Molecular identification and antifungal susceptibility profiles of Candida dubliniensis and Candida africana isolated from vulvovaginal candidiasis: A single‐centre experience in Iran

Abstract Background Vulvovaginal candidiasis (VVC) is a common and debilitating long‐term illness affecting million women worldwide. This disease is caused mainly by Candida albicans and a lesser extent by other species, including the two phylogenetically closely related pathogens Candida africana and Candida dubliniensis. Objectives In this study, we report detailed molecular epidemiological data about the occurrence of these two pathogenic yeasts in Iranian patients affected by VVC, or its chronic recurrent form (RVVC), and provide, for the first time, data on the antifungal activity of two new drugs, efinaconazole (EFN) and luliconazole (LUL). Methods A total of 133 vaginal yeast isolates, presumptively identified as C albicans by phenotypic and restriction analysis of rDNA, were further analysed by using a specific molecular method targeting the HWP1 gene. All C africana and C dubliniensis isolates were also tested for their in vitro susceptibility to a panel of modern and classical antifungal drugs. Results and Conclusions Based on the molecular results, among 133 germ‐tube positive isolates, we identify 119 C albicans (89.47%), 11 C africana (8.27%) and 3 C dubliniensis (2.26%) isolates. C africana and C dubliniensis showed low MIC values for most of the antifungal drugs tested, especially for EFN and LUL, which exhibited a remarkable antifungal activity. High MIC values were observed only for nystatin and terbinafine. Although C albicans remains the most common Candida species recovered from Iranian VVC/RVVC patients, our data show that its prevalence may be slightly overestimated due to the presence of difficult‐to‐identify closely related yeast, especially C africana.


| INTRODUC TI ON
Vulvovaginal candidiasis (VVC) is one of the most common and widespread human fungal infection in the world that affects 70%-75% of women at least once in their lifetime, especially in childbearing age. 1 According to recent estimates, 2 up to 9% of young women experience recurrent episodes (4 or more) of VVC (RVVC) each year, which result in hundreds of millions of people affected globally, and in a huge economic loss estimated to be in tens of billions of dollars. 2 The infection is caused by some species belonging to Candida genus (phylum Ascomycota; sub-phylum Saccharomycotina; order: Saccharomycetales), a complex and heterogeneous group of yeastlike fungi widely distributed in nature. 3 This genus contains at least 300 taxa 4 but, of the over 40 species known as etiological agents of candidiasis, 5 only 5 (Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei) are responsible for more than 95% of the infections occurring in humans. 6 C albicans is the most pathogenic species of the group and remains the most frequently isolated yeast from clinical specimens, including vaginal swabs. 7,8 In fact, over 90% of VVC cases are attributable to this species while the remaining ones are caused by several other species, such as C glabrata, C tropicalis and C parapsilosis, which are collectively referred to as non-albicans Candida species. 8 Until 1995, C albicans was considered the only germ-tube and chlamydospores positive Candida species infecting humans, but molecular comparative studies, performed on some atypical Irish C albicans strains, revealed the existence of high levels of genetic diversity which led to the description of a new phylogenetically related species, called Candida dubliniensis. 9 In the same year, other unusual germ-tube positive C albicans isolates were recovered from African patients with vaginitis, 10 and subsequently proposed as representatives of a novel species, Candida africana, 11 on the basis of some unusual phenotypes clearly different from those of typical C albicans isolates, for example the lack of chlamydospores production. 12 However, although the status of new species has been genetically confirmed for C dubliniensis, for C africana the taxonomic position is still controversial 13 and some authors consider it as a biovar of C albicans. 12 Nevertheless, C africana represents undoubtedly one the most intriguing evolutionary lineage within the global C albicans population 14,15 and recent genome-wide studies 16,17 have tried to clarify its evolutionary history by drawing an interesting and fascinating picture on the origin and evolution of the members of the C albicans clade, including the well-known and synonymised Candida stellatoidea. 13 Interestingly, these studies suggested that C albicans probably originated from an ancestral hybridisation event between two divergent lineages, which also generated C africana and C stellatoidea making their taxonomic position in the clade even more uncertain and complicated. 13 The clinical and epidemiological implications of these findings are very important because members of the C albicans clade, as well as the non-hybrid species C dubliniensis, 17 despite exhibiting some lineage-specific phenotypes, they may still be misidentified as the same species by conventional mycological methods. 12,13 Furthermore, the paucity of specific drug susceptibility data for these genetically distinct Candida lineages makes it difficult to choose the appropriate therapeutic treatment for VVC/RVCC caused by them and contributes to the increase of azole-resistant strains. 18 Although C africana and C dubliniensis are widely distributed across the world, 9,15,19 there are limited data on the extent of RVVC caused by these pathogenic yeasts in Iranian women. 20 Recent studies have shown that most of vaginal C africana isolates from Iran exhibit resistance to various antifungal drugs, 21,22 and ~28% of world isolates appear to be resistant to itraconazole. 23 Furthermore, although C africana is generally associated with vaginal infections, in Iran, this pathogen has also been reported as a cause of paediatric candiduria, 24 and/or oropharyngeal candidiasis in cancer patients. 25 From these latter patients, fluconazole-and amphotericin B-resistant isolates have been also recovered. 25 These epidemiological findings support the hypothesis that C africana prevalence, and/or drug resistance, can be geographically variable 23   Iranian patients presenting signs and symptoms of VVC who referred to clinics of gynaecology in Jahrom city, south of Iran. Swabs terbinafine. Although C albicans remains the most common Candida species recovered from Iranian VVC/RVVC patients, our data show that its prevalence may be slightly overestimated due to the presence of difficult-to-identify closely related yeast, especially C africana.

| Genomic DNA extraction, molecular identification and partial HWP1 gene sequencing
In this study, a preliminary molecular screening of the yeast isolates was accomplished using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of ribosomal internal transcribed regions (ITS). 27,28 Total genomic DNA was extracted from each isolate using the glass-bead phenol-chloroform method according to a previous study. 29 Purified DNA samples were then subjected to PCR-RFLP analysis as previously described in Gharaghani et al. 27 Briefly, the ITS-rDNA regions were amplified using 0.5 μM of the fungal universal primers ITS1 and ITS4, 4 μl of DNA template, 12.5 μl of 2× Taq DNA master mix (Ampliqon) and PCR-grade water to make a final reaction volume of 25 μl. 30 PCR cycles included initial denaturation at 94°C for Subsequently, the digested products were separated and visualised on a 2% (wt/vol) agarose gel stained with ethidium bromide (0.5 μg/ ml) and photographed under ultraviolet light at 305 nm. Final species identification was done by comparing the obtained RFLP patterns with those of reference strains used in other studies. 27,28 In order to correctly identify each member of the C albicans clade, all isolates were further characterised by using a simple PCR-based assay developed for rapid and specific identification of these pathogens. 12,31 This method is based on partial amplification of the HWP1 gene using only a single pair of primers: CR-f-GCTACCACTTCAGAATCATCATC-3′ and CR-r-GCACCTTCAGTCGTAGAGACG. 31 The reaction mixture and conditions used for in vitro amplification were the same as those previously described by Romeo and Criseo, 2008. 31 PCR products were separated on a 1.3% (wt/vol) agarose gel and, according to the size of the amplicon produced, Candida isolates were classified as follows: C albicans (~941 bp), C stellatoidea (~800 bp), C africana (~740 bp) and C dubliniensis (~569 bp). 12,31 All HWP1 fragments obtained from isolates identified as C africana and C dubliniensis, including three random selected C albicans isolates, were also sequenced using the same CR-f/CR-r primer set However, unlike typical C albicans isolates, C africana usually exhibits slower growth rates at different temperatures. 16  with MspI restriction endonuclease, 28 confirming that the PCR-RFLP method employed was not able to discriminate between C albicans, C dubliniensis and C africana. 28  The results of the in vitro antifungal susceptibility testing for C africana and C dubliniensis are shown in Table 2. The geometric mean MIC against all tested isolates was 0.019 and 0.024 μg/ml for the two novel drugs EFN and LUL, respectively (Table 2). However, in general, except for nystatin and terbinafine, all other antifungal agents tested showed low MIC values and therefore all the isolates were considered susceptible to these drugs ( Table 2). F I G U R E 1 Phylogenetic analysis of C albicans, Cafricana and Cdubliniensis based on HWP1 gene sequences obtained in this and other studies. The GenBank accession numbers for each sequence used in the analysis are indicated in the tree. The evolutionary history was inferred using the maximum-likelihood method based on the Tamura-Nei substitution model. Numbers at the nodes represent bootstrap values expressed as percentages of 1000 replicates (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) and C dubliniensis (12)(13)(14)

| DISCUSS ION
Vaginal candidiasis is still one of the most common human fungal infections worldwide that affects an estimated 138 million women each year, a number that is destined to increase by more than 20 million over the next 10 years. 2 In Iran, over 2.7 million women, aged between 15 and 50 years, suffer from RVVC but current epidemiological data are not sufficient to establish with certainty the real burden of this disease in this country. 36

TA B L E 2 In vitro susceptibilities of 14
Candida isolates against eleven antifungal agents we believe that the denomination of a 'species complex' is no longer appropriate to indicate these pathogenic yeasts and therefore, according to Mixão et al, 2020, 13 there is an urgent need to clarify the taxonomic position of C albicans and its related lineages.
Collectively, in this study, C dubliniensis and C africana make up 10.5% (14/133) of VVC/RVVC cases caused by germ-tube positive Candida isolates and, in this context, C africana makes the most significant contribution (11/14 non-albicans isolates; ~78.6%) which is in line with previous studies, including its high ability to cause mainly vaginal infections. 12,15,23,35,41 Conversely, C dubliniensis has been frequently associated with oral infections in HIV-positive patients, 9,12 and our data confirm its very low prevalence in VVC/RVVC patients  Table 2) against all our Candida isolates, confirming the excellent in vitro activity of these two new antifungal drugs against a broad spectrum of pathogenic fungi. [59][60][61][62] However, it should be pointed out that in our study, the susceptibility test was performed using a pH 7 culture medium as described by standard CLSI guidelines. 33,34 This condition may not reflect the real susceptibility of isolates normally exposed, or adapted, to the vaginal acidic environment (normal pH between 3.8 and 4.5). 63 Therefore, a sensitivity test with culture medium at pH≃4 should be evaluated in future studies against these vaginal Candida pathogens.
In conclusion, although C albicans remains the main causative agent of vaginal infections in Iranian patients, a considerable part of these cases can be attributed to other Candida pathogens that are very difficult to identify using traditional identification methods. 12,39,42 Nevertheless, based on our data, and several previous studies, 12,23,35,37,41,58,64 the differentiation between C albicans, C africana and C dubliniensis appears to be clinically irrelevant, as discrimination of these pathogens should not affect therapeutic choices commonly used for the treatment of VVC/RVVC. However, several drug-resistant C africana isolates, or strains with reduced susceptibility to different classes of antimycotics, 12,22,23 have been recently reported, and we also confirmed the occurrence of isolates with high MIC values for NYS and TRB drugs. Therefore, further epidemiological studies on a global scale will be useful to establish the real prevalence, and the role, of C africana in vaginal infections as well as understanding the extent of antifungal resistance in this pathogenic yeast.

ACK N OWLED G EM ENTS
This study was supported by Jahrom University of Medical Sciences, Jahrom, Iran (grant no. 98001518). The authors thank the participants for their involvement in this project.