Sinha Pallavi, Gupta Anamika, Prakash Pradyot, Anupurba Shampa, Tripathi Rajneesh, Srivastava G N
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, 221 005, Varanasi, India.
Departmrnt of TB and Respiratory Diseases, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
BMC Infect Dis. 2016 Mar 12;16:123. doi: 10.1186/s12879-016-1450-1.
Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy.
The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing.
Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5%) and 19 (29.2%) cases were culture positive for M. tuberculosis. Based on CRS, 450 patients had "clinical TB" (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed "non-TB" cases. For culture, the sensitivity was low, 79.3% for pulmonary and 54.3% for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1% for pulmonary and 91.4% for extra-pulmonary TB cases with specificity of 100% and 93.3% respectively.
Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB.
由于缺乏可用于资源匮乏地区的简单、快速且经济高效的诊断技术，全球结核病负担的控制受到阻碍。为了便于直接从临床标本中早期诊断结核病，我们已对巢式多重聚合酶链反应（nested multiplex PCR）的使用进行了标准化和验证，该方法针对结核分枝杆菌复合群和非结核分枝杆菌（NTM）特有的基因片段IS6110、MTP40和32kDα抗原编码基因，与涂片显微镜检查、固体培养和单步多重聚合酶链反应进行了比较。与由微生物学结果（涂片和培养）、临床、放射学和细胞病理学发现、临床治疗以及抗结核治疗反应组成的综合参考标准（CRS）相比，对结果进行了评估。
对巢式多重聚合酶链反应（nMPCR）检测方法在600例（535例肺部标本和65例肺外标本）临床疑似结核病病例中的实用性进行了评估。所有标本均进行涂片、培养、单步多重聚合酶链反应和巢式多重聚合酶链反应检测。
在535例筛查的肺部标本和65例肺外标本中，分别有329例（61.5%）和19例（29.2%）结核分枝杆菌培养呈阳性。根据CRS，450例患者患有“临床结核病”（确诊结核病、可能结核病和疑似结核病）。其余150例被确认为“非结核病”病例。对于培养，敏感性较低，肺部病例为79.3%，肺外病例为54.3%。以综合参考标准作为金标准，评估了nMPCR检测的敏感性和特异性结果。nMPCR检测方法对肺部结核病病例的敏感性为97.1%，对肺外结核病病例为91.4%，特异性分别为100%和93.3%。
使用三种基因引物的巢式多重聚合酶链反应是一种快速、可靠且高度敏感和特异的诊断技术，可用于从NTM基因组中检测和区分结核分枝杆菌复合群，对诊断菌量少的样本很有用。发现巢式多重聚合酶链反应检测方法在评估结核病诊断方面优于单步多重聚合酶链反应。
