| Objective To compare the detection effects and the accordance of three drug sensitivity test(DST) methods based on bacterial culture for testing the sensitivity of Mycobacterium tuberculosis(M. tuberculosis) to ethambutol(EMB); To analyze the correlations between the molecular characteristics and EMB drug resistance of M. tuberculosis; To investigate the correlations between emb CAB mutations and EMB resistance level, thereby provide a reliable basis for establishing new rapid and accurate molecular diagnosis methods to test EMB resistance of M. tuberculosis. Methods(1) We detected the sensitivity of 126 clinical M. tuberculosis isolates to EMB by means of L-J proportion method(L-J method), BACTEC MGIT 960 system(960 system) and microplate Alamar blue assay(MABA),respectively, and in addition, the results were compared interactively.(2) The genome DNA of M. tuberculosis isolates were extracted by CTAB method, in which the aiming fragments of the genes associated with EMB resistance of M. tuberculosis were amplified by polymerase chain reaction(PCR) and the PCR products were sequenced for blast.(3) MIC values of EMB against M. tuberculosis were tested by MABA, for the correlation analysis between emb CAB mutations and EMB resistance level. Results(1) The total concordance rate of the results of the three methods was 75.4%. If taking the results of L-J proportion method(L-J method) as the reference, the sensitivity, specificity and consistency of 960 system and MABA were 62.8 %(49/78), 100.0 %(48/48), 77.0%(97/126) and 82.1%(64/78), 97.9%(47/48), 88.1%(111/126), respectively; If the results of 960 system were taken as the reference, the sensitivity, specificity and consistency of L-J method and MABA were 100%(49/49), 62.3%(48/77), 77.0%(97/126) and 98.0%(48/49), 77.9%(60/77), 85.7%(108/126), respectively; If the results of MABA were set as the reference, the sensitivity, specificity and consistency of L-J proportion method and 960 system were 98.5%, 77.0% and 88.1%; 73.8%, 98.4% and 85.7%, respectively. Both L-J method and 960 system were superbly consistent with MABA, L-J method and 960 system were in moderately accordance.(2) Seventy-eight M. tuberculosis isolates were mutated in emb CAB operon, the mutation rate was 61.9%, in which seventy-five isolates mutated in emb B, especially in emb B306, sixteen isolates mutated in emb A as well as the upstream region of it, two isolates were detected harbor mutations in emb C. The sensitivity, specificity and concordance rate of emb B306-497 mutations as molecular marker of EMB resistance diagnosis were 79.5%, 72.9% and 77.0%, respectively.(3) Among the 14 isolates which presented MIC < 5.0μg/ml, 6 isolates were emb B mutants, the MIC values were enhanced along with the increasing rate of multiple emb AB mutations. Conclusions(1) The three methods used in this study were in satisfying concordance for testing the sensitivity of M. tuberculosis to EMB, MABA was demonstrated a more appropriate method for the clinical laboratory application.(2) emb B and emb A upstream region were statistically correlative with EMB resistance while emb C was in contrast. Using emb B306-497 mutations as the molecular marker were better for diagnosis of M. tuberculosis EMB resistance. Multiple mutated isolates were resistant to more numbers of drugs, while the mutation rates had no correlation with Beijing genotype isolates.(3) emb AB multiple mutations would increase the EMB resistance level of M. tuberculosis. Low level of EMB resistance has poor correlation with the gene mutations, there might be other mechanisms causing M. tuberculosis EMB resistance which needed further research in future. |
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