OJMM> Vol.1 No.1, December 2011

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

DownloadDownload as PDF (Size:117KB)  HTML    PP. 1-6  

ABSTRACT

16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.

Cite this paper

N. Gadsby, A. Onen, S. Phillips, L. Tysall, S. Breusch, H. Simpson, J. Dave, E. Czarniak and K. Templeton, "Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens," Open Journal of Medical Microbiology, Vol. 1 No. 1, 2011, pp. 1-6. doi: 10.4236/ojmm.2011.11001.

References

[1] F. Fenollar, V. Roux, A. Stein, M. Drancourt and D. Raoult, “Analysis of 525 Samples to Determine the Usefulness of PCR Amplification and Sequencing of the 16S rRNA Gene for Diagnosis of Bone and Joint Infections,” Journal of Clinical Microbiology, Vol. 44, No. 3, 2006, pp. 1018-1028. doi:10.1128/JCM.44.3.1018-1028.2006
[2] A. L. Rosey, E. Abachin, G. Quesnes, C. Cadilhac, Z. Pejin, C. Glorion, P. Berche and A Ferroni, “Development of a Broad-Range 16S rDNA Real-Time PCR for the Diagnosis of Septic Arthritis in Children,” Journal of Microbiological Methods, Vol. 68, No. 1, 2007, pp. 88-93. doi:10.1016/j.mimet.2006.06.010
[3] P. E. Fournier, F. Thuny, H. Richet, H. Lepidi, J. P. Casalta, J. P. Arzouni, M. Maurin, M. Célard, J. L. Mainardi, T. Caus, F. Collart, G. Habib and D. Raoult, “Comprehensive Diagnostic Strategy for Blood Culture-Negative Endocarditis: A Prospective Study of 819 New Cases,” Clinical Infectious Diseases, Vol. 51, No. 2, 2010, pp. 131- 140. doi:10.1086/653675
[4] K. A. Harris and J. C. Hartley, “Development of Broad- Range 16S rDNA PCR for Use in the Routine Diagnostic Clinical Microbiology Service,” Journal of Medical Microbiology, Vol. 52, No. 8, 2003, pp. 685-691. doi:10.1099/jmm.0.05213-0
[5] A. Ahmadian, M. Ehn and S. Hober, “Pyrosequencing: History, Biochemistry and Future,” Clinica Chimica Acta, Vol. 363, No. 1-2, 2006, pp. 83-94. doi:10.1016/j.cccn.2005.04.038
[6] N. Kobayashi, T. W. Bauer, M. J. Tuohy, I. H. Lieberman, V. Krebs, D. Togawa, T. Fujishiro and G. W. Procop, “The Comparison of Pyrosequencing Molecular Gram Stain, Culture and Conventional Gram Stain for Diagnosing Orthopaedic Infections,” Journal of Orthopaedic Research, Vol. 24, No. 8, 2006, pp. 1641-1649. doi:10.1002/jor.20202
[7] J. A. Jordan, A. R. Butchko and M. B. Durso, “Use of Pyrosequencing of 16S rRNA Fragments to Differentiate between Bacteria Responsible for Neonatal Sepsis,” The Journal of Molecular Diagnostics, Vol. 7, No. 1, 2005, pp. 105-110. doi:10.1016/S1525-1578(10)60015-3
[8] R. A. Luna, L. R. Fasciano, S. C. Jones, B. L. Boyanton, T. T. Ton and J. Versalovic, “DNA Pyrosequencing- Based Bacterial Pathogen Identification in a Pediatric Hospital Setting,” Journal of Clinical Microbiology, Vol. 45, No. 9, 2007, pp. 2985-2992. doi:10.1128/JCM.00630-07
[9] J. A. Jordan, J. Jones-Laughner and M. B. Durso, “Utility of Pyrosequencing in Identifying Bacteria Directly from Positive Blood Culture Bottles,” Journal of Clinical Microbiology, Vol. 47, No. 2, 2009, pp. 368-372. doi:10.1128/JCM.01991-08
[10] J. Lu, R. Yu, Y. Yan, J. Zhang and X. Ren, “Use of Pyromark Q96 ID Pyrosequencing System in Identifying Bacterial Pathogen Directly with Urine Specimens for Diagnosis of Urinary Tract Infections,” Journal of Microbiological Methods, Vol. 86, No. 1, 2011, pp. 78-81. doi:10.1016/j.mimet.2011.03.016
[11] C. Arnold, L. Westland, G. Mowat, A. Underwood, J. Magee and S. Gharbia, “Single-Nucleotide Polymorphism- Based Differentiation and Drug Resistance Detection in Mycobacterium Tuberculosis from Isolates or Directly from Sputum,” Clinical Microbiology and Infection, Vol. 11, No. 2, 2005, pp. 122-130. doi:10.1111/j.1469-0691.2004.01034.x
[12] R. A. Bright, D. K. Shay, B. Shu, N. J. Cox and A. I. Klimov, “Adamantane Resistance among Influenza A Viruses Isolated Early during the 2005-2006 Influenza Season in the United States,” Journal of the American Medical Association, Vol. 295, No. 8, 2006, pp. 891-894. doi:10.1001/jama.295.8.joc60020
[13] C. Schabereiter-Gurtner, M. Nehr, P. Apfalter, A. Makristathis, M. L. Rotter and A. M. Hirschl, “Evaluation of a Protocol for Molecular Broad-Range Diagnosis of Culture-Negative Bacterial Infections in Clinical Routine Diagnosis,” Journal of Applied Microbiology, Vol. 104, No. 4, 2008, pp.1228-1237. doi:10.1111/j.1365-2672.2007.03648.x
[14] C. E. Corless, M. Guiver, R. Borrow, V. Edwards-Jones, E. B. Kaczmarski and A. J. Fox, “Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR,” Journal of Clinical Microbiology, Vol. 38, No. 5, 2000, pp. 1747-1752.
[15] S. Chakravorty, D. Helb, M. Burday, N. Connell and D. Alland, “A Detailed Analysis of 16S Ribosomal RNA Gene Segments for the Diagnosis of Pathogenic Bacteria,” Journal of Microbiological Methods, Vol. 69, No. 2, 2007, pp. 330-339. doi:10.1016/j.mimet.2007.02.005
[16] O. Kommedal, K. Lekang, N. Langeland and H. G. Wiker, “Characterization of Polybacterial Clinical Samples Using a Set of Group-Specific Broad-Range Primers Targeting the 16S rRNA Gene Followed by DNA Sequencing and RipSeq Analysis,” Journal of Medical Microbiology, Vol. 60, No. 7, 2011, pp. 927-936. doi:10.1099/jmm.0.028373-0
[17] S. Yang, P. Ramachandran, A. Hardick, Y. H. Hsieh, C. Quianzon, M. Kuroki, J. Hardick, A. Kecojevic, A. Abeygunawardena, J. Zenilman, J. Melendez, V. Doshi, C. Gaydos and R. E. Rothman, “Rapid PCR-Based Diagnosis of Septic Arthritis by Early Gram-Type Classification and Pathogen Identification,” Journal of Clinical Microbiology, Vol. 46, No. 4, 2008, pp. 1386-1390. doi:10.1128/JCM.02305-07
[18] J. Jalava, M. Skurnik, A. Toivanen, P. Toivanen and E. Eerola, “Bacterial PCR in the Diagnosis of Joint Infection,” Annals of the Rheumatic Diseases, Vol. 60, No. 3, 2001, pp. 287-289. doi:10.1136/ard.60.3.287
[19] H. Bonilla, R. Kepley, J. Pawlak, B. Belian, A. Raynor and L. D. Saravolatz, “Rapid Diagnosis of Septic Arthritis Using 16S rDNA PCR: A Comparison of 3 Methods,” Diagnostic Microbiology & Infectious Disease, Vol. 69, No. 4, 2011, pp. 390-395. doi:10.1016/j.diagmicrobio.2010.11.010

  
comments powered by Disqus

Copyright © 2014 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.