Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Candida glabrata, Candida parapsilosis and Candida rugosa from two major hospitals in Kuala Lumpur, Malaysia
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The purpose of this study is to characterize 3 non-albicans Candida spp. that were collected from two major hospitals in a densely populated area of Kuala Lumpur for their susceptibilities to azole and genetic background. Fifteen non-albicans Candida clinical isolates in two major hospitals in Kuala Lumpur area of Malaysia were collected by convenience sampling during 2007 and 2010. The genetic diversity of 15 non-albicans Candida species comprising C. glabrata (n = 5), C. parapsilosis (n = 5) and C. rugosa (n = 5) were assessed by RAPD-PCR typing. Strains were initially identified using biochemical tests and CHROMagar Candida medium. Fluconazole and voriconazole susceptibilities were determined by E-test method. Commercial kits were used for DNA extraction and amplification with RAPD primers (OPA02, OPA03 and OPA08). PCR conditions were optimized and simultaneous identification was possible by agarose gel electrophoresis of PCR products and the bands obtained were analyzed using BioNumerics Applied Maths v.6.6 software. The RAPD primers used in this study generated 100% polymorphic profile. Cluster analysis using the RAPD-PCR profile showed 12.5-25% similarity among the strains. The genetic diversity was based on the strain susceptibility towards both the azoles, site of isolation and place according to their unique banding patterns. In contrast, strains susceptible to azoles were found to be genetically similar with clonal dissimilarity. The use of OPA02, OPA03 and OPA08 primers in differentiating non-albicans Candida spp. underscores the higher resolution of RAPD-PCR as a reliable tool for strain/species level differentiation.Keywords:
Candida rugosa
Candida parapsilosis
Candida glabrata
Agarose gel electrophoresis
DNA profiling
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Random Amplification of Polymorphic DNA (RAPDPCR) is a powerful discriminative assay comparing several clinical isolates by amplifying the target DNA genome by a group of short random primers. In clinical mycology the infections with Candida species increase, especially Candida albicans and their complications become a serious problem, So this work aims to determine the genetic typing of clinical C. Albicans isolated from ICU patient's (urine, blood, vaginal secretions, and bronchoalveolar lavage) who existing in Elarreish hospital, Egypt based on RAPD-PCR assay. To investigate their genetic relationships.
Totally one hundred samples of 40 samples of urine, 30 samples of blood, 20 samples of vaginal secretions and 10 samples of Bronchoalveolar lavage were collected. About 50 different species of Candida were isolated, 28 species out of them were identified by standard-taxonomic criteria and molecular technique as Candida albicans (n =28). Candida albicans represents 14 samples of urine (35%), 7 samples of blood (23%), 3 samples of vaginal secretions (10%), and 4 samples of bronchoalveolar lavage (40%).
Genetic relevance of the different 28 Candida albicans was analyzed by (RAPD) assay using 6 different primers OPA1, OPA2, OPA3, OPA7, OPA8, and OPA9. Only three primers OPA3, OPA7, and OPA9 allow discrimination among the Candida albicans isolates. PCR bands were scored as present or absent and the genetic similarity between them was determined using Jaccard's coefficient(Sj). Dendrograms constructed using the UPGMA method showed that 12, 14, and 20 different genotypes can be identified by OPA3, OPA7, and OPA9 primers respectively since they showed a high degree of genetic heterogeneity. Data indicated that RAPD analysis is useful for providing genotypic characteristics for C. albicans in the epidemiological investigation.
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Candida krusei
Candida glabrata
Germ tube
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Objective To genotype 9 candida Clinical isolates by PCR ITS1-ITS2 and RAPD methods, and compare the results of these two methods. Methods Nine Candida species identified by YBC Test Kit included 7 C.albicans,1 C.tropicalias and 1 C.glabrates. PCR ITS1-ITS2 and RAPD methods were used to genotype 9 Candida species which were isolated from the oral cavity. Results Seven C.albicans were divided into 2 genotypes by PCR ITS1-ITS2,and 2 Candida clinical isolates were identified as 1 C.tropicalias and 1 C.glabrates. Seven C.albicans were divided into 6 genotypes by RAPD,two isolates speculated as non- C.albicans. Conclusion The results of the genotypes between the two methods were not consistent completely. The results suggest that the PCR ITS1-ITS2 method is useful to genotype candida.
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Candida species is currently recognized as the major cause of a variety of human and animal fungal infections globally. Since the world has become a global village, the knowledge of local epidemiology of Candida albicans and the evaluation of its diversity is important and will help in understanding and controlling their transmission globally. The present study was conducted to evaluate the genetic relatedness between human and animal Candida albicans isolates in Nigeria. The C. albicans isolates (n=15) were typed by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) using three primers (P4, OPA-03 and OPE-18) after identification, antifungal susceptibility and virulence profile screening using standardized methods. A high degree of separation of human and animal C. albicans isolates into distinct genotypes was clearly evident. Some degree of genotypic relatedness was seen but at varying distance of separation. The highest level of genetic similarities (84%) was observed between C. albicans recovered from cow faeces and that recovered from human high vaginal swab, suggesting that they were related but not completely identical. Moreover, no clonal relationship was found. Though this is a preliminary study, we observed different banding patterns suggesting a high degree of discriminatory power for the three primers used. It further confirms the usefulness of RAPD-PCR in molecular typing of Candida isolates. Although no clonal relationship was seen and considering the sample size in this study, the possibility of transfer cannot be ruled out when humans are exposed to animals that harbor virulent and resistant strains or when such animals are eaten.
DNA profiling
Molecular Epidemiology
Genetic Variability
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Thirteen strains of the genus Candida were isolated from catheter, urine and surgical wounds from individual patients of the Santa Casa de Misericórdia, Belo Horizonte, MG, Brazil. Ten strains were characterized as Candida albicans, two as Candida glabrata, and one as Candida parapsilosis. Isolates were evaluated for molecular relatedness by random amplified polymorphic DNA technique using 15 primers. The analysis of the genomic DNA obtained revealed a low intraspecific polymorphism and did not allow for the differentiation between strains of the same species obtained from distinct clinical sources (catheter, urine and surgical wounds). The RAPD profiles generated were able to differentiate among the species of Candida albicans, Candida parapsilosis and Candida glabrata strains isolated in this study.
Candida parapsilosis
Candida glabrata
genomic DNA
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Candida parapsilosis
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Objective To approach the spectrum of pathogen of candidal vaginitis and analyze the randomly amplified polymorphism DNA (RAPD) of isolated pathogen,and provide basis for clinical diagnosis and epidemiological surveillance of candidal vaginitis.Methods The specimens of vaginal discharge of 106 patients suspected with candidal vaginitis were collected.Pathogenic fungal were isolated and identified.10 random primers (RAPD 1-10) were used to make RAPD analysis on the isolates.Results ①72 strains of Candida were isolated,among which C.albicans was 56 strains (77.78%),non-Candida albicans Candida (NCAC) species was 16 strains (22.22%) including 6 strains of C. glabrata (8.33%),4 strains of C. tropicalis (5.56%),1 strains of C. krusei (1.39%) and 5 strains of other Candida sp. (6.94%).②RAPD analysis result showed that 2 primers (RAPD2 and RAPD5) could have better clear and stabile specific pattern bandings with evident interspecies genetic variability and intraspecies genetic resemblance.Conclusion C.albicans is the main pathogen of candidal vaginitis.RAPD2 and RAPD5 are suitable for identification and typing of C.albicans and C.tropicalis.
Candida krusei
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Candida parapsilosis
Candida krusei
Candida glabrata
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