Matthieu Million1,2, Maxime Gaudin1,2, Cléa Melenotte1,2, Lionel Chasson3, Sophie Edouard1,2, Constance Verdonk4, Elsa Prudent1,2, Bernard Amphoux1,2, Stéphane Meresse3, Richard Dorent4, Hubert Lepidi1,2,5, Bernard La Scola1,2, Jean-Pierre Gorvel3, Christelle Desnues1,2,6, Didier Raoult1,2. 1. Institut Hospitalo-Universitaire (IHU) -Méditerranée Infection, Marseille. 2. Aix Marseille Univ, Institut de recherche pour le développement (IRD), Assistance Publique Hôpitaux de Marseille, Unité Microbes Evolution Phylogenie et Infections (MEPHI). 3. Aix Marseille Univ, Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre d'Immunologie de Marseille-Luminy (CIML). 4. Department of Cardiology, Hôpital Bichat, Assistance Publique - Hôpitaux de Paris (APHP), Paris. 5. Service d'anatomie et cytologie pathologique et de neuropathologie, Centre Hospitalo-Universitaire (CHU) Timone, Assistance Publique Hôpitaux de Marseille,, France. 6. Aix Marseille Univ, Université de Toulon, Centre National de la Recherche Scientifique (CNRS), Institut de Recherche pour le Développement (IRD), Mediterranean Institute of Oceanography, France.
Abstract
BACKGROUND: Etiological diagnosis is a key to therapeutic adaptation and improved prognosis, particularly for infections such as endocarditis. In blood culture-negative endocarditis (BCNE), 22% of cases remain undiagnosed despite an updated comprehensive syndromic approach. This prompted us to develop a new diagnostic approach. METHODS: Eleven valves from 10 BCNE patients were analyzed using a method that combines human RNA bait-depletion with phi29 DNA polymerase-based multiple displacement amplification and shotgun DNA sequencing. An additional case in which a microbe was serendipitously visualized by immunofluorescence was analyzed using the same method, but after laser capture microdissection. RESULTS: Background DNA prevented any diagnosis in cases analyzed without microdissection because the majority of sequences were contaminants. Moraxella sequences were dramatically enriched in the stained microdissected region of the additional case. A consensus genome sequence of 2.4 Mbp covering more than 94% of the Moraxella osloensis KSH reference genome was reconstructed with 234X average coverage. Several antibiotic-resistance genes were observed. Etiological diagnosis was confirmed using Western blot and specific polymerase chain reaction with sequencing on a different valve sample. CONCLUSIONS: Microdissection could be a key to the metagenomic diagnosis of infectious diseases when a microbe is visualized but remains unidentified despite an updated optimal approach. Moraxella osloensis should be tested in blood culture-negative endocarditis.
BACKGROUND: Etiological diagnosis is a key to therapeutic adaptation and improved prognosis, particularly for infections such as endocarditis. In blood culture-negative endocarditis (BCNE), 22% of cases remain undiagnosed despite an updated comprehensive syndromic approach. This prompted us to develop a new diagnostic approach. METHODS: Eleven valves from 10 BCNE patients were analyzed using a method that combines human RNA bait-depletion with phi29 DNA polymerase-based multiple displacement amplification and shotgun DNA sequencing. An additional case in which a microbe was serendipitously visualized by immunofluorescence was analyzed using the same method, but after laser capture microdissection. RESULTS: Background DNA prevented any diagnosis in cases analyzed without microdissection because the majority of sequences were contaminants. Moraxella sequences were dramatically enriched in the stained microdissected region of the additional case. A consensus genome sequence of 2.4 Mbp covering more than 94% of the Moraxella osloensis KSH reference genome was reconstructed with 234X average coverage. Several antibiotic-resistance genes were observed. Etiological diagnosis was confirmed using Western blot and specific polymerase chain reaction with sequencing on a different valve sample. CONCLUSIONS: Microdissection could be a key to the metagenomic diagnosis of infectious diseases when a microbe is visualized but remains unidentified despite an updated optimal approach. Moraxella osloensis should be tested in blood culture-negative endocarditis.
Authors: Laure Flurin; Matthew J Wolf; Cody R Fisher; Edison J Cano Cevallos; James J Vaillant; Bobbi S Pritt; Daniel C DeSimone; Robin Patel Journal: J Clin Microbiol Date: 2022-08-30 Impact factor: 11.677
Authors: Paula Santibáñez; Concepción García-García; Aránzazu Portillo; Sonia Santibáñez; Lara García-Álvarez; María de Toro; José A Oteo Journal: Pathogens Date: 2021-12-29