MycobacteriologyEvaluation of the BDProbeTec ET system as screening tool in the direct detection of mycobacterium tuberculosis complex in respiratory specimens
Introduction
Tuberculosis (TB) is an increasing health problem worldwide, especially in developing countries. The spread of HIV/AIDS and the emergence of multidrug-resistant TB are contributing to the worsening impact of this disease. Up to one third of the world's population is estimated by the World Health Organization (WHO) to be infected with Mycobacterium tuberculosis and it is estimated that between 2002 and 2020, approximately 1 billion people will be newly infected, over 150 million people will develop the disease, and 36 million people will die of TB if control is not further strengthened (World Health Organization, 2002).
TB control is based on early detection by acid-fast bacilli (AFB) through AFB stain and culture of mycobacteria using liquid and/or solid media. AFB smear results are available in hours or less but the technique has poor sensitivity and can not distinguish among different species of mycobacteria (Peterson et al., 1999). Although culture is the gold standard with excellent sensitivity, while the liquid media have significantly reduced the detection time, it requires on the average 2-3 weeks to obtain results for slow growing mycobacteria and up to 6 weeks to finalize negative specimens. Because of the low sensitivity of AFB microscopy it is possible that in some cases tuberculosis will not be early detected, which can cause the patient to become an important source of transmission, particularly if culture is not performed. Tuberculosis transmission from smear negative patients is estimated to be approximately 17% (Behr et al., 1999).
Methods based on direct nucleic acid testing (NAT) are more sensitive than microscopy and have reduced the time to detection of Mycobacterium tuberculosis complex (MTC). Polymerase chain reaction (PCR), transcript-mediated assay (TMA), and ligase chain reaction (LCR) were used as molecular tools in the diagnosis of tuberculosis Bergmann and Woods, 1996, Dalovisio et al., 1996, Ichiyama et al., 1996, Moore and Curry, 1995, Wobeser et al., 1996, Bergmann et al., 1999, Gamboa et al., 1998, Piersimoni et al., 1998, Bennedsen et al., 1996, Jonas et al., 1993, Jungkind et al., 1996, Lindbrathen et al., 1997, Pfyffer et al., 1996. Strand Displacement Amplification (SDA) is an isothermal method for the detection of IS6110, a specific target for MTC Ichiyama et al., 1997, Pfyffer et al., 1999. The BD ProbeTec ET system (Becton Dickinson) uses SDA in combination with real time fluorescence detection of amplified product, thus making results available in a few hours (Little et al., 1999). The BD ProbeTec ET system is approved for direct detection of MTC DNA (DTB test) from decontaminated, digested clinical respiratory specimens such as sputa, induced sputa, bronchial washings, and other respiratory specimens. The DTB test is not available in the USA but is commercially available for diagnostic purposes in Europe and recently in Asia. We have evaluated the BD ProbeTec ET System for the direct detection of MTC in respiratory specimens from patients with suspected TB.
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Materials and methods
A total of 502 clinical specimens (435 sputa, 39 broncheoalveolar lavages (BAL), 27 pleural fluids and one pulmonar aspirate) from 266 patients (257 with suspected tuberculosis and 9 receiving anti-tuberculosis treatment), were included in the study. A variable number of specimens per patient was received: more than three per patient in 13 cases, three in 72 cases, two in 43 cases, and only one in 138 cases. Specimens were tested at the Clinical Microbiology Laboratory, Puerto Real University
Results
Thirty-nine from 502 respiratory specimens collected from 23 patients were positive with any method (ZN microscopy, culture on LJ slant, culture in MGIT broth, and/or BDProbeTec ET). Only one specimen, which was negative in all methods, was inhibited in BDProbeTec ET. Results are summarized in Table 1. Twenty-two specimens were ZN positive. Mycobacteria grew from 33 specimens (32 Mycobacterium tuberculosis and 1 Mycobacterium chelonae); 32 MTC isolates grew in MGIT liquid medium and 26 grew on
Discussion
SDA is a molecular technique based on isothermal amplification of DNA, using a two-enzyme system (restriction enzyme and DNA polymerase) (Walker et al., 1992). More recently, new thermophilic restriction endonuclease (BsoBI) and DNA polymerase (exo'Bca) were incorporated (Spargo et al., 1996), as well as fluorescence polarization detection (Walker et al., 1996) which was applied to the diagnosis of Mycobacterium tuberculosis using the IS6110 insertion sequence (Walker & Linn, 1996). Down et al.
Acknowledgements
We are indebted to André De Bock, Yolanda Gonzalo, and Fernando García (Becton Dickinson) for their valuable help in the preparation of the manuscript. We thank Becton Dickinson (Madrid, Spain) for providing reagents and instrumentation. This study was partially presented at the 42nd ICAAC, San Diego, CA, 2002.
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Performance of the BD ProbeTec ET direct detection assay for the analysis of Mycobacterium tuberculosis in respiratory and non-respiratory clinical specimens
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