| Objective To understand the epidemiological distribution characteristics of isoniazid-resistant Mycobacterium tuberculosis(MTB)in Guangxi,to explore the influence factors of INH resistance.To understand the genes mutation characteristics,lineage distribution and genotype composition of INH-resistant MTB.So as to provide a theoretical basis for the development of INH-resistant tuberculosis(TB)prevention and treatment measures in Guangxi,and at the same time lay a foundation for future molecular epidemiology research and drug resistance mechanism.Methods Guilin,Guigang,Baise,Chongzuo and Fangchenggang were selected as the research sites and the monitoring method was used to collect patients,and 21 counties(cities,districts)in 5 cities were selected by pure random sampling method.From January to December 2017,a total of 1479MTB strains that were registered and treated in the local tuberculosis prevention and treatment institutions at various monitoring points in accordance with the case inclusion criteria,complete questionnaire information and positively cultured were continuously enrolled in the study.The ratio method recommended by World Health Organization was used to drug susceptibility test to INH.Random number table method was used to extract 101 strains and 516strains from INH resistant strains and sensitive strains respectively,and the DNA of the strains was extracted for whole genome sequencing.The sequencing results were compared with the H37Rv sequence of the standard strain in the NCBI database.Python 2.7 was used to process the original data and 43 interval sequences were extracted by Blast sequence alignment.The phylogenetic tree was constructed by MEGA 7 and modified it on the i TOL website.Epidata 3.1was used for double-entry and verification of all questionnaires,and SPSS 22.0was used for statistical analysis.The t test,F analysis or rank sum test was used to compare of measurement data,chi-square test or Fisher exact probability method was used to compare of classification data,and the unconditional logistic regression model was used to analyze the influence of 13 factors such as gender and age on INH resistance.Results 1.Epidemic characteristics of isoniazid-resistant MTB in Guangxi:A total of 1479 strains were collected,including 397 strains(26.84%)from Guilin,401 strains(27.11%)from Guigang,231 strains(15.62%)from Baise,316 strains(21.37%)from Chongzuo and 134 strains(9.06%)from Fangchenggang.There were 129 strains identified as INH resistant by drug susceptibility testing(DST),and the total detection rate of INH resistance was8.72%(129/1479),and the detection rates of INH resistance were 7.25%(94/1296)and 19.14%(35/183),respectively.The INH resistance detection rate among seasons was no statistical difference(χ~2=3.063,P=0.382).It was given priority to with the young and middle-aged male and farmers accounted for the highest proportion(84.50%).The INH resistance detection rate in border area(Baise,Chongzuo,Fangchenggang)(10.72%)was higher than the non-border area(Guilin,Guigang)(7.02%),the difference was statistically significant(χ~2=6.325,P=0.012).2.Factors related to isoniazid resistance:Univariate analysis results showed that,except for region,nationality,registration classification,number of lung fields affected by lesions(P<0.05),there was no statistical correlation between other factors(including gender,age)and the occurrence of INH resistance(P>0.05).Multivariate analysis results showed that three factors were statistical correlation with the occurrence of INH-resistant MTB:region,registration classification and number of lung fields involved(P<0.05).The risk factors for INH resistance were patients in border area,patients undergoing re-treatment and patients with lung field number more than three lesions,whose OR were(95%CI:1.03～2.16),2.65(95%CI:1.72～4.08)and 1.56(95%CI:1.06～2.31)respectively.By comparing INH resistance and its influencing factors in border and non-border area,it was found that only ethnic groups and HIV infection status had statistical differences in the distribution of INH resistance in two regions(P<0.05),and re-treatment was a risk factor for INH resistance in two regions.3.Gene mutation characteristics and lineage analysis of isoniazid-resistant MTB in Guangxi:In the 101 isoniazid-resistant MTB strains,there were 85strains which contained INH resistance related gene mutation,meaning that the agreement rate between DST and the prediction of drug resistance was 84.16%(85/101).There were totally 4 kinds of INH resistance mutations were found,including kat G,ahp C＿promoter,fab G1＿promoter and inh A gene,and kat G gene mutation rate was highest among them,accounting for 90.59%(77/85),which mainly for the 315 locus mutation.Kat G 315 locus mutation rate was 84.71%(72/85).In the 516 strains of INH sensitive MTB strains,there were 35 strains(6.78%,35/516)which contained INH resistance related gene mutation.There were 5 kinds of INH resistance mutations were found,including kat G,kas A,fab G1＿promoter,ahp C＿promoter and inh A gene,and kat G gene mutation rate was the highest,accounting for 51.43%(18/35),which mainly for the 315 locus mutation.Kat G 315 locus mutation rate was 40.00%(14/35).Kat G 315 locus mutation in INH resistant and susceptible strains of distribution difference was statistically significant(χ~2=331.117,P<0.001).With DST as the gold standard,analysing kat G 315 locus mutation forecast INH susceptibility,the results show that the sensitivity of detection kat G 315 locus mutation was 71.29%,and the specificity was 97.29%,positive predictive value was 83.72%,negative predictive value was 94.54%,Youden’s index was 0.69,Kappa value was 0.73.The Receiver operating characteristic curve(ROC)was plotted and the area under the ROC curve was found to be 0.84.The area under the curve was tested and the result showed statistical significance(P<0.001).The lineage analysis results of 101 INH resistant strains showed that Linage 2(L2)were the strains with the highest proportion,accounting for 59.41%.Among the strains with kat G gene mutation,L2 accounted for the largest proportion.The genotyping results by spacer oligonucleotide typing showed that Beijing genotype strains accounted for 54.46%(55/101)and non-Beijing genotype strains accounted for45.54%(46/101).Non-Beijing genotype strains included T,U,H,CAS family and newly discovered genotypes,accounting for 20.79%,6.93%,2.97%,0.99%and 7.92%,respectively.Conclusion Although the INH-resistant rate of MTB in Guangxi is lower than national level,it is still higher than other economically developed countries and regions such as Singapore and Shanghai,prompting us to pay attention to the problem of INH-resistant MTB.The occurrence of INH-resistant TB is mainly in young and middle-aged males,and farmers were in the majority.The detection rate of INH-resistant in border area is higher than that in non-border area.Re-treatment,border area,and lung field number more than three lesions are three risk factors for INH resistance,which should be paid more attention to.Re-treatment is a risk factor for INH resistance in border and non-border areas.Among all INH-resistant related genes found in Guangxi,kat G gene mutation rate is the highest,especially the mutation of 315 locus.The sensitivity of the kat G 315 locus mutation to predict INH susceptibility is 71.29%,and the specificity is 97.29%.The detection of this locus mutation may be considered as an auxiliary method for screening,diagnosis and elimination of INH-resistant in Guangxi,which has important guiding value for the prevention and control of INH-resistant TB.Isoniazid-resistant MTB is mainly composed of L2,and Beijing genotype strains are mainly prevalent.Among strains with kat G gene mutation,L2 accounts for the largest proportion,which should be focused on. |
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