1887

Abstract

This study evaluated conventional methods, GLC and three molecular tests, including 16S rRNA sequencing, for the identification of mycobacteria, and the experiences of the authors with the integration of these methods into a diagnostic clinical laboratory were also recorded. Of 1067 clinical isolates of mycobacteria identified by conventional tests, 365 were tested by Accuprobe hybridization assays and PCRs specific for (MTB) complex or complex (MAC), 202 were tested by 16S rRNA sequencing, and 142 were tested by GLC. Three runs of all tests were performed on a weekly basis. The identifications for 209 MTB complex and 118 MAC isolates obtained by species-specific PCR were in complete agreement with AccuProbe hybridization and conventional test results. The 16S rRNA sequence-based identification, at a similarity of ⩾99 %, for 132 of 142 isolates was concordant with the identifications made by the biochemical methods, and for 134 isolates was concordant with the identifications made by GLC at species, group or complex level. 16S rRNA sequencing resulted in fewer incorrectly identified or unidentified organisms than GLC or conventional tests. For the slowly growing non-tuberculous mycobacteria, the mean turnaround times for identification were 4–5 days for 16S rRNA sequencing, 14–29 days for GLC and 22–23 days for conventional methods. Considering the large proportion of some species among clinical isolates, a strategy of initial screening with species-specific PCR (or AccuProbe assays) for the MTB complex and MAC, followed by direct sequencing of the strains that yield negative results, should make 16S rRNA sequencing more affordable for routine application in diagnostic laboratories.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.46298-0
2006-05-01
2019-10-20
Loading full text...

Full text loading...

/deliver/fulltext/jmm/55/5/529.html?itemId=/content/journal/jmm/10.1099/jmm.0.46298-0&mimeType=html&fmt=ahah

References

  1. Arnold, L. J., Jr, Hammond, P. W., Wiese, W. A. & Nelson, N. C. ( 1989; ). Assay formats involving acridinium-ester-labeled DNA probes. Clin Chem 35, 1588–1594.
    [Google Scholar]
  2. Brown-Elliott, B. A. & Wallace, R. J., Jr ( 2002; ). Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev 15, 716–746.[CrossRef]
    [Google Scholar]
  3. Brown-Elliott, B. A., Griffith, D. E. & Wallace, R. J., Jr ( 2002; ). Newly described or emerging human species of nontuberculous mycobacteria. Infect Dis Clin North Am 16, 187–220.[CrossRef]
    [Google Scholar]
  4. Chan, C. M., Yuen, K. Y., Chan, K. S., Yam, W. C., Yim, K. H., Ng, W. F. & Ng, M. H. ( 1996; ). Single-tube nested PCR in the diagnosis of tuberculosis. J Clin Pathol 49, 290–294.[CrossRef]
    [Google Scholar]
  5. Chou, S., Chedore, P. & Kasatiya, S. ( 1998; ). Use of gas chromatographic fatty acid and mycolic acid cleavage product determination to differentiate among Mycobacterium genavense, Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium tuberculosis. J Clin Microbiol 36, 577–579.
    [Google Scholar]
  6. Clarridge, J. E., III ( 2004; ). Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 17, 840–862.[CrossRef]
    [Google Scholar]
  7. Cloud, J. L., Neal, H., Rosenberry, R., Turenne, C. Y., Jama, M., Hillyard, D. R. & Carroll, K. C. ( 2002; ). Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J Clin Microbiol 40, 400–406.[CrossRef]
    [Google Scholar]
  8. Drancourt, M., Bollet, C., Carlioz, A., Martelin, R., Gayral, J. P. & Raoult, D. ( 2000; ). 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 38, 3623–3630.
    [Google Scholar]
  9. El Amin, N. M., Hanson, H. S., Pettersson, B., Petrini, B. & Von Stedingk, L. V. ( 2000; ). Identification of non-tuberculous mycobacteria: 16S rRNA gene sequence analysis vs. conventional methods. Scand J Infect Dis 32, 47–50.[CrossRef]
    [Google Scholar]
  10. Goto, M., Oka, S., Okuzumi, K., Kimura, S. & Shimada, K. ( 1991; ). Evaluation of acridinium-ester-labeled DNA probes for identification of Mycobacterium tuberculosis and Mycobacterium avium-Mycobacterium intracellulare complex in culture. J Clin Microbiol 29, 2473–2476.
    [Google Scholar]
  11. 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]
    [Google Scholar]
  12. Holberg-Petersen, M., Steinbakk, M., Figenschau, K. J., Jantzen, E., Eng, J. & Melby, K. K. ( 1999; ). Identification of clinical isolates of Mycobacterium spp. by sequence analysis of the 16S ribosomal RNA gene. Experience from a clinical laboratory. Acta Pathol Microbiol Immunol Scand 107, 231–239.[CrossRef]
    [Google Scholar]
  13. Kirschner, P., Springer, B., Vogel, U., Meier, A., Wrede, A., Kiekenbeck, M., Bange, F. C. & Bottger, E. C. ( 1993; ). Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 31, 2882–2889.
    [Google Scholar]
  14. Larsson, L., Jantzen, E. & Johnsson, J. ( 1985; ). Gas chromatographic fatty acid profiles for characterisation of mycobacteria: an interlaboratory methodological evaluation. Eur J Clin Microbiol 4, 483–487.[CrossRef]
    [Google Scholar]
  15. Lebrun, L., Espinasse, F., Poveda, J. D. & Vincent-Levy-Frebault, V. ( 1992; ). Evaluation of nonradioactive DNA probes for identification of mycobacteria. J Clin Microbiol 30, 2476–2478.
    [Google Scholar]
  16. Li, Z., Bai, G. H., von Reyn, C. F., Marino, P., Brennan, M. J., Gine, N. & Morris, S. L. ( 1996; ). Rapid detection of Mycobacterium avium in stool samples from AIDS patients by immunomagnetic PCR. J Clin Microbiol 34, 1903–1907.
    [Google Scholar]
  17. Padilla, E., Gonzalez, V., Manterola, J. M. & 8 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]
    [Google Scholar]
  18. Park, Y. K., Bai, G. H. & Kim, S. J. ( 2000; ). Restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolated from countries in the western Pacific region. J Clin Microbiol 38, 191–197.
    [Google Scholar]
  19. Patel, S., Yates, M. & Saunders, N. A. ( 1997; ). PCR-enzyme-linked immunosorbent assay and partial rRNA gene sequencing: a rational approach to identifying mycobacteria. J Clin Microbiol 35, 2375–2380.
    [Google Scholar]
  20. Patel, J. B., Leonard, D. G., Pan, X., Musser, J. M., Berman, R. E. & Nachamkin, I. ( 2000; ). Sequence-based identification of Mycobacterium species using the MicroSeq 500 16S rDNA bacterial identification system. J Clin Microbiol 38, 246–251.
    [Google Scholar]
  21. Pfyffer, G. E., Brown-Elliott, B. A. & Wallace, R. J. ( 2003; ). Mycobacterium: general characteristics, isolation, and staining procedures. In Manual of Clinical Microbiology, pp. 532–559. Edited by P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller & R. H. Yolken. Washington, DC: American Society for Microbiology.
  22. Richter, E., Weizenegger, M., Fahr, A. M. & Rusch-Gerdes, S. ( 2004; ). Usefulness of the GenoType MTBC assay for differentiating species of the Mycobacterium tuberculosis complex in cultures obtained from clinical specimens. J Clin Microbiol 42, 4303–4306.[CrossRef]
    [Google Scholar]
  23. Sarkola, A., Makinen, J., Marjamaki, M., Marttila, H. J., Viljanen, M. K. & Soini, H. ( 2004; ). Prospective evaluation of the GenoType assay for routine identification of mycobacteria. Eur J Clin Microbiol Infect Dis 23, 642–645.
    [Google Scholar]
  24. Torkko, P., Katila, M. L. & Kontro, M. ( 2003; ). Gas-chromatographic lipid profiles in identification of currently known slowly growing environmental mycobacteria. J Med Microbiol 52, 315–323.[CrossRef]
    [Google Scholar]
  25. Vincent, V., Brown-Elliott, B. A., Jost, K. C., Jr & Wallace, R. J. ( 2003; ). Mycobacterium: phenotypic and genotypic identification. In Manual of Clinical Microbiology, pp. 560–587. Edited by P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller & R. H. Yolken. Washington, DC: American Society for Microbiology.
  26. Witebsky, F. G. & Kruczak-Filipov, P. ( 1996; ). Identification of mycobacteria by conventional methods. Clin Lab Med 16, 569–601.
    [Google Scholar]
  27. Woo, P. C., Ng, K. H., Lau, S. K., Yip, K. T., Fung, A. M., Leung, K. W., Tam, D. M., Que, T. L. & Yuen, K. Y. ( 2003; ). Usefulness of the MicroSeq 500 16S ribosomal DNA-based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. J Clin Microbiol 41, 1996–2001.[CrossRef]
    [Google Scholar]
  28. Yakrus, M. A., Hernandez, S. M., Floyd, M. M., Sikes, D., Butler, W. R. & Metchock, B. ( 2001; ). Comparison of methods for identification of Mycobacterium abscessus and M. chelonae isolates. J Clin Microbiol 39, 4103–4110.[CrossRef]
    [Google Scholar]
  29. Yam, W. C., Yuen, K. Y. & Seto, W. H. ( 1998; ). Direct detection of Mycobacterium tuberculosis in respiratory specimens using an automated DNA amplification assay and a single tube nested polymerase chain reaction. Clin Chem Lab Med 36, 597–599.
    [Google Scholar]
  30. Yam, W. C., Tam, C. M., Leung, C. C. & 8 other authors ( 2004; ). Direct detection of rifampin-resistant Mycobacterium tuberculosis in respiratory specimens by PCR-DNA sequencing. J Clin Microbiol 42, 4438–4443.[CrossRef]
    [Google Scholar]
  31. Yuen, K. Y., Yam, W. C., Wong, L. P. & Seto, W. H. ( 1997; ). Comparison of two automated DNA amplification systems with a manual one-tube nested PCR assay for diagnosis of pulmonary tuberculosis. J Clin Microbiol 35, 1385–1389.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.46298-0
Loading
/content/journal/jmm/10.1099/jmm.0.46298-0
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