The Characteristics Of Phenotype And Genotype Of Multidrug-resistant Tuberculosis Isolates

Tuberculosis had become one of the toughest global public health problems. Estimates are that one third of the world’s population is infected with Mycobacterium tuberculosis. There were an estimated8.8million incident cases of TB and1.45million deaths globally in WHO2010report. The increasing drug-resistant tuberculosis including multidrug-resistant and extensively drug-resistant tuberculosis challenges the control of global tuberculosis. To know the characteristics of phenotype and genotype of MDR, we achieved the goals by MGIT960and molecular line probe assay. The mixed infection detected by molecular line probe will be confirmed by genotyping method.Part I. The characteristics of drug-resistant phenotype in MDR-TB strainsIn this study, the MDR-TB confirmed by proportion method from Shanghai and Nanjing were enrolled randomly. The susceptibility of M. tuberculosis to isoniazid (INH), rifampin (RFP), ethambutol (EMB), ofloxacin (OFX), moxifloxacin (MFX) and amikacin (AMK) detect by MGIT960assay. The susceptibility of M. tuberculosis to pyrazinamide (PZA) detects by MGIT960, Wayne assay and microplates assay modified from microscope observation drug-susceptibility assay. Out of94MDR-TB strains,34were resistant to EMB,16resistant to AMK,51resistant to OFX and52resistant to MFX. The rate of drug resistant to EMB, AMK, OFX and MFX by BD MGIT960assay were36.2%,17.0%,54.3%and55.3%, respectively. Among these isolates,13were XDR-TB.53isolates were selected from the94strains randomly for PZA susceptibility test by MGIT960and Wayne assay. Of53isolates,26were resistant to PZA by MGIT960and26were PZase negative by Wayne assay.There were12strains which didn’t grow in deadline by microplate assay and got the same results after repeat. Out of53strains,41got the results from the fifth to the fourteenth day. Take the MGIT960assay as refenence, the concordance rate was97.6%. These suggest that MDR-TB possess high drug-resistant rate to Quino-lones and PZA while to AMK much lower. For the results getting from the PZA microplates assay were reliable, it could be used in clinical lab.Part Ⅱ. The characteristics of drug-resistant genotype in MDR-TB strainsThe molecular characteristics of relative drug resistant genes were detected by molecular line probe and gene sequencing methods. Out of94MTB,81were resistant to INH and90resistant to RFP using GenoType MTBDRplus kit. Compared with MGIT960, the concordance rate of GenoType MTBDRplus was86.2%and95.7%respectively. Taking GenoType MTBDRslkit to detect the relative drug-resistant genes, we found that31strains were resistant to EMB,14were resistant to AMK,52were resistant to OFX and MFX. Taking MGIT960results as reference, the sensitivity of GenoType MTBDRsl detecting the susceptibility of EMB、 AMK、 OFX and MFX to94isolates were47.1%、81.3%、94.1%、94.2%, respectively. The specificity were75%、98.7%、90.7%、92.9%, respectively. There were23mixed infection isolates detected by GenoType MTBDRplus and GenoType MTBDRslkits. In13XDR-TB,6were mixed infection by the two kits. There were7undetectable mutations sites strains after gene probe hybridization. The mutation sites of rpoB focused on S531L chiefly in Shanghai and Nanjing TB strains.There were5strains with mutation at D516V in Shanghai but Nanjing didn’t have. The mutation sites of katG were located at S315TS chiefly. The number of strains with M306V mutation sites in embB genes from Nanjing was much than Shanghai’s.It seems that Nanjing mixed infection strains were more than Shanghai strains.There were30strains which had different mutation sites in pncA genes by sequencing method. The pncA mutations were highly diverse and scattered along the gene, which is a characteristic quique to PZA among drug-resistance mutations in M. tuberculosis. We can draw the conclution that GenoType MTBDRplus had good sensitivity and specificity for RFP susceptibility test and GenoType MTBDRsl was ideal ability for Quinolones test and there were being some hot spots in rpoB and katG mutation sites and differences among the distribution of drug-resistant genes in different regions.Part Ⅲ. The analysis for rare mutation sites and mixed infectionIn the94MDR-TB strains, there were7strains which we couldn’t read out the mutation sites and23strains were mixed infection. To analyze these strains, we use the gene sequencing and VNTR genotyping method to detect them. Sequencing the7strains found that all of them exist one or more mutations which didn’t enrolled on the strip except the inhA gene. After the VNTR genotyping test,13isolates were identified as mixed infection. In23mixed infection strains,5were non-Beijing genotype and1were mixed with Beijing and non-Beiing genotype. It can be suggested that the probes coaded in the strip were common mutations in clinic. Although the line probes couldn’t detect where the rare mutation located, it could confirm the strains with mutation site. Some XDR-TB may caused by2or more different genotypes M. tuberculosis.