Detection of TR34/L98H CYP51A Mutation through Passive Surveillance for Azole-Resistant Aspergillus fumigatus in the United States from 2015 to 2017.

The emergence of azole-resistant Aspergillus fumigatus has become a clinical problem in many parts of the world. Several amino acid mutations in the azole target protein Cyp51Ap contribute to this resistance, with the most concerning being the environmentally derived TR34/L98H and TR46/Y121F/T289A mutations. Here, we performed passive surveillance to assess a sample of the A. fumigatus population in the United States for the presence of these mutations. We found 1.4% of those isolates to exhibit elevated MIC via broth microdilution, and five of those isolates harbored the TR34/L98H mutation.

Role of FKS Mutations in Candida glabrata: MIC values, echinocandin resistance, and multidrug resistance.

Candida glabrata is the second leading cause of candidemia in U.S. hospitals. Current guidelines suggest that an echinocandin be used as the primary therapy for the treatment of C. glabrata disease due to the high rate of resistance to fluconazole. Recent case reports indicate that C. glabrata resistance to echinocandins may be increasing. We performed susceptibility testing on 1,380 isolates of C. glabrata collected between 2008 and 2013 from four U.S. cities, Atlanta, Baltimore, Knoxville, and Portland. Our analysis showed that 3.1%, 3.3%, and 3.6% of the isolates were resistant to anidulafungin, caspofungin, and micafungin, respectively. We screened 1,032 of these isolates, including all 77 that had either a resistant or intermediate MIC value with respect to at least one echinocandin, for mutations in the hot spot regions of FKS1 and FKS2, the major mechanism of echinocandin resistance. Fifty-one isolates were identified with hot spot mutations, 16 in FKS1 and 35 in FKS2. All of the isolates with an FKS mutation except one were resistant to at least one echinocandin by susceptibility testing. Of the isolates resistant to at least one echinocandin, 36% were also resistant to fluconazole. Echinocandin resistance among U.S. C. glabrata isolates is a concern, especially in light of the fact that one-third of those isolates may be multidrug resistant. Further monitoring of U.S. C. glabrata isolates for echinocandin resistance is warranted.

Development of a Luminex-based multiplex assay for detection of mutations conferring resistance to Echinocandins in Candida glabrata.

Echinocandins are the recommended treatment for invasive candidiasis due to Candida glabrata. Resistance to echinocandins is known to be caused by nonsynonymous mutations in the hot spot-1 (HS1) regions of the FKS1 and FKS2 genes, which encode a subunit of the ß-1,3-glucan synthase, the target of echinocandins. Here, we describe the development of a microsphere-based assay using Luminex MagPix technology to identify mutations in the FKS1 HS1 and FKS2 HS1 domains, which confer in vitro echinocandin resistance in C. glabrata isolates. The assay is rapid and can be performed with high throughput. The assay was validated using 102 isolates that had FKS1 HS1 and FKS2 HS1 domains previously characterized by DNA sequencing. The assay was 100% concordant with DNA sequencing results. The assay was then used for high-throughput screening of 1,032 C. glabrata surveillance isolates. Sixteen new isolates with mutations, including a mutation that was new to our collection (del659F), were identified. This assay provides a rapid and cost-effective way to screen C. glabrata isolates for echinocandin resistance.

Validation of 24-hour flucytosine MIC determination by comparison with 48-hour determination by the Clinical and Laboratory Standards Institute M27-A3 broth microdilution reference method.

Flucytosine and itraconazole are the only antifungal agents for which the Clinical Laboratory and Standards Institute recommendations include MIC breakpoint readings at 48 h only. Here we show good essential and categorical agreement between the flucytosine MIC readings at 48 and 24 h for Candida species.

Candida lusitaniae MICs to the echinocandins are elevated but FKS-mediated resistance is rare.

MIC values were generated for caspofungin, micafungin, and anidulafungin against 106 isolates of C. lusitaniae, and these values were compared to established epidemiologic cutoff values. The majority of isolates were wild type both by MIC value as well as by FKS1 hotspot sequencing. Although C. lusitaniae isolates have MIC values to the echinocandins that are elevated compared to other common species, with regard to known mechanisms of resistance to the echinocandins, isolates with MIC values at or below the epidemiological cutoff values of 0.5 and 1 µg/mL for micafungin and anidulafungin, respectively, should be considered wild type.

Evidence for aneuploidy and recombination in the human commensal yeast Candida parapsilosis.

Isolates of Candida parapsilosis, including representatives of the three major sub-species groups, were screened for single nucleotide polymorphisms (SNPs) by sequencing five independent loci totaling 4kb per isolate. Group I isolates were highly conserved and in some cases, group I alleles were found in group II and III strains. Unique alleles were also associated with groups II and III, consistent with earlier observations of intergroup divergence. There was no heterozygosity in any strain, and a FACS analysis demonstrated that for all three groups nuclei are variant in size, ranging from 0.5 to 1.0 x the size of other diploid yeast genomes. This suggests that natural isolates of C. parapsilosis are aneuploid, with some isolates being essentially haploid. Taken collectively with the observation of group I alleles within group II and III strains, we propose that some form of recombination is occurring between groups.

The human commensal yeast, Candida albicans, has an ancient origin.

Candida albicans, the primary causative agent of candidiasis, is a ubiquitous member of the human flora and is capable of causing severe invasive disease. Despite its importance as a human pathogen, little is known concerning those factors creating and maintaining genetic diversity within the species and how extant strains reflect their evolutionary history. Based on nucleotide polymorphism frequencies, we estimated the time to a most recent common ancestor for the species to be about 3-16 million years, with variation due to molecular clock calibration. As C. albicans genotypes have broad geographic associations, this suggests that the origins of DNA sequence variation in extant populations coincided with early hominid evolution. This is consistent with an emerging view of a genetically complex organism that is able to survive under host immunity as an obligate commensal species.