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Standardization of a TaqMan-Based Real-Time PCR for the Detection of Mycobacterium tuberculosis-Complex in Human Sputum

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  • Instituto de Medicina Tropical Alexander von Humboldt, Lima, Peru; Universidad Peruana Cayetano Heredia, Lima, Perú; Infectious Diseases and Immunity, Imperial College London, and Wellcome Trust Imperial College Centre for Global Health, London,United Kingdom; IFHAD: Innovation For Health And Development, London, United Kingdom; Institute of Tropical Medicine, Antwerp-Belgium; University of Antwerp, Belgium

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Löwenstein-Jensen medium and tested by qPCR for the small mobile genetic element IS6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost ∼5US$ per sample and provided same-day results compared with 2–6 weeks for culture.

Author Notes

* Address correspondence to Francesca Barletta, Instituto de Medicina Tropical Alexander von Humboldt Molecular Epidemiology Unit - TB Av. Honorio Delgado No, 430, San Martin de Porres. E-mail: francescabarletta@yahoo.es

Financial support: This study was funded by the Belgian Directorate-General for Development Cooperation (DGDC) through an institutional collaboration between the Institute of Tropical Medicine in Antwerp, Belgium and the Instituto de Medicina Tropical Alexander von Humboldt in Lima, Peru. CAE and some sample collection and microbiology were funded by the Wellcome Trust and the charity IFHAD: Innovation For Health And Development. CAE acknowledges support from the Imperial College Biomedical Research Centre and by the Joint Global Health Trials consortium (MRC, DFID, and Wellcome Trust).

Authors' addresses: Francesca Barletta, Instituto de Medicina Tropical Alexander von Humboldt Molecular Epidemiology, Lima, Peru, E-mail: francescabarletta@yahoo.es. Koen Vandelannoote, Prince Leopold Institute of Tropical Medicine, Mycobacteriology, Antwerp, Belgium, E-mail: kvandelannoote@itg.be. Jimena Collantes and Jorge Arévalo, Universidad Peruana Cayetano Heredia, Instituto de Medicina Tropical Lima, Lima, Peru, and Epidemiologia Molecular, Lima, Peru, E-mails: jimenacollantes@hotmail.com and biomoljazz@gmail.com. Carlton A. Evans, Imperial College, Department of Infectious Disease and Immunity, Wellcome Centre for Tropical Medicine, London, UK, E-mail: carlton.evans@ifhad.org. Leen Rigouts, University of Antwerp, BMW, Antwerp, Belgium, E-mail: lrigouts@itg.be.

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