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Article

Trends on Aspergillus Epidemiology—Perspectives from a National Reference Laboratory Surveillance Program

by
Raquel Sabino
1,*,
Paulo Gonçalves
1,2,
Aryse Martins Melo
1,3,
Daniela Simões
1,
Mariana Oliveira
1,
Mariana Francisco
1,
Carla Viegas
4,5,6,
Dinah Carvalho
7,
Carlos Martins
7,
Teresa Ferreira
8,
Cristina Toscano
9,
Helena Simões
1 and
Cristina Veríssimo
1
1
Infectious Diseases Department, National Health Institute Dr. Ricardo Jorge, 1649-016 Lisbon, Portugal
2
European Programme for Public Health Microbiology Training (EUPHEM), European Centre for Disease Prevention and Control, 16973 Solna, Sweden
3
Programa de Pós-Graduação em Microbiologia e Parasitologia, Instituto de Biologia, Universidade Federal de Pelotas, Avenida Eliseu Maciel, Pelotas 96010-610, Brazil
4
H&TRC—Health & Technology Research Center, ESTeSL—Escola Superior de Tecnologia da Saúde, Instituto Politécnico de Lisboa, 1990-096 Lisbon, Portugal
5
NOVA National School of Public Health, Public Health Research Centre, Universidade NOVA de Lisboa, 1600-560 Lisbon, Portugal
6
Comprehensive Health Research Center (CHRC), 1169-056 Lisbon, Portugal
7
Centro Hospitalar Universitário Lisboa Norte EPE, 1649-028 Lisbon, Portugal
8
Centro Hospitalar Universitário Lisboa Central, 1050-099 Lisbon, Portugal
9
Microbiology Laboratory, Centro Hospitalar Lisboa Ocidental, Hospital Egas Moniz, 1349-019 Lisbon, Portugal
*
Author to whom correspondence should be addressed.
J. Fungi 2021, 7(1), 28; https://doi.org/10.3390/jof7010028
Submission received: 31 October 2020 / Revised: 23 December 2020 / Accepted: 30 December 2020 / Published: 6 January 2021
(This article belongs to the Special Issue Aspergillus Infection)
Versions Notes

Abstract

:
Identification of Aspergillus to species level is important since sibling species may display variable susceptibilities to multiple antifungal drugs and also because correct identification contributes to improve the knowledge of epidemiological studies. Two retrospective laboratory studies were conducted on Aspergillus surveillance at the Portuguese National Mycology Reference Laboratory. The first, covering the period 2017–2018, aimed to study the molecular epidemiology of 256 Aspergillus isolates obtained from patients with respiratory, subcutaneous, or systemic infections and from environmental samples. The second, using our entire collection of clinical and environmental A. fumigatus isolates (N = 337), collected between 2012 and 2019, aimed to determine the frequency of azole-resistant A. fumigatus isolates. Aspergillus fumigatus sensu stricto was the most frequent species in both clinical and environmental samples. Overall, and considering all Aspergillus sections identified, a high frequency of cryptic species was detected, based on beta-tubulin or calmodulin sequencing (37% in clinical and 51% in environmental isolates). Regarding all Fumigati isolates recovered from 2012–2019, the frequency of cryptic species was 5.3% (18/337), with the identification of A. felis (complex), A. lentulus, A. udagawae, A. hiratsukae, and A. oerlinghauensis. To determine the frequency of azole resistance of A. fumigatus, isolates were screened for azole resistance using azole-agars, and 53 possible resistant isolates were tested by the CLSI microdilution reference method. Nine A. fumigatus sensu stricto and six Fumigati cryptic isolates showed high minimal inhibitory concentrations to itraconazole, voriconazole, and/or posaconazole. Real-time PCR to detect cyp51A mutations and sequencing of cyp51A gene and its promoter were performed. The overall frequency of resistance to azoles in A. fumigatus sensu stricto was 3.0%. With this retrospective analysis, we were able to detect one azole-resistant G54R mutant A. fumigatus environmental isolate, collected in 2015. The TR34/L98H mutation, linked to environmental transmission route of azole resistance, was the most frequently detected mutation (N = 4; 1.4%). Our findings underline the demand for correct identification and susceptibility testing of Aspergillus isolates.

1. Introduction

Invasive aspergillosis (IA) affects about 300,000 patients per year and more than 30 million patients are at risk [1]. The genus Aspergillus is composed of several hundred species, some of which are considered to be potentially pathogenic, causing serious infections with a fatality rate that can reach 50% if treated and more than 99% if not treated [1]. The main pathologies associated with infections by Aspergillus affect the lungs, including allergic bronchopulmonary aspergillosis (ABPA), and chronic pulmonary aspergillosis (CPA), but the infection can also spread to other organs leading to the development of IA [2]. Patients at high risk of acquiring IA generally suffer from severe granulocytopenia (leukemia, bone marrow, or solid organ transplant patients). In addition, prolonged use of corticosteroids, diabetes, severe burns, and major surgery are considered predisposing risk factors for IA [3].
Molecular studies [4,5] have shown the limitation of conventional identification through morphological characteristics to distinguish Aspergillus species. In fact, an isolate identified using morphological methodologies can only be included in one of the “species sections”, such as Fumigati, Flavi, Nidulantes, Usti, or Terrei. Species that are morphologically identical and only distinguishable from each other using molecular methodologies are called cryptic species. Molecular differences also reflect a difference in susceptibility to antifungals, with cryptic species being in general less susceptible [4].
The Aspergillus species most frequently isolated in the clinical context are A. fumigatus, A. flavus, A. niger, and A. terreus. The Fumigati section, composed of A. fumigatus sensu stricto and its cryptic species, is the most frequently isolated from clinical samples and is also frequently isolated from environmental sources. Moreover, the prevalence of clinical isolates of the Fumigati section with azole resistance has been increasing [6]. The proposed reasons for this include the development of resistance by prolonged antifungal prophylaxis or therapy. However, the inhalation of environmental strains resistant to antifungals is currently one of the biggest and most recent concerns of the scientific community [6,7,8].
It has been shown that azole-resistant A. fumigatus isolates have, for the most part, a resistance mechanism mediated by the cyp51A gene. Depending on the specific mutation, they may show resistance to one azole or to any azole [6]. TR34/L98H and TR46/Y121F/T289A are the most common mutations associated with environmental pan-azole resistance. However, other mutations in the promoter of the cyp51A gene, as TR53, TR463, and TR464, have also been described as from environmental origin [9]. A recently described mutation in the promoter of the cyp51A gene is TR120, probably more associated with prolonged azole exposure [10]. Other mutations at various positions of the cyp51A gene such as G54, M220, P216, G138, and G448 have also been associated with resistance, of which the G54 and M220 mutations are the most common [11,12].
Azoles are the first line of prophylaxis and treatment of infections by A. fumigatus and, therefore, there is a high concern inherent to treatment failure. Resistance to triazoles can severely limit treatment options and be associated with worse patient prognosis [13].
The European Center for Disease Prevention and Control (ECDC) published in 2013 [14] a recommendation for epidemiological surveillance to be carried out, to collect information at the local level, both in clinical and environmental context. In Portugal, few reports on Aspergillus prevalence and resistance have been published [15,16]. As such, we hypothesized that the reported prevalence of azole-resistant A. fumigatus may not represent the true prevalence of azole resistance and, therefore, surveillance studies are warranted.
Here, we report the results from the laboratory-based national surveillance program for Aspergillus that is established in our country since 2012, coordinated by the National Reference Laboratory for Mycology at the National Institute of Health Dr. Ricardo Jorge in Lisbon. Our laboratory provides reference diagnostic services to several hospitals from the National Health Service and for with a wide range of microbiological and clinical specialties from different regions of Portugal. In the context of this program, our laboratory receives clinical and environmental specimens or isolates for identification and antifungal susceptibility profiling of Aspergillus. Thus, with the two retrospective laboratory studies here presented, we are able to evaluate the frequency and diversity of Aspergillus species and the resistance profile to azoles of A. fumigatus at national level and, in doing so, to contribute to informed decisions and policies on the control of aspergillosis and on the use of azole antifungals, both in medicine and in the environment.

2. Materials and Methods

2.1. Aspergillus Identification and Diversity

During 2017 and 2018, Aspergillus isolates were obtained from clinical (respiratory, subcutaneous, or systemic) and environmental (soil, air, and surfaces from different sources, including hospitals, dwellings, and greenhouses) samples, analyzed at the Mycology National Reference Laboratory or received from collaborating institutions in the context of the Aspergillus surveillance program.
All isolates included in the surveillance program were assigned with a unique and sequential designation (VA1, VA2, etc.). All isolates were plated for growth as single colonies on malt extract agar with chloramphenicol (MEA). The morphological identification of the Aspergillus section was carried out based on macro and microscopic characteristics and using identification atlases [17,18].
Molecular identification of the species was carried out by partial sequencing the genes encoding calmodulin or beta-tubulin. Genomic DNA was prepared from each isolate using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.
Amplifications and sequencing of the partial beta-tubulin and calmodulin genes were performed as described previously [19]. The resultant nucleotide sequences were edited with Chromas Lite v 2.01 and aligned with CLUSTALX v 2.1 programs. Edited sequences were further compared with sequences deposited in the GenBank (Bethesda, MD, USA) and WI-KNAW Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands) databases, in order to achieve the identification to species level, accepted when the obtained homology was ≥98%.
For both groups of clinical and environmental samples, species richness (total number of species), their abundance and diversity were evaluated through the Simpson diversity index (D = 1 − {[Σn(n − 1)]/[ΣN(N − 1)]}, where n is the total number of organisms of a particular species and N is the total number of organisms of all species; the index ranges from 0 to 1, the greater the value, the greater the diversity in the group), and the Shannon diversity index ( H ′ = Σ p i ln p i , where pi is the proportion of individuals of species; the more unequal the abundance of species, the smaller the index; if abundance is primarily concentrated into one species, the index will be close to zero) [20].

2.2. Azole Susceptibility Profiling

For the study of azole-resistance patterns in A. fumigatus in Portugal, our entire 2012–2019 collection of isolates identified as belonging to Fumigati section was analyzed, which included both clinical and environmental isolates from different regions of the country.
Clinical isolates were obtained from biological specimens collected from different sites (mainly from the respiratory tract), from patients from 23 independent clinical institutions. Environmental isolates were collected from soil, air, and surfaces from previous referred sources (hospitals, dwellings, and greenhouses) but also from thermal spas, animal production farms, and a waste sorting plant.
The pattern of susceptibility of the isolates to antifungal agents was carried out using an initial screening media for azole resistance. Plates of Sabouraud dextrose agar (SDA) (Oxoid, Hampshire, UK) supplemented with concentrations of 4 mg/mL of itraconazole (ICZ), 1 mg/mL (until 2017) [8] and 2 mg/mL (2018 to present) of voriconazole (VCZ) or 0.5 mg/mL of posaconazole (PCZ) were used [21,22,23]. Fresh conidia from a 7-day-old culture grown on MEA were suspended in saline solution at a turbidity equivalent to a 0.5 McFarland standard. Plates were inoculated by swabbing and incubated at 37 °C for 48 h. To control strain viability, an SDA plate without chloramphenicol was used. The reference strain Aspergillus fumigatus ATCC 204305 was used as negative control and a pan-azole resistant strain TR34/L98H (kindly provided by Professor Jacques Meis, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands) was used as positive control. Aspergillus growth was observed visually and classified as “Negative (−)” (no growth, susceptible), “Relevant growth (+)” (growth similar to positive control) and “Residual growth (±)” (growth of only one or few small colonies). Strains that were not able to grow in the azole-supplemented plates were considered as susceptible to the respective azoles, according to EUCAST guidelines [23]. In case of doubt or to confirm the screening results (for residual or relevant growths, respectively), the antifungal susceptibility profiles were determined by broth microdilution reference method for susceptibility testing of molds. The M38-A2 protocol from the Clinical and Laboratory Standards Institute was applied for determining the Minimal Inhibitory Concentrations (MIC) for ICZ, VCZ, PCZ. The final concentrations of drugs in the wells ranged from 0.0156 to 8 μg/mL for all. An internal control strain (Aspergillus flavus ATCC 204304) with known susceptibility was included as a positive control for MIC determination. SDA plates were inoculated with the final inoculum, incubated at 35 C, and examined after 48 h of incubation to check the inoculum concentration. Absence of visual growth defined the MIC. Isolates with high MICs were tested in triplicate to confirm the obtained results. Breakpoints for mold testing have not been established by the CLSI except for voriconazole and A. fumigatus. The CLSI epidemiological cut-off values (ECVs) used were 1 mg/L for ICZ, and 0.5 mg/L for PCZ and, for VCZ, 1 mg/L as breakpoint [24,25,26].
Working breakpoints were assigned according to Moin et al. [27] as follows: Itraconazole: susceptible (MIC ≤ 1 mg/L), intermediate (MIC = 2 mg/L), and resistant (MIC ≥ 4 mg/L); voriconazole: susceptible (MIC ≤ 1 mg/L), and resistant (MIC ≥ 2 mg/L); posaconazole: susceptible (MIC ≤ 0.5 mg/L), and resistant (MIC ≥ 1 mg/L).
The AsperGenius® Resistance multiplex real-time polymerase chain reaction (PCR) assay to detect TR34/L98H, and TR46/T289A/Y121F mutations and the AsperGenius® G54/M220 RUO PCR assay to detect G54/M220 mutations (PathoNostics, Maastricht, The Netherlands) were used to test the isolates with a MIC for voriconazole, posaconazole, and/or itraconazole above the mentioned ECVs. These assays were performed on the Qiagen RotorGene Q instrument (Qiagen, Hilden, Germany) following the manufacturer’s instructions. To confirm the real-time PCR results and to check the existence of other mutations, point mutations, and tandem repeats, the cyp51A gene and its promoter were sequenced according to Prigitano et al. and Mellado et al. [28,29].
The programs GeneStudio ™ Professional Edition version 2.2.0.0 was used to edit the resultant nucleotide sequences and MEGA version 10.0.5 was used to align them. Edited sequences were compared with sequences deposited in the GenBank database (Bethesda, MD, USA).

3. Results

3.1. Molecular Epidemiology

From the 256 isolates collected during the study period (2017–2018), 156 were from clinical (human) sources and 99 from different environmental sources.
One hundred and forty-four clinical isolates (92.3%) were from respiratory specimens, including bronchial/bronchoalveolar lavage (N = 109), bronchial secretions (N = 21), sputum (N = 12), and bronchial aspirate (N = 2). Other samples included chest drain pus (N = 1), tissue sample (N = 1), and clinical samples from unknown body source (N = 10). These clinical samples were collected from 129 patients (58 males, 66 females, 5 not known), with ages ranging from 17 to 88 years old at 14 hospital centers distributed throughout the country. The clinical information associated with the biological samples sent to the laboratory was scarce, but the majority of the patients presented bronchiectasis in their image exams (N = 32). Other clinical information available was: cavitary lung lesions/nodules/abscess/infiltrates (N = 11), respiratory infections/pneumonia (N = 9), HIV +/previous or active tuberculosis (N = 9); asthma (N = 2), invasive aspergillosis (N = 2), neoplasms (N = 3), cystic fibrosis (N = 1), chronic obstructive pulmonary disease (N = 1), diabetes (N = 1), and admission at the ICU, surgery, or infectiology units (N = 4). In 54 cases, no clinical information was provided to the laboratory.
A. fumigatus sensu stricto was the most frequent species in both clinical and environmental sources. Among the 156 clinical isolates, eight different Aspergillus sections were identified [Fumigati (N = 70), Nigri (N = 40), Terrei (N = 13), Flavi (N = 11), Circumdati (N = 2), Clavati (N = 1), Aspergilli (N = 1), and Nidulantes (N = 13)], the latter now including previous section Versicolores) [30]. A total of 25 different species were identified (Table 1).
Twenty clinical isolates were identified only as Aspergillus sp. by morphological methods; all were collected from respiratory samples (bronchial/ bronchoalveolar lavage and bronchial secretions) (Table 1).
From those, 15 were identified by calmodulin and/or beta-tubulin sequencing, 11 of which were identified as cryptic species, some of them described as less susceptible to antifungals (as A. lentulus). In five isolates, no PCR product was obtained. Molecular identification to species level was obtained in 139 isolates. In total, cryptic species represented 36.7% (51/139) of the total clinical isolates.
More than one Aspergillus species were isolated from the clinical samples of 19 patients (Table 2). Three patients (#6, #9, and #17) were infected/colonized by A. fumigatus sensu stricto and its cryptic species A. lentulus. This cryptic species was also isolated from patient #13, together with A. niger and A. terreus. Seven patients (#6, #7, #8, #10, #11, #13, and #19) were simultaneously colonized/infected with A. terreus together with other species.
The 99 isolates were recovered from hospital environment (air, surfaces) (N = 17), soil (N = 1), air conditioning filters from different settings (N = 22), dwellings (air, surfaces) (N = 20), and air from agricultural environments (N = 19). Among these environmental isolates, eight different Aspergillus sections were identified [Fumigati (N = 39), Nigri (N = 21), Usti (N = 19), Nidulantes (N = 12), Flavi (N = 2), Circundati (N = 3), Clavati (N = 1), and Flavipedes (N = 2)]. Within these sections, 18 different species were identified (Table 1). Cryptic species represented 51.5% (50/99) of the total isolates.
Species diversity was compared between clinical and environmental isolates. Simpson index showed greater species diversity in environmental samples compared with clinical samples (D = 0.82 versus D = 0.76, respectively). On the other hand, the abundance of a specific species is slightly greater in clinical samples when compared to environmental samples (Shannon index = −2.07 versus −2.12, respectively).

3.2. Characterization of the Resistance Pattern of Isolates from Fumigati Section

A total of 337 isolates belonging Fumigati section were collected in the period between 2012 and 2019 (Table 3).
Among those, the frequency of cryptic species within this section was 5.3% (18/337). Twenty-four Fumigati isolates lost their viability and it was not possible to test them regarding their antifungal susceptibility. From the remaining, relevant and residual growth in azole-resistance screening media were obtained for 46 out of 296 sensu stricto isolates and for 7 out of 17 Fumigati cryptic species. Per published epidemiological cut-off values established using the CLSI M38A2 broth microdilution method, 4.4% of A. fumigatus sensu stricto isolates would be considered non-wild type to itraconazole (MIC >1 mg/L), 2.0% to voriconazole (MIC > 1 mg/L) and 2.0% to posaconazole (MIC > 0.5 mg/L) (Table 4).
Overall, all isolates considered as resistant or non-wildtype by microdilution grew in azole screening media (Table 4). It was not possible to run the MICs of two isolates (1 A. fumigatus sensu stricto and 1 A. lentulus), since they were not able to be recovered in culture.
Isolates that showed elevated MICs to azoles were screened by multiplex Aspergillus PCR kit for detection of specific mutations or sequenced the cyp51A gene and its promoter.
The overall prevalence of resistance to azoles in A. fumigatus sensu stricto was 3.0% (four clinical—VA85, VA303, VA328, and VA346—and five environmental isolates—VA86, VA95, VA299CP, VA873CP, VA1209CP) (Table 4). Regarding clinical isolates, the amplification of the cyp51A gene of the VA85 isolate was inhibited, preventing any conclusions regarding the resistance mechanism involved (high MICs for PCZ and VCZ, intermediate for ICZ). Although displaying MICs higher than the proposed ECVs, no mutations were found in the cyp51A gene of VA328 isolate (high MICs for ICZ and PCZ, wild type for VCZ), whereas cyp51A sequencing of VA346 isolate (high MIC for PCZ, intermediate for ICZ, susceptible to VCZ) showed the presence of the following mutations: F46Y, M172V, N248T, D255E. In the VA303 isolate (pan-azole resistant), the TR34/L98H mutations were detected. For the environmental isolates considered as non-wild types, the TR34/L98H was also detected in the VA299CP, VA873CP, VA1209CP isolates, conferring pan-azole resistance. The multiplex Aspergillus PCR kit M220-G54 and the cyp51A gene sequencing allowed the detection of a G54R mutation in the VA86 isolate, conferring in vitro resistance to ICZ and PCZ. Although displaying MICs higher than the proposed ECVs for either ICZ, VCZ, and PCZ, no mutation was found in the cyp51A gene of the VA95 isolate.
The TR34/L98H was the most frequently detected mutation (in 1.4% of the isolates), found in three environmental and one clinical isolate. In all studied cases, data obtained by the real time Aspergillus multiplex PCR were corroborated by sequencing of the cyp51A gene and its promoter.
Regarding only the Fumigati cryptic species, its overall resistance to azoles was 33.3% (all resistant isolates were from clinical sources—VA116, VA118, VA144, VA199, and VA254) (Table 4).

4. Discussion

In this study, we analyzed the Aspergillus distribution in clinical and environmental samples during a two-year period, from 2017 to 2018, and also the patterns of A. fumigatus azole-resistance using isolates from the surveillance program established by our Reference Laboratory, from 2012 to 2019.
Correct species identification within Aspergillus genus requires partial sequencing of the beta-tubulin or calmodulin genes, methods that are not available in most laboratories. This lack of identification to species level may have important consequences as cryptic species often display some level of intrinsic resistance to azoles and other antifungal drugs. Some of the clinical species identified during our study, such as A. lentulus, A. udagawae, A. felis, or A. tubigensis have been associated with refractory cases of IA [31,32,33].
As expected, Fumigati was the most frequently isolated section from our clinical samples. The same was observed in several epidemiological studies in other countries [5,34].
In a relevant percentage of cases, diagnosis of invasive aspergillosis is not done based on microbiological data and relies on suggestive imagology and/or positive fungal biomarkers such as galactomanan, β-1,3-glucan [35]. Hence, the actual contribution of Aspergillus spp. other than A. fumigatus (namely cryptic species) in clinical setting is unknown [36].
A high percentage (37%) of cryptic species was detected in our study in clinical isolates. To the best of our knowledge, this proportion of cryptic species of Aspergillus was the highest reported in literature, even doubling what was previously reported in a previous study from our group [19]. In another Portuguese study [16], this value was much lower (37.0% versus 7.5%). These differences may reflect the geographical source of the isolates and their epidemiological status. In both studies, the most frequent cryptic species was A. welwitschiae. In a study published by Pinto et al. [16], the 7.5% prevalence of cryptic species was distributed by the A. niger section (3.1%) and by the A. fumigatus section (2.2%). We found 84% of cryptic species among our clinical isolates belonging to Nigri section, and 5.2% belonging to the Fumigati section. In Spain, the percentage of clinical isolates identified as cryptic species belonging to Fumigati section was lower (2.2%) [37]. When observing fungal growth, several points should be observed to determine contamination, such as specimen type, amount and growth’s localization of the culture, pattern of growth, and number of cultures positive to the same organism [38].
Regarding mixed infections/colonizations, A. terreus was found frequently associated with other species, even with a pan-azole resistant A. fumigatus (patient #19). In a study published by Zoran et al. [39], posaconazole-resistance in A. terreus isolates is higher than 10%. Given the reduced susceptibility of A. terreus to amphotericin B [40], the management of those infections may be more difficult. Simultaneous colonizations/infections of A. terreus together with other species should be taken into consideration in the evaluation of therapeutic approaches. According to recent guidelines [41], species identification at the complex level should be carried out for clinically relevant isolates since some species are intrinsically resistant to either azoles or amphotericin B. Moreover, Aspergillus colonies from a single sample may harbor different resistance profiles and therefore it is advisable to perform multiple colony testing (up to five colonies) in order to increase sensitivity for azole-resistance detection [41].
Interestingly, several of the identified cryptic species, especially A. lentulus, were isolated together with A. fumigatus sensu stricto or with other species (Table 2). This has also been reported in other studies [42]. According to several authors [34,43,44,45], cryptic Fumigati species have been recognized as occasional causes of invasive aspergillosis in 3 to 6% of cases. Noteworthy, in our surveillance study, several of these cryptic species could not be identified by culture morphology, but only through molecular methods, and five isolates could not even be characterized at section level, having been referred as Aspergillus sp. (Table 1).
Infections due to A. lentulus or other A. fumigatus-related species are associated with a particularly high mortality rate, about 60% [36]. These species are often less susceptible to azoles [44,46], which is also reported in our study. Their actual prevalence may be underestimated because of their lack of recognition by conventional diagnostic approaches. Therefore, IDSA guideline on diagnosis and management of aspergillosis [47] recommends (strong recommendation and a high-quality evidence) that species identification by molecular methods should be performed in isolates with atypical growth or concerns for resistance.
Our results showed an even higher frequency of cryptic species in environmental isolates, as corroborated by Simpson index value. As already shown in several studies [48,49,50,51], the environment represents the major source of Aspergillus isolates causing infections. Exposure to Aspergillus may occur at home, in agricultural/animal production, at the workplace, and during periods of hospitalization, among others. The knowledge of the Aspergillus epidemiology of these environments may be important given that different fungal species may lead to different clinical manifestations and outcomes, depending on the host susceptibility, as well [52].
In 2017, the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) recommend the susceptibility testing of Aspergillus on isolates causing invasive disease in patients from regions where resistance is found in surveillance programs [41]. However, this recommendation brings concern regarding the paucity of surveillance programs that perform mold susceptibility testing. Thus, this study aimed to present an estimate of the frequency of azoles resistant isolates of A. fumigatus in Portugal. According to our data, the estimated overall frequency of resistance in A. fumigatus sensu stricto was of 3.0%. Such a value differs from previous Portuguese studies. Amorim et al. [53] reported a <1% prevalence of itraconazole resistance in 159 isolates of A. fumigatus collected from cystic fibrosis patients receiving azole antifungal therapy. More recently, in a study on susceptibility of environmental A. fumigatus collected in the North of Portugal [54], 21.8% of isolates were resistant to itraconazole, 38.2% to posaconazole, and none of them were resistant to voriconazole. These differences may be explained by the date of collection of the isolates and by the geographical region from where the isolates were obtained.
According to a recent Spanish survey [37], 6.6% of patients carried azole-resistant A. fumigatus sensu lato. A similar percentage was obtained with our data, 4.8%.
The frequency of azole resistant A. fumigatus varies worldwide, ranging from 1.75% in India to 10%–20% in Europe, probably due to different usage of azole fungicides [27].
Azole-containing plates are easy to use in a routine mycology laboratory, offering the possibility of screening large collections of clinical strains at low cost [19]. When comparing the data obtained by azole screening with data obtained by the reference method, false negatives were rare (only three isolates with MIC > 0.5 mg/L that did not grow on PCZ-agar and one with MIC > 1 mg/L that did not grow on VCZ-agar). However, the multi-azole resistant isolates bearing the mutations TR34/L98H and G54R were detected in the screening media. According to Arendrup et al. [55], the sensitivity of azole screening media in detecting strains harboring the mutations G54E/R/V/W, G54R + N284K, M220I/K/T/V, M220I + V101F, TR34/L98H, and TR46/Y121F/T289A cyp51A mutations is 99%.
On the other hand, false positives were frequent but in the majority of the isolates considered as having “residual” growth, MICs were below the ECVs values. Morio et al. [56] refer that the use of a concentration of 4 mg/L itraconazole, which is above the ECV for A. fumigatus might be a limitation, as some non-susceptible isolates could be missed.
The Clinical Laboratory Standards Institute (CLSI) is developing susceptibility breakpoints for the triazoles and A. fumigatus to provide a tool to guide and optimize treatment. Recently, CLSI set breakpoints for voriconazole and A. fumigatus. These results identified voriconazole MIC values linked to treatment failure (≥2 μg/mL) and further identified susceptibility at concentrations ≤ 0.5 μg/mL. Regarding posaconazole, Espinel-Ingroff et al. [26] observed an overlap between MICs for non-mutant and mutant isolates was more evident with the ECVs of 0.5 μg/mL and, for that reason, we selected this value as ECV. Seven A. fumigatus sensu stricto isolates showed a MIC = 2 μg/mL to ICZ and three isolates showed a MIC = 2 μg/mL to VCZ. The latter value was considered as intermediate by CLSI. According to the most recent EUCAST guidelines [57], for some organism-agent combinations (as for A. fumigatus and itraconazole and voriconazole), results may be in an area designated the Area of Technical Uncertainty (ATU), where the interpretation is uncertain. In these cases, results should be reported as resistant with the comment that in some clinical situations (non-invasive infections) itraconazole can be used provided sufficient exposure is ensured. As an example, VA350 isolate showed MIC = 2 μg/mL to ICZ but no cyp51A mutation was found.
Among our A. fumigatus isolates collected from 2012 to 2019 and available for molecular testing, three harbored the TR34/L98H alterations in the cyp51A gene. These isolates were collected between 2018 and 2019, which may show an increase in the frequency of circulation of triazole-resistant A. fumigatus. The first clinical A. fumigatus isolates resistant to azoles detected in Portugal were described in 2018 [14]. According to our retrospective analysis, our first resistant isolate (mutation G54R) was collected in 2015, which reveals that azole resistant environmental isolates emerged in our country earlier than what was believed until now. Sharma et al. [58] also found resistant environmental isolates associated with this mutation. Isolate VA346 showed the presence of the following mutations: F46Y, M172V, N248T, D255E. These mutations (plus E427K) have been described to have different azole susceptibility profiles and to be azole susceptible or resistant, depending on the authors, but in all cases, they have higher azole MICs than A. fumigatus strains with wild-type cyp51A [59]. The same first four mutations were also found (together with E427K) in a pan-azole resistant Portuguese isolate analyzed by Monteiro et al. [54].
Mutation N248K was detected in two isolates that grew in azole screening media, but their MICs were lower than the proposed ECVs. As in other studies, N248K amino acid substitutions were previously reported in azole-susceptible A. fumigatus, suggesting that these alterations are SNPs that do not confer phenotypic resistance [60].
No mutations were detected in the cyp51A gene in the of the triazole-resistant VA95 isolate. As we have only screened for mutations on the cyp51A, it is possible that that resistance may be due to additional mechanisms of azole drug tolerance, such as mutations in hapE gene, decreased absorption of azole, or increased expression of efflux pumps [61].
Limitations of this study include the fact that the sequencing protocol used does not allow for the complete sequencing of the cyp51A gene (reaching only to the codon 365). Thus, mutations occurring at the end of this gene may not be detected. Furthermore, it was not possible to amplify calmodulin or beta-tubulin genes of several isolates, which resulted in an incomplete classification of our set of isolates. The isolates included in this study were not collected systematically from all regions of the country, which may lead to a bias in their geographical distribution. The majority of the isolates were obtained from samples that were not accompanied by enough clinical information, making the differentiation between colonization and infection difficult. Finally, when interpreting the resistance percentage reported here, we should also consider that, in clinical practice, a sizeable number of patients have culture-negative aspergillosis. As such, a positive selection bias toward resistance may occur, as sampling procedures are more likely to be undertaken for patients for whom antifungal therapy fails. Additionally, since the National Reference laboratory receives isolates voluntarily, a bias towards less common species (difficult to identify) and towards resistant isolates can occur, which can explain higher rates of cryptic species and may led to the overestimation of the resistance rate, in comparison to unbiased non-selected clinical isolates.
Considering the before mentioned limitations, it becomes clear that studies aiming at using a representative set of isolates and that may account for all these potential confounders are much needed, in order to have a more accurate estimation of the resistance profile in our country.
In summary, this study highlights the importance of the implementation of a national surveillance network for Aspergillus. These findings are important contributions to raise the awareness to the importance of Aspergillus epidemiological distribution and to the surveillance of azole resistance in A. fumigatus in different Portuguese institutions, environments, and from diverse geographic locations. Understanding the prevalence of azole resistant isolates is important to guide clinical and public health decision-making.

5. Conclusions

The understanding shifts in the epidemiology of Aspergillus and of local resistance patterns is needed to manage therapeutic approaches. From all studied Aspergillus, a very high percentage of cryptic species was found. In our collection of Fumigati isolates, more than 5% of them were cryptic species and from those almost all showed resistance to azoles. From A. fumigatus sensu stricto, 3% showed resistance to azoles, being the TR34/L98H the most frequent mutation.

Author Contributions

R.S.: Conception, design of the work, and writing of original draft preparation; R.S., A.M.M., P.G., D.S., M.O., M.F., H.S., C.V. (Cristina Veríssimo) and C.V. (Carla Viegas): Data acquisition, analysis, and interpretation; C.V. (Carla Viegas), C.M., D.S., and M.F.: Collection of environmental isolates; D.C., T.F., C.T., H.S., and C.V. (Cristina Veríssimo): Collection of clinical isolates; P.G., A.M.M., D.S., M.O., and C.V. (Carla Viegas): Draft reviewing and editing; C.V. (Cristina Veríssimo): Critical revision of the article. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Acknowledgments

Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisboa, Portugal, for funding the project “Programa de Vigilância de Aspergillus”. PathoNostics (Maastricht, The Netherlands), for sponsoring the AsperGenius® G54/M220 RUO PCR assay. HortoSimões Sociedade Agrícola Lda (Lisboa, Portugal) for allowing the collection of environmental isolates associated to agricultural environments. All the Health institutions enrolled in this Programme for contributing with isolates. Some environmental isolates used in this study were obtained in a work supported by FCT—Fundação para Ciência e Tecnologia, project EXPOsE (02/SAICT/2016—Project nº 23222) and Instituto Politécnico de Lisboa, Lisbon, Portugal, which funded the Project “Waste FRPD” (IPL/2018/WasteFRPD_ESTeSL).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Available online: http://www.life-worldwide.org/fungal-diseases/invasive-aspergillosis (accessed on 3 October 2020).
  2. Kosmidis, C.; Denning, D.W. The clinical spectrum of pulmonary aspergillosis. Thorax 2015, 70, 270–277. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Baddley, J.W. Clinical risk factors for invasive aspergillosis. Med. Mycol. 2011, 49 (Suppl. 1), S7–S12. [Google Scholar] [CrossRef] [PubMed]
  4. Balajee, S.A.; Houbraken, J.; Verweij, P.E.; Hong, S.B.; Yaghuchi, T.; Varga, J.; Samson, R.A. Aspergillus species identification in the clinical setting. Stud. Mycol. 2007, 59, 39–46. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Kontoyiannis, D.P.; Marr, K.A.; Park, B.J.; Alexander, B.D.; Anaissie, E.J.; Walsh, T.J.; Ito, J.; Andes, D.R.; Baddley, J.W.; Brown, J.M.; et al. Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, 2001-2006: Overview of the Transplant-Associated Infection Surveillance Network (TRANSNET) Database. Clin. Infect. Dis. 2010, 50, 1091–1100. [Google Scholar] [CrossRef] [PubMed]
  6. Snelders, E.; van der Lee, H.A.; Kuijpers, J.; Rijs, A.J.; Varga, J.; Samson, R.A.; Mellado, E.; Donders, A.R.; Melchers, W.J.; Verweij, P.E. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med. 2008, 5, e219. [Google Scholar] [CrossRef]
  7. Verweij, P.E.; Snelders, E.; Kema, G.H.; Mellado, E.; Melchers, W.J. Azole resistance in Aspergillus fumigatus: A side-effect of environmental fungicide use? Lancet Infect. Dis. 2009, 9, 789–795. [Google Scholar] [CrossRef]
  8. Mortensen, K.L.; Mellado, E.; Lass-Flörl, C.; Rodriguez-Tudela, J.L.; Johansen, H.K.; Arendrup, M.C. Environmental study of azole-resistant Aspergillus fumigatus and other aspergilli in Austria, Denmark, and Spain. Antimicrob. Agents Chemother. 2010, 54, 4545–4549. [Google Scholar] [CrossRef] [Green Version]
  9. Zhang, J.; Snelders, E.; Zwaan, B.J.; Schoustra, S.E.; Meis, J.F.; van Dijk, K.; Hagen, F.; van der Beek, M.T.; Kampinga, G.A.; Zoll, J.; et al. A novel environmental azole resistance mutation in Aspergillus fumigatus and a possible role of sexual reproduction in its emergence. mBio 2017, 8, e00791-17. [Google Scholar] [CrossRef] [Green Version]
  10. Hare, R.K.; Gertsen, J.B.; Astvad, K.; Degn, K.B.; Løkke, A.; Stegger, M.; Andersen, P.S.; Kristensen, L.; Arendrup, M.C. In Vivo Selection of a Unique Tandem Repeat Mediated Azole Resistance Mechanism (TR120) in Aspergillus fumigatus cyp51A, Denmark. Emerg. Infect. Dis. 2019, 25, 577–580. [Google Scholar] [CrossRef] [Green Version]
  11. Meis, J.F.; Chowdhary, A.; Rhodes, J.L.; Fisher, M.C.; Verweij, P.E. Clinical implications of globally emerging azole resistance in Aspergillus fumigatus. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2016, 5, 371. [Google Scholar] [CrossRef] [Green Version]
  12. Verweij, P.E.; Chowdhary, A.; Melchers, W.J.; Meis, J.F. Azole Resistance in Aspergillus fumigatus: Can We Retain the Clinical Use of Mold-Active Antifungal Azoles? Clin. Infect. Dis. 2016, 62, 362–368. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Snelders, E.; Karawajczyk, A.; Schaftenaar, G.; Verweij, P.E.; Melchers, W.J. Azole Resistance Profile of Amino Acid Changes in Aspergillus fumigatus CYP51A Based on Protein Homology Modeling. Antimicrob. Agents Chemother. 2010, 54, 2425–2430. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. European Centre for Disease Prevention and Control. Risk Assessment on the Impact of Environmental Usage of Triazoles on the Development and Spread of Resistance to Medical Triazoles in Aspergillus Species; ECDC: Stockholm, Sweden, 2013. [Google Scholar]
  15. Monteiro, C.; Faria, M.A.; Pinheiro, D.; Lameiras, C.; Pinto, E. First description of clinical Aspergillus fumigatus cyp51A TR46/Y121F/T289A mutant in Portugal. J. Glob. Antimicrob. Resist. 2018, 13, 190–191. [Google Scholar] [CrossRef] [PubMed]
  16. Pinto, E.; Monteiro, C.; Maia, M.; Faria, M.A.; Lopes, V.; Lameiras, C.; Pinheiro, D. Aspergillus Species and Antifungals Susceptibility in Clinical Setting in the North of Portugal: Cryptic Species and Emerging Azoles Resistance in A. fumigatus. Front. Microbiol. 2018, 23, 1656. [Google Scholar] [CrossRef] [Green Version]
  17. Campbell, C.K.; Johnson, E.M.; Philpot, C.M.; Warnock, D.W. Identification of Pathogenic Fungi; Public Health Laboratory Service: London, UK, 1996. [Google Scholar]
  18. de Hoog, D.; Guarro, J.; Gene, G.; Figueras, M. Atlas of Clinical Fungi—The Ultimate Benchtool for Diagnosis; Version 4.1.4; Utr Centraalbureau voor Schimmelcultures: Utrecht, The Netherlands, 2016. [Google Scholar]
  19. Sabino, R.; Veríssimo, C.; Parada, H.; Brandão, J.; Viegas, C.; Carolino, E.; Clemons, K.V.; Stevens, D.A. Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources. Med. Mycol. 2014, 52, 519–529. [Google Scholar] [CrossRef] [Green Version]
  20. Kim, B.R.; Shin, J.; Guevarra, R.; Lee, J.H.; Kim, D.W.; Seol, K.H.; Lee, J.H.; Kim, H.B.; Isaacson, R. Deciphering diversity indices for better understanding of the microbial communities. J. Microbiol. Biotechnol. 2017, 27, 2089–2093. [Google Scholar] [CrossRef] [Green Version]
  21. Guinea, J.; Verweij, P.E.; Meletiadis, J.; Mouton, J.W.; Barchiesi, F.; Arendrup, M.C.; Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). How to: EUCAST recommendations on the screening procedure E.Def 10.1 for the detection of azole resistance in Aspergillus fumigatus isolates using four-well azole-containing agar plates. Clin. Microbiol. Infect. 2019, 25, 681–687. [Google Scholar] [CrossRef]
  22. Dudakova, A.; Spiess, B.; Tangwattanachuleeporn, M.; Sasse, C.; Buchheidt, D.; Weig, M.; Groß, U.; Bader, O. Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species. Clin. Microbiol. Rev. 2017, 30, 1065–1091. [Google Scholar] [CrossRef] [Green Version]
  23. Arendrup, M.C.; Meletiadis, J.; Mouton, J.W.; Lagrou, K.; Hamal, P.; Guinea, J.; Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). EUCAST Definitive Document E. DEF. 9.3.1. Method for the Determination of Broth Dilution Minimum Inhibitory Concentrations of Antifungal Agents for Conidia Forming Moulds; European Committee on Antimicrobial Susceptibility Testing: London, UK, 2017. [Google Scholar]
  24. Meletiadis, J.; Mavridou, E.; Melchers, W.J.; Mouton, J.W.; Verweij, P.E. Epidemiological cutoff values for azoles and Aspergillus fumigatus based on a novel mathematical approach incorporating cyp51A sequence analysis. Antimicrob. Agents Chemother. 2012, 56, 2524–2529. [Google Scholar] [CrossRef] [Green Version]
  25. CLSI. Epidemiological Cutoff Values for Antifungal Susceptibility Testing, 3rd ed.; CLSI Supplement M59; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2020. [Google Scholar]
  26. Espinel-Ingroff, A.; Diekema, D.J.; Fothergill, A.; Johnson, E.; Pelaez, T.; Pfaller, M.A.; Rinaldi, M.G.; Canton, E.; Turnidge, J. Wild-type MIC distributions and epidemiological cutoff values for the triazoles and six Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document). J. Clin. Microbiol. 2010, 48, 3251–3257. [Google Scholar] [CrossRef] [Green Version]
  27. Moin, S.; Farooqi, J.; Jabeen, K.; Laiq, S.; Zafar, A. Screening for triazole resistance in clinically significant Aspergillus species; report from Pakistan. Antimicrob. Resist. Infect. Control. 2020, 9, 62. [Google Scholar] [CrossRef] [PubMed]
  28. Prigitano, A.; Venier, V.; Cogliati, M.; De Lorenzis, G.; Esposto, M.; Tortorano, A. Azole-resistant Aspergillus fumigatus in the environment of Northern Italy, May 2011 to June 2012. Eurosurveillance 2014, 19, 20747. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  29. Mellado, E.; Diaz-Guerra, T.; Cuenca-Estrella, M.; Rodriguez-Tudela, J. Identification of Two Different 14-α Sterol Demethylase-Related Genes (cyp51A and cyp51B) in Aspergillus fumigatus and Other Aspergillus species. J. Clin. Microbiol. 2001, 39, 2431–2438. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Hubka, V.; Nováková, A.; Peterson, S.W. A reappraisal of Aspergillus section Nidulantes with descriptions of two new sterigmatocystin-producing species. Plant Syst. Evol. 2016, 302, 1267–1299. [Google Scholar] [CrossRef]
  31. Park, S.Y.; Yoon, J.A.; Kim, S.H. Voriconazole-refractory invasive aspergillosis. Korean J. Intern Med. 2017, 32, 805–812. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Vinh, D.C.; Shea, Y.R.; Sugui, J.A.; Parrilla-Castellar, E.R.; Freeman, A.F.; Campbell, J.W.; Pittaluga, S.; Jones, P.A.; Zelazny, A.; Kleiner, D.; et al. Invasive aspergillosis due to Neosartorya udagawae. Clin. Infect. Dis. 2009, 49, 102–111. [Google Scholar] [CrossRef] [Green Version]
  33. Harada, S.; Ohkushi, D.; Nakano, K.; Mitani, H.; Yamamoto, Y.; Kamei, K.; Hayama, B. Fatal invasive pulmonary aspergillosis caused by voriconazole-resistant Aspergillus tubingensis in a patient with solid tumor. J. Infect. Chemother. 2020, 26, 3. [Google Scholar] [CrossRef] [PubMed]
  34. Alastruey-Izquierdo, A.; Mellado, E.; Peláez, T.; Pemán, J.; Zapico, S.; Alvarez, M.; Rodríguez-Tudela, J.L.; Cuenca-Estrella, M.; FILPOP Study Group. FILPOP Study Group. Population-based survey of filamentous fungi and antifungal resistance in Spain (FILPOP Study). Antimicrob. Agents Chemother. 2013, 57, 3380–3387. [Google Scholar] [CrossRef] [Green Version]
  35. Donnelly, J.P.; Chen, S.C.; Kauffman, C.A.; Steinbach, W.J.; Baddley, J.W.; Verweij, P.E.; Clancy, C.J.; Wingard, J.R.; Lockhart, S.R.; Groll, A.H.; et al. Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin. Infect. Dis. 2020, 71, 1367–1376. [Google Scholar] [CrossRef] [Green Version]
  36. Lamoth, F. Aspergillus fumigatus-Related Species in Clinical Practice. Front. Microbiol. 2016, 7, 683. [Google Scholar] [CrossRef] [Green Version]
  37. Escribano, P.; Rodríguez-Sánchez, B.; Díaz-García, J.; Martín-Gómez, M.T.; Ibáñez, E.; Rodríguez-Mayo, M.; Peláez, T.; García-Gómez de la Pedrosa, E.; Tejero-García, R.; Marimón, J.M.; et al. Azole resistance survey on clinical Aspergillus fumigatus isolates in Spain. Clin. Microbiol. Infect. 2020. [Google Scholar] [CrossRef] [PubMed]
  38. Larkin, P.M.K.; Multani, A.; Beaird, O.E.; Dayo, A.J.; Fishbein, G.A.; Yang, S. A Collaborative Tale of Diagnosing and Treating Chronic Pulmonary Aspergillosis, from the Perspectives of Clinical Microbiologists, Surgical Pathologists, and Infectious Disease Clinicians. J. Fungi 2020, 6, 106. [Google Scholar] [CrossRef] [PubMed]
  39. Zoran, T.; Sartori, B.; Sappl, L.; Aigner, M.; Sánchez-Reus, F.; Rezusta, A.; Chowdhary, A.; Taj-Aldeen, S.J.; Arendrup, M.C.; Oliveri, S.; et al. Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon? Front. Microbiol. 2018, 9, 516. [Google Scholar] [CrossRef] [PubMed]
  40. Posch, W.; Blatzer, M.; Wilflingseder, D.; Lass-Flörl, C. Aspergillus terreus: Novel lessons learned on amphotericin B resistance. Med. Mycol. 2018, 56 (Suppl. 1), 73–82. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  41. Ullmann, A.J.; Aguado, J.M.; Arikan-Akdagli, S.; Denning, D.W.; Groll, A.H.; Lagrou, K.; Lass-Flörl, C.; Lewis, R.E.; Munoz, P.; Verweij, P.E.; et al. Diagnosis and management of Aspergillus diseases: Executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin. Microbiol. Infect. 2018, 24 (Suppl. 1), e1–e38. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Montenegro, G.; Sánchez Puch, S.; Jewtuchowicz, V.M.; Pinoni, M.V.; Relloso, S.; Temporitti, E.; Iovannitti, C.A.; Mujica, M.T. Phenotypic and genotypic characterization of Aspergillus lentulus and Aspergillus fumigatus isolates in a patient with probable invasive aspergillosis. J. Med. Microbiol. 2009, 58 Pt 3, 391–395. [Google Scholar] [CrossRef]
  43. Balajee, S.A.; Kano, R.; Baddley, J.W.; Moser, S.A.; Marr, K.A.; Alexander, B.D.; Andes, D.; Kontoyiannis, D.P.; Perrone, G.; Peterson, S.; et al. Molecular identification of Aspergillus species collected for the Transplant-Associated Infection Surveillance Network. J. Clin. Microbiol. 2009, 47, 3138–3141. [Google Scholar] [CrossRef] [Green Version]
  44. Alastruey-Izquierdo, A.; Alcazar-Fuoli, L.; Cuenca-Estrella, M. Antifungal susceptibility profile of cryptic species of Aspergillus. Mycopathologia 2014, 178, 427–433. [Google Scholar] [CrossRef]
  45. Escribano, P.; Peláez, T.; Muñoz, P.; Bouza, E.; Guineam, J. Is azole resistance in Aspergillus fumigatus a problem in Spain? Antimicrob. Agents Chemother. 2013, 57, 2815–2820. [Google Scholar] [CrossRef] [Green Version]
  46. Sabino, R.; Carolino, E.; Veríssimo, C.; Martinez, M.; Clemons, K.V.; Stevens, D.A. Antifungal susceptibility of 175 Aspergillus isolates from various clinical and environmental sources. Med. Mycol. 2016, 54, 740–756. [Google Scholar] [CrossRef] [Green Version]
  47. Patterson, T.F.; Thompson, G.R., III; Denning, D.W.; Fishman, J.A.; Hadley, S.; Herbrecht, R.; Kontoyiannis, D.P.; Marr, K.A.; Morrison, V.A.; Nguyen, M.H.; et al. Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America. Clin. Infect. Dis. 2016, 63, e1–e60. [Google Scholar] [CrossRef] [PubMed]
  48. Warris, A.; Verweij, P.E. Clinical implications of environmental sources for Aspergillus. Med. Mycol. 2005, 43 (Suppl. 1), S59–S65. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Richardson, M.; Rautemaa-Richardson, R. Exposure to Aspergillus in Home and Healthcare Facilities’ Water Environments: Focus on Biofilms. Microorganisms 2019, 7, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Puértolas-Balint, F.; Rossen, J.; Oliveira Dos Santos, C.; Chlebowicz, M.; Raangs, E.C.; van Putten, M.L.; Sola-Campoy, P.J.; Han, L.; Schmidt, M.; García-Cobos, S. Revealing the Virulence Potential of Clinical and Environmental Aspergillus fumigatus Isolates Using Whole-Genome Sequencing. Front. Microbiol. 2019, 10, 1970. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Park, J.H.; Ryu, S.H.; Lee, J.Y.; Kim, H.J.; Kwak, S.H.; Jung, J.; Lee, J.; Sung, H.; Kim, S.H. Airborne fungal spores and invasive aspergillosis in hematologic units in a tertiary hospital during construction: A prospective cohort study. Antimicrob. Resist. Infect. Control. 2019, 8, 88. [Google Scholar] [CrossRef] [PubMed]
  52. Sabino, R.; Veríssimo, C.; Viegas, C.; Viegas, S.; Brandão, J.; Alves-Correia, M.; Borrego, L.M.; Clemons, K.V.; Stevens, D.A.; Richardson, M. The role of occupational Aspergillus exposure in the development of diseases. Med. Mycol. 2019, 57 (Suppl. 2), S196–S205. [Google Scholar] [CrossRef]
  53. Amorim, A.; Guedes-Vaz, L.; Araujo, R. Susceptibility to five antifungals of Aspergillus fumigatus strains isolated from chronically colonised cystic fibrosis patients receiving azole therapy. Int. J. Antimicrob. Agents 2010, 35, 396–399. [Google Scholar] [CrossRef] [Green Version]
  54. Monteiro, C.; Pinheiro, D.; Maia, M.; Faria, M.A.; Lameiras, C.; Pinto, E. Aspergillus species collected from environmental air samples in Portugal-molecular identification, antifungal susceptibility and sequencing of cyp51A gene on A. fumigatus sensu stricto itraconazole resistant. J. Appl. Microbiol. 2019, 126, 1140–1148. [Google Scholar] [CrossRef]
  55. Arendrup, M.C.; Verweij, P.E.; Mouton, J.W.; Lagrou, K.; Meletiadis, J. Multicentre validation of 4-well azole agar plates as a screening method for detection of clinically relevant azole-resistant Aspergillus fumigatus. J. Antimicrob. Chemother. 2017, 72, 3325–3333. [Google Scholar] [CrossRef]
  56. Morio, F.; Aubin, G.G.; Danner-Boucher, I.; Haloun, A.; Sacchetto, E.; Garcia-Hermoso, D.; Bretagne, S.; Miegeville, M.; Le Pape, P. High prevalence of triazole resistance in Aspergillus fumigatus, especially mediated by TR/L98H, in a French cohort of patients with cystic fibrosis. J. Antimicrob. Chemother. 2012, 67, 1870–1873. [Google Scholar] [CrossRef] [Green Version]
  57. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs for Antifungal Agents, Version 10.0. 2020. Available online: http://www.eucast.org/astoffungi/clinicalbreakpointsforantifungals/ (accessed on 5 January 2021).
  58. Sharma, C.; Hagen, F.; Moroti, R.; Meis, J.F.; Chowdhary, A. Triazole-resistant Aspergillus fumigatus harbouring G54 mutation: Is it de novo or environmentally acquired? J. Glob. Antimicrob. Resist. 2015, 3, 69–74. [Google Scholar] [CrossRef] [PubMed]
  59. Garcia-Rubio, R.; Alcazar-Fuoli, L.; Monteiro, M.C.; Monzon, S.; Cuesta, I.; Pelaez, T.; Mellado, E. Insight into the Significance of Aspergillus fumigatus cyp51A Polymorphisms. Antimicrob. Agents Chemother. 2018, 62, e00241-18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Shalhoub, S.; Luong, M.L.; Howard, S.J.; Richardson, S.; Singer, L.G.; Chaparro, C.; Keshavjee, S.; Akinlolu, Y.; Rotstein, C.; Mazzulli, T.; et al. Rate of cyp51A mutation in Aspergillus fumigatus among lung transplant recipients with targeted prophylaxis. J. Antimicrob. Chemother. 2015, 70, 1064–1067. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Buil, J.B.; Hagen, F.; Chowdhary, A.; Verweij, P.E.; Meis, J.F. Itraconazole, Voriconazole, and Posaconazole CLSI MIC Distributions for Wild-Type and Azole-Resistant Aspergillus fumigatus Isolates. J. Fungi 2018, 4, 103. [Google Scholar] [CrossRef] [Green Version]
Table
Table 1. Clinical and environmental Aspergillus species collected at the National Reference Laboratory in the scope of the Aspergillus surveillance program (2017–2018).
Table 1. Clinical and environmental Aspergillus species collected at the National Reference Laboratory in the scope of the Aspergillus surveillance program (2017–2018).
SectionClinical IsolatesEnvironmental Isolates
SpeciesNTotalSpecies (N)NTotal
FumigatiA. fumigatus sensu stricto61 70A. fumigatus sensu stricto3939
A. lentulus4
A. felis/A. pseudofelis2
A. udagawae2
Not identified to species level 1
FlaviA. flavus sensu stricto8 11 2
A. sergii/transmontaneensis1
Not identified to species level2
A. nomius2
NidulantesA. nidulans sensu stricto 12
A. delacroixii
A. minutus /insuetus
A. teneensis A. teneensis1
A. jensenii
A. fructus1A. fructus8
A. tabacinus1A. tabacinus1
A. creber/A. paulaauensis1
A. versicolor sensu stricto/A. tabacinus1
Not identified to species level 1
A. sidowii1
A. protuberus1
NigriA. niger sensu stricto540A. niger sensu stricto521
A. welwitschiae22 A. welwitschiae7
A. tubigensis4 A. tubigensis9
A. brasiliensis1
Not identified to species level8
ClavatiA. giganteus11
A. clavatus sensu stricto11
TerreiA. terreus sensu stricto 12 13
A. alabamensis1
CircumdatiA. ochraceopetaliformis/A. flocculosus (N = 1)12 3
A. westerdijkiae (N = 1)1A. westerdijkiae1
A. melleus2
AspergilliA. chevalierii (N = 1) 1
Flavipedes A. iizukae22
Usti A. ustus sensu stricto319
A. insuetus6
A. minutus8
A. germanicus2
Not identified to section levelAspergillus sp. 5
TOTAL 156 99
Table
Table 2. Patients infected/colonized by more than one Aspergillus species.
Table 2. Patients infected/colonized by more than one Aspergillus species.
Patient #IsolateProductSpecies
1VA141BALA. fumigatus sensu stricto
VA142BLA. welwitschiae
2VA151BALA. welwitschiae
VA152A. terreus sensu stricto
3VA153BLA. fumigatus sensu stricto
VA154A. welwitschiae
4VA159BALA. welwitschiae
VA160BLA. teneensis
5VA191BALA. fumigatus sensu stricto
VA192A. welwitschiae
6VA193BALA. terreus sensu stricto
VA194A. fumigatus sensu stricto
VA198BLA. fumigatus sensu stricto
VA199A. lentulus
7VA228BALA. fumigatus sensu stricto
VA229A. terreus sensu stricto
8VA230BLA. niger (section)
VA231A. terreus sensu stricto
9VA242BALA. lentulus
VA243A. fumigatus sensu stricto
10VA244Respiratory sampleA. fumigatus sensu stricto
VA245A. terreus sensu stricto
11VA251Respiratory sampleA. fumigatus sensu stricto
VA252A. terreus sensu stricto
12VA272BLAspergillus sp.
VA273A. fumigatus sensu stricto
VA274BALA. giganteus
VA275Aspergillus sp.
13VA276BALA. terreus sensu stricto
VA277BALA. lentulus
VA278A. niger (section)
14VA280BLA. flavus (section)
VA289BALA. fumigatus sensu stricto
15VA281BLA. flavus sensu stricto
VA285A. fumigatus sensu stricto
16VA296Bronchial
secretions		A. fumigatus sensu stricto
VA297A. chevalierii
17VA298Bronchial
secretions		A. fumigatus sensu stricto
VA299A. lentulus
18VA301BALA. niger (section)
VA304A. fumigatus sensu stricto
19VA302BLA. terreus sensu stricto
VA303A. fumigatus sensu stricto (TR34/L98H mutant)
Legend: BAL—Brochoalveolar lavage; BL—Bronchic Lavage.
Table
Table 3. Aspergillus species from Fumigati section collected in the period 2012–2019.
Table 3. Aspergillus species from Fumigati section collected in the period 2012–2019.
Species Number of IsolatesSource
A. fumigatus sensu stricto319Clinical and environmental
A. lentulus8Clinical (respiratory samples)
A. felis/parafelis/pseudofelis5Clinical (respiratory samples)
A. hiratsukae1Environmental (hospital environment)
A. udagawae3Clinical (respiratory samples)
A. oerlinghauensis1Clinical (ear exudate)
Table
Table 4. Characterization of the isolates presenting growth on azole screening media.
Table 4. Characterization of the isolates presenting growth on azole screening media.
Azole Screening MediaMinimal Inhibitory Concentration (mg/L)cyp51A Mutation
IsolateYear
Isolation		Geographic Location
NUTS II		Location CodeSourceBiological SampleAgeGenderClinical InformationAspergillus Section Molecular
Identification		ICZVCZPCZICZVCZPCZ
VA72013Lisbon1ClinicalSputum22MCystic fibrosisFumigatiA. fumigatus sensu stricto±-±10.50.5N.P.
VA82013Lisbon1ClinicalBronchial secretions39MCystic fibrosisFumigatiA. fumigatus sensu stricto+-±0.50.250.25N.P.
VA92013Lisbon1ClinicalSputum63MCystic fibrosisFumigatiA. fumigatus sensu stricto+--0.50.50.25N.P.
VA102013Lisbon1ClinicalN.R.73FCystic fibrosisFumigatiA. fumigatus sensu stricto+--0.250.50.125N.P.
VA162013Lisbon1ClinicalSputum16FCystic fibrosisFumigatiA. fumigatus sensu stricto±--0.1250.250.0625N.P.
VA212013Lisbon1ClinicalBronchoalveolar lavage48MLiver transplantFumigatiA. fumigatus sensu stricto±--10.50.5N.P.
VA352014North2ClinicalBronchial aspirate67FN.R.FumigatiA. fumigatus sensu stricto+--10.50.5N.P.
VA372014Lisbon3EnvironmentN.R.N.A.N.A.N.A.FumigatiA. fumigatus sensu stricto±--0.50.50.5N.P.
VA442014North2ClinicalBronchial aspirate79MSolid TumorFumigatiA. fumigatus sensu stricto±-±0.50.50.5N.P.
VA462014North2ClinicalBronchial aspirate88FN.R.FumigatiA. fumigatus sensu stricto±-±0.50.250.5N.P.
VA542015Lisbon4ClinicalN.R.N.R.N.R.N.R.FumigatiA. fumigatus sensu stricto±-±10.50.5N.P.
VA552015Lisbon4ClinicalBronchial secretions58FChronic Liver diseaseFumigatiA. fumigatus sensu stricto±-±10.50.5N.P.
VA632015Lisbon4ClinicalSputum64MSarcoidosisFumigatiA. fumigatus sensu stricto±-±20.50.5N.P.
VA652015Lisbon4ClinicalBronchial secretions21MPolytraumatizedFumigatiA. fumigatus sensu stricto±--0.50.250.5N.P.
VA672015Lisbon4ClinicalSputum55MTuberculosisFumigatiA. fumigatus sensu stricto±-±0.50.250.5N.P.
VA702015Lisbon4ClinicalBronchial secretions78MChronic obstructivepulmonary disease FumigatiA. fumigatus sensu stricto±--10.50.5N.P.
VA712015Lisbon4ClinicalN.R.N.R.N.R.N.R.FumigatiA. fumigatus sensu stricto+-±10.50.5N.P.
VA732015Lisbon4ClinicalBronchoalveolar lavage69MDiabetesFumigatiA. fumigatus sensu stricto+-±110.5N.P.
VA742015Lisbon4ClinicalBronchial secretions81FSepsisFumigatiA. fumigatus sensu stricto±-±10.50.5N.P.
VA762015Lisbon4ClinicalBroncoalveolar lavage41FPulmonary emphysemaFumigatiA. fumigatus sensu stricto±--10.250.5N.P.
VA772015Lisbon4ClinicalPulmonary tissue57MHIV+FumigatiA. fumigatus sensu stricto±--10.250.125N.P.
VA782015Lisbon4ClinicalBronchial secretions57MHIV+FumigatiA. fumigatus sensu stricto--±10.50.5N.P.
VA832015Lisbon4ClinicalPeritoneal fluid82MLiver cirrhosisFumigatiA. fumigatus sensu stricto+--10.250.5N.P.
VA852015North5ClinicalBronchial lavage40MTuberculosis and AspergillomaFumigatiA. fumigatus sensu stricto+±+221No amplification
VA862015Centre6EnvironmentSwine airN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto+-+>80.52G54R
VA952015Lisbon7EnvironmentAirN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto+±+221No mutation detected
VA1372016Lisbon8ClinicalBronchial secretions87MN.R.FumigatiA. fumigatus sensu stricto±-±10.250.125N.P.
VA1162016North2ClinicalBronchial secretions70FN.R.FumigatiA. lentulus+±+281N.P.
VA1182016North3ClinicalSputum80MBronchiectasis FumigatiA. lentulus+++2>81N.P.
HSMA672017Lisbon1EnvironmentHospital surfaceN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto+--10.250.5N.P.
VA1462017Leiria9ClinicalBronchoalveolar lavage45MHIV+ FumigatiA. fumigatus sensu stricto+--20.50.5 N.P.
VA1492017North5ClinicalBronchoalveolar lavage59FBronchiectasis FumigatiA. fumigatus sensu stricto+--10.50.5N.P.
VA1762017North6ClinicalBronchial lavage67MPulmonary infectionFumigatiA. fumigatus sensu stricto±--0.250.030.03N.P.
VA1442017North4ClinicalBronchial aspirate57MN.R.FumigatiA. felis+++4>81N.P.
VA1822017Lisbon4ClinicalN.R.N.R.FHIV+, pulmonary aspergillosis,
previous tuberculosis		FumigatiA. felis++-210.25N.P.
VA1992017North6ClinicalBronchial lavage62MCavitated pneumoniaAspergillus sp.A. lentulus+++221N.P.
VA2442018Lisbon8ClinicalBronchial secretions75FN.R.FumigatiA. fumigatus sensu stricto±±-110.5N.P.
VA2852018North6ClinicalBronchial lavage83FPulmonary abscessFumigatiA. fumigatus sensu stricto±--10.250.5N.P.
VA3032018North6ClinicalBronchial lavage65MBronchiectasis FumigatiA. fumigatus sensu stricto+ + + >882TR34/L98H
VA2422018North7ClinicalBronchoalveolar lavage48MBronchiectasis Aspergillus sp. A. lentulus±±-LVN.P.
VA2542018Lisbon7ClinicalEar exudate14F N.R.FumigatiA. oerlinghauensis++-442N.P.
VA3282019North6ClinicalBronchoalveolar lavage84MN.R.FumigatiA. fumigatus sensu stricto±--40.51No mutation detected
VA3462019Centre9ClinicalBronchial secretions67MN.R.FumigatiA. fumigatus sensu stricto±--20.51F46Y, M172V, N248T, D255E
VA3502019North6ClinicalBronchial lavage56MBronchiectasis FumigatiA. fumigatus sensu stricto±--20.50.5No mutation detected
VA299CP2019Alentejo10EnvironmentCowshed airN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto+ + + 442TR34/L98H
VA873CP2019Lisbon11EnvironmentFRPD fom waste sorting industry’ workersN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto+ + + 421TR34/L98H
VA978CP2019Lisbon11EnvironmentFRPD fom waste sorting industry’ workersN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto-+ -10.250.25No mutation detected
VA1161CP2019Lisbon12EnvironmentThermal SPAN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto+ --LVN.P.
V1207CP2019Lisbon11EnvironmentFRPD fom waste sorting industry’ workersN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto--+ 10.50.5No mutation detected
VA1209CP2019Lisbon11EnvironmentFRPD fom waste sorting industry’ workersN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto+ -+ 841TR34/L98H
VA1215CP2019Lisbon11EnvironmentFRPD fom waste sorting industry’ workersN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto-+ + 10.250.125N248K
VA1216CP2019Lisbon11EnvironmentFRPD fom waste sorting industry’ workersN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto-+ -10.250.25N248K
VA610CP2019North13EnvironmentHospital airN.A.N.A.N.A.FumigatiA. fumigatus sensu stricto±--20.50.5 No mutation detected
N.A.: Not applicable; N.R.: Not referred; N.P.: Not performed; FRPD: Filtering respiratory protective devices; LV: Lost of strain viability.
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Sabino, R.; Gonçalves, P.; Martins Melo, A.; Simões, D.; Oliveira, M.; Francisco, M.; Viegas, C.; Carvalho, D.; Martins, C.; Ferreira, T.; et al. Trends on Aspergillus Epidemiology—Perspectives from a National Reference Laboratory Surveillance Program. J. Fungi 2021, 7, 28. https://doi.org/10.3390/jof7010028

AMA Style

Sabino R, Gonçalves P, Martins Melo A, Simões D, Oliveira M, Francisco M, Viegas C, Carvalho D, Martins C, Ferreira T, et al. Trends on Aspergillus Epidemiology—Perspectives from a National Reference Laboratory Surveillance Program. Journal of Fungi. 2021; 7(1):28. https://doi.org/10.3390/jof7010028

Chicago/Turabian Style

Sabino, Raquel, Paulo Gonçalves, Aryse Martins Melo, Daniela Simões, Mariana Oliveira, Mariana Francisco, Carla Viegas, Dinah Carvalho, Carlos Martins, Teresa Ferreira, and et al. 2021. "Trends on Aspergillus Epidemiology—Perspectives from a National Reference Laboratory Surveillance Program" Journal of Fungi 7, no. 1: 28. https://doi.org/10.3390/jof7010028

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