High-resolution Multilocus Sequence Typing Of Chlamydia Trachomatis Among STD Clinic Outpatients In Guangxi And Jiangsu Provinces And The Epidemic Study For Prevalence Of New Variant Of Chlamydia Trachomatis Among Female Sex Workers In Southern China

Chapter One:High-resolution multi-locus sequence typing of urogenital Chlamydia trachomatis in STD clinics outpatients in Jiangsu and Guangxi in ChinaBackgroud:Chlamydia trachomatis is the most common bacterial sexually transmitted infections (STIs) in the world. Approximately 105.7 million new Chlamydia trachomatis occurred worldwide in 2008 acrrording to the data of WHO reports. The discriminatory power of traditional ompA genotyping method is unsufficient for epidemiological purpose. MLST (multi-locus sequence typing) is a nucleotide sequence based approach for the unambiguous characterization of isolates of bacteria and other organisms via the internet. It’s used to monitor the molecular epidemiology of pathogen and also to help understand the evolution and population biology of bacteria.Objective:To enrich genetic background information of Chlamydia trachomatis in the MLST database and to help understand the molecular characteristics of Chlamydia trachomatis in China.Methods:Swabs and demographic data were collected from 99 Chlamydia trachomatis-infected patients and 16 follow-up patients who were recruited from selected STI clinics respectively from Jiangsu Province and Guangxi Zhuang region. DNA was extracted from those samples by using QIAxtractor full automatic nucleic acid purification instrument and amplified by nested PCR. DNA amplification products were sequenced and analyzed for the regions ompA, CT046 (hctB), CT058, CT144, CT172 and CT682 (pbpB). Allele numbers were assigned by comparing the sequence at each locus to all known corresponding alleles available in the MLST database (http://mlstdb.bmc.uu.se). Allele profiles are expressed as sequence type (ST). Comparison for Chlamydia STs from different areas were used BioNumerics 7 to generate a minimum spanning tree, x2 test was used to analyze the relationship between different Chlamydia trachomatis STs and clinical symptoms. Meanwhile, the situation of co-infection with Neisseria gonorrhoeae was analyzed too.Results:The DNA of Chlamydia trachomatis was extracted from six follow-up samples (37.5%,6/16). Of 105 Chlamydia-positive samples,90 samples (85.71%) were available for further typing analysis by MLST. We found seven different ompA genovars and 26 different MLST types, of which 5 STs were new to the publicly available Chlamydia trachomatis MLST database at the time of writing. There was no obvious difference in Chlamydia trachomatis genovar distributions between two provinces (Jiangsu and Guangxi), but significant difference was found in Chinese strains and Italy strains. Among 6 follow-up Chlamydia-positive samples three samples were identified as Chlamydia re-infection, and the rest were failed to obtained full MLST data. Our results indicated that the genotype J was less likely to have co-infection with Neisseria gonorrhoeae, but meanwhile E-3 and F-110 strains were found have higher co-infection rates with Neisseria gonorrhoeae. After control of the impact of Neisseria gonorrhoeae infection, we investigated that abnormal secretions were mainly caused by Genotype J and E, urethral discomfort were mainly caused by Genotype E and F, and the genotype J caused urethral discomfort was significantly lower than other genovars. In addition, D-35, F-110 and E-3 strains were more prone to cause urethral discomfort. However, further research is needed to provide rich evidences and experiences to instruct clinical practice.Conclusions:More than one third of follow-up patients were chlamydia positive. The prevalent STs of Chlamydia trachomatis in China were different to Italy. Half of Chlamydia-positive follow-up samples (3/6) were identified as re-infected. Different STs of Strains had various clinical manifestations and different co-infection rates with Neisseria gonorrhoeae.Chapter Two:Prevalence of new variant of Chlamydia trachomatis among female sex workers in southern China and the evaluation for Abbott Real Time CT/NG assay in this groupIn 2006, a new variant of Chlamydia trachomatis (nvCT) was reported in Sweden. The nvCT contains a 377-base pair (bp) deletion in the cryptic plasmid that covers the single targets originally utilized in the NAATs (nucleic acid amplification testing methods) from Abbott Laboratories (Abbott m2000) and Roche Diagnostics (Amplicor, Cobas Amplicor, Cobas TaqMan48). The nvCT caused many thousands of falsely negative diagnoses in Sweden. Since 1999 our laboratory used Roche amplicor to estimate the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae infections. So the prevalence of domestic nvCT and its impact on China’s public health also are not clear.Objective:To analyze the possible presence of new variant of Chlamydia trachomatis (nvCT) among female sex workers (FSWs) in China and evaluate the performance of the Abbott Real-Time CT/NG assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in this group in China.Methods:Cervical swabs from 997 participants were blindly detected by the Abbott Real-Time CT/NG assay on the automated m2000 molecular platform and Roche Cobas Amplicor CT/NG assay. Discrepant analyses were confirmed by the Qiagen care CT PCR assay. The sample was defined as candidate nvCT-positive if it was Chlamydia trachomatis positive in the Abbott m2000 assay, but Chlamydia trachomatis negative in the other two assays.Results:No nvCT specimens were identified in this study.25 specimens that were discordant for Chlamydia trachomatis and 26 specimens that were discordant for Neisseria gonorrhoeae between the two assays were resolved by Qiagen care CT & NG PCR assays. The sensitivity and specificity, respectively, for Abbott m2000 assay were 92.59% and 100% for Chlamydia trachomatis and 95.45% and 99.90% for NG. The positive predictive value (PPV) and negative predictive value (NPV) of Abbott m2000 assay were100% and 98.52% for Chlamydia trachomatis and 95.5% and 99.90% for Neisseria gonorrhoeae, respectively.Conclusion:There is currently no evidence that nvCT is present in FSWs in China. Abbott Real-Time CT/NG assay were more specify for Chlamydia trachomatis and Neisseria gonorrhoeae detection, however, its sensitivity for these two pathogens were a little bit lower than Roche Cobas Amplicor CT/NG assay. Abbott Real-Time CT/NG assay had higher PPV for Neisseria gonorrhoeae detection than Roche Cobas Amplicor CT/NG assay; it would be more suitable for screening for population with low prevalence Neisseria gonorrhoeae.Chapter Three:The present situation for Chlamydia trachomatis detection in ChinaObjective:To investigate the present situation of Chlamydia trachomatis testing and the detection ability in China.Method:Analyzed the 2015 Chlamydia trachomatis quality control feedback results and selected 36 medical units in five provinces to finish the questionnaire about Chlamydia trachomatis diagnostic testing.Results:Rapid immune chromatography was used to detect Chlamydia trachomatis in 214 laboratories (79.3%) of 277 participating laboratories, and in 27(84.4%) study units of 36 participating study units. Four medical units did not conduct any diagnostic testing for Chlamydia trachomatis. The sensitivity of rapid immune chromatography to detect weakly positive specimens was 26.64%. The positivity of rapid immune chromatography (1.22%) was significant lower than nucleic acid assay (6.16%). From January to June in 2015,34462 cases were detected for Chlamydia in 23 comprehensive hospitals, while 48219 cases in 9 maternal and child health hospitals. The Chlamydia screening numbers in maternal and child health hospitals were more than those in comprehensive hospitals.Conclusion:At present the rapid immune chromatograph method is widely applied to detect Chlamydia tracomatis in China. However, it seems that the sensitivity of this method have not been satisfied with the detection of weak chlamydia tracomatis positive spcimens. Therefore those detect approach with high sensitivity such as nucleic acid assay should be extensitively used to test Chlamydia trachomatis. Additionally, comprehensive hospitals should increase the screening of chlamydia tracomatis in high risk population.