Molecular Diagnosis Of Isoniazid-resistant Mutations In Mycobacterium Tuberculosis With Real-time PCR

The high prevalence rate of drug-resistant Mycobacterium tuberculosis (MTB) is the major challenge in control and prevention of tuberculosis. As one of the mostly often used first-line drug for TB treatment, isoniazide (INH)-resistant TB has high morbidity and mortality. The aim of this thesis is to establish new systems for detection of the INH-resistant mutations and explore their applications in clinical settings. The thesis is composed of five parts:a review on drug-resistant TB, its diagnosis and treatment (Chapter1), establishment of a real-time PCR-based melting curve analysis method for detection of INH-resistant mutations (Chapter2), a multicenter validation study on the newly established method (Chapter3), an epidemiology study on INH-resistant TB in two local cities (Chapter4) and finally, development of a quantitative method for the katG S315T mutation (Chapter5).Chapter1. IntroductionFirstly, the epidemiology of TB and drug-resistant TB were described, the biological background of tubercle bacillus and TB diagnostic methods were reviewed. Secondly, the mechanisms of INH action and resistance were discussed. Existing methods and their future trend in the detection of drug-resistant mutations were described. The working principle of probe melting analysis (PMA) and its use in mutation detection was illustrated. Finally, the aim, contents and meaningfulness of this thesis were given.Chapter2. Establishment and evaluation of PMA for INH-resistant mutation detection.INH resistance associated genes are katG315, inhA promoter, ahpC promoter and inhA94. A2-tube,2-color PMA was established targeting these four gene loci. By using our own extraction method and under the optimal experimental conditions, the limit of detection of the assay was3genome copies per reaction. The melting temperature (Tm) between wild type and the mutant types were larger than2℃and the SD were smaller than1℃(n=6). Consequently, all mutations could be efficiently detected regardless of the loci. The specificity was100%in the detection of reference panel and the detection ability for the heteroresistance was30%. The results with a panel of non-tuberculosis mycobacteria (NTM) from NIFDC showed that none NTM could display signal in all the four detection windows. The established assay could be used on three different modes of machines and the results showed no significant difference.Chapter3. A multicenter validation of the PMA methodClinical isolates (1096) collected from hospitals of Beijing, Henan and Shenzhen were used to validate the above described PMA in a double blind format. The comparison method was proportional method for drug susceptibility testing (culture method) and confirmation method was sequencing. The results showed that the clinical sensitivity of PMA was90.8%(397/437) and the specificity was96.4%(635/659) according to the culture method. The sequencing results confirmed the rightness of all the mutations detected by PMA including397INH-resistant isolates and24susceptible isolates. Forty INH-resistant isolates were detected as wild type by PMA. Sequencing analysis displayed that4of them were heteroresistance,5had mutations uncovered by PMA and31had no mutation in the four loci. Of the23types of mutation detected by PMA and identified by sequencing in INH-resistant isolates, the most common mutation types were katG S315T AGCâ†'ACC, inhA promoter-15Câ†'T, katG S315N AGCâ†'AAC, and ahpC promoter-10Câ†'T which accounted for87.4%(382/437) in INH-resistant isolates. No mutations were detected in inhA94loci. PMA successfully detected76.5%(13/17) heteroresistant samples. In conclusion, PMA could accurately detect the mutations, had good concordance with the culture method, and therefore would be suitable for clinical use.Chapter4. Epidemiological study on INH-resistant TB in Xiamen and ZhangzhouIn this study,785clinical isolates were collected in Xiamen and Zhangzhou from June2006to December2009and146smear positive sputum samples were collected in Xiamen from October2008and December2009. All the samples were subjected to PMA for detection of INH-resistant mutations. The mutant samples were confirmed by sequencing analysis. Totally,910valid samples from833patients were successfully analyzed. The results showed that the overall mutation rate in the population was19.4%(162/833), which was close to the mutation rate of18.9%in China and higher than that of13.9%worldwide. Of the14mutation types detected, katG S315T (AGCâ†'ACC) accounted for48%(77/162,72patients had single mutation while5patients had compound mutations), inhA promoter-15Câ†'T accounted for30%(48/162,41patients had single mutation while7patients had compound mutations), and katG S315N (AGCâ†'AAC) accounted for8%(13/162,11patients had single mutation and2patients had compound mutations), and altogether they contributed to85%(138/162) and were typically predominant mutations, which was in concordance with both domestic and international report. The katG S315T (AGCâ†'ACC) was reported to be associated with high-level drug resistance, multidrug resistance and extensively drug resistance, indicating that9.2%(77/833) of the patients might need treatment with second line drugs. inhA promoter-15Câ†'T was associated with low-level drug resistance, this portion of patients (5.8%,48/833) could be treated with high dose INH but not with either ethionamide or protionamide. Consequently, this epidemiological study was helpful not only for understanding the situation of drug-resistant TB in these regions but also for choosing suitable treatment strategy for the patients and, in the end, benefiting prevention of drug-resistant TB.Chapter5. Quantification of mutations in INH-resistant M.tuberculosis by real-time PAPReal-time tracking of the occurrence and accumulation of drug-resistant mutation in TB was valuable in guiding treatment. In the early beginning the mutant occurs at extremely low abundance, and the process of continuous accumulation can be only followed by a quantitative measurement. For this purpose, we developed a method that could selectively quantify rare mutations. Pyrophosphorolysis-activated polymerization (PAP) is an extremely specific method for rare mutation detection. By borrowing the concept of real-time PCR detection, we developed a real-time PAP method to quantify katG S315T (AGCâ†'ACC) mutation, which is the most common mutation in INH-resistant MTB. Two real-time PAP systems were established for both the wild type and the mutant by using the fluorogenic dye. With plasmid DNA, the limit of detection of the two systems was10copies per reaction, specificity was5×105and5×104copies, respectively, and the selectivity was1:105. The real-time PAP systems were used to analyze71smear-positive sputum samples, and the results were compared with both sequencing and a probe-based real-time PCR method. The results showed that fully concordant results were obtained in64samples with three methods. Of the7discrepant samples,6samples were found to have mixed wild-type and mutant MTB of different level (0.07%-94.2%), and5of them were detected as mutant by real-time PCR and2were detected as mutant by sequencing. One sample missed by both real-time PAP and real-time PCR was found to harbor a different mutation, i.e., katG S315N (AGCâ†'AAC). The above results demonstrated that real-time PAP could detect and quantify rare mutation, and thus has great potential to track the process of the mutation accumulation and help to choose an optimal treatment strategy.