Voriconazole MICs Research Articles

Gene Amplification of CYP51B: a New Mechanism of Resistance to Azole Compounds in Trichophyton indotineae.

ABSTRACTTrichophyton indotineae causes dermatophytosis that is resistant to terbinafine and azole compounds. The aim of this study was to determine the mechanisms of resistance to itraconazole (ITC) and voriconazole (VRC) in strains of T. indotineae. Two azole-sensitive strains (ITC MIC < 0.125 μg/mL; VRC MIC < 0.06 μg/mL) and four azole-resistant strains (ITC MIC ≥ 0.5 μg/mL; VRC MIC ≥ 0.5 μg/mL) were used for the investigation. The expression of MDR genes encoding multidrug transporters of the ABC family for which orthologs have been identified in Trichophyton rubrum and those of CYP51A and CYP51B encoding the targets of azole antifungal compounds were compared between susceptible and resistant strains. TinMDR3 and TinCYP51B were overexpressed in T. indotineae resistant strains. Only small differences in susceptibility were observed between TinMDR3 disruptants and parental strains overexpressing TinMDR3. Whole-genome sequencing of resistant strains revealed the creation of a variable number of TinCYP51B tandem repeats at the specific position of their genomes in three resistant strains. Downregulation of TinCYP51B by RNA interference (RNAi) restored the susceptibility of azole-resistant strains. In contrast, overexpression of TinCYP51B cDNA conferred resistance to a susceptible strain of T. indotineae. In conclusion, the reduced sensitivity of T. indotineae strains to azoles is mainly due to the overexpression of TinCYP51B resulting from additional copies of this gene.

A Pragmatic Approach to Susceptibility Classification of Yeasts without EUCAST Clinical Breakpoints.

EUCAST has established clinical breakpoints for the six most common Candida species and Cryptococcus neoformans but not for less common yeasts because sufficient evidence is lacking. Consequently, the question “How to interpret the MIC?” for other yeasts often arises. We propose a pragmatic classification for amphotericin B, anidulafungin, fluconazole, and voriconazole MICs against 30 different rare yeasts. This classification takes advantage of MIC data for more than 4000 isolates generated in the EUCAST Development Laboratory for Fungi validated by alignment to published EUCAST MIC data. The classification relies on the following two important assumptions: first, that when isolates are genetically related, pathogenicity and intrinsic susceptibility patterns may be similar; and second, that even if species are not phylogenetically related, the rare yeasts will likely respond to therapy, provided the MIC is comparable to that against wild-type isolates of more prevalent susceptible species because rare yeasts are most likely “rare” due to a lower pathogenicity. In addition, the treatment recommendations available in the current guidelines based on the in vivo efficacy data and clinical experience are taken into consideration. Needless to say, it is of utmost importance (a) to ascertain that the species identification is correct (using MALDI-TOF or sequencing), and (b) to re-test the isolate once or twice to confirm that the MIC is representative for the isolate (because of the inherent variability in MIC determinations). We hope this pragmatic guidance is helpful until evidence-based EUCAST breakpoints can be formally established.

Delineation of the Direct Contribution of Candida auris ERG11 Mutations to Clinical Triazole Resistance.

ABSTRACTResistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris.IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11. However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

Phylogenetic Distribution of csp1 Types in Aspergillus fumigatus and Their Correlates to Azole Antifungal Drug Resistance.

ABSTRACTIn Aspergillus fumigatus, the repetitive region of the csp1 gene is one of the most frequently used loci for intraspecies typing of this human pathogenic mold. Using PCR amplification and Sanger sequencing of only a single marker, csp1 typing is readily available to most laboratories and highly reproducible. Here, I evaluate the usefulness of the csp1 marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. After resolving nomenclature conflicts from published studies and adding novel csp1 types, the number of known types now adds up to 38. Their distribution mostly correlates with A. fumigatus population structure, and they are also meaningful for narrowly defined cases of azole resistance phenotypes. Isolates carrying the pandemic resistance allele TR34/L98H show signs of interclade crossing of strains with t02 or t04A, into the t11 clade. Furthermore, absolute differences in voriconazole MIC values between t02/t04B versus t11 TR34/L98H isolates indicate that the genetic background of resistance mutations may have a pivotal role in cross-resistance phenotypes and, thus, clinical outcome and environmental selection. Despite the general genetic similarity of isolates with identical csp1 types, outcrossing into other clades is also observed. The csp1 type alone, therefore, does not sufficiently discriminate genetic clades to be used as the sole marker in epidemiologic studies.IMPORTANCE Aspergillus fumigatus is a ubiquitously distributed saprophytic mold and a leading cause of invasive aspergillosis in human hosts. Pandemic azole-resistant strains have emerged on a global scale, which are thought to be propagated through use of azole-based fungicides in agriculture. To perform epidemiologic studies, genetic typing of large cohorts is key. Here, I evaluate the usefulness of the frequently used csp1 marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. The phylogenetic distribution of csp1 types mostly correlates with A. fumigatus population structure and is also meaningful for narrowly defined cases of azole resistance phenotypes. Nevertheless, outcrossing of csp1 into other clades is also observed. The csp1 type alone, therefore, does not sufficiently discriminate genetic clades and should not be used as the sole marker in epidemiologic studies.

Efficacy of Voriconazole, Isavuconazole, Fluconazole, and Anidulafungin in the Treatment of Emerging Candida auris Using an Immunocompromised Murine Model of Disseminated Candidiasis

Antifungal activity of anidulafungin, voriconazole, isavuconazole, and fluconazole in the treatment of Candida auris was determined in vitro and in vivo. MICs for anidulafungin, voriconazole, isavuconazole, fluconazole, and amphotericin B were 0.5, 1, >64, 0.25, and 4 μg/ml, respectively. Significant in vivo efficacy was observed in the anidulafungin- and voriconazole-treated groups in survival and reduction in kidney tissue fungal burden compared to that in the untreated group (P values of <0.001 and 0.044, respectively). Our data showed that anidulafungin and voriconazole had comparable efficacies against C. auris, whereas isavuconazole did not show significant in vivo activity.

Intra- and inter-laboratory comparison of mDixon and FastFung broths for Malassezia antifungal susceptibility testing.

Malassezia spp. antifungal susceptibility testing (AFST) capacities are limited by the lack of efficient and standardised AFST procedure, mainly because of the fastidious cultivation of these yeast. This study aimed to compare the FastFung broth (FFB) to modified Dixon broth (mDIXB) for the in vitro AFST of Malassezia spp. Fluconazole, ketoconazole, voriconazole and terbinafine MICs against a 19 Malassezia strains, including 6M furfur, 4M pachydermatis, 5M sympodialis and 4M slooffiae. The essential agreement (EA) between the two assays, and the intra- and inter-laboratory agreement of each assay were assessed. The MIC data obtained in our study were comparable to those reported in the literature. FFB showed to enhance Malassezia growth and displayed 100% (±2-fold dilution) EAs demonstrating similar performances to mDIXB. In addition, the MIC data obtained by using the FFB were reproducible between laboratories with EAs ranging from 94.7% to 100%. Therefore, FFB is a suitable alternative to mDXB for Malassezia spp. AFST.

In Vitro Antifungal Drug Resistance Profiles of Clinically Relevant Members of the Mucorales (Mucoromycota) Especially with the Newer Triazoles.

Mucoromycoses (infections caused by members of the order Mucorales, phylum Mucoromycota [ex-Zygomycota]) are highly destructive, rapidly progressive infections, with dire prognoses especially when they occur in immunocompromised hosts. Current treatment guidelines recommend liposomal formulations of amphotericin B with adjunctive surgery as first line therapy, with the newer triazoles posaconazole or isavuconazole as alternative treatments, or as salvage therapy. Among the many organisms belonging to this order, a limited number of species in the genera Rhizopus, Mucor, Lichtheimia and Rhizomucor are responsible for most cases of human infection. Here, we present the minimum inhibitory concentration data (MICs) for amphotericin B, posaconazole, isavuconazole, itraconazole and voriconazole with a panel of over 300 isolates of the five most common agents of human infection (Lichtheimia corymbifera, Rhizopus arrhizus, R. microsporus, Rhizomucor pusillus and Mucor spp.) determined using the CLSI broth microdilution method. In agreement with previous studies, the most active antifungal drug for all Mucorales was amphotericin B, with MICs within the range that would predict susceptibility with Aspergillus fumigatus. Conversely, MICs for voriconazole against all species tested were high, and above the range associated with clinical efficacy with A. fumigatus. Interestingly, whilst isavuconazole and posaconazole MIC distributions indicated in vitro activity against some members of the Mucorales, activity was species-dependent for both agents. These data underscore the importance of accurate identification of the causative agents of mucoromycosis, coupled with antifungal susceptibility testing of individual isolates, in determining the optimal treatment of infections caused by these aggressive opportunistic human fungal pathogens.

Hmg1 Gene Mutation Prevalence in Triazole-Resistant Aspergillus fumigatus Clinical Isolates.

Recently, mutations in the 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase-encoding gene (hmg1), a gene involved in ergosterol production, were associated with triazole-resistance in Aspergillus fumigatus. In this study, we determined the prevalence and characteristics of hmg1 mutations in a collection of clinical triazole-resistant A. fumigatus isolates collected during 2001–2019 from two international mycology reference centers: the Belgian National Reference Center for Mycosis and the Center of Expertise in Mycology Radboudumc/CWZ. Clinical isolates with and without cyp51A gene mutations and randomly selected wild-type (WT) controls were included. Isolates were characterized by in vitro susceptibility testing, cyp51A and hmg1 sequencing, and short tandem repeat typing. Available clinical records were analyzed for previous triazole exposure. In 23 isolates (24%) of the 95 triazole-resistant A. fumigatus isolates, hmg1 gene mutations were observed; including 5/23 (22%) isolates without cyp51A gene mutations and 18/72 (25%) with cyp51A mutations. Four previously described hmg1 gene mutations (E105K, G307R/D, G466V, and S541G) and two novel mutations (W273S and L304P) were found; 4/23 (17%) in the sterol-sensing-domain region. No triazole-antifungal exposure was reported in 75% (9/12) of patients harboring an isolate with hmg1 gene mutations. Three of 39 WT isolates (8%) contained a hmg1 gene mutation; E105K (2-isolates) and S541G. Hmg1 gene mutations were predominantly found in A. fumigatus with cyp51A mutations with voriconazole MICs ≥ 8 mg/L.

Nationwide surveillance of azole-resistant Aspergillus fumigatus environmental isolates in Greece: detection of pan-azole resistance associated with the TR46/Y121F/T289A cyp51A mutation.

Acquired azole resistance (AR) in Aspergillus fumigatus emphasizes the importance of the One Health multisectorial approach. The prevalence of azole-resistant A. fumigatus in the environment of Greece is unknown. Between October 2016 and September 2017, a total of 716 soil samples were collected from 23 provinces and screened for AR using azole-containing agar plates. Recovered isolates were macro-/microscopically identified and colonies were counted. Azole susceptibility testing of A. fumigatus species complex (SC) isolates was performed (EUCAST E.DEF9.3.1). Azole-resistant A. fumigatus isolates were subjected to confirmatory molecular identification and sequencing of the cyp51A gene. No yeasts were recovered, while multiple moulds grew on 695 (97%) samples. Overall, zygomycetes (most non-Mucor genera) grew on 432 (60%) samples, while Aspergillus spp. grew on 500 (70%) [410 (57%) Aspergillus niger SC; 120 (17%) Aspergillus terreus SC; 101 (14%) A. fumigatus SC; 34 (5%) Aspergillus flavus SC]. The mean ± SD soil load of Aspergillus spp. was 2.23 ± 0.41 log10 cfu/g (no differences among species). No azole-resistant non-A. fumigatus spp. isolate was detected. Itraconazole, voriconazole, isavuconazole and posaconazole MIC50/MIC90 (MIC range) of A. fumigatus SC strains were 0.25/0.5 (0.25 to >8), 0.5/1 (0.25 to >8), 1/1 (0.125 to >8) and 0.06/0.125 (0.06-1) mg/L, respectively. Overall, 1/500 (0.2%) of Aspergillus isolates, and 1/101 (1%) of A. fumigatus SC isolates, was pan-azole-resistant (itraconazole, voriconazole, isavuconazole and posaconazole MIC >8, >8, >8 and 1 mg/L, respectively). The resistant isolate was recovered from organically grown raisin grapes treated with homemade compost and it was an A. fumigatus sensu stricto isolate harbouring the TR46/Y121F/T289A mutation. The soil's load was higher compared with azole-susceptible strains (3.74 versus 2.09 log10 cfu/g). This is the first known report of environmental pan-azole-resistant A. fumigatus in Greece. Since data on Greek clinical isolates are lacking, this finding must alarm the systematic local surveillance of AR in medical settings.

Paradoxal Trends in Azole-Resistant Aspergillus fumigatus in a National Multicenter Surveillance Program, the Netherlands, 2013-2018.

We investigated the prevalence of azole resistance of Aspergillus fumigatus isolates in the Netherlands by screening clinical A. fumigatus isolates for azole resistance during 2013–2018. We analyzed azole-resistant isolates phenotypically by in vitro susceptibility testing and for the presence of resistance mutations in the Cyp51A gene. Over the 6-year period, 508 (11%) of 4,496 culture-positive patients harbored an azole-resistant isolate. Resistance frequency increased from 7.6% (95% CI 5.9%–9.8%) in 2013 (58/760 patients) to 14.7% (95% CI 12.3%–17.4%) in 2018 (112/764 patients) (p = 0.0001). TR34/L98H (69%) and TR46/Y121F/T289A (17%) accounted for 86% of Cyp51A mutations. However, the mean voriconazole MIC of TR34/L98H isolates decreased from 8 mg/L (2013) to 2 mg/L (2018), and the voriconazole-resistance frequency was 34% lower in 2018 than in 2013 (p = 0.0001). Our survey showed changing azole phenotypes in TR34/L98H isolates, which hampers the use of current PCR-based resistance tests.

Revision of EUCAST breakpoints: consequences for susceptibility of contemporary Danish mould isolates to isavuconazole and comparators.

EUCAST recently revised the definition of the 'I' category from 'intermediate' to 'susceptible, increased exposure'. Consequently, all current antifungal breakpoints have been reviewed and revised breakpoints (v 10.0) have been released. We investigated isavuconazole and comparator MICs (mg/L) against contemporary moulds and the consequences of the breakpoint revision for susceptibility classification. Six hundred and ninety-six Aspergillus and 46 other moulds were included. EUCAST E.Def 10.1 azole resistance screening was performed for Aspergillus fumigatus and E.Def 9.3.1 testing of non-susceptible A. fumigatus and other moulds. Most non-wildtype/resistant isolates underwent cyp51A sequencing. Isavuconazole MIC50/MIC90s were ≤1/≤2 mg/L for Aspergillus flavus, A. fumigatus and Aspergillus nidulans versus 2/4 mg/L for Aspergillus niger and 2/16 mg/L for Aspergillus terreus. For the remaining moulds, MICs were highest for Fusarium (16 to >16 mg/L), lowest for dermatophytes (0.06-0.5 mg/L) and in between for Mucorales and others (1 to >16 mg/L). A very strong isavuconazole-voriconazole MIC correlation was found for A. fumigatus (Pearson r = 0.888) and itraconazole-posaconazole correlation for A. fumigatus (r = 0.905) and A. terreus (r = 0.848). For A. fumigatus, the revised breakpoints lowered isavuconazole resistance (22.6% to 7.7%, P < 0.0001) and increased voriconazole resistance (3.8% to 6.7%, P = 0.025), resulting in similar resistance rates across the four azoles (range: 6.7%-7.7%). For A. terreus, isavuconazole resistance remained unchanged (81.3%) and higher than itraconazole (43.8%, P = 0.004) and posaconazole (53.1%, P = 0.03) resistance. Azole cross-resistance was found in 24/24, 13/20 and 4/90 isolates, and Cyp51A alterations in 16/18, 1/7 and 2/4 sequenced isolates with isavuconazole MICs of >4, 4 and 2 mg/L, respectively. Isavuconazole displays broad anti-mould activity. The revised breakpoints result in fewer misclassifications of wildtype isolates without compromising detection of resistant mutants.

Diclofenac exhibits synergism with azoles against planktonic cells and biofilms of Candida tropicalis

This study aimed to evaluate the effect of diclofenac on minimum inhibitory concentrations of antifungals against planktonic cells and biofilms of Candida tropicalis. Susceptibility testing of planktonic cells was evaluated using the broth microdilution assay and checkerboard method. Biofilm formation by C. tropicalis in the presence of diclofenac, alone or in combination with antifungals, was also evaluated, and scanning electron microscope (SEM) and confocal microscope (CLSM) analyses were performed. Diclofenac showed an MIC of 1024 μg ml−1 against planktonic cells. The MICs of fluconazole and voriconazole against azole-resistant isolates were reduced 8- to 32-fold and 16- to 256-fold, respectively, when in combination with diclofenac. When in combination with fluconazole or voriconazole, diclofenac reduced the antifungal concentration necessary to inhibit C. tropicalis biofilm formation. In conclusion, diclofenac presents synergism with fluconazole and voriconazole against resistant C. tropicalis strains and improves the activity of these azole drugs against biofilm formation.

Toward Harmonization of Voriconazole CLSI and EUCAST Breakpoints for Candida albicans Using a Validated In Vitro Pharmacokinetic/Pharmacodynamic Model.

CLSI and EUCAST susceptibility breakpoints for voriconazole and Candida albicans differ by one dilution (≤0.125 and ≤0.06 mg/liter, respectively) whereas the epidemiological cutoff values for EUCAST (ECOFF) and CLSI (ECV) are the same (0.03 mg/liter). We therefore determined the pharmacokinetic/pharmacodynamic (PK/PD) breakpoints of voriconazole against C. albicans for both methodologies with an in vitro PK/PD model, which was validated using existing animal PK/PD data. Four clinical wild-type and non-wild-type C. albicans isolates (voriconazole MICs, 0.008 to 0.125 mg/liter) were tested in an in vitro PK/PD model. For validation purposes, mouse PK were simulated and in vitro PD were compared with in vivo outcomes. Human PK were simulated, and the exposure-effect relationship area under the concentration-time curve for the free, unbound fraction of a drug from 0 to 24 h (fAUC0-24)/MIC was described for EUCAST and CLSI 24/48-h methods. PK/PD breakpoints were determined using the fAUC0-24/MIC associated with half-maximal activity (EI50) and Monte Carlo simulation analysis. The in vitro 24-h PD EI50 values of voriconazole against C. albicans were 2.5 to 5 (1.5 to 17) fAUC/MIC. However, the 72-h PD were higher at 133 (51 to 347) fAUC/MIC for EUCAST and 94 (35 to 252) fAUC/MIC for CLSI. The mean (95% confidence interval) probability of target attainment (PTA) was 100% (95 to 100%), 97% (72 to 100%), 83% (35 to 99%), and 49% (8 to 91%) for EUCAST and 100% (97 to 100%), 99% (85 to 100%), 91% (52 to 100%), and 68% (17 to 96%) for CLSI for MICs of 0.03, 0.06, 0.125, and 0.25 mg/liter, respectively. Significantly, >95% PTA values were found for EUCAST/CLSI MICs of ≤0.03 mg/liter. For MICs of 0.06 to 0.125 mg/liter, trough levels 1 to 4 mg/liter would be required to attain the PK/PD target. A PK/PD breakpoint of C. albicans voriconazole at the ECOFF/ECV of 0.03 mg/liter was determined for both the EUCAST and CLSI methods, indicating the need for breakpoint harmonization for the reference methodologies.

Alarming India-wide phenomenon of antifungal resistance in dermatophytes: A multicentre study.

An alarming increase in recalcitrant dermatophytosis has been witnessed in India over the past decade. Drug resistance may play a major role in this scenario. The aim of the present study was to determine the prevalence of in vitro resistance to terbinafine, itraconazole and voriconazole in dermatophytes, and to identify underlying mutations in the fungal squalene epoxidase (SQLE) gene. We analysed skin samples from 402 patients originating from eight locations in India. Fungi were identified by microbiological and molecular methods, tested for antifungal susceptibility (terbinafine, itraconazole, voriconazole), and investigated for missense mutations in SQLE. Trichophyton (T.) mentagrophytes internal transcribed spacer (ITS) Type VIII was found in 314 (78%) samples. Eighteen (5%) samples harboured species identified up to the T interdigitale/mentagrophytes complex, and T rubrum was detected in 19 (5%) samples. 71% of isolates were resistant to terbinafine. The amino acid substitution Phe397Leu in the squalene epoxidase of resistant T mentagrophytes was highly prevalent (91%). Two novel substitutions in resistant Trichophyton strains, Ser395Pro and Ser443Pro, were discovered. The substitution Ala448Thr was found in terbinafine-sensitive and terbinafine-resistant isolates but was associated with increased MICs of itraconazole and voriconazole. The high frequencies of terbinafine resistance in dermatophytes are worrisome and demand monitoring and further research. Squalene epoxidase substitutions between Leu393 and Ser443 could serve as markers of resistance in the future.

Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes.

Terbinafine resistance is increasingly reported in Trichophyton, rendering susceptibility testing particularly important in non-responding cases. We performed a multicentre evaluation of six EUCAST-based methods. Ten laboratories susceptibility tested terbinafine, itraconazole, voriconazole and amorolfine against a blinded panel of 38 terbinafine WT and target gene mutant isolates. E.Def 9.3.1 modifications included: medium with/without addition of chloramphenicol and cycloheximide (CC), incubation at 25°C to 28°C for 5-7 days and three MIC endpoints [visually and spectrophotometrically (90%/50% inhibition)], generating 7829 MICs. Quality control (QC) strains were Aspergillus flavus ATCC 204304 and CNM-CM1813. Eyeball, ECOFFinder (where ECOFF stands for epidemiological cut-off) and derivatization WT upper limits (WT-ULs), very major errors (VMEs; mutants with MICs ≤WT-ULs) and major errors (MEs; WT isolates with MICs >WT-ULs) were determined. MICs fell within the QC ranges for ATCC 204304/CNM-CM1813 for 100%/96% (voriconazole) and 84%/84% (itraconazole), respectively. Terbinafine MICs fell within 0.25-1 mg/L for 96%/92%, suggesting high reproducibility. Across the six methods, the number of terbinafine MEs varied from 2 to 4 (2.6%-5.2%) for Trichophyton rubrum and from 0 to 2 (0%-2.0%) for Trichophyton interdigitale. Modes for WT and mutant populations were at least seven 2-fold dilutions apart in all cases. Excluding one I121M/V237I T. rubrum mutant and two mixed WT/mutant T. interdigitale specimens, the numbers of VMEs were as follows: T. rubrum: CC visual, 1/67 (1.5%); CC spectrophotometric 90% inhibition, 3/59 (5.1%); and CC spectrophotometric 50% inhibition, 1/67 (1.5%); and T. interdigitale: none. Voriconazole and amorolfine MICs were quite uniform, but trailing growth complicated determination of itraconazole visual and spectrophotometric 90% inhibition MIC. Although none of the laboratories was experienced in dermatophyte testing, error rates were low. We recommend the CC spectrophotometric 50% inhibition method and provide QC ranges and WT-ULs for WT/non-WT classification.

In Vitro Evaluation of Antifungal Drug Combinations against Multidrug-Resistant Candida auris Isolates from New York Outbreak.

Since 2016, New York hospitals and health care facilities have faced an unprecedented outbreak of the pathogenic yeast Candida auris We tested over 1,000 C. auris isolates from affected facilities and found high resistance to fluconazole (MIC > 256 mg/liter) and variable resistance to other antifungal drugs. Therefore, we tested if two-drug combinations are effective in vitro against multidrug-resistant C. auris Broth microdilution antifungal combination plates were custom manufactured by TREK Diagnostic System. We used 100% inhibition endpoints for the drug combination as reported earlier for the intra- and interlaboratory agreements against Candida species. The results were derived from 12,960 readings, for 15 C. auris isolates tested against 864 two-drug antifungal combinations for nine antifungal drugs. Flucytosine (5FC) at 1.0 mg/liter potentiated the most combinations. For nine C. auris isolates resistant to amphotericin B (AMB; MIC ≥ 2.0 mg/liter), AMB-5FC (0.25/1.0 mg/liter) yielded 100% inhibition. Six C. auris isolates resistant to three echinocandins (anidulafungin [AFG], MIC ≥ 4.0 mg/liter; caspofungin [CAS], MIC ≥ 2.0 mg/liter; and micafungin [MFG], MIC ≥ 4.0 mg/liter) were 100% inhibited by AFG-5FC and CAS-5FC (0.0078/1 mg/liter) and MFG-5FC (0.12/1 mg/liter). None of the combinations were effective for C. auris 18-1 and 18-13 (fluconazole [FLC] > 256 mg/liter, 5FC > 32 mg/liter) except MFG-5FC (0.1/0.06 mg/liter). Thirteen isolates with a high voriconazole (VRC) MIC (>2 mg/liter) were 100% inhibited by the VRC-5FC (0.015/1 mg/liter). The simplified two-drug combination susceptibility test format would permit laboratories to provide clinicians and public health experts with additional data to manage multidrug-resistant C. auris.

First azole-resistant Aspergillus fumigatus isolates with the environmental TR46 /Y121F/T289A mutation in Iran.

SummaryBackgroundAzole resistance in Aspergillus fumigatus is an emerging problem and reported from all continents. As triazole antifungals are the mainstay of therapy in the management of invasive aspergillosis, azole‐resistant A fumigatus has become a major medical concern and with complicated clinical management.ObjectiveScreening of environmental presence of azole‐resistant A fumigatus in Iran.MethodsCompost from Northern Iran, collected between 2017 and 2018, was screened for the presence of azole‐resistant A fumigatus with azole‐containing agar. Phenotypic MICs were obtained from selected, molecularly confirmed isolates. cyp51A gene sequencing and genotyping of azole‐resistant isolates were done.ResultsAmong 300 compost samples, three A fumigatus isolates had high voriconazole MICs (≥16 mg/L) and harboured the TR46/Y121F/T289A mutation in the cyp51A gene. Microsatellite typing of these isolates showed that two strains had the same allele across all nine examined microsatellite loci and were genotypically related to Indian azole‐resistant strains. The other isolate had a different genotype.ConclusionThis is the first report of A fumigatus with TR46/Y121F/T289A mutation from the region. Monitoring and surveillance of antifungal susceptibility of clinical A fumigatus is warranted in Iran and elsewhere in the region.

2118. Antifungal Susceptibility Testing of Clinical Isolates of Aspergillus in Mexico

BackgroundInvasive aspergillosis (IA) is a leading cause of morbidity and mortality in immunocompromised hosts. Triazole resistance in Aspergillus fumigatus is emerging globally. We performed antifungal susceptibility testing (AST) in Aspergillus isolates from Mexico and evaluated risk factors associated with 6 week mortality, including the MICs.MethodsAspergillus isolates from clinical samples were collected in a tertiary care center from 2014 to 2018. Species-level identification and broth microdilution following the CLSI M38 method were performed. MICs were interpreted according to epidemiological cutoff values. PCR and cyp51A gene sequencing were performed in A. fumigatus isolates with voriconazole (VRC) MIC >1 µg/mL. Data from the medical record were obtained to classify patients according to the MSG/EORTC criteria. The relationship between the MICs and 6-week mortality was described. Multivariate analysis of factors associated with six week mortality was performed.ResultsAST was performed on 85 Aspergillus isolates: 60/85 from patients with IA, 15/85 from patients with Aspergillus colonization, 2 patients with Aspergilloma, 1 with chronic otitis media and 1 with endophtalmitis. Information from 6 patients was unavailable. VRC MIC > 1 µg/mL was found in 3/38 (7.8%) A. fumigatus, from two patients with IA. Both had a TR34/L98H mutation in the cyp51A gene. Amphotericin B (AmB) MICs ≥ 2 were found in 16/49 (32%) A. fumigatus, 10/15 (66%) A. flavus and 1/14 (8%) A. niger. Forty-one patients with IA were treated: 29/41 (71%) with VRC or posaconazole, 7/41 (17%) with AmB and 5 with combination therapy. Overall, 6-week mortality was 30/49 (61.2%) among patients with IA; 2/2 (100%) when VRC MIC >1 µg/mL and 12/19 (63%) when AmB > 2 µg/mL, of which only 4 patients received initial treatment with AmB. Age older than 65 years (OR 11.8; 95% CI 1.14–123) and hepatic failure (OR 7.9; 95% CI 1.22–50.9) were independently associated with 6-week mortality in multivariate analysis.ConclusionWe found a VRC MIC >1 µg/mL prevalence of 7.8% among A. fumigatus and a high prevalence of AmB MIC ≥ 2 among clinical isolates of Aspergillus in Mexico. Elevated mortality was seen in IA among older patients with hepatic failure. Larger epidemiological studies are warranted.Disclosures All authors: No reported disclosures.

Cyp51A Mutations, Extrolite Profiles, and Antifungal Susceptibility in Clinical and Environmental Isolates of the Aspergillus viridinutans Species Complex.

The past decade has seen an increase in aspergillosis in humans and animals due to Aspergillus viridinutans species complex members. Azole resistance is common to these infections, carrying a poor prognosis. cyp51A gene mutations are the main cause of acquired azole resistance in Aspergillus fumigatus This study aimed to determine if the azole-resistant phenotype in A. viridinutans complex members is associated with cyp51A mutations or extrolite profiles. The cyp51A gene of clinical and environmental isolates was amplified using novel primers, antifungal susceptibility was tested using the Clinical and Laboratory Standards Institute methodology, and extrolite profiling was performed using agar plug extraction. Very high azole MICs were detected in 84% of the isolates (31/37). The MICs of the newer antifungals luliconazole and olorofim (F901318) were low for all isolates. cyp51A sequences revealed 113 nonsynonymous mutations compared to the sequence of wild-type A. fumigatus M172A/V and D255G, previously associated with A. fumigatus azole resistance, were common among all isolates but were not correlated with azole MICs. Two environmental isolates with nonsusceptibility to itraconazole and high MICs of voriconazole and isavuconazole harbored G138C, previously associated with azole-resistant A. fumigatus Some novel mutations were identified only among isolates with high azole MICs. However, cyp51A homology modeling did not cause a significant protein structure change for these mutations. There was no correlation between extrolite patterns and susceptibility. For A. viridinutans complex isolates, cyp51A mutations and the extrolites that they produced were not major causes of antifungal resistance. Luliconazole and olorofim show promise for treating azole-resistant infections caused by these cryptic species.

A rare case of blood stream infection due to Candida ciferii in an immuno-compromised patient

Invasive infections related to yeast are increasingly observed in immune-compromised patients in hospitals.Fungal infections have increased morbidity and mortality and prolonged hospital stay which can lead to rise in medical care costs. Non-albicans Candida species have been increasingly found as causative agents in human infections with important therapeutic implications. We present a case of a 37-year-old, female patient, known case of B cell Acute Lymphoblastic Leukaemia admitted in a tertiary care hospital in central India for supportive care and chemotherapy. Patient was responding well to chemotherapy. On post induction day 20, she complained of high-grade fever with abdominal pain.Two sets of blood culture were sent to Microbiology Diagnostic Laboratory for diagnosis. She was started on Injection Magnex Forte (Cefoperazone-Sulbactum) empirically.The Gram stain of positive blood culture showed Gram positive budding yeast like cells in all four bottles.The organism was identified as C. ciferrii on Vitek 2 with 95% identification.Antibiotic susceptibility testing showed sensitive to Amphotericin B MIC ≤0.25 and voriconazole MIC ≤0.12. It was resistant to fluconazole MIC ≥64 μg/ml.