Assessment of fluorescent amplified fragment length polymorphism analysis for epidemiological genotyping of Legionella pneumophila serogroup 1

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Epidemiological genotyping of Legionella pneumophila serogroup 1 by fluorescent-AFLP: and inter-platform comparative study

This study compared the reproducibility and epidemiological concordance of double-enzyme fluorescent-Amplified Fragment Length Polymorphism (f-AFLP) analysis for genotyping of Legionella pneumophila serogroup 1. F-AFLP was performed on three different platforms (one gel- and two capillary-based) in different laboratories from three Countries (DE, UK, IT): (i) an ALF Express (Amersham Pharmacia), (ii) a CEQ 8000 DNA Analysis System (Beckman Coulter), and (iii) an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). A well-characterised set of 50 strains of L. pneumophila serogroup 1 was used (Phase II collection), which had previously been analysed by a standardised non-fluorescent single-enzyme AFLP protocol used by members of the European Working Group for Legionella Infections (EWGLI) for the epidemiological typing of L. pneumophila. F-AFLP data were imported into BioNumerics (Applied Maths) and analysed using the Pearson correlation similarity coefficient using a range of parameters. Dendrogram outputs were converted to arbitrary types, after selection of a specified percentage similarity threshold, and results were compared to those obtained using the standard non-fluorescent method. The results were broadly concordant with those generated by non-fluorescent AFLP. Using optimised settings for each f-AFLP method to analyse the panel of 50 strains, epidemiological concordance (E) values of 1.00 and reproducibility (R) values of 1.00 were obtained and the number of types ranged from 9-15, compared to E=1.00 and R=1.00, with 16 types, for the non-fluorescent protocol. This study demonstrates the potential of f-AFLP to type strains of L. pneumophila sg 1 on all three platforms, although inter-platform comparison of f-AFLP data was not achieved. We conclude that F-AFLP analysis may have a role in the fingerprinting of multiple isolates in legionella outbreak investigation. However. further work demonstrating intercentre reproducibility, epidemiological concordance and discrimatory index, using the same platform, is required prior to type designation and the development of identification librarie