Adaptation of Amoeba Plate Test To Recover Legionella Strains from Clinical Samples

The isolation of Legionella from respiratory samples is the gold standard for diagnosis of Legionnaires' disease (LD) and enables epidemiological studies and outbreak investigations. The purpose of this work was to adapt and to evaluate the performance of an amoebic coculture procedure (the amoeba plate test [APT]) for the recovery of Legionella strains from respiratory samples, in comparison with axenic culture and liquid-based amoebic coculture (LAC). Axenic culture, LAC, and APT were prospectively performed with 133 respiratory samples from patients with LD. The sensitivities and times to results for the three techniques were compared. Using the three techniques, Legionella strains were isolated in 46.6% (n = 62) of the 133 respiratory samples. The sensitivity of axenic culture was 42.9% (n = 57), that of LAC was 30.1% (n = 40), and that of APT was 36.1% (n = 48). Seven samples were positive by axenic culture only; for those samples, there were <10 colonies in total. Five samples, all sputum samples, were positive by an amoebic procedure only (5/5 samples by APT and 2/5 samples by LAC); all had overgrowth by oropharyngeal flora with axenic culture. The combination of axenic culture with APT yielded a maximal isolation rate (i.e., 46.6%). Overall, the APT significantly reduced the median time for Legionella identification to 4 days, compared with 7 days for LAC (P < 0.0001). The results of this study support the substitution of LAC by APT, which could be implemented as a second-line technique for culture-negative samples and samples with microbial overgrowth, especially sputum samples. The findings provide a logical basis for further studies in both clinical and environmental settings.