The Investigational Fungal Cyp51 Inhibitor VT-1129 Demonstrates Potent In Vitro Activity against Cryptococcus neoformans and Cryptococcus gattii.

Thein vitroactivities of the novel fungal Cyp51 inhibitor VT-1129 were evaluated against a large panel ofCryptococcus neoformansandCryptococcus gattiiisolates. VT-1129 demonstrated potent activities against bothCryptococcusspecies as demonstrated by low MIC50and MIC90values. ForC. gattii, thein vitropotency was maintained against all genotypes. In addition, significantly lower geometric mean MICs were observed for VT-1129 than for fluconazole againstC. neoformans, including isolates with reduced fluconazole susceptibility.

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.

Species identification and antifungal susceptibility testing of Candida bloodstream isolates from population-based surveillance studies in two U.S. cities from 2008 to 2011.

Between 2008 and 2011, population-based candidemia surveillance was conducted in Atlanta, GA, and Baltimore, MD. Surveillance had been previously performed in Atlanta in 1992 to 1993 and in Baltimore in 1998 to 2000, making this the first population-based candidemia surveillance conducted over multiple time points in the United States. From 2,675 identified cases of candidemia in the current surveillance, 2,329 Candida isolates were collected. Candida albicans no longer comprised the majority of isolates but remained the most frequently isolated species (38%), followed by Candida glabrata (29%), Candida parapsilosis (17%), and Candida tropicalis (10%). The species distribution has changed over time; in both Atlanta and Baltimore the proportion of C. albicans isolates decreased, and the proportion of C. glabrata isolates increased, while the proportion of C. parapsilosis isolates increased in Baltimore only. There were 98 multispecies episodes, with C. albicans and C. glabrata the most frequently encountered combination. The new species-specific CLSI Candida MIC breakpoints were applied to these data. With the exception of C. glabrata (11.9% resistant), resistance to fluconazole was very low (2.3% of isolates for C. albicans, 6.2% for C. tropicalis, and 4.1% for C. parapsilosis). There was no change in the proportion of fluconazole resistance between surveillance periods. Overall echinocandin resistance was low (1% of isolates) but was higher for C. glabrata isolates, ranging from 2.1% isolates resistant to caspofungin in Baltimore to 3.1% isolates resistant to anidulafungin in Atlanta. Given the increase at both sites and the higher echinocandin resistance, C. glabrata should be closely monitored in future surveillance.

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.

Epidemiologic cutoff values for triazole drugs in Cryptococcus gattii: correlation of molecular type and in vitro susceptibility.

Cryptococcus gattii causes infection in tropical and subtropical regions worldwide but has garnered increased attention since its 1999 emergence in North America. C. gattii can be divided into 4 molecular types that may represent cryptic species. Recent evidence has shown that azole antifungal MIC values differ among these molecular types. We tested a large collection of C. gattii isolates for susceptibility to 4 azole drugs. We found that isolates of molecular type VGII have the highest geometric mean (GM) fluconazole MIC values (8.6 µg/mL), while isolates of molecular type VGI have the lowest (1.7 µg/mL). For fluconazole, itraconazole, and voriconazole GM MIC values, VGI < VGIII < VGIV < VGII. The GM MIC values for posaconazole were similarly represented across molecular types, with the exception that VGII < VGIII and VGIV. We used the MIC values to establish preliminary epidemiologic cutoff values for each azole and molecular type of C. gattii.