Monitoring Antifungal Resistance in a Global Collection of Invasive Yeasts and Molds: Application of CLSI Epidemiological Cutoff Values and Whole-Genome Sequencing Analysis for Detection of Azole Resistance in Candida albicans

Abstract

The activity of 7 antifungal agents against 3,557 invasive yeasts and molds collected in 29 countries worldwide in 2014 and 2015 was evaluated. Epidemiological cutoff values (ECVs) published in the Clinical and Laboratory Standards Institute (CLSI) M59 document were applied for species with no clinical breakpoints. Echinocandin susceptibility rates were 95.9% to 100.0% for the 5 most common species, except for the rates for to anidulafungin (88.7% susceptible, 100.0% wild type). Rates of fluconazole resistance ranged from 8.0% for to 0.4% for Seven species displayed 100.0% wild-type amphotericin B MIC results, and and exhibited wild-type echinocandin MIC results. The highest fluconazole, voriconazole, and posaconazole MIC values for var. were 8 μg/ml, 0.12 μg/ml, and 0.25 μg/ml, respectively. isolates were 100.0% wild type for caspofungin and amphotericin B, but 3 (0.8%) of these isolates were non-wild type to itraconazole (2 isolates) or voriconazole (1 isolate). Mutations in hot spot (HS) regions were detected among 13/20 isolates displaying echinocandin MICs greater than the ECV (16 of these 20 isolates were ). Most isolates carrying mutations in HS regions were resistant to 2 or more echinocandins. Five fluconazole-nonsusceptible isolates were submitted to whole-genome sequencing analysis. Gain-of-function, heterozygous, and homozygous mutations were observed in 1 isolate each. One isolate displayed MDR1 promoter allele alterations associated with azole resistance. Elevated levels of expression of MDR1 or CDR2 were observed in 3 isolates and 1 isolate, respectively. Echinocandin and azole resistance is still uncommon among contemporary fungal isolates; however, mechanisms of resistance to antifungals were observed among spp., showing that resistance can emerge and monitoring is warranted.

Invasive Candida bovina Infection, France

“…For other antifungal agents, interpretation of MICs is complicated in the absence of specific clinical breakpoints. However, MICs were comparable to those of common species (7). In the case of this patient, catheter removal and caspofungin treatment led to recovery.…”

“…Among azoles, fluconazole showed MICs of 2 mg/L, considered susceptible because the breakpoint for all non-C. glabrata Candida is 2 mg/L. However, this MIC is higher than those of common Candida, such as C. albicans (MIC 90 <0.25 mg/L) (7). For other antifungal agents, interpretation of MICs is complicated in the absence of specific clinical breakpoints.…”

A New Age in Molecular Diagnostics for Invasive Fungal Disease: Are We Ready?

“…The exception may be in C. glabrata where azole resistance is associated with mutations in the transcription factor pdr1 (Ferrari et al, 2009), or if targeted DNA sequencing is performed for a mutation already known to confer resistance. Resistance mutations are broadly distributed throughout the genome and as such, NGS approaches have been studied for the detection of mutations in genes associated with azole resistance including in ERG11, ERG3, TAC1, and CgPDR1 (Garnaud et al, 2015;Biswas et al, 2017;Castanheira et al, 2017). NGS further has potential to detect novel mutations implicated in phenotypic resistance that may otherwise be missed by targeted DNA sequencing.…”

In Vitro Activity of APX001A (Manogepix) and Comparator Agents against 1,706 Fungal Isolates Collected during an International Surveillance Program in 2017

“…Candida sp. isolates showing echinocandin MIC values above ECVs were subjected to whole-genome sequencing (29). Specifically, total genomic DNA was used as the input material for library construction using the Nextera XT library construction protocol and index kit (Illumina, San Diego, CA, USA), following the manufacturer's instructions.…”

“…Concurrent with the increasing number of invasive fungal infections, antifungal surveillance programs have become important in defining the species distribution and resistance patterns of the responsible pathogens, providing needed information for appropriate empirical antifungal treatment (23)(24)(25)(26)(27)(28). The SENTRY Antimicrobial Surveillance Program is a global program (https://www.jmilabs.com/sentry-surveillance -program) that has been ongoing for more than 20 years (from 1997 to 2019) and collects, in each calendar year, consecutive invasive isolates of Candida, Aspergillus, and other opportunistic fungi from medical centers located in North America, Europe, Latin America, and the Asia-Pacific region (1,(29)(30)(31). Applying modern methods for species identification (e.g., sequence-based identification and matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]), testing of antifungal susceptibility, and characterization of antifungal resistance mechanisms provides a level of standardization and clarity that makes these observations useful in the ongoing fight against resistance (1,4,9,25,29,(32)(33)(34).…”