Use of cost effective and rapid molecular tools for identification of Candida species, opportunistic pathogens

Background and Purpose : Candidiasis is a widespread fungal infection caused by different Candida species. Rapid identification of Candida species in clinical laboratory is becoming increasingly important since the identification and discrimination of ethological agents for early treatment. We aimed at molecular identification of commonly Candida species isolated from clinical samples by using both PCR-RFLP assay and amplification of hwp1 gene. Materials and Methods: Clinical samples comprising of vaginal specimens ,cutaneous, sputum, bronchoalveolar lavage(BAL,( and blood cultures were recovered from suspected patients. Candida isolates were initially identified phenotypically and confirmed by molecular approaches based on restriction fragment length polymorphism (PCR-RFLP (with MspI restriction enzyme. Amplification of hwp1 gene was performed for discrimination of C. albicans from C. dubliniensis and C. africana. Results: The most abundant species were C. albicans (n=67; 44.6 %), C. glabrata (n=10; 20 %), C. tropicalis (n=20; 13.3 %), C. krusei (n=12; 8 %), C. parapsilosis (n=11; 7.3 %). Out of 67 C. albicans species, 6 species identified as C. dubliniensis and 4 species identified as C. africana. Conclusion: High frequency of non-albicans Candida species and differences in levels of susceptibility to the antifungal agents are important issues in medicine .Therefore, to manage the Candida-related infections properly, molecular diagnostic methods would be fast, reliable and even cost-effective approaches for identification of Candida species.


Introduction
andidiasis is a widespread fungal infection caused by different Candida species especially in patients infected with human immunodeficiency virus (HIV), immunocompromised individuals, intravenous drug abusers, patients undergoing hematopoietic stem cell and solid organ transplant recipients, and those who present underlying valvular heart diseases or prolonged use of intravenous catheters [1]. Although Candida albicans is responsible for up to 78 % of nosocomial candidiasis [2], reports describing cases of Candida infections by non-albicans Candida species have been also documented [3]. Consequently, it is important to identify the causative organism to the species level correctly. Rapid identification of Candida species in clinical laboratory is becoming increasingly important since the identification and discrimination of etho-logical agents for early treatment, and preventing the invasion is highly recommended [4]. Due to the high degree of phenotypic similarity between Candida species, identification problems are imminent. Conventional approaches for identification down to the species level are not very effective in detecting yeast [5]. Therefore, Alternative molecular assay with high specificity, reproducibility and sensitivity are necessary [6,7]. The objective of current study was to identify the common Candida species isolated from clinical samples by using both polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method and amplification of hwp1 gene.

DNA Extraction
Genomic DNA was extracted, using glass bead disruption. Briefly, 300 µl of lyses buffer (10 mM Tris, 1 mM EDTA (pH= 8), 1 % SDS, 100 mM NaCl, 2 % triton X-100), 300 µl of phenol chloroform (1:1) solution and equal to 300 µl of 0.5 mm diameter glass beads, were added to yeast. After 5 min of vigorous shaking which followed by 5 min centrifugation at 10000 rpm, the supernatant was isolated and transferred to a new tube and equal volume of chloroform was added, mixed gently, centrifuged and its supernatant was transferred to a new tube. For alcohol precipitation, 0.1 mL volume sodium acetate (pH= 5.2) and 2.5 mL volume of cold absolute ethanol were added and the mixture was gently shaken and centrifuged at 10000 rpm for 10 min at 4°C. After washing with 70 % ethanol, the pellets re-suspended in 100 µl TE buffer (10 mM Tris, 1 mM EDTA) and were stored at -20°C prior to use.

Partial amplification of the hwp1 gene
Definitive species identification and discrimination of all members of the C. albicans species complex (C. albicans, C. dubliniensis, C. africana, and C. stellatoidea) was performed by the amplification of the hyphal wall protein 1 (hwp1) gene as described by Romeo and Criseo [8]. PCR amplification of hwp1 gene was achieved using the forward, 5′-GCTACCACTTCAGAATCATCATC-3′ and reverse, 5′-GCACCTTCAGTCGTA GAGACG-3′ primer pairs. The partial amplification of the hwp1 gene was used to distinguish C. albicans isolates (941 bp) from atypical form i.e., C. africana (750 bp) and C. dubliniensis (569bp). However, results were confirmed by sequencing of the ITS rDNA regions using previously described primers [9]. Briefly, sequencing was performed on an ABI 3730xl automatic sequencer (Applied Biosystems, Foster City, CA) and adjusted using the SeqMan of Lasergene software (DNAStar Inc., USA.) and compared with GenBank database (http://www.ncbi.nlm.nih.gov/).

Discussion
Recently, the increased incidence of severe candidiasis has been attributed to the enlargement of the population of high risk patients, which is resulted from advances achieved by modern medicine, immunosuppressives, and broadspectrum antibacterial drugs [10]. Despite the advances in medical interventions and the development of new antifungal agents, the mortality rate remains high [2]. Because of high degree of phenotypic similarity among species of Candida, identification problems are imminent [11]. Identification down to the species level based on conventional criteria is time consuming and need expertise. This report provides the species distribution of clinical isolates of Candida species with molecular identification. In this study, C. albicans was the most common species associated with Candida infection (44.6 %), followed by C. glabrata (20 %) which is in agreement with other studies [2,12,13], however the overall percentage of non-albicans species (55.4 %) was higher than in previous reports [13]. Shokohi et al. and Papon et al. mentioned C. glabrata as the most common non-C. albicans species in their investigation [14,15]. However, Alborzi et al. showed that he most abundant species isolated from Iranian patients were as follow; C. albicans (50 %), C. glabrata (21.4 %), C. dubliniensis (13.3 %), C. krusei (9.8%), C. kefyr (3.1 %), C. parapsilosis (1.6 %), and C. tropicalis (0.8 %) [16]. These differences in rates might be attributable to differences in the representation of the populations studied and ecological and climatological factors. The present study found that Candida species isolated from patients identified as C. dubliniensis (n=6) and C. africana (n=4). Solimani et al, also showed that 7.1 % of species from C. albicans identified as C. dubliniensis, however C. africana strains were not found [17]. In addition, Yazdanparast et al. believe that based on molecular tools, C. africana constituted 4.38 % of the vaginal isolates of the C. albicans species complex [11]. In this study, we performed PCR-RFLP assay and amplification of hwp1 gene to identify the clinically important Candida species and we found that C. albicans as predominant species isolated from this clinical samples [6,17,18].

Conclusion
High frequency of non albicans Candida species and differences in levels of susceptibility to the antifungal agents are important issues in medicine. Therefore, to manage the Candidarelated infections properly, molecular diagnostic methods would be fast, reliable and even costeffective approaches for identification of Candida species.