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.

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. Candida species continue to be a leading cause of bloodstream infection in U.S. hospitals, especially in intensive care units (1, 2). Although the antifungal armamentarium is limited, there are good options for the treatment of Candida species, especially with the arrival of the newest antifungal agents, the echinocandins (3, 4). The echinocandins are intravenously administered agents with a favorable safety profile. As inhibitors of 1,3-␤-D glucan synthase in the cell wall, they have a mechanism of action different from that of the older azole antifungals, which act to disrupt ergosterol (cell membrane) synthesis. This alternate mechanism of action allows the echinocandins to be effective against Candida isolates that are azole resistant. Early studies of in vitro susceptibility showed resistance to echinocandins to be extremely low for all Candida species (5, 6).Candida glabrata has recently become the second-most-frequent cause of candidemia in the United States, surpassing C. parapsilosis and C. tropicalis (6-8). While the ultimate cause for this increase in the prevalence of C. glabrata is unknown, the increase might be related to C. glabrata's higher incidence of resistance to fluconazole in comparison to most other Candida species (6-9). Because of the increased probability of fluconazole resistance, echinocandins are recommended as first-line therapy against C. glabrata (4). Alarmingly, C. glabrata is the first species of Candida for which measurable resistance to echinocandins has been detected (6, 10). Case reports of echinocandin-resistant C. glabrata following echin...

Candida glabrata is the second leading cause of candidemia in the United States. Its high-level resistance to triazole antifungal drugs has led to the increased use of the echinocandin class of antifungal agents for primary therapy of these infections. We monitored C. glabrata bloodstream isolates from a population-based surveillance study for elevated echinocandin MIC values (MICs of >0.25 g/ml). From the 490 C. glabrata isolates that were screened, we identified 16 isolates with an elevated MIC value (2.9% of isolates from Atlanta and 2.0% of isolates from Baltimore) for one or more of the echinocandin drugs caspofungin, anidulafungin, and micafungin. All of the isolates with elevated MIC values had a mutation in the previously identified hot spot 1 of either the glucan synthase FKS1 (n ‫؍‬ 2) or FKS2 (n ‫؍‬ 14) gene. No mutations were detected in hot spot 2 of either FKS1 or FKS2. The predominant mutation was mutation of FKS2-encoded serine 663 to proline (S663P), found in 10 of the isolates with elevated echinocandin MICs. Two of the mutations, R631G for FKS1 and R665G for FKS2, have not been reported previously for C. glabrata. Multilocus sequence typing indicated that the predominance of the S663P mutation was not due to the clonal spread of a single sequence type. With a rising number of echinocandin therapy failures reported, it is important to continue to monitor rates of elevated echinocandin MIC values and the associated mutations.

Specific oligonucleotide probes were developed to identify medically important fungi that display yeast-like morphology in vivo. Universal fungal primers ITS1 and ITS4, directed to the conserved regions of ribosomal DNA, were used to amplify DNA from Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, Penicillium marneffei, Sporothrix schenckii, Cryptococcus neoformans, five Candida species, and Pneumocystis carinii. Specific oligonucleotide probes to identify these fungi, as well as a probe to detect all dimorphic, systemic pathogens, were developed. PCR amplicons were detected colorimetrically in an enzyme immunoassay format. The dimorphic probe hybridized with DNA from H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis, and P. marneffei but not with DNA from nondimorphic fungi. Specific probes for H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis, P. marneffei, S. schenckii, C. neoformans, and P. carinii hybridized with homologous but not heterologous DNA. Minor cross-reactivity was observed for the B. dermititidis probe used against C. immitis DNA and for the H. capsulatum probe used against Candida albicans DNA. However, the C. immitis probe did not cross-react with B. dermititidis DNA, nor did the dimorphic probe hybridize with C. albicans DNA. Therefore, these fungi could be differentiated by a process of elimination. In conclusion, probes developed to yeast-like pathogens were found to be highly specific and should prove to be useful in differentiating these organisms in the clinical setting.

The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera, Aspergillus sp. The Clinical and Laboratory Standards Institute (CLSI; formerly the NCCLS) Subcommittee on Antifungal Susceptibility Tests has developed reproducible procedures for antifungal susceptibility testing of molds by the broth microdilution method (M38-A document) (3). An agar diffusion method has been developed for yeasts by disk diffusion methodology (CLSI M44-A document for fluconazole and voriconazole) (2, 4, 6). Reference guidelines are not available for mold disk diffusion testing. However, although infections caused by molds are not as common as yeast infections, an increased incidence of systemic infections caused by Aspergillus and more recently the zygomycetes and other species (Aspergillus, Fusarium, and Scedosporium) has been documented (16). Therefore, there is a need for an easier and more economical standard method to test the susceptibility of mold isolates to available antifungal agents.The overall objective of this study was to identify standard testing guidelines for disk testing of molds (i) by determining the correlation between zone diameters in millimeters by a disk diffusion method that were read at each of three incubation times with broth microdilution reference MICs (CLSI M38-A method) or MECs (minimum effective concentrations, caspofungin) (3), (ii) by determining the reproducibility of replicate zone diameters obtained on 3 different days and under different testing conditions by the disk diffusion method, and (iii) by determining the performance of the disk diffusion method in identifying resistant isolates. This study evaluated the following 18 mold species (555 isolates Because MIC or MEC breakpoints are not available for mold testing, isolates were grouped as susceptible (MIC or MEC, Յ1 g/ml), intermediate (MIC or MEC, 2 g/ml) and resistant (MIC or MEC, Ն4 g/ml) to determine the performance of the disk diffusion method for identifying resistant isolates. These categorical breakpoints were chosen to enable this determination; supporting clinical data are not available. These breakpoints have not been approved by the CLSI, the FDA, or the pharmaceutical companies. MATERIALS AND METHODSStudy design. Two of the five laboratories received the same panel of 72 isolates, including 4 isolates of each of the 18 species evaluated in the study and 2 quality control (QC) isolates. Each isolate was tested with amphotericin B, caspofungin, itraconazole, posaconazole, and voriconazole by both the broth microdilution M38-A and disk diffusion assays on 3 different days to obtain reproducibility data. In addition, three of the five laboratories tested a total of 483 isolates, including ...