Enhancement of culture of Legionella longbeachae from respiratory samples using immunomagnetic separation and antimicrobial decontamination

Background Legionella longbeachae is the commonest Legionella species identified in patients with community acquired pneumonia in New Zealand. Isolation of the organism on culture is the gold standard for the diagnosis of Legionnaires disease, but has poor sensitivity (40%) compared with qPCR. We have developed a selective decontamination process using glycine, vancomycin, polymyxin and cycloheximide (GVPC) with immunomagnetic separation (IMS) for culturing L. longbeachae. Methods A polyclonal antibody specific for L. longbeachae was produced from New Zealand white rabbits and coupled to tosylactivated magnetic beads. Stored L. longbeachae qPCR positive respiratory samples were retrieved from -80°C storage for testing. One portion of test samples were mixed with GVPC and the antibody beads complex, separated, washed and cultured on MWY agar. Another portion was exposed to HCl:KCl acidic buffer (pH 2.2) before incubation on MWY agar. qPCR used probes specific for the ITS (internal transcribed spacer) region of the L. longbeachae genome. Results Cultures were positive in 10/53 (19%) samples after acid wash and 26/53 (49%) after GVPC-IMS (p 0.001). Growth of contaminants was rare. The mean qPCR Ct were lower in culture positive samples after acid wash than the culture negative samples (mean 29.9 v 34.8; difference 4.9 95% CI +/- 2.9; p =0.001) but not after GVPC-IMS (mean 33.0 v 34.7; difference 1.7 95% CI +/- 2.48; p = 0.16). Conclusions The sensitivity of culture for L. longbeachae in respiratory specimens may be improved by using GVPC-IMS rather than acid wash for decontamination but this should be confirmed in a prospective study of fresh specimens.