Highly Polymorphic Microsatellite for Identification of Candida albicans Strains

Abstract

The polymorphism of a new microsatellite locus (CAI) was investigated in a total of 114 Candida albicans strains, including 73 independent clinical isolates, multiple isolates from the same patient, isolates from several episodes of recurrent vulvovaginal infections, and two reference strains. PCR genotyping was performed automatically, using a fluorescence-labeled primer, and in the 73 independent isolates, 26 alleles and 44 different genotypes were identified, resulting in a discriminatory power of 0.97. CAI was revealed to be species specific and showed a low mutation rate, since no amplification product was obtained when testing other pathogenic Candida species and no genotype differences were observed when testing over 300 generations. When applying this microsatellite to the identification of strains isolated from recurrent vulvovaginal infections in eight patients, it was found that 13 out of 15 episodes were due to the same strain. When multiple isolates, obtained from the same patient and plated simultaneously, were typed for CAI, the same genotype was found in each case, confirming that the infecting population was clonal. Moreover, the same genotype appeared in isolates from the rectum and the vagina, revealing that the former could be a reservoir of potentially pathogenic strains. This new microsatellite proves to be a valuable tool to differentiate C. albicans strains. Furthermore, when compared to other molecular genotyping techniques, CAI proved to be very simple, highly efficient, and reproducible, being suitable for low-quantity and very-degraded samples and for application in large-scale epidemiological studies.It is known that opportunistic yeast pathogens are common residents of the mucosal surfaces of the gastrointestinal tract, genitourinary system, and oral cavity in warm-blooded animals. Although several yeast species can be associated with infection, the predominant causal agent of candidiasis is Candida albicans. This yeast causes several infections in humans, including a wide variety of life-threatening conditions triggered by bloodstream infections, especially in immunocompromised patients. Since pathogenicity and antifungal susceptibility often vary among strains, a rapid and accurate identification of the disease-causing strains of C. albicans is crucial for clinical treatment and epidemiological studies.Advances in molecular biology in the last 2 decades have allowed the development of rapid molecular genotyping techniques for clinical and epidemiological analysis. Several molecular typing methods have been developed to differentiate C. albicans strains, including electrophoretic karyotyping (2), the use of species-specific probes such as Ca3 or 27A in restriction enzyme analysis (20,23,27,29,32,33,35), and PCR-based methods (1,10,21,24,28,37). More recently, short tandem repeats (STRs) or microsatellites have assumed increasing importance as molecular markers in fields so diverse as oncogenetics, population genetics, and strain identification and characterization. They occur in se...

A prospective analysis of the genotypic diversity and dynamics of the Candida albicans colonizing flora in neutropenic patients with de novo acute leukemia

“…To address this question, we used a molecular typing method based on four microsatellite markers separately shown to be suitable for epidemiological studies. 24,25 These markers are located on different chromosomes, are stable over many generations, and are highly discriminative. Bretagne et al observed a discriminatory power of 0.97 using a combination of the EF3, HIS3 and CDC3 microsatellites while Sampaio et al obtained a similar value using CAI alone.…”

“…23 Clones were genotyped using four microsatellite markers: one set of three loci referred to as EF3, CDC3, HIS3, located on chromosomes 5, 1 and 2, respectively, 24 and one additional marker referred to as CAI, located on chromosome 4. 25 Amplification reactions were performed using primer sequences and thermal cycling parameters described elsewhere. 24,25 Fragment size analysis of the polymerase chain reaction fragments was performed by automated fluorescent capillary electrophoresis as previously described.…”

“…25 Amplification reactions were performed using primer sequences and thermal cycling parameters described elsewhere. 24,25 Fragment size analysis of the polymerase chain reaction fragments was performed by automated fluorescent capillary electrophoresis as previously described. 23 To ensure reproducibility of the results, the reference strain ATCC 26278 was run as a control in each series.…”

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

“…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).…”

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

“…The amplification of species-specific microsatellites by PCR has been successfully applied in molecular epidemiology and population studies to differentiate between C. albicans strains [8] [9]. The microsatellite loci CAI (CAA repeats), CAIII (GAA repeats), and CAVI (TAAA repeats) present in the C. albicans genome have been used for genotyping clinical isolates.…”