Digital PCR assay detection of circulating Mycobacterium tuberculosis DNA in pulmonary tuberculosis patient plasma

• Published in: Tuberculosis Vol. 99; pp. 47 - 53
• Main Authors: Ushio, Ryota, Yamamoto, Masaki, Nakashima, Kentaro, Watanabe, Hiroki, Nagai, Kenjiro, Shibata, Yuji, Tashiro, Ken, Tsukahara, Toshinori, Nagakura, Hideyuki, Horita, Nobuyuki, Sato, Takashi, Shinkai, Masaharu, Kudo, Makoto, Ueda, Atsuhisa, Kaneko, Takeshi
• Format: Journal Article
• Language: English
• Published: Scotland Elsevier Ltd 01.07.2016; Elsevier BV
• Subjects: Adult; Aged; Aged, 80 and over; Case-Control Studies; Circulating DNA; Digital PCR; DNA Copy Number Variations; DNA Gyrase; DNA Gyrase - genetics; DNA, Bacterial; DNA, Bacterial - blood; Female; Gene Dosage; Genetic Markers; gyrB; Humans; Infectious Disease; IS6110; Male; Middle Aged; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Plasma; Polymerase Chain Reaction; Polymerase Chain Reaction - methods; Predictive Value of Tests; Pulmonary/Respiratory; Reproducibility of Results; Tuberculosis; Tuberculosis, Pulmonary; Tuberculosis, Pulmonary - blood; Tuberculosis, Pulmonary - diagnosis; Tuberculosis, Pulmonary - microbiology; Young Adult; IS6110; Plasma; gyrB; Digital PCR; Tuberculosis; Circulating DNA
• ISSN: 1472-9792; 1873-281X; 1873-281X
• DOI: 10.1016/j.tube.2016.04.004

Nucleic acid amplification tests are a major diagnostic tool for pulmonary tuberculosis (PTB). Recently, digital PCR (dPCR) assay has improved sensitivity for the detection of small copy numbers of target molecules. The aim of this study is to explore the utility of dPCR for detecting Mycobacterium tuberculosis (MTB) DNA in PTB patient plasma. Total DNA was purified from plasma samples of newly diagnosed sputum smear-positive PTB patients. Copy numbers of MTB-specific genes in the samples were quantified with dPCR assays targeted for IS6110 or gyrB. A total of 33 PTB patients were enrolled. Significant differences between PTB patients and controls were observed in copy numbers of both targets: IS6110 mean ± SD, 144.8 ± 538.3 vs 0.44 ± 0.49 (copies/20 μL, p = 0.004; Mann–Whitney U test) and gyrB mean ± SD, 359.0 ± 2116 vs 0.07 ± 0.28 (copies/20 μL, p = 0.011; Mann–Whitney U test), respectively. This test had sensitivities of 65% or 29% and a specificity of 93% or 100% with the IS6110-targeted or gyrB-targeted assays, respectively. A dPCR assay successfully detected MTB DNA in PTB patient plasma. This minimally invasive and accurate method has potential to become an alternative diagnostic option.