1887

Abstract

Non-tuberculous mycobacteria (NTM) are increasingly important opportunistic pathogens responsible for a variety of clinical diseases. The aim of this study was to evaluate a novel technique, real-time PCR coupled with high-resolution melting analysis (real-time PCR-HRMA), for NTM identification. Two pairs of unique primers targeted to the 16S rRNA gene and the 16S–23S internal transcribed spacer region were selected for further evaluation. A total of 149 mycobacterial clinical isolates were subjected to analysis using the real-time PCR-HRMA system. Overall, 134 NTM identified by the 16S rRNA full-gene sequencing method were categorized into four major groups: complex, group, and group. Of the 134 prevalent mycobacterial isolates, 101 mycobacteria (75.4 %) could be identified correctly by the real-time PCR-HRMA system. The individual sensitivities for the complex, group, and groups were 90.9, 89.1, 100 and 36.8 %, respectively. The specificity of identifying these groups varied from 96.4 to 100 %. When identification failed, mostly it was attributable to various species in the group. The real-time PCR-HRMA system is therefore a rapid and sensitive method for identifying prevalent NTM in a clinical laboratory.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.042424-0
2012-07-01
2020-08-12
Loading full text...

Full text loading...

/deliver/fulltext/jmm/61/7/944.html?itemId=/content/journal/jmm/10.1099/jmm.0.042424-0&mimeType=html&fmt=ahah

References

  1. Ajitkumar P., Barkema H. W., De Buck J. 2012; Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences. Vet Microbiol 155:332–340[PubMed] [CrossRef]
    [Google Scholar]
  2. Chen X., Kong F., Wang Q., Li C., Zhang J., Gilbert G. L. 2011; Rapid detection of isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis clinical isolates using high-resolution melting analysis. J Clin Microbiol 49:3450–3457 [CrossRef][PubMed]
    [Google Scholar]
  3. Cheng J.-C., Huang C.-L., Lin C.-C., Chen C.-C., Chang Y.-C., Chang S.-S., Tseng C.-P. 2006; Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clin Chem 52:1997–2004 [CrossRef][PubMed]
    [Google Scholar]
  4. Choi G. E., Lee S. M., Yi J., Hwang S. H., Kim H. H., Lee E. Y., Cho E. H., Kim J. H., Kim H.-J., Chang C. L. 2010; High-resolution melting curve analysis for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates. J Clin Microbiol 48:3893–3898 [CrossRef][PubMed]
    [Google Scholar]
  5. Douarre P. E., Cashman W., Buckley J., Coffey A., O’Mahony J. M. 2012; High resolution melting PCR to differentiate Mycobacterium avium subsp. paratuberculosis “cattle type” and “sheep type”. J Microbiol Methods 88:172–174 [CrossRef][PubMed]
    [Google Scholar]
  6. Erali M., Voelkerding K. V., Wittwer C. T. 2008; High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85:50–58 [CrossRef][PubMed]
    [Google Scholar]
  7. Foongladda S., Pholwat S., Eampokalap B., Kiratisin P., Sutthent R. 2009; Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth. J Mol Diagn 11:42–48 [CrossRef][PubMed]
    [Google Scholar]
  8. Gopinath K., Singh S. 2010; Non-tuberculous mycobacteria in TB-endemic countries: are we neglecting the danger?. PLoS Negl Trop Dis 4:e615 [CrossRef][PubMed]
    [Google Scholar]
  9. Hall L., Doerr K. A., Wohlfiel S. L., Roberts G. D. 2003; Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J Clin Microbiol 41:1447–1453 [CrossRef][PubMed]
    [Google Scholar]
  10. Herrmann M. G., Durtschi J. D., Wittwer C. T., Voelkerding K. V. 2007; Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin Chem 53:1544–1548 [CrossRef][PubMed]
    [Google Scholar]
  11. Hoek K. G., Gey van Pittius N. C., Moolman-Smook H., Carelse-Tofa K., Jordaan A., van der Spuy G. D., Streicher E., Victor T. C., van Helden P. D., Warren R. M. 2008; Fluorometric assay for testing rifampin susceptibility of Mycobacterium tuberculosis complex. J Clin Microbiol 46:1369–1373 [CrossRef][PubMed]
    [Google Scholar]
  12. Kim K., Lee H., Lee M.-K., Lee S.-A., Shim T.-S., Lim S. Y., Koh W.-J., Yim J.-J., Munkhtsetseg B. other authors 2010; Development and application of multiprobe real-time PCR method targeting the hsp65 gene for differentiation of Mycobacterium species from isolates and sputum specimens. J Clin Microbiol 48:3073–3080 [CrossRef][PubMed]
    [Google Scholar]
  13. Lane D. J., Pace B., Olsen G. J., Stahl D. A., Sogin M. L., Pace N. R. 1985; Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 82:6955–6959 [CrossRef][PubMed]
    [Google Scholar]
  14. Lebrun L., Weill F. X., Lafendi L., Houriez F., Casanova F., Gutierrez M. C., Ingrand D., Lagrange P., Vincent V., Herrmann J. L. 2005; Use of the INNO-LiPA-MYCOBACTERIA assay (version 2) for identification of Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex isolates. J Clin Microbiol 43:2567–2574 [CrossRef][PubMed]
    [Google Scholar]
  15. Leung K. L., Yip C. W., Cheung W. F., Lo A. C., Ko W. M., Kam K. M. 2009; Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory. J Appl Microbiol 107:1433–1439 [CrossRef][PubMed]
    [Google Scholar]
  16. Li H., Turhan V., Chokhani L., Stratton C. W., Dunbar S. A., Tang Y.-W. 2009; Identification and differentiation of clinically relevant Mycobacterium species directly from acid-fast bacillus-positive culture broth. J Clin Microbiol 47:3814–3820 [CrossRef][PubMed]
    [Google Scholar]
  17. Lim S. Y., Kim B.-J., Lee M.-K., Kim K. 2008; Development of a real-time PCR-based method for rapid differential identification of Mycobacterium species. Lett Appl Microbiol 46:101–106[PubMed]
    [Google Scholar]
  18. Lin Y.-C., Lin Y.-C., Liu T.-C., Chang J.-G., Lee H.-H. 2011; High-resolution melting curve (HRM) analysis to establish CYP21A2 mutations converted from the CYP21A1P in congenital adrenal hyperplasia. Clin Chim Acta 412:1918–1923 [CrossRef][PubMed]
    [Google Scholar]
  19. Lu P.-L., Yang Y.-C., Huang S.-C., Jenh Y.-S., Lin Y.-C., Huang H.-H., Chang T.-C. 2011; Evaluation of the Bactec MGIT 960 system in combination with the MGIT TBc identification test for detection of Mycobacterium tuberculosis complex in respiratory specimens. J Clin Microbiol 49:2290–2292 [CrossRef][PubMed]
    [Google Scholar]
  20. Padilla E., González V., Manterola J. M., Pérez A., Quesada M. D., Gordillo S., Vilaplana C., Pallarés M. A., Molinos S. other authors 2004; Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and genotype Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with mycobacterial strains. J Clin Microbiol 42:3083–3088 [CrossRef][PubMed]
    [Google Scholar]
  21. Pang Y., Zhou Y., Wang S., Lu J., Lu B., He G., Wang L., Zhao Y. 2011; A novel method based on high resolution melting (HRM) analysis for MIRU-VNTR genotyping of Mycobacterium tuberculosis . J Microbiol Methods 86:291–297 [CrossRef][PubMed]
    [Google Scholar]
  22. Pietzka A. T., Indra A., Stöger A., Zeinzinger J., Konrad M., Hasenberger P., Allerberger F., Ruppitsch W. 2009; Rapid identification of multidrug-resistant Mycobacterium tuberculosis isolates by rpoB gene scanning using high-resolution melting curve PCR analysis. J Antimicrob Chemother 63:1121–1127 [CrossRef][PubMed]
    [Google Scholar]
  23. Quezel-Guerraz N. M., Arriaza M. M., Avila J. A., Sánchez-Yebra Romera W. E., Martínez-Lirola M. J. Indal-TB Group 2010; Evaluation of the Speed-oligo® Mycobacteria assay for identification of Mycobacterium spp. from fresh liquid and solid cultures of human clinical samples. Diagn Microbiol Infect Dis 68:123–131 [CrossRef][PubMed]
    [Google Scholar]
  24. Ramirez M. V., Cowart K. C., Campbell P. J., Morlock G. P., Sikes D., Winchell J. M., Posey J. E. 2010; Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis. J Clin Microbiol 48:4003–4009 [CrossRef][PubMed]
    [Google Scholar]
  25. Reischl U., Pulz M., Ehret W., Wolf H. 1994; PCR-based detection of mycobacteria in sputum samples using a simple and reliable DNA extraction protocol. Biotechniques 17:844–845[PubMed]
    [Google Scholar]
  26. Ricchi M., Barbieri G., Cammi G., Garbarino C. A., Arrigoni N. 2011; High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis. FEMS Microbiol Lett 323:151–154 [CrossRef][PubMed]
    [Google Scholar]
  27. Richardson E. T., Samson D., Banaei N. 2009; Rapid Identification of Mycobacterium tuberculosis and nontuberculous mycobacteria by multiplex, real-time PCR. J Clin Microbiol 47:1497–1502 [CrossRef][PubMed]
    [Google Scholar]
  28. Roth A., Reischl U., Streubel A., Naumann L., Kroppenstedt R. M., Habicht M., Fischer M., Mauch H. 2000; Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases. J Clin Microbiol 38:1094–1104[PubMed]
    [Google Scholar]
  29. Said H. M., Ismail N., Osman A., Velsman C., Hoosen A. A. 2011; Evaluation of TBc identification immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in samples from broth cultures. J Clin Microbiol 49:1939–1942 [CrossRef][PubMed]
    [Google Scholar]
  30. Shrestha N. K., Tuohy M. J., Hall G. S., Reischl U., Gordon S. M., Procop G. W. 2003; Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR. J Clin Microbiol 41:5121–5126 [CrossRef][PubMed]
    [Google Scholar]
  31. Slinger R., Bellfoy D., Desjardins M., Chan F. 2007; High-resolution melting assay for the detection of gyrA mutations causing quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi. Diagn Microbiol Infect Dis 57:455–458 [CrossRef][PubMed]
    [Google Scholar]
  32. Sun J.-R., Lee S.-Y., Perng C.-L., Lu J.-J. 2009; Detecting Mycobacterium tuberculosis in Bactec MGIT 960 cultures by inhouse IS6110-based PCR assay in routine clinical practice. J Formos Med Assoc 108:119–125 [CrossRef][PubMed]
    [Google Scholar]
  33. Tortoli E. 2009; Clinical manifestations of nontuberculous mycobacteria infections. Clin Microbiol Infect 15:906–910 [CrossRef][PubMed]
    [Google Scholar]
  34. Tully C. C., Hinkle M. K., McCall S., Griffith M. E., Murray C. K., Hospenthal D. R. 2011; Efficacy of minocycline and tigecycline in a hamster model of leptospirosis. Diagn Microbiol Infect Dis 71:366–369 [CrossRef][PubMed]
    [Google Scholar]
  35. Wang H., Yue J., Han M., Yang J., Zhao Y. 2010; Rapid method for identification of six common species of mycobacteria based on multiplex SNP analysis. J Clin Microbiol 48:247–250 [CrossRef][PubMed]
    [Google Scholar]
  36. Won H., Rothman R., Ramachandran P., Hsieh Y. H., Kecojevic A., Carroll K. C., Aird D., Gaydos C., Yang S. 2010; Rapid identification of bacterial pathogens in positive blood culture bottles by use of a broad-based PCR assay coupled with high-resolution melt analysis. J Clin Microbiol 48:3410–3413 [CrossRef][PubMed]
    [Google Scholar]
  37. Wu T.-L., Chia J.-H., Kuo A.-J., Su L.-H., Wu T.-S., Lai H.-C. 2008; Rapid identification of mycobacteria from smear-positive sputum samples by nested PCR-restriction fragment length polymorphism analysis. J Clin Microbiol 46:3591–3594 [CrossRef][PubMed]
    [Google Scholar]
  38. Yang S., Ramachandran P., Rothman R., Hsieh Y. H., Hardick A., Won H., Kecojevic A., Jackman J., Gaydos C. 2009; Rapid identification of biothreat and other clinically relevant bacterial species by use of universal PCR coupled with high-resolution melting analysis. J Clin Microbiol 47:2252–2255 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.042424-0
Loading
/content/journal/jmm/10.1099/jmm.0.042424-0
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited this month Most Cited RSS feed

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