A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study

Abstract

OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.

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.…”

“…In this scenario, the aim has been to demonstrate potential environmental sources of infection, thereby allowing timely intervention, in order to prevent further infection. Many such methods have been developed and applied with the purpose of discriminating between epidemiologically unrelated strains of L. pneumophila, particularly those belonging to serogroup (sg) 1 (Edelstein et al, 1986;Fry et al, 1999;Saunders et al, 1990;Schoonmaker et al, 1992;van Ketel et al, 1984).…”

“…In this scenario, the aim has been to demonstrate potential environmental sources of infection, thereby allowing timely intervention, in order to prevent further infection. Many such methods have been developed and applied with the purpose of discriminating between epidemiologically unrelated strains of L. pneumophila, particularly those belonging to serogroup (sg) 1 (Edelstein et al, 1986;Fry et al, 1999; Saunders et al, 1990;Schoonmaker et al, 1992;van Ketel et al, 1984).The utility of the multi-locus sequence typing (MLST) approach, in which sequences from five to 10 housekeeping genes are determined, in the identification of major bacterial lineages associated with invasive disease was first described in 1998 (Enright & Spratt, 1998;Maiden et al, 1998). Since then, this robust and portable technique (or variations of it) has been applied to the study of genetic diversity and clonal expansion, and to the long-term epidemiological analysis of microbial populations (see http://pubmlst.org/ and http://www.mlst.net/) (Giske et al, 2006;Paraskevopoulos et al, 2006;Vassileva et al, 2006).…”

Variable genetic element typing: a quick method for epidemiological subtyping of Legionella pneumophila

“…For public health authorities it is essential to detect the source of infection promptly by comparing clinical and environmental strains of L. pneumophila to conduct decontamination measures and to prevent further cases. For this numerous phenotypic and genotypic typing methods have been applied to the epidemiological typing of L. pneumophila in the recent years [4]. This include monoclonal antibody (MAb) subgrouping as a rapid screening method [3]and genotyping methods such as amplified fragment length polymorphism (AFLP) [4], pulsed-field gel electrophoresis (PFGE) [4;5] and sequence based typing [6][7][8].…”

“…For this numerous phenotypic and genotypic typing methods have been applied to the epidemiological typing of L. pneumophila in the recent years [4]. This include monoclonal antibody (MAb) subgrouping as a rapid screening method [3]and genotyping methods such as amplified fragment length polymorphism (AFLP) [4], pulsed-field gel electrophoresis (PFGE) [4;5] and sequence based typing [6][7][8]. In contrast to band based typing methods that are difficult to standardize the developed sequence based typing (SBT) has the potential of excellent typeability, interlaboratory reproducibility and epidemiologic concordance [9].…”