New Microsatellite Multiplex PCR for Candida albicans Strain Typing Reveals Microevolutionary Changes

Abstract

Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions. Candida albicans, the most common fungal pathogen, is a commensal yeast that belongs to the normal microbial population of the mouth, vagina, and gastrointestinal tract in humans. However, in people with a variety of transient or permanent immunocompromised conditions, including transplant recipients, chemotherapy patients, underweight neonates, and human immunodeficiency virus-infected individuals, it may become an invasive pathogen (30,56). Infections by opportunistic fungal agents are a major medical problem due to the growing number of immunocompromised patients with risk factors for such infections. Moreover, this problem is exacerbated by the fact that only a limited array of antifungal drugs is available and by the growing resistance among clinical isolates (39,46,55). The development of techniques and strategies that can accurately differentiate clinical isolates is of great relevance. These techniques should provide the ability to differentiate among strains responsible for clinical infections, as well as to trace their epidemiological pathways. Several molecular methods have been used to differentiate C. albicans strains, including electrophoretic karyotyping (2), the use of species-specific probes such as Ca3 or 27A in restriction enzyme analysis (28,35,36,43), P...

Microevolution of <i>Candida albicans</i> Isolate from a Patient with Mucocutaneous Candidiasis and HIV Infection

“…The observed allele size variation was apparently due to the deletion of five dinucleotide units (from 38 to 33). This suggests adaptation to environmental conditions and confirms a microevolution event [10]. Different studies have reported that microsatellite markers have high discriminatory power and reproducibility and are useful for studying the genetic diversity of Candida spp.…”

“…The genetic relationship among the isolates was established with three polymorphic microsatellites: CAI, CAIII, and CAVI. Microsatellite-based genotyping was performed by multiplex PCR using automated fluorescent genotyping (Applied Biosystems & Laragen Inc., USA) according to the protocol by Sampaio et al [10]. For each marker and for a given isolate, one or two bands were observed.…”

“…Genotyping with microsatellites multiplex PCR was performed in a 25-µl final volume of PCR buffer (20 mM Tris HCl, pH 8.4, 50 mM KCl), 0.2 mM dNTPs, 2 mM MgCl2, 50 ng of genomic DNA, and 0.5 U of Taq polymerase (Applied Biosystems, USA). Forward primers used in the PCR amplification were labelled with 6-carboxyfluorescein (6-FAM) (for CAI and CAIII) and hexachlorofluorescein (HEX) (for CAVI) fluorochromes [10]. The PCR products were run on an ABI 310 Genetic Analyser (Applied Biosystems) together with the GeneScan-500 (TAMRA) size standard (Applied Biosystems).…”

“…High similarity among samples suggests adaptation to environmental conditions and the occurrence of microevolutionary processes [10]. Herein, the three C. albicans isolates from the patient were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).…”

Microsatellite Typing of Catheter-Associated Candida albicans Strains

“…These different multilocus genotypes are used to distinguish strains within a single species (Applied Biosystems [AB], 2005). Microsatellite markers provide the possibility to discriminate strains of the same species and to trace their epidemiological pathways (Botterel et al, 2001;Sampaio et al, 2005).…”