rDNA-RFLP identification of Candida species in immunocompromised and seriously diseased patients.

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from São Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS1-ITS4, for the amplification of ITS1 and ITS2 regions, including the gene for the 5.8 s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups.

Pinto PM ，Resende MA ，Koga-Ito CY ，Ferreira JA ，Tendler M ... -  《CANADIAN JOURNAL OF MICROBIOLOGY》

Identification of Candida species using PCR-RFLP in cancer patients in Iran.

Opportunistic infections caused by Non-Candida albicans. have been increasing. Traditional methods that are used to identify clinical isolates of Candida species are time-consuming and not appropriate for rapid, accurate and reliable identification. To identify Candida spp isolated from cancer patients using PCR-restriction enzyme. Using universal primers, ITS1 and ITS4, in this study, we could amplify ITS1-5.8S-ITS2 rDNA regions at both 80 clinical isolates and 3 standard strains. The PCR products were digested with two restriction enzymes MspI and BlnI separately. We successfully identified all isolated species using two restriction enzymes (MspI, BlnI). Candida albicans was the most common species (77.5%), followed by C. glabrata (15%), C. tropicalis (5%), C. krusei (2.5%). Although the primers and enzyme had the ability to identify C. parapsilosis, C. guilliermondii, C. dubliniensis, present isolates did not include these among identified ones. RFLP-PCR using ITSI and ITS4 primers and restriction enzyme is a rapid, easy, reliable and also applicable method in clinical laboratory for identification of medically important Candida spp.

Shokohi T ，Hashemi Soteh MB ，Saltanat Pouri Z ，Hedayati MT ，Mayahi S ... -  《-》

Molecular identification of yeast species associated with 'Hamei'--a traditional starter used for rice wine production in Manipur, India.

In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called 'Hamei' is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four 'Hamei' samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database. ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with 'Hamei' were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1-K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from different samples. Other karyotypes K2-K7 were very unique with less than 80% similarity. Finally using mitochondrial DNA restriction analysis (mt-DNA RFLP), S. cerevisiae strains belonging to the major karyotype K1 were distinctly differentiated with highly polymorphic bands by Hinf I and Hae III endonucleases.

Jeyaram K ，Singh WM ，Capece A ，Romano P ... -  《INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》

Identification of medically important Candida species by polymerase chain reaction-restriction fragment length polymorphism analysis of the rDNA ITS1 and ITS2 regions.

We aimed to identify the distribution of species in candidal strains isolated from clinical samples and restriction fragment length polymorphism (RFLP) method based on Msp I and Bln I restrictive enzyme cuts of polymerase chain reaction (PCR) products after the amplification of ITS1 and ITS2 regions of rDNA genotypically. One hundred and fifty candidal strains isolated from various clinical samples were studies/ included. Phenotypic species assessment was performed using automated VITEK-2 system and kit used with the biochemical tests. Common genomic region amplification peculiar to candidal strains was carried out using ITS1 and ITS2 primer pairs. After the amplification, PCR products were cut with Msp I and Bln I restriction enzymes for species identification. The majority of Candida isolates were isolated from urine (78.6%) while other isolates were composed of strains isolated from swab, wound, blood and other samples by 11.3%, 3.3%, 2% and 4.7%, respectively. The result of RFLP analysis carried out with Msp I and Bln I restriction enzymes showed that candidal strains were Candida albicans by 45.3%, Candida glabrata by 19.3%, Candida tropicalis by 14.6%, Candida parapsilosis by 5.3%, Candida krusei by 5.3%, Candida lusitaniae by 0.6% and other candidal strains by 9.3%. When the ability to identify Candida to species level of phenotypic and PCR-RFLP methods was assessed, a great difference was found between these two methods. It may be argued that Msp I and Bln I restriction enzyme fragments can be used in the identification of medically important Candida species. Further studies are needed to develop this kind of restriction profile to be used in the identification of candidal strains.

Bayraktar S ，Duran N ，Duran GG ，Eryilmaz N ，Aslan H ，Önlen C ，Özer B ... -  《-》

[Molecular identification of Candida albicans and Candida dubliniensis strains isolated from clinical samples].

Candida dubliniensis is a recently identified opportunistic pathogen, which has close phylogenetic relation with Candida albicans. The aim of this study was the genotypic differentiation of 55 germ tube-positive Candida strains isolated from clinical specimens (30 blood, 25 throat swab specimens). The isolates were phenotypically identified by API ID 32C system, and genotypically identified by using polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) method. Initially ITS2 region has been amplified by using universal fungal primers with PCR. After amplification and purification of approximately 340 base pair products, they were treated with species-specific restriction enzymes (MspA1 I and BsmAI for C. albicans and C. dubliniensis, respectively). As a result, API ID 32C system identified 52 (94.5%) isolates as C. albicans and 3 (5.5%) isolates as C. dubliniensis, whereas PCR-RFLP analysis yielded 50 (90.9%) C. albicans and 5 (9.1%) C. dubliniensis. It can be concluded that PCR-RFLP method may be used for the differentiation of C. dubliniensis and C. albicans isolates in clinical samples.

Toraman ZA ，Bulut Y ，Yilmaz M ，Ozdarendeli A ... -  《MIKROBIYOLOJI BULTENI》