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 SfiI: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using PstI/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 SfiI 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 SfiI 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.