Mixed Infections Of Mycobacterium Tuberculosis In Tuberculosis Patients In Shanghai, China

Tuberculosis is an old zoonosis, it was selected as emergent global public health problems by the WHO together with HIV and malaria. Tuberculosis is also one of the most important infectious diseases in China. Mycobacterium tuberculosis is the bacterium that causes tuberculosis, mostly propagated by respiratory passage. Because of the long stage of latency, traditional epidemiology method is very limited in the studies of propagation of Mycobacterium tuberculosis. Since nineteen twenties, molecular epidemiology which is based on using molecular biology method to differentiate different bacterium and combined with traditional epidemiology, offered better tools for the control of tuberculosis. In this study, using molecular genotyping method, we focused on the mixed infections of Mycobacterium tuberculosis, gave a new sight on the infection and propagation of Mycobacterium tuberculosis.Infection with multiple strains of M. tuberculosis, distinguishable by their different genotypes, within the same patient before, during or after successful treatment is defined as a mixed infection. Information about the number and rate of mixed infections are important for several reasons. First, a high frequency of mixed infections could indicate there is a lot of transmission of M. tuberculosis. Mixed infections could explain conflicting laboratory drug susceptibility test results on specimens from the same patient. In clinical practice, mixed infections could explain heterogeneous clinical responses to anti-tuberculosis therapy.We applied a 7 loci Variable-Number-Tandem-Repeats (VNTR-7) analysis method to identify mixed infections of Mycobacterium tuberculosis and to estimate the rate of mixed infections among pulmonary tuberculosis patients in Shanghai, China. Mtub21, QUB-18, QUB-11a, QUB-11b, MIRU26, VNTR3820 and QUB-26, these 7 loci were included in this study. From 1999 to 2004, 22 tuberculosis patients who had two isolates conserved in the strain reservoir of Shanghai CDC, and the two isolates had different drug resistant patterns were selected. We get the first isolate of each patient and inoculated on L-J solid medium, after 4 weeks we harvested the genome DNA by the boiling method, and we further inoculated the bacterium on middlebrook 7H11 plates. For each isolate, we randomly selected 30 colonies after 4 to 6 weeks. Using VNTR genotyping method, we genotyped the 22 isolates and 30 colonies for each isolate. The results showed that, two out of 22 isolates had mixed infections of Mycobacterium tuberculosis, directly genotyping the isolates and from the colonies of each isolate had the same results. So we were able to using this method to identifying mixed infections directly from the isolates.We used the validated VNTR-7 method to genotype an isolate from each of the 249 from pulmonary tuberculosis patients reported from the Songjiang and Chongming districts in Shanghai during 2006. We identified 14 patients with mixed infections, and the estimated rate of mixed infections was 5.6% (14/249,95% CI 3.1%-9.2%). Mixed infections were observed more frequently among tuberculosis patients undergoing retreatment (15.6%) than among new cases (4.1%) (p<0.05), and among tuberculosis patients whose disease was caused by non-Beijing genotype strains (12.5%) versus Beijing genotype strains (3.5%) (p<0.05). The VNTR-7 method is a highly sensitive, practical tool with relatively high discriminatory power, making it useful for studying mixed infections.