Division of Infectious Disease, Department of Internal Medicine, Kaohsiung Medical University Hospital, 100 TzYou 1st Rd., Kaohsiung 80756, Taiwan.
R&D Department, TAUNS Laboratories, Inc., 761-1 Kamishima, Izunokuni, Shizuoka 410-2325, Japan.
EBioMedicine. 2020 Oct;60:103007. doi: 10.1016/j.ebiom.2020.103007. Epub 2020 Sep 16.
Nucleic acid amplification tests (NAATs) are widely used to diagnose tuberculosis (TB), but cannot discriminate live bacilli from dead bacilli. Live bacilli can be isolated by culture methods, but this is time-consuming. We developed a de novo TB diagnostic method that detects only live bacilli with high sensitivity within hours.
A prospective study was performed in Taiwan from 2017 to 2018. Sputum was collected consecutively from 1102 patients with suspected TB infection. The sputum was pretreated and heated at 46°C for 1 h to induce the secretion of MPT64 protein from live Mycobacterium tuberculosis. MPT64 was detected with our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide (thio-NAD) cycling. We compared our data with those obtained using a culture test (MGIT), a smear test (Kinyoun staining), and a NAAT (Xpert).
The limit of detection for MPT64 in our culture-free ultrasensitive ELISA was 2.0 × 10 moles/assay. When the criterion for a positive response was set as an absorbance value ≥17 mAbs, this value corresponded to ca. 330 CFU/mL in the culture method - almost the same high-detection sensitivity as the culture method. To confirm that MPT64 is secreted from only live bacilli, M. bovis BCG was killed using 8 μg/mL rifampicin and then heated. Following this procedure, our method detected no MPT64. Our rapid ultra-sensitive ELISA-based method required only 5 h to complete. Comparing the results of our method with those of culture tests for 944 specimens revealed a sensitivity of 86.9% (93/107, 95% CI: 79.0-92.7%) and a specificity of 92.0% (770/837, 95% CI: 89.9-93.7%). The performance data were not significantly different (McNemar's test, P = 0.887) from those of the Xpert tests. In addition, at a ≥1+ titer in the smear test, the positive predictive value of our culture-free ultrasensitive ELISA tests was in a good agreement with that of the culture tests. Furthermore, our culture-free ultrasensitive ELISA test had better validity for drug effectiveness examination than Xpert tests because our test detected only live bacilli.
Our culture-free ultrasensitive ELISA method detects only live TB bacilli with high sensitivity within hours, allowing for rapid diagnosis of TB and monitoring drug efficacy.
Matching Planner Program from JST (VP29117939087), the A-STEP Program from JST (AS3015096U), Waseda University grants for Specific Research Projects (2017A-015 and 2019C-123), the Precise Measurement Technology Promotion Foundation to E.I.
核酸扩增检测（NAAT）广泛用于诊断结核病（TB），但无法区分活细菌和死细菌。可以通过培养方法分离活细菌，但这很耗时。我们开发了一种新的 TB 诊断方法，可在数小时内以高灵敏度检测仅活细菌。
2017 年至 2018 年在台湾进行了一项前瞻性研究。连续收集了 1102 例疑似结核感染患者的痰液。痰液经过预处理并在 46°C 下加热 1 小时，以诱导活结核分枝杆菌分泌 MPT64 蛋白。我们使用我们的超敏酶联免疫吸附试验（ELISA）与硫代烟酰胺腺嘌呤二核苷酸（thio-NAD）循环结合来检测 MPT64。我们将我们的数据与培养试验（MGIT）、涂片试验（金胺染色）和 NAAT（Xpert）的结果进行了比较。
我们无培养超敏 ELISA 中 MPT64 的检测限为 2.0×10 摩尔/测定。当将阳性反应的标准设置为吸光度值≥17 mAbs 时，这对应于培养方法中的约 330 CFU/mL-几乎与培养方法一样高的检测灵敏度。为了确认 MPT64 仅从活细菌中分泌出来，使用 8 μg/mL 利福平杀死牛分枝杆菌，然后加热。经过此程序，我们的方法未检测到 MPT64。我们的快速超敏 ELISA 方法仅需 5 小时即可完成。将我们的方法与 944 个标本的培养试验结果进行比较，显示敏感性为 86.9%（93/107，95%CI：79.0-92.7%），特异性为 92.0%（770/837，95%CI：89.9-93.7%）。性能数据（McNemar 检验，P=0.887）与 Xpert 试验无显著差异。此外，在涂片试验的≥1+滴度时，我们无培养超敏 ELISA 试验的阳性预测值与培养试验非常吻合。此外，由于我们的试验仅检测活细菌，因此我们的无培养超敏 ELISA 试验在检查药物疗效方面比 Xpert 试验更有效。
我们的无培养超敏 ELISA 方法可在数小时内以高灵敏度检测仅活 TB 细菌，从而可快速诊断结核病并监测药物疗效。
日本科学技术振兴机构（JST）的配套计划（VP29117939087）、日本科学技术振兴机构的 A-STEP 计划（AS3015096U）、早稻田大学特定研究项目补助金（2017A-015 和 2019C-123）、精密测量技术促进基金会给 E.I.的补助金。
