| China is one of the countries with a high burden of tuberculosis(TB)in the world.The number of people with tuberculosis in China ranks third in the world.Tuberculosis is one of the four major infectious diseases controlled by China.The epidemic of tuberculosis,especially drug-resistant tuberculosis,is responsible for the economic development and people's health in China.The TB epidemic was mainly caused by 62%of Mycobacterium tuberculosis(MTB)Beijing genotype strains.In recent years,China has adopted many effective measures to control tuberculosis.However,TB is still one of the serious public health problems and social problems in China.In order to further effectively control tuberculosis and reduce the prevalence and mortality,we need to study the genetic variation,evolution of MTB,and the epidemiological patterns of tuberculosis,to understand the genetic characteristics of Beijing genotype strains.And the law of transmission has a positive role in promoting effective prevention and control of tuberculosis.At present,there are still many problems in the field of tuberculosis control in China that have not been systematically studied.For example,the origin,evolution,and prevalence of the major epidemic strains of MTB in China are unclear,and the mutation pattern of resistance-related genes associated with tuberculosis in China is not Clearly,the mathematical model for the occurrence and spread of tuberculosis in China has not yet been established.There is a lack of systematic research on the virulence,pathogenicity,and drug resistance of the major epidemic strains of tuberculosis in China,as well as the epidemiological and transmission laws.If we can conduct in-depth research on the above issues,it will provide scientific basis for further revision and improvement of our country's TB prevention and control strategies,and provide guidance for the implementation of tuberculosis prevention and control measures.ObjectiveTo validate and evaluate the combination method of SNP and VNTR for genotyping of MTB Beijing genotype strains and understand the prevalence and transmission of MTB Beijing genotype strains by using this genotyping method from the national cross-sectional and regional dynamic monitoring studies,further to analyze the association between drug susceptibility and genotype.Finally,to explore the genetic characteristics of Beijing genotype strains via GWAS for the successful reason of transmission.Method1.The combination method of SNP and VNTR-15 was performed on MTB strains collected from 70 surveillance sites in 31 provinces across the country and was evaluated by comparison with the HGI of VNTR-15 and VNTR-24 for genotyping of MTB Beijing genotype strains.2.Nationwide analysis of the prevalence and transmission of Beijing genotype strains and related factors in transmission by using the combination method of SNP and VNTR-15.3.Regional(continuously 3 years)dynamic monitoring of the prevalence and transmission of Beijing genotype strains and related factors in transmission by using the combination method of SNP and VNTR-15.4.The drug susceptibility test was used to detect the drug-resistant phenotype of MTB.Genetic polymorphisms of MTB in Sichuan province were analyzed by the combination method of SNP and VNTR-15.And the correlation between drug susceptibility and genotypes was analyzed.5.Whole genome sequencing was applied to understand the genetic characteristics of clustered Beijing genotypes and analyze the role of genetic mechanisms in their widespread transmission.Result1.For Beijing genotype strains,the resolution of combined SNP and VNTR-15 genotyping methods(HGI=0.9950)was higher than that of VNTR-15(HGI=0.9915)and VNTR-24(HGI=0.9943);for modern Beijing genotype strains,the resolution(HGI=0.9917)of combined method is also higher than that of VNTR-15(HGI=0.9859)and VNTR-24(HGI=0.9905);for ancient Beijing genotype strains,the resolution of VNTR-24(HGI=0.9989)was higher than that of the combined SNP and VNTR-15 genotyping method(HGI=0.9980)and VNTR-15 method(HGI=0.9978).2.The prevalence rate of MTB Beijing genotype strains in China was 73.69%(1731/2349),among which the prevalence rate of modern Beijing genotype strains was 76.43%(1323/1731)in China.And the prevalence rate of ancient Beijing genotype strains was 23.57%(408/1731).Modern Beijing genotype strains were the dominant genotype in China.Through univariate analysis,the results showed that the distribution of Beijing genotypes and their subtypes was uneven across the country(P<0.001:P<0.001).Beijing genotype strains had stronger transmission than non-Beijing genotype strains(P<0.001);modern Beijing genotype strains had stronger transmission(P<0.001)than ancient Beijing genotype strains.In addition,the prevalence of Beijing genotype strains was associated with MDR(P=0.028)and streptomycin resistance(P<0.001).3.The prevalence rate of MTB Beijing genotype strains in Sichuan province was 55.51%(489/881),among which the prevalence rate of modern Beijing genotype strains is 70.96%(347/881),and the prevalence rate of ancient Beijing genotype strains was 29.04%(142/881).The dominant strains in Sichuan province were modern Beijing genotype strains.The prevalence of Beijing genotypes and its subtype strains was not statistically different in Sichuan provinces(P=0.117;P=0.311).Beijing genotype strains had a higher infection rate in retreatment patients,which was approximately twice that of non-Beijing genotype strains(P=0.009,OR=1.984);Beijing genotype strains were more prone to recent transmission.And the recent transmission power is about 3 times that of non-Beijing genotype strains(P=0.005,OR=2.880).Comparing modern Beijing genotypes with ancient Beijing genotypes,the results showed that male patients are more likely to infect with modern Beijing strains(P=0.035)than female tuberculosis patients;patients with different age groups had different probability of infecting modern Beijing genotype strains and ancient Beijing genotype strains(P=0.007).The prevalence of Beijing genotypes and its subtypes did not change significantly between 3 years(P=0.621;P=0.739);and the recent transmission in 2015 was significantly higher than 2013 and 2014(P<0.001);the recent transmission rate of Beijing genotype strains in 2015 was higher than in 2013 and 2014;subtypes Bmyc10 have an absolute advantage in Beijing genotype strains.4.Among the tuberculosis patients in Sichuan Province,male patients are more likely to have rifampicin resistance(P=0.034)and multidrug resistance(P=0.024)than female patients.In addition,patients who experienced recent transmission(clusters)were more likely to develop rifampicin resistance(P=0.007),ethambutol resistance(P=0.007),and multidrug resistance(P=0.002).Beijing genotype strains were more prone to rifampicin resistance(P=0.038),isoniazid resistance(P=0.036),ethambutol resistance(P=0.003)and multi-drug resistance(P=0.006)than non-Beijing genotype strains,indicates that there is a correlation between the prevalence of Beijing genotype strains and drug susceptibility in Sichuan province.The strains of subtype Bmyc210 and subtype Bmyc10 were statistically different in SM resistance,INH resistance,RFP resistance and MDR.5.The detection of SNPs and InDel in East Asian branches(Beijing genotype families)of MTB was significantly higher than that in other branches;the SNP hotspots in Beijing genotypes were mainly distributed in genes related to PE/PPE protein family;the genetic evolution distances between Beijing genotypes and H37Rv standard strains are relatively far apart;the genetic distances between modern Beijing genotype strains are relatively close,and the genetic distances between ancient Beijing genotype strains are far away.Conclusion1.The VNTR and SNP typing are complementary to the study of the epidemic and transmission pattern of Beijing genotype strains.The combination method of SNP and VNTR-15 genotyping method have high resolution for MTB Beijing genotypes and modern subtype strains.It is recommended to use the method for analysis of recent transmission patterns and risk factors in regions where Beijing genotype strains or modern Beijing subtypes predominate.In areas where ancient Beijing genotype strains are the predominant strains,the VNTR-24 typing method is recommended.2.Modern Beijing genotype strains are dominant strains in China;Beijing genotypes and their modern subtypes are distributed differently in different regions of the country;Beijing genotype strains are associated with SM resistance and MDR,and Beijing genotype strains have more potential for transmission,suggesting that Beijing genotypes have the dual advantages of drug resistance and transmission.At the same time,there are statistical differences between modern Beijing genotype strains and ancient Beijing genotype strains in terms of their transmission potential and streptomycin resistance.Modern Beijing genotype strains have more advantages.3.MTB modern Beijing genotype strains are dominant strains in Sichuan province;Beijing genotype strains are also associated with clustering and drug resistance,and are more prevalent in retreatment cases,indicating that Beijing genotype strains are more easier to spread due to stronger latency,and drug resistance provides it with a longer time advantage of spreading infection.Modern Beijing genotype strains are the predominant strains in Sichuan province;Beijing genotype strains have a positive correlation with clustering and drug resistance at the same time,indicating that Beijing genotype strains in this area have double advantages,and drug resistance provides them with the advantage of longer infectious time;male and drug resistance are risk factors for recent transmission(clusters);subtype Bmyc10 strains is more likely than other Beijing genotype strains to have recent transmission,and its phenotypic changes accumulated by gene mutations may be the reason for its increased adaptability.4.Mycobacterium tuberculosis isolates from Sichuan Province of China have different degrees of resistance to isoniazid,streptomycin,rifampicin,ethambutol,ofloxacin,and kanamycin.Except Dazhu County,the proportion of primary drug-resistant tuberculosis in the remaining 4 monitoring sites is higher than that of acquired drug-resistant tuberculosis.Compared with non-Beijing genotypes,Beijing genotypes are positively associated with rifampicin resistance,isoniazid resistance,ethambutol resistance,and multidrug resistance.Bmyc210 subtype strains are more susceptible to SM resistance,INH resistance,RFP resistance and MDR.5.The Beijing genotype strains have high mutations in genes of the PE/PPE protein family,which may be the reason that it evade the host immune system and cause widespread epidemic.Modern Beijing genotypes are relatively recent evolutionary subtypes compared to ancient Beijing genotypes and may be more prone to spread(clusters). |
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