Etest ECVs/ECOFFs for Detection of Resistance in Prevalent and Three Nonprevalent Candida spp. to Triazoles and Amphotericin B and Aspergillus spp. to Caspofungin: Further Assessment of Modal Variability.

Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs), or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs, and ECVs for some fungal species. Although the concentration gradient strip bioMérieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We reevaluated and consolidated Etest data points from three previous studies and included new data. We defined ECOFFinder Etest ECVs for three sets of species-agent combinations: fluconazole, posaconazole, and voriconazole and 9 Candida spp.; amphotericin B and 3 nonprevalent Candida spp.; and caspofungin and 4 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, and South Africa) included (antifungal agent dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled, and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.), amphotericin B (3 less-prevalent Candida spp.), and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 non-WT, 4 of 6 species).

Re-examination of species limits in Aspergillus section Flavipedes using advanced species delimitation methods and description of four new species.

Since the last revision in 2015, the taxonomy of section Flavipedes evolved rapidly along with the availability of new species delimitation techniques. This study aims to re-evaluate the species boundaries of section Flavipedes members using modern delimitation methods applied to an extended set of strains (n = 90) collected from various environments. The analysis used DNA sequences of three house-keeping genes (benA, CaM, RPB2) and consisted of two steps: application of several single-locus (GMYC, bGMYC, PTP, bPTP) and multi-locus (STACEY) species delimitation methods to sort the isolates into putative species, which were subsequently validated using DELINEATE software that was applied for the first time in fungal taxonomy. As a result, four new species are introduced, i.e. A. alboluteus, A. alboviridis, A. inusitatus and A. lanuginosus, and A. capensis is synonymized with A. iizukae. Phenotypic analyses were performed for the new species and their relatives, and the results showed that the growth parameters at different temperatures and colonies characteristics were useful for differentiation of these taxa. The revised section harbors 18 species, most of them are known from soil. However, the most common species from the section are ecologically diverse, occurring in the indoor environment (six species), clinical samples (five species), food and feed (four species), droppings (four species) and other less common substrates/environments. Due to the occurrence of section Flavipedes species in the clinical material/hospital environment, we also evaluated the susceptibility of 67 strains to six antifungals (amphotericin B, itraconazole, posaconazole, voriconazole, isavuconazole, terbinafine) using the reference EUCAST method. These results showed some potentially clinically relevant differences in susceptibility between species. For example, MICs higher than those observed for A. fumigatus wild-type were found for both triazoles and amphotericin B for A. ardalensis, A. iizukae, and A. spelaeus whereas A. lanuginosus, A. luppiae, A. movilensis, A. neoflavipes, A. olivimuriae and A. suttoniae were comparable to or more susceptible as A. fumigatus. Finally, terbinafine was in vitro active against all species except A. alboviridis.

How to interpret MICs of antifungal compounds according to the revised clinical breakpoints v. 10.0 European committee on antimicrobial susceptibility testing (EUCAST).

BACKGROUND: EUCAST has revised the definition of the susceptibility category I from 'Intermediate' to 'Susceptible, Increased exposure'. This implies that I can be used where the drug concentration at the site of infection is high, either because of dose escalation or through other means to ensure efficacy. Consequently, I is no longer used as a buffer zone to prevent technical factors from causing misclassifications and discrepancies in interpretations. Instead, an Area of Technical Uncertainty (ATU) has been introduced for MICs that cannot be categorized without additional information as a warning to the laboratory that decision on how to act has to be made. To implement these changes, the EUCAST-AFST (Subcommittee on Antifungal Susceptibility Testing) reviewed all, and revised some, clinical antifungal breakpoints. OBJECTIVES: The aim was to present an overview of the current antifungal breakpoints and supporting evidence behind the changes. SOURCES: This document is based on the ten recently updated EUCAST rationale documents, clinical breakpoint and breakpoint ECOFF documents. CONTENT: The following breakpoints (in mg/L) have been revised or established for Candida species: micafungin against C. albicans (ATU = 0.03); amphotericin B (S ≤/> R = 1/1), fluconazole (S ≤/> R = 2/4), itraconazole (S ≤/> R = 0.06/0.06), posaconazole (S ≤/> R = 0.06/0.06) and voriconazole (S ≤/> R = 0.06/0.25) against C. dubliniensis; fluconazole against C. glabrata (S ≤/> R = 0.001/16); and anidulafungin (S ≤/> R = 4/4) and micafungin (S ≤/> R = 2/2) against C. parapsilosis. For Aspergillus, new or revised breakpoints include itraconazole (ATU = 2) and isavuconazole against A. flavus (S ≤/> R = 1/2, ATU = 2); amphotericin B (S ≤/> R = 1/1), isavuconazole (S ≤ /> R = 1/2, ATU = 2), itraconazole (S ≤/> R = 1/1, ATU = 2), posaconazole (ATU = 0.25) and voriconazole (S ≤/> R = 1/1, ATU = 2) against A. fumigatus; itraconazole (S ≤/> R = 1/1, ATU = 2) and voriconazole (S ≤/> R = 1/1, ATU = 2) against A. nidulans; amphotericin B against A. niger (S ≤/> R = 1/1); and itraconazole (S ≤/> R = 1/1, ATU = 2) and posaconazole (ATU = 0.25) against A. terreus. IMPLICATIONS: EUCAST-AFST has released ten new documents summarizing existing and new breakpoints and MIC ranges for control strains. A failure to adopt the breakpoint changes may lead to misclassifications and suboptimal or inappropriate therapy of patients with fungal infections.

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.

BACKGROUND: The emergence of azole-resistant Aspergillus fumigatus isolates is a matter of significant concern in Europe, with countries reporting resistance rates (which can be as high as 30%) in hospitalized patients. Consequently, the treatment guidelines in The Netherlands, the country with the highest documented prevalence of azole-resistant A. fumigatus, has just been revised to now recommend initial therapy with combination therapy until the susceptibility pattern is known. Therefore, susceptibility testing of clinically relevant isolates has been strongly recommended in the ESCMID-EFISG aspergillosis guidelines. Furthermore, mixed azole-susceptible and azole-resistant (isogenic as well as non-isogenic) infections have been reported to occur, which implies that colonies of clinical cultures may harbour various phenotypes of azole susceptibility. OBJECTIVES: The EUCAST-AFST (European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing) has released a new screening method document (E.Def 10.1) for the detection of azole-resistant A. fumigatus isolates and updated the QC tables for antifungal susceptibility testing with associated QC endpoints. This review described in detail how to perform the screening test. SOURCES: This "How to document" is based on the EUCAST azole agar screening method document E.Def 10.1 and the QC tables for antifungal susceptibility testing document, v 2.0 (available at http://www.eucast.org/ast_of_fungi/qcafsttables/) CONTENTS: The method is based on the inoculation of azole-containing and azole-free agars and visual determination of fungal growth after one and two days of incubation. It can easily be implemented in routine laboratories of clinical microbiology and has been validated for simultaneous testing of up to five A. fumigatus colonies using itraconazole and voriconazole (mandatory), and posaconazole (optional). IMPLICATIONS: This easy-to-use screening procedure for the detection of azole resistance in clinical A. fumigatus isolates will allow rapid testing in the daily routine of the microbiology laboratory and thus facilitate earlier appropriate therapy.

High incidence of candidaemia in a nationwide cohort: Underlying diseases, risk factors and mortality.

BACKGROUND: Denmark has a high incidence rate of candidaemia. A Nordic study suggested a higher Danish prevalence of haematological malignancies as an underlying reason. This nationwide study ascertained clinical characteristics of Danish candidaemia patients and investigated potential factors contributing to the high incidence and mortality. METHODS: Microbiological and clinical data for candidaemia patients in 2010-2011 were retrieved. 30-day mortality was estimated by hazard ratios (HR) with 95% confidence intervals (CI, Cox regression). RESULTS: Data were available for 912/973 candidaemia episodes (93.7%). Intensive care unit (ICU) held the largest share of patients (43.2%). Prevalent host factors were multi-morbidity (≥2 underlying diseases, 74.2%) and gastrointestinal disease (52.5%). Haematological disease was infrequent (7.8%). Risk factors included antibiotic exposure (90.5%), CVC (71.9%) and Candida colonisation (66.7%). 30-day mortality was 43.4%, and 53.6% in ICU. Mortality was lower for patients with recent abdominal surgery (HR 0.70, 95% CI: 0.54-0.92). CONCLUSION: A substantial prevalence of multi-morbidity and a high 30-day mortality was found. We hypothesise, that an increasing population of severely ill patients with prolonged supportive treatment and microbiological testing may in part explain the high candidaemia incidence in Denmark. Nationwide studies are warranted to clarify this issue.

Implications of the EUCAST Trailing Phenomenon in Candida tropicalis for the In Vivo Susceptibility in Invertebrate and Murine Models.

Candida tropicalis isolates often display reduced but persistent growth (trailing) over a broad fluconazole concentration range during EUCAST susceptibility testing. Whereas weak trailing (<25% of the positive growth control) is common and found not to impair fluconazole efficacy, we investigated if more pronounced trailing impacted treatment efficacy. Fluconazole efficacy against two weakly (≤25% growth), two moderately (26% to 50% growth), and one heavily (>70% growth) trailing resistant isolate and one resistant (100% growth) isolate were investigated in vitro and in vivo (in a Galleria mellonella survival model and two nonlethal murine models). CDR1 expression levels and ERG11 sequences were characterized. The survival in fluconazole-treated G. mellonella was inversely correlated with the degree of trailing (71% to 9% survival in treatment groups). In mice, resistant and heavily trailing isolates responded poorly to fluconazole treatment. CDR1 expression was significantly higher in trailing and resistant isolates than in wild-type isolates (1.4-fold to 10-fold higher). All isolates exhibited ERG11 wild-type alleles. Heavily trailing isolates were less responsive to fluconazole in all in vivo models, indicating an impact on fluconazole efficacy. CDR1 upregulation may have contributed to the observed differences. Moderately trailing isolates responded less well to fluconazole in larvae only. This confirms clinical data suggesting fluconazole is effective against infections with such isolates in less severely ill patients and supports the current 50% growth endpoint for susceptibility testing. However, it is still unclear if the gradual loss of efficacy observed for moderately trailing isolates in the larva model may be a reason for concern in selected vulnerable patient populations.

Multicentre determination of rezafungin (CD101) susceptibility of Candida species by the EUCAST method.

OBJECTIVES: Rezafungin (CD101) is a new long-acting echinocandin allowing weekly dosing, currently undergoing phase-II clinical trials for invasive candidiasis. The aim of this study was to assess rezafungin's in vitro activity against the most frequent Candida species following the EUCAST methodology. METHODS: The susceptibility of 2018 clinical Candida isolates was determined at four European laboratories. In parallel, six control strains were repeatedly tested. Wild-type upper limits (WT-ULs), defined as the MIC value where the wild-type distribution ends, were determined following the principles for EUCAST ECOFF-setting. RESULTS: The lowest rezafungin MICs (geometric MIC (GM-MIC), MIC range (mg/L)) were observed for C. albicans (0.016, 0.002-0.125) and the highest for C. parapsilosis (1.657, 0.063->4). MICs for the remaining species were in between these values (GM-MICs 0.048-0.055). Visual and statistical WT-ULs were identical for C. glabrata (0.125), C. krusei (0.125), C. parapsilosis (4), and C. tropicalis (0.25). If adopting these WT-ULs for classification into WT and non-WT populations, 1/413 C. glabrata, 1/402 C. krusei, 1/398 C. parapsilosis, and 1/402 C. tropicalis isolates were categorized as non-WT, all of which derived from Laboratory 1. For C. albicans unexplained laboratory variation was observed (WT-UL: 0.063-0.125 in Laboratories 1 and 2 versus 0.016 in Laboratories 3 and 4). A similar systematic difference was observed comparing the MICs for the three C. albicans QC strains, specifically, obtained in Laboratories 1and 2 with those in Laboratories 3 and 4. DISCUSSION: Rezafungin displayed species-specific activity similar to other echinocandins. Interlaboratory variation was observed for the most susceptible species C. albicans clinical and QC strains, an observation that warrants further investigation.

Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline.

The European Society for Clinical Microbiology and Infectious Diseases, the European Confederation of Medical Mycology and the European Respiratory Society Joint Clinical Guidelines focus on diagnosis and management of aspergillosis. Of the numerous recommendations, a few are summarized here. Chest computed tomography as well as bronchoscopy with bronchoalveolar lavage (BAL) in patients with suspicion of pulmonary invasive aspergillosis (IA) are strongly recommended. For diagnosis, direct microscopy, preferably using optical brighteners, histopathology and culture are strongly recommended. Serum and BAL galactomannan measures are recommended as markers for the diagnosis of IA. PCR should be considered in conjunction with other diagnostic tests. Pathogen identification to species complex level is strongly recommended for all clinically relevant Aspergillus isolates; antifungal susceptibility testing should be performed in patients with invasive disease in regions with resistance found in contemporary surveillance programmes. Isavuconazole and voriconazole are the preferred agents for first-line treatment of pulmonary IA, whereas liposomal amphotericin B is moderately supported. Combinations of antifungals as primary treatment options are not recommended. Therapeutic drug monitoring is strongly recommended for patients receiving posaconazole suspension or any form of voriconazole for IA treatment, and in refractory disease, where a personalized approach considering reversal of predisposing factors, switching drug class and surgical intervention is also strongly recommended. Primary prophylaxis with posaconazole is strongly recommended in patients with acute myelogenous leukaemia or myelodysplastic syndrome receiving induction chemotherapy. Secondary prophylaxis is strongly recommended in high-risk patients. We strongly recommend treatment duration based on clinical improvement, degree of immunosuppression and response on imaging.

Update from a 12-Year Nationwide Fungemia Surveillance: Increasing Intrinsic and Acquired Resistance Causes Concern.

New data from the years 2012 to 2015 from the Danish National Fungemia Surveillance are reported, and epidemiological trends are investigated in a 12-year perspective (2004 to 2015). During 2012 to 2015, 1,900 of 1,939 (98%) fungal bloodstream isolates were included. The average incidence was 8.4/100,000 inhabitants, and this appears to represent a stabilizing trend after the increase to 10.1/100,000 in 2011. The incidence was higher in males than females (10.0 versus 6.8) and in patients above 50 years, and those changes were mainly driven by an increasing incidence among 80-to-89-year-old males (65.3/100,000 in 2014 to 2015). The proportion of Candida albicans isolates decreased from 2004 to 2015 (64.4% to 42.4%) in parallel with a doubling of the proportion of Candida glabrata isolates (16.5% to 34.6%, P < 0.0001). C. glabrata was more common among females (34.0% versus 30.4% in males). Following an increase in 2004 to 2011, the annual drug use stabilized during the last 2 to 3 years of that time period but remained higher than in other Nordic countries. This was particularly true for the fluconazole and itraconazole use in the primary health care sector, which exceeded the combined national levels of use of these compounds in each of the other Nordic countries. Fluconazole susceptibility decreased (68.5%, 65.2%, and 60.6% in 2004 to 2007, 2008 to 2011, and 2012 to 2015, respectively, P < 0.0001), and echinocandin resistance emerged in Candida (0%, 0.6%, and 1.7%, respectively, P < 0.001). Amphotericin B susceptibility remained high (98.7%). Among 16 (2.7%) echinocandin-resistant C. glabrata isolates (2012 to 2015), 13 harbored FKS mutations and 5 (31%) were multidrug resistant. The epidemiological changes and the increased incidence of intrinsic and acquired resistance emphasize the importance of continued surveillance and of strengthened focus on antifungal stewardship.

Emergence of Aspergillus fumigatus azole resistance in azole-naïve patients with chronic obstructive pulmonary disease and their homes.

Azole-resistant Aspergillus fumigatus (ARAF) has been reported in patients with chronic obstructive pulmonary disease (COPD) but has not been specifically assessed so far. Here, we evaluated ARAF prevalence in azole-naïve COPD patients and their homes, and assessed whether CYP51A mutations were similar in clinical and environmental reservoirs. Sixty respiratory samples from 41 COPD patients with acute exacerbation and environmental samples from 36 of these patient's homes were prospectively collected. A. fumigatus was detected in respiratory samples from 11 of 41 patients (27%) and in 15 of 36 domiciles (42%). Cyp51A sequencing and selection on itraconazole medium of clinical (n = 68) and environmental (n = 48) isolates yielded ARAF detection in 1 of 11 A. fumigatus colonized patients with COPD (9%) and 2 of 15 A. fumigatus-positive patient's homes (13%). The clinical isolate had no CYP51A mutation. Two environmental isolates from two patients harbored TR34 /L98H mutation, and one had an H285Y mutation. Coexistence of different cyp51A genotypes and/or azole resistance profiles was detected in 3 of 8 respiratory and 2 of 10 environmental samples with more than one isolate, confirming the need for a systematic screening of all clinically relevant isolates. The high prevalence of ARAF in patients with COPD and their homes supports the need for further studies to assess the prevalence of azole resistance in patients with Aspergillus diseases in Northern France.

Evaluation of the in vitro activity of isavuconazole and comparator voriconazole against 2635 contemporary clinical Candida and Aspergillus isolates.

OBJECTIVE: The in vitro activity of isavuconazole was determined for 1677 Candida and 958 Aspergillus isolates from 2012 to 2014 with voriconazole as comparator. METHODS: Aspergillus isolates were screened for resistance using azole-agar. Aspergillus isolates that screened positive and all Candida isolates underwent EUCAST broth microdilution testing. Isolates were categorized as wild-type (wt) or non-wt, adopting EUCAST epidemiological cut-off values (ECOFFs) (where available) or wt upper limits (wtULs; two two-fold dilutions above the MIC50). The CYP51A gene was sequenced for non-wt Aspergillus fumigatus isolates. Itraconazole and posaconazole MICs were determined for selected Aspergillus isolates with isavuconazole MIC ≥2 mg/L. RESULTS: Isavuconazole MIC50 (range) (mg/L) against Candida species were: Candida albicans: ≤0.03 (≤0.03 to >4), Candida dubliniensis: ≤0.03 (≤0.03), Candida glabrata: ≤0.03 (≤0.03-4), Candida krusei: 0.06 (≤0.03-0.5), Candida parapsilosis: ≤0.03 (≤0.03-0.06), Candida tropicalis: ≤0.03 (≤0.03 to >4), Saccharomyces cerevisiae (anamorph: Candida robusta): ≤0.03 (≤0.03-0.5). Non-wt isavuconazole/voriconazole MICs were found for C. albicans: 0.8/1.0%, C. dubliniensis: 0/1.8%, C. glabrata: 14.9/9.5%, C. krusei: 2.7/1.4%, C. parapsilosis: 1.7/1.8%, C. tropicalis: 14.3/19.1% and S. cerevisiae: 10.0/0%. Isavuconazole MIC50 (range) (mg/L) against Aspergillus species were: A. fumigatus: 1 (≤0.125 to >16), Aspergillus niger: 2 (1-8), Aspergillus terreus: 1 (0.25-8), Aspergillus flavus: 1 (0.5-2), Aspergillus nidulans: ≤0.125 (≤0.125-0.25). Non-wt isavuconazole/voriconazole MICs were found for 13.7/15.2% A. fumigatus, 4.9/0% A. niger and 48.2/22.2% A. terreus. CONCLUSION: Isavuconazole displayed broad in vitro activity, similar to that of voriconazole. Up to 15% of C. glabrata, C. tropicalis and A. fumigatus isolates were non-wt, reflecting increased resistance at a reference centre and technical issues. Significant CYP51A alterations were reliably detected applying the isavuconazole breakpoint.

Comparison of EUCAST and CLSI Reference Microdilution MICs of Eight Antifungal Compounds for Candida auris and Associated Tentative Epidemiological Cutoff Values.

Candida auris is an emerging multidrug-resistant yeast. So far, all but two susceptibility testing studies have examined ≤50 isolates, mostly with the CLSI method. We investigated CLSI and EUCAST MICs for 123 C. auris isolates and eight antifungals and evaluated various methods for epidemiological cutoff (ECOFF) determinations. MICs (in milligrams per liter) were determined using CLSI method M27-A3, and the EUCAST E.Def 7.3. ANOVA analysis of variance with Bonferroni's multiple-comparison test and Pearson analysis were used on log2 MICs (significance at P values of <0.05). The percent agreement (within ±0 to ±2 2-fold dilutions) between the methods was calculated. ECOFFs were determined visually, statistically (using the ECOFF Finder program and MicDat1.23 software with 95% to 99% endpoints), and via the derivatization method (dECOFFs). The CLSI and EUCAST MIC distributions were wide, with several peaks for all compounds except amphotericin B, suggesting possible acquired resistance. Modal MIC, geometric MIC, MIC50, and MIC90 values were ≤1 2-fold dilutions apart, and no significant differences were found. The quantitative agreement was best for amphotericin B (80%/97% within ±1/±2 dilutions) and lowest for isavuconazole and anidulafungin (58%/76% to 75% within ±1/±2 dilutions). We found that 90.2%/100% of the isolates were amphotericin B susceptible based on CLSI/EUCAST methods, respectively (i.e., with MICs of ≤1 mg/liter), and 100%/97.6% were fluconazole nonsusceptible by CLSI/EUCAST (MICs > 2). The ECOFFs (in milligrams per liter) were similar across the three different methods for itraconazole (ranges for CLSI/EUCAST, 0.25 to 0.5/0.5 to 1), posaconazole (0.125/0.125 to 0.25), amphotericin B (0.25 to 0.5/1 to 2), micafungin (0.25 to 0.5), and anidulafungin (0.25 to 0.5/0.25 to 1). In contrast, the estimated ECOFFs were dependent on the method applied for voriconazole (1 to 32) and isavuconazole (0.125 to 4). CLSI and EUCAST MICs were remarkably similar and confirmed uniform fluconazole resistance and variable acquired resistance to the other agents.

A prospective international Aspergillus terreus survey: an EFISG, ISHAM and ECMM joint study.

OBJECTIVES: A prospective international multicentre surveillance study was conducted to investigate the prevalence and amphotericin B susceptibility of Aspergillus terreus species complex infections. METHODS: A total of 370 cases from 21 countries were evaluated. RESULTS: The overall prevalence of A. terreus species complex among the investigated patients with mould-positive cultures was 5.2% (370/7116). Amphotericin B MICs ranged from 0.125 to 32 mg/L, (median 8 mg/L). CONCLUSIONS: Aspergillus terreus species complex infections cause a wide spectrum of aspergillosis and the majority of cryptic species display high amphotericin B MICs.

Multicenter Study of Method-Dependent Epidemiological Cutoff Values for Detection of Resistance in Candida spp. and Aspergillus spp. to Amphotericin B and Echinocandins for the Etest Agar Diffusion Method.

Method-dependent Etest epidemiological cutoff values (ECVs) are not available for susceptibility testing of either Candida or Aspergillus species with amphotericin B or echinocandins. In addition, reference caspofungin MICs for Candida spp. are unreliable. Candida and Aspergillus species wild-type (WT) Etest MIC distributions (microorganisms in a species-drug combination with no detectable phenotypic resistance) were established for 4,341 Candida albicans, 113 C. dubliniensis, 1,683 C. glabrata species complex (SC), 709 C. krusei, 767 C. parapsilosis SC, 796 C. tropicalis, 1,637 Aspergillus fumigatus SC, 238 A. flavus SC, 321 A. niger SC, and 247 A. terreus SC isolates. Etest MICs from 15 laboratories (in Argentina, Europe, Mexico, South Africa, and the United States) were pooled to establish Etest ECVs. Anidulafungin, caspofungin, micafungin, and amphotericin B ECVs (in micrograms per milliliter) encompassing ≥97.5% of the statistically modeled population were 0.016, 0.5, 0.03, and 1 for C. albicans; 0.03, 1, 0.03, and 2 for C. glabrata SC; 0.06, 1, 0.25, and 4 for C. krusei; 8, 4, 2, and 2 for C. parapsilosis SC; and 0.03, 1, 0.12, and 2 for C. tropicalis The amphotericin B ECV was 0.25 µg/ml for C. dubliniensis and 2, 8, 2, and 16 µg/ml for the complexes of A. fumigatus, A. flavus, A. niger, and A. terreus, respectively. While anidulafungin Etest ECVs classified 92% of the Candida fks mutants evaluated as non-WT, the performance was lower for caspofungin (75%) and micafungin (84%) cutoffs. Finally, although anidulafungin (as an echinocandin surrogate susceptibility marker) and amphotericin B ECVs should identify Candida and Aspergillus isolates with reduced susceptibility to these agents using the Etest, these ECVs will not categorize a fungal isolate as susceptible or resistant, as breakpoints do.

Spectrophotometric reading of EUCAST antifungal susceptibility testing of Aspergillus fumigatus.

OBJECTIVES: Given the increasing number of antifungal drugs and the emergence of resistant Aspergillus isolates, objective, automated and high-throughput antifungal susceptibility testing is important. The EUCAST E.Def 9.3 reference method for MIC determination of Aspergillus species relies on visual reading. Spectrophotometric reading was not adopted because of concern that non-uniform filamentous growth might lead to unreliable and non-reproducible results. We therefore evaluated spectrophotometric reading for the determination of MICs of antifungal azoles against Aspergillus fumigatus. METHODS: Eighty-eight clinical isolates of A. fumigatus were tested against four medical azoles (posaconazole, voriconazole, itraconazole, isavuconazole) and one agricultural azole (tebuconazole) with EUCAST E.Def 9.3. The visually determined MICs (complete inhibition of growth) were compared with spectrophotometrically determined MICs and essential (±1 twofold dilution) and categorical (susceptible/intermediate/resistant or wild-type/non-wild-type) agreement was calculated. Spectrophotometric data were analysed with regression analysis using the Emax model, and the effective concentration corresponding to 5% (EC5) was estimated. RESULTS: Using the 5% cut-off, high essential (92%-97%) and categorical (93%-99%) agreement (<6% errors) was found between spectrophotometric and visual MICs. The EC5 also correlated with the visually determined MICs with an essential agreement of 83%-96% and a categorical agreement of 90%-100% (<5% errors). CONCLUSIONS: Spectrophotometric determination of MICs of antifungal drugs may increase objectivity, and allow automation and high-throughput of EUCAST E.Def 9.3 antifungal susceptibility testing of Aspergillus species.

Azole-resistant Aspergillus fumigatus in Denmark: a laboratory-based study on resistance mechanisms and genotypes.

Azole-resistant Aspergillus fumigatus originating from the environment as well as induced during therapy are continuously emerging in Danish clinical settings. We performed a laboratory-based retrospective study (2010-2014) of azole resistance and genetic relationship of A. fumigatus at the national mycology reference laboratory of Denmark. A total of 1162 clinical and 133 environmental A. fumigatus isolates were identified by morphology, thermotolerance and/or ß-tubulin sequencing. Screening for azole resistance was carried out using azole agar, and resistant isolates were susceptibility tested by the EUCAST (European Committee on Antimicrobial Susceptibility Testing) E.Def 9.2 reference method and CYP51A sequenced. Genotyping was performed for outbreak investigation and, when appropriate, short tandem repeat Aspergillus fumigatus microsatellite assay. All 133 environmental A. fumigatus isolates were azole susceptible. However, from 2010 to 2014, there was an increasing prevalence of azole resistance (from 1.4 to 6% isolates (p <0.001) and 1.8 to 4% patients (p <0.05)) among the clinical isolates, with the well-known environmental CYP51A variant TR34/L98H responsible for >50% of the azole resistance mechanisms. Among 184 Danish A. fumigatus isolates, 120 unique genotypes were identified and compared to a collection of 1822 international genotypes. Seven (5.8%) Danish genotypes were shared between isolates within Denmark but with different origin, 19 (15.8%) were shared with foreign genotypes, and two (11.8%) of 17 genotypes of isolates carrying the TR34/L98H resistance mechanisms were identical to two Dutch TR34/L98H isolates. Our findings underlines the demand for correct identification and susceptibility testing of clinical mould isolates. Furthermore, although complex, genotyping supported the hypotheses regarding clonal expansion and the potential of a single origin for the TR34/L98H clone.

EUCAST technical note on isavuconazole breakpoints for Aspergillus, itraconazole breakpoints for Candida and updates for the antifungal susceptibility testing method documents.

The Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST) has determined breakpoints for isavuconazole and Aspergillus and for itraconazole and Candida spp., released a new document summarizing existing and new minimum inhibitory concentration ranges for quality control strains and revised the method documents for yeast and mould susceptibility testing. This technical note is based on the EUCAST isavuconazole and itraconazole rationale documents, version 1.0 of the routine and extended internal quality control for antifungal susceptibility testing as recommended by EUCAST, and the E.Def 7.3, E.Def 9.2 and E.Def 9.3 method documents (http://www.eucast.org).

Posttreatment Antifungal Resistance among Colonizing Candida Isolates in Candidemia Patients: Results from a Systematic Multicenter Study.

The prevalence of intrinsic and acquired resistance among colonizing Candida isolates from patients after candidemia was investigated systematically in a 1-year nationwide study. Patients were treated at the discretion of the treating physician. Oral swabs were obtained after treatment. Species distributions and MIC data were investigated for blood and posttreatment oral isolates from patients exposed to either azoles or echinocandins for <7 or ≥ 7 days. Species identification was confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and internal transcribed spacer (ITS) sequencing, susceptibility was examined by EUCAST EDef 7.2 methodology, echinocandin resistance was examined by FKS sequencing, and genetic relatedness was examined by multilocus sequence typing (MLST). One hundred ninety-three episodes provided 205 blood and 220 oral isolates. MLST analysis demonstrated a genetic relationship for 90% of all paired blood and oral isolates. Patients exposed to azoles for ≥ 7 days (n = 93) had a significantly larger proportion of species intrinsically less susceptible to azoles (particularly Candida glabrata) among oral isolates than among initial blood isolates (36.6% versus 12.9%; P < 0.001). A similar shift toward species less susceptible to echinocandins among 85 patients exposed to echinocandins for ≥ 7 days was not observed (4.8% of oral isolates versus 3.2% of blood isolates; P > 0.5). Acquired resistance in Candida albicans was rare (<5%). However, acquired resistance to fluconazole (29.4%; P < 0.05) and anidulafungin (21.6%; P < 0.05) was common in C. glabrata isolates from patients exposed to either azoles or echinocandins. Our findings suggest that the colonizing mucosal microbiota may be an unrecognized reservoir of resistant Candida species, especially C. glabrata, following treatment for candidemia. The resistance rates were high, raising concern in general for patients exposed to antifungal drugs.

Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus.

To investigate azole resistance in clinical Aspergillus isolates, we conducted prospective multicenter international surveillance. A total of 3,788 Aspergillus isolates were screened in 22 centers from 19 countries. Azole-resistant A. fumigatus was more frequently found (3.2% prevalence) than previously acknowledged, causing resistant invasive and noninvasive aspergillosis and severely compromising clinical use of azoles.