A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity

Wen Hung Wang, Rikiya Takeuchi, Shu Huei Jain, Yong Huang Jiang, Sonoko Watanuki, Yoshiharu Ohtaki, Kazunari Nakaishi, Satoshi Watabe, Po Liang Lu*, Etsuro Ito*


研究成果: Article査読

23 被引用数 (Scopus)


Background: 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. Methods: 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). Findings: The limit of detection for MPT64 in our culture-free ultrasensitive ELISA was 2.0 × 10−19 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. Interpretation: 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. Funding: 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.

出版ステータスPublished - 2020 10月

ASJC Scopus subject areas

  • 生化学、遺伝学、分子生物学一般


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