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Background: Rapid and accurate detection of drug resistance in Mycobacterium tuberculosis is critical for effective control of tuberculosis (TB). Herein, we established a novel, low cost strategy having high accuracy and speed for the detection of M. tuberculosis drug resistance, using gene splicing by overlap extension PCR (SOE PCR). Methods: The SOE PCR assay and Sanger sequencing are designed and constructed to detect mutations of rpoB, embB, katG, and inhA promoter, which have been considered as the major contributors to rifampicin (RFP), isoniazid (INH), and ethambutol (EMB) resistance in M. tuberculosis. One hundred and eight M. tuberculosis isolates came from mycobacterial cultures of TB cases at Chongqing Public Health Medical Center in China from December 2018 to April 2019, of which 56 isolates were tested with the GeneXpert MTB/RIF assay. Performance evaluation of the SOE PCR technique was compared with traditional mycobacterial culture and drug susceptibility testing (DST) or GeneXpert MTB/RIF among these isolates. Kappa identity test was used to analyze the consistency of the different diagnostic methods. Results: We found that the mutations of S531L, S315T and M306V were most prevalent for RFP, INH and EMB resistance, respectively, in the 108 M. tuberculosis isolates. Compared with phenotypic DST, the sensitivity and specificity of the SOE PCR assay for resistance detection were 100.00% and 88.00% for RFP, 94.64% and 94.23% for INH, and 68.97% and 79.75% for EMB, respectively. Compared with the GeneXpert MTB/RIF, the SOE PCR method was completely consistent with results of the GeneXpert MTB/RIF, with a concordance of 100% for resistance to RFP. Conclusions: In present study, a novel SOE PCR diagnostic method was successfully developed for the accurate detection of M. tuberculosis drug resistance. Our results using this method have a high consistency with that of traditional phenotypic DST or GeneXpert MTB/RIF, and SOE PCR testing in clinical isolates can also be conducted rapidly and simultaneously for detection of drug resistance to RFP, EMB, and INH. [ABSTRACT FROM AUTHOR]

Background: By 2014 although tuberculosis (TB) incidence had fallen by an average of 1.5% per year since 2000 and was 18% lower than the level of in 2000, 1.5 million people died for TB in that year. One of reason was that drug resistant Mycobacterium tuberculosis (DRTB) spread. This study aims to determine drug resistant characteristics and genotype of DRTB that isolated from patients in a tuberculosis referral hospital of southwest China.Methods: Five hundred thirty-eight drug resistant tuberculosis samples were collected from July 2013 to March 2015. All the isolates were identified by genomic deletions in region of difference 105 (RD105) and genotyped by mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-VNTR). Polymorphism and cluster analysis of each locus was carried out using Bionumerics Version 3.0 and phyloviz software.Results: Five hundred thirty-eight TB strains included 503 Mycobacterium tuberculosis (MTB) isolates and 35 non Mycobacterium tuberculosis (NMTB) isolates. Of 503 isolates Beijing family type was 447 (88.9%, 447/503) and non-Beijing family type was 56 (11.1%, 56/503). Five hundred three DRTB isolates were divided into 345 genotypes, of which 265 isolates were single genotype and the remaining 238 strains were classified into 80 clusters with cluster rate of 47.3% and cluster ratio of 31.4%. Sixty-nine clusters belonged to Beijing family with cluster rate was 48.3% and clustering ratio was 32.9%. The non - Beijing family had 11 clusters with a cluster rate of 39.3% and the clustering ratio of 19.6%. Beijing genotype had a significant correlation with the age (P < 0.05), the retreatment patients (P < 0.05) and the city of Chongqing (P < 0.05), not with gender (P > 0.05). In the 9 Beijing genotype clusters each cluster contained some patients who lived in the same region.Conclusions: Beijing genotype was the predominant in the patients with DRTB in our hospital. In Chongqing retreatment patients with Beijing genotype MTB may be patient with DRTB. Drug resistance test (DST), regular medication and strict follow-up are very important for patients with Beijing genotype MTB. In Chongqing control and treatment of DRTB should be paid attention. Transmission and relations of patients with DRTB need to be further research. [ABSTRACT FROM AUTHOR]