【作者】 朱琳；

【导师】 吴淑燕；

【作者基本信息】 苏州大学 ， 病原生物学， 2018， 硕士

【摘要】 目的:回顾性分析连云港地区2014至2016年期间肺结核患者的结核分枝杆菌药物敏感试验及其对利福平和异烟肼相关耐药基因的检测结果。了解连云港地区结核分枝杆菌的耐药现状和耐药基因的突变情况,为临床有效防治结核病提供参考。方法:1.数据来源:2014年1月1日到2016年12月31日期间,连云港市县结核病防治机构送检疑似肺结核病患者痰涂片阳性标本1747例,分别对患者年龄、性别、初治和复治情况、以及结核分枝杆菌培养和药物敏感试验、耐药基因芯片检测结果进行数据统计。2.结核分枝杆菌培养、鉴定和药物敏感试验:痰标本用罗氏培养基培养,结核分枝杆菌的鉴定用对硝基苯甲酸法;比例法药物敏感试验检测对异烟肼(isoniazid,INH)、利福平(rifampicin,RFP)、卡那霉素(kanamycin,KM)和氧氟沙星(ofloxacin,OFX)4种药物的耐药性。3.基因芯片分析:对1747例标本利用基因芯片技术分析耐RFP结核分枝杆菌的rpoB基因和耐INH结核分枝杆菌的kat G和inh A基因上的突变位点。4.统计学分析:采用SPSS20.0软件进行统计分析,计数资料组间比较采用x~2检验,P<0.05表示差异具有统计学意义,P<0.01表示差异具有显著统计学意义。结果:1.连云港地区肺结核流行情况:在1747例标本中1307例结核分枝杆菌培养结果阳性,阳性率为74.81%。三年中初治患者总例数为1114例(85.23%),复治患者总例数193例(14.77%)。男性患者总例数为1017例(77.81%),女性患者总例数为290例(22.19%),男女比例为3.51:1(1017/290)。发病年龄1~94岁,平均年龄51.1岁,以年龄21~30岁区间患者人数最多,其次为61~70岁区间。2.连云港地区结核分枝杆菌耐药情况:(1)2014至2016年总耐药率为16.14%,耐药率逐年下降(20.39%、14.04%、13.55%),x~2=9.67,P=0.008<0.05,差异有统计学意义。(2)2014至2016年中结核分枝杆菌对四种药物耐药率从高到低依次为INH(9.72%)、OFX(6.27%)、RFP(6.04%)、KM(1.07%),3年间结核分枝杆菌对OFX的耐药率逐年降低(8.58%、5.08%、4.91%),差异具有统计学意义(x~2=6.52,P=0.04<0.05);结核分枝杆菌对INH、RFP和KM的耐药率3年中没有统计学差异,但有下降趋势。(3)初治患者所分离的结核分枝杆菌耐药率为14.00%(17.42%、13.39%、10.80%)、复治患者所分离的结核分枝杆菌耐药率为28.50%(37.14%、19.15%、26.32%),明显高于初治患者,x~2=25.53,P=0.000<0.05,差异有统计学意义。(4)3年间耐多药率平均为3.67%(3.86%、3.87%、3.27%)和广泛耐药率平均为0.31%(0.43%、0.24%、0.23%),复治组耐多药率(9.33%)显著高于初治组(2.69%),x~2=20.46,P=0.000<0.01。3.连云港地区结核分枝杆菌耐药基因的突变情况:(1)结核分枝杆菌对RFP耐药突变位点以rpoB531(46.75%)和rpoB526(19.48%)为主;最常见的突变类型为531(TCG→TTG)和526(CAC→TAC)(2)对INH耐药突变位点以kat G315(78.64%)为主,最常见的突变类型为315(AGC→ACC)。4.结核分枝杆菌比例法药敏试验与耐药基因芯片法比较:(1)两种方法分别检测结核分枝杆菌对RFP及INH的耐药率均未见显著差异(x~2=0.749,P=0.387>0.05;x~2=0.794,P=0.373>0.05)。(2)芯片法检测结核分枝杆菌对RFP的耐药性敏感度为80.30%、特异度为97.93%,与比例法药敏试验结果的符合度为96.94%;芯片法检测结核分枝杆菌对INH耐药性的敏感度为63.81%、特异度为97.01%,与比例法药敏试验结果的符合度为94.56%。结论:1.连云港地区肺结核患者人数男性高于女性,21~30岁年龄区间患者人数最多。2.2014年到2016年结核分枝杆菌对药物的总体耐药率呈下降趋势,特别是对OFX的耐药率具有统计学差异;复治患者所分离的结核分枝杆菌耐多药率显著高于初治患者。3.连云港地区结核分枝杆菌RFP耐药突变位点以rpoB531和rpoB526为主,最常见的突变类型为531(TCG→TTG)和526(CAC→TAC);对INH耐药突变位点以kat G 315为主,最常见的突变类型为315(AGC→ACC)。4.比例法药物敏感试验和基因芯片法检测结核分枝杆菌对RFP和INH的耐药性结果没有显著差异,基因芯片法可作为早期筛查结核分枝杆菌耐药性的快速有效方法。更多还原

【Abstract】 Objective:The drug susceptibility test of Mycobacterium tuberculosis(M.tuberculosis)in patients with pulmonary tuberculosis during the period from 2014 to 2016 in Lianyungang and the related test results of rifampicin and isoniazid resistance genes were analyzed retrospectively.To provide reference for the effective prevention and treatment of M.tuberculosis in clinic.Methods:1.Source of data.From January 1,2014 to December 31,2016,Tuberculosis Prevention and Control Agency of Lianyungang County sent 1747 sputum smear specimens of suspected pulmonary tuberculosis patients.The data of age,gender,initial treatment and retreatment,the culture of M.tuberculosis culture and drug susceptibility testing,as well as the test results of drug-resistant gene chip were analyzed.2.M.tuberculosis culture,identification and drug susceptibility test.The sputum specimens were cultured in Roche medium and identified by p-nitrobenzoic acid method.Ratio drug sensitive test was used to detect M.tuberculosis resistance to isoniazid(INH),rifampicin(RFP),kanamycin(KM)and ofloxacin(OFX).3.Gene chip analysis.The rop B gene of RFP-resistant M.tuberculosis and kat G and inh A genes of RFP-resistant were analyzed by gene chip technique.4.Statistical analysis.SPSS20.0 software was used for statistical analysis,continuous measures were compared using the chi-square test.P < 0.05 indicates statistical difference,P < 0.01 indicates significant statistical difference.Results:1.Epidemic situation of M.tuberculosis in Lianyungang.In 1747 samples,1307 M.tuberculosis cultures were positive,with a positive rate of 74.81%.In three years,the number of initial treated patients was 1114(85.23%),and the number of retreatment patients was 193(14.77%).The number of male and female patients was 1017(77.81%)and 290(22.19%),respectively the male to female ratio was 3.51:1(1017/290).The ages of patient were from 1 to 94 years and the average age was 51.1 years.The number of patients with age 21 to 30 years interval was the most,followed by the 61 to 70-years interval.2.Drug resistance of M.tuberculosis in Lianyungang.(1)From 2014 to 2016,the total resistant rate was 16.14%,and the drug resistant rate is decreasing during three years(20.39%,14.04%,13.55%),x~2 = 9.67,P = 0.008 < 0.05,and the difference was statistically significant.(2)From 2014 to 2016,the resistance rates of M.tuberculosis to four drugs were as follows: INH(9.72%),OFX(6.27%),RFP(6.04%),and KM(1.07%).Among them,the resistance rate of OFX statistically decreased(8.58%,5.08%,4.91%),x~2 = 6.52,P = 0.04 < 0.05.The resistant rate of RFP and INH was not statistically different in 3 years,but there was a downward trend.(3)The drug-resistant rate of M.tuberculosis isolated from initial treated patients was 14.00%(17.42%,13.39%,10.80%).The drug-resistant rate of M.tuberculosis isolated from retreated patients was was 28.50%(37.14%,19.15%,26.32%),which was significantly higher than that of initial treated patients,x~2 = 25.53,P = 0.000 < 0.05.(4)In the three years,the average MDR-TB rate was 3.67%(3.86%,3.87%,3.27%)and the XDR-TB rate was 0.31%(0.43%,0.24%,0.23%).The MDR-TB rate in the retreatment group(9.33%)was significantly higher than that in the initial treated group(2.69%),x~2 = 20.46,P = 0.000 < 0.01.3.The mutations of drug resistance genes of M.tuberculosis to RFP and INH.(1)Gene mutation sites for RFP resistance were mainly at rpoB531(46.75%)and rpoB526(19.48%),and the most common mutations were 531(TCG→TTG)and 526(CAC→TAC).(2)Gene mutation sites for INH resistance was predominantly kat G315(78.64%),and the most common mutation was 315(AGC→ACC).4.The comparative study of ratio drug sensitive test and drug-resistance gene chip in detecting M.tuberculosis.(1)There was no significant difference between the two methods in the detection of M.tuberculosis resistance to RFP and INH(x~2 = 0.749,P = 0.387 > 0.05;x~2 = 0.794,P = 0.373 > 0.05).(2)The sensitivity of RFP detected by the chip method was 80.30%,the specificity was 97.93%,and the coincidence was 96.94%.(3)The sensitivity of INH detected by the chip method was 63.81%,the specificity was 97.01%,and the coincidence was 94.56%.Conclusions:1.Male patients with tuberculosis are higher than females.The largest number of patients is between the ages of 21 and 30.2.The total drug resistance rate of M.tuberculosis showed a decreasing trend from 2014 to 2016,especially the resistance rate of OFX was statistically significant.The MDR-TB rate of M.tuberculosis isolated from retreatment patients was significantly higher than that of in initial treated patients.3.The RFP resistance mutation sites of M.tuberculosis were mainly rpoB531 and rpoB526,and the most common mutation types were 531(TCG→TTG)and 526(CAC→TAC);The INH resistance mutation site was mainly kat G315,and the most common mutation type was 315(AGC→ACC).4.There was no significant difference between ratio drug sensitive test and gene chip method in detecting M.tuberculosis resistance to RFP and INH.Gene chip could be a rapid and effective method for early screening of M.tuberculosis drug resistance.更多还原