Microsatellite Length Polymorphism for DNA-Based Typing of Candida albicans Isolated from HIV Positive Patients in Tehran, Iran

Abstract

Background: The importance of Candida albicans as the most common cause of fungal infections in humans is undeniable. Genotyping methods have been developed as useful tools to differentiate between fungal strains isolated from various infections. Several molecular typing methods have been described for C. albicans, and fragment length analysis of microsatellites called microsatellite fragment length polymorphism (MLP) is one of the most accurate genotyping methods. Objectives: The present study aimed at evaluating the genetic diversity and genetic relationships among C. albicans isolates recovered from HIV-positive patients with oral candidiasis in Iran using MLP. Methods: We analyzed 30 isolates of C. albicans obtained from HIV-positive patients in Tehran. Genotypes of C. albicans isolates associated with oropharyngeal candidiasis were determined using microsatellite length polymorphism analysis. Three loci including EF3, CDC3, and HIS3 were amplified using multiplex PCR. After amplification, the product was run on an automated single-capillary genetic analyzer, and band sizes (known as alleles) were calculated with gene scan mapper. Results: PCR MLP typing of the 30 isolates under study yielded 27 different profiles, and the discriminatory power index was obtained as 0.993. Ten alleles and 18 different combinations were detected for the EF3 gene, 7 alleles and 18 combinations for the EF3 gene, and 10 alleles and 14 combinations for the HIS3 gene. Only 2 isolates were homozygous in all the 3 loci. To identify the origin of superficial infections in 6 patients, C. albicans isolates from the superficial as well as oral samples were simultaneously genotyped. Results showed the identity of genotypes in 4 of these patients. For 1 patient, the C. albicans genotype of the nails was different from the genotype observed in the oral cavity, which raised the possibility of an exogenous source for the superficial infection. Also, there were changes at only 1 or 2 alleles, which represented microevolution in some isolates. Conclusions: The high variation of genotypes throughout the population of C. albicans suggested that the microsatellite fragment length polymorphism using multiplex PCR-based system provided high-speed genotyping, indicating its usefulness in molecular epidemiological evaluation.

Genotypic diversity and antifungal susceptibility pattern of Candida albicans species isolated from hospitalized paediatric patients with urinary tract infection in Iran

“…that the microsatellite analysis of C. albicans isolates by targeting these markers was highly effective. These DP values are similar to those reported by Farahbakhsh et al (2018). They described 27 independent genotypes for 30 C. albicans isolates originating from oropharyngeal candidiasis (DP = 0Á993).…”

“…To amplify the three loci, multiplex PCRs were carried out in a volume of 25 µl containing 12Á5 µl Multiplex Master Mix (Amplicon, Odense, Denmark), 1 µl of each primer (5 pmole EF3, 2 pmole HIS3 and CDC3) and 3 µl of genomic DNA. Multiplex PCR was performed as the method described by Farahbakhsh et al (2018). The amplification reaction included initial denaturation at 95°C for 10 min, 30 cycles for denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 and a final extension at 72°C for 30 min.…”

“…It was also found that Candida genotypes may correlate with geographical distribution and antifungal resistance (Costa-de-Oliveira et al 2011;da Silva-Rocha et al 2014). The three loci of EF3, CDC3 and HIS3 are the most popular loci for the analysis of microsatellite regions and represent the high variability of genotypes in the population of C. albicans (Botterel et al 2001;Eloy et al 2006;Shi et al 2007;Farahbakhsh et al 2018). The genetic similarity was observed between some strains of C. albicans from different fungemia patients suggested the likelihood that they had the same sources (hospital-acquired) (Costa- de-Oliveira et al 2011).…”

“…2007; Farahbakhsh et al . 2018). The genetic similarity was observed between some strains of C. albicans from different fungemia patients suggested the likelihood that they had the same sources (hospital‐acquired) (Costa‐de‐Oliveira et al .…”

Genotyping of Candida albicans isolated from animals using 25S ribosomal DNA and ALT repeats polymorphism in repetitive sequence

“…DNA extraction was performed using a physicochemical method previously described [24]. For genotyping on the basis of 25S rDNA and RPS sequences, genomic DNA was amplified in a reaction mixture (25 µl) containing 1.75 mM magnesium chloride, 0.2 mM dNTPmix, 1U Taq DNA polymerase (Fermentase), 50 pmol of primers (i.e., CA-INT-L and CA-INT-R or AS-I), and 2 µl DNA template.…”