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

Abstract

This study assessed the reproducibility and epidemiological concordance of double-enzyme fluorescent amplified fragment length polymorphism (fAFLP) analysis for genotyping of Legionella pneumophila serogroup (sg) 1. fAFLP fragment analysis was performed on three different sequencing platforms (one gel- and two capillary-based) in different laboratories with a well-characterised set of 50 strains of L. pneumophila sg 1. fAFLP data were analysed with the Pearson correlation similarity coefficient, using a range of parameters, and dendrogram outputs were converted to arbitrary types after selection of a specified percentage similarity threshold. The results obtained were compared with those obtained by the standard non-fluorescent AFLP method and were found to be broadly concordant. Using optimised settings for each fAFLP method to analyse the panel of 50 strains, epidemiological concordance (E) and reproducibility (R) values of 1.00 were obtained, and the number of types ranged from nine to 15, compared with E=1.00 and R=1.00, with 16 types, for the non-fluorescent AFLP protocol. The study demonstrated the potential of fAFLP for typing strains of L. pneumophila sg 1 on all three platforms; however, inter-platform comparison of fAFLP data was not achieved. fAFLP analysis may have a role in the fingerprinting of multiple isolates during Legionella outbreak investigations, but further work is required before type designations and identification libraries can be developed.

Guidelines for the validation and application of typing methods for use in bacterial epidemiology

“…For some bacterial species, databases have been developed and inter‐centre reproducibility assessed [220–222,224]. Recent and, as yet, unpublished studies have revealed that AFLP may also suffer from the absence of inter‐centre reproducibility, especially when different electrophoresis platforms are being employed [220,221,223]. For Acinetobacter spp., as illustrated in Fig.…”

External Quality Assessment of Molecular Typing of Staphylococcus aureus Isolates by a Network of Laboratories

“…Because of limited discrimination, isolates grouped together by indistinguishable RAPD/rep-PCR profiles should be confirmed as genotypically related either by further PCR testing with an additional primer(s) or, preferably, by another genotyping technique. Despite previous studies showing the good performance of AFLP analysis for typing L. pneumophila (14,15), Acinetobacter baumannii (6,21), and S. aureus (20), the performance of AFLP as used by a single center also showed suboptimal discrimination for S. aureus typing in this survey.…”

“…National and international EQA programs have been developed for antimicrobial susceptibility testing (3,17,29). Multicenter studies have examined the interlaboratory reproducibility of newly developed methods for genotyping of pathogens of public health importance, including S. aureus (1,4,5,9,22,30,32), Legionella pneumophila (14,15), and Mycobacterium tuberculosis (2). However, EQA programs for molecular typing of nosocomial pathogens as a support to hospital surveillance and infection control policy have not been described.…”

Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms.

“…• The use in molecular typing in epidemiology, especially in short-term outbreaks (Antonishyn et al, 2000;Borst et al, 2003;Ball et al, 2004;Fry et al, 2005;Tavanti et al, 2007). • Genome mapping and linkage analyses -AFLPs were used to estimate polymorphism rates and to construct AFLP based linkage maps (Martinelli et al, 2005;Kumar et al, 2013).…”