| Tuberculosis, a chronic infectious disease, remains a serious problem of public health worldwide. In China, tuberculosis is one of key infectious diseases. Under DOTs (Directly observed treatment, short-course) strategy, most patients with tuberculosis are cured. However some patients with tuberculosis who complete adequete course of treatment with the standard drugs develop recurrent tuberculosis. Patients with recurrent tuberculosis have high rate of drug resistance and severe complication, leading to a low rate of treatment success and advanced trasmission of Mycobacterium tuberculosis. So recurrent tuberculosis is a significant problem for tuberculosis control programs. Meanwhile, the emergence of drug resistant tuberculosis, especially multi-drug resistant tuberculsis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) poses another great challenge for tuberculosis control worldwide.Recurrent tuberculosis is an important index for the effectiveness of tuberculosis control program. Evaluation of recurrent tuberculosis in China could help to assess the effectiveness of Chinese tuberculosis control program and improve the tuberuclosis control strategy. On the other hand, recurrent tuberculosis could be cuased by endogenous reactivation or exogenous reinfection. Identification of the cause of recurrence has significant implication for evaluation of treatment regimen and for development of tuberculosis vaccine. We performed a retropective study in Shanghai and found that exogenous reinfection (67.6%,25/37) is the main cause of recurrent tuberculosis. However, the smaple size of the study was small, and the result need to be further evaluated. Therefore, the aim of part one of the present study was to assess the epidemiologic characteristics of recurrence among31,323patients with tuberculosis during2000-2008in Shanghai, China, and to determine the cause of recurrent tuberculosis using variable number of tandem repeats (VNTR) genotyping.From epidemiologic analysis, we found that the proportion of recurrece was relative low, only4.0%of the patient developed recurrent tuberculosis within ten years following treatment completion. However, the incidence of recurrence (0.756per100person-years, an incidence of756per100,000inhabitants per year) among patients with tuberulosis was higher than the incidence of tuberculosis (40per100,000inhabitants per year) among general population. Ninty percent of recurrence occurred within5years after completion of treatment. These results suggesting that patient with a history of tuberculosis must be considered as a group at risk of having tuberculosis despite having correctly completed treatment, and tricter follow-up and stricter post-treatment controls, especially within5years, should be implemented to detect and treat recurent tuberculosis early.In order to determine the cause of recurrent tuberculosis, we used VNTR genotyping to differentiate Mycobacterium tuberculosis isoloates from patients who experienced2successive tuberculosis episode. We found that exogenous reinfection (44.7%,42/94) with a different strain of Mycobacterium tuberculosis is an important cause of recurrence. High exogenous reinfection among recurrent tuberculosis suggested that recent transmission of Mycobacterium tuberculosis was common. Our findings indicated that primary infection with Mycobacterium tuberculosis could not confer protective immunity against a second infetion. Therefore, the development of improved vaccines against tuberculosis will be especially challenging and the need for better understanding of the host defenses against tuberculosis particularly critical.We used Cox multi-variate analysis to detemine the risk factors associated with recurrent tuberculosis. We found that patients who were male (aHR=1.5, P<0.001),>30years (aHR=1.3, P=0.003), had cavitory (aHR=1.4, P<0.001), had diabetes (aHR=1.4, P=0.001), were smear positive (aHR=1.4, P<0.001), had any drug resistance (aHR=1.6, P<0.001) and had MDR (aHR=3.0, P<0.001) were more likely to have recurrence. Then we combined results of genotyping and used Logistic univariate anlysis to identify the factors associated with endogenous reactivation or exogenous reinfection. We found that cavitory (OR=3.2, P=0.009) and MDR (OR=7.2, P=0.0394) were significantly associated with endogenous reactivation. Our findings suggested that different intervention should be implemented among tuberculosis patients with different cause of recurrence.We also analyzed the phenotype of drug resistance among patients with recurrent tuberculosis. We found that23.8%(12/52) of recurrence caused by endogenous reactivation acquired advanced drug resistance during treatment. Among7patients with infection of MDR in the first tuberculosis episode,6patients had endogenous reactivation, suggesting that the effectiveness of treatment for drug resistant tuberculosis was poor. Among recurrent tuberculosis with drug resistance, there were4clusters of Mycobacterium tuberculosis containing12patients, suggesting recent transmission of Mycobacterium tuberculosis with drug resistance. Control of drug resistant tuberuclosis should be of great urgency. Early detection and rapid dianosis of drug resistance are very important for interruption of transmission and appropriate treatment of drug-resistant tuberculosis. The accuracy of rapid molecular diagnostics is dependent largely on the strength of the association between a specific Mycobacterium tuberculosis gene mutation and the phenotypic resistance of the isolate with that mutaion. Therefore, the aim of part two of the present study was to determine the prevalence of drug resistance to second-line anti-tuberculosis drugs and the molecular characteristics of mutations coferring drug resistance of Mycobacterium tuberculosis.To determine the prevalence of resistance to second-line anti-tuberculosis drugs among Mycobacterium tuberculosis, we performed drug susceptibility testing (ofloxacin, moxifloxacin, kanamycin, amikacin, capreomycin, protionamide, and P-aminosalicylic acid) for410clinical strains with resistance to first-line drugs. We found that the prevelance of resistance to second-line anti-tuberculosis drugs among Mycobacterium tuberculosis in Shanghai was high. Among410strains,25.4%had any resistance to second-line drugs, meanwhile among MDR-TB,52.0%had any resistance to second-line drugs, suggesting that drug susceptibility testing for second-line drugs should routinely performed for early detection of resistance and for prompt treatment for tuberculosis patients with drug resistance.Demonstrating the molecular characteristics of specific gene mutation is the basis of development of molecular diagnostics. We sequeced gyrA, rrs, and eis promoter among410clinical strains of Mycobacterium tuberculosis. We found that sensitivity and specificity of gyrA for fluoroquinolone resistance were90%, and99%, respectively. The sensitivity of combination of rrs and eis for kanamycin resistance was54.3%. The sensitivity of rrs for amikacin and capreomycin were76.9%, and64.7%, respectively. The specificity of rrs and eis for kanamycin amikacin and capreomycin was97%. The most frequent mutation in the quenolone resistance determing region (QRDR) of gyrA was Asp94(56.9%,29/51), followed by Ala90(29.4%,15/51) and Ser91(3.9%,2/51). The most frequent mutations in rrs was A1401G, while the most frequent mutations in eis promoter was G(-37)T. Our findings suggested that gyrA could be an ideal molucular marker for fluoroquinolone resistance. We also found that Beijing genotype strains were more likely to have gyrA mutations (OR=4.9,P=0.004). The mechanism needs to be demonstrated.In conclusion, this is the first stuty to investigate the epidemiologic chrateristics of recurrent tuberculosis in Shanghai, China. We also confirmed the role of exogenous reinfection for recurrence, and identified the risk factors associated with recurrence. The present study also analyzed the phenotypic and genotypic pattern of resistance to second-line drugs of Mycobacterium tuberculosis. Our findings had important implication for tuberculosis control program, for development of vaccines for tuberculosis, and for the development of molecular diagnostics of resistance. |
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