Participants of this study were women with signs and symptoms of VVC, who were admitted to the gynecology clinic in Mahdieh Educational Hospital in Tehran, Iran during a 2-year period from March 2006 to 2008. Participants who experienced the signs of VVC less than four times (including their latest admission to the clinic) in the previous year were categorized in nonrecurrent VVC (NRVVC), and those who experienced the vaginal itch and secretions equal or more than four times in the previous year were considered recurrent VVC (RVVC) after being confirmed by finding three positive cultures of VVC within a year in their medical records. Specimens were collected from the first 100 consecutive patients with NRVVC and likewise for the first 100 consecutive patients with RVVC. Each patient was tested only once at her first visit to the clinic during the two-year period of the study. Out of a total 200 collected specimens, 25 were excluded from the study due to contamination or having no signs of fungal growth. Hence, the final number of specimens was 175. To further evaluate the risk factors for VVC, we also asked our participants to complete a questionnaire regarding their demographic and behavioral characteristics. This study was approved by Iran's National Research Ethics Committee. All of the patients signed a written consent form before participating in the study and patients' confidentiality was strictly protected. Furthermore, no complication was seen after taking the vaginal samples.
Vaginal sampling of the participants performed by using a sterile swab by the principle researcher and was cultured simultaneously onto sabouraud dextrose agar medium.
Candida species colonies were also identified by germ tube formation in serum, chlamydospore production on CMA, and carbohydrate absorption using the API 20
C-AUX kit (bioM'erieux, Paris, France), results of which have already been published elsewhere [
15]. Colonies were placed in transport medium at room temperature before being processed for PCR analysis.
DNA extraction and purification were performed using a Genomic DNA Extraction kit (AccuPrep Bioneer Corporation) based on the guidelines. Multiplex PCR was performed using the PCR premix kit (AccuPower Bioneer Corporation) with a total reaction volume of a 50
μL consists of 10
mM Tris-HCl (pH 8.3), 40
mM KCl, 1.5
mM MgCl
2, 0.8
mM deoxyribonucleoside triphosphates (0.2
mM each), 4
μM primers (0.16
μM each), and Taq DNA polymerase (1
U). The primers used in this reaction were synthesized at TAGC (Berlin, Germany) including universal primers (ITS1 [5′TCCGTAGGTGAACCTGCGG-3′] and ITS2 [5′GCTGCGTTCTTCATCGATGC-3′]) [
12] and
C.
albicans-specific primers (CA3 [5′-GGTTTGCTTGAAAGACGGTAG-3′] and CA4 [5′-AGTTTGAAGATATACGTGGTAG-3′]). A conserved portion of the 18S
rDNA region, the adjacent ITS1, and a portion of the 28S
rDNA region were amplified, using the ITS1 and ITS2 primers. A portion of the ITS2 region of
C.
albicans was amplified by including CA3 and CA4 in the PCR mixture. PCR amplification process was carried out with an Eppendorf thermal cycler under the following conditions: initial denaturation (94°C, 3 min); 35 cycles of denaturation (94°C, 1
min), annealing (60°C, 1
min), and extension (72°C, 1
min); final extension (72°C, 5
min). PCR products were analyzed by electrophoresis through a 2% agarose gel (Roche) containing ethidium bromide (Sigma), and UV visualization were performed according to the protocols provided (UVdoc, GAS9000, England). The length of the bands was measured by UVIsoft software. Positive controls were included in each PCR experiment and consisted of one strain of each
C.
albicans ATCC14053 (218 or 219, and 110
bp),
C.
glabrata CBS2175 (482 or 483
bp),
C.
parapsilosis CBS2195 (229
bp),
C.
tropicalis CBS94 (218
bp), and
C.
krusei CBS573 (182
bp). Pyrogen-free water was used as negative control. Two DNA bands were identified for
C.
albicans while one DNA band was corresponded to other
Candida species. An assessment of the different species of RVVC and NRVVC was also made. Descriptive statistics, chi square test, and Spearman's correlation were used to analyze the data.
