Investigation of Legionella pneumophila and other Legionella species in atypical pneumonia patients

Abstract

ABSTRACT Objective: The aim of this study is to investigate Legionella species in 50 patients with atypical pneumonia, using culture, urinary antigen test and molecular techniques. Methods: Non-selective BCYE-α media (Oxoid, England) and selective BMPA media (Oxoid, England) were used to isolate Legionella spp. from respiratory tract samples. The urinary samples of the patients were tested with the Alere BinaxNOW Legionella Urinary Antigen Card (Abbott, US) test to identify the presence of L. pneumophila serogroup 1 specific bacterial antigen. All respiratory tractsamples were tested with a commercial Duplicα RealTime Legionella pneumophila 23S rRNA specific region detection kit (Euroclone Diagnostica, Italy) and two home-made PCR methods. Home-made gel electrophoresis PCR tests were performed using Leg primers designed from 16S ribosomal RNA gene partial sequences for Legionella spp and primers targeting the Lmip (macrophage infectivity potentiator) gene for L. pneumophila. In the home-made real-time PCR test, primers targeting the lipopolysaccharide (lps) biosynthesis gene of L. pneumophila serogroup-1, the L. pneumophila mip gene, and the Legionella spp DNA region encoded by the 16S ribosomal RNA gene were used. Results: The commercial Real-time PCR assay identifed the sequence of L. pneumophila 23S rRNA gene specific region in seven respiratory tract samples. Five samples were detected as Legionella spp. in home-made gel electrophoresis-based PCR and home-made Real-time PCR assay. Hovewer, all samples tested negative in the urinary antigen card test for L. pneumophila serogroup 1. Conclusion: We conclude that the PCR positivities with three different molecular methods indicate that Legionella species other than L. pneumophila serogroup 1 should be investigated in the patients with atypical pneumonia using molecular methods. Also, our study demonstrates the significance of PCR methods in the investigation of Legionella species in clinical samples taken from patients with negative test results for L. pneumophila serogroup 1 specific urinary antigen test, but who are clinically considered to have Legionella pneumoniae.