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Journal of Clinical Microbiology, October 2003, p. 4565-4572, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4565-4572.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Rapid Diagnosis of Mycobacterial Infections and Quantitation of Mycobacterium tuberculosis Load by Two Real-Time Calibrated PCR Assays

Francesco Broccolo,1 Paolo Scarpellini,2 Giuseppe Locatelli,1,{dagger} Anna Zingale,2 Anna M. Brambilla,2 Paola Cichero,3 Leonardo A. Sechi,4 Adriano Lazzarin,2 Paolo Lusso,1 and Mauro S. Malnati1*

Unit of Human Virology,1 Division of Infectious Diseases,2 Department of Microbiology, San Raffaele Scientific Institute, Milan,3 Department of Biomedical Science, Division of Clinical and Experimental Microbiology, University of Sassari, Sassari, Italy4

Received 28 March 2003/ Returned for modification 8 June 2003/ Accepted 5 July 2003

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.

* Corresponding author. Mailing address: Unit of Human Virology, Via Olgettina 58, DIBIT, San Raffaele Scientific Institute, 20132 Milan, Italy. Phone: 39-02-2643-4903. Fax: 39-02-2643-4905. E-mail: malnati.mauro{at}hsr.it.

{dagger} Present address: Pharmacia, Pharmacology Dept., Gene Expression Unit, 20014 Nerviano, Milan, Italy.

Journal of Clinical Microbiology, October 2003, p. 4565-4572, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4565-4572.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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