Evaluation Of Rapid Identification System Of Mycobacteria And Detection Of Drug Resistance Mutations Of Common Non-tuberculous Mycobacteria

M.tuberculosis has killed about 1 billion people infected in the past two centuries,and is still one of the top ten causes of death in the world.Although the global infection rate of NTM is lower than that of M.tuberculosis,due to treatment methods of NTM are different from M.tuberculosis,the limitations of species identification method,and the incidence of drug resistance increases year by year,as a result,the clinical incidence of NTM significantly increases.Therefore,the accurate species identification and accurate detection of drug resistance of mycobacterium are very important.In addition,MABC is the strongest pathogenicity and the strongest resistance to chemotherapy drugs of rapidly growing mycobacteria,and recent studies have shown that MABC can spread from person to person.And the three subspecies of MABC have different drug resistance to clarithromycin,therefore,the subspecies identification of MABC is of guiding significance for clinical treatment.In the first chapter,first of all,this paper briefly introduced the biological characteristics and global epidemiology about mycobacteria.Secondly,common species identification methods of mycobacteria and our laboratory established common mycobacteria rapid identification method were introduced.Then,the taxonomic evolution process and effectively species identification method of MABC had carried on the summary.In the end,the common drug resistance mechanisms and drug resistance detection methods of NTM were summarized.In addition,the purpose,content,and significance of this research were summarized.In the second chapter,we used 812 clinical sputum samples from suspected pulmonary tuberculosis patients to evaluate the effect of MeltPro Myco assay,and the sensitivity,specificity,and accuracy of this method were calculated by comparing with the verification method.Results showed that in the 14 species of mycobacteria detected by MeltPro Myco assay,the sensitivity of M.tuberculosis,M.intracellular,MABC,and M.fortuitum were 78.0%(255/327),88.5%(23/26),93.8%(15/16),and 75.0%(3/4),respectively.The sensitivity of the other 10 species were 100%,and the specificity of the 14 species were≥99.9%.Secondly,the accuracy of 275 liquid culture samples detected by MeltPro Myco assay was 99.6%(274/275).In addition,we compared the results of 812 clinical sputum samples detected by MeltPro Myco assay with its species identification results after cultured,and it was concluded that the sensitivity of MeltPro Myco assay was 81.4%(342/420),the specificity was 100%,the accuracy was 90.4%(734/812),and the Kappa value was 0.809,while the culture positive rate of MGIT 960 system was 65.0%(273/420),the specificity was 99.5%(390/392),the accuracy was 79.4%(645/812),and the Kappa value was 0.637.Therefore,this method can basically meet the needs of direct detection of sputum samples in clinical practice and it is expected to be promoted.In the third chapter,A single reaction 3-color subspecies identification method of MABC was established.We used 234 clinical liquid culture samples to evaluate the method,and took rpoB gene sequencing and multi-gene combined sequencing as the control method.The results showed that the sensitivity of MAS A,MASM,and MASB were 99.3%(139/140),100%(88/88),and 100%(2/2),respectively,and specificity were 100%(90/90),99.3%(141/142),and 100%(228/228),respectively,and accuracy was 99.6%(229/230).This method can be used to identify subspecies of liquid culture samples of MABC,and it is expected to be applied in clinical practice.In the fourth chapter,A single reaction 4-color method that can rapidly detect drugresistant mutations of common NTM was established.We used this method to detect 179 liquid culture samples of MABC,and took the AST as the gold standard.The sensitivity and specificity of the method detecting acquired CLA resistance were 71.4%(5/7)and 100%(172/172),and the sensitivity and specificity of the method detecting induced CLA resistance were 100%(77/77)and 97.0%(99/102).The accuracy of the method was 98.3%(176/179)compared with the AST,and the Kappa value was 0.966.However,the sample size of the detection is small.In the future,we will collect more clinical samples to make a more accurate clinical evaluation of the method,and more NTM will be included in the system for detection of drug-resistant mutations,so as to provide a reliable clinical screening method for NTM resistance.