Centre for Biotechnology, Maharshi Dayanand University (MDU), Rohtak, India.
Department of Orthopaedics, University of Health Sciences (UHS), Rohtak, India.
Lett Appl Microbiol. 2022 Jan;74(1):17-26. doi: 10.1111/lam.13567. Epub 2021 Nov 5.
Diagnosis of osteoarticular tuberculosis (OATB) exhibits serious challenges owing to paucibacillary nature of specimens and localization of disease at sites that are difficult to access. We recently developed indirect immuno-PCR (I-PCR) and real-time I-PCR (RT-I-PCR) assays for the detection of mycobacterial antigen 85 complex (Ag85) in OATB patients. Detection limits for the purified Ag85 protein were found to be 1 and 41 fg ml by I-PCR and RT-I-PCR, respectively, which were at least 10 -fold lower than respective ELISA. While spiking synovial fluids of non-TB control subjects with the purified Ag85 protein, LODs of 100 and 120 fg ml were obtained by I-PCR and RT-I-PCR, respectively, thus demonstrating the sample matrix effect. Sensitivities of 87·5 and 70·5% were observed in bodily fluids of confirmed (n = 8) and clinically suspected (n = 51) OATB cases, respectively, by I-PCR, with a specificity of 93·9% (n = 33). Markedly, the sensitivities obtained by I-PCR/RT-I-PCR were significantly higher (P < 0·05-0·01) than ELISA and GeneXpert assay (n = 30). However, no substantial difference in sensitivity was observed between the I-PCR and RT-I-PCR assays. After further improving the accuracy of I-PCR, this test may lead to development of an attractive diagnostic kit.
骨关节炎结核（OATB）的诊断由于标本的少菌性和疾病部位难以触及而存在严重挑战。我们最近开发了间接免疫聚合酶链反应（I-PCR）和实时 I-PCR（RT-I-PCR）检测 OATB 患者分枝杆菌抗原 85 复合物（Ag85）的方法。通过 I-PCR 和 RT-I-PCR 检测纯化的 Ag85 蛋白的检测限分别为 1 和 41 fg ml-1，比各自的 ELISA 至少低 10 倍。在向非结核对照受试者的滑膜液中加入纯化的 Ag85 蛋白时，通过 I-PCR 和 RT-I-PCR 分别获得 100 和 120 fg ml-1 的 LOD，从而证明了样品基质效应。通过 I-PCR 分别在确诊（n=8）和临床疑似（n=51）OATB 病例的体液中观察到 87.5%和 70.5%的敏感性，特异性为 93.9%（n=33）。值得注意的是，I-PCR/RT-I-PCR 获得的敏感性明显高于 ELISA 和 GeneXpert 检测（n=30）（P<0.05-0.01）。然而，I-PCR 和 RT-I-PCR 检测之间未观察到敏感性的实质性差异。在进一步提高 I-PCR 的准确性后，该测试可能会开发出一种有吸引力的诊断试剂盒。
