Detection and epidemiologic subtyping of Legionella pneumophilia using DNA-based molecular methods

Abstract: Legionella pneumophila causes 2 - 6 % of hospitalised pneumonia cases. Several community- and hospital-acquired outbreaks have been reported over the years. Legionnaires' disease (LD) or legionellosis is also a frequent cause of pneumonia amongst travellers. Conventional methods for diagnosing legionella infection include culture, detection of antibodies (serology) and urinary antigen. These methods, however, either lack full sensitivity or specificity. Legionellae are found in water distribution systems, especially hot water, and are able to multiply intracellularly in free living protozoa. Thus, the bacteria are transmitted to humans either by aerosols or by microaspiration. It is therefore important to link the patient isolate to possible environmental sources. In this thesis a nested format for polymerase chain reaction (PCR) was constructed for detecting L. pneumophila in clinical specimens. The method, which was highly sensitive and specific, was found to be more rapid than earlier PCR methods. In a multicentre study, conducted in collaboration with the European Working Group on Legionella Infections (EWGLI), 14 different DNA-based methods were evaluated for typing 114 related and unrelated isolates of L. pneumophila serogroup (sg) 1. The group favoured amplified fragment length polymorphism analysis (AFLP) because of superior reproducibility and epidemiological concordance. In a second phase of the study the use of AFLP was assessed using a standard protocol. In a third phase, 31 AFLP genotypes were designated from a European culture collection of 130 isolates and a proficiency panel was distributed among the participating laboratories. An international database of defined L. pneumophila genotypes was established for the first time. AFLP and to some extent macro-restriction followed by pulsed-field gel electrophoresis (PFGE) was used for the investigation of the molecular epidemiology in four Swedish nosocomial clusters. The genotypes of the causative L. pneumophila strains were different, but the three sg 1 genotypes were found to be widely distributed geographically in Sweden. In these studies, monoclonal antibody (MAb) 3/1positive and negative phenotypes were found inside the same genotypic clusters. For the first time this was proven to be due to a genetic event, which was not reflected in the fingerprinting patterns. The (MAb) 3/1-negative strains thus lacked the lipopolysaccharide associated gene (lag1). Also, for the first time a legionella outbreak was shown to be caused by strains belonging to two different serogroups, sg 4 and 10, that possessed a common fingerprinting pattern. MAb subtyping and genotyping methods should be used together and in conjunction with clinical and epidemiological data in investigations of Legionnaires' disease.