Molecular Epidemiological Study On The Prevalence And Transmission Of Drug Resistant TB In Rural Area Of Eastern China

Since 1990s,DOTS based strategy has been expanded to most of areas in China, providing the free anti-YB therapy to more than 1,300 million infectious pulmonary tuberculosis patients and subsequently helping China reaching the 70%of cure rate set by WHO in 2006.But considering the disease burden,China remains to be one of countries with the highest TB burden,only second to India.China has owned 16%of incident TB cases(1.58/9.88million) worldwide,with smear positive TB rate and active TB rate being respectively 46/100,000 and 101/100,000 in 2004.The control of tuberculosis in China has been further complicated and threatened by an increasing incidence of drug-resistant tuberculosis especially including mulit-drug-resistant tuberculosis(MDR-TB) in last ten years.MDR-TB is the pattem of drug resistance to at least isoniazid and rifampicin.A recent global survey of MDR-TB incidence estimated that an average of 2.7%of all new cases with no TB history,18.5%of previously treated and 4.3%of all cases had MDR-TB in 2004[2].Indeed,the MDR-TB incidence estimates for China in 2004 was considerably higher than the global average with 5.3%among cases with no history of TB,27.2%among previous treated cases.The 4^th TB epidemiological study reported that 18.7%patients were resistant to at least one of the first-line anti-TB drugs and 6% of TB patients had MDR-TB.The monitoring data from different sources has demonstrated the hotspot of drug resistant TB in some Chinese areas.Drug resistant TB could directly cause the high prevalence and mortality of TB and meanwhile the elongation of treatment period and increase in treatment cost.Setting in two DOTS covered rural counties respectively from Jiangsu and Zhejiang provinces,this study attempted to describe the prevalence and transmission of drug resistant TB in rural area of Eastem China from multi-displinary perspective in terms of field epidemiology and molecular epidemiology:to describe the prevalence of drug resistant TB and socioeconomic factors influencing its occurrence by the cross sectional study;to investigate the genetic diversity of prevailing M.TB strain and explore the transmission mechanism of drug resistant TB by combining all the available genotyping methods including MIRU, Spoligotying and IS6110;to identify the drug resistance genotype and its association with phenotype by direct DNA sequencing;to determine the transmission pattern of drug resistant TB strain in rural China through tracing the clustered cases:to study the contribution of Beijing genotype and its predominant member to the genotype and phenotype of drug resistant TB in rural China and uncover the mechanism behind them.1.A cross-sectional study was conducted in two rural counties in Eastern China: Deqing with over 10 years' DOTS implementation and Guanyun under its second year of DOTS.The subjects were all culture-positive pulmonary TB patients newly diagnosed or retreated during 12 months of 2004 to 2005.The proportion method was used for drug susceptibility testing.Among the 399 subjects,283 were new TB cases and 116 were previously treated.The rates of overall resistance in new cases were 50.4%(67) and 63.3%(95) respectively in Deqing and Guanyun,and 67.3%(33) and 83.6%(56) respectively in previously treated cases.The rates of MDR-TB in new cases were 3.8%(5) in Deqing and 14.7%(22) in Guanyun(p=0.0018),and 16.3% (8) and 34.3%(23) in previously treated cases(p=0.0305).Furthermore,the comparision of socio-demographic and medical characteristics was made between 58 MDR-TB patients,193 other drug resistant and 148 pan-drug sensitive TB patients.Compared to pan-drug sensitive patients,both in two counties, previous treatment history(≥4weeks/＜4weeks:Deqing:OR:4.980;95%CI:1.509-8.031;Guanyun:OR:3.941;95%CI:2.043-12.48),case contact(yes/no:Deqing: OR:7.841;95%CI:1.712-25.30;Guanyun:OR:4.801;95%CI:1.321-7.243) and individual income(high/low income:Deqing:OR:0.794;95%CI:0.038-0.927; Guanyun:OR:0.150;95%CI:0.043-0.891) were associated with MDRTB.Only in Guanyun with shorter duration NTP-DOTs,were age(30～year/60～year group:OR: 2.748;95%CI:1.047-6.974) and smear positive(smear positive/negative:OR:5.721; 95%CI:2.107-17.17) related to MDRTB.There was higher proportion of MDRTB among male patients(male/female:OR:2.084,95%CI:1.061-6.349).It was concluded that Socioeconomic(age,sex and individual income) and clinic(case contact and previous treatment history) characteristics were risk factors related to presence of drug resistance TB.The vulnerable population(the poor and the elder) should become the concern of the TB control.2.DNA sequencing on the hotspot of resistance related genes was applied to investigate the drug-resistance genotype and its association with the corresponding drug resistance phenotype.81(61.8%) of 131 INH resistant isolates had single-nucleotide substitution in codon 315 of katG with 315ACC(Thr) responsible for 49.6%of INH-resistance.Drug resistance to RIF was mostly due to the genetic mutation of rpoB gene,where the single-nucleotide substitution in codon 516,526 and 531 took account respectively for 12.3%,29.2%and 56.9%.81.0%isolates with rpoB531Set were MDR-TB.Meanwhile,the DNA sequencing analysis of rpsL43, revealed resistance mutations in 60 of 115 SM resistant isolates totally due to a single base change(43rpsLAGG).While spot mutation related to mono-drug SM resistance represented a largest proportion(51.3%).Among 20 of 42 EMB-resistant isolate containing the genetic mutation in embB306,most of embB306 mutations were observed in the EMB-resistant M.TB isolate simultaneously resistant to other first-line anti-TB drugs.3.Three genotyping methods,including mycobacterial interspersed repetitive unit(MIRU) typing,Spoligotyping,and IS6110 restriction fragment length polymorphism(RFLP) typing,were used in this study.All 351 MTB isolates were first molecularly characterized by Spoligotyping and MIRU.Each MIRU cluster was further genotyping by IS6110 RFLP(i.e.MIRU plus IS6110) for clustering analysis. Spoligotyping identified 243 isolates(69.2%) that presented the Beijing family Spoligotype.MIRU typing determined 220 genotypes from all isolates in this study, including 183 unique patterns and 37 clusters(including 168 isolates).The largest MIRU cluster contained 38(10.8%) isolates,sharing an identical MIRU genotype 223325173533,which has been referred to as the "Shandong cluster" in a previous study.Again,all isolates from the "Shandong cluster" were members of the Beijing family.Shandong cluster was further distinguished into 9 sub-clusters by IS6110 RFLP.The largest MIRU/IS6110 cluster had 4 isolates.4.Based on the MIRU-IS6110 pattern,the 62 isolates was defined as the clustered and 161 as the unique.The comparison of host's socio-demographic and medical characteristics between these two groups revealed the drug resistant isolates in Guanyun were more likely to be clustered compared to Deqing(34.1%.vs.18.2%; OR:3.311;95%CI:1.498-7.319).In addition,the population aged 30-60 year was in high risk of recent transmission causing the disease(30～year/60～year age group: 34.7%.vs.19.0%;OR:2.326;95%CI:1.039-5.393).The smear positive was the factor independent of disease due to the recent transmission((Postive/Negative.32.2%.vs. 18.9%;OR:2.242;95%CI:1.010-4.977).The Shandong cluster was more likely to develop the disease through the recent transmission(67.7%.vs.15.3%;OR:6.681; 95%CI:3.258-15.02).5.Those with the clustered isolated was investigated to describe the transmission pattern of drug resistant TB through case tracing.The transmission pattern of drug resistant TB was presented by the sporadic distribution in small group.The population aged 30-60 years and the previously treated patients were the main sources of infection.Meanwhile,the causal contact contributed a lot to the recent transmission of TB.6.Targeting the 243 Beijing isolates available,this part of study was to investigate the contribution of Beijing family and its predominant member to drug resistance genotype and phenotype.The attempt was made to explore the mechanism behind the connection between Beijing family and drug resistance in terms of the polymorphism of repair gene and bands of IS6110 insertion.The Binary Logistic Regression Model adjusted by age,sex,county of the subjects was applied to analyze the association between drug resistance,gene mutation and genotypes.Beijing family isolates presented increased risks to be MDR-TB(OR:2.723,95%CI:1.351-6.733) and have katG and rpoB mutations(OR:2.553,95%CI:1.031-6.324) compared with non-Beijing family isolates.Clustering analysis by MIRU plus IS6110 RFLP revealed that Beijing family isolates were more likely to be clustered.The repair gene and bands of IS6110 insertions proven to play a role in the acquiring the genotype and phenotype of drug resistance.