| [Objective] First, Based on the previous study, through the statistics of March,2009to March,2011STD clinic persistent genital Chlamydial infection(GCI) patients clinical data, analysis of patients with persistent GCI related factors were made and evidence were provided for better controlling genital Chlamydial infection to improve the quality of life. Second, To analyze the association HLA-DQA1gene polymorphism with persistent GCI susceptibility in order to find possible predisposing genes. Trying to explain the immuno-pathway, to achieve the prompting of the high-risk group of persistent GCI, then strengthening the early prophylaxis. Third, To observe the effects of exogenous indole on the growth of the Chlamydia trachomatis(C.t) serotype E-UW-5/Cx laboratory strains and clinical strains for better controlling persistent genital Chlamydial infection in clinics.[Methods] First, To evaluate the clinical features including general data, the distribution of the types of occupation, educational level, the occupation of the sex partners, the concurrent infections of the patients and summerize the epidemiological characteristics of the patients with persistent genital Chlamydia infection in Tianjin by analyzing the clinical statistics of the GCI venereal outpatients in Tianjin Medical University General Hospital during March2009to March2011. Second, EDTA anticoagulant whole blood from the cubital vein of patients which were divided into three groups:the one with persistent GCI patients, general GCI patients and healthy people.2ml whole blood was collected for each patient. There were80patients for each group. Genome DNA were extracted from the blood samples using TKM method. The distribution of alleles and genotype of HLA-DQA1and HLA-DQB1were detected by polymerase chain reaction(PCR) and gene sequencing technique. Then the gene polymorphism of the persistent genital Chlamydial infection was studied to screen the susceptibility alleles and analyze the correlation between HLA-DQA1, HLA-DQB1allele and the persistent genital Chlamydia infection. Third, To observe the effects of exogenous indole on the growth of the Chlamydia trachomatis(C.t) serotype E-UW-5/Cx laboratory strains and clinical strains.(一) Tryptophan-starved McCoy monolayers in96-well plates were infected with reference serovars E and D and clinical isolates at an moi of0.1. Infected cells were fed with DMEM without L-tryptophan. This medium is referred to as DMEM-10(-Trp). The medium was replaced at20-24hours after infection with complete DMEM-10, DMEM-10(-Trp), or DMEM-10(-Trp) plus10μM indole. Monolayers were fixed in methanol at48hours after infection and stained by iodine to enumerate inclusion forming units (IFUs). The monolayers were examined for inclusions by inverted microscopy.(二) Monolayers of McCoy cells in6-well plates were inoculated with reference serovars E and D and clinical isolates EBs at an moi of3-5. The infected wells were incubated with complete DMEM-10as a control or with complete DMEM-10further supplemented with5ng/ml recombinant human IFN-y with or without the addition of50μM indole. Following incubation for48-72hours at37℃, the culture supernatant was collected and the McCoy cells were lysed with cold distilled H2O. Aliquots of the combined McCoy cell lysates and culture medium were used to infect fresh McCoy cell monolayers for enumeration of recoverable inclusion forming units (IFUs).[Result] First, There were1599cases of patient with general GCI in the sexually transmitted diseases clinics in Tianjin Medical University General Hospital during the March2009and March2011, cases varied from724to875, demonstrating a slight increasing trend of the disease.74.79%of the patients aged20-39years old whowere sexually active. Sex distribution showed that the male patients outnumbered the female ones, accounting for68.38%(x2=8.1479,P=0.0043) of the patients. Most of them were married, accounting for76.50%. Most of the patients with general infection own secondary school educational level, accounting for41.45%(x2=4.9627,P=0.0259). Of all kinds of occupations, workers made the greatest percentage of them, about41.67%(%2=6.8248, P=0.0090). The sexual partners mainly worked as the attendants in the hotels, restaurants, bath centers, singing rooms and other sex workers which accounted for30.56%(x2=6.1601, P=0.0131). Most of the patients were local residents, accounting for64.29%(x2=0.5866, P=0.4437). There were158cases of persistent GCI during March2009and March2011. The20-39age group owned the highest incidence rate which is75.86%(x2=0.0574, P=0.8107), as for gender distribution, the probability of female patients suffering from persistent infection was higher than the male ones, the incidence rate was13.27%(x2=17.9217, P=0.0001). Most of the persistent infection patients were married, accounting for81.90%(x2=1.5584, P=0.2119), the people with Bachelor Degree who were well-educated accounted for38.79%(x2=4.1177, P=0.0424) of the patients. Of all types of occupations, the commercial occupation was the most common one with persistent infection, accounting for36.21%(%2=6.4013, P=0.0114), the sexual partners of the persistent infection patients mainly worked as the attendants in the hotels, restaurants, bath centers, singing rooms and other sex workers or the spouse, accounting for31.03%(x2=5.4969, P=0.0190). Most of the patients were local, accounting for68.10%(%2=0.5866, P=0.4437), the percentage of the patients complicated with syphilis, candida albicans, mycoplasms and multiple infection was higher in persistent infection group than the one of general infection group(x2=4.9614, P=0.0259). Second, The frequency of HLA-DQA1*0102allele, HLA-DQA1*0501allele and HLA-DQB1*0602allele in patients with persistent genital Chlamydial infection was22.5%,5%and45%, respectively. While the frequency of HLA-DQA1*0102allele, HLA-DQA1*0501allele and HLA-DQB1*0602allele in patients with general genital Chlamydial infection was5%,20%and15%, respectively. Then the frequency of HLA-DQA1*0102allele, HLA-DQA1*0501allele and HLA-DQB1*0602allele in healthy controls was2.5%,17.5%and22.5%, respectively. It was found that the frequency of DQA1*0102(x2=14.6286, P<0.001) and DQB1*0602(x2=14.0800, P<0.001) was significantly increased in patients with persistent genital Chlamydial infection, while the frequency of DQA1*0501was significantly decreased (x2=6.2598, P<0.05). Third, Tryptophan-starved reference serovar E and clinical isolates were fed with complete DMEM-10, DMEM-10(-Trp) and DMEM-10(-Trp) plus10μM indole, respectively. The number of inclusion for group of DMEM-10(-Trp) was significantly decreased than the other two groups (P<0.01). There were no significant difference between the group of DMEM-10and group of DMEM-10(-Trp)+IND (P>0.05). The laboratory reference serovar E and clinical isolates were incubated with complete DMEM-10as a control or with complete DMEM-10further supplemented with5ng/ml recombinant human IFN-y with or without the addition of50μM indole, respectively. The number of inclusion for group DMEM-10+IFN was significantly decreased than the other two groups (P<0.01). There were no significant difference between the group of DMEM-10and group of DMEM-10+IFN+IND (P>0.05). Hence, the result demonstrated that genital strain E maintains a functional synthase capable of using indole for tryptophan biosynthesis to escape IFN-y-mediated eradication and thus establish persistent infection. Moreover, functional enzyme activity was directly correlated to IFN-y resistance through an indole rescue mechanism. The similar results were achieved by the experiments with serovar D laboratory and clinical strains.[Conclusions] First, Most of the persistent GCI patients aged20-39years old who are sexually active. Female patients owns the higher incidence rate than the male ones. Most of them are married and well-educated. Of all types of occupations, the commercial occupation is the most common one with persistent infection. The sexual partners of the persistent infection patients mainly work as the attendants in the hotels, restaurants, bath centers, singing rooms and other sex workers or the spouse. Most of the patients are local residents, the percentage of the patients complicated with syphilis, Candida albicans, mycoplasms and multiple infection is higher in persistent infection group than the one of general infection group. Second, HLA-DQA1*0102and HLA-DQB1*0602allele may be the susceptible gene or have close linkage with susceptible genes. While HLA-DQA1*0501allele may have protective effects on development of persistent genital Chlamydial infection in the Chinese. HLA can be used as a genetic marker, reminding us of the group with high-risk to develop chronic persistent genital Chlamydial infection, then strengthening the earley prophylaxis to the chronic persistent genital Chlamydial infection. Third, Tryptophan-starved reference serovar E and clinical isolates maintains a functional synthase capable of using exogenous indole for tryptophan biosynthesis to escape IFN-γ-mediated eradication and thus establish persistent infection. |
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