环介导等温扩增技术检测痰样结核分枝杆菌临床价值评价

Clinical value of the loop-mediated isothermal amplification assay for direct detection of Mycobacterium tuberculosis in the sputum

目的 评价环介导等温扩增技术（LAMP）检测痰标本中结核分枝杆菌的效果和临床价值。 方法 收集深圳市慢性病防治中心2013年4—9月350例结核病专科门诊就诊者的痰标本，其中肺结核患者216例（初诊患者93例，其中涂阳患者41例，涂阴患者52例；随访治疗患者123例），非结核病患者134例（含非结核分枝杆菌感染11例）。用涂片法、罗氏培养法、实时PCR法和LAMP法检测样本中的结核分枝杆菌，以临床诊断为标准，用SPSS 17.0软件进行统计学分析，计算LAMP对初诊患者的检出率和检测敏感度和特异度。不同方法之间检测效率的比较采用χ2检验，P〈0.05为差异有统计学意义。LAMP与实时PCR法检测的一致性评价采用Kappa检验。 结果 上述4种方法检测初诊肺结核患者痰样的敏感度分别为44.1%（41/93）、52.7%（49/93）、48.4%（45/93）、48.4%（45/93），LAMP检测敏感度与实时PCR一致；高于涂片法，低于罗氏培养法，但差异均无统计学意义（χ^2=0.75，P〉0.05；χ^2=0.75，P〉0.05）。LAMP对初诊涂阳患者的总检出率为90.2%（37/41），对初诊涂阴患者的检出率为15.4%（8/52），诊断肺结核的特异度为100.0%（134/134）。对123例治疗随访的患者，涂片、培养、实时PCR 和LAMP 4种方法单次检测的阳性率分别为26.0%（32/123）、14.6%（18/123）、31.7%（39/123）和30.9%（38/123）。LAMP和实时PCR检测总体一致率为96.9%（339/350），Kappa值为0.91。 结论 LAMP检测痰样本结核分枝杆菌用于结核病诊断具有良好的特异度，总体检测效能和实时PCR具有很高一致性，具有临床应用价值。

Objective To evaluate the value of loop-mediated isothermal amplification （LAMP） assay for the detection of Mycobacterium tuberculosis in the sputum. Methods The sputum samples were collected from 350 patients from The Tuberculosis （TB） Clinic of Shenzhen Center for Chronic Disease Control between April and September 2013, which including 216 cases of TB and 134 cases of pseudophthisis. Of 216 TB patients, 93 were the cases newly diagnosed, including 41 smear-positive cases and 52 smear-negative cases; 123 were the cases follow-up therapy. There are 11 cases with nontuberculous Mycobacterium infection in pseudophthisis. The specimens were simultaneously detected using four methods （smear microscopy, L-J culture, real-time PCR and LAMP）. The clinical dia-gnoses were used as the standard method, the sensitivity and specificity of LAMP method in newly diagnosed patients were calculated by SPSS 17.0. The detection efficiency between different methods was compared usingχ^2 test. P〈0.05 was considered as significant difference. The concordance of LAMP and real time PCR method was evaluated by Kappa test. Results The sensitivities of smear microscopy, L-J culture, LAMP and real-time PCR in newly diagnosed TB patients were 44.1%（41/93）, 52.7%（49/93）, 48.4%（45/93）and 48.4%（45/93）, respectively. There were statistic differences among four methods （P〉0.05）. The sensitivity of LAMP was 90.2% （37/41） in smear-positive TB patients, while the sensitivity in smear-negative TB patients was 15.4%（8/52）, and the specificity of LAMP was 100.0% （134/134）. The positive rates of a single test using smear microscopy, L-J culture, real-time PCR and LAMP for 123 treated patients were 26.0%（32/123）, 14.6%（18/123）, 31.7%（39/123） and 30.9%（38/123）, respectively. The concordance of LAMP and real-time PCR was 96.9%（339/350）. The Kappa value was 0.91. Conclusion LAMP was a specific and simple method for the diagnosis of TB , and the performance of LAMP was high consistency with real-time PCR.