Microsatellite Marker Analysis as a Typing System for Candida glabrata

Abstract

Candida glabrata is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers, RPM2, MTI, and ERG3, in order to obtain a rapid genotyping method for C. glabrata. One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other Candida species and 138 independent C. glabrata clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since C. glabrata is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the RPM2, MTI, and ERG3 loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for C. glabrata. A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning C. glabrata. However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.Epidemiological surveillance of candidemia shows that although Candida albicans remains the main species, Candida glabrata is the second leading species recovered from blood cultures in Europe and in the United States (20, 28), especially in intensive care units (9). The same phenomenon is observed for recurrent vaginitis (25). Concern arises about this increase in the incidence of C. glabrata infections because of its frequent decreased sensitivity to azoles compared to C. albicans (14,22) and the high mortality rate of bloodstream infections (28,29).Investigations for C. glabrata have not been as extensive as for C. albicans. The epidemiology is thought to be similar to C. albicans (10). However, few tools are available to study the source of contamination, cross-contamination of patients at risk, and the biology of this yeast, which has, unlike C. albicans, a haploid genome (10). Studies of population structure have begun to emerge using enzymatic and randomly amplified polymorphic DNA typing (6) or sequencing of the cytochrome c oxidase subunit 2 gene (24). Recently, a technique based on the sequencing of six variable-locus genes, or multilocus sequence typing (MLST), has been proposed as a powerful typing method (7). Microsatellites represent another class of genetic markers, defined as short tandem repeats of two to six nucleotides, known to be highly polymorphic (30). Primarily developed for human genetics, they are used for fungi (15) and more specifically for pathogenic fungi such as C. albicans (3-5, 19, 23, 26) and Aspergillus fumigatus (1, 2, 16, 17).We descri...

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Rapid Typing of Candida glabrata

“…By combining four of them, a discriminatory power (D) of 0.902, higher than those previously published, was achieved (12). As D values higher than 0.90 are needed for the accurate typing of epidemiologically related isolates (12,16), microsatellite-based MLVA seems to be an attractive method for large epidemiological surveys and appears to be as discriminant as MLST (D ϭ 0.898 computed from data obtained by Dodgson et al [9]). Because of their specificity and typeability, microsatellite markers can also be (1,5,18).…”

“…However, the distribution of alleles was irregular and several alleles were prominent, since three multilocus genotypes represented 52% of the isolates studied. Other studies using microsatellite or MLST data have highlighted the fact that distinct genetic clades of C. glabrata prevail in different geographical regions (8,9,12). Our unrelated isolates were collected from patients from a restricted geographical area, which could partially explain the unequal distribution of genotypes in the population studied.…”

“…Microsatellite polymorphism analysis has been widely used for typing fungi (3,4,11,15,18,25), and this could be an alternative, easy-to-perform, reproducible method suitable for large-scale studies of C. glabrata epidemiology. Recently, Foulet et al described three polymorphic microsatellite markers to investigate the delineation of clinical C. glabrata isolates (12). The discriminatory power of this method was good but not optimal.…”

Clonal Population of Flucytosine-ResistantCandida tropicalisfrom Blood Cultures, Paris, France

“…Each allele was named after the length of PCR fragments. Isolates for which 1 signal was observed for a given locus on the electromorph were considered homozygous for this locus by analogy with what is reported for another diploid yeast, C. albicans (8).…”

Recognition of Yeast Species from Gene Sequence Comparisons

“…RAPD analysis has also been used to resolve populations in various species, such as S. cerevisiae, S. bayanus var. uvarum, C. albicans and C. glabrata [65][66][67][68][69][70][71][72]. One concern in using pattern-based identification techniques is reproducibility between laboratories, because small differences in PCR conditions may impact the species-specific patterns that serve as reference.…”