Metagenomic Analysis of Microdissected Valvular Tissue for Etiologic Diagnosis of Blood Culture Negative Endocarditis

BACKGROUND: Etiological diagnosis is one of the keys 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 prompting us to develop a new diagnostic approach. METHODS: Eleven valves of 10 BCNE patients were analyzed using a method combining human RNA bait-depletion followed by phi29 DNA polymerase-based multiple displacement amplification and shotgun DNA sequencing. An additional case, in which the microbe was serendipitously visualized by immunofluorescence, was analyzed by the same method, but after laser capture microdissection (LCM). RESULTS: Background DNA prevents any diagnosis in the cases analyzed without microdissection because majority sequences were contaminants. Moraxella sequences were dramatically enriched in the stained microdissected region of the additional case. A consensus genome sequence of 2.4Mbp covering more than 94% of the Moraxella osloensis KSH reference genome was reconstructed with a 234X average coverage. Several antibiotic resistance genes were observed. Etiological diagnosis was confirmed by western blot and specific PCR with sequencing on a different valve sample. CONCLUSION: 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. M. osloensis should be tested in blood-culture negative endocarditis.