Analysis Of Yeast Species Of Patients With Recurrent Vulvovaginal Candidiasis

BackgroundVulvovaginal candidosis (VVC) is one of the most common vaginal infections and accounts for 40%-50% of all cases of infectious vulvovaginitis. Seventy percent to 75% of sexually active women will experience at least 1 episode of VVC in their lifetime, and 5%-8% of adult women have recurrent VVC (RVVC), which is defined as≥4 episodes per year. Because of its repeated recurrence and prolonged unhealed, it seriously interfere with women's physical and mental health. There are some factors related to the occurrence of RVVC generally:①the existence of sub-clinical yeast;②enhanced pathogen virulence;③intestinal infection;④sexually transmitted;⑤weakening of host defense function.RVVC becomes much more refractory because of the wide range and repeated applications of antifungal agents, which affects the quality of patients'life. Due to the increase in pathogenic Candida species and the deficiency sensitivity or have a natural resistance to common medicine of anti-Candida in C. glabrata, C. krusei, it has brought new issues to the diagnosis and treatment of Candida infections, so identification of pathogenic strains of Candida is of great significance to trace infection sources and assist in diagnosis and treatment. C. albicans is the most common causative organism for VVC. Because of its multi-variability, C. albicans can survive in many anatomic site of the host and has its own specific system to adapt to the environment. This means that the disease spectrum of C. albicans and other Candida species is much more widely than most other symbiotic microbes. It has been found that the Candida genotype infected by patients with RVVC has not changed, which always infects with strains of the same clone. Someone has biotyped and genotyped of C. albicans isolated from different anatomical of 2 continuous episodes, and the results suggested that the majority infections of RVVC were endogenous. The isolates from the patients and their male partners'vulva are of the same or highly relevant, which indicated that the dominant strains of the vagina can not only cause to its own repeated infection but also can be transmitted to their sexual partners. But some studies have found that treatment of intestinal infection and sexual partners wound not reduce the recurrence of RVVC. Therefore, it is necessary to study further the C. albicans from different parts in order to understand whether the genetype of strains are the same.Conventional taxonomy of yeasts such as the API20 and Bio-Log systerm mainly relies on cellular morphology and physiological properties characterized by a standardized set of fermentation, assimilation and other biochemical tests. Sometimes it difficult to ensure the accuracy of identification to rely on physiological and biochemical reactions. The development of molecular biology techniques provides a more effective means for the classification of Candida at the DNA level. The molecular biology methods, such as the homology or relevance of DNA-DNA (or DNA-RNA), rRNA/rDNA sequence analysis, PFGE karyotype analysis, random amplified polymorphic of DNA fragments make wide range of applications in the taxonomic study of Candida, improving our understanding of Candida to a great degree. In which the sequence analysis of ribosomal RNA (rRNA) or ribosomal DNA (rDNA) is playing an increasingly important role in the taxonomic study of yeast. The rDNA gene of Candida is repetitive sequence of beaded, about 100 to 200 copies, including the gene of coding 28sDNA,18sDNA and 5.8sDNA. The intervening spacer region among these sequences is internal transcribed spacers (ITS), which consisting of two parts ITSI and 1TS2. Because of rapid evolution and qualities of species-specific and intraspecific conservative, ITS is good for species classified, but high sequencing cost limits its application.Single-strand conformation polymorphism (SSCP) technology makes the detection technology widely use. SSCP analysis is one of the most common methods to detect genetic mutation. The theory is based on the secondary or tertiary conformation with sequence-specific of single-stranded DNA. The variation of one or more of the bases can affect its conformation, and the changes of conformation is performed as change of migration rate of single chain DNA in non-denaturing polyacrylamide gel electrophoresis. Therefore, through heating or denaturant, the double-stranded DNA becomes to single-chain, and then we can distinguish the mutants and non-mutant by various single-chain mobility of sample in non-denaturing polyacrylamide gel electrophoresis, achieve the purpose of isolates.There are many methods for typing C. albicans, of which micro-satellite sequence polymorphism analysis method is based on PCR amplification of DNA fingerprint analysis. Micro-satellite that is short tandem repeats (STR) or short tandem repeats (short sequence repeats, SSR) refers to repeat unit of a few nucleotides (mostly 2-4) of DNA sequences. Tt has a significant difference between different strains of the same species in number of repetition of oligonucleotide, so microsatellite markers can be used for molecular typing of strains. The CAI microsatellite sequences in non-coding region on 4th chromosome of C. albicans are highly polymorphic. It can achieve a resolution of 0.97 through scaning the gene of these area, which is the maximum resolution by far for point analysis. And it will have a good application prospects in the analysis of genotype of C. albicans because CAI area has a high specificity and reproducibility.ObjectiveIn order to explore the mechanisms of RVVC relapse, to investigate whether there was a link between RVVC and the genotype of C. albicans, we collected the discharge of vagina,anus,oral cavity of patient with RVVC and the glans of their sexual partners, identified the species of Candida and the genotypes of C. albicans which was major pathogenic Candida.Materials and Methods1,55 patients diagnosed as RVVC had been selected in our experiment in out-patient of Zhujiang Hospital from July 2008 to October 2008. They were not history of diabetes, long-term use of antibiotics and immunosuppressive agents aged from 20-45 years, mean age (29±7) years. Patient'conditions have been scored according to scoring criteria about the norm in diagnosis and treatment of vulvovaginal candidiasis, which was made by Group of Infectious Disease, Society of Obstetrics and Gynecology, Chinese Medical Association.2,Samples were collected from the discharge of vagina, rectum, oral cavity in 55 patients and the discharge of balanus in 45 sexual partners of patients with sterile cotton swab. All the samples with 10%KOH were examined by microscope, and we can see the hyphae or spores with high power lens, cultured directly on Sabouraud-chloramphenicel glucose agar and incubated at 28℃for 48-72 hours. Single cheese-like colony was selected to culture on YPD medium for three times, a single colony was inoculated into wort medium for activation. 3,The total DNA of Candida was extracted by using of enzymatic digestion. A multiple PCR was used to amplify the ITS. And the strains with representative pattern of ITS-SSCP were analysed by D1/D2 sequence analysis. Then we can identify the species of Candida by comparing the sequence and the known sequences of Candida in the EMBL/DDBJ/GenBank.4,All of the C. albicans were analysed by CAI-SSCP, and the strains with different SSCP patterns were amplfity by the primers CAIF (5'fluorescently labeled with 6-carboxyfluorescein) and CAIR. The fluorescently labeled PCR products were run in an ABI 370 analyzer. Fragment sizes were determined automatically using the GeneScan 3.7 analysis software. The CAI genotypes were determined at the criteria of the repeated fragment (27-38) of two allele loci in the CAI area of the standard strain ATCC90028.5, SPSS13.0 statistical package were used to statistical analysis. The chi-square test was used to compare the difference of the rate between two groups; The difference of groups of measurement data was compared using two sample t test. These tests were of statistical significance by P<0.05.Results1,The positive rates Candida isolated from the vagina,oral and rectum of patients with RVVC and the balanus of their sexual partners were100.0%(55/55),61.8%(34/55),1.8%(1/55),60.0%(27/45) respectively. Among 55 samples from vagina of patients with RVVC, C. albicans was found in 45 patients (81.8%), C. glabrata in 8(14.5%), C. tropicalis and C. parapsilosis in 1 (1.8%)respectively; there were 29 strains (82.4%) of C. albicans,4 strains (11.8%) of C. glabrata,1 strain(2.9%) of C. parapsilosis from rectum secretions; only 1 strain of C. albicans was isolated from the secretions of oral; Among 45 samples from balanus of the sexual partners, C. albicans was found in 22 (81.5%), C. glabrata in 4 (14.8%), C. tropicalis in 1 (3.7%). There was no significantly different distribution of the Candida species among the strains which isolated from the vigina, anel and glan (x2=2.371, P=0.984).2,There were 12 genotypes of 45 C. albicans, with genotypes 30-45(12 strains, 26.7%) and 32-46(16 strains,35.6%) being the most common, accounting for 62.2% totally, showing the distribution of apparent advantages. Compared with the proportion reported by the predecessor in 28 strains of uncomplicated VVC, there was significantly difference (62.2%vs.35.7%; x2=4.860, P=0.027).3,There were 12 genotypes of C. albicans isolated from anal secretions of patients with RVVC, and the coincidence of C. albicans genotype between the discharge from vagina and rectum were 86.2% in the same person. There were 10 genotypes of C. albicans isolated from the balanus secretion of the sexual partners', anymore half of the C. albicans genotypes of the discharge from the vagina of RVVC patients were same as the discharge from the balanus of her sexual partner.4,There were no significantly difference in clinical score between dominant genotype and non-dominant genotype of C. albicans corresponding with RVVC (t=0.238, P=0.813).Conclusions1. There was no difference in distribution of Candida from the vagina,rectum of patients with RWC and balanus of the sexual partners. C.albicans was still the dominant stain and C. glabrata was the more common in non-albicans.2,The genotypes of CAI 30-45 and CAI 32-46 of C.albicans were likely to have a stronger virulence to the vaginal mucous membrane. But there was no correlation between the dominant genotype and the severity of clinical symptoms in patients with RVVC, suggested that there was some relation between these genotypes and the recurrence of RVVC.3. Intestinal infections and sexual transmission may be play a role of the recurrence of RVVC, so it was valuable of systemic management and simultaneous treatment of the sexual partners.