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

Abstract

A multiple-locus variable-number tandem-repeat analysis (MLVA) using six microsatellite markers was assessed in 127 Candida glabrata isolates. Thirty-seven different genotypes, stable both in vitro and in vivo, were observed. The highest discriminatory power (D ‫؍‬ 0.902) was reached by using only four markers. MLVA seems to be relevant for C. glabrata typing.Candida glabrata has recently emerged as a major pathogen, causing mucosal and systemic infections (10,17). Several methods, such as electrophoretic karyotyping, restriction enzyme analysis, Southern blotting with probes, randomly amplified polymorphic DNA, and multilocus sequence typing (MLST), have been used to distinguish and type C. glabrata isolates (2,7,8,14,21,28). 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. The aim of our study was to assess a microsatellite-based multiple-locus variable-number tandemrepeat analysis (MLVA) using new markers for C. glabrata typing.One hundred twenty-seven C. glabrata strains were analyzed, including four reference strains, 98 independent clinical isolates, and 25 epidemiologically related isolates. These 25 were from the blood cultures and peripheral site isolates of eight patients with C. glabrata candidemia. Genomic DNA was extracted by boiling with Chelex resin as previously described (6, 23). Six microsatellite markers were selected from the C. glabrata DNA sequences available in the GenBank database (29). Primer sequences were designed with Primer3 software (24), and locations in the C. glabrata genome were determined with the Genolevures database (http://cbi.labri.fr/Genolevures/) ( Table 1). For each primer set, PCRs were performed with a 20-l final volume containing 1 l of DNA, each deoxynucleoside triphosphate at 200 M, a forward primer and a 5Ј-dyelabeled reverse primer at 0.25 M each, and 1 U of Taq DNA polymerase (Promega, Madison, WI). The amplification conditions were 5 min at 95°C, followed by 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 45 s and then a final step of 72°C for 10 min. For any given isolate, amplicons of each PCR were pooled before multiplex fragment sizing with a Ceq 8000 Genetic Analyzer (Beckman Coulter, Fullerton, CA). Strain IHEM 9670 was run as a control in each experiment. As allele sizing depends on dyes and the analyzer used for electrophoresis, results were expressed as the exact size of the sequence (determined by sequencing of each representative allele for each locus) to allow further interlaboratory comparisons of MLVA results (13,20). The reproducibility and stability of the method were assessed as described elsewhere (25) . Primer specificity was also tested against the sequ...

Evaluation of Polymorphic Locus Sequence Typing for Candida glabrata Epidemiology

“…Correspondingly, the diversity indexes (excluding epidemiological replicates; see below) were 0.94 and 0.91. These values are comparable to those obtained with other typing methods and strain sets (14,24,32,34,35).…”

“…Candidate loci were then evaluated by amplification and sequencing from small panels of unrelated strains, and CgMT-M (on chromosome M, upstream of CAGL0M00902g) emerged as the most promising. This locus was independently identified by Grenouillet et al (35) as one of the four most informative targets within their MLVA scheme, which assesses length variation alone. A partial alignment of CgMT-M sequences from representative strains is shown in Fig.…”

“…The most widely applied include (i) multilocus sequence typing (MLST), which analyzes 6 relatively conserved housekeeping loci for single nucleotide polymorphisms (SNPs) (29,30), (ii) pulsed-field gel electrophoresis (PFGE), which compares total DNA banding patterns with or without restriction enzyme digestion (14,23,31), (iii) multilocus variable-number tandem-repeat analysis (MLVA, also known as microsatellite analysis), which examines length variation in 6 to 9 PCR-amplified loci that contain polymorphic tandem repeats (32)(33)(34)(35), and (iv) random amplification of polymorphic DNA (RAPD), which compares banding patterns following PCR with a nonspecific primer (26,36). In general, these methods are comparable in their strain resolution, achieving diversity indexes of ca.…”

Multilocus Microsatellite Markers for Molecular Typing of Candida glabrata : Application to Analysis of Genetic Relationships between Bloodstream and Digestive System Isolates

“…However, this method requires the use of radioactive elements, and the sequences of the probes are not available. Sequence-based methods such as multilocus sequence typing (MLST) and multilocus microsatellite analysis have been recently proposed (15,19,21), but in all cases one could hope for better discriminatory power.…”

Multicenter Collaborative Study for Standardization ofCandida albicansGenotyping Using a Polymorphic Microsatellite Marker

“…It has proved its efficacy in genotyping large collections of samples in epidemiological studies of not only C. albicans but also other yeasts, such as Candida glabrata (10,13), Candida tropicalis (6), and filamentous fungi, such as Aspergillus fumigatus or Penicillium marneffei (1,7,17). However, interlaboratory exchange of microsatellite data can be difficult, because the use of different equipment and diverse capillary electrophoresis conditions can lead to altered assessment of PCR fragment lengths.…”