Detection And Clinical Significance Of Heteroresistance In Mycobacterium Tuberculosis

Objective: To understand the occurrence of heteroresistance of Mycobacterium tuberculosis,the proportion of heterogeneous resistance,and the reason of heteroresistance,the sequence of anti-tuberculosis drug resistance gene were by analyzed and VNTR was applied for genotyping.The ultimate goal of this study is to provide a theoretical basis for the development and adjustment of clinical treatment plans.Methods: Double-peak mutations(A two-base sequence map appears at the same site)were detected in the sequencing maps of 2645 rifampin-resistant gene(rpoA,rpoB,rpoC and rpoZ),isoniazid resistance gene(katG and inhA gene),pyrazinamide resistance gene(pncA gene),and ethambutol resistance gene(embA,embB and embC),and streptomycin-resistant gene(rrs and rpsL).Then,the mutation regions were compared with the sequence of the standard strain H37 Rv in the NCBI database to examine non-synonymous double-peak mutations(heteroresistance).Heteroresistance-associated bacterial strains were resuscitated.The strains were identified by VNTR for genotype analyses.In order to analyze the causes of heteroresistance.Results: Among 2465 clinical isolates of Mycobacterium tuberculosis in this study,there are 39 cases of double-peak mutation of rifampicin-resistant genes.Comparising with NCBI database,31 cases of non-synonymous double-peak mutations were found in the sequence diagrams of 2060 cases of rifampicin-resistant genes,the mutation rate was 1.50%(31/2060);8 cases of non-synonymous double-peak mutations were found in the sequence diagrams of 243 cases of pyrazinamide-resistant genes,the mutation rate was 1.50% 3.29%(8/243).Among the double-peak mutation of rifampicin-resistant genes,2 cases of rpoA,17 cases of rpoB,10 cases of rpoC,2 cases of rpoZ gene mutations were detected.1 case of rpoB gene double-peak mutations in rifampicin resistance-determining region(RRDR)was found(Locating at 531 locus,the mutation type was TCG531TTG);Among the 31 cases of double-peak mutations of rifampin--resistant genes,28 cases were phenotypic resistant and 3 cases were phenotypic sensitivity.15 novel mutations were found,including:ACC171TCC,GAG213 GCG,AAG278ACG,TTC329 TTA,GAG644GCG,GTG646 ATG,TTC671TGC,CCG1131 GCG,GAG49GCG,GCC710 ACC,ATC1046AAC,CTT68 GTT,GTC70GGC,GTA21 GCA,and CCA68 GCA.Among 25 non-synonymous bimodal mutations,VNTR genotyping detection shows simple infections of the heteroresistance.Conclusion: There are 31 cases of heteroresistance are detected in 2060 cases of rifampicin resistance gene sequencing,with a drug resistance rate of 1.50%;8 cases of heteroresistance were detected in 243 cases of pyrazinamide resistance gene sequencing,with a drug resistance rate of 3.29%;Confirmed by VNTR typing,the bimodal mutations in this study were dominated by the differentiation of single bacteria which developing into drug-resistant and susceptible strains.The bimodal mutations more often appear in the phenotypically resistant and retreatment Mycobacterium tuberculosis patients.