1887

Abstract

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 PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of was developed. The assays reproducibly detected 25 fg DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The and IS 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 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. PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS PCR performed at almost equivalent levels. 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.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.47265-0
2007-10-01
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/jmm/56/10/1356.html?itemId=/content/journal/jmm/10.1099/jmm.0.47265-0&mimeType=html&fmt=ahah

References

  1. Akhtar, M., Bretzel, G., Boulahbal, F., Dawson, D., Fattorini, L., Feldmann, K., Frieden, T., Havelková, M., de Kantor, I. N. & other authors ( 2000; ). Technical Guide: Sputum Examination For Tuberculosis by Direct Microscopy in Low Income Countries, 5th edn. Paris: International Union Against Tuberculosis and Lung Disease.
  2. Altman, D. G. ( 1991; ). Some common problems in medical research. In Practical Statistics for Medical Research, pp. 396–439. London: Chapman & Hall.
  3. Beige, J., Lokies, J., Schaberg, T., Finckh, U., Fischer, M., Mauch, H., Lode, H., Kohler, B. & Rolfs, A. ( 1995; ). Clinical evaluation of a Mycobacterium tuberculosis PCR assay. J Clin Microbiol 33, 90–95.
    [Google Scholar]
  4. Bennedsen, J., Thomsen, V. O., Pfyffer, G. E., Funke, G., Feldmann, K., Beneke, A., Jenkins, P. A., Hegginbothom, M., Fahr, A. & other authors ( 1996; ). Utility of PCR in diagnosing pulmonary tuberculosis. J Clin Microbiol 34, 1407–1411.
    [Google Scholar]
  5. Chakravorty, S. & Tyagi, J. S. ( 2005; ). Novel multipurpose methodology for detection of mycobacteria in pulmonary and extrapulmonary specimens by smear microscopy, culture, and PCR. J Clin Microbiol 43, 2697–2702.[CrossRef]
    [Google Scholar]
  6. Chakravorty, S., Dudeja, M., Hanif, M. & Tyagi, J. S. ( 2005a; ). Utility of USP smear microscopy, culture and PCR in the diagnosis of pulmonary tuberculosis. J Clin Microbiol 43, 2703–2708.[CrossRef]
    [Google Scholar]
  7. Chakravorty, S., Sen, M. K. & Tyagi, J. S. ( 2005b; ). Diagnosis of extrapulmonary tuberculosis by smear, culture and PCR using universal sample processing technology. J Clin Microbiol 43, 4357–4362.[CrossRef]
    [Google Scholar]
  8. Chakravorty, S., Pathak, D., Dudeja, M., Haldar, S., Hanif, M. & Tyagi, J. S. ( 2006; ). PCR amplification of shorter fragments from the devR (Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis. FEMS Microbiol Lett 257, 306–311.[CrossRef]
    [Google Scholar]
  9. Eisenach, K. D. ( 1998; ). Molecular diagnostics. In Mycobacterium: Molecular Biology and Virulence, pp. 161–179. Edited by C. Ratledge & J. Dale. Oxford: Blackwell Science.
  10. Haldar, S., De Majumdar, S., Chakravorty, S., Tyagi, J. S., Bhalla, M. & Sen, M. K. ( 2005; ). Detection of acid-fast bacilli in postlysis debris of clinical specimens improves the reliability of PCR. J Clin Microbiol 43, 3580–3581.[CrossRef]
    [Google Scholar]
  11. Hellyer, T. J., DesJardin, L. E., Assaf, M. K., Bates, J. H., Cave, M. D. & Eisenach, K. D. ( 1996; ). Specificity of IS6110-based amplification assays for Mycobacterium tuberculosis complex. J Clin Microbiol 34, 2843–2846.
    [Google Scholar]
  12. Katoch, V. M. ( 2004; ). Infections due to non-tuberculous mycobacteria (NTM). Indian J Med Res 120, 290–304.
    [Google Scholar]
  13. Li, Q.-G., Liang, J.-X., Luan, G.-Y., Zhang, Y. & Wang, K. ( 2000; ). Molecular beacon-based homogeneous fluorescence PCR assay for the diagnosis of infectious diseases. Anal Sci 16, 245–248.[CrossRef]
    [Google Scholar]
  14. Narayanan, S., Parandaman, V., Narayanan, P. R., Venkatesan, P., Girish, C., Mahadevan, S. & Rajajee, S. ( 2001; ). Evaluation of PCR using TRC(4) and IS6110 primers in detection of tuberculous meningitis. J Clin Microbiol 39, 2006–2008.[CrossRef]
    [Google Scholar]
  15. Piatek, A. S., Tyagi, S., Pol, A. C., Telenti, A., Miller, L. P., Kramer, F. R. & Alland, D. ( 1998; ). Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat Biotechnol 16, 359–363.[CrossRef]
    [Google Scholar]
  16. Radhakrishnan, I., Manju, Y. K., Kumar, R. A. & Mundayoor, S. ( 2001; ). Implications of low frequency of IS6110 in fingerprinting field isolates of Mycobacterium tuberculosis from Kerala, India. J Clin Microbiol 39, 1683 [CrossRef]
    [Google Scholar]
  17. Singh, K. K., Nair, M. D., Radhakrishnan, K. & Tyagi, J. S. ( 1999; ). Utility of PCR assay in diagnosis of en-plaque tuberculoma of the brain. J Clin Microbiol 37, 467–470.
    [Google Scholar]
  18. Singh, K. K., Muralidhar, M., Kumar, A., Chattopadhyaya, T. K., Kapila, K., Singh, M. K., Sharma, S. K., Jain, N. K. & Tyagi, J. S. ( 2000; ). Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy. J Clin Pathol 53, 355–361.[CrossRef]
    [Google Scholar]
  19. Tsourkas, A. & Bao, G. ( 2003; ). Shedding light on health and disease using molecular beacons. Brief Funct Genomic Proteomic 1, 372–384.[CrossRef]
    [Google Scholar]
  20. Tyagi, S. & Kramer, F. R. ( 1996; ). Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14, 303–308.[CrossRef]
    [Google Scholar]
  21. WHO ( 2005; ). Global Tuberculosis Control Surveillance, Planning, Financing. Geneva: World Health Organization.
  22. Zhang, J. H., Chung, T. D. & Oldenburg, K. R. ( 1999; ). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4, 67–73.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.47265-0
Loading
/content/journal/jmm/10.1099/jmm.0.47265-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error