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.Tuberculous meningitis (TBM) is the severest form of infection of Mycobacterium tuberculosis, causing death or severe neurological defects in more than half of those affected in spite of antituberculosis treatment (ATT) (1,2,8,18). The diagnosis of TBM remains a complex issue, because the most widely used conventional bacteriological detection methods, such as direct smear for acid-fast bacilli (AFB) and culture for M. tuberculosis, are unable to rapidly detect M. tuberculosis with sufficient sensitivity in the acute phase of TBM (3-13, 18, 19). In 2006, we designed a novel internally controlled quantitative nested real-time PCR (QNRT-PCR) assay based on TaqMan PCR (Applied Biosystems) (15). Moreover, based on this original QNRT-PCR (OR-QNRT-PCR) assay, an improved wide-range QNRT-PCR (WR-QNRT-PCR) assay was developed (17). For use as a "calibrator" in WR-QNRT-PCR assay, a new internal control was constructed (17).In the preliminary experiments, the WR-QNRT-PCR assay demonstrated significantly improved quantitative accuracy and had a wide detection range (1 to 10 5 copies) compared to what was seen for the OR-QNRT-PCR assay (17).In this study, we tried to quantitatively detect M. tuberculosis DNA in actual cerebrospinal fluid (CSF) samples by using the WR-QNRT-PCR assay. In addition, the clinical usefulness of this novel assay technique for the rapid and accurate diagnosis of TBM and for assessing the clinical course of TBM was examined. MATERIALS AND METHODSThis study was approv...

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