title: Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: clinical application for diagnosis of tuberculous meningitis.
abstract: Although the "gold standard" for diagnosis of tuberculous meningitis (TBM) is bacterial isolation of Mycobacterium tuberculosis, there are still several complex issues. Recently, we developed an internally controlled novel wide-range quantitative nested real-time PCR (WR-QNRT-PCR) assay for M. tuberculosis DNA in order to rapidly diagnose TBM. For use as an internal control calibrator to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. Due to the development of the NM-plasmid, the WR-QNRT-PCR assay demonstrated statistically significant accuracy over a wide detection range (1 to 10(5) copies). In clinical applications, the WR-QNRT-PCR assay revealed sufficiently high sensitivity (95.8%) and specificity (100%) for 24 clinically suspected TBM patients. In conditional logistic regression analysis, a copy number of M. tuberculosis DNA (per 1 ml of cerebrospinal fluid) of >8,000 was an independent risk factor for poor prognosis for TBM (i.e., death) (odds ratio, 16.142; 95% confidence interval, 1.191 to 218.79; P value, 0.0365). In addition, the copy numbers demonstrated by analysis of variance statistically significant alterations (P < 0.01) during the clinical treatment course for 10 suspected TBM patients. In simple regression analysis, the significant correlation (R(2) = 0.597; P < 0.0001) was demonstrated between copy number and clinical stage of TBM. We consider the WR-QNRT-PCR assay to be a useful and advanced assay technique for assessing the clinical treatment course of TBM.
authors.affiliation: Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan. teruyuk@med.nihon-u.ac.jp
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Takahashi
authors.forename: Masato
authors.initials: M
authors.lastname: Tamura
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Asami
authors.forename: Eiko
authors.initials: E
authors.lastname: Kitamura
authors.forename: Kosuke
authors.initials: K
authors.lastname: Saito
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Suzuki
authors.forename: Sachiko Nonaka
authors.initials: SN
authors.lastname: Takahashi
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Shigemasa
authors.initials: S
authors.lastname: Sawada
authors.forename: Eise
authors.initials: E
authors.lastname: Yokoyama
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takasu
mesh_terms: D000328:Adult; D000368:Aged; D015169:Colony Count, Microbial / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D016015:Logistic Models; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011379:Prognosis; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014390:Tuberculosis, Meningeal / Q000175:diagnosis* / Q000382:microbiology / Q000473:pathology
chemical_list: D004269:DNA, Bacterial