molecular typing tools for the investigation of outbreaks of infections with Candida

Brittany S.

Abstract: 

The traceability of infections Candida spp. in order to elucidate contamination grouped to decide on possible preventive measures benefited in recent years from new molecular biology techniques.Some techniques such as pulsed fields or RAPD are no longer relevant. Conversely, two methods are emerging, both based on PCR, microsatellite markers of the art and the study of SNPs in the technique called MLST (Multi Locus Sequence Typing).

Microsatellites are defined as tandem repeats of short fragments of DNA, 2 to 6 nucleotides long. The discrimination between both isolates is the length of the amplified fragments. Two primers are designed in the flanking parts of the microsatellite and one of the primers is labeled with a fluorophore. Following amplification, the DNA fragments are analyzed during their migration in capillary sequence able to distinguish differences in the order of one base pair. The marking of the primers to assign a length to the analyzed fragment. For diploid yeast, such Candida albicans and C.tropicalis , two peaks are expected heterozygous isolates (1, 6) . For haploid species, such as C.glabrata , a single peak is expected (8, 11) . If additional peaks are observed, the first explanation is the presence of an initial mixture. Next microsatellites, the number of alleles at a given locus is variable but often high. Most publications report a discriminatory power close to 1 (the maximum) when 3 or 4 loci are considered. This method is highly reproducible but has some limitations in the transfer of data between laboratories due to conditions very dependent migration of the material used(12) . This drawback can be solved by the use of allelic ladders (5, 10) .

The other well-established method for genotyping yeast is the MLST technique. This is based on the amplification and sequencing of several loci of genes conserved within a given species, usually so-called "housekeeping" genes. Databases are already available (http://www.mlst.net/) for several species including C. albicans (2, 3) , C. tropicalis (13) and C. glabrata (7) . The main advantage is the character not or little questionable, easily comparable data sequences between laboratories. The comparison between the MLST techniques and microsatellites show for C. albicans 7 MLST loci have a comparable discriminating power analysis of microsatellite 3 (9) . For the study of SNPs, other methods are possible as technical HRM (High-Resolution Melting) (4) .

Reliable methods, broadband, reproducible and easily codified providing data for comparisons between experiments and between laboratories are now available to any lab with access to a sequencer. These techniques should be preferred for the exploration of cross-contamination and outbreaks of yeast infections in a hospital setting.

2010

Full conference title: 

Réunion Interdisciplinaire de Chimiothérapie Anti Infectieuse
    • RICAI 30th (2010)