| Objective:To understand the status of drug resistance of Mycobacterium tuberculosis to first-line anti-tuberculosis drugs in Qinghai,and explore whether the mutation of Mycobacterium tuberculosis resistance genes have any difference in sensitive or resistant strains.Methods:198 strains of Mycobacterium tuberculosis isolated from Qinghai area were cultured in modified L-J medium,using proportional method for drug resistance testing,analysis of the relationship between drug resistance and demographic characteristics in the area,and analyze the results.Mycobacterium tuberculosis DNA was extracted by traditional boiling method,Mycobacterium tuberculosis genes of kat G,pre-inh A,oxy R-ahp C integrate,rpo B,emb B,rps L and gid B were amplified,mutations in M.tuberculosis-resistant and susceptible strains were described,and the results were statistically analyzed.The value of gene sequencing method diagnosing M.tuberculosis were judged based on sensitivity,specificity,negative likelihood ratio,positive likelihood ratio and kappa.Results:The total drug resistance rate resulted from 198 strains of Mycobacterium tuberculosis was 58.59%(116/198).Four kinds of first-line drug resistance rates from high to low in turn Isoniazid(45.96%,91/198)>Rifampicin(43.94%,87/198)>streptomycin(40.91%,81/198)>ethambutol(30.30%,60/198).The drug resistance of Mycobacterium tuberculosis in Qinghai was found different from areas and ages(χ~2=5.236,P=0.022;χ~2=8.796,P=0.032),but no differences were seen related to gender and ethnicity(χ~2=0.871,P=0.351;χ~2=0.011,P=0.916).Sequencing 196 strains of Mycobacterium tuberculosis kat G gene,which was resistance of Isoniazid,among 89 strains of M.tuberculosis resistant to isoniazid,the kat G315 locus mutations was 61.80%(55/89),the mutation rate of kat G315 in 107 isoniazid sensitive strains was 3.74%(4/107),mutations in the kat G315 locus differed in sensitive or resistant Mycobacterium tuberculosis(χ~2=40.251,P<0.001).Nineteen strains of M.tuberculosis were mutated in the oxy R-ahp C integrate with a mutation rate of 9.69%(19/196).There were 17 resistant strains with a mutation rate of 19.10%(17/89).Two of the susceptible strains were mutated with a mutation rate of1.87%,mutations in the oxy R-ahp C integrate was different in M.tuberculosis-susceptible or resistant strains(χ~2=13.456,P<0.001).Seven strains of Mycobacterium tuberculosis mutated in pre-inh A,the mutation rate was 3.57%,three strains of resistant bacteria mutated,the mutation rate was3.37(3/89),four strains of susceptible bacteria had a mutation,the mutation rate was 3.74%(4/107),the mutation was no difference in pre-inh A of M.tuberculosis-sensitive or drug-resistant strains(χ~2=0.019,P=0.890).Among the 196 sequenced M.tuberculosis strains,43.37%(85/196)strains were resistant to rifampicin,56.63%were(111/196)sensitive strains to rifampicin.A total of 72 strains were mutated,the mutation rate was36.74%(72/196).Among 85 resistant strains,the gene mutation rate was81.18%(69/85),111 strains of susceptible strains gene mutation rate of 2.70%(3/111),Mycobacterium tuberculosis was resistant or susceptible to rifampin related to the mutation of rpo B gene(χ~2=124.199,P<0.001).196 M.tuberculosis ethambutol resistant rate was 30.10%(59/196),The sensitive strains were 69.90%(137/196),The mutation rate of emb B gene was20.41%(40/196).The mutation rate of emb B gene in 59 EMB-resistant strains was 45.76%(27/59).The mutation rate of emb B gene of 137 EMB-sensitive strains was 9.49%(13/137),Mutations in the emb B gene were associated with resistance or susceptibility to M.tuberculosis(χ~2=33.406,P<0.001).Among 196 strains of Mycobacterium tuberculosis,streptomycin-resistant strains accounted for 40.82%(80/196),80 strains of drug-resistant Mycobacterium tuberculosis rps L gene mutation rate was 56.25%(45/80).Streptomycin-sensitive strains accounted for 59.18%(116/196),116strains of drug sensitive Mycobacterium tuberculosis rps L gene mutation rate was 19.83%(23/116),Streptomycin-resistant or susceptible strains were associated with mutations in the rps L gene(χ~2=27.722,P<0.001).sequencing 192 strains of M.tuberculosis gid B gene,10 strains of M.tuberculosis did not have gene mutation,accounting for 5.21%(9/192).182strains of M.tuberculosis had gene mutation,the mutation rate was 94.79%(182/192).173 strains simultaneously gid BE92D and gid BA205A mutations,69 strains were gid BE92D and gid BA205A simultaneously mutations in 78resistant strains,the mutation rate was 88.46%(69/78).104 were strains of gid BE92D and gid BA205A simultaneousl mutations in 114 resistant strains,the mutation rate was 91.23%(104/114).Mutations of gid BE92D and gid BA205A were not related to M.tuberculosis streptomycin resistance(χ~2=0.398,P=0.528).Conclusion:Drug-resistant of Mycobacterium tuberculosis in Qinghai area was serious,higher than the national level.The resistance of Mycobacterium tuberculosis resistant to First-line anti-TB drugs relates with the gene mutations.Detection of drug-resistant mutations locus can quickly diagnose Mycobacterium tuberculosis drug resistance. |
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