Molecular Epidemiological Studies On Genital Infections With Chlamydia Trachomatis In Women At High Risk

Genital Chlamydia trachomatis (C. trachomatis) infection is one of the most prevalent sexually transmitted diseases (STDs) in the world. In the recent five years, reported number and incidence of genital C. trachomatis infection has increased dramatically to be the highest among all reported STDs in many areas or countries, such as North America, Europe and Southeast Asia. After its invasion, C. trachomatis can induce type-specific cellular and humoral immune response, and the infected individual can only produce a too weak and temporal anti-infection capability to clean the pathogens in vivo, or protect the body against potential infections of other types of C. trachomatis in the future, resulting in the persist sub-clinical infection or relapsing infection of genital C. trachomatis. A serial of clinical manifestations can be observed in cases of genital C. trachomatis infection. For instance, non-gonococcal urethritis (NGU), post-gonococcal urethritis, epididymitis, prostatitis can occur in males, and cervicitis, urethritis, pelvic inflammation disease in females. In addition, pregnant women with genital C. trachomatis infection can deliver the pathogen to their neonates through reproductive tract, causing severe fatal infections such as neonatal conjunctivitis and Chlamydial pneumonia. C. trachomatis infection leads to tube-based infertility, extra-uterine pregnancy and other inconvertible complications if a timely treatment is not given. Results from many studies have shown a large number of individuals with asymptomatic infection or sub-clinical infection with a slight clinical manifestation. Such conditions can cause the pernicious consequences because of the delay in detection, diagnosis and treatment of the infections. According to the data from World Health Organization and reports from several countries, incidence of genital C. trachomatis infection is not only the highest one among all STDs, but also shows the rapid increase. Though a lot of prevention and intervention strategies have been implemented against genital C. trachomaits infections as well as other STDs, the efficacy is not as good as expected. Taking into account these challenges, after innovative approaches have been developed, for instance, a population based C. trachomatis screening program has been introduced in UK; a molecular epidemiology study on genital C. trachomatis genotyping based on ompl PCR-RFLP has been explored in counties (or areas) like Japan, Thailand, Korea, India and Taiwan, in order to find out new strageties to improve C. trachomatis prevention and control. In Chinese STD surveillance system, genital C. trachomatis infection is not considered as a separate disease in the list of seven notifiable STDs, but included into NGU for case-reporting. With these limitations, it is therefore not possible to precisely estimate the C. trachoamtis infection rate, incidence and disease burden, or evaluate the effectiveness of strategies against C. trachomatis infections. According to data from Chinese national STD reporting system in resent five years, the incidence of NGU, mainly caused by C. trachornatis, is the highest among all reported STDs, indicating that C. trachomatis infection in China is the same challenge as that in many other countries. With these considerations, the current study is proposed with following contents.Chapter 1. Collection and Identification of A～K and L1～L3 C. trachomatis reference strainsWe set up storage center of C. trachomatis reference strains through two channels. SerotypeA,B,C,D,F,G,H,I,J,K,L1,L2,L3 strains were donated by US CDC, and serotype E was bought from ATCC. According to the C. trachomatis characteristics of lacking enzymes for synthesizing ATP energy, the survival depends upon the host cell energy and nutrients. Therefore, C. trachomatis is a strict cytozoic microorganism. Regarding the characteristics and the current condition in our lab, and using McCoy cell as the host cell and 1640 culture medium containing 10% bovine serum with 1%o cycloheximide as the medium, all fifteen reference strains were successfully cultured, and elementary bodies were observed under microscope by using iodine staining method. Then we designed the specific primers for ompl gene amplification, and identified the strains by PCR-RFLP. With the above mentioned efforts, we built up a relatively completed reference C. trachomaits serotype collection in China. Chapter 2. C. trachomaits Plasmid PCR and Detection MethodAlmost all isolates of C. trachomatis have cryptic plasmid. And all plasmids from human C. trachomatis isolates are extremely similar, with a variation of less than 1% nueleotide sequence. The plasmid DNA has about 7,500 nucleotides in size with eight open reading frames (ORFs). Among which five ORFs are functionally coded for proteins of more than 100 amino acids with short non-coding sequences between some of them. All Chlamydia plasmids have four 22-base-pair tandem repeats in the intergenic region between ORFs 1 and 8, where the origin of replication is located. C. trachomaits has 7-10 copies of cryptic plasmid gene and 1 copy of ompl gene, the plasmid PCR is more sensitive than omp1 PCR in detecting C. trachomatis infection in the same samples, even there is a micro-density of plasmid gene in those samples. Based on the primers in Roche Amplicor CT/NG Preparation Kit and research result from Bass, we designed CP24 and CP27 primers, and successfully amplified plasmid gene from reference strains and clinical specimens.Chapter 3. C. trachomaits Ompl PCR-RFLP MethodMajor Outer Membrane Protein (MOMP) is the principal protein among all C. trachomatis membrane proteins. Sixty percent molecular weight of total membrane proteins is attributed to MOMP. The MOMP is also the important immuno-dominant surface antigen of C. trachomatis, with antigenic determinants located across four symmetrically spaces variable domains (VD1～VD4), which are flanked and interspaced by five constant domains (CDs). C. trachomatis has been classified into 15 basic serotypes based on immunogenic epitope analysis of the MOMP through monoclonal and polyclonal antibodies. Variable domains are coded by ompl gene. Convenience and effective genotyping methods of PCR-RFLP or DNA sequencing based on the 1.1kb ompl gene in length are suitable for large scale of epidemiological study on C. trachomatis genotyping and can describe the serovar distribution in terms of population, time trend and geographical locations. Because there is only one copy of ompl gene in C. trachomatis, we need a high sensitive ompl PCR and detection method. In this chapter, we optimized PCR-RFLP assay, including DNA abstraction, selection of primers for primary and nested PCR, establishment of PCR reaction system for improving the sensitivity of ompl amplification. In addition, we found that the plasmid gene can also be amplified in the ompl PCR condition, so we set plasmid PCR as a positive control in each ompl gene amplification.Chapter 4 PCR-RFLP genotyping study on a ease with lymphogranuloma venereumA female case of lymphogranuloma venereum (LGV) was confirmatofily diagnosed by an aggregated analysis of clinical data and laboratory testing results of lymphnode biopsy, vaginoscopy as well as rapid test with Clearview C. trachomatis. Then we carried out the ompl amplification, Alu I digestion, polyacrylamide gel eleetrophoresis, and then designed a pair of primers to amplify and sequence the ompl CD3, VD4 and CD5, finally we identified an infection of C. trachomatis serotype L3 in this patient.Chapter 5 Clinical epidemiology study on C. trachomatis genotypingTo our understanding, our survey is one of the first epidemiological studies in China focusing on distribution analysis of genital Chlamydia trachomatis serotype in the females at high-risk using ompl gene-based restriction fragment length polymorphism (RFLP) analysis. A total of 1770 cervical swab samples from women attending sexually transmitted disease clinics and the female sex workers in six cities (Shenzhen and Guangzhou in southern China, Nanjing and Shanghai in eastern China, and Narming and Chengdu in southwestem China) were collected for serovar genotyping. We also collected 324 urethral swab specimens in male patients attending Nanning STD clinics. Among 324 samples, 92.5% (37/40) plasmid positive samples were successfully genotyped by PCR-RFLP. Among 240 C. trachomatis plasmid-positive samples was 94.2% (226/240) were successfully amplified for ornpl genes. Serotypes E (n=63; 27.9%), F (n=53; 23.5%), G (n=28; 12.4%), and D (n=25; 11.1%) were most prevalent serovars. Though there was no significant difference in the geographic distribution of the serovars, serotype E was predominant in the South (32.1%) and East (27.1%), while serotype F was predominant in the Southwest (28.3%). Serotype F infection was associated with young age and single status. Serovar G was associated with lower abdominal pain; 47.5% of asymptomatic patients were infected with serovar E. These results provide information on distribution of genital C. trachomatis serotypes among women at high-risk in China and indicate that these women, including those who are asymptomatic can be infected with multiple serovars of C. trachomatis, revealing exposure to multiple sources of infections. Although the generalizations are limited by our small sample size, our results showing the clinical correlations with genotypes are informative.