New Polymorphic Microsatellite Markers Able To Distinguish among Candida parapsilosis Sensu Stricto Isolates

Abstract

Among the Candida species causing bloodstream infections, Candida parapsilosis is one of the most frequently isolated. The objective of the present work was the identification of new microsatellite loci able to distinguish among C. parapsilosis isolates. DNA sequences with trinucleotide repeats were selected from the C. parapsilosis genome database. PCR primer sets flanking the microsatellite repeats were designed and tested with 20 independent isolates. On the basis of the amplification efficiency, specificity, and observed polymorphism, four of the sequences were selected for strain typing. Two hundred thirty-three independent C. parapsilosis sensu stricto isolates were genotyped by using these markers. The polymorphic loci exhibited from 20 to 42 alleles and 39 to 92 genotypes. In a multiplex analysis, 192 genotypes were obtained and the combined discriminatory power of the four microsatellites was 0.99. Reproducibility was demonstrated by submission of subcultures of 4 isolates each, in triplicate, interspersed with unique numbers among a group of 30 isolates for blind testing. Comparison of the genotypes obtained by microsatellite analysis and those obtained by randomly amplified polymorphic DNA analysis, restriction fragment length polymorphism analysis, and internal transcribed sequence grouping was performed and showed that the microsatellite method could distinguish individual isolates; none of the other methods could do that. Related species, C. orthopsilosis and C. metapsilosis, were not confused with C. parapsilosis sensu stricto. These new microsatellites are a valuable tool for use for the differentiation of C. parapsilosis sensu stricto strains, vital in epidemiology to answer questions of strain relatedness and determine pathways of transmission.

Different scenarios for Candida parapsilosis fungaemia reveal high numbers of mixed C. parapsilosis and Candida orthopsilosis infections

“…Considering only independent isolates (the first C. parapsilosis strain isolated from each patient), the combined DP of the multiplex was 0.99, which is in agreement with previous studies (DiabElschahawi et al, 2012;Sabino et al, 2010). In this study, isolates with the same allelic combination at all microsatellite markers correspond to genetically similar strains and were defined as identical, while isolates with distinct allelic combinations at more than one locus correspond to genetically dissimilar strains and were considered different strains.…”

“…Cells for colony PCR were prepared as described by Vaz et al (2011). The primers, PCR amplification conditions and the allele size determination by GENESCAN (version 3.7) analysis after a run on an ABI PRISM 310 genetic analyser (Applied Biosystems) were as described previously (Sabino et al, 2010) with some modifications as described by Vaz et al (2011), namely the redesign of one primer. Fragment sizes were determined automatically using GENESCAN 3.5 analysis software.…”

“…Genotyping isolates with the microsatellite multiplex failed to amplify all markers in 39 isolates. Since this multiplex strategy is specific to C. parapsilosis sensu stricto (Sabino et al, 2010), all the isolates were sequenced. Sequencing showed that 37 strains were Candida orthopsilosis, two were Candida metapsilosis and the remaining 61 were C. parapsilosis (Table 1).…”

“…The discrimination of C. parapsilosis strains is fundamental not only for the rapid identification of the strains involved in the infection but also to clarify nosocomial cross-transmission and possible routes of transmission in hospital settings (van Asbeck et al, 2009). Since the description of a microsatellite multiplex strategy for C. parapsilosis strain differentiation (Sabino et al, 2010), this method has been applied in several studies mainly regarding outbreaks (Diab-Elschahawi et al, 2012;Romeo et al, 2013;Vaz et al, 2011) and is described as the most discriminatory method for C. parapsilosis strain differentiation. Herein, we used this method to genotype presumed C. parapsilosis isolates involved in fungaemia episodes from three hospitals in Brazil and to determine the patterns of relatedness of the isolates, including the pair identified in the bloodstream/catheter.…”

Microsatellite Genotyping Clarified Conspicuous Accumulation of Candida parapsilosis at a Cardiothoracic Surgery Intensive Care Unit

“…A panel of six short tandem repeat (STR) markers was used for genotyping the C. parapsilosis isolates. Three trinucleotide repeat markers as described earlier by Sabino et al (17) were amplified in a multiplex PCR. For these markers, we redesigned the amplification markers to generate shorter PCR products and to amplify all three in one multiplex PCR.…”