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

Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan,¹ Division of Neurology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan,² Division of Nephrology and Endocrinology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan,³ Department of Internal Medicine, Nihon University Nerima-Hikarigaoka Hospital, Tokyo, Japan,⁴ Department of Public Health, Nihon University School of Medicine, Tokyo, Japan⁵

Received 16 November 2007/ Returned for modification 22 January 2008/ Accepted 27 February 2008

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.

Published ahead of print on 12 March 2008.