Differentiation of Candida albicans complex species isolated from invasive and non-invasive infections using HWP1 gene size polymorphism

Background and Purpose: Taxonomy of Candida is controversial and has changed due to the investigation of the novel species. Candida africana and Candida dubliniensis are new members of the C. albicans complex that are currently gaining both clinical and epidemiologic significance. This study aimed to report the prevalence of C. africana among the strains isolated from patients using hyphal wall protein 1 (HWP1) gene size polymorphism. Materials and Methods: In total, 235 yeasts confirmed as C. albicans complex based on chromogenic media and internal transcribed spacers sequencing isolated from various clinical forms of invasive and non-invasive candidiasis mainly candidemia were re-identified using HWP1 gene polymorphisms. The HWP1-polymerase chain reaction amplicons were re-confirmed by sequencing and BLAST analysis. Results: Based on the HWP1 gene size polymorphism, 223 strains were identified as C. albicans (94.89%) from which 7 isolates produced two DNA fragments (850 and 941 bp). The C. dubliniensis (n=4, 1.7%), C. africana (n=1, 0.42%), and mix of C. albicans and C. africana (n=7, 2.97%) were also identified. Conclusion: It can be said that C. albicans remains the most common Candida species, while C. dubliniensis and C. africana are rarely found among the patient isolates. Due to limited information on the molecular epidemiology of this novel yeast, more studies using molecular methods are recommended.


Introduction
ncidence rate of infections caused by various yeasts species has increased considerably in the past decades [1]. Classification of Candida albicans as the most common cause of invasive fungal infections has been subjected to significant changes describing new species, such as Candida dubliniensis and Candida africana, as the cryptic species complexes [2,3]. According to the limited number of studies performed to date, C. africana is reported to have a strong association with human genitals, and it is rarely isolated from other body sites [4]. Accordingly, C. dubliniensis and C. africana have received less attention, compared to C. albicans and there is a lack of experimental and clinical evidence about their pathogenic potential.
According to the previous studies, C. dubliniensis and C. africana are inherently susceptible to azole and polyene antifungal drugs. However, some reports have shown that the antifungal susceptibility patterns of C. africana and C. dubliniensis are slightly different from those of C. albicans [5,6]. Moreover, based on previous studies, some C. africana isolates have been classified as resistant to itraconazole, fluconazole, voriconazole, clotrimazole, 5-flucytosine, and Terbinafine [4,7,8]. Echinocandins is the first-line antifungal drug for the treatment of Candida infections and has shown prolonged post antifungal effect and concentration-dependent killing activity against the majority of Candida species, including the C. albicans complex [9,10]. Phenotypic characteristics do not allow differentiation between the members of closely related C. albicans complex species. More reliable tests are based on molecular techniques, such as specific polymerase chain reaction (PCR) amplification of the hyphal wall protein 1 (HWP1) gene [11]. The HWP1 gene has been proposed as the molecular target for discriminating between C. albicans species complex based on its size polymorphism as it has 941/850 base pair (bp) for C. albicans, 569 bp for C. dubliniensis, and ∼700 bp for C. africana [11].
Although epidemiological and clinical data suggest that C. africana has a worldwide distribution, little is known about the frequency of Candida isolated from systemic candidiasis in Iran [12]. Hence, this study was carried out to investigate the microbial epidemiology of C. albicans complex species among different clinical specimens, especially those strains isolated from systemic candidiasis.

Candida albicans complex isolates and strains
The majority of Candida samples had already been isolated from the patients with systemic candidiasis admitted to the neonatal and pediatric ICUs of Children's Medical Centre, Tehran, Iran, and identified as C. albicans mostly by internal transcribed spacers sequencing and/or matrix-assisted laser desorption ionization-time of flight [13]. In addition, a part of the samples was isolated from vulvovaginal candidiasis and candiduria from the patients in Al-Zahra Hospital, Isfahan, Iran. The C. albicans (ATCC 64553), C. dubliniensis (ATCC 2018), and two isolates of C. africana (GenBank accession number: MG434677 and MG434680) were used as the positive controls.

Molecular identification
The colonies conserved at -20 ºC freezer were subcultured on CHROMagar Candida [15], and DNA was extracted from a single colony by boiling method [14]. A fragment of the HWP1 gene was amplified using CR-f (5'-GCT ACC ACT TCA GAA TCA TCATC-3') and CR-r (5'-GCA CCT TCA GTC GTA GAG ACG-3') primers [11] in the following thermal conditions: 5 min at 95 ºC, followed by 35 cycles of 40 s at 94 ºC, 45 s at 60 ºC, and 60 s at 72 ºC as well as a final extension of 5 min at 72 ºC. The reaction mixture contained 7.5 μL of 2 master mix (Ampliqon, Denmark), 0.33 μM of each primer, and 2 μl of DNA in a total volume of 15 μl. It should be mentioned that appropriate positive and negative controls were used for each PCR run.
An aliquot of 5 μl of each sample was added to 1.5% agarose gel containing 0.5 μg/ml of ethidium bromide. It was electrophoresed for 90 min in 120 V and visualized under UV light documentation. Species identification was performed based on the size polymorphism of the HWP1 gene in different species, i.e. C. albicans (∼940/850 bp), C. dubliniensis (∼570 bp), and C. africana (∼700 bp) [11,16]. The HWP1-PCR product identified as C. africana was subjected to sequencing with the above-mentioned forward primer and the result was analyzed by Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast).

Results
In this study, a total of 235 Candida albicans isolates were re-identified based on HWP1 gene polymorphisms. The isolates were collected from patients with systemic candidiasis (n=150), vulvovaginal candidiasis (n=60), and candiduria (n=25). The age of patients with candidiasis ranged from 1 to 78 years and the majority of them were female (n=154, 65.53 %).
Based on HWP1 gene amplification, the species distribution was as follows: C. albicans (n=223, 94.89%), from which 7 isolates produced two different DNA fragments (850 and 941 bp), C. dubliniensis (n=4, 1.7%), C. africana (n=1, 0.42%), and the mix of C. albicans and C. africana (n=7, 2.97%) (Figure 1). The amplicon of the single pure C. africana isolate was subjected to PCR-sequencing. The obtained sequences showed 99.71% identity with an isolate of C. africana (MN817936.1) with an Evalue of 99.42 and 100% coverage and the sequence was inserted in GenBank under accession numbers MZ578437. It is noteworthy that BLAST analysis of the obtained sequence confirmed the identity.
This C. africana was obtained from the urine specimen of a 45-year-old female with diabetes. All isolates of C. dubliniensis and all samples with a mix of C. albicans and C. africana were collected from the patients with systemic candidiasis. The results of the molecular analysis are shown in Table 1.

Discussion
The incidence of mild to severe fungal infections has dramatically increased worldwide in the last several decades. Fungal species distribution varies owing to the hospital, hospitalization unit, and geographical area [17]. Invasive candidiasis is a considerable cause of morbidity and mortality, especially amongst patients suffering from immunodeficiency [18]. The C. albicans complex is one of the major fungal groups, which is involved in more than 50% of Candida infections, pointing out their significant prevalence among human beings [19].
The differences in adherence ability, pathogenicity, and biofilm formation observed between C. albicans and C. africana highlight the necessity of discriminating them in clinical laboratories [20]. Therefore, this study aimed to identify the archived cryptic specimens belonging to the C. albicans complex isolated from clinical samples of hospitalized patients to demonstrate the existence of species that are not routinely identified and reported.
The HWP1 is a particular target for differentiation of the C. albicans complex species i.e. C. albicans, C. dubliniensis, and C. africana [7]. In the present study, C. albicans (94.89%) was the predominant species among the 235 isolates, which is in line with the results of some previous studies [12,16,[21][22][23][24]. In the present research, the majority (97.3 %) of C. albicans with 941 bp DNA fragments were homozygous, while 2.97% (n=7) of them produced two DNA fragments of 850 and 941 bp, demonstrating heterozygosity at HWP1 locus. The 850 bp DNA fragment is considered a novel allele of the HWP1 gene [22].
Nouraei et al. [25] evaluated the exoenzyme activity of 60 C. albicans species consisting of 30 homozygous and 30 heterozygous strains. They found that the homozygous strains of C. albicans had more phospholipase and proteinase exoenzyme activity than heterozygous strains in different ranges, while no significant statistical differences were observed between the strains in terms of virulence factors. Further studies are needed to clarify the probabilistic pathogenic role of these homozygous or heterozygous strains.
In this study, C. africana (3.4%) had a higher prevalence rate than C. dubliniensis (1.7%). This result corroborates those of the previous research performed in Iran [7,12,[26][27][28]. However, the higher isolation rate of C. dubliniensis over C. africana has also been reported in other studies [21,29,30]. In a study conducted by Romeo et al. [31], the frequency of C. africana (7.2%) was higher than that of C. dubliniensis (2.9%) among the Candida strains isolated from 498 clinical specimens collected from various patient groups [31]. The C. dubliniensis is less prevalent than C. albicans and shows phenotypic similarities with C. albicans, which may invade sterile body sites, such as mucosal surfaces, blood, central nervous system, and pleural fluid, with mortality rates similar to C. albicans [32][33][34][35].
Although C. africana has a worldwide distribution, an epidemiological meta-analysis showed that its overall prevalence rates in Iran and Honduras were higher, compared to other countries worldwide [5]. Shokoohi et al. [26] reported that one of the largest clusters of C. africana isolates was from Iran with a prevalence rate similar to those reported from some other countries indicating that this yeast may be more locally or regionally prevalent [26] Hana et al. [21] reviewed the global epidemiological status of C. africana reported between 2010 and 2019 from more than 11 [21].
Based on the results of global epidemiological studies, most of the C. africana strains have been isolated from vulvovaginal specimens [36]. However, in agreement with the study conducted by Yazdanpanah et al. [37] and Gumral et al. [38], the results of our assay revealed that no C. africana was recovered from the vaginal specimens. The distribution of C. africana may be partially based on geographical variation, although a larger number of vulvovaginal samples are needed to confirm this hypothesis. We also identified C. africana among the patients with candiduria, suggesting that this fungus can also be associated with a wider clinical spectrum [31].
In this investigation, molecular identification demonstrated seven co-infections by C. africana and C. albicans in patients with systemic candidiasis. An attempt to discriminate species in mixed infection/colonization, especially in children, is important for clinicians as they could differ both in virulence and spectrum of antifungal. Consequently, the lack of specific microbiological data could force physicians to empirically treat life-threatening mycoses with broad-spectrum antifungal medications, which would impact the existing issues with antifungal resistance. cost-effective method for the differentiation of C. africana and C. dubliniensis from C. albicans, C. africana was detected in 3.4% of the isolates. This means that this species is not uncommon in Iranian patients.