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Legionella pneumophila LPS to evaluate urinary antigen tests

Anne-Gaëlle Ranc^a,b,c,d,e,⁎, Margot Carpentier^a, Laëtitia Beraud^a, Ghislaine Descours^a,b,c,d,e,

Christophe Ginevra^a,b,c,d,e, Elodie Maisonneuve^f, Julien Verdon^f, Jean-Marc Berjeaud^f,

Gérard Linaa,b,c,d,e, Sophie Jarrauda,b,c,d,e

aHospices Civils de Lyon, Centre, National de Référence des Légionelles, France

bCIRI, Centre International de Recherche en Infectiologie, Université de Lyon, Lyon, France

cINSERM U1111, Lyon, France Ecole Normale Supérieure de Lyon, Lyon, France

dUniversité Lyon 1, Lyon, France

eCNRS, UMR 5308, Lyon, France

fUniversité de Poitiers, Laboratoire Ecologie et Biologie des Interactions, UMR CNRS, 7267, Poitiers, France

a b s t r a c t a r t i c l e i n f o

Article history: Received 7 April 2017

Received in revised form 16 June 2017 Accepted 17 June 2017 Available online xxxx Keywords: Urinary antigen LPS Pontiac mAb3/1 Legionella pneumophila

Three urinary antigen tests were compared using purified Legionella pneumophila (Lp) LPS. For Lp serogroup1, Sofia®FIA and Binax®EIA limits of detection (LOD) were similar; that of BinaxNOW® lower. For all tests the LOD was higher with LPS from non-Pontiac compared to Pontiac-strains. The LOD was variable for other Lp serogroups.

© 2017 Elsevier Inc. All rights reserved.

Urinary antigen detection is the main diagnosis method for Legion-naires' disease (LD), a severe pneumonia due to Legionella pneumophila (Lp), and accounts for 70–80% of Legionella diagnostic tests performed (ECDC, 2014). The antigen detected is bacteria lipopolysaccharide (LPS) (Williams and Lever, 1995), which diversity in structure and anti-genicity is the basis for Lp serogroups identification. According to the re-action with the monoclonal antibody mAb3/1 of Dresden panel (Helbig et al., 1995; Joly et al., 1986), Lp serogroup 1 (Lp1) can be separated into 9 different subgroups clustered into two major groups of strains: Ponti-ac or mAb3/1-positive (Philadelphia, Knoxville, Benidorm, France/Al-lentown) and non-Pontiac or mAb3/1-negative (Bellingham, Oxford, OLDA, Heysham, Camperdown). Commercial urinary antigen tests (UATs) were developed to detect Lp1 LD, and due to a low availability of samples from patients with LD, the evaluation of UAT on different types of Lp strains remains difficult. The aim of our study was to evalu-ate a new method to compare the limit of detection (LOD) of 3 commer-cial UATs by using extracted LPS.

The bacterial strains used in this study belong to 15 serogroups of Lp (strain from serogroup 16 not available) and to 9 subgroups of Lp1. The reference strain or equivalent was chosen based onJoly et al. (1986)and

submitted to chemical extraction adapted fromLuck and Helbig (2013) in order to extract the LPS (one representative strain for each subgroup or serogroup). Strains were grown for 48 to 72 h on buffered charcoal yeast extract (BCYE) at 37 °C in 2.5% CO2. Buffered yeast extract (BYE) broth was inoculated with a bacterial suspension (DO600nm= 0.1) and incubated for 48 h under shaking (80 rpm). Bacterial broth was then centrifuged for 5 min at 1000 g and bacteria resuspended in sterile water were inactivated by heat at 100 °C for 30 min. The suspension was mixed with the same volume of lysis buffer (0.625 M Tris, 40 g/L SDS, 20% glycerol, 10% beta-mercaptoethanol) and incubated at 100 °C for 10 min. Proteinase K was added (1μg of proteinase K for 10 μg of pro-tein), and the mix was incubated for at least 1 h. LPS was precipitated by MgCl20.375 M in 95% ethanol overnight at−20 °C. After centrifugation (30 min, 1000 g, 4 °C) and drying, the LPS was resuspended in water (1 V) and precipitated 2 h by acetone (4 V) at−20 °C, then centrifuged (30 min, 1000×g, 4 °C) and dried to give purified LPS. Traces of protein were checked by Bradford protein assay and DNA contamination by agarose gel analysis. The LPS amount was indirectly defined by 2-keto-3-deoxyoctonate (KDO) determination as described byKarkhanis et al. (1978). This provides a standardized method to compare UAT LODs. A pool of sterile urine that tested negative with the three UATs was constituted. Afixed amount of LPS was added to this urine pool. LPS preparations were simultaneously tested once using BinaxNOW®

Dans le document Phenol Soluble Modulins et lipopolysaccharide de Legionella pneumophila : rôle dans la réponse immunitaire innée (Page 109-117)