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.

Figure 1

Selection of eligible patients for the serological evaluation study. Only individuals with ≥2 paired sera were selected for this study (grey boxes). Non-LD patients and healthy controls formed the non-cases. Also shown are the serological results for the 116 CAP patients with negative Legionella culture, PCR, and/or UAT. Four LD cases (three diagnosed with UAT during the acute phase and one with positive serology and negative UAT) were admitted to other hospitals in Norway and are not included in the figure. CAP: community-acquired pneumonia. UAT: Legionella urinary antigen test. LD: Legionnaires’ disease (confirmed by Legionella culture, PCR and/or UAT). Non-LD: CAP of non-Legionella aetiology.

Figure 2

Cases of Legionnaires’ disease and community-acquired pneumonia (CAP) by date of admission to the hospital.

Figure 3

IgG and IgM binding to the outbreak strain with sera from UAT negative LD cases. The immunoblots show IgG and IgM antibody binding, respectively, with sera from 25 UAT-negative cases to whole cells of the L. pneumophila serogroup 1 outbreak strain. Individual cases are identified by numbers above the nitrocellulose strips, and the upper and lower arrows to the right show the positions of proteins of molecular masses of approx. 80 kDa and 25 kDa, respectively. Strip a: binding of a monoclonal antibody to serogroup 1 L. pneumophila (Lp 1 from the Dresden panel [26]); strip b: IgM antibody reactions of serum from case no. 25 from another experiment with proteinase K treated cells, showing the ladder-like LPS antibody responses. IgG and IgM binding intensities of each serum to LPS are rated below the strips as + (strong), (+) (weak), and – none. Each 12% acrylamide gel was loaded with whole cells from the outbreak strain, corresponding to 2 μg protein/strip, and the strips were incubated with 1:200 serum dilutions. UAT: urine antigen test.