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.

Comparison of multilocus sequence typing and Ca3 fingerprinting for molecular subtyping epidemiologically-related clinical isolates of Candida albicans.

Southern hybridization with the complex probe Ca3 is a well established tool for molecular subtyping of Candida albicans. Multilocus sequence typing (MLST) is a DNA sequence-based subtyping method recently applied to C. albicans and shown to have a high degree of intraspecies discriminatory power. However, its utility for studying the molecular epidemiology of sequential isolates from recurrent disease has not been established. We compared Ca3 Southern hybridization and MLST using seven housekeeping genes (CaAAT1a, CaACC1, CaADP1, CaPMI, CaSYA1, CaVPS13, CaZWF1b) for their ability to discriminate among 37 C. albicans isolates from recurrent cases of oropharyngeal candidiasis (OPC) in ten HIV-positive patients from India and the US. Among the 37 isolates, MLST identified 23 distinct genotypes (index of diversity = 97%); Ca3 Southern hybridization identified 21 distinct genotypes (index of diversity = 95%). Both methods clustered isolates into seven genetically-related groups and, with one exception, isolates that were indistinguishable by MLST were indistinguishable or highly related by Ca3 Southern hybridization. These results demonstrate that MLST performs equally well or better compared to Ca3 Southern hybridization for defining genetic-relatedness of sequential C. albicans isolates from recurrent cases of OPC in HIV-positive patients.

Evaluation of amphotericin B interpretive breakpoints for Candida bloodstream isolates by correlation with therapeutic outcome.

One hundred seven Candida bloodstream isolates (51 C. albicans, 24 C. glabrata, 13 C. parapsilosis, 13 C. tropicalis, 2 C. dubliniensis, 2 C. krusei, and 2 C. lusitaniae strains) from patients treated with amphotericin B alone underwent in vitro susceptibility testing against amphotericin B using five different methods. Fifty-four isolates were from patients who failed treatment, defined as death 7 to 14 days after the incident candidemia episode, having persistent fever of >or=5 days' duration after the date of the incident candidemia, or the recurrence of fever after two consecutive afebrile days while on antifungal treatment. MICs were determined by using the Clinical Laboratory Standards Institute (formally National Committee for Clinical Laboratory Standards) broth microdilution procedure with two media and by using Etest. Minimum fungicidal concentrations (MFCs) were also measured in two media. Broth microdilution tests with RPMI 1640 medium generated a restricted range of MICs (0.125 to 1 microg/ml); the corresponding MFC values ranged from 0.5 to 4 microg/ml. Broth microdilution tests with antibiotic medium 3 produced a broader distribution of MIC and MFC results (0.015 to 0.25 microg/ml and 0.06 to 2 microg/ml, respectively). Etest produced the widest distribution of MICs (0.094 to 2 microg/ml). However, none of the test formats studied generated results that significantly correlated with therapeutic success or failure.

Environmental influences on the phenology and abundance of flowering by Androsace septentrionalis (Primulaceae).

We studied the timing and abundance of flowering by Androsace septentrionalis L. (Primulaceae), an indeterminate winter annual or short-lived perennial, in 2 × 2 m plots at the Rocky Mountain Biological Laboratory in Colorado, USA, from 1982 to 2000. Flowers were counted every other day for most or all of the growing season in seven plots in a rocky meadow habitat and nine plots in a wet meadow habitat. The phenology and abundance of flowering were both highly variable, with mean dates of first flowering ranging from 16 May to 12 July and maximum daily counts of flowers ranging from 1 to 1187. Snowmelt date was the primary determinant of timing of flowering. For rocky meadow plots, the previous year's summer precipitation and the current year's average minimum temperature in May had significant effects on maximum number of flowers produced, but no environmental variable we considered was significantly correlated with flower abundance in the wet meadow plots. Length of flowering in individual plots ranged from 2 to 85 d, and many plot-years had both primary (about 1 mo) and secondary (about 10-12 d) flowering periods. The predicted increase in variability of precipitation accompanying climate change will affect negatively the long-term abundance and persistence of this species at our study site.

Comparative evaluation of NCCLS M27-A and EUCAST broth microdilution procedures for antifungal susceptibility testing of candida species.

A two-laboratory study was performed to evaluate the correlation between the NCCLS M27-A and EUCAST microdilution procedures for antifungal testing of Candida spp. A panel of 109 bloodstream isolates was tested against amphotericin B, flucytosine, fluconazole, and itraconazole. Overall, the agreement was 92% and the intraclass correlation coefficient was 0.90 (P < 0.05).

Fungemia caused by Zygoascus hellenicus in an allogeneic stem cell transplant recipient.

Zygoascus hellenicus (Candida hellenica) was isolated from a blood culture from a patient who had received an allogeneic stem cell transplant. The isolate displayed an antifungal susceptibility pattern of decreased susceptibility to fluconazole and itraconazole, high susceptibility to voriconazole, and low susceptibility to caspofungin. The organism was misidentified by a commercial yeast identification system. This is the first reported case of human infection with this rare ascomycetous yeast.

Comparison of visual and spectrophotometric methods of broth microdilution MIC end point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of fluconazole and itraconazole against trailing and nontrailing Candida isolates.

Visual determination of MIC end points for azole antifungal agents can be complicated by the trailing growth phenomenon. To determine the incidence of trailing growth, we performed testing of in vitro susceptibility to fluconazole and itraconazole using the National Committee for Clinical Laboratory Standards broth microdilution M27-A reference procedure and 944 bloodstream isolates of seven Candida spp., obtained through active population-based surveillance between 1998 and 2000. Of 429 C. albicans isolates, 78 (18.2%) showed trailing growth at 48 h in tests with fluconazole, and 70 (16.3%) showed trailing in tests with itraconazole. Of 118 C. tropicalis isolates, 70 (59.3%) showed trailing growth in tests with fluconazole, and 35 (29.7%) showed trailing in tests with itraconazole. Trailing growth was not observed with any of the other five Candida spp. tested (C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, and C. parapsilosis). To confirm whether or not isolates that showed trailing growth in fluconazole and/or itraconazole were resistant in vitro to these agents, all isolates that showed trailing growth were retested by the sterol quantitation method, which measures cellular ergosterol content rather than growth inhibition after exposure to azoles. By this method, none of the trailing isolates was resistant in vitro to fluconazole or itraconazole. For both agents, a 24-h visual end point or a spectrophotometric end point of 50% reduction in growth relative to the growth control after 24 or 48 h of incubation correlated most closely with the result of sterol quantitation. Our results indicate that MIC results determined by either of these end point rules may be more predictive of in vivo outcome for isolates that give unclear visual end points at 48 h due to trailing growth.

Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program.

To determine the incidence of Candida bloodstream infections (BSI) and antifungal drug resistance, population-based active laboratory surveillance was conducted from October 1998 through September 2000 in two areas of the United States (Baltimore, Md., and the state of Connecticut; combined population, 4.7 million). A total of 1,143 cases were detected, for an average adjusted annual incidence of 10 per 100,000 population or 1.5 per 10,000 hospital days. In 28% of patients, Candida BSI developed prior to or on the day of admission; only 36% of patients were in an intensive care unit at the time of diagnosis. No fewer than 78% of patients had a central catheter in place at the time of diagnosis, and 50% had undergone surgery within the previous 3 months. Candida albicans comprised 45% of the isolates, followed by C. glabrata (24%), C. parapsilosis (13%), and C. tropicalis (12%). Only 1.2% of C. albicans isolates were resistant to fluconazole (MIC, > or = 64 microg/ml), compared to 7% of C. glabrata isolates and 6% of C. tropicalis isolates. Only 0.9% of C. albicans isolates were resistant to itraconazole (MIC, > or = 1 micro g/ml), compared to 19.5% of C. glabrata isolates and 6% of C. tropicalis isolates. Only 4.3% of C. albicans isolates were resistant to flucytosine (MIC, > or = 32 microg/ml), compared to < 1% of C. parapsilosis and C. tropicalis isolates and no C. glabrata isolates. As determined by E-test, the MICs of amphotericin B were > or = 0.38 microg/ml for 10% of Candida isolates, > or =1 microg/ml for 1.7% of isolates, and > or = 2 microg/ml for 0.4% of isolates. Our findings highlight changes in the epidemiology of Candida BSI in the 1990s and provide a basis upon which to conduct further studies of selected high-risk subpopulations.

Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species.

Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual "prominent growth reduction" (MIC(2)) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode +/- one dilution range across all 17 laboratories. MIC(2) at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.