon asahii in a Captive Plumed Basilisk (Basiliscus plumifrons)

by Chieh Lo, Chu-Lin Kang, Pei-Lun Sun, Pin-Huan Yu and Wen-Ta Li

J. Fungi 2021, 7(12), 1003; https://doi.org/10.3390/jof7121003 - 24 Nov 2021

Cited by 2 | Viewed by 2204

Abstract

Trichosporon spp. are heavily arthroconidiating fungi and widely distributed in nature. Due to the similar fungal morphology, confusion among Trichosporon spp., Geotrichum spp., and Nannizziopsis spp. in reptiles is apparent and cannot be overlooked. Although few reptile Trichosporon isolates have been examined using the newer speciation criteria, the information on Trichosporon asahii in reptiles is still scarce. In the present study, we report the case of disseminated fungal infection and fungemia caused by T. asahii in a captive plumed basilisk (Basiliscus plumifrons). Multiple 0.2–0.5 cm, irregularly shaped, ulcerative nodules on the left hind foot were observed. The animal died due to the non-responsiveness to treatment. A microscopic evaluation revealed the fungal infection that primarily affected the left hind foot and right lung lobe with fungal embolisms in the lung and liver. The molecular identification of the fungal species by the DNA sequences of the ITS regions and D1/D2 gene from the fungal culture and ITS regions, from formalin-fixed paraffin-embedded (FFPE) lung tissues, were completely matched to those of T. asahii. The current report describes the first confirmed case of disseminated fungal infection and fungemia caused by T. asahii in a captive plumed basilisk. Full article

(This article belongs to the Special Issue Molecular Tissue Diagnosis of Fungal Infections)

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