S M Cho1, S Shin2, Y Kim2, W Song3, S G Hong1, S H Jeong2, M S Kang4, K A Lee5. 1. Department of Laboratory Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Republic of Korea. 2. Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea. 3. Department of Laboratory Medicine, Hallym University College of Medicine, Seoul, Republic of Korea. 4. Department of Laboratory Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Republic of Korea. Electronic address: olive@chamc.co.kr. 5. Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea. Electronic address: KAL1119@yuhs.ac.
Abstract
OBJECTIVES: The rapid diagnosis of tuberculosis (TB) is important for patient treatment and infection control. Current molecular diagnostic techniques for TB have insufficient sensitivity to detect samples with low bacterial loads. The sensitivity of molecular testing depends on not only the performance of the assay technique but also the nucleic acid extraction method. Here, we present a novel approach using exosomal DNA (exoDNA) and droplet digital PCR (ddPCR) platforms to detect Mycobacterium tuberculosis DNA in clinical samples. METHODS: The ddPCR platform targeting IS6110 was evaluated in parallel using total DNA and exoDNA. The clinical performance of ddPCR method was assessed with 190 respiratory samples from patients with suspected pulmonary TB. RESULTS: Compared with mycobacterial culture, sensitivity and specificity of ddPCR were 61.5% (95% CI 44.6-76.6%) and 98.0% (95% CI 94.3-99.6%) using total DNA, and 76.9% (95% CI 60.7-88.9%) and 98.0% (95% CI 94.3-99.6%) using exoDNA, respectively. Among 15 culture-positive specimens with low concentrations of target molecules (2~99 positive droplets with exoDNA), only 53.3% (8/15), 46.7% (7/15), and 26.7% (4/15) of cases were detected using ddPCR with total DNA, real-time PCR with exoDNA, and real-time PCR with total DNA, respectively. DISCUSSION: Our platform using ddPCR and exoDNA has the potential to provide sensitive and accurate methodology for TB diagnosis.
OBJECTIVES: The rapid diagnosis of tuberculosis (TB) is important for patient treatment and infection control. Current molecular diagnostic techniques for TB have insufficient sensitivity to detect samples with low bacterial loads. The sensitivity of molecular testing depends on not only the performance of the assay technique but also the nucleic acid extraction method. Here, we present a novel approach using exosomal DNA (exoDNA) and droplet digital PCR (ddPCR) platforms to detect Mycobacterium tuberculosis DNA in clinical samples. METHODS: The ddPCR platform targeting IS6110 was evaluated in parallel using total DNA and exoDNA. The clinical performance of ddPCR method was assessed with 190 respiratory samples from patients with suspected pulmonary TB. RESULTS: Compared with mycobacterial culture, sensitivity and specificity of ddPCR were 61.5% (95% CI 44.6-76.6%) and 98.0% (95% CI 94.3-99.6%) using total DNA, and 76.9% (95% CI 60.7-88.9%) and 98.0% (95% CI 94.3-99.6%) using exoDNA, respectively. Among 15 culture-positive specimens with low concentrations of target molecules (2~99 positive droplets with exoDNA), only 53.3% (8/15), 46.7% (7/15), and 26.7% (4/15) of cases were detected using ddPCR with total DNA, real-time PCR with exoDNA, and real-time PCR with total DNA, respectively. DISCUSSION: Our platform using ddPCR and exoDNA has the potential to provide sensitive and accurate methodology for TB diagnosis.
Authors: Geng Lu; XinRui Jiang; Anni Wu; Jiawei Zhou; Hengjun Liu; Fei He; Qiuling Zhang; Ke Zen; Shuangshuang Gu; Jun Wang Journal: Front Microbiol Date: 2021-04-15 Impact factor: 5.640