Assessment of the in vitro kinetic activity of caspofungin against Candida glabrata.

Echinocandins have become the drug of choice in infections caused by Candida glabrata. The objective of this study was to evaluate the in vitro activity of caspofungin alone and in combination against C. glabrata. In vitro assays demonstrated that caspofungin alone showed excellent fungicidal activity against C. glabrata, including fluconazole-resistant strains. The combination of caspofungin and azole antifungals showed potential synergy against C. glabrata. Overall, caspofungin demonstrated excellent in vitro activity, alone and in combination, against strains of C. glabrata.

Growth inhibition of Candida albicans by human vaginal epithelial cells.

Vulvovaginal candidiasis (VVC) is a common mucosal infection caused by Candida species in women of childbearing age. Although acute VVC affects a large number of women and is often precipitated by hormonal fluctuations involving high estrogen levels, recurrent VVC (RVVC) affects another 5%-10% of women without any known predisposing factors. We have recently reported that vaginal epithelial cells from nonhuman primates and mice inhibit the growth of Candida albicans in vitro, which may represent an innate host defense mechanism against C. albicans at the vaginal mucosa. In the present study, we show that vaginal epithelial cells collected from healthy women with no history of VVC also exhibit anti-Candida activity, with no differences in activity at various stages of the menstrual cycle. Women diagnosed with RVVC, on the other hand, have reduced epithelial cell anti-Candida activity. These results are further evidence that vaginal epithelial cells provide an innate host resistance mechanism against Candida and that reduced activity may contribute to RVVC.

In vitro susceptibilities of Candida and Aspergillus species to Melaleuca alternafolia (tea tree) oil.

Candida species are an important cause of opportunistic infection in the oral cavity of immunocompromised patients, especially HIV infected patients. Melaleuca oil obtained commercially was investigated since it is known to have broad antifungal properties. The in-vitro susceptibilities of Aspergillus and susceptible and resistant Candida species were performed utilizing serial dilutions in microtiter plates with Sabouraud dextrose agar and the commercial preparation of Melaleuca. As a comparator, in vitro susceptibilities to amphotericin B and fluconazole were also determined using the broth microdilution technique. The results demonstrate that Melaleuca inhibited the Candida species. However, the growth of Aspergillus was not inhibited at the concentrations tested. Thus, preparations containing Melaleuca alternafolia may be a useful alternative for superficial candidal infections. In fact, it may be a useful alternative regimen for advanced HIV-positive patients with oropharyngeal candidiasis refractory to fluconazole. However, controlled clinical studies to evaluate its efficacy are still needed.

Evaluation of DNA-based typing procedures for strain categorization of Candida spp.

DNA-based procedures have replaced earlier epidemiologic methodologies that relied on nonreproducible and insensitive measurements of phenotypic characteristics to identify a specific strain as the source of infection. The reliability (interlaboratory percent agreement for strain delineation) and sensitivity (recognition of subtle strain-to-strain variation) of similar DNA-based typing systems by different laboratories were evaluated. Ten isolates (five epidemiologic-related and five unrelated strains each) of Candida albicans, C. lusitaniae, C. parapsilosis, C. tropicalis, and Candida (Torulopsis) glabrata were characterized in a blinded fashion by three laboratories. All 50 isolates were subtyped in each laboratory by electrophoretic karyotyping (EK) analysis using contour-clamped homogenous electric field (CHEF) electrophoresis protocols. In addition, two laboratories also performed restriction endonuclease analysis of genomic DNA (REAG) using the restriction endonucleases SfiI and BssHII followed by CHEF electrophoresis separation of resulting fragments. DNA strain identification of the 50 isolates by the three different laboratories using similar CHEF methodologies demonstrated the following species-dependent, interlaboratory reproducibility: C. tropicalis (82%), C. parapsilosis (83%), C. albicans (90%), C. lusitaniae (93%), and C. glabrata (100%). In addition, agreement was higher by the CHEF method (83 to 100%), when compared with the strain types identified by the REAG (60 to 100%) method. Five to seven strains of each Candida species evaluated were detected by the different methodologies used for this study. This study indicates that these procedures are relatively discriminatory and reliable tools to study strain-to-strain variations in epidemiologic evaluations of these yeasts.

Stable phenotypic resistance of Candida species to amphotericin B conferred by preexposure to subinhibitory levels of azoles.

The fungicidal activity of amphotericin B (AmB) was quantitated for several Candida species. Candida albicans and C. tropicalis were consistently susceptible to AmB, with less than 1% survivors after 6 h of exposure to AmB. C. parapsilosis and variants of C. lusitaniae and C. guilliermondii were the most resistant, demonstrating 50 to 90% survivors in this time period and as high as 1% survival after a 24-h exposure time. All Candida species were killed (<1% survivors) after 24 h of exposure to AmB. In contrast, overnight exposure to either fluconazole or itraconazole resulted in pronounced increases in resistance to subsequent exposures to AmB. Most dramatically, C. albicans was able to grow in AmB cultures after azole preexposure. Several other Candida species did not grow in AmB but showed little or no reduction in viability after up to 24 h in AmB. Depending on the growth conditions, Candida cells preexposed to azoles may retain AmB resistance for days after the azoles have been removed. If this in vitro antagonism applies to the clinical setting, treatment of patients with certain antifungal combinations may not be beneficial. The ability of some Candida isolates to survive transient exposures to AmB was not reflected in the in vitro susceptibility changes as measured by standard MIC assays. This finding should be considered in studies attempting to correlate patient outcome with in vitro susceptibilities of clinical fungal isolates. Patients who fail to respond to AmB may be infected with isolates that are classified as susceptible by standard in vitro assays but that may be resistant to transient antifungal exposures which may be more relevant in the clinical setting.

Identification of Candida species by randomly amplified polymorphic DNA fingerprinting of colony lysates.

We have characterized a method that produces simple yet diagnostic fingerprints that are unique to isolates of Candida species. DNA from individual colonies can be amplified from crude single-colony lysates. Randomly amplified polymorphic DNA (RAPD) fingerprints generated from a single primer correctly identified the species of most (>98%) of the isolates identified with CHROMagar Candida plates as non-Candida albicans Candida species. RAPD fingerprints were much more informative than the plates, since they distinguished between all tested species and required less time. Most (91%) of these identifications agreed with those assigned by API 20C tests. In almost every incident of species identity mismatch, electrophoretic karyotyping showed that the RAPD fingerprint was correct. This underscores the improved objectivity and reliability of this method over those of conventional diagnostic tools. The identities of approximately 30% of C. albicans isolates identified in clinical laboratories by positive germ tube tests are not verified by either testing on CHROMagar Candida plates or RAPD fingerprinting. Data suggest that clinical isolates conventionally identified as C. albicans in clinical settings are heterogeneous, consisting of both misidentified and atypical yeasts. RAPD fingerprints obtained from primary culture plate colonies allows for rapid, highly accurate determinations of Candida species, hence permitting earlier selection of appropriate antifungal agents in the clinical setting.

In vitro activity of a new pneumocandin antifungal, L-743,872, against azole-susceptible and -resistant Candida species.

The in vitro activity of a new pneumocandin, L-743,872, was evaluated with 108 strains of Candida and compared with the activities of various antifungals. L-743,872 demonstrated the best activity against azole-susceptible and -resistant strains of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. kefyr and less activity against C. krusei, C. lusitaniae, and C. guilliermondii.

Use of electrophoretic karyotyping in the evaluation of Candida infections in a neonatal intensive-care unit.

OBJECTIVE: To evaluate a possible common-source outbreak of Candida infections in the neonatal intensive-care unit. Systemic Candida infections increased from 6 to 11 cases (0.71 to 1.34 per 1,000 patient-days). In addition, Candida parapsilosis infections increased from 1 in 1992 to 10 in 1993. DESIGN AND SETTING: Tertiary-care, teaching, pediatric institution with a 40-bed neonatal intensive-care unit (NICU). Clinical characteristics, associated conditions, and antimicrobial therapy were obtained from the medical records of all NICU patients with positive blood cultures for Candida during 1992 and 1993. Nineteen Candida isolates from 15 infants were studied retrospectively using contour-clamped homogeneous electric-field (CHEF) electrophoresis. RESULTS: CHEF revealed eight karyotypes of C parapsilosis. Five isolates recovered from four patients shared one karyotype. The remaining isolates from seven infants all had distinctly different karyotypes. CONCLUSIONS: The evidence was insufficient to implicate a single source of infection, even though four patients in the same unit had identical strain types. However, identical strains of C parapsilosis were associated geographically, suggesting that nosocomial acquisition of C parapsilosis through indirect patient contact in the NICU was possible. The CHEF technique yields unique patterns that may be used to delineate clinical isolates and to study the molecular epidemiology of candidal infections.

Invasive Candida guilliermondii infection: in vitro susceptibility studies and molecular analysis.

Candida guilliermondii is rarely isolated from humans. We describe a case of disseminated C. guilliermondii with associated purulent pericarditis, despite high-dose amphotericin B (AmB), in a 19-year-old female with aplastic anemia who underwent BMT. In vitro susceptibility studies of the 13 clinical isolates, two control strains and one environmental isolate revealed a minimum inhibitory concentration (MIC) range of (0.19-1.56 micrograms/ml) for AmB and (1.25-10 micrograms/ml) for fluconazole. Pulsed-field gradient gel electrophoresis was performed to evaluate possible similarities between strains. This case is significant for several reasons, the high degree and prolonged duration of fungemia despite high-dose AmB and concomitant flucytosine, the change in in vitro susceptibility during therapy, the initial misidentification of the yeast isolate, and the invasiveness of the organism. The poor response to therapy may have been due to the severe and sustained neutropenia and the high MICs of C. guilliermondii to AmB.

Saccharomyces cerevisiae vaginitis: transmission from yeast used in baking.

OBJECTIVE: To determine whether vaginitis due to Saccharomyces cerevisiae can be caused by exposure to exogenous sources of baker's yeast. METHODS: Eight women with S cerevisiae vaginitis were identified from a cohort of women referred for the evaluation of chronic vaginal symptoms. In those with high-level exposure to exogenous sources of S cerevisiae, isolates from the vagina and those sources were sent in a blinded fashion for contour-clamped homogeneous electric-field electrophoresis. RESULTS: Four women from a cohort of approximately 750 referred patients had high-level exposures to S cerevisiae. In one of these patients, electrophoresis analysis revealed similarities between the strains isolated from her vagina, her husband's fingers, and the yeast he used in his pizza shop. CONCLUSION: Saccharomyces cerevisiae vaginitis can be the result of the inoculation of this yeast from exogenous sources.