Study Of The Rapid Diagnosis Of Mycobacterium Tuberculosis And Drug Resistant Mycobacterium

BackgroundTuberculosis (TB) is the major health threat globally, and China is one of the 22 high-burden countries for TB in the world. China has the world's second highest burden of TB cases with more than 4.5 million cases of TB, and 130,000 deaths due to TB every year. Furthermore, almost half of population is already infected with Mycobacterium tuberculosis (M.tb) in China. The emergence of drug resistant TB, especially multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB, pose a substantial threat to TB control programs, and become an urgent issue to be solved. Mycobacterium tuberculosis grows slowly, which has constrained the timelyness of clinical diagnosis and treatment of patients. Thus, there are urgent demands for a rapid diagnostic tool with good specificity, sensitivety and simplicity.Research PurposeWe have evaluated the sensitivity and specificity for Mycobacterium tuberculosis (TB) Isothermal Amplification-DNA Strip Diagnostic Kit assay and Diagnostic kit for detection of Drug Resistance Mutations in Mycobacterium tuberculosis assay to do rapid diagnosis of tuberculosis and drug resistance. These two have been proved be sample, rapid and reliable and we recommanding these assasys to Basic Medical Institutions for rapid diagnosis of tuberculosis and drug resistance. The applications of these methods will be helpful for initiate effective treatment rigimens for TB patients and is of great importance for preventing transmissions.Research DesignIn order to evaluate the application of TB Isothermal Amplification-DNA Strip Diagnostic Kit in the TB patients,465 sputum specimens from Shanghai TB hospital patients were collected. All the sampled sputum specimens were tested by TB Isothermal Amplification-DNA Strip Diagnostic kit, AFB, L-J and BD960 simultaneously, and the sensitivity and specificity were analyzed and compared between them. To evaluate the application of Diagnostic kit using asymmetric PCR amplification technique for detection of drug resistance mutations in the TB patients, another 158 MDR isolates were randomly selected from 322 MDR TB cases collected at the Shanghai Municipal Center for Disease Control and Prevention (Shanghai CDC) from March 2004 to November 2008. The sampled 158 strains were tested by the Diagnostic kit for detection of Drug Resistance Mutations in Mycobacterium tuberculosis, and compared with the sequencing data.Results1. Evaluation of Mycobacterium tuberculosis (TB) Isothermal Amplification-DNA Strip Diagnostic Kit for Rapid Detection of the TB patientsIn 465 sputum samples from Mycobacterium tuberculosis (TB) patients, 22 were polluted during the operation,10 cases were non-tuberculous mycobacterial, to reflect the comparability between methodologies, the contaminated specimens and NTM were excluded. In remaining 433 sputum samples from TB patients, The positive detection rates of AFB, L-J, MGIT/960 and Isothermal Amplification-DNA Strip Diagnostic Kit were 37.4%(162/433),47.1%(204/433),51.3%(222/433) and 45.0%(195/433) respectively. In this study, the time spent for AFB, MGIT/960, L-J and Isothermal Amplification-DNA Strip Diagnostic Kit were 4 hours,13 days,30 days, and 4 hours respectively. Using BD960 as the "gold standard", the sensitivity of AFB, L-J, and Isothermal Amplification-DNA Strip Diagnostic Kit were 70.7% (157/222),89.6%(199/222), and 83.8%(186/222) respectively. The specificity of AFB, L-J, and Isothermal Amplification-DNA Strip Diagnostic Kit were 97.6%(206/211), 97.6%(206/211), and 95.7%(202/211) respectively. Statistical significant differences in above results were observed. The Kappa Consistency for AFB, L-J and Isothermal Amplification-DNA Strip Diagnostic Kit were 67.9%,87.1% and 79.7% respectively. The sensitivity of L-J and TB Isothermal Amplification-DNA Strip Diagnostic kit for smear-negative but BD 960 culture-positive specimens were 70.8%(46/65) and 78.5% (51/65), and the specificity were 98.0%(202/206) and 98.5%(203/206) respectively, with statistically significant difference. The Kappa Consistency of L-J and Isothermal Amplification-DNA Strip Diagnostic Kit in patients with smear-negative but BD 960 culture-positive were 74.7% and 81.7%. Results from this study suggest that TB Isothermal Amplification-DNA Strip Diagnostic Kit has a relatively high sensitivity and specificity to detect Mycobacterium tuberculosis especially for the smear negative but culture positive cases. The procedure of this method is simple, rapid and not required for special equipment and could be easily applied in the general laboratory in Medical Institutions.2. Evaluation of Diagnostic kit for detection of Drug Resistance Mutations in Mycobacterium tuberculosisIn this study, we developed a novel real-time PCR method based on melting curve analysis of dual-labeled probes. Six probes targeting rpoB 81bp core region, katG315, inhA promoter, ahpC promoter and embB306 were designed and tested with clinical isolates. First,10 multi-drug resistant (MDR) strains with a wide mutation spectrum were used to analyze the melting temperature (Tm) deviations of different mutations by multiplex real-time PCR. All mutations can be detected by significant Tm reductions compared to the wild-type. Then three duplex real-time PCR reactions, with two probes in each, were developed to detect mutations with 158 MDR strains. Comparing with the sequencing data, all mutations targeted by the six probes were detected with 100% sensitivity and 100% specificity. Our method provided a new way to rapidly detect drug resistant mutations of M.tuberculosis. Compared to other real-time PCR methods, ours needs fewer probes. The duplex reactions with probes labeled by the same fluorophore guaranteed this assay can be run on single-channel instruments. In conclusion, we have developed a new and widely applicable real-time PCR assay to detect drug-resistant mutations of M.tuberculosis.