Genotyping by PCR

Identification of Legionella pneumophila and subtyping it by Sequence-based typing (SBT) for Serogroups 1-14. If isolate is a L. pneumophila Serogroup 1, then further subtyping by a monoclonal antibody (mAb) panel is done.

Pure culture.

BCYE plates, slants, or charcoal transport swabs are acceptable. Isolated colonies on BCYE plate are preferred.

Store samples between room temperature and 37^oC until shipped for testing. Ship at room temperature.

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

Suspected Legionnaires' Disease or Pontiac Fever.

Completed Special Bacteriology requisition form detailing all patient information and relevant clinical information. If possible, attach lab results that have already been done at local or provincial laboratories.

All patient and strain history must be included. Detection in clinical material available upon special request only. Please contact the Special Bacteriology Laboratory for approval before sending specimens.

Legionella pneumophila is identified by direct fluorescent assays (DFA) using the BioRad MONOFLUO L. pneumophila IFA kit, the ProLab Diagnostics L. pneumophila Direct Fluorescent Antibody Serogroup 1-14 kit, as well as the Serogroup 1 kit. Determining the serogroup is done by latex agglutination using ProLab Diagnostics Latex Agglutination Serogroups 1-14 antibody kits. L. pneumophila Serogroup 1 is typed using the Dresden Legionella LPS mAb subtyping panel. All serogroups are further typed using SBT. This involves the amplification and sequencing of seven target genes, including some under diversifying pressure, for the purpose of strain typing. It is an adaptation of multi-locus sequence typing (MLST), where all gene targets are non-selective (eg. house-keeping genes).

25 calendar days. In cases where staff or resources are limited final report may be delayed and status of request will be forwarded in a preliminary report. Final report is sent once all results are complete.

1. Gaia, V., N. K. Fry, B. Afshar, P. C. Luck, H. Meugnier, J. Etienne, R. Peduzzi, and T. G. Harrison. 2005. May. Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila. J. Clin. Microbiol. 43:2047-2052
2. Gilmour, M. W., K. Bernard, D. M. Tracz, A. B. Olson, C. R. Corbett, T. Burdz, B. Ng, D. Wiebe, G. Broukhanski, P. Boleszczuk, P. Tang, F. Jamieson, G. Van Domselaar, F. A. Plummer, and J. D. Berry. 2007. Mar. Molecular typing of a Legionella pneumophila outbreak in Ontario, Canada. J. Med. Microbiol. 56:336-341
3. Helbig, J. H., S. Bernander, M. Castellani Pastoris, J. Etienne, V. Gaia, S. Lauwers, D. Lindsay, P. C. Luck, T. Marques, S. Mentula, M. F. Peeters, C. Pelaz, M. Struelens, S. A. Uldum, G. Wewalka, and T. G. Harrison. 2002. 10. Pan-European study on culture-proven Legionnaires' disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups. Eur. J. Clin. Microbiol. Infect. Dis. 21:710-716
4. Isenberg, Henry D., Clinical Microbiology Procedures Handbook, "Direct Immunofluorescent-Antibody Examination for Legionella spp.," Section 9.5, American Society for Microbiology, 1992
5. Ratzow, S., V. Gaia, J. H. Helbig, N. K. Fry, and P. C. Luck. 2007. Jun. Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains. J. Clin. Microbiol. 45:1965-1968
6. Wilkinson, H. W. 1987. Hospital-Laboratory Diagnosis of Legionella infections. Centers for Disease Control, Atlanta, GA.