Molecular identification and distribution profile of Candida species isolated from Iranian patients.

A total of 855 yeast strains isolated from different clinical specimens, mainly nail (42%) and vulva-vagina (25%) were identified by a set of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Genomic DNA was extracted from fresh colonies using Whatman FTA Card technology. PCR assays were performed on the complete ribosomal DNA internal transcribed spacer (rDNA-ITS) region for all isolates and species identification was carried out through their specific electrophoretic profiles after digestion with the enzyme MspI. Those isolates suspected as Candida parapsilosis group were then subjected to amplification of the secondary alcohol dehydrogenase (SADH) gene and restriction digestion with NlaIII enzyme. In total, 71.1% of the strains were obtained from females and 28.9% from males. The age group of 31-40 years consisted of the highest frequency of patients with candidiasis. Candida albicans was the predominant species (58.6%) followed by C. parapsilosis (11.0%), C. glabrata (8.3%), C. tropicalis (7.0%), C. kefyr (5.8%), C. krusei (4.4%), C. orthopsilosis (2.1%), and C. guilliermondii (0.6%). A few strains of C. lusitaniae, C. rugosa, C. intermedia, C. inconspicua, C. neoformans and S. cerevisiae were isolated. We could not identify 8 (0.9%) isolates. Candida albicans remains the most frequently species isolated from Iranian patients; however, the number of non-C. albicans Candida species looks to be increasing. The simple and reliable PCR-RFLP system used in the study has the potential to identify most clinically isolated yeasts.

Isolation and molecular characterization of clinical and environmental dematiaceous fungi and relatives from Iran.

Background and Purpose: The frequency and genetic diversity of black fungi in environmental and clinical settings have not been fully studied in Iran. This study aimed to identify and evaluate intra- and inter-species DNA sequence variation and also understand the phylogenetic relationships of melanized fungi and relatives isolated from different geographical regions of Iran. Materials and Methods: In total, 111 clinical and environmental strains of dematiaceous fungi were isolated, and their internal transcribed spacer ribosomal DNA (rDNA) regions were sequenced and analyzed. Results: An inter-species nucleotide sequence diversity rate of 1 to 464 nucleotides was observed between the species. Intra-species differences were found in the strains of Alternaria alternata, Cladosporium cladosporioides, Alternaria tenuissima, Curvularia spicifera, Aureobasidium pullulans, Curvularia hawaiiensis, Neoscytalidium dimidiatum, Alternaria terricola, Alternaria chlamydospora, Didymella glomerata, and Drechslera dematioidea by 0-59, 0-22, 0-4, 0-4, 0-3, 0-2, 0-2, 0-2, 0-2, 0-1, and 0-1 nucleotide, respectively. Conclusion: The internal transcribed spacer rDNA is useful for the discrimination of several taxa of dematiaceous fungi. However, a better understanding of the taxonomy of species of Alternaria requires a larger rDNA region or a library of other gene sequences.

Utilization of size polymorphism in ITS1 and ITS2 regions for identification of pathogenic yeast species.

PURPOSE: Despite the existence of a variety of available yeast-identification strategies, easier and more cost-effective methods are required for routine use in clinical laboratories. The internal transcribed spacer (ITS) regions of fungal rRNA genes exhibit variable sizes depending on the yeast species. In the present study, fragment size polymorphism (FSP) analysis of the ITS1 and ITS2 regions for identification of the clinically most important yeast species was assessed. METHODOLOGY: The ITS1 and ITS2 regions of 190 strains, including isolates of 31 standard strains and 159 clinical isolates, were separately PCR amplified with two primer sets: ITS1-ITS2 and ITS3-ITS4. PCR products were mixed and the two-band electrophoretic pattern of each sample was analysed according to the size of the ITS regions as predicted from the GenBank database. RESULTS: Using this method and avoiding expensive tools such as sequencing or capillary electrophoresis, we were able to differentiate nearly all pathogenic yeast species, including Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida guilliermondii, Candida kefyr, Candida lusitaniae, Candida rugosa, Cryptococcus neoformans and Saccharomyces cerevisiae. The method showed limited discriminatory power to differentiate species of the Candida parapsilosis complex. Differentiation of Candida albicans and Candida tropicalis needs already identified controls. CONCLUSION: FSP method benefits from advantages such as lower cost, higher speed and wider range of species than some commercial yeast-identification methods. We consider this method as one of the easiest molecular approaches for identifying a wide range of human pathogenic yeast species, applicable to both diagnostic and epidemiological purposes.

Comparison of CHROMagar, polymerase chain reaction-restriction fragment length polymorphism, and polymerase chain reaction-fragment size for the identification of Candida species.

BACKGROUND AND PURPOSE: The epidemiological alteration in the distribution of Candida species, as well as the significantly increasing trend of either intrinsic or acquired resistance of some of these fungi highlights the need for a reliable method for the identification of the species. Polymerase chain reaction (PCR) is one of the methods facilitating the quick and precise identification of Candida species. The aim of this study was to compare the efficiency of CHROMagar, PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-fragment size polymorphism (PCR-FSP) assays in the identification of Candida species to determine the benefits and limitations of these methods. MATERIALS AND METHODS: This study was conducted on 107 Candida strains, including 20 standard strains and 87 clinical isolates. The identification of the isolates was accomplished by using CHROMagar as a conventional method. The PCR-RFLP assay was performed on the entire internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), and the consequent enzymatic digestion was compared with PCR-FSP results in which ITS1 and ITS2 regions were separately PCR amplified. In both molecular assays, yeast identification was carried out through the specific electrophoretic profiles of the PCR products. RESULTS: According to the results, the utilization of CHROMagar resulted in the identification of 29 (33.3%) Candida isolates, while the PCR-RFLP and PCR-FSP facilitated the identification of 83 (95.4%) and 80 (91.9%) clinical isolates, respectively. The obtained concordances between CHROMagar and PCR-RFLP, between CHROMagar and PCR-FSP, as well as between PCR-RFLP and PCR-FSP were 0.23, 0.20, and 0.77, respectively. CONCLUSION: The recognition of the benefits and limitations of PCR methods allows for the selection of the most efficient technique for a fast and correct differentiation. The PCR-RFLP and PCR-FSP assays had satisfactory concordance. The PCR-FSP provides a rapid, technically simple, and cost-effective method for the identification of Candida species. Nevertheless, to accurately differentiate among the taxonomically related species, PCR-RFLP should be implemented.

A Molecular Epidemiological Survey of Clinically Important Dermatophytes in Iran Based on Specific RFLP Profiles of Beta-tubulin Gene.

BACKGROUND: Surveillance of dermatophytosis is essential to determine the likely changes in etiological trends and distribution profile of this infection. In this study beta tubulin gene (BT2), was used as the first time in a PCR-RFLP format to clarify the distribution of dermatophytosis agents in some parts of Iran. METHODS: A total of 603 clinical isolates was obtained from 500 patients in Tehran, Isfahan, Mazandaran and Guilan provinces. The isolates were identified using macro/micro-morphological criteria and electrophoretic patterns of PCR amplicons of BT2after digestion with each of the restriction enzymes FatI, HpyCH4V, MwoI and Alw21I. RESULTS: Among the patients, 59.2% were male and 40.8% female. The most prevalent clinical form was tinea pedis (42.4%), followed by tinea cruris (24.2%), tinea unguium (12.3%), tinea corporis (10.8%), tinea faciei (4%), tinea manuum (3.14%), tinea capitis (3%) and tinea barbae (0.16%), respectively. Trichophyton interdigitale ranked the first, followed by T. rubrum, Epidermophyton floccosum, Microsporum canis, T. tonsurans, T. erinacei and T. violaceum (each 0.49%) and the less frequent species were T. schoenleinii, M. gypseum and T.anamorph of Arthroderma benhamiae (each 0.16%). A case of scalp infection by E. floccosum was an exceptional event in the study. No case of T. verrucosum was found. CONCLUSION: Trichophyton species and E. floccosum are yet the predominant agents of infection in Iran, while Microsporum species are decreasing. T. interdigitale and Tinea pedis remain as the most causal agent and clinical form of dermatophytosis, respectively. It seems that BT2 can be a useful genetic marker for epidemiological survey of common pathogenic dermatophytes.