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 1 and 2. Results of susceptibility to standard antifungal drugs were as follows: fluconazole: 29 isolates (58%) susceptible, 5 isolates (10%) susceptible-dose dependent or intermediate and 16 isolates (32%) resistance; ketoconazole: 31 isolates (62%) susceptible, 5 isolates (10%) susceptible-dose dependent or intermediate and 14 isolates (28%) resistance; amphotericin B: 48 isolates (96%) susceptible, 2 isolates (4%) susceptible-dose dependent or intermediate; clotrimazole: 35 isolates (70%) susceptible, 8 isolates (16%) susceptible-dose dependent or intermediate and 7 isolates (14%) resistance; flucytosine: 50 isolates (100%) resistance and nystatin: 50 isolates (100%) susceptible.
The inhibition zone diameters of fluconazole ranged from 4±1.8 to 40±3.8 mm (mean value: 23.0±11.7 mm), 7±1.0 to 37±1.8 mm (mean value: 24.8±9.4 mm) for ketoconazole, 14±08 to 24±08 mm (mean value: 17.2±2.1 mm) for amphotericin B, 7±4.2 to 40.0 mm (mean value: 24.8±8.7 mm) for clotrimazole and 25±0.0 to 33±0.0 mm (mean value: 26.6±2.0 mm) for nystatin. In addition, all isolates were resistant to flucytosine (no inhibition zone was observed).
Using the primer CALB, C. albicans isolates yielded RAPD profiles with one strong band, with molecular size of 273 bp. On the basis of RAPD-PCR profiles (Fig. 1) and similarity coefficient ≥ 90%, genotypes containing from 3 to 17 isolates, which encompassed 30 (58.82%) isolates and 13 (25.49%) genotypic particular strains. The first cluster, which was the smallest one, composed of ramification a (ATCC strain) and a' (C32 and C35 strains), the second cluster composed of ramification 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' ramification. There were no significant 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 findings 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 finger 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 specific 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 profiles 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 profile and similarity coefficient ≥ 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 defined including 11 cluster with 80% similarly coefficient(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 coefficient ≥ 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 fluconazole 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 fluconazole had less activity against Candida species(19). Over all, fluconazole 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 fluconazole resistance could be explained by the fact that azoles, especially fluconazole, have been used for prophylaxis or incompletely treatment of oral candidiasis in HIV-infected patients and resistance to fluconazole are common during AIDS related complex(21).
Limitations
- Despite all efforts, 50 isolates as the sample size is not efficient 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.