Novel Wide-Range Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA: Clinical Application for Diagnosis of Tuberculous Meningitis

• Published in: Journal of Clinical Microbiology Vol. 46; no. 5; pp. 1698 - 1707
• Main Authors: Takahashi, Teruyuki, Tamura, Masato, Asami, Yukihiro, Kitamura, Eiko, Saito, Kosuke, Suzuki, Tsukasa, Takahashi, Sachiko Nonaka, Matsumoto, Koichi, Sawada, Shigemasa, Yokoyama, Eise, Takasu, Toshiaki
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-05-2008; American Society for Microbiology (ASM)
• Subjects: Adult; Aged; Bacterial diseases; Bacteriology; Biological and medical sciences; Colony Count, Microbial - methods; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Female; Fundamental and applied biological sciences. Psychology; Human bacterial diseases; Humans; Infectious diseases; Logistic Models; Male; Medical sciences; Microbiology; Middle Aged; Miscellaneous; Mycobacteriology and Aerobic Actinomycetes; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - isolation & purification; Plasmids; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Prognosis; Reference Standards; Sensitivity and Specificity; Tuberculosis and atypical mycobacterial infections; Tuberculosis, Meningeal - diagnosis; Tuberculosis, Meningeal - microbiology; Tuberculosis, Meningeal - pathology; Nested polymerase chain reaction; Microbiology; Mycobacterial infection; Real time; Tuberculous meningitis; Infection; Tuberculosis; Mycobacterium tuberculosis; Mycobacteriales; Central nervous system disease; Bacteriosis; Mycobacteriaceae; Bacteria; Actinomycetes; Diagnosis; Quantitative analysis
• ISSN: 0095-1137; 1098-660X; 1098-5530
• DOI: 10.1128/JCM.02214-07

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 10⁵ 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 (R² = 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.