基因芯片技术在宜昌地区结核分枝杆菌耐药情况监测中的应用

The use of gene chips to determine the resistance of Mycobacterium tuberculosis in the City of Yichang,Hubei Province,China

目的利用基因芯片技术检测宜昌地区结核分枝杆菌感染及耐药情况,为本地区结核病预防和控制提供依据。方法收集2013年7月-2014年7月采自宜昌地区共1 200例抗酸染色阳性患者临床标本,其中痰标本746份,菌液320份,分离菌株134份。对1 200例患者采取性别,年龄分组的方法分析本地区结核分枝杆菌感染分布及流行趋势;采用基因芯片技术检测利福平耐药相关ropB基因511（T→C）、513（C→A）516（G→T,A→T,A→G）、526（C→T,C→G,A→T,A→G）、531（C→T,C→G）、533（T→C）位点及异烟肼耐药相关KatG基因的315（G→C,G→A）位点和Inhat基因的-15（G→T）位点突变情况。以比例法药敏试验为金标准,计算基因芯片法的检出率,敏感度和特异度。结果基因芯片法检测利福平异烟肼敏感968例,单耐利福平31例,单耐异烟肼63例,利福平异烟肼双耐57例,分枝杆菌属感染39例,未检出42例。药敏试验显示本地区结核分枝杆菌对利福平、异烟肼的总耐药率为13.49%;男性占73.90%,女性占26.09%;≤20岁占2.50%（28/1119）,21～岁占15.46%（173/1119）,41～岁占40.21%（450/1119）,〉60岁占41.82%（468/1119）。基因芯片检测利福平耐药相关ropB基因突变率最高的位点为531（突变率32.35%）、其次是511（突变率20.6%）;异烟肼耐药相关KatG基因突变位点为315（突变率54.69%）,ihat突变位点为-15（突变率43.75%）。检出KatG和ihat双突变1例,占突变株的1.56%。与比例法药敏试验相比,基因芯片法检测利福平/异烟肼耐药株的符合率分别是90.90%（1089/1198）和97.41%（1167/1197）,检测利福平耐药株的敏感度为90.62%（29/32）,特异度为98.62%（1060/1166）;检测异烟肼株的敏感度84.0%（63/75）,特异度98.39%（1104/1122）。结论宜昌地区结核分枝杆菌对利福平和异烟肼的总耐药率低于全国平均水平,男性结核分枝杆菌感染者远高于女性,且随着年龄增大感染率及耐药率增高。本地区结核分枝杆菌耐药株的突变位点与其他地区有较大差异。

Objective To study the state and prevalence of drug-resistant tuberculosis in Yichang, Hubei Province in order to provide a basis for effective control of drug-resistant tuberculosis. Methods Twelve hundred clinical specimens were collected from patients with TB in the Yichang area. All specimens tested positive for acid-fast bacilli according to microscopy. Specimens included 746 sputum samples, 320 bacterial suspensions, and 134 isolates. The 1 200 patients were divided into several groups based on their sex and age to analyze the distribution and prevalence of TB in the area. Biochips were used to detect mutations in the ropB gene （associated with resistance to rifampicin） at locus 511 （T-→C）, locus 513 （C→A）, locus 516 （C-→T, C-→G, A-→T, or A-→G）, locus 531 （C-→T, C-→G）, or locus 533（T-→C）. Bio- chips were also used to detect mutations in the KatG gene at locus 315 （G-→C or G-→A） and mutations in the inhA gene （both of which are associated with resistance to isoniazid） at locus 15 （G→T）. A drug susceptibility test using the pro- portion method was performed to serve as a gold standard to calculate sensitivity and specificity and overall concordance with biochip results. Results Bioehips revealed that 968 specimens were sensitive to rifampicin and isoniazid, 31 speci- mens were rifampicin-resistant, 63 specimens were isoniazid-resistant, 57 specimens were resistant to both rifampicin and isoniazid, 39 specimens contained mycobacteria, and 42 specimens did not contain mycobacteria. Statistical analysis indi-cated that resistance to first-line anti-TB drugs （rifampicin and isoniazid） was 13.49 %. Of the patients affected, 73.91 were male and 26.09% were female. Of the patients affected, 2.50%（28/1119） were under the age of 20, 15.46%（173/1119） were age 21--40, 40.21% （450/1119） were age 41--60, and 41.82% （468/1119） were over the age of 60. Biochips revealed that the most prevalent mutations in rpoB were at eodon 531 （32.35%） and at codon 511 （20. 6%）. Mutations in KatG were only at eodon 315 （54.69%） and mutations in inhA were only at codon 15 （43.75%）. Suscepti- bility testing using the proportion method served as the gold standard. The rate of concordance between gene chips and the proportion method in terms of detecting drug resistance to rifampicinisoniazid and multi-drug resistance was 90.90G （1089/1198） and 97.41% （1167/1197）. The gene chip had a sensitivity of 90.62% and a specificity of 98.62% at detecting sensitivity to rifampin and a sensitivity of 84.0% and a specificity of 98.39% at detecting sensitivity to isoniazid. Conclusion Compared to the national average, M. tuberculosis in the City of Yiehang is less resistant to rifampicin and i- soniazid. M. tuberculosis is far more prevalent in men than in women, and the rate of infection and drug resistance in- crease with age. The sites of mutations in M. tuberculosis from the City of Yiehang differ vastly from those in M. tuber- culosis from other areas. These findings have provided a basis for doctors to properly use anti-tuberculosis drugs,