Can Digital PCR Enhance the Detection of Legionella pneumophila in Legionnaires' Disease Cases?

The laboratory diagnosis of Legionnaires' disease (LD) remains a significant challenge due to the limitations of current methods. Most LD cases are diagnosed through the detection of Legionella urine antigen using commercial assays that are usually specific just for Legionella pneumophila (Lp) serogroup 1. Isolating Lp through culture is rarely done and difficult to achieve, especially due to two important issues: the low sensitivity of the method and the fact that it is difficult to obtain respiratory secretions from a significant proportion of patients. Real-time PCR (qPCR) has significantly improved LD diagnosis, indicating that molecular methods based on Legionella DNA detection can be highly effective. This study aimed to adapt a qPCR assay to a digital PCR (dPCR) assay, taking advantage of the more sensitive and more sophisticated dPCR technology. In particular, the study focused on 42 respiratory specimens, including bronchoalveolar lavage, tracheal aspirates, and sputum samples, with a particular focus on culture-negative specimens presumed to contain low concentrations of Legionella DNA, which were considered particularly suitable for assessing the potential of dPCR to improve the diagnosis of Legionnaires' disease. The results of culture, qPCR, and dPCR of respiratory samples were compared with each other and with data from urinary antigen tests (UATs) available for the corresponding patients. Overall, dPCR detected Legionella DNA in additional respiratory specimens compared with qPCR, corresponding to a 19% increase in the number of qPCR-negative specimens yielding a positive dPCR result. dPCR also detected Legionella DNA in specimens from patients with negative urinary antigen results. These findings suggest that dPCR may have potential as a complementary molecular approach for improving Legionella DNA detection in respiratory specimens, particularly in culture-negative samples. However, the clinical significance of dPCR-positive/qPCR-negative results could not be independently confirmed in this study and requires further investigation.