Legionella in clinical specimens and hospital water supply facilities: molecular detection and genotyping of the isolates

To evaluate genus- and species-specific polymerase chain reactions [PCRs] for the detection of the genus Legionella and the species Legionella pneumophila in clinical specimens and hospital water supplies, and to establish a simple and reproducible random amplification of polymorphic DNA [RAPD]-PCR technique for genotyping of Legionella. A total of 70 respiratory tract specimens [bronchoalveolar lavage: n = 46; endotracheal secretions: n = 9; sputum: n = 15] from patients with atypical pneumonia, and 283 environmental samples [water: 20; swabs: 263] collected from water storage and supply facilities of the Mubarak Al-Kabeer Hospital, Kuwait, were tested by culture and genus-specific PCR for the detection of Legionella. The L pneumophila isolates were subsequently typed by serology and RAPD-PCR using serotype-specific sera and arbitrary primers, respectively. Of the 70 clinical samples, culture yielded 2 [2.9%] whereas genus-specific PCR detected Legionella in 20 [28.6%] samples. The 2 culture-positive specimens were also positive for L.-pneumophila-specific PCR. Testing of swab and water samples by culture and genus-specific PCR yielded 61 [21.6%] and 67 [23.7%] positive samples, respectively. All of the 61 culture-positive samples were also positive by genus-specific PCR and 45 of them were positive for L.-pneumophila-specific PCR. Serological typing of 43 L pneumophila isolates showed that 8 of these belonged to serotype 1 and 35 to serotype 3; however, RAPD-PCR analyses demonstrated polymorphisms among the isolates of both serotypes. A higher association between PCR and culture was observed for the environmental samples than for the clinical samples. The application of genus- and species-specific PCRs and RAPD is useful in the detection and typing of Legionella in clinical and environmental samples

Comparison of multiplex PCR and culture for detection of Legionellae in cooling tower water samples.

We compared multiplex polymerase chain reaction (PCR) and culture for detecting the presence of Legionella pneumophila and Legionella spp in cooling tower water samples. Multiplex PCR was performed after phenol extraction of DNA from the samples. The set of primers for the PCR assay involved the 5S rRNA (Legionella spp) and the mip (macrophage infectivity potentiator gene, specific for L. pneumophila) genes as target sequences for amplification. Both the sensitivity and the specificity of the PCR assay were 100% when the 5S rRNA gene was used as target sequence. Isolation of Legionellae from the samples was observed only with the PCR-positive samples. We propose that PCR be used as a screening test before attempting to culture Legionellae from cooling tower water samples.