Implementation of molecular techniques in the diagnosis of legionnaires’ disease and in the investigation of legionella outbreaks

Background. Legionnaires’ Disease (LD) is a mild to severe, potentially lethal, respiratory syndrome caused by members of the Legionella genus, in particular L. pneumophila serogroup (sg) 1 alone causes about 95% of culture confirmed cases. The infection is usually acquired by inhalation of aerosols originating from contaminated fresh water sources, consequently typing of both clinical and environmental isolates is crucial to rapidly identify the possible source and prevent further cases. Legionellae are difficult to isolate by culture, moreover as respiratory samples are not available for up to 65% patients, alternative techniques are needed to diagnose LD and maximise the amount of typing data that can be obtained to aid investigations. Urinary antigen detection and serology provide very limited information regarding the infecting strain, while the advent of PCR and Sanger sequencing has allowed reliable diagnostic and typing methods to be introduced.

Objectives. The aim of this study was to improve existing diagnostic and typing molecular assays, and to develop new ones to further standardise diagnostic and typing procedures across members of the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Legionella Infections (ESGLI). Utility of the assays was assessed in both routine and outbreak scenarios.

Methods. A wide range of both in silico and in vitro experiments were used to design and validate specific oligonucleotides to improve detection and typing of L. pneumophila. Genomic DNA was manually extracted and prepared for Whole Genome Sequencing (WGS) using Illumina platforms. A bioinformatic approach was used to design a WGS based typing scheme and decipher the evolution of L. pneumophila sg1 Sequence Type (ST) 47, a major disease-causing strain.

Results. A real-time PCR detecting L. pneumophila and sg1 specific targets was validated with international colleagues and made available to ESGLI members. Sequence based typing was improved and expanded, and specific typing guidelines produced. A 50 gene core-genome MLST was identified as the best approach to improve the current typing method. ST47 was shown to be a ‘chimera’ between ST109 and ST62, and a specific real-time PCR was designed to detect this strain.

Conclusions. The results of this study allowed researchers to obtain faster and more accurate diagnosis of LD, and L. pneumophila typing data from both isolates and primary samples. A metagenomics approach is presently under evaluation to obtain typing results by WGS directly from clinical and environmental samples.