Sonication of heart valves detects more bacteria in infective endocarditis

Abstract

Optimal antimicrobial treatment of infective endocarditis requires identification and susceptibility patterns of pathogens. Sonication of explanted heart valves could increase the identification and culture of pathogens, as shown in prosthetic joint and pacemaker/ICD infections. We tested 26 explanted heart valves from 20 patients with active definite endocarditis for added diagnostic value of sonication to the standard microbiological workup in a prospective diagnostic proof of concept study. Two sonication protocols (broth enrichment vs. centrifugation) were compared in an additional 35 negative control valves for contamination rates. We selected sonication/centrifugation based on acceptable false positive rates (11.4%; 4/35). Sonication/enrichment yielded many false positive results in negative controls (28.6%; 10/35), mainly Propionibacterium acnes (next-generation sequencing excluded technical problems). Compared to direct culture only, adding sonication/centrifugation (including molecular testing) significantly increased the diagnostic yield from 6/26 to 17/26 valves (p = 0.003). Most importantly, culture positives almost doubled (from 6 to 10), providing unique quantitative information about antimicrobial susceptibility. Even if direct molecular testing was added to the standard workup, sonication/centrifugation provided additional diagnostic information in a significant number of valves (8/26; 31%; p = 0.013). We concluded that sonication/centrifugation added relevant diagnostic information in the workup of heart valves with infective endocarditis, with acceptable contamination rates.

Figure 1

Inclusion and exclusion of heart valves in study. Bio = biological prosthetic valve; mechano = mechanical prosthetic valve; n = number. *Diagnosis of active definite endocarditis was made for n = 23 valves presurgically (on antimicrobial therapy for a median duration of 27 [range 4–54] days) and for n = 3 valves surgically (antimicrobial therapy started thereafter); ^†Surgical indication of negative controls: valves stenosis (n = 33), valve insufficiency (n = 1), valve prosthesis too small due to growth of patient (n = 1).

Figure 2

Microbiological workflow of heart valves (active endocarditis and negative controls). Centrifugation = centrifugation of sonication fluid and thereafter culture of the sediment on solid media - BA, CHOC, and BBA agar plates; Direct culture = pressing of aberrant looking parts of the valve onto the solid media BA, CHOC, and BBA plates; Enrichment = direct culture of sonication fluid on solid media - BA, CHOC, and BBA agar plates - as well as enrichment of sonication fluid in blood culture bottles; Molecular testing (direct) = in this study comprising 16S-PCR performed directly on aberrant looking parts of the valve; Molecular testing (sonication) = in this study comprising 16S-PCR performed on the sediment of sonication fluid retrieved after centrifugation; Sonication = the sonication procedure of the explanted heart valve. Hereafter, the sonication fluid was handled according to two different protocols: enrichment and centrifugation; Standard = standard workup, including Gram-stain and direct culture on the solid media BA, CHOC, and BBA agar plates.

Figure 3

Negative control valves (n = 35 in total) with the identification of contaminating microorganisms by various microbiological analyses. C = centrifugation of sonication fluid and thereafter culture of the sediment on solid media - BA (blood agar +5% sheep blood), CHOC (chocolate agar), and BBA (Brucella blood agar +5% sheep blood) agar plates; E = enrichment, including direct culture of sonication fluid on solid media (BA, CHOC, and BBA plates) as well as enrichment of sonication fluid in blood culture bottles; M(D) = molecular testing, in this study comprising 16S-PCR performed directly on aberrant looking parts of the valve; M(S) = molecular testing, in this study compromising 16S-PCR performed on the sediment of sonication fluid retrieved after centrifugation; S = sonication, includes the sonication procedure of the explanted heart valve, hereafter the sonication fluid was handled according to two different protocols (enrichment and centrifugation); ST = standard work-up, including direct culture on solid media (BA, CHOC, and BBA plates).

Figure 4

Heart valves (n = 26 in total) from patients with active endocarditis tested positive for culture and/or 16S-PCR. (a) Absolute number of valves – positive valves are counted separately for culture, 16S-PCR and in total, and thus one valve can be counted multiple times, (b) relative number of valves – n = 26 valves in each circle with each valve counted once for the qualitative best possible identification of its pathogen: 1. Culture, qualitatively the best option providing identification of the microorganism and antimicrobial susceptibility testing; 2. 16S-PCR, qualitatively the least option providing identification of the microorganism, only.