Dataset Information

Multilocus microsatellite markers for molecular typing of Candida tropicalis isolates.

ABSTRACT: BACKGROUND: Candida tropicalis is considered to be the leading pathogen causing nosocomial fungemia and hepatosplenic fungal infections in patients with cancer, particularly those with leukemia. Microsatellite-based typing methods using sets of genetic markers have been developed and reported for population structure analysis of C. albicans, C. glabrata, and C. parapsilosis, but no studies have been published for genetic analysis of C. tropicalis. The objective of this study was to develop new microsatellite loci that have the ability to distinguish among C. tropicalis isolates. RESULTS: DNA sequences containing over 10 bi- or tri-nucleotide repeats were selected from the C. tropicalis genome database. Thirty PCR primers sets specific for the microsatellite loci were designed and tested using eight clinically independent isolates. According to the amplification efficiency, specificity, and observed polymorphisms, eight markers were selected for further population structure analysis and molecular typing. Sixty-five independent C. tropicalis isolates were genotyped using these 8 markers. Based on these analyses, six microsatellite loci were confirmed, although two loci were found to be with unstable flanking areas. The six polymorphic loci displayed 4-22 alleles and 7-27 genotypes. The discriminatory power of the six loci ranged from 0.70 to 0.95. Genotyping results obtained by microsatellite analysis were compared to PCR-fingerprinting and multi-locus sequence typing (MLST). The comparisons showed that microsatellite analysis and MLST had the similar discriminatory power for C. tropicalis, which were more powerful than PCR-fingerprinting. CONCLUSIONS: This is the first attempt to develop new microsatellite loci for C. tropicalis. These newly developed markers will be a valuable resource for the differentiation of C. tropicalis isolates. More C. tropicalis isolates will need to be sequenced and analyzed in order to fully show the potential of these newly developed microsatellite markers.

SUBMITTER: Wu Y

PROVIDER: S-EPMC4247128 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Multilocus microsatellite markers for molecular typing of Candida tropicalis isolates.

Wu Yuan Y Zhou Hai-jian HJ Che Jie J Li Wen-ge WG Bian Fu-ning FN Yu Shuan-bao SB Zhang Li-juan LJ Lu Jinxing J

BMC microbiology 20141120

<h4>Background</h4>Candida tropicalis is considered to be the leading pathogen causing nosocomial fungemia and hepatosplenic fungal infections in patients with cancer, particularly those with leukemia. Microsatellite-based typing methods using sets of genetic markers have been developed and reported for population structure analysis of C. albicans, C. glabrata, and C. parapsilosis, but no studies have been published for genetic analysis of C. tropicalis. The objective of this study was to develo ...[more]

PMID: 25410579

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Project description:Leishmania infantum is the main etiological agent of both visceral and cutaneous clinical forms of leishmaniasis in the Mediterranean area. Leishmania/HIV coinfection in this area is characterized by a chronic course and frequent recurrences of clinical episodes. The present study using Multilocus Microsatellite Typing (MLMT) analysis, a highly discriminative tool, aimed to genetically characterize L. infantum isolates taken from monitored Leishmania/HIV coinfected patients presenting successive clinical episodes.In this study, by the analysis of 20 microsatellite loci, we studied the MLMT profiles of 25?L. infantum isolates from 8 Leishmania/HIV coinfected patients who had experienced several clinical episodes. Two to seven isolates per patient were taken before and after treatment, during clinical and non-clinical episodes, with time intervals of 6 days to 29 months. Genetic diversity, clustering and phenetic analyses were performed.MLMT enabled us to study the genetic characteristics of the 25?L. infantum isolates, differentiating 18 genotypes, corresponding to a genotypic diversity of 0.72. Fifteen genotypes were unique in the total sample set and only 3 were repeated, 2 of which were detected in different patients. Both clustering and phylogenetic analyses provided insights into the genetic links between the isolates; in five patients isolates showed clear genetic links: either the genotype was exactly the same or only slightly different. In contrast, the isolates of the other three patients were dispersed in different clusters and some could be the result of mixing between populations.Our data indicated a great MLMT variability between isolates from coinfected patients and no predominant genotype was observed. Despite this, almost all clinical episodes could be interpreted as a relapse rather than a reinfection. The results showed that diverse factors like an intrapatient evolution over time or culture bias could influence the parasite population detected in the patient, making it difficult to differentiate between relapse and reinfection.

Project description:Candida parapsilosis, a pathogenic yeast, is composed of three newly designated genomic species that are physiologically and morphologically indistinguishable. Nosocomial infections caused by group I C. parapsilosis are often associated with the breakdown of infection control practices and the contamination of medical devices, solutions, and indwelling catheters. Due to the low levels of nucleotide sequence variation that are observed, an investigation of the size polymorphisms in loci harboring microsatellite repeat sequences was applied for the typing of C. parapsilosis group I isolates. PCR primer sets that flank the microsatellite repeats for seven loci were designed. Following amplification by PCR, the size of each amplification product was determined automatically by capillary electrophoresis. A total of 42 C. parapsilosis group I isolates were typed by microsatellite analysis, and their profiles were compared to the hybridization profiles obtained by use of the Cp3-13 DNA probe. A high degree of discrimination (discriminatory power = 0.971) was observed by microsatellite analysis. The number of different alleles per locus ranged from 14 for locus B to 5 for locus C. Microsatellite analysis detected 30 different microsatellite genotypes, with 24 genotypes represented by a single isolate. Comparison of the genotypes obtained by microsatellite analysis and those obtained by analysis of the Cp3-13 hybridization profiles showed that they were similar, and these methods were able to identify related and unrelated isolates. Some discrepancies were observed between the methods and may be due to higher mutation rates and/or homoplasy by microsatellite markers. Identical results were observed between microsatellite analysis and Cp3-13 DNA hybridization profile analysis for C. parapsilosis isolates obtained from two patients, demonstrating the reproducibilities of the methods in vivo. Identical microsatellite profiles were observed for isolates displaying different phenotypic switching morphologies. Indistinguishable Cp3-13 DNA hybridization profiles were observed for six epidemiologically related isolates; however, only three of six primary isolates had identical microsatellite profiles. Size variation at a single locus was observed for three of six isolates obtained either after the outbreak period or from a different body site, suggesting the potential of the method to detect microevolutionary events. Interestingly, for most loci a single allele per strain was observed; in contrast, two alleles per locus were observed for some strains, and consistent with the findings for natural isolates, some isolates may be aneuploid. Due to the potential for high throughput, reproducibility, and discrimination, microsatellite analysis may provide a robust and efficient method for the genotyping of large numbers of C. parapsilosis group I isolates.

Dataset Information

Multilocus microsatellite markers for molecular typing of Candida tropicalis isolates.

Publications

Multilocus microsatellite markers for molecular typing of Candida tropicalis isolates.

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