Identification of Candida species Isolated From Vulvovaginal Candidiasis Patients by Chromgen agar and PCR-RFLP Method

This study focuses on diagnosis of Candida species causing Vulvovaginal Candidiasis using phenotype and genotype analyzing methods, and frequencies of candida species also using Vulvovaginal Candidiasis patients. 130 samples (100 from patients and 30 from non infected women) were collected and cultured on biological media. Identifying the yeasts, initially some phenotypic experiments were carried out such as germ tube, from motion of pseudohyphae and clamydospores in CMA+TW80 medium, API20 candida and CHROMagar Candida. Genomic DNA of all species were extracted and analyzed with PCR and subsequent Polymerase Chain Reaction Restriction Fragments Length Polymorphism (PCR-RFLP) methods. Frequency of C. albicans, C. krusei, C. tropicalis , C. parapsilosis and C. glabrata were 46.4%, 31%, 18%, 7.2%, and 1.8%, respectively.The ITS1-ITS4 region was amplified and the Restriction enzyme Msp1 digests this region and was used to identify of candida species .Electrophoretically ribosomal DNA of C. albicans, C. krusei, C. tropicalis and C. glabrata produced two bands whereas the C. parapsilosis gave one band.


Introduction:
Vulvovaginal candidiasis (VVC) is an insidious that affects a large porportio in of women of all ages, and 5 to 8 of affected women experience recurrent VVC (RVVC) [1], and it is a common problem in women and may affect their physical and emotional health, as well as relationships with their partners [2].There are two forms of RVVC: primary RVVC is idiopathic with unknown predisposing factors, secondary RVVC is the occurrence of frequent episodes of acute VVC because of certain predisposing factors such as hormone replacement therapy or diabetes mellitus [3].VVC is caused by overgrowth of Candida yeast species in the vagina and is characterized by curd-like vaginal discharge, itching, and erythema [4].

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Vol. 13(2)2016 3:3 common cause of candidiasis, but other species are not uncommon [5].Candida albicans account for 70 to 90 of all VVC cases, with a recent emergence of nonalbicans species [6].The rise in VVC infection, more specifically in those caused by non-albicans species, could be due to several factors, ranging from an increase in over-the -counter antifungal use to an increase in high-risk patient populations (i.e., diabetics and menopausal women).Candida glabrata is the primary non-albicans species emerging in VVC, accounting for up to 14% of infection in immune-competent women [6].Candida glabrata was found to be the primary species isolated from diabetic (61.3%) and elderly (51.2%) patient with VVC [7].The detection of Candida in vaginal swabs is corelated with the age of patients.It was shown that women under 35 years old have the highest rate of detectable Candida compared to the other groups, The detection rate of non-albicans Candida increased 2.75 folds in the age group of 26 to 35 years.[8] the incidence of VVC in pregnant women was 3.5 fold higher than that of non-pregnant women.It continued to increase in the third trimester of pregnancy [8].Pregnancy has been known to be associated with depressed aspects of cell-mediated immunity that permit fatal retention.Moreover, the hormonal changed milieu of the vagina during pregnancy enhances Candida colonization and serves as a risk factor for symptomatic expression [9] .Delay in speciation of candida isolates by conventional methods and resistance to antifungal drugs (especially fluconazole, amphotericin B, etc.) in various Candida species are some of the factors responsible for the increase in morbidity and mortality due to candidemia.So, the rapid detection and Identification of Candida isolates is very important for the proper management of patients having candidemia [10].The RFLP-PCR using the restriction enzyme MpsI is a good rapid identification method that identifies the most important Candida spp isolated from patients and recommends further studies to develop new methods using different restriction enzymes to increase the range of identified candida spp [11].This study aims to focuses on diagnosis on Candida species based on phenotypic and genotypic approaches and analysis of frequency of Candida species in vulvovaginalcandidiasis patients.

Materials and Methods:
Patients: One hundred of high vaginal swabs were taken from 100 married women, 30 of them were nonpregnant (N.P) and 70 were pregnant (P.) women.They were suffering from vulvovaginal candidiasis in addition to 30 healthy controls.Sample were taken during the period from first of June 2012 till the end of April 2013, under the supervision of specialized gynecologist in the bent Al-Huda hospital, Thi Qar.A special questionnaire was prepared for each individual.The swabs were incubated in Sabouraud's dextrose agar (SDA) with chloramphenicol (0.5 mg/ml) at 37°C for 48 h .(underaerobic conditions) and in CHROMagar ™ Candida (CHRO Magar, France) at 35°C for 48 h for production of species-specific colors.Different chromogenic culture media are capable of distinguishing C. albicans from other clinically important yeast strains are commercially available.Such media distinguish Candida strains from other yeast strains on the basis of the color changes produced by the Candida colonies, which are measured using pH indicators and by fermentation of specific compounds or chromogenic substrates for the presumptive identification of C. albicans, C. tropicalis, and C. krusei [12].10% KOH preparation and Gram stain for 3:4 microscopic examination of pseudohyphae and yeast cell forms.Carbohydrate assimilation tests was used.Fresh yeast colonies were incubated with rabbit serum at 37°C for 3 h to test for germ tube formation.Development of filamentousform cells and chlamydospore formation were evaluated by culturing the yeast isolates on Dalmau plates (cornmeal-Tween 80 agar) at 30°C for 48 h [13].and identification using API 20 AUX (Bionereux, Paris, France).Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed using specific primers for the molecular identification of Candida spp.

Restriction enzyme analysis:
A volume of 25 μL of PCR products were digested directly and individually by the restriction enzyme MspI .For each restriction digestion reaction, 5 μL of the amplified PCR product was digested with 1.5 μL of restriction enzyme buffer, 0.5 μl of the restriction enzyme MspI, and 8 μL of Deionizer distilled water; the reaction mixture 15 μL was incubated at 37°C for 120 min.Separation of the digested fragments was visualized on 2% agarose gel run in TBE buffer at 100 V for 45 min, and stained with 0.5 μg ml-1 ethidium bromide [15].

Results and Discussion:
Isolation of candida spp.We identified the different Candida spp.from women infected with VVC and healthy women as control group, by using the restriction enzyme MspI.(PCR-RFLP assay),chromogen agar, Biochemical tests and API20 C. albicans were the most commonly identified species (41.1).This result agrees with AL-Hashime who isolated 60 isolate of Candida albicans from 120 Non-pregnant infected women with volvovaginal candidiasis [16] and with Roudbary et al who indicated that Candida albicans is the most dominant species compare with other species [17].Candida albicans is the most abundant isolated microorganism from Volvovaginal Candidiasis patients with frequency of 47.2% [18].

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Habibeh et al. [19] have shown that C. albicans 53.64% as the major causative agents of Vulvovaginal Candidiasis.This because Candida albicans have high ability to adherence on epithelial cells and its ability to produce germ tube in infected tissue ,and high product to protein digestive enzymes and phospholipase enzymes [20].The color of Candida albicans colonies on CHROMagar Candida was green, while Candida tropicalis was blue and Candida krusei was pink fuzzy as in figure (1).