Clinical Significance of a 16S-rDNA Analysis of Heart Valves in Patients with Infective Endocarditis: a Retrospective Study

ABSTRACT A substantial proportion of patients with infective endocarditis (IE) are subjected to heart valve surgery. Microbiological findings on valves are important both for diagnostics and for tailored antibiotic therapy, post-operatively. The aims of this study were to describe microbiological findings on surgically removed valves and to examine the diagnostic benefits of 16S-rDNA PCR and sequencing (16S-analysis). Adult patients who were subjected to heart valve surgery for IE between 2012 and 2021 at Skåne University Hospital, Lund, where a 16S-analysis had been performed on the valve, constituted the study population. Data were gathered from medical records, and the results from blood cultures, valve cultures, and 16S-analyses of valves were compared. A diagnostic benefit was defined as providing an agent in blood culture negative endocarditis, providing a new agent in episodes with positive blood cultures, or confirming one of the findings in episodes with a discrepancy between blood and valve cultures. 279 episodes in 272 patients were included in the final analysis. Blood cultures were positive in 259 episodes (94%), valve cultures in 60 episodes (22%), and 16S-analyses in 227 episodes (81%). Concordance between the blood cultures and the 16S-analysis was found in 214 episodes (77%). The 16S-analyses provided a diagnostic benefit in 25 (9.0%) of the episodes. In blood culture negative endocarditis, the 16S-analyses had a diagnostic benefit in 15 (75%) of the episodes. A 16S-analysis should be routinely performed on surgically removed valves in blood culture negative endocarditis. In patients with positive blood cultures, 16S-analysis may also be considered, as a diagnostic benefit was provided in some patients. IMPORTANCE This work demonstrates that it can be of importance to perform both cultures and analysis using 16S-rDNA PCR and sequencing of valves excised from patients undergoing surgery for infective endocarditis. 16S-analysis may help both to establish a microbiological etiology in cases of blood culture negative endocarditis and to provide help in situations where there are discrepancies between valve and blood cultures. In addition, our results show a high degree of concordance between blood cultures and 16S-analyses, indicating that the latter has a high sensitivity and specificity for the etiological diagnosis of endocarditis in patients who were subjected to heart valve surgery.

Please describe the statistical tests you performed as you report significance levels. Please describe the method of heart valve sampling (biopsy, instrument, size of biopsy, any details would be good to know). The type of agar plates is described superficially, what type of blood agar? For instance, chocolate agar is usually required for the HACEK group, but not mentioned here. What is the "fastidious anaerobic agar"; please contact the laboratory for details and edit this chapter. 16S amplification/sequencing: How did you detect a mixed infection (e.g. S. aureus together with S. agalactiae or E. faecalis), as we can usually hardly resolve a mixed 16S sequence?

Results
Viridans streptococci are a frequently used term in the clinical evaluation of a microbiological pathogen, but should be extended here to the -as far as possible -exact species (possibly as appendix material). The same applies to HACEK group, enterococci and ß-haemolytic streptococci; please specify.

Discussion.
Please expand the interesting discussion on the contamination risk of valve cultures to include the two findings of Acinetobacter spp. and Pseudomonas oryzihabitans (I suspect all water contamination; similar as "Environmental bacteria").
Otherwise well done.
Reviewer #2 (Comments for the Author): The study by Johansson et al investigates the microbiology of infective endocarditis at the authors' institution and the benefit provided by 16S rDNA analysis of surgically removed heart valves. The study will be of considerable interest to some readers. The article is generally well written, but I believe it would benefit from the changes suggested below. 1) Line 98: It may be helpful to add "(culture or 16S)" after "valve analysis" so that the reader understands that "one" and "the other" are referring to the different analyses performed on valves rather than to different valves.
2) Line 108: If I understand this sentence, the meaning would be clearer if the word "Previously" was replaced with "Prior to that," to indicate that the authors are referring to practices prior to 2019.
3) Line 112: The meaning of this statement is unclear. Presumably the valve tissue was homogenized in some fashion. Could the authors elaborate? Also, was there a set procedure for determining the portion of valve tissue allocated to culture vs. PCR? 4) Line 120-1: Can the authors provide a reference for these primers? It would be especially helpful if the paper cited characterized primer specificity. 5) Line 123: How did the authors determine when lysozyme and lysostaphin were needed? Was this based on failure of the PCR without them, the species (based on culture results), or some other criterion? 6) Lines 125-6: Which database was used--a custom database created in-house, a general nucleotide database, or a public database devoted to rRNA sequences? 7) Line 140: Although the finding that valve cultures were far more frequently negative than blood cultures or 16S valve analyses is apparently not novel, identification of the cause of this difference would be a valuable contribution. Have the authors examined whether there was a difference in the duration of antibiotic treatment prior to attempted valve culture for the samples that were positive vs. negative? Such a relationship might be apparent for the patient population as a whole or when broken down by organism. 8) Line 147 and elsewhere: The results of statistical tests are provided, but the tests employed have not been identified. 9) Lines 252-4: I don't understand why 16S analysis not being performed on 3 patients with BCNE indicates a less severe bias than originally thought. Are the authors suggesting that this number is smaller than they had expected, larger, or is there some other reasoning?
The authors claimed that 16S-analysis should be routinely performed on surgically removed valves in blood culture-negative endocarditis. In patients with positive blood cultures, 16S analysis may also be considered since a diagnostic benefit was provided in some patients. However, I have some concerns. 1. What are immune cells (eg. macrophages) abundance and response in patients with culture-negative and positive endocarditis? 2. Please do a small prospective analysis (ie. 10 culture-negative: 10 positive endocarditis patients) to validate your retrospective study findings. 3. if possible, please collect the patients' PBMC and heart surgery samples to do RNA-seq to investigate the immune responses and mechanisms, which may be potential targets to treat the patients.

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