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J Med Microbiol 56 (2007), 1356-1362; DOI: 10.1099/jmm.0.47265-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons

Sagarika Haldar1, Soumitesh Chakravorty1,{dagger}, Manpreet Bhalla2, Shyamasree De Majumdar1 and Jaya Sivaswami Tyagi1

1 Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

2 Lala Ram Sarup Institute of TB and Respiratory Diseases, New Delhi 110030, India

Correspondence
Jaya Sivaswami Tyagi
jstyagi{at}aiims.ac.in

Received 2 March 2007
Accepted 21 June 2007

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the ‘gold’ standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a cost-effective manner.

Abbreviations: FAM, 6-carboxyfluorescein; ISBCN, IS6110 beacon; TB, tuberculosis; TO, target oligonucleotide; USP, universal sample processing.

{dagger}Present address: Division of Infectious Diseases, Department of Medicine and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103, USA.




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