Evaluation Of Rapid Detection Assay And Treatment Outcome Analysis Of Drug-resistant Tuberculosis

In recent years, the threats of the epidemic of drug resistant tuberculosis (TB), especially the occurrence of multidrug resistant-tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), have brought big challenges to TB control. In order to successfully control the spread of drug resistant TB, suitable diagnostic methods and effective treatment strategies are essential. Genetic mutations are the molecular mechanisms of the development of drug resistance of mycobacteria tuberculosis (MTB) and the accumulation of these mutations result in MDR-TB and XDR-TB. Previous studies have found several drug resistance related mutations, including mutations in rpoB related to rifampin resistance, mutations in katG and inhA related to isoniazide resistance, mutations in embB related to Ethambutol resistance, mutations in rrs, eis and tlyA related to aminoglycosides resistance, and mutations in gyrA and gyrB related to fluoroquinolones resistance. With the development of researches on the mechanism of TB drug resistance, more mutations have been found and progress have been made in molecular detection assays. As the gold standard of drug resistant TB diagnosis, the conventional phenotypic detection assay (drug susceptibility testing, DST), has higher sensitivity and specificity, However, it takes more detection time, requires strict laboratory condition and may pose biological threats to lab operators. The molecular drug resistant detection based on gene mutations could overcome the limitations of such conventional DSTs. WHO has recommended the line probe assays to detect drug resistance of TB, but it has not been systematically evaluated in China. Therefore, we performed a study to assess value of GenoType MTBDRplus and MTBDRsl in diagnose the drug resistance of MTB circled in Jiangsu Province. In addition, we used DNA sequencing method to explore other mutations and confirm the existence of hetero-resistance.The control of drug resistant tuberculosis relies on not only timely and accurate diagnosis but also effective treatments. Though the absolute number of MDR-TB patients under standard treatment has increased, the proportion and the completion rate of treatment was low while the proportion of withdrawing and information missing was high. Patients in the previous studies were mainly recruited prior to publication of the standard treatment guideline amended by WHO. Data of treatment outcomes of drug resistant TB are limited. Thus, we recruited MDR-TB and XDR-TB patients treated in Zhenjiang City, followed their outcomes and analyzed the determinants.Part IEvaluation of rapid detection assay in diagnosis of drug resistant tuberculosis[Objective] To evaluate the value of line probe assays in rapid diagnosis of drug resistant TB.[Methods] 112 strains were collected from Jiangsu, followed by culture, strain identification, conventional DST and DNA extraction. GenoType MTBDRplus and MTBDRs7 were used to detect drug resistance to RFP, INH, OFX, Km and EMB by comparing with conventional DST. Strains with unclear results, discordant results between the two assays, and unclear mutations were further sequenced.[Results] After excluding 13 strains being identified as non tuberculosis mycobacteria (NTM),99 MTB strains were involved in the subsequent analysis. The sensitivity and specificity of GenoType MTBDRplus were 100% and 50% for RFP respectively, and 86.11% and 47.06% for INH respectively. The most common mutation combinations detected by GenoType MTBDRplus were rpoBWT8 omission +MUT3 presence, katGWT omission+MUT1 presence and inhAWTl omission+ MUT1 presence. The sensitivity of GenoType MTBDRsl for OFX, Km and EMB were 94.74%,62.50% and 58.82%, respectively. The specificity for OFX, Km and EMB were 92.59%,98.81% and 91.67%, respectively. The most common mutations detected by GenoType MTBDRsl were gyrA WT3 omission+MUT3C presence, rrsMUTl presence, embBWT omission+MUTIB presence and embBWT omission +MUT1A presence. Sequencing analysis found several uncommon mutations at rpoB504,512,513,515,516,518,522,535,562,572 codon; katG317,299 codon; inhA-26,-34; gyrB422,435,450,473,511 codon; rrs1491; eis-10 and embB319,407, 410 codon.[Conclusion] GenoType MTBDRplus and GenoType MTBDRs/could be applied for the rapid detection of drug resistance to RFP and OFX. However, the role of GenoType MTBDRplus for INH resistance detection has not been confirmed since the current results were different from previous reports. It can’t be widely applied until being validated in China. In addition, since the mechanism of Km and EMB resistance were not completely identified, GenoType MTBDRsl for detecting the resistance to Km and EMB is not suitable for clinical application currently. The correlation between uncommon mutations identified in this study and drug resistance needs to be confirmed in the following studies.Part IITreatment outcome analysis of drug resistant tuberculosis[Objective] To describe the treatment outcomes of patients with drug resistant TB, and to explore the potential determinants, with aims to provide recommendations for TB control.[Methods] 103 MDR-TB and XDR-TB patients treated in the Third Hospital of Zhenjiang from July 2009 to March 2013 were recruited in this study. These patients were involved in the Global Fund program for drug resistant TB. The treatment outcomes were identified by sputum culture. Time of hospitalization, and duration of treatment were collected. Outcomes and the determinants were analyzed.[Results] There were 94 (91.26%) MDR-TB patients and 9 (8.74%) XDR-TB patients involved in this study, with the male to female ratio of 3:1. The average age was 50.02±13.88 years. The median diagnostic delay for drug resistance was 31 days. The median duration of treatment was 730 days and hospitalization was 62 days, respectively. Patients older than 60 years old or with XDR-TB had a shorter treatment duration time.46 (48.94%) patients had positive outcomes while 48 (51.06%) had worse outcomes, including 10 (10.64%) deaths. XDR-TB cases and patients older than 60 years old had higher risks for worse outcomes.[Conclusion] The median duration of treatment for MDR-TB patients in the study setting was similar to the original guideline, but patients with XDR-TB receive shorter treatment time. The outcomes of MDR-TB and XDR-TB were unsatisfied. Development of new anti-tuberculosis drugs, implement of better therapeutic strategies and improvement of treatment compliance should be enhanced in order to achieve the goal of TB control.