Utility of Fluoroplate Candida for the rapid identification of Candida albicans

BACKGROUND: Candida albicans infections are frequent in immunocompromised patients and a prompt diagnosis could favor an early and proper antifungal treatment. The rapid identification of clinical yeast isolates facilitate this diagnosis. METHODS: The utility of Fluoroplate Candida ready-to-use plates for Candida albicans rapid identification was evaluated with 653 clinical isolates from 23 yeast species, including 307 C. albicans plated onto Fluoroplate Candida agar (Merck, Germany). Rapid identification of C. albicans was based on the hydrolysis of 4-methylumbelliferyl-N-acetyl-beta-D-galactosaminide by the galactosaminidase activity of C. albicans producing white fluorescent colonies under ultraviolet light. Identification on Fluoroplate Candida was confirmed by germ tube, chlamydoconidia formation and API-ATB ID 32C assays. RESULTS: Three hundred and five of 306 isolates showing fluorescent colonies were C. albicans and one was Candida glabrata (false positive). The rest of the isolates showed colonies without fluorescence and with the exception of two false negatives, these isolates were identified as non-C. albicans by other methods. CONCLUSIONS: Fluoroplate Candida allows a rapid and excellent identification of C. albicans showing a sensitivity and specificity of 99.3 and 99.7%, respectively.     

Rapid identification of Candida species with species-specific DNA probes

Rapid identification of Candida species has become more important because of an increase in infections caused by species other than Candida albicans, including species innately resistant to azole antifungal drugs. We previously developed a PCR assay with an enzyme immunoassay (EIA) format to detect amplicons from the five most common Candida species by using universal fungal primers and species-specific probes directed to the ITS2 region of the gene for rRNA. We designed probes to detect seven additional Candida species (C. guilliermondii, C. kefyr, C. lambica, C. lusitaniae, C. pelliculosa, C. rugosa, and C. zeylanoides) included in the API 20C sugar assimilation panel, five probes for species not identified by API 20C (C. haemulonii, C. norvegica, C. norvegensis, C. utilis, and C. viswanathii), and a probe for the newly described species C. dubliniensis, creating a panel of 18 Candida species probes. The PCR-EIA correctly identified multiple strains of each species tested, including five identified as C. albicans by the currently available API 20C database but determined to be C. dubliniensis by genotypic and nonroutine phenotypic characteristics. Species identification time was reduced from a mean of 3.5 days by conventional identification methods to 7 h by the PCR-EIA. This method is simple, rapid, and feasible for identifying Candida species in clinical laboratories that utilize molecular identification techniques and provides a novel method to differentiate the new species, C. dubliniensis, from C. albicans.     

Unfortunate in vitro selection of resistant Candida albicans with severe clinical consequences

Cutaneous angiosarcoma is a rare aggressive tumour of capillary and lymphatic endothelial cell origin. It presents as multiple purple and red papules and nodules on the head and neck or the extremities. We report an 86-year-old woman with angiosarcoma arising on her chronically lymphoedematous right leg. The lymphoedema, secondary to chronic immobility, had developed gradually over 40 years. No other family members had lymphoedema. The patient presented with a plaque of friable tumour tissue on the lower right leg and dorsum of the foot, and satellite lesions on the knee and groin which initially appeared to be petechial haemorrhages. The satellite lesions in the groin grew into tumour nodules. There was no evidence of a preceding malignancy, nor any operative intervention to the affected limb or abdomen. Histological examination of all tumour specimens revealed moderately to poorly differentiated angiosarcoma. She died within 5 months of the first appearance of the skin nodules.     

Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR

Fluconazole-resistant Candida albicans, a cause of recurrent oropharyngeal candidiasis in patients with human immunodeficiency virus infection, has recently emerged as a cause of candidiasis in patients receiving cancer chemotherapy and marrow transplantation (MT). In this study, we performed detailed molecular analyses of a series of C. albicans isolates from an MT patient who developed disseminated candidiasis caused by an azole-resistant strain 2 weeks after initiation of fluconazole prophylaxis (K. A. Marr, T. C. White, J. A. H. vanBurik, and R. A. Bowden, Clin. Infect. Dis. 25:908-910, 1997). DNA sequence analysis of the gene (ERG11) for the azole target enzyme, lanosterol demethylase, revealed no difference between sensitive and resistant isolates. A sterol biosynthesis assay revealed no difference in sterol intermediates between the sensitive and resistant isolates. Northern blotting, performed to quantify mRNA levels of genes encoding enzymes in the ergosterol biosynthesis pathway (ERG7, ERG9, and ERG11) and genes encoding efflux pumps (MDR1, ABC1, YCF, and CDR), revealed that azole resistance in this series is associated with increased mRNA levels for members of the ATP binding cassette (ABC) transporter superfamily, CDR genes. Serial growth of resistant isolates in azole-free media resulted in an increased susceptibility to azole drugs and corresponding decreased mRNA levels for the CDR genes. These results suggest that C. albicans can become transiently resistant to azole drugs rapidly after exposure to fluconazole, in association with increased expression of ABC transporter efflux pumps.     

Comparison of the new API Candida system to the ID 32C system for identification of clinically important yeast species

API Candida was evaluated in comparison with the ID 32C system for the identification of 619 yeast isolates. The sensitivity of API Candida for the identification of the 15 species it claims to identify with and without additional tests was 97.4% (593 of 609) and 75.2% (458 of 609), respectively. The API Candida system is easy to use and rapid (result in 18 to 24 h).     

Gas-chromatographic identification of drugs in gastric aspirate samples: a rapid screening procedure for emergency toxicology

Gastric aspirate has not been used frequently in the identification of ingested drugs. Because there is only qualitative significance in such a sample, a new method is described that facilitates identification. A 1-ml gastric aspirate sample is extracted with 100 mul of chloroform and analyzed directly by gas chromatography. Seventeen drugs commonly involved in overdose cases are included in this 25-min procedure. The gastric drug screen has been applied to more than 500 patients, and results are described.     

Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains

From each of two AIDS patients with oropharyngeal candidiasis, five Candida albicans isolates from recurrent episodes of infection which became gradually resistant against fluconazole during antimycotic treatment were analyzed for molecular changes responsible for drug resistance. In both patients, a single C. albicans strain was responsible for the recurrent infections, but the CARE-2 fingerprint pattern of the isolates exhibited minor genetic alterations, indicating that microevolution of the strains took place during fluconazole therapy. In the isolates from patient 1, enhanced mRNA levels of the MDR1 gene, encoding a multiple drug resistance protein from the superfamily of major facilitators, and constitutive high expression of the ERG11 gene, coding for the drug target enzyme sterol 14alpha-demethylase, correlated with a stepwise development of fluconazole resistance. The resistant strains exhibited reduced accumulation of fluconazole and, for the last in the series, a slight increase in drug needed to inhibit sterol 14alpha-demethylation in vitro. In the isolates from patient 2, increased MDR1 mRNA levels and the change from heterozygosity to homozygosity for a mutant form of the ERG11 gene correlated with continuously decreased drug susceptibility. In this series, reduced drug accumulation and increased resistance in the target enzyme activity, sterol 14alpha-demethylase, were observed. These results demonstrate that different molecular mechanisms contribute to a gradual development of fluconazole resistance in C. albicans.     

A disc plate assay for characterization of the effect of interaction between polyenes and azoles on growth of Candida albicans CA12

A simple, reproducible and efficient assay was described for visual demonstration that triazole fluconazole interfered with the anti-Candida albicans activity of polyene amphotericin B. The assay also indicated the existence of more than one antifungal mechanism involving imidazole ketoconazole.     

Specific immunohistochemical identification of Candida albicans in paraffin-embedded tissue with a new monoclonal antibody (1B12)

BACKGROUND: Substantial progress has been made in the treatment of acute myeloid leukemia in the last two decades. We wanted to evaluate the outcome of intensive chemotherapy and the influence of recent therapy changes in underprivileged patients treated in a large urban public university hospital. METHODS: The records of all patients treated for acute myeloid leukemia from 1980 to 1993 were analyzed. RESULTS: 109 patients were identified; 41 did not receive any treatment for the leukemia because of infectious and/or hemorrhagic complications of advanced disease. Median survival in this group was 4 days. The other 68 patients received one of two induction protocols: TAD from 1980 to 1985 (n = 23) and ara-C plus daunorubicin from 1985 to 1992 (n = 45). The complete remission rate was 56%, disease-free survival 24% and overall survival 15% at 13 years. Overall survival was better for patients treated with ara-C plus daunorubicin than with TAD (19% versus 8%, p = 0.01). This is attributed to a reduction in infection mortality after ceftazidime and amikacin replaced cephalotin, carbenicillin and amikacin as the antibiotic regimen. CONCLUSIONS: The most effective intervention in our population would probably be an improvement in the primary health care system, so that earlier diagnosis could allow the treatment of a larger fraction of patients.     

Effects of tetracycline hydrochloride and chlorhexidine gluconate on Candida albicans. An in vitro study

This study examined the effects of tetracycline hydrochloride (TCN) and chlorhexidine gluconate (CHX) on the growth and viability of Candida albicans. Subcultures of Candida albicans on Sabouraud's agar, were divided into 5 treatment groups: group 1, untreated control; group 2, 0.12% CHX; group 3, 3.0 mg/ml TCN adjusted to pH 4.5; groups 4 and 5, sodium azide free Tris buffer adjusted to pH 4.5 and pH 7.4, respectively. All groups were incubated for 10 days, and sampled and subcultured daily to determine the viability of each group. Additional samples from group 2 (day 4), group 4 (day 7) and all groups at day 10 were selected for SEM and TEM examination. Visual, SEM and TEM results showed that for groups 1, 3, 4, and 5 there was a heavy and constant uniform growth of Candida albicans throughout the period of the study. However, group 2 (CHX), showed decreasing viability and attachment from day 3 to day 10, with SEM and TEM revealing decreased blastospores and profound changes in the ultrastructural morphology, indicating inhibition of normal cell growth and replication. These results show that TCN even when used at high concentrations, in vitro, will allow uninhibited growth of Candida albicans whereas CHX inhibits cell growth and replication.     

A novel technique for assessment of adherence of Candida albicans to solid surfaces

A novel approach for the assessment of adherence of Candida albicans to translucent acrylic material is described. The method uses the inverted microscope to visualise yeast adhering to acrylic surfaces while the test material remains immersed in buffer. Adherent cells were not subjected to surface tension forces that can occur during drying processes, so that an even distribution of yeast with no aggregation occurred. The process of counting attached yeast was subsequently performed without difficulty. From the 11 C albicans isolates examined, two groups were evident with respect to acrylic adherence: one group of four isolates with an adherence level of 400 yeast/mm2 acrylic, and one group of seven isolates with adherence levels of 1000 yeast/mm2 acrylic.     

PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples

A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.     

IQ-memory discrepancies in normal and clinical samples.

The potential utility of IQ—Memory Index discrepancy scores derived from the Wechsler Adult Intelligence Scale—Revised (WAIS—R) and the Wechsler Memory Scale—Revised (WMS—R; D. Wechsler, 1987) was examined in a clinical sample, whose scores were then compared to those of subjects from the WMS—R standardization sample. The clinical sample included patients with diagnoses associated with memory deficits. Discrepancy scores between Full-Scale IQ and the Delayed Memory Index differentiated the groups, but material-specific discrepancies between IQ scores and immediate recall memory scores did not. The largest mean discrepancy and the greatest prevalence of scores beyond a criterion score of 15 were found in patients with presumed Alzheimer's disease. Issues related to limitations in the application of such discrepancy scores are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Inhibition of 2,3-oxidosqualene-lanosterol cyclase in Candida albicans by pyridinium ion-based inhibitors

The N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpyridinium and N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpicolinium cations were evaluated for their ability to inhibit 2,3-oxidosqualene-lanosterol cyclase activity in Candida albicans. Both compounds inhibited fungal growth, were fungicidal, and resulted in the accumulation of squalene epoxide concurrent with a decrease in ergosterol, monomethyl sterols, and lanosterol, as was expected for the specific inhibition of 2,3-oxidosqualene-lanosterol cyclase activity. These compounds are electron-poor aromatic mimics of a monocyclized transition state or high-energy intermediate formed from oxidosqualene, which may explain their selective action.     

Diagnostic contribution of abnormal delayed-type hypersensitivity to Candida albicans. Characterization test by activation of cells sensitized to successive dilutions of Candida

By measuring the activation of different cell models (lymphocytes and lymphocytic subsets) in the presence of Candida albicans with flow cytometry reading, it is possible to show that successive dilutions of Candida albicans can lead to lymphocyte activation in abnormally-sensitized subjects. In a first trial, 10 subjects were tested in duplicate. The decrease of activity of the dilutions does not appear to be regular in relation to the progression of the dilutions. The activity of the dilutions wanes relatively rapidly with the first dilutions, then recurs later very distinctly, at the 6th dilution, then ebbs, then reappears in similar manner at the 9th, the 14th, and finally, the 19th dilution. Cell reactivity appears to differ depending on the subject. It can be represented through the calculated slope of the regression line, for each series of data. It therefore appears feasible to determine a threshold of reactivity and a scale of sensitivity, to make it possible to specify the degree of abnormal reactivity existing at a given time for a given subject. The constancy of the activity of the different dilutions tested, on 10 cultures of a single cell suspension, is especially well demonstrated in the second trial, showing unusually small standard deviations. Thus, the question arises as to the exact nature of the observed phenomenon and of its analysis from a physical-chemical point of view, with regard to the pharmacological effect of successive dilutions of Candida albicans.     

Effects of the combination of ketoconazole and calmodulin inhibitors against Candida albicans in vitro. Short communication

The susceptibility of 66 strains of Candida albicans from patients was tested against ketoconazole (Ktz), chlorpromazine (Chl) levomepromazine (Lev), haloperidol (Hal) and the combination of Ktz with these calmodulin inhibitors, using Sabouraud's broth. The minimal inhibitory concentrations (MICs) for 66 strains of C. albicans were as follows: Chl 192 +/- 11.4 micrograms/ml, Lev 306 +/- 16.4 micrograms/ml, Hal 464 +/- 13.8 micrograms/ml compared with Ktz 34.46 +/- 3.9 micrograms/ml. The combination of Ktz and calmodulin inhibitors in various ratios (1:1,1:2,2:1) was found to exert synergistic effect and the mean values of the combinations were: Ktz+Chl 3.45 +/- 0.35, 3.78 + 0.36, 5.58 + 0.4 micrograms/ml; Ktz+Lev 10.8 +/- 2.19, 9.7 +/- 2.23, 10.5 + 2 micrograms/ml; Ktz+Hal 6.4 +/- 1.7, 6.8 +/- 1.6, 7.28 +/- 1.5 micrograms/ml. These results were significantly different (p < 0.001) from those of ketoconazole. These findings indicate that some calmodulin inhibitors increase the antifungal activity of Ktz against C. albicans in vitro.     

Comparison of the rapid yeast plus panel with the API20C yeast system for identification of clinically significant isolates of Candida species

The RapID Yeast Plus system (Innovative Diagnostic Systems, Norcross, Ga.) is a qualitative micromethod employing conventional tests and single-substrate chromogenic tests and having a 4-h incubation period. This system was compared with the API20C (bioMerieux Vitek, Hazelwood, Mo.) system, a 24- to 72-h carbohydrate assimilation method. One hundred thirty-three clinical yeast isolates, including 57 of Candida albicans, 26 of Candida tropicalis, 23 of Candida glabrata, and 27 of other yeasts, were tested by both methods. When discrepancies occurred, isolates were further tested by the Automated Yeast Biochemical Card (bioMerieux Vitek). Germ tube production and microscopic morphology were used as needed to definitively identify yeast isolates. The RapID Yeast Plus system correctly identified 125 yeast isolates, with an overall accuracy of 94% (125 of 133). Excellent correlation was found in the recognition of the three yeasts most commonly isolated from human sources. The test was 99% (105 of 106 isolates) accurate with C. albicans, C. tropicalis, and C. glabrata. The RapID Yeast Plus system compares favorably with the API20C system and provides a simple, accurate alternative to conventional assimilation methods for the rapid identification of the most commonly encountered isolates of Candida species.     

Comparative evaluation of macrodilution and chromogenic agar screening for determining fluconazole susceptibility of Candida albicans

A simple screening method for fluconazole susceptibility using CHROMagar Candida with fluconazole was compared with the National Committee for Clinical Laboratory Standards (NCCLS) macrobroth method. In this agar dilution method, susceptible Candida albicans colonies are smaller on medium with fluconazole than on fluconazole-free medium. Yeasts with decreased susceptibility have normal-sized colonies on medium containing fluconazole. On agar with 16 micrograms of fluconazole per ml, 32 of 34 strains with NCCLS MICs of > or = 16 micrograms/ml were correctly predicted, as were 66 of 68 with MICs of < 16, an agreement of 96%. On agar with 8 micrograms of fluconazole per ml, 38 of 41 isolates with MICs of > or = 8 were correctly predicted, as were 59 of 61 isolates with MICs of < 8, an agreement of 95%. This agar dilution methods appears to highly correlate with NCCLS macrobroth methods for detection of C. albicans and may be an effective screen for fluconazole susceptibility.