Genetic Diversity and Antifungal Susceptibility of Candida Albicans Strains Isolated from Lranian HIV+ Patients With Oral Candidiasis


 Objective: The objectives of this study were to investigate the antifungal susceptibility and genetic diversity of oral Candida albicans strains isolated from HIV+ patients with oropharyngeal candidiasis. A total of 50 C. albicans isolates were cultured on Sabouraud glucose agar containing chloramohenicol. The antifungal susceptibility of C. albicans against fluconazole, clotrimazole, nystatin, amphotericin B, ketoconazole and flucytosine was assessed using disc diffusion method. The genetic diversity of different C. albicans strains was determined using random amplified polymorphic DNA technique. Results: The inhibition zones ranged from 4±1.8 to 40±3.8 mm for fluconazole, 7±1.0 to 37±1.8 mm for ketoconazole, 14±0.8 to24±0.8 mm for amphotericin B, 25±0.0 to 33±0.0 mm for nystatin and 7±4.2 to 40±0.0 mm for clotrimazole. At 90% similarity, three distinct groups were observed. The smallest cluster composed of 3 of 50 C. albicans isolates, whereas the largest cluster composed of 17 of 50 isolates. Of 50 C. albicans isolates, 32%, 28% and 14% were resistant to fluconazole, ketoconazole and clotrimazole, respectively. There were no significant differences among antifungal susceptibility of different C. albicans strains from three genotype clusters.


Introduction
Candida albicans (C. albicans) has been known as one of the most prominent clinical pathogen, as well as in primary and secondary immunocompromised patients (1). Oropharyngeal candidiasis (OPC) is frequently complicated situation of predisposing factors such as hematologic malignancy, acquired immunode ciency syndrome (AIDS), Nezelof syndrome, zink and iron de ciency. OPC, as the independent predictor of immunode ciency in AIDS patients, increases the mortality and morbidity among these patients; consequently, it requires prompt therapy and precise diagnosis (2). C. albicans, only species recovered from up to 70% of HIV-infected individuals, is one of the most common cause of mucosal yeast infection in human. Totally, 93% of untreated HIV patients harbour OPC at least with one frequent recurrences per year (3). Recently, the prevalence of oral Candida infections in HIV patients has been decreased. Two factors have been described this phenomenon. First, overuse of antifungal agents, particulary the azole antibiotics. Second, the introduction of highly active antiretroviral therapy has resulted in a signi cant decrease in the incidence of a number of opportunistic diseases and the mortality of AIDS (4). Resistance of Candida species to the azole antifungals (as the main challenge antibiotic susceptibility) is the most prevalent type of resistance to antifungals in these patients. Since azoles, particularly uconazule, have been used for prophylaxis or treatment of AIDS patients, resistance to this antifungal agent is common during AIDS-related complex (5). Resistance can be caused by an alteration of the target enzyme, the cytochrome P-450 lanosterol 14 α-demethylase, mediated by ERG11 gene, or the failure of azole antifungal agents to accumulate inside the yeast cell as a consequence of enhanced drug e ux, mediated by MDR and CDR genes (6).
In recent years, RAPD analysis has been increasingly used as molecular method for population genetics and genotyping of different organisms (7). The purposes of the current study were to determine the antifungal susceptibility and the genetic diversity of C. albicans isolates collected from oral cavity of Iranian HIV + patients with OPC.

Microorganisms
This study was performed on 50 C. albicans isolates obtained from oral cavity of HIV + patients, from October to November 2011 at the AIDS Research and Training Center of Imam Khomeini Hospital, Tehran, Iran. We used a wet mount with 10% KOH preparation and Giemsa stain for microscopic examination of pseudo-hyphae and yeast cell forms. In addition, all samples were cultured on Sabouraud glucose agar (SGA; 20 g/l glucose, 10 g/l peptone, 20 g/l agar, pH: 5.6) containing 0.05% chloramphenicol (Merck Co., Darmstadt, Germany). The cultures were incubated at 37 oC and examined daily for one week.
Identi cation of C. albicans isolates were performed on the basis of germ tube test, colony color on Chrom agar (Paris, France Company), sugar fermentation and assimilation tests by RAPID yeast plus system (Remel Inc., USA) and internal transcribed spacer (ITS) primer pairs (CALB1 and CALB2).

Antifungal drugs
All standard antifungal discs including uconazole (25 µg/disc), clotrimazole (10 µg/disc), nystatin (50 µg /disc), amphotericin B (20 µg/disc), ketoconazole (10 µg/disc) and ucytosine (1 µg/disc) were obtained from Oxoid (Hampshire, UK). The agar disc diffusion method was employed for the determination of anti-C. albicans activity as described by the National Committee for Clinical Laboratory Standard (8). Brie y, a suspension of C. albicans (106 cell/ml) was spread on Muller-Hinton agar containing 2% glucose and 0.5 µg/ml methylene blue dye. Standard antifungal discs were placed on the inoculated plates. These plates were incubated at 37 oC for 48 h. The diameter of the inhibition zones was measured in millimeter (mm) and the results were interpreted based on comparison to standards.
Antifungal susceptibility assay was performed in duplicate.

DNA extraction
All samples were cultured on SGA at 37 oC for 48 h. Genomic DNA was extracted as previously described (9) and puri ed using a commercial DNA puri cation kit (Ultraclean Microbial DNA Isolation kit, MO BIO, USA) according to manufactuterer`s structures. DNA concentration and purity were determined by optical density at 260 nm and ratio OD 260 /280 nm determinations, respectively.

PCR assay and RAPD analysis
Diagnostic PCR analysis was performed with the oligonucleotide primers CALB1: TTTATCAACTTGTCACACCAGA and CALB2: ATCCCGCCTTACCACTACCG (The GenBank accession number refers to primers L47111, L28817) with amplify at 450 bp fragment within the 5.8 s ribosomal RNA gene (10). Ampli cation reactions were done in a nal volume of 25 µl containing 2.5 µl of reaction buffer (10X), 1.5 mM Mgcl2 (50 mM), 0.2 mM dNTP (10 mM), 0.5 µM (each) primers, 0.5 u of Taq DNA polymerase and 2 µl of genomic DNA template. Ampli cation was carried out using a Techne Tc-512 thermo cycler (Techne, UK), Initial denaturation was at 96 °C for 5 min, as follows: 35 cycles of 30 s for denaturation at 94 °C, 30 s for annealing at 55 °C and 30 s primer extension at 72 °C, followed by a terminal extension at 72 °C for 15 min. The PCR products were electrophoresed on 1.5% agarose gel for 1 h at 80v and stained with ethidium bromide (2 µg/ml) to visualize uorescent band while using UV in the gel document system (Biorad, UK).
RAPD-PCR was performed with RSD12: 5′-GGTCCGTGTTTCAAGACG-3′ primer (11). Each reaction mixture contained 2.5 µl of reaction buffer (10X), 2.5 mM Mgcl2, 200 mM dNTPs mix,1.25 µM of primer RSD12, 1U of Taq DNA polymerase and 100-400 ng of C. albicans DNA as template in nal volume of 25 ml. PCR ampli cation program for RSD12 primer involved 1 cycle at 95 °C for 5 min, then 40 cycles as follows: 30 s for denaturation at 94 °C, 2 min for annealing at 57 °C and a nal extension step 72 °C for 2 min in a TC-512 thermocycler (Techne, Cambridge, UK). The PCR products were analysed by electrophoresis on 1.5% agarose gel at 70 V for 80 min in TBE buffer (1x) and stained in a 0.5 mg/ml ethidium bromide solution for 15 min and photographed by CCD Video Camera. DNA banding patterns were analysed using the GelCompar software package, version 6 (Applied Math, Belgium).

Statistical analysis
The chi-square test and t-test using SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA) were performed to statistical analysis. A phenogram was constructed by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) after determining the association coe cients by the simple matching method.

Results
Antifungal susceptibility and genetic diversity of 50 clinical isolate of C. albicans were evaluated by disc diffusion methods and RAPD analysis, respectively. The antifungal susceptibilities of C. albicans strains from oral cavity of understudy Iranian HIV + patients were shown in Tables  to ucytosine (no inhibition zone was observed).
Using the primer CALB, C. albicans isolates yielded RAPD pro les with one strong band, with molecular size of 273 bp. On the basis of RAPD-PCR pro les (Fig. 1) and similarity coe cient ≥ 90%, genotypes containing from 3 to 17 isolates, which encompassed 30 (58.82%) isolates and 13 (25.49%) genotypic particular strains. The rst cluster, which was the smallest one, composed of rami cation a (ATCC strain) and a ' (C32 and C35 strains), the second cluster composed of rami cation b and b ' as shown in Figure 1, and the third one (the largest cluster) composed of 17 of 50 isolates which distributed in c and c ' rami cation. There were no signi cant differences among the anti-C. albicans susceptibility of different genotype clusters.
C. albicans has been known as one of the most frequently yeasts obtained from HIV-infected in Iran and the world (12). The ndings of the current study revealed genetic diversity and antifungal susceptibility in the genotype groups of C. albicans strains isolated from oral cavity of HIV + patients. Genetic nger typing of C. albicans has been studied in different countries (13). But there is no information about the correlation between the antifungal susceptibility and genetic diversity of C. albicans isolated from HIV + individuals (14). Regarding the speci c primer pair for C. albicans CALB1 and CALB2, it yielded approximately molecular size of 273 bp. The molecular size of this study is same with the results of previous study that CALB1 and CALB2 produced amplicon size of approximately 273 bp. These results are in line with that of Sharifzadeh et al. (2013) as well (15). The results of this study using with primer RSD12 indicated different genetic pro les between C. albicans strains with various antifungal susceptibility patterns. Whereas it was not possible in our study to show that different susceptibility to various antifungal agents is attributable to the genetic diversity of the understudy strains. Regarding the RSD12 primer pro le and similarity coe cient ≥ 90% , genotypes consisted of 3 to 17 isolates, which were encompassed 30 (58.82%) isolates and 13 (25.49%) genotypic particular strains. Hamzehee et al.
(2019) carried out RAPD-PCR method for estimating the strains of C. albicans, 46 genotypes were de ned including 11 cluster with 80% similarly coe cient (16). Sun et al. (2009) used primer RSD6 and also the same RSD12 assess genetic diversity of C. albicans isolates from root canal infection and found 31 genotypes among the 37 isolates (17). RSD10 and RSD12 primers to were used to determine the clonal viability of 443 C. albicans strains obtained from 16 HIV-infected individuals. These isolates formed clusters comprising 2 or more strains at value of a similarity coe cient ≥ 80%(18). Our results are in agreement with mentioned previous reports as well. Considering to our results, amphotericin B and nystatin were the most effective antifungal drugs, and uconazole had the poorest activity. Fluconazole, in spite of using drug potency up to 25 mg per disc, had poor activity on isolates tested. There are many studies indicating that uconazole had less activity against Candida species (19). Over all, uconazole resistance, in spite of using drug potency up to 25 µg per disc, was 32%. These higher rates of resistance are not in accordance with those observed in Mexico, Brazil, United Kingdom and other studies which reported lower rates of resistance to the antifungal (20). The reason for higher uconazole resistance could be explained by the fact that azoles, especially uconazole, have been used for prophylaxis or incompletely treatment of oral candidiasis in HIV-infected patients and resistance to uconazole are common during AIDS related complex (21).

Limitations
Despite all efforts, 50 isolates as the sample size is not e cient to study the genetic relatedness and anti-fungal susceptibility of C. albicans isolated from HIV + patients.
RAPD is a strong genotyping method; however, it is not precise enough because of lack of reproduciblity. Other genotyping methods are suggested to be implemented.