Highly Sensitive Detection Of Isoniazid-resistant Mutations In Mycobacterium Tuberculosis And Rapid Identification Of Mycobacterium Species

Tuberculosis is one of the public health problems that seriously endanger human health worldwide.With the in-depth study of the drug resistance mechanisms of Mycobacterium tuberculosis,the rapid identification of Mycobacterium tuberculosis and its drug resistance using gene detection methods is significantly important for guiding the tuberculosis treatment.However,the sensitivity of detecting heteroresistance for current molecular methods does not match that of traditional drug susceptibility testing by which 1%resistant bacteria was defined.Besides,the infections caused by nontuberculous mycobacteria have gradually increased in recent years,whose clinical manifestation resembles that of tuberculosis.However,the treatment regimens for both are different.Rapid diagnosis for nontuberculous mycobacteria diseases and distinguishing them from tuberculosis is particularly important for the treatment of mycobacterial diseases.Therefore,this thesis focuses on the highly sensitive detection of Mycobacterium tuberculosis heteroresistance,and the rapid identification of clinically relevant Mycobacterium species.In chapter one,we first briefly introduces the epidemiology of tuberculosis worldwide,the diagnosis and treatment of drug-resistant tuberculosis,and the overview and identification methods of nontuberculous mycobacteria diseases.Then,we systematically introduced the method of melting curve analysis,including its principle,characteristics,and its suitable fluorescent probes applied in the detection of drug-resistant tuberculosis.Finally,we put forward the research purpose,content and significance of this thesis.In chapter two,we developed a melting-curve analysis-based approach,termed DeepMelt,that can detect less-abundant mutants through selective clamping of the wild-type in mixed populations.The singleplex DeepMelt assay was able to detect 0.01%katG S315T in 105 MTB genomes/μL.Based on the DeepMelt principle,we established multiplex DeepMelt TB/INH assay to detect isoniazid-resistant mutations in the katG 315 codon,the inhA promoter,and the ahpC promoter.The DeepMelt TB/INH assay could detect 1%of mutant species in the four loci associated with isoniazid resistance in 104 MTB genomes/μL.The DeepMelt TB/INH assay was tested on a panel of DNA extracted from 602 pre-characterized clinical isolates.Using the 1%proportion method as the gold standard,compared with the MeltPro TB/INH kit,the sensitivity was found to be increased from 93.6%(176/188,95%confidence interval[CI]=89.2-96.3%)to 95.7%(170/188,95%CI=91.8-97.8%).Further evaluation of 109 smear-positive sputum specimens increased the sensitivity from 83.3%(20/24,95%CI=64,2-93.3%)to 91.7%(22/24,95%CI=74.2-97.7%).In both cases,the specificity remained nearly unchanged.Moreover,all the 20 heteroresistant samples found in this study were confirmed by DNA sequencing,and even partially by digital PCR,which proved that the DeepMelt TB/INH assay has a high sensitivity for heteroresistance detection.In chapter three,we developed a "fluorescence-melting temperature"two-dimensional labeling analysis-based MeltPro Myco assay that allows identification of clinically relevant 19 Mycobacterium species in a single reaction within 3 h.The MeltPro Myco assay correctly identified 51 reference mycobacterial strains,and showed no cross-reaction with 16 non-mycobacterial strains.The limit of detection was 300 bacilli/mL,and 1%of minor species could be detected in the case of mixed infections.Finally,365 clinical isolates were used to evaluate its clinical performance.The results showed that the sensitivity of the system to detect mycobacteria was higher than 95%,and the specificity was higher than 99%.Considering its rapidity,ease of use,and accuracy,we concluded that our real-time assay could be used for identification of clinically common mycobacteria.In chapter four,to investigate mycobacterial species distribution in different regions,a total of 1163 clinical isolates collected from five regions including Guangzhou,Xiamen,Henan,Heilongjiang,and Xinjiang were tested using the MeltPro Myco assay established in Chapter three.The results showed that the nontuberculous mycobacteria isolated rates in Guangzhou,Xiamen,Henan,Heilongjiang,and Xinjiang were 33.6%,4.2%,7.4%,0%,and 8.5%,respectively.Moreover,South China(Guangzhou and Xiamen)showed a higher incidence rate and richer species of nontuberculous mycobacteria(135/567,23.8%;12 species)in comparison with that of North-Central China(Henan,Heilongjiang,and Xinjiang)(23/596,3.9%;2 species).Furthermore,94 smear-positive specimens collected from Guangzhou Chest hospital were tested by the MeltPro Myco assay.The results showed that the nontuberculous mycobacteria isolated rate was 25.5%.To verify the results obtained by the MeltPro Myco assay,the Mycobacterium tuberculosis complex detection kit,DNA sequencing or Spoligotyping methods were used as control methods.The results showed that the concordance rates between the MeltPro Myco assay and the control methods in the detection of 1163 clinical isolates and 94 sputum specimens were 99.7%and 100%,respectively.Therefore,the MeltPro Myco assay could be tested on clinical isolates and smear-positive sputum specimens.It could be expectedly implemented as a screening tool in tuberculosis-endemic regions.