Assessment of Intercentre Reproducibility and Epidemiological Concordance of Legionella pneumophila Serogroup 1 Genotyping by Amplified Fragment Length Polymorphism Analysis

Abstract

The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a "reproducibility" panel (n=20) and an "epidemiologically related" panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each centre following a previously determined standard protocol. Results were analysed by the participants, using gel analysis software where available, and submitted to the coordinating centre. The coordinating centre reanalysed all results visually and selected data-sets with gel analysis software. Data analysis by participants yielded reproducibility (R) values of 0.20-1.00 and epidemiological concordance (E) values of 0.11-1.00, with 6 to 34 types. Following visual analysis by the coordinating centre, R=0.78-1.00, and E=0.67-1.00, with 10-20 types. Analysis of three data-sets by the coordinating centre using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1.00) and epidemiologically concordant (E=1.00), with good discrimination. The electropherograms generated are amenable to computer-aided analysis, but strict adherence to a previously defined laboratory protocol is required. Following designation of representative type strains and patterns, this method will be adopted by the European Working Group on Legionella Infections as the first internationally standardised typing method for use in the investigation of travel-associated Legionella infections.

Clonal population structure of Legionella pneumophila inferred from allelic profiling

“…Data were obtained from L. pneumophila isolates from the following sources: (i) the EU Legionella culture collection (Fry et al, 1999(Fry et al, , 2000(Fry et al, , 2002Gaia et al, 2003Gaia et al, , 2005; (ii) the authors' strain collection following investigation of legionellosis (communityacquired, nosocomial or travel-associated) in the UK from 1977 to 2006; (iii) the National Collection of Type Cultures, Health Protection Agency Centre for Infections, London, UK; or from other published data (Amemura-Maekawa et al, 2005;Benson et al, 2006;Bernander et al, 2006;Coscollá & González-Candelas, 2007;Fendukly et al, 2007;Gilmour et al, 2007;Ratzow et al, 2007;Wong et al, 2006). Only data from isolates considered epidemiologically unrelated to any other, or those subsequently shown to be unrelated to the index case isolate in each cluster, were included.…”

Consensus Sequence-Based Scheme for Epidemiological Typing of Clinical and Environmental Isolates ofLegionella pneumophila

“…Members of The European Working Group for Legionella Infections (EWGLI) have previously evaluated a number of genotypic methods for the epidemiological typing of L. pneumophila, which is particularly applicable to cases of travel-associated legionellosis. The aim of such studies has been to achieve a standardized protocol that allows the exchange of typing data rather than strains across countries and borders, the latter of which is increasingly difficult, costly, and time-consuming (8)(9)(10). One of these methods, a singleendonuclease, amplified fragment length polymorphism analysis method by which the patterns are resolved by standard agarose electrophoresis (8,34), was adopted as an international standard (9) and is widely used by the members of EWGLI.…”

“…This collection of isolates was established by members of the EWGLI to facilitate epidemiological typing studies. Each isolate has previously been extensively characterized, and details for these isolates have been described previously (8)(9)(10)(11). The epidemiologically related panel (panel 2) comprised five sets of epidemiologically related isolates and two replicates of the same isolate.…”

Legionella longbeachae serogroup 1 infections linked to potting compost

“…The cultures of L. longbeachae sg 1 were identified by polyclonal IFA serology (Wilkinson et al, 1981) and mip gene speciation (Ratcliff et al, 1998) and then genotyped by amplified fragment length polymorphism (AFLP) using the standard European Working Group for Legionella Infection method (Fry et al, 2000).…”