Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays

Abstract

Background: In May 2005, a long-distance outbreak of Legionnaires' disease (LD) caused by Legionella pneumophila serogroup 1 occurred in south-east Norway. The initial outbreak investigation without serology identified 56 laboratory-confirmed LD cases of whom 10 died. However, 116 patients with community-acquired pneumonia might belong to the outbreak based on epidemiological investigations, but acute laboratory tests other than serology were negative or not performed. To assess the true extent of the outbreak, we evaluated two serological assays in order to reclassify the 116 patients with indeterminate case status. Methods: Two polyvalent antibody tests, a serogroup 1-6 immunofluorescence assay (IFA) and a serogroup 1-7 enzyme-linked immunosorbent assay (ELISA) were used. They were evaluated with cases defined as culture-or urinary antigen positive LD patients (n = 40) and non-cases defined as confirmed non-LD patients (n = 39) and healthy control subjects (n = 62). The 116 patients, who were negative in culture, polymerase chain reaction and/or urinary antigen tests, were analysed by the same serological assays. Antibodies to the outbreak strain were determined by immunoblotting. Results: In the evaluation study, the sensitivity and specificity of a ≥4-fold IFA titre change was 38% and 100%, respectively, with corresponding values of 30% and 99% for seroconversion in ELISA. A single high positive IFA titre yielded sensitivity and specificity of 73% and 97%, respectively, with corresponding values of 68% and 96% for a single high immunoglobulin (Ig) G and/or IgM in ELISA. Based on this evaluation, the following serological testing identified 47 more LD cases, and the outbreak thus comprised 103 cases with a case fatality rate of 10%. About the same proportion (70%) of the urinary antigen positive and negative LD cases had antibodies to the serogroup-specific lipopolysaccharide of the outbreak strain. In addition to the 103 LD cases, Legionella infection could not be verified or excluded in 32 patients based on epidemiology and/or lack of microbiological sampling. Conclusions: The acute-phase tests (culture, polymerase chain reaction, and urinary antigen) identified less than 55% of the 103 patients in this outbreak. Serological testing thus remains an important supplement for diagnosis of LD and for determination of outbreak cases.

PCR method for the rapid detection and discrimination of Legionella spp. based on the amplification of pcs, pmtA, and 16S rRNA genes

“…Bacteria were cultured on buffered charcoal yeast agar plates (BCYE; Oxoid, Basingstoke, Hampshire, UK) for 3 days at 37°C (L. drancourtii and L. lytica were grown at 32°C) in humid atmosphere and 5 % CO 2 . Escherichia coli DH5α (Sambrook et al 1989) and Pseudomonas aeruginosa ATCC 27853 were used as negative controls. These two bacterial strains were cultured overnight at 37°C in Luria-Bertani broth (LB, Sigma-Aldrich).…”

“…Another disadvantage of serological testing is the inability to accurately detect all Legionella species and serogroups. Serological testing is a valuable epidemiological tool and remains an important supplement for diagnosis of LD and for determination of outbreak cases (Simonsen et al 2015).…”