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Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays

Broccolo, Francesco and Scarpellini, Paolo and Locatelli, Giuseppe and Zingale, Anna and Brambilla, Anna M. and Cichero, Paola and Sechi, Leonardo Antonio and Lazzarin, Adriano and Lusso, Paolo and Malnati, Mauro S. (2003) Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays. Journal of Clinical Microbiology, Vol. 41 (10), p. 4565-4572. eISSN 1098-660X. Article.

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DOI: 10.1128/JCM.41.10.4565-4572.2003

Abstract

Sensitive and specific techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS6110 multicopy element and on the senX3-regX3 intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of Mycobacterium recognition: broader for the IS6110-based assay and restricted to the M. tuberculosis complex for the senX3-regX3-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the M. tuberculosis DNA load measured by the senX3-regX3-based assay. In addition, reduced levels of M. tuberculosis DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens.

Item Type:Article
ID Code:3890
Status:Published
Refereed:Yes
Uncontrolled Keywords:Mycobacterium tuberculosis, mycobacterial infection, diagnosis
Subjects:Area 06 - Scienze mediche > MED/07 Microbiologia e microbiologia clinica
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze biomediche
Publisher:American Society for Microbiology
eISSN:1098-660X
Deposited On:17 May 2010 16:22

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