Genotyping of candida albicans isolates obtained from vulvovaginal candidiasis patients in Zanjan, Iran, based on ABC and RPS typing systems

Background and Purpose: Genotyping of pathogenic microorganisms is important for epidemiological studies and the adoption of appropriate strategies to control infectious diseases. In this regard, the present study aimed to genotype Candida albicans strains isolated from vulvovaginal candidiasis (VVC) patients using combined ABC type (25SrDNA) and repetitive sequence (RPS) typing systems. using combined typing systems of ABC type (25SrDNA) and repetitive sequence (RPS). Materials and Methods: In total, 140 patients with VVC were investigated. Vaginal discharges were collected on Sabouraud dextrose agar and identified by CHROMagar. After species identification, a polymerase chain reaction system targeting 25S rDNA as well as ALT repeats in the RPS was designed to determine C. albicans genotypes. The dendrogram was constructed by zero-one matrix data based on the combination of ABC and RPS typing systems. Statistical analysis of data was performed in SPSS software (version 23). Results: In total, 41 (29.3%) Candida isolates were obtained from 140 VVC patients. The most common Candida species that were identified included C. glabrata (56.1%) and C. albicans (39%). Genotype A3 with five isolates (31.25%) had the highest frequency, followed by B2/3 with three isolates (18.3%), A3/4, C3/4, and B3/4 with two isolates (12.5%), and C2/3 and C3 with one isolate (6.25%), respectively. No significant association was found between the genotypes and antifungal resistance (P<0.05). Conclusion: The results showed that non-albicans Candida species are more prevalent in VVC patients, compared to C. albicans. The results also indicated that ABC and RPS typings are useful for rapid genotyping and differentiation of C. albicans isolates in regional and small-scale studies.


Introduction
here are more than 20 Candida species that are known as opportunistic human pathogens. Today, Candida infections have increased due to the increase in the use of aggressive medical interventions and the number of immunosuppressed patients. Candida albicans is the most important pathogen of the Candida genus, which could cause mucocutaneous or systemic infections in healthy people or patients with underlying diseases, such as immunosuppression [1]. Although C. albicans is still known as the main pathogen of this genus, there is a pattern change from albicans to non-albicans infections, especially C. glabrata and C. tropicalis infections [2].
One of these infections is vulvovaginal candidiasis (VVC) whose severity and frequency could be different among different people. It has been estimated that 75% of women experience vaginitis at least once in their lifetime which negatively affects their social and work lives [3]. The VVC is the second most common infection after bacterial vaginitis. Candida albicans has been reported as the most common cause of VVC worldwide. However, the increasing prevalence of vaginitis caused by non-albicans Candida species is a public health concern due to their tendency to develop resistance to azoles [4][5][6].
It is necessary to use reliable and reproducible molecular typing methods in epidemiological studies of Candida infections. It is possible to genetically classify clinical isolates in terms of drug susceptibility patterns, type of infection, and the site of anatomical involvement based on genetic diversity and polymorphism [7]. The ABC typing based on ribosomal sequences of the 25 S rDNA region has been used in several studies in the genotyping of C. albicans isolates into A, B, C, D, and E types [8][9][10].
Repetitive sequences (RPSs) were found in all C. albicans chromosomes except for chromosome 3 [11]. Each RPS region has a short tandem repeating unit of 172 bp, which is known as ALT. The number of ALT repeats in RPS is different for each chromosome, causing variation in RPSs electrophoresis fragment size [12]. The combined analysis of both ABC and RPS could improve the discriminatory power of genotyping [13].
Epidemiological studies of pathogenic microorganisms are important for the adoption of appropriate strategies to control infectious diseases [14,15]. Given the importance of genotyping in epidemiological studies, ABC and RPS typing systems were applied to determine the genotypes and identify the population structure of C. albicans strains isolated from vaginal candidiasis. Moreover, the results were compared with the genotyping profile of isolates obtained from oropharyngeal candidiasis samples in a previous study.

Strains
In this study, 41 Candida colonies were collected from 140 VVC patients referring to three health centers in Zanjan, Iran in 2019. In total, 16 isolates were identified as C. albicans. The isolates were evaluated in terms of their genotypes using ABC and RPS genotyping systems.

DNA extraction and polymerase chain reaction conditions
The DNA extraction was performed using the conventional phenol-chloroform extraction method [16]. The ABC typing of C. albicans isolates based on the 25s rDNA region sequence was performed using the primer pairs of CA-INT-L and CA-INT-R. Moreover, two further primers, ASDcF and pCSCR, were used to determine genotypes based on ALT repeats [14,17]. Details of the primers and the expected band size of polymerase chain reaction (PCR) products have been described previously [13].
The PCR amplification conditions were similar to those reported in previous studies [6]. Briefly, the volume of the reaction mixture was 25 µL which consisted of 1 µL (4 ng) of DNA template, 1.5 µL of each forward and reverse primer (5 mM), 12.5 µL of master mix (SinaClon Bioscience Co., Karaj, Iran), and 8.5 µL of distilled water. Thermal cycling started at 97 ˚C for 5 min, followed by 30 cycles of denaturation at 94 ˚C for 30 s, annealing at 60 ˚C for 30 s, extension at 72 ˚C for 40 s, and a final extension at 72 ˚C for 5 min.
It should be noted that all reactions were performed using a thermal cycler (SimpliAmp; Applied biosystem; Cat No: A24811). The PCR products were electrophoresis on 1.5% agarose gel in TBE buffer.

Phylogenetic analysis
First, the zero-one matrix was prepared by electrophoresis banding patterns. Phylogenetic analysis was performed based on zero-one matrix data via the unweighted pair group method with arithmetic averages (UPGMA) using an online tool (www.genoms.urv.cat/upgma).
The discriminatory power of ABC and RPS typing systems was measured by Simpson's diversity index using an online tool (http://insilico.ehu.eus/) [20]. This index demonstrates the ability of typing methods in differentiating between species.

Statistical analysis
Statistical analysis of collected data was carried out in SPSS software (version 23). The associations between genotypes and antifungal susceptibility were evaluated using Fisher's exact test. The P values < .05 were considered statistically significant.

Results
In this study, genotyping of 16 clinical isolates of C. albicans was performed by ABC and RPS typing. As shown in Table 1, the obtained PCR products using specific pairs of primers (CA-INT-L and CA-INT-R as well as ASDcF-pCSCR) provided distinct band patterns to differentiate genotypes. In this study, ABC typing of C. albicans isolates based on the 25s rDNA region sequence presented three genotypes, namely A, B, and C. Genotype A with seven isolates (43.7%) was the most frequent genotype, followed by genotype B (n=5, 31.3%) and genotype C (n=4, 25%). In addition, the banding pattern of RPS typing showed that genotypes 3 and 3/4 had the highest and equal frequencies (n=6, 37.5%), followed by genotype 2/3 (n=4, 25%). The distribution of genotypes based on the combined ABC and RPS typing is summarized in Table 1.  As shown in Figure 1, the UPGMA dendrogram was constructed based on the combined ABC and RPS (ALT repeats) typing data using an online tool (www.genoms.urv.cat/upgma) Based on this phylogenetic tree, the studied isolates were divided into seven clades with distinct genotypes. Genotype A3 with five isolates (31.3%) was the most prevalent type, followed by genotype B2/3 (n=3, 18.3%), genotypes A3/4, C3/4, and B3/4 (n=2, 12.5%), and genotypes C3 and C2/3 (n=1, 6.3%).
The discriminatory power of typing systems was calculated according to Simpson's diversity index using an online tool (http://insilico.ehu.eus/) [20]. According to the results, 16 clinical isolates of C. albicans were classified into seven distinct genotypes, indicating a discriminatory power index of 0.8667.

Antifungal susceptibility testing
In-vitro antifungal susceptibility tests to fluconazole, ketoconazole, and itraconazole were performed using the CLSI broth micro-dilution method. These drugs are the most commonly and widely used drugs in cases of vaginal candidiasis. The results of drug sensitivity testing are shown in Table 2.

Discussion
In this study, genotyping of clinical isolates of C. albicans were conducted based on ABC and RPS typing method. The frequency distribution of C. albicans isolates in terms of ABC typing was as follows: genotype A with seven (43.75%) isolates, genotype B with five (31.25%) isolates, and genotype C with four (25%) isolates. Candida dubliniensis electrophoretic band pattern was not observed in any of the samples. In addition, the frequency distribution of C. albicans isolates in terms of ALT repeats was as follows: ALT3 and ALT 3/4 with six isolates apiece  (2006) reported that the highest frequency of C. albicans isolates was related to genotype A3, followed by genotypes A3/4 and B3 [8]. In a study, a significant association was found between ABC genotypes and blood invasion, and genotype A had a higher prevalence in blood samples [22].  in their study performed in Japan found that among 179 clinical isolates, genotype A with 92 isolates had the highest frequency, followed by genotypes B and C with 38 and 6 isolates, respectively. They also found that based on ALT repeat typing, type three had the highest frequency [13]. However, the frequency rates of genotypes A, B, and C vary in different studies which could be due to the diversity of isolates in different studies. Several studies have suggested that genotypic differences in C. albicans isolates may be related to the site of infection [25][26][27][28][29][30].
In drug susceptibility testing of Candida isolates, it was found that drug resistance had no significant correlation with ABC and RPS genotypes, which is in accordance with the results of our previous report [6]. Studies have shown that there is a genotypic association between ABC genotypes and resistance to flucytosine; accordingly, genotype A is less sensitive and genotype B is more sensitive to flucytosine [14,29,30].
The isolates obtained from vaginal discharge samples in this study were compared with those obtained from oral lesions in our previous study in terms of the frequency of genotypes [6]. The results showed that in both studies, genotype A3 had the highest prevalence. The frequency of genotypes B3 and C3 in vaginal discharge samples was zero (0.0%) and one case (6.3%), respectively. Nevertheless, in oral lesion samples, the frequency of B3 and C3 genotypes was six isolates (19.35%) [6]. Furthermore, 16 isolates from vaginal discharge samples with seven distinct genotypes had a DP of 0.8667, while 31 isolates from oral lesions with seven separate genotypes had a DP of 0.7634. A comparison of the results of these two studies shows that the genotypic diversity is higher in isolates obtained from vaginal discharge samples, compared to those obtained from oral lesions.
Studies have shown that there is no significant correlation between genotypic diversity and the anatomical origins of isolates [8,31,32]. Moreover, it has been found that there is no clear genotypic relationship between blood and non-blood isolates of C. albicans [33].
Results of a study performed by Hattori et al. (2006) showed that the strains isolated from infectious and non-infectious areas (e.g., mouth and feces) had a similar genotype in each patient. This finding strengthens the possibility of endogenous C. albicans infections [8]. According to the aforementioned report, C. albicans has a unique genotype in each person, and this genotype is the same even in different anatomical areas. In that study, no genotypic differences were observed between C. albicans strains isolated from the same individual over 6 months [8].
Xiao-dong et al. (2008) used EcoRI and ClaI enzymes to treat the 780 bp PCR product of the RPS region of C. albicans strains isolated from skin and vaginal discharge samples using the restriction fragment length polymorphism method [34]. In their study, 10 distinct genotypes were identified, and no significant genotypic differences were observed between the two groups. However, C. albicans strains isolated from different skin regions of each patient had the same genotypic characteristics [34]. In contrast, Xu et al. (1999) showed that multiple genotypes of the same species could colonize different areas of the host body, indicating the dynamics of the colonization process in the host [25].
The codon adaptation index gene analysis and single-strand conformational polymorphism genotyping of C. albicans isolates have shown that most VVC-associated isolates have specific genotypes [35]. Moroever, the genotypic distribution of C. albicans strains isolated from VVC, balanitis, and nongenital infections was investigated in a study by Li in 2008. The findings revealed that some vaginopathic C. albicans isolates exhibited high virulence and vaginal tropism and that the possibility of sexual transmission of vaginal infections was very high. These results clarify the importance of species differences in the etiology of VVC, the relationship between specific genotypes and genital infections, as well as the importance of genotyping in the clinical diagnosis and treatment of VVC.
Nevertheless, there is no clear correlation between different typing methods, and no reliable genotyping technique has been provided to classify or differentiate pathogenic and drug-resistant strains yet. Nevertheless, genotyping using sequencing techniques, such as multilocus sequence typing, is considered an ideal but costly method with high discriminatory power and clear results [36][37][38][39].

Conclusion
The RPS/ALT typing method is a simple, fast, and affordable method that could be used in epidemiological studies and the management of Candida infections. Besides, by using this technique, C. albicans could be distinguished from C. dubliniensis and C. stellatoidea based on the number of ALT repetitions in RPS [8,13,23].