Combination of antibody-based screening methods for the identification and quantification of Legionella pneumophila in process water.

Legionnaires' disease is a severe and often fatal pneumonia primarily caused by the inhalation of aerosols contaminated with the waterborne pathogen Legionella pneumophila serogroup 1. While conventional culture-based detection is often standard for monitoring evaporative cooling systems, the required 10-day cultivation period delays necessary risk assessments. To overcome this time barrier, we applied a novel approach to the flow-based chemiluminescence sandwich microarray immunoassay using cryopreserved Legionella pneumophila standards for rapid and culture-independent serotyping. The microarray features nine monoclonal and one polyclonal capture antibodies and successfully characterized the highly virulent Pontiac group at a concentration down to 105 total cells per 100 mL and specifically identified the highly infectious Philadelphia subtype at lower concentrations as a strain of the Pontiac group. However, some limitations of direct subtyping remain. To address the challenge, a novel workflow combining immunomagnetic separation and flow cytometry with the chemiluminescence sandwich microarray immunoassay was developed. Since viability is maintained after flow cytometric quantification, the cells can be subsequently cultured for a brief period, and single colonies can be used for subtyping. As before, we spiked real process water samples containing complex mixtures of four different subtypes using the cryopreserved Legionella pneumophila standards. This combined approach demonstrated diagnostic sensitivity, and successful reduction of the total processing time. The method enables both the precise, early quantification of Legionella pneumophila and detailed subtyping for demanded and exceeded action limits within real and complex process water matrices.