Determinants Of Drug-resistant Tuberculosis And Molecular Epidemiological Study In Jiangsu Province

Objective: The fourth national epidemiological sampling survey of tuberculosis (TB)showed, the44.5%(550million) of the population infected with Mycobacteriumtuberculosis(M.TB), higher than the global infection level. In recent years,multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis havebrought a huge challenge to the prevention and control work. Drug resistance ofM.TB to the four first-line anti-tuberculosis drugs (INH, RFP, EMB and SM) wereassociated with the mutation of rpoB, katG, rpsL and embB gene respectively. Since1995van Soolingen D found the Beijing family is unique in Beijing, China, whichattract worldwide attention, a lot studies were taken focus on this family. Thisresearch plans (1) to describe the baseline data and epidemiological characteristics ofdrug-resistant M.TB strains circulating in Jiangsu Province, and to provide policymakers with recommendations for prevention and control of tuberculosis.(2) todescribe the genotype characteristics of M.TB and its association with the drugresistant phenotype.To explore the molecular mechanisms of drug-resistant, so as toprovide evidences for the rapid diagnosis and screening of drug-resistant M.TB.(3) toanalyze the M.TB DNA fingerprint by using the spoligotyping method, and to furtherexplore the relationship between Beijing family genotypes and the drug resistance ofM.TB.Methods:(1) According to the guidelines of drug-resistant tuberculosis surveillancerecommended by World Health Organization/International Union AgainstTuberculosis and Lung Disease Federation (WHO/IUATLD), we selected30counties(districts) from Jiangsu Province as the study sites by using cluster sampling method.Sputum smear-positive tuberculosis patients reported during June and July2010,were continuously recruited. Diagnosis was further confirmed by sputum culture. Proportional method which is recommended by WHO/IUATLD was used todetermine the sensitivity to the four first-line anti-tuberculosis drugs, we furhteranalyzed the drug resistant spectrum and possible risk factors;(2) The mutations ofrifampin resistance related rpoB gene and isoniazid resistance related katG gene wereamplified and sequenced. We analyzed the association between genotypes andphenotypes of drug resistance;(3) We used spoligotyping method to study themolecular characteristics of M.TB strains, and explored the relationship between theBeijing family strains and anti-tuberculosis drug resistance.Results:(1) Of all the260patients, there were216(83.1%) new cases and44(16.9%)previously treated ones, with the male to female ratio of2.25:1. Drug resistant testindicated that186(71.5%) strains were sensitive to all the four anti-tuberculosisdrugs, and74strains were resistant to at least one drug. The total resistanceproportion was28.5%and the MDR (at least resistant to INH and RFP at the sametime) proportion was34(13.1%). There were17(6.5%) strains resistant to all thefour first-line drugs. Among new cases,51(23.6%) were resistant to at least one drug,while among previously treated ones, the proportion was52.3%(P<0.001). Theproportion of MDR-TB was10.2%in new cases and27.3%in previously treated ones,respectively (P=0.002). Of the new cases,8cases (3.7%) were resistant to all thefirst-line drugs, while of the previously treated ones, it was20.5%(P<0.001). Weanalyzed the rrole of gender, age, treatment history, history of BCG vaccination andmigrantion in the risk of MDR-TB and found that treatment history was significantlyassociated with MDR. Compared with the new cases, previously treated ones had3.40times risk for MDR, with the adjusted OR of3.40(95%CI:1.42-8.13).(2) Wesequenced255M.TB strains and found that28isolates (11.0%) had point mutations atthe81bp core area of rpoB gene. The main mutation was rpoB531TTG (46.4%).Besides, we found two simultaneous point mutations at rpoB gene in seven strains.For katG gene,11.8%strains had mutations. All mutations were located at katG315position, with the mutation form of katG315AGC-ACC. rpoB gene mutations alloccurred in the rifampicin resistant strains and katG gene mutations all occurred inthe isoniazid resistant strains with high sensitivity and specificity were100%for both. (3) By BioNumerics5.0clustering analysis, the spoligotyping of260M.TB strainspresented significant polymorphisms. The largest cluster containing209M.TB strainswas belonged to Beijing family, which accounted for80.3%(209/260). Logisticregression analysis revealed that Beijing family strains had increased risks forMDR-TB, with the adjusted OR of12.76(95%CI:1.60-101.94).Conclusions: The high prevalence of drug resistance has been a major challenge forTB control. Prevention and control of drug-resistant tuberculosis should beemphasized on the implementation of the DOT (direct observed therapy). To increasethe treatment compliance and treatment success rate, reduce the occurrence of drugresistance tuberculosis. RpoB and katG gene mutations are an important molecularmechanism of M.TB to rifampicin and isoniazid resistance. Molecularepidemiological studies of M.TB are useful for understanding the mechanisms ofdrug resistance, and can help develop tools for early diagnosis of drug-resistant TB.Beijing family strains may increase risks for MDR-TB, adjusted OR=12.76(95%CI:1.60-101.94). It is suggested that further monitoring were needed to the Beijingfamily strain, and continue to research the mechanism for the spread of the Beijingfamily strains.