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OBJECTIVES: Several studies have documented the potent in vitro activity of caspofungin against Candida spp. This is of special concern for Candida glabrata infections that are often resistant to many azole antifungal agents and, consequently, difficult to treat. The aim of the present study was to expand the data on the in vitro activity of caspofungin against azole-resistant isolates of C. glabrata. METHODS: A total of 50 clinical isolates of C. glabrata were tested for susceptibility to caspofungin. The isolates were cross-resistant to multiple azoles, including fluconazole, itraconazole, ketoconazole and voriconazole. Expression of the resistance-related CgCDR1 and CgCDR2 genes was evaluated by quantitative RT-PCR analysis. The MICs of caspofungin were determined by using the National Committee for Clinical Laboratory Standards M27-A2 reference method. RESULTS: C. glabrata isolates exhibited increased expression of the CDR efflux pump(s), and this was in accordance with their high-level azole resistance. In contrast, all the isolates were highly susceptible to caspofungin (100% of isolates were inhibited at

Nosocomial Candida bloodstream infections rank among infections with highest mortality rates. A retrospective cohort analysis was conducted at Catholic University Hospital to estimate the risk factors for mortality of patients with candidemia. We reviewed records for patients with a Candida bloodstream infection over a 5-year period (January 2000 through December 2004). Two hundred ninety-four patients (42.1% male; mean age ± standard deviation, 65 ± 12 years) were studied. Patients most commonly were admitted with a surgical diagnosis (162 patients [55.1%]), had a central venous catheter (213 [72.4%]), cancer (118 [40.1%]), or diabetes (58 [19.7%]). One hundred fifty-four (52.3%) patients died within 30 days. Of 294 patients, 168 (57.1%) were infected by Candida albicans , 64 (21.7%) by Candida parapsilosis , 28 (9.5%) by Candida tropicalis , and 26 (8.8%) by Candida glabrata . When fungal isolates were tested for biofilm formation capacity, biofilm production was most commonly observed for isolates of C. tropicalis (20 of 28 patients [71.4%]), followed by C. glabrata (6 of 26 [23.1%]), C. albicans (38 of 168 [22.6%]), and C. parapsilosis (14 of 64 [21.8%]). Multivariable analysis identified inadequate antifungal therapy (odds ratio [OR], 2.35; 95% confidence interval [95% CI], 1.09 to 5.10; P = 0.03), infection with overall biofilm-forming Candida species (OR, 2.33; 95% CI, 1.26 to 4.30; P = 0.007), and Acute Physiology and Chronic Health Evaluation III scores (OR, 1.03; 95% CI, 1.01 to 1.15; P < 0.001) as independent predictors of mortality. Notably, if mortality was analyzed according to the different biofilm-forming Candida species studied, only infections caused by C. albicans ( P < 0.001) and C. parapsilosis ( P = 0.003) correlated with increased mortality. Together with well-established factors, Candida biofilm production was therefore shown to be associated with greater mortality of patients with candidemia, probably by preventing complete organism eradication from the blood.

Candida colonization is an important predictor for development of invasive fungal infection (IFI). We investigated whether early detection of Candida mannan (Mn) in bronchoalveolar lavage fluid (BALF) reduces IFI among preterm infants.We conducted an observational study of infants with gestational age ofor =28 weeks, where a group undergoing Candida surveillance cultures (pre-Mn detection group) was compared with a group defined after the initiation of routine use of Candida Mn detection in BALF (Mn detection group). Antifungal treatment was started based on positive microbiologic (surveillance culture or Mn-antigen assay) results.No significant differences were detected when the groups were compared for several predictors of IFI. IFI was observed for 12 (23%) of 51 infants in the pre-Mn detection group, and for 0 (0%) of 29 infants in the Mn detection group (P = 0.003). Surveillance cultures in the pre-Mn detection group became positive at 15.0 +/- 7.2 days after birth, whereas the mean age at time of positive Mn antigen results in the Mn detection group was 4.3 +/- 3.1 days (P0.0001). Among 16 infants positive for surveillance cultures, 12 (75%) developed IFI (P0.0001).This study suggests that Candida Mn detection in BALF may be useful for earlier identification and preemptive therapy targeting preterm infants at high risk of IFI.

The Vitek 2 yeast susceptibility test was evaluated by testing 122 Candida isolates against fluconazole and voriconazole. Excellent categorical agreement with the CLSI broth microdilution method was observed (97.5% for both the azoles). Moreover, the Vitek 2 system was able to identify all but 2 of 59 investigated fluconazole-resistant organisms.

Candida surveillance cultures were obtained from 51 neonatal intensive care unit patients. Sixteen infants showing positive cultures developed subsequent Candida infection, whereas it did not occur for the 35 infants with negative cultures. Fifteen of 16 infants (

Summary Our previous investigation on Candida glabrata azole-resistant isolates identified two isolates with unaltered expression of CgCDR1/CgCDR2, but with upregulation of another ATP-binding cassette trans- porter, CgSNQ2, which is a gene highly similar to ScSNQ2 from Saccharomyces cerevisiae. One of the two isolates (BPY55) was used here to elucidate this phenomenon. Disruption of CgSNQ2 in BPY55 decreased azole resistance, whereas reintroduction of the gene in a CgSNQ2 deletion mutant fully reversed this effect. Expression of CgSNQ2 in a S. cerevisiae strain lacking PDR5 mediated not only resistance to azoles but also to 4-nitroquinoline N-oxide, which is a ScSNQ2-specific substrate. A putative gain-of-function mutation, P822L, was iden- tified in CgPDR1 from BPY55. Disruption of CgPDR1 in BPY55 conferred enhanced azole susceptibility and eliminated CgSNQ2 expression, whereas intro- duction of the mutated allele in a susceptible strain where CgPDR1 had been disrupted conferred azole resistance and CgSNQ2 upregulation, indicating that CgSNQ2 was controlled by CgPDR1. Finally, CgSNQ2 was shown to be involved in the in vivo response to fluconazole. Together, our data first demonstrate that CgSNQ2 contributes to the devel- opment of CgPDR1-dependent azole resistance in C. glabrata. The overlapping in function and regula- tion between CgSNQ2 and ScSNQ2 further highlight the relationship between S. cerevisiae and C. glabrata.

We have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene AFR1 in Cryptococcus neoformans is involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of AFR1 in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of C. neoformans , strain BPY22; an afr1 mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an AFR1- overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22- and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the AFR1 gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of C. neoformans .

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

The role of Candida glabrata in fungaemia is attributed in part to its reduced susceptibility to azoles, usually due to altered expression of genes encoding drug efflux pumps. The aims of this study were to identify risk factors for fungaemia due to C. glabrata isolates with decreased susceptibility to fluconazole and to analyse the response to antifungal treatment and the clinical outcome of C. glabrata infections in hospitalized patients.A retrospective case-case-control study was conducted at a university hospital from 2000 to 2006. Three patient groups were studied: 14 patients infected by a fluconazole-less-susceptible isolate [susceptible-dose-dependent (SDD) or resistant]; 21 patients infected by a fluconazole-susceptible (FS) isolate; and 70 uninfected controls. We measured expression of the drug efflux pump-encoding CgCDR1 and CgCDR2 genes in isolates of the two infected groups using quantitative real-time PCR.Multivariable analysis found that patients with prior fluconazole use [odds ratio (OR) 12.24, 95% confidence intervals (CIs) 1.77-84.39, P = 0.01], diabetes (OR 10.47, 95% CI 1.96-55.96, P = 0.006) and a central venous catheter (CVC) (OR 8.48, 95% CI 1.82-39.36, P = 0.006) were more likely to develop fungaemia due to a less-susceptible isolate. Previous surgery (OR 7.73, 95% CI 2.18-27.41, P = 0.002) was an independent risk factor for fungaemia due to a susceptible isolate, in addition to the presence of a CVC (OR 5.48, 95% CI 1.69-17.72, P = 0.004). The crude 30 day mortality rate was high for both case groups. Seven patients received inadequate antifungal treatment, including five infected by a fluconazole-resistant isolate but empirically treated with fluconazole; six of these seven patients died. Expression of the CgCDR genes was up-regulated in all fluconazole-resistant and, to a lesser extent, SDD isolates, but not in the FS isolates.Our data suggest that when candidaemia is suspected or detected, a more broad-spectrum antifungal drug (i.e. echinocandins or amphotericin B) should be considered as initial treatment for patients with prior azole exposure.