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Susceptibility

Species Distribution and Comparison between EUCAST and Gradient Concentration Strips Methods for Antifungal Susceptibility Testing of 112 Aspergillus Section Nigri Isolates

B. Carrara, R. Richards, S. Imbert, F. Morio, M. Sasso, N. Zahr, A. C. Normand, P. Le Pape, L. Lachaud, S. Ranque, D. Maubon, R. Piarroux, A. Fekkar
B. Carrara
aAP-HP, Groupe Hospitalier La Pitié-Salpêtrière, Service de Parasitologie Mycologie, Paris, France
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R. Richards
aAP-HP, Groupe Hospitalier La Pitié-Salpêtrière, Service de Parasitologie Mycologie, Paris, France
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S. Imbert
aAP-HP, Groupe Hospitalier La Pitié-Salpêtrière, Service de Parasitologie Mycologie, Paris, France
bSorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses, Cimi-Paris, Paris, France
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F. Morio
cLaboratoire de Parasitologie Mycologie, CHU Nantes, Nantes, France
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M. Sasso
dLaboratoire de Parasitologie Mycologie, CHU Nîmes, Nîmes, France
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N. Zahr
eAP-HP, Groupe Hospitalier La Pitié-Salpêtrière, Service de Pharmacologie, Paris, France
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A. C. Normand
aAP-HP, Groupe Hospitalier La Pitié-Salpêtrière, Service de Parasitologie Mycologie, Paris, France
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P. Le Pape
cLaboratoire de Parasitologie Mycologie, CHU Nantes, Nantes, France
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L. Lachaud
fLaboratoire de Parasitologie Mycologie, Université de Montpellier et CHU de Montpellier, Montpellier France
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S. Ranque
gAix Marseille Univ, IRD, AP-HM, SSA, VITROME, IHU-Méditerranée Infection, Marseille, France
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  • ORCID record for S. Ranque
D. Maubon
hLaboratoire de Parasitologie Mycologie, CHU Grenoble, Grenoble, France
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R. Piarroux
aAP-HP, Groupe Hospitalier La Pitié-Salpêtrière, Service de Parasitologie Mycologie, Paris, France
iSorbonne Université, INSERM, Institut Pierre Louis d’Epidemiologie et de Santé Publique, Paris, France
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A. Fekkar
aAP-HP, Groupe Hospitalier La Pitié-Salpêtrière, Service de Parasitologie Mycologie, Paris, France
bSorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses, Cimi-Paris, Paris, France
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DOI: 10.1128/AAC.02510-19
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ABSTRACT

Aspergillus niger, the third species responsible for invasive aspergillosis, has been considered as a homogeneous species until DNA-based identification uncovered many cryptic species. These species have been recently reclassified into the Aspergillus section Nigri. However, little is yet known among the section Nigri about the species distribution and the antifungal susceptibility pattern of each cryptic species. A total of 112 clinical isolates collected from 5 teaching hospitals in France and phenotypically identified as A. niger were analyzed. Identification to the species level was carried out by nucleotide sequence analysis. The MICs of itraconazole, voriconazole, posaconazole, isavuconazole, and amphotericin B were determined by both the EUCAST and gradient concentration strip methods. Aspergillus tubingensis (n = 51, 45.5%) and Aspergillus welwitschiae (n = 50, 44.6%) were the most common species while A. niger accounted for only 6.3% (n = 7). The MICs of azole drugs were higher for A. tubingensis than for A. welwitschiae. The MIC of amphotericin B was 2 mg/liter or less for all isolates. Importantly, MICs determined by EUCAST showed no correlation with those determined by the gradient concentration strip method, with the latter being lower than the former (Spearman’s rank correlation tests ranging from 0.01 to 0.25 depending on the antifungal agent; P > 0.4). In conclusion, A. niger should be considered as a minority species in the section Nigri. The differences in MICs between species for different azoles underline the importance of accurate identification. Significant divergences in the determination of MIC between EUCAST and the gradient concentration strip methods require further investigation.

INTRODUCTION

The opportunistic molds Aspergillus spp. have been for years classified according to macro-microscopic examination. In this setting, Aspergillus niger, the third species responsible for invasive aspergillosis and the most prevalent agent of otomycosis, has been considered as a homogeneous species. However, the advent of molecular methods has uncovered many cryptic species (e.g., Aspergillus tubingensis or Aspergillus brasiliensis) formerly identified as A. niger, and new species are regularly described (1). To accommodate this situation, all of these species have been then reclassified into the Aspergillus section Nigri. Yet, little is known in the section Nigri about the species repartition and their in vitro antifungal susceptibility profiles, though variation in antifungal susceptibility between cryptic species has been demonstrated for other sections (2). Determination of MICs is, therefore, a very important issue. Finally, classification is a long dynamic process that leads to the renaming or disappearance of some species names, such as Aspergillus welwitschiae, which has replaced Aspergillus awamori. Some studies focusing on the accurate species distribution and in vitro antifungal susceptibility of Aspergillus section Nigri clinical isolates are available (3–8). In addition, azole drug MICs seems to be highly heterogeneous. This could be related to the variety of techniques (e.g., solid medium versus liquid medium), reference methodologies (CLSI or EUCAST), interlaboratory variabilities, as well as the existence of cryptic species that can bear different susceptibility patterns. This phenomenon exists for all fungi but seems particularly exacerbated for the section Nigri. This work aims to better understand the distribution of Aspergillus section Nigri species in clinical samples as well as the susceptibility profile of each cryptic species to antifungal drugs using the EUCAST methodology and gradient concentration strip methods.

(Part of this data was presented as a poster communication during the 27th European Congress of Clinical Microbiology and Infectious Diseases, Vienna, Austria.)

RESULTS

Species repartition and clinical origin.Molecular identification was available for all 112 isolates. Aspergillus tubingensis (n = 51, 45.5%) and A. welwitschiae (n = 50, 44.6%) were the most prevalent species (Fig. 1A). They were found in the five centers in equal proportion. Importantly, Aspergillus niger accounted for only 6.3% (n = 7). Two isolates of Aspergillus neoniger, one isolate of Aspergillus japonicus, and one isolate of Aspergillus brasiliensis were also identified. Isolates were mainly isolated from respiratory samples (60.7%; n = 68), i.e., bronchoalveolar lavage or sputum (Fig. 1B). The second most frequent source was the external auditory canal (30.5%; n = 34), while samples from the ear, nose, and throat sphere accounted for 4.5% (n = 5). It should be noted that 3 isolates were found in stool samples. No evidence of a different distribution of the clinical site by species was found, although a nonstatistically significant trend was observed for more A. welwitschiae in ear samples (20/50) than A. tubingensis (12/51; P = 0.07 by chi-square test). Five patients had isolates from two different species.

FIG 1
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FIG 1

Species repartition (A) and origin (B) of 112 Aspergillus section Nigri clinical isolates from five hospitals in France.

MICs determined by the EUCAST.All values of the Candida parapsilosis control strain ATCC 22019 were within the target range. MICs show significant variations across the different species (Table 1). Azoles displayed higher MICs for A. tubingensis than for A. welwitschiae or A. niger. Geometric mean of the MIC of voriconazole was 1.54 mg/liter for A. tubingensis and 0.77 mg/ml for A. welwitschiae (P < 0.0001 by Student test). For posaconazole, itraconazole, and isavuconazole, geometric means were also about 2-fold higher for A. tubingensis than for A. welwitschiae; all differences were statistically significant (P < 0.0001). Of note, MICs were higher for isavuconazole than for either voriconazole or posaconazole, whatever the species considered. Considering all of the species, for 5.3% of the isolates (6/112), MICs were above the epidemiological cutoff (ECOFF) for voriconazole (2 mg/liter), 28.6% (32/112) for itraconazole (4 mg/liter), and 34.8% (39/112) for isavuconazole (4 mg/liter). There was a higher proportion of these non-wild-type isolates among A. tubingensis in comparison with that among A. welwitschiae. Among the 112 isolates tested for amphotericin B by EUCAST, 91 isolates (81.3%) were susceptible (MIC ≤ 1 mg/ml), 21 (18.7%) were intermediate (MIC = 2 mg/liter), but none were resistant (MIC > 2 mg/liter). For amphotericin B, repartition of A. tubingensis and A. welwitschiae within the susceptible and intermediate groups was similar (P = 0.6 by chi-square test), and contrary to azole drugs, MICs of amphotericin B were not statistically different between A. tubingensis and A. welwitschiae isolates although a trend was observed (geometric means, 0.9 mg/liter versus 1.18 mg/liter; P = 0.06).

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TABLE 1

MIC values, range, and geometric means determined by EUCAST and Etest method for Aspergillus section Nigri isolatesa

MICs determined by gradient concentration strips.In line with the EUCAST, MICs determined by gradient concentration strips (Table 1) were higher for A. tubingensis than for A. welwitschiae for both itraconazole (geometric means, 0.9 mg/liter versus 0.61 mg/liter; P = 0.005) and isavuconazole (geometric means, 0.35 mg/liter versus 0.27 mg/liter; P < 0.05). However, no statistically significant difference between A. tubingensis and A. welwitschiae was seen for voriconazole (geometric means, 0.06 mg/liter versus 0.05 mg/liter; P = 0.27) and posaconazole (geometric means, 0.1 mg/liter versus 0.08 mg/liter; P = 0.5). MICs of amphotericin B were below 1 mg/ml for all isolates. A comparison of previously reported MICs by EUCAST, CLSI, or Etest methods for A. tubingensis and A. welwitschiae isolates is shown in Table 2.

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TABLE 2

Comparison of previously reported MICs for A. tubingensis and A. welwitschiae isolates

Correlation between MICs of the azole class.When considering the values determined by EUCAST, MIC values for azole drugs showed poor correlations. Voriconazole and posaconazole values had a Spearman’s rank correlation rho of 0.26, which is particularly low and is indicative of absence of correlation (r = 0.26; P < 0.01). Correlations between voriconazole and itraconazole (r = 0.43; P < 0.0001) and between posaconazole and isavuconazole (r = 0.48; P < 0.0001) were also weak. Only voriconazole and isavuconazole had a rho factor above 0.5 (r = 0.55; P < 0.0001). On the contrary, MIC values for azoles determined by gradient concentration strips were more correlated each other; voriconazole and posaconazole showed a strong highly statistically significant correlation (r = 0.617; P < 0.00001). Similar results were observed when comparing posaconazole and isavuconazole (r = 0.67; P < 0.00001) or voriconazole and itraconazole (r = 0.564; P < 0.00001).

Comparison between values determined by EUCAST and gradient concentration strips.MICs determined by EUCAST and by gradient concentration strips showed very different values, with the latter being much lower than the former. The results obtained show the absence of correlation between the EUCAST method and the gradient concentration strip method for amphotericin B MICs (r = 0.016; P = 0.88 by Spearman’s rank correlation test). Yet the essential agreement (±2-fold dilutions) was 76.7% (69/90). For azoles, nonsignificant correlation was found whatever the drug tested. Spearman’s rank correlation tests were 0.01 (P = 0.9) for itraconazole, 0.06 (P = 0.54) for voriconazole, 0.08 (P = 0.42) for posaconazole, and 0.25 for isavuconazole (P = 0.9). Also, essential agreement was extremely weak for voriconazole with 14.4% (13/90) and isavuconazole with 18.5% (15/81) and low for posaconazole (48.9%; 44/90) and itraconazole (66.7%; 60/90). The percentage of major errors (ME) was 0% for all of the antifungal drugs, whereas the percentage of very major errors (VME) was between 0% and 50%, depending the antifungal drug and the species concerned (Table 1). These surprising results led us to perform once again the determination of MICs of the different azole drugs for 40 isolates (35.7%). The two determinations were in line with one another. Comparison between the first and second determination shows a categorical agreement of 100% for voriconazole, 97.5% for posaconazole, 95% for isavuconazole, but only 82.5% for itraconazole. Intriguingly, for itraconazole, some isolates changed dramatically from 1 to 16 mg/liter or from 16 to 1 mg/liter (Table 3).

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TABLE 3

Comparison of MIC values for 40 Aspergillus section Nigri isolates obtained in two distinct measurements according to the EUCAST methoda

Acidification of RPMI medium by Aspergillus section Nigri.During experiments, we observed that the liquid medium used for MIC determination by EUCAST methodology turned from light red into yellow in the presence of the fungus, reflecting an acidification of the medium. Aspergillus niger is a well-known agent of the fermentation industry and is notably used for the production of citric acid. After 48 h of incubation, we measured the pH in the medium and noticed that it diminished at 4 for A. welwitschiae, A. tubingensis, and A. niger while it remained at 7 for an Aspergillus fumigatus sensu stricto clinical isolates used as control. Of note, the RPMI solid medium used for Etest also showed some variations of its color, but pH could not be determined. Therefore, we increased the concentration of the morpholinepropanesulfonic acid (MOPS) buffer in the medium up to 150 μM. This led to less acidification after 48 h of growth (pH 6) but had no impact on the MICs (data not shown). We also investigate whether acidification or specific metabolism of Aspergillus section Nigri could lead to degradation of azole drugs. For that, known concentrations of voriconazole and posaconazole were mixed for 4 h at 37°C with a culture supernatant of A. tubingensis or A. welwitschiae grown beforehand for 48 h at 37°C with no antifungal drug. No effect on drug concentrations (determined by validated high-performance liquid chromatography-tandem mass spectrometry assay) was observed (data not shown).

DISCUSSION

Aspergillus section Nigri is the most prevalent agent of otomycosis and the third agent of invasive aspergillosis, mainly of respiratory origin (9). Other localizations, such as cutaneous infection, have also been reported (10), and it is also frequently found in the environment. In a recent study in Italy, among a screening of azole-resistant isolates, Aspergillus section Nigri was more frequently found than other species, notably A. fumigatus (11). In the present study, the majority of isolates were found in the respiratory tract and then in the external auditory canal. Isolates were selected in chronological order by each center, so there is no bias according to clinical implication. As previously reported by others (12, 13), our results indicate that A. tubingensis is far more frequently isolated than A. niger. Therefore, our study reinforces that, in contrast to other parts of the world (8, 14), A. niger seems to be uncommon in Europe.

The MICs determined according to EUCAST method showed that A. tubingensis isolates are frequently resistant to azole drugs as reported by others (7). Moreover, for itraconazole, a bimodal distribution was observed, which was not evidenced using the Etest method. For several isolates, we also observed for this drug some previously described paradoxical growth in EUCAST (i.e., presence of fungus in the well containing 8 mg/liter and absence of growth at lower concentrations) (8) that was not taken into account for the MIC determination. In a study using the CLSI M38-A2 method for MIC determination of 42 isolates belonging to the section Nigri, the authors also found that MICs of azole drugs were higher for A. tubingensis than for A. welwitschiae (called A. awamori in this study) (6). The authors hypothesized that Q228R substitution could confer posaconazole resistance, but this amino acid substitution was detected more recently in both resistant and nonresistant isolates by Hashimoto et al. who analyzed the entire cyp51A gene in a collection of 115 Aspergillus section Nigri isolates from Japan (14). In this later work, the authors also detected many other substitutions, none of which could be related to azole resistance (14). Intriguingly, Aspergillus section Nigri isolates with high MICs for azoles do not share a common mechanism of resistance with A. fumigatus, i.e., mutation of the cyp51 gene. The mechanisms that confer high MIC values remain unknown. However, local and systemic treatments based on azoles have been used successfully for treatment of aspergillosis due to section Nigri isolates (15). We also found that isavuconazole MICs, the most recent broad-spectrum azole drug, were higher than those determined for voriconazole or posaconazole. This is in accordance with the isavuconazole ECOFF proposed by the EUCAST for A. niger being higher if compared with that of voriconazole and also higher than that for others species, such as A. fumigatus or Aspergillus flavus (16). In addition, higher MIC values of isavuconazole in comparison with those of voriconazole or posaconazole for isolates of A. welwitschiae have also been reported (17).

Strikingly, we observed a very significant difference between MIC values determined by EUCAST and those determined using Etest. As the MIC values determined by EUCAST were unexpectedly high, we tested again 40 randomly selected isolates and found coherent results and good reproducibility for azoles, except for itraconazole with some isolates showing changes from 1 to 16 mg/liter or from 16 to 1 mg/liter without clear explanation. Strikingly, it is also with this molecule that we observed a bimodal distribution and a paradoxical regrowth effect at high itraconazole concentrations. Other authors reported this phenomenon as well as trailing growth leading to discrepancies between different broth microdilution methods (8, 18). Finally, our results are also in line with other works, which report discrepancies and/or higher MICs of azoles with broth microdilution methods in comparison with those of the Etest method (2, 19, 20). Moreover, no correlation was seen between the methods for some antifungals. Yet, very few studies reported comparison between the reference method approach (EUCAST or CLSI) and the Etest method. Howard et al. performed both of the methods and found that the geometric mean of MICs of itraconazole were nearly 3.5 times higher for EUCAST methods than for the Etest method (2.79 mg/liter versus 0.77 mg/liter), which is in accordance with our results (2). Thus, this was a modified EUCAST method with a lower fungal inoculum that could have minimized the MIC values. The authors do not conclude, however, if there was or was not some correlation between the two methods.

As the EUCAST reference method in liquid medium is subjected to high pH variations in relation with the particular metabolism of the section Nigri and pH has been shown to impact antifungal drug activity (21), the hypothesis that higher MICs for section Nigri species than for others species, such as A. fumigatus, is an analytical bias due to acidification of the medium is valid and noteworthy. By the way, Howard et al. (2), although they do not measure the pH, reported similar observations. However, under our experimental conditions, the pH and/or the fungal metabolites do not seem to have any effect on azole concentrations. It might, however, impact the entrance of the drug into the fungal cell. Our attempt to counterweigh this acidification did not influence the MIC values. So, the discrepancy of MICs observed between EUCAST and Etest methods is, for now, still a question to solve. It might be of interest to set up in vivo experiments using animals or alternative models (such as Galleria) that will provide data regarding the in vivo outcome of Aspergillus section Nigri infections according to the administered triazole. Such an approach would probably help to uncover the reasons behind the discrepancies observed, for some isolates of the present study, between the EUCAST and Etest methods.

Finally, our results indicate the importance of a precise identification of the Aspergillus section Nigri isolates using either DNA sequencing or matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry backed by accurate databases of reference spectra (22). Thus, this study is, to the best of our knowledge, the biggest series determining the MIC values of Aspergillus section Nigri isolates by both a microplate dilution reference method and a commercial agar diffusion method, unravelling unexpected results that deserve further investigations.

In conclusion, among the section Nigri, A. tubingensis and A. welwitschiae are the most prevalent species. Our results indicate the importance of a sharp identification of the Aspergillus section Nigri isolates to improve epidemiological knowledge and underlines the necessity of performing MIC determination. Yet, important differences between the methods exist, underlying the necessity to investigate the effect of acid production by Aspergillus section Nigri on drug efficacy by both in vitro and in vivo approaches.

MATERIALS AND METHODS

Isolates.A total of 112 isolates from clinical samples (102 patients) collected in 5 teaching hospitals in France across the country (Grenoble, Marseille, Nantes, Nîmes, and Paris) and identified phenotypically as Aspergillus section Nigri were randomly selected and sent to the mycology laboratory of La Pitié-Salpêtrière Hospital for this retrospective study. These hospitals have between 1,800 and 2,500 beds, with medical, surgical, and intensive care units.

Identification.Identification to the species level was ensured by nucleotide sequencing of a part of the calmodulin and β-tubulin genes. Nucleotide sequences were compared to those available on the reference site www.aspergilluspenicillium.org. Resulting DNA sequences were compared to those of the reference strains inside the section Nigri by establishing maximum likelihood trees, for each gene individually, using the MEGA7 software.

MIC assays.MICs were determined using the EUCAST standardized methodology and gradient concentration strip methods according to the manufacturer’s protocol. Gradient concentration strip methods were performed using MIC test strips for isavuconazole (Liofilchem, Roseto degli Abruzzi, Italy) and Etest strips (bioMérieux, Marcy l’Etoile, France) for itraconazole, voriconazole, posaconazole, and amphotericin B. The Candida parapsilosis strain ATCC 22019 or the A. fumigatus strain ATCC 204305 was used for an internal quality check in each EUCAST microplate. For the EUCAST technique, the plates were read at 48 h of incubation by visual reading; the MIC was the first well with a complete lack of growth (i.e., full inhibition). For the Etest technique, plates were read at 48 h according to the manufacturer’s recommendation. For amphotericin B, breakpoints (≤1 mg/ml, susceptible; >2 mg/liter, resistant) available on the EUCAST website (http://www.eucast.org) were used. For azoles, results obtained by EUCAST were analyzed according to epidemiological cutoff (ECOFF) values (4 mg/liter for itraconazole, 2 mg/liter for voriconazole, 4 mg/liter for isavuconazole, and 0.5 mg/liter for posaconazole) according to Arendrup et al. (23). Results obtained by Etest were analyzed according to ECOFF (4 mg/liter for itraconazole, 1 mg/liter for voriconazole, 0.5 mg/liter for posaconazole, and 2 mg/liter for amphotericin B) published by Espinel-Ingroff et al. (19, 24). Essential agreement (EA) between methods was considered to be achieved when the MIC values were within ±2 dilutions. Comparison between EUCAST and Etest methods also allows the determination of the percentage of very major errors (VME; or false susceptibility), major errors (ME; or false resistance), and minor errors (mE; or misclassification of intermediate results).

Statistical analysis.GraphPad Prism 6 was used for statistical analyses (GraphPad Software, La Jolla, CA). Correlation was assessed by Spearman’s rank correlation. A P value below 0.05 was considered significant.

ACKNOWLEDGMENTS

This work was granted by MSD. The data are the property of the Assistance Publique–Hôpitaux de Paris. The decision to publish was made by the authors and not by MSD. MSD was not implicated in the writing of the manuscript.

During the past 5 years, A. Fekkar has received research grants from MSD and Astellas; travel grants from Gilead, MSD, Pfizer, and Astellas; and speaker’s fees from Gilead and MSD.

Ethical approval was not required.

FOOTNOTES

    • Received 16 December 2019.
    • Returned for modification 11 February 2020.
    • Accepted 15 April 2020.
    • Accepted manuscript posted online 20 April 2020.
  • Copyright © 2020 American Society for Microbiology.

All Rights Reserved.

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Species Distribution and Comparison between EUCAST and Gradient Concentration Strips Methods for Antifungal Susceptibility Testing of 112 Aspergillus Section Nigri Isolates
B. Carrara, R. Richards, S. Imbert, F. Morio, M. Sasso, N. Zahr, A. C. Normand, P. Le Pape, L. Lachaud, S. Ranque, D. Maubon, R. Piarroux, A. Fekkar
Antimicrobial Agents and Chemotherapy Jun 2020, 64 (7) e02510-19; DOI: 10.1128/AAC.02510-19

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Species Distribution and Comparison between EUCAST and Gradient Concentration Strips Methods for Antifungal Susceptibility Testing of 112 Aspergillus Section Nigri Isolates
B. Carrara, R. Richards, S. Imbert, F. Morio, M. Sasso, N. Zahr, A. C. Normand, P. Le Pape, L. Lachaud, S. Ranque, D. Maubon, R. Piarroux, A. Fekkar
Antimicrobial Agents and Chemotherapy Jun 2020, 64 (7) e02510-19; DOI: 10.1128/AAC.02510-19
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  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
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KEYWORDS

antifungal susceptibility testing
Aspergillus section Nigri
species complex
cryptic species
invasive aspergillosis
antifungal drugs
azoles
amphotericin B
antifungal resistance

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