Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: clinical application for diagnosis of tuberculous meningitis.
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.
Takahashi
Teruyuki
T
Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan. teruyuk@med.nihon-u.ac.jp
Tamura
Masato
M
Asami
Yukihiro
Y
Kitamura
Eiko
E
Saito
Kosuke
K
Suzuki
Tsukasa
T
Takahashi
Sachiko Nonaka
SN
Matsumoto
Koichi
K
Sawada
Shigemasa
S
Yokoyama
Eise
E
Takasu
Toshiaki
T
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
D004269:DNA, Bacterial