1887

Abstract

We explored the efficiency of , the gene encoding a putative ABC drug transporter specific for the complex (MTBC), as a diagnostic marker.

A 190 bp region of and a 300 bp region of were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (=50) was confirmed by sequencing. Clinical isolates of (=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (=13) and non-mycobacterial (=8) reference strains were included in the study.

The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of as a diagnostic marker for MTBC.

The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.000440
2017-03-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jmm/66/3/371.html?itemId=/content/journal/jmm/10.1099/jmm.0.000440&mimeType=html&fmt=ahah

References

  1. Drobniewski F, Cooke M, Jordan J, Casali N, Mugwagwa T et al. Systematic review, meta-analysis and economic modelling of molecular diagnostic tests for antibiotic resistance in tuberculosis. Health Technol Assess 2015; 19:1–188 [View Article][PubMed]
    [Google Scholar]
  2. Pai M, Schito M. Tuberculosis diagnostics in 2015: landscape, priorities, needs, and prospects. J Infect Dis 2015; 211:S21–S28 [View Article][PubMed]
    [Google Scholar]
  3. World Health Organization Global tuberculosis report. WHO/HTM/TB/2014.0. Geneva, Switzerland: World Health Organization; 2014
  4. Alvarez J, de Juan L, Bezos J, Romero B, Sáez JL et al. Interference of paratuberculosis with the diagnosis of tuberculosis in a goat flock with a natural mixed infection. Vet Microbiol 2008; 128:72–80 [View Article][PubMed]
    [Google Scholar]
  5. Sharma K, Sharma A, Modi M, Singh G, Kaur H et al. PCR detection of co-infection with Mycobacterium tuberculosis and Mycobacterium avium in AIDS patients with meningitis. J Med Microbiol 2012; 61:1789–1791 [View Article][PubMed]
    [Google Scholar]
  6. Kent PT, Kubica GP. A Guide for the Level III Laboratory Atlanta, GA: Centers for Disease Control; 1985
    [Google Scholar]
  7. Springer B, Stockman L, Teschner K, Roberts GD, Böttger EC. Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods. J Clin Microbiol 1996; 34:296–303[PubMed]
    [Google Scholar]
  8. Ringuet H, Akoua-Koffi C, Honore S, Varnerot A, Vincent V et al. hsp65 sequencing for identification of rapidly growing mycobacteria. J Clin Microbiol 1999; 37:852–857[PubMed]
    [Google Scholar]
  9. Adékambi T, Drancourt M. Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int J Syst Evol Microbiol 2004; 54:2095–2105 [View Article][PubMed]
    [Google Scholar]
  10. Ong CS, Ngeow YF, Yap SF, Tay ST. Evaluation of PCR–RFLP analysis targeting hsp65 and rpoB genes for the typing of mycobacterial isolates in Malaysia. J Med Microbiol 2010; 59:1311–1316 [View Article][PubMed]
    [Google Scholar]
  11. Park MY, Kim YJ, Hwang SH, Kim HH, Lee EY et al. Evaluation of an immunochromatographic assay kit for rapid identification of Mycobacterium tuberculosis complex in clinical isolates. J Clin Microbiol 2009; 47:481–484 [View Article][PubMed]
    [Google Scholar]
  12. Hopprich R, Shephard L, Taing B, Kralj S, Smith A et al. Evaluation of (SD) MPT64 antigen rapid test, for fast and accurate identification of Mycobacterium tuberculosis complex. Pathology 2012; 44:642–643 [View Article][PubMed]
    [Google Scholar]
  13. Varma-Basil M, Garima K, Pathak R, Dwivedi SK, Narang A et al. Development of a novel PCR restriction analysis of the hsp65 gene as a rapid method to screen for the Mycobacterium tuberculosis complex and nontuberculous mycobacteria in high-burden countries. J Clin Microbiol 2013; 51:1165–1170 [View Article][PubMed]
    [Google Scholar]
  14. Tuberculist 2013; Mycobacterium tuberculosis H37Rv. www.tuberculist.epfl.ch
  15. National Center for Biotechnology information. U.S. National Library of Medicine 2016; Basic Local Alignment Search Tool. http://blast.ncbi.nlm.nih.gov/Blast.cgi
  16. Joshi KR, Dhiman H, Scaria V. tbvar: a comprehensive genome variation resource for Mycobacterium tuberculosis. Database (Oxford) 2014; 2014:bat083 [View Article][PubMed]
    [Google Scholar]
  17. Mcnabb A, Eisler D, Adie K, Amos M, Rodrigues M et al. Assessment of partial sequencing of the 65-kilodalton heat shock protein gene (hsp65) for routine identification of Mycobacterium species isolated from clinical sources. J Clin Microbiol 2004; 42:3000–3011 [View Article][PubMed]
    [Google Scholar]
  18. KT Clearinghouse 2000; Centre for evidence based medicine: stats calculator. http://ktclearinghouse.ca/cebm/practise/ca/calculators/statscale
  19. Teachepi – A Website Resource for Learning and Teaching Epidemiology 2012; Epidemiology calculators and tools. www.teachepi.org/resources/epict.htm
  20. Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007; 175:367–416 [View Article][PubMed]
    [Google Scholar]
  21. Lima DM, Bollela VR, Jácomo BJ, Martinez R, Lopes da Fonseca BA. Identification of Mycobacterium species in contaminated cultures by polymerase chain reaction. Chest 2005; 127:1283–1288 [View Article][PubMed]
    [Google Scholar]
  22. Dasgupta N, Kapur V, Singh KK, das TK, Sachdeva S et al. Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis. Tuber Lung Dis 2000; 80:141–159 [View Article][PubMed]
    [Google Scholar]
  23. Broccolo F, Scarpellini P, Locatelli G, Zingale A, Brambilla AM et al. Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays. J Clin Microbiol 2003; 41:4565–4572 [View Article][PubMed]
    [Google Scholar]
  24. Kim BJ, Hong SK, Lee KH, Yun YJ, Kim EC et al. Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB). J Clin Microbiol 2004; 42:1308–1312 [View Article][PubMed]
    [Google Scholar]
  25. Chakravorty S, Tyagi JS. Novel multipurpose methodology for detection of mycobacteria in pulmonary and extrapulmonary specimens by smear microscopy, culture, and PCR. J Clin Microbiol 2005; 43:2697–2702 [View Article][PubMed]
    [Google Scholar]
  26. Nimesh M, Joon D, Pathak AK, Saluja D. Comparative study of diagnostic accuracy of established PCR assays and in-house developed sdaA PCR method for detection of Mycobacterium tuberculosis in symptomatic patients with pulmonary tuberculosis. J Infect 2013; 67:399–407 [View Article][PubMed]
    [Google Scholar]
  27. Moatter T, Mirza S, Siddiqui MS, Soomro IN. Detection of Mycobacterium tuberculosis in paraffin embedded intestinal tissue specimens by polymerase chain reaction: characterization of IS6110 element negative strains. J Pak Med Assoc 1998; 48:174–178[PubMed]
    [Google Scholar]
  28. Radhakrishnan I, Manju YK, Kumar RA, Mundayoor S. Implications of low frequency of IS6110 in fingerprinting field isolates of Mycobacterium tuberculosis from Kerala, India. J Clin Microbiol 2001; 39:1683 [View Article][PubMed]
    [Google Scholar]
  29. Chakravorty S, Pathak D, Dudeja M, Haldar S, Hanif M et al. PCR amplification of shorter fragments from the devR (Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis. FEMS Microbiol Lett 2006; 257:306–311 [View Article][PubMed]
    [Google Scholar]
  30. Kim H, Kim SH, Shim TS, Kim MN, Bai GH et al. Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int J Syst Evol Microbiol 2005; 55:1649–1656 [View Article][PubMed]
    [Google Scholar]
  31. Varma-Basil M, Pathak R, Singh K, Dwivedi SK, Garima K et al. Direct early identification of Mycobacterium tuberculosis by PCR-restriction fragment length polymorphism analysis from clinical samples. Jpn J Infect Dis 2010; 63:55–57[PubMed]
    [Google Scholar]
  32. Zakham F, Bazoui H, Akrim M, Lemrabet S, Lahlou O et al. Evaluation of conventional molecular diagnosis of Mycobacterium tuberculosis in clinical specimens from Morocco. J Infect Dev Ctries 2012; 6:40–45[PubMed]
    [Google Scholar]
  33. Macente S, Fujimura Leite CQ, Santos AC, Siqueira VL, Machado LN et al. Evaluation of hsp65 nested PCR-restriction analysis (PRA) for diagnosing tuberculosis in a high burden country. Biomed Res Int 2013; 2013:391549 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.000440
Loading
/content/journal/jmm/10.1099/jmm.0.000440
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