Analysis Of The Infection Source And Genotype Of Candida Albicans For Recurrent Vulvovaginal Candidiasis

BackgroundVulvovaginal candidosis (VVC) is one of the most common vaginal infections and accounts for40%-50%of all cases of infectious vulvovaginitis. Seventy percent to75%of sexually active women will experience at least one episode of VVC in their lifetime. Vulvovaginal candidosis was divided into uncomplicated VVC and complicated VVC based on the epidemiology, microbiology, clinical manifestations and host performance. Complicated VVC including severe VVC, non-Candida albicans VVC, recurrent VVC (RVVC), VVC with diabetes, VVC with pregnancy and VVC with immunocompromised. RVVC is defined as four episodes per year or more than four times. Because of its repeated recurrence and prolonged unhealed, it seriously interfere with women’s physical and mental health.There are a variety factors related to the occurrence of RVVC and its pathogenesis has not yet entirely clear. According to the etiology, factors can be divided into two groups:the host and pathogen. At present, different researches showed a lot of reports in these two areas. In recent decades, animal and clinical studies are were displayed the results:adaptive immunity may not be the major protective immune mechanism, natural immunity is considered to be the main defense mechanism of mucosal Candida infections. In terms of the pathogen, the source and pathogenicity of the pathogen become the hot and focus of all researches.For the pathogens that cause recurrent VVC is still an uncertain controversy all over the world. Different studies showed different results, this may be due to the particularity of the reproductive system and extensive colonization of Candida in body. There are three aspects of the hypothesis about the recurrent VVC:①The existence of vaginal sub-clinical yeast:Candida is one of the normal floras of the vaginal microenvironment,5-10%of asymptomatic populations for carrier. The genotype analysis of strains from the same asymptomatic person of different anatomic sites and different individuals of the same anatomic site showed that the strains from the same individual of different anatomical parts have different genotypes and the genotypes of strains isolated from different individuals of the same anatomic site are same or similar. Foreign scholars were studied the biotyping and genotyping of Candida albican strains which were isolated from different anatomic sites in patients with twice recurrent episodes and results showed that most VVC recurrence is endogenous. Meanwhile there was found a vaginal colonization strain in USA. Scholars in our country analysis the relationship of the genotype of Candida alb cans and its colonized site according to molecular genetics pointed out that the micro-environment of the planting site selective colonization and vaginal isolates was vaginal specific. Other countries studies found that after antifungal therapy,20-25%VVC patients were infection negative followed by the vaginal secretions of fungi detected, if the detected was taken30days after treatment, most patients again showed positive; and the pathogenic Candida species treatment before and after the two acute attacks more than2/3are the same. These residual strains of low concentration are likely to lead to the next relapse. Genotype analysis of Candida albican from asymptomatic carrier and vaginitis patients found that two groups of flora genotype distribution of similarity, suggesting the occurrence of VVC is infection of the vaginal flora. At present, some academics have suggested that the endometrial may be a storage pool of Candida, with the shedding of the endometrial during menstrual cycle and cause infection, but without any experimental data to support this argument.②Intestinal infection:Candida as Conditional pathogens parasitic in the oral cavity, gastrointestinal tract, vagina, and other parts of the mucosal folds. Researchers have proposed the anus can become the initial source of vaginal Candida colonization, and isolates of Candida from the rectum and vagina with the same or similar character. The intestine is a long-lasting storage of Candida vaginitis pathogens. The recent analysis found that the genotype distribution of Candida albicans isolated from the genital and other parts of the same patient has a significant difference.③Sexually transmitted:Candida albican is the main pathogen of the male genital Candida infections, sexcual intercourse or other sexual activities can cause cross-infection of Candida has not been conclusive. The genital candidiasis is not classified in the context of sexually transmitted diseases in our country.The study of RV-VC patients and their husbands found Ca3type Candida albican from Vulvovaginal can be isolated from male at least one part of body. The analysis of Candida strains from RVVC patients and asymptomatic sexual partners of different anatomical parts showed that most asymptomatic sexual partners can be isolated the same genotype strain with RVVC patients. Studies have shown that the genital of asymptomatic partner of VVC patient carry Candidas as four times as sexual partners of non-VVC patient carrying. Another study showed20%sexual partners of RVVC patients had the penile Candida infection, and infected the same strain of Candida with RVVC parients, and the genotype distribution of Candida albican between Candida balanitis and Candida vaginitis was very similar, even more has the same dominant genotype. Prompting from another aspect that the male genitalia and vaginitis pathogenic Candida albicans exists the possibility of sexual transmission. But one research showed the treatment of sexual partners will not affect the cure rate and the relapse rate of RVVC.With the development of the molecular genetics research on Candida, to explore the relationship of Candida albicans pathogenicity and disease type from gene level become a trend, and a lot of works were done by researchers from different countries and regions. The beginning is the strain fingerprint analysis in1985by Burnie et al. the genotyping method of Candida albicans continue to make breakthrough with using restriction fragment length polymorphism analysis and microsatellite analysis. The micro-satellite sequence polymorphism analysis method is based on polymerase chain reaction(PCR) amplification of DNA fingerprint analysis. Micro-satellite that is short tandem repeats (STR) or short tandem repeats (SSR) refers to repeat unit of a few nucleotides (mostly2-4) of DNA sequences. There is a big difference between different strains of the same species in number of repetition of oligonucleotide, so microsatellite marker can be used for molecular typing of strains. Sampaio reported the CAI microsatellite sequences in non-coding region on fourth chromosome of Candida albican are highly polymorphic. It can achieve a resolution of0.97through scaning the gene of these area, which is the maximum resolution by far for point analysis. Single-strand conformation polymorphism (SSCP) technology makes the detection technology widely used. 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 change 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, 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 and with high sensitivity. Introduce SSCP techlonogy into the genotype analysis of the CA1region of Candida albican and combined with the GeneScan technology were economic, convenient and accurate in genotyping analysis of large sample clinical source of pathogenic Candida albican.In summary, the source of RVVC recurrent infection is vaginal endogenous recurrence or exogenous infections? Sexually transmitted whether is an important route of transmission of the onset and recurrence in RVVC patients? Some scholars at home and abroad have done researches in this regards, various hypotheses still need more in-depth research evidence to support. In this study, by the analysis the characteristics of pathogenic Candida species from different recurrent episodes of RVVC patients, and the genotype distribution of Candida albican associated with RVVC and sporadic VVC and asymptomatic person, designed to further explore the etiology of recurrence for RVVC and the relationship between the genetype of Candida albicans and recurrence of RVVC.ObjectiveIn order to identify the main pathogen Candida strains of RVVC through analysis the Candida species distribution of recurrent episodes from vaginal secretions; To study the genotype distribution of Candida albicans between episodes of RVVC patients and assess the relationship of the clinical symptoms severity of RVVC patients and pathogen species and Candida albican genotype; Investigate the possible infection routes of RVVC recurrence in the pathogens and the relationship of genotype variability of Candida albican and RVVC.Materials and Methods1、The study inclusion criteria:The ages of the patients varied from18to50years (with an average of32.5years). Women who were diabetic, HIV-positive, or pregnant were excluded.The diagnostic criteria of RVVC and sporadic VVC:patients suffered four or more episodes of VVC per year were recorded as RVVC and less than four episodes of VVC per year called sporadic VVC by re-attend or telephone follow-up. A total of80RVV^66VVC patients attending the clinics of the Gynecologic Department, Southern University Zhujiang Hospital (southern China) during the period from September2009to October2010were enrolled in this study. Meanwhile, to culture vaginal discharge from298healthy women without clinical symptoms and signs in June-August2010who take physical examination in Physical examination center, and make sure each patient vaginal secretions microscopy negative. Informed consent was obtained from the patient before entry. History of similar episodes was recorded about every patient. Vaginal swabs were taken and the women were norm-treated, patient has a return visit one or two week after treatment to make sure whether cure. Women were cured and be asked to re-attend if their symptoms recurred. The vaginal swabs were repeated at each visit.We scored the severity of clinical symptoms 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、Specimen collection:Samples were collected from the discharge of vagina with sterile cotton swab. The samples from vagina with10%KOH were examined by microscope, and we can see the hyphae or spores with high power lens. Direct inoculation of the fresh samples in enrichment YPD liquid medium with chloramphenicol to the liquid turbid, the turbidity bacteria liquid plated on Sabouraud-chloramphenicel glucose agar and incubated at28℃for48-72hours. Then the single cheese-like colony was selected to culture on YPD medium, after three times repeated purification, a single colony was inoculated into wort medium for activation.3、Strain identification:The total DNA of Candida was extracted by using of enzymatic digestion. A multiple PCR was employed to amplify the ITS1. And the strains with representative pattern of ITS-SSCP were analysed by ITS or D1/D2sequence analysis. Then we can identify the species of Candida by comparing the sequence with the known sequences of Candida in the EMBL/DDBJ/GenBank.4、Candida albican genotype analysis: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 with6-carboxyfluorescein) and CAIR. The fluorescently labeled PCR products were run in an ABI370analyzer. Fragment sizes were determined automatically using the GeneScan3.7analysis software. The CAI genotype was determined at the criteria of the repeated fragment (27-38) of two allele loci in the CAI area of the standard strain ATCC90028.5、SPSS13.0statistical package used for statistical analysis. The chi-square test was used to compare the difference of the rate between two groups, if theoretical number is less than5using calibration, if the theoretical value is less than one using the exact probability method; 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 RVVC patient、VVC patient and Asymptomatic person were100.0%(132/132),100%(66/66),9.7%(29/298), respectively. Among132strains from vagina of RVVC patients, C. albicans was found in89cases (67.4%), C. glabrata in38cases (28.0%), C. parapsilosis in2cases (1.8%),C. guilliermondii in1case(0.8%), C. nivariensis in2cases (1.5%) Issatchenkia orientalis in1case (0.8%) respectively; Among the Candida strains, including2to4specimens from recurrent episodes of RVVC patients.66strains were cultured from sporadic VVC patients, there were51strains (77.3%) of C. albicans,14strains (21.2%) of C. glabrata,1strain(2.9%) of C. tropicalis; For asymptomatic person,29strains were cultured including13strains (44.8%) of C.albican,13strains (44.8%) of C.glabrata,2strains (6.9%) of C. parapsilosis and1strain (3.4%) of C. tropicalis. There was significantly different distribution of the Candida species among the strains which isolated from the three group (χ2=9.653, P=0.008).C.albican is still the main pathogens in RVVC and VVC patients. However, There was no significantly different distribution of the C.albican and no-C.albican in RVVC and VVC patients (χ2=2.060, P=0.151)2、There were38CAI genotypes of153C. albicans,140C. albicans from VVC and RVVC with genotypes30-45(45strains,32.1%)、32-46(23strains,16.4%) being the most common, some genotypes(30-32-43-47) were only one or two three-base fragment in CAI fragment repeat different with genotypes30-45and32-46, all of them accounted for66.0%, which was23.08%(3/13) in asymptomatic person. There was significantly different dominant genotype distribution of C.albicna in VVC and Asymptomatic person (χ2=9.676, P=0.002). Besides, dominant genotype of C.albicna from RVVC patient were72.0%(64/89), and from VVC patients were66.7%(34/51), the difference has no statistically significance(χ2=0.424, P=0.515).3,36patients diagnosed with RVVC have been followed up to isolated symptomatic recurrences strains, six persons complicated three times samples and five patients have four samples, the others be followed up two times.67Candida albicans and21non-Candida albicans were isolated.20CAI genotypes were recognized in the67strains from recurrent episodes of RVVC. Genotype30-45and32-46respectively accounted for23strains (34.3%) and11strains (16.4%), genotype32-47was7strains (104%), The other17genotype identified comprised1-3strains. The Candida species identical trend between initial and recurrences was77.8%(28/36), including25pair of Candida albicans and3pairs of Candida glabrata. Strains were isolated from two recurrent episodes of one patient with identical CAI genotypes in19cases, account for76%(19/25). The dominant genotypes in these samples still were30-45(8patients),32-46(4patients),32-47(2patients), the other genotypes were record in one patient.4, Among the198VVC patients, Including95severe VVC and103mild-moderate VVC patients. To the95severe VVC patients, C. albican were isolated from59cases (62.1%), C. glabrata from31cases (32.6%), C. parapsilosis from1case (1.0%), C. tropicalis from1case (1.0%), and other yeasts from3cases (3.1%). In the103mild-moderate VVC patients, C. albican was isolated from81cases (78.6%), C. glabrata from21cases (20.4%), and C. parapsilosis from1case (1.0%). The infection of Candida albicans and non-Candida albicans in severe and mild-moderate VVC patients showing a significant difference (χ2=6.524, P=0.011). the severity of RVVC and sporadic VVC patients study showed that there was no Significantly difference (χ2=0.496, P=0481)5、The genotype distribution of Candida albicnas from59severe VVC patients and81mild-moderate patients, including2dominant genotypes30-45(30strains,49.2%) and32-46(8strains,13.6%) in severe VVC patients, and the two dominant genotype were (15strains,18.5%) and (15strains,18.5%) in mild-moderate VVC patients. Meanwhile, genotype (30-32～44-47) which was only one or two three-base fragment in CAI fragment repeat different with genotypes30-45and32-46were14strains and16strains in severe and mild-moderate VVC patients. The dominant genotype of Candida albicans accounted for88.1%(52/59) and56.8%(46/81) in two groups. There was Significantly difference in severe and mild-moderate VVC patients (χ2=15.971, P=0.000)Conclusion 1、We found that Candida albicans alone remain the most common pathogenic strain for VVC, there was a higher rate infection of non-C.albican VVC.2、The genotype distribution of Candida albicans isolated from asymptomatic population was similar to vaginitis patients, which is different with the Candida albicans isolated from non-genital colonization of the body, explaining the importance of the vaginal endogenous infection.3、Genotype distributions were more concentrated in dominant genotypes30-45and32-46of Candida albicans isolated from vaginal, suggested there is a relationship between genotype of C.albicans and vaginisis.4、Strains isolated from initial and recurrent episodes of RVVC patients are identical in more than two-thirds of recurrences, The analysis of the genetic relatedness of C.albican strains pretreatment and recurrent episodes post-treatment demonstrated that the same genotypic C.albican strains maintenance in the majority of women. Our study supported the" vaginal relapse" hypothesis in RVVC patients and there is a relationship between dominant genotype of C.albican and vaginisis.5、Our study suggests a correlation between infection by the Candida strains and the severity of VVC. Patients with severe VVC had a lower infection rate by C.albican than those with mild-moderated VVC. The genotype distribution of C. albican was related to the severity of VVC. The dominant genotype was found more frequently in strains isolated from patients with severe VVC than in strains from patients with mild-moderate VVC, supported the associated with genotype with pathogenicity of C.albican.