| SectionI Study on characteristics of Mycobacterium tuberculosis drug resistance spectrum in ChinaChina is one of the 22 countries with high TB burden,and the Chinese government has always been involved in extensive efforts to prevent and control TB.The incidence and infection of tuberculosis,especially drug-resistant tuberculosis,is still a major threat to the national economic development and people's health.The fifth national tuberculosis epidemiological sampling survey in 2010 suggested that there were 4.99 million tubercular patients in China,with 1.3 million new TB case,and TB caused an estimated 50000 deaths.For many years,mycobacteriu,m tuberculosis has always killed more people than any other infectious agent.WHO and IUATLD launched a global program for surveillance of drug resistance from 1994 to 2007,disclosed that the total drug resistance rate was 20.0%on average and the multidrug resistance rate was 5.3%on average.Chinese government conducted a national survey of drug-resistant tuberculosis(2007-2008),the result displayed approximately 1 of 10(8.3%)had MDR-TB,5.7%with new cases of tuberculosis and 25.6%with previously treated cases had multidrug-resistant(MDR)tuberculosis,there were an estimate of 120 thousand new MDR cases every year,China have always been on the list of "Countries and Districts need more attention"proposed by WHO due to the severe situation of drug resistance tuberculosis.In 2014,WHO set a target to "END TB”by 2035,and the global emergence of MDR-TB,floating population and TB/HIV co-infection pose three major challenges,MDR-TB control will directly affect the success of "END TB"strategyBy popularizing and implementing the Directly Observed Treatment Short-course(DOTS)strategy,the epidemic situation of tuberculosis in China has been effectively controlled,and the TB incidence dropped to 60.53 per 100000 people in 2017.However,tuberculosis remains one of the most serious public health and social problems,highlighting the need for enhanced strategies to reduce TB incidence and mortality.The success of TB control depends on the development of better preventative,diagnostic,and therapeutic interventions,and better understand of TB prevalence and drug resistant situation in China.In consideration of the severe drug-resistant tuberculosis epidemic situation,the main task for TB control at present is to control and prevent drug-resistant tuberculosis.A national surveillance survey of drug-resistant tuberculosis can help to determine the proportion of tuberculosis cases that are resistant to drugs,estimate the incidence of drug-resistant tuberculosis,and identify the factors that are linked to drug-resistant tuberculosis,especially to MDR tuberculosis,furthermore,it can also help formulate,improve and evaluate the national TB control strategy,To help China develop a national response to drug-resistant tuberculosis,we conducted a national surveillance survey of drug-resistant tuberculosis in 2013.MALDI-TOF-MS was applied to identify mycobacteria species,and then MTBC strains were subjected to drug susceptibility tests using microporous plates containing 12 drugs and MGIT 960 liquid culture for PZA.Thus,we determined the drug resistance rate of mycobacterium tuberculosis to 13 drugs,estimated the incidence rate of drug-resistant tuberculosis,and analyzed the risk factors that are linked to drug-resistant tuberculosis,especially to MDR-TB.In this survey,26.8%of the mycobacterium tuberculosis were resistant to at least one anti-tuberculosis drugs.Overall,7.25%of the tuberculosis were MDR-TB,with 5.35%of new case and 25.64%of previously treated case.Among the patients with MDR tuberculosis,6.93%of the tuberculosis were XDR-TB.By statistical analysis,we found people with previously treated,live in cities(towns)and age range from 30 to 45 are more likely to be drug-resistant tuberculosis.When compared with 2007-2008 national surveillance survey of drug-resistant tuberculosis,the results proved the severity of TB drug-resistant has declined,this decrease was resulted from the significant efforts of the Chinese government to prevent and control TB by launching a series of policies in past years.But China still has a serious epidemic of drug-resistant tuberculosis,effective prevention and treatment for drug-resistant tuberculosis is still neededIn this study,MALDI-TOF MS and Mycobacterium tuberculosis MIC plate were used to conduct species identification and drug susceptibility testing of mycobacterium tuberculosis,and this survey present a comprehensive view of the epidemic characteristics of drug-resistance tuberculosis in China,including four first-line and nine second-line anti-tuberculosis drugs,providing a scientific basis for the formulation of drug-resistant tuberculosis control policies.In addition,all strains were subjected on whole genome sequencing,which laid a foundation for the prediction of drug sensitivity by WGS and the analysis of the principal of mycobacterium tuberculosis transmission in China.Section ? Establishment of LAMP-based method for detection of Mycobacterium tuberculosis complexTB diagnosis is based on examination of etiology(including bacteriology and molecular biology),and epidemiological history,clinical manifestations and chest imaging results are also included in the analysis to confirm diagnosis.Mycobacterium tuberculosis complex(MTBC)is the causative agent of pulmonary tuberculosis,early,rapid and accurate detect MTBC is crucial to tuberculosis control and prevention.However,traditional bacteriological methods,including Ziehl-Neelsen(ZN)stain and solid culture,both exhibit some drawbacks.For past decades,many TB diagnostic methods have been developed,but few of them show promise for the detection of MTBC in sputum samples,with sensitive,rapid and convenient delivery of results.Loop-mediated isothermal amplification(LAMP),as the popular isothermal amplification technique,which has the the advantages of rapid amplification,high sensitivity,high specificity,simple operation and easy detectionIS6110 and mpb64 are two genomic genes found only in MTBC,numerous of nucleic acid-based molecular methods targeted IS6110 or mpb64 have been developed to detect MTBC,which were proved to have excellent detection efficiency.However,many studies have found IS6110 or mpb64 are naturally absent in some MTBC strains,thus,false negative result may appear if only target one of them(IS6110 or mpb64)In this study,we developed a multi-LAMP target both IS6110 and mpb64 combined with hydroxynaphthol blue(HNB)to detect MTBC,termed MTBC-MLAMP-HNB Moreover,during testing of 94 clinical samples,the sensitivity and specificity of MTBC-MLAMP-HNB results were 93.7%and 98.3%,respectively,when compared to results obtained using MGIT liquid culture method.Therefore,the MTBC-MLAMP-HNB method developed in this study holds promise for application as an effective point-of-care test to detect M.tuberculosis complex.Section III Development of multi-LAMP-based method for detection of Mycobacterium abscessus complexThe prevalence of pulmonary infection due to Mycobacterium abscessus complex(MABC)is increasing worldwide.MABC is one of the most common NTM isolated from patient with chronic pulmonary disease,which has similar clinical symptoms to TB Treatment of MABC infections is complicated because of its innate multidrug resistance that can render even combination antibiotic treatment ineffective Based on divergence of rpoB sequences,M.abscessus complex is thought to be comprised of three species-Mycobacterium abscessus,Mycobacterium massiliense and Mycobacterium bolletii.Of the three,M.bolletii is rarely isolated,M.abscessus and M.massiliense are major pathogens that cause post-surgical wound infection and chronic pulmonary disease.Although they are closely related subspecies of M.abscessus complex,their infections are associated with different drug-resistance and cure rate.In the present study,a loop-mediated isothermal amplification(LAMP)coupled with lateral flow dipstick(LFD)method was developed to simultaneous detect M.abscessus and M.massiliense,via specific erm(41)gene.The amplification was carried out at 65 ? for only 60 min,and the results could be visualized on a lateral flow strip.Positive results only occurred in M.abscessus and M.massiliense,no cross-reaction with other mycobacterial species was observed.Therefore,the cost-effective MABC(M.abscessus complex)-LAMP-LFD method developed here was able to correct the diagnose of M.abscessus and M.massiliense infection in a short time.Thus,this method could be used to guide clinicians in treatment of M.abscessus group infections. |
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