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Sequencing is increasingly used for infective endocarditis (IE) diagnosis. Here, the performance of 16S rRNA gene PCR/sequencing of heart valves utilized in routine clinical practice was compared with conventional IE diagnostics. Subjects whose heart valves were sent to the clinical microbiology laboratory for 16S rRNA gene PCR/sequencing from August 2020 through February 2022 were studied. A PCR assay targeting V1 to V3 regions of the 16S rRNA gene was performed, followed by Sanger and/or next-generation sequencing (NGS) (using an Illumina MiSeq), or reported as negative, depending on an algorithm that included the PCR cycle threshold value. Fifty-four subjects, including 40 with IE, three with cured IE, and 11 with noninfective valvular disease, were studied. Thirty-one positive results, 11 from NGS and 20 from Sanger sequencing, were generated from analysis of 16S rRNA gene sequence(s). Positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 55% and 75%, respectively ( P  = 0.06). In those with prior antibiotic exposure, positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 11% and 76%, respectively ( P < 0.001). Overall, 61% of blood culture-negative IE subjects had positive valve 16S rRNA gene PCR/sequencing results. 16S rRNA gene-based PCR/sequencing of heart valves is a useful diagnostic tool for pathogen identification in patients with blood culture-negative IE undergoing valve surgery in routine clinical practice., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

Objective: Detection of the intracellular bacterium Coxiella burnetii, causative agent of chronic Q fever, is notoriously difficult. Diagnosis of and duration of antibiotic treatment for chronic Q fever is partly determined by detection of the bacterium with polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) might be a promising technique for detecting C. burnetii in tissue samples from chronic Q fever patients, but its value in comparison with PCR is uncertain. We aim to assess the value of FISH for detecting C. burnetii in tissue of chronic Q fever patients., Methods: FISH and PCR were performed on tissue samples from Dutch chronic Q fever patients collected during surgery or autopsy. Sensitivity, specificity, and overall diagnostic accuracy were calculated. Additionally, data on patient and disease characteristics were collected from electronic medical records., Results: In total, 49 tissue samples from mainly vascular walls, heart valves, or placentas, obtained from 39 chronic Q fever patients, were examined by FISH and PCR. The sensitivity and specificity of FISH compared to PCR for detecting C. burnetii in tissue samples from chronic Q fever patients was 45.2% (95% confidence interval (CI), 27.3% - 64.0%) and 84.6% (95% CI, 54.6% - 98.1%), respectively. The overall diagnostic accuracy was 56.8% (95% CI, 42.2% - 72.3%). Two C. burnetii PCR negative placentas were FISH positive. Four FISH results (8.2%) were deemed inconclusive because of autofluorescence., Conclusion: With an overall diagnostic accuracy of 57.8%, we conclude that FISH has limited value in the routine diagnostics of chronic Q fever., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Immunohistochemistry (IHC) using monoclonal and polyclonal antibodies is a useful diagnostic method for detecting pathogen antigens in fixed tissues, complementing the direct diagnosis of infectious diseases by PCR and culture on fresh tissues. It was first implemented in a seminal publication by Albert Coons in 1941., Main Body: Of 14,198 publications retrieved from the PubMed, Google, Google Scholar and Science Direct databases up to December 2021, 230 were selected for a review of IHC techniques, protocols and results. The methodological evolutions of IHC and its application to the diagnosis of infectious diseases, more specifically lice-borne diseases, sexually transmitted diseases and skin infections, were critically examined. A total of 59 different pathogens have been detected once in 22 different tissues and organs; and yet non-cultured, fastidious and intracellular pathogens accounted for the vast majority of pathogens detected by IHC. Auto-IHC, incorporating patient serum as the primary antibody, applied to diseased heart valves surgically collected from blood culture-negative endocarditis patients, detected unidentified Gram-positive cocci and microorganisms which were subsequently identified as Coxiella burnetii, Bartonella quintana, Bartonella henselae and Tropheryma whipplei. The application of IHC to ancient tissues dated between the ends of the Ptolemaic period to over 70 years ago, have also contributed to paleomicrobiology diagnoses., Conclusion: IHC plays an important role in diagnostic of infectious diseases in tissue samples. Paleo-auto-IHC derived from auto-IHC, is under development for detecting non-identified pathogens from ancient specimens., (© 2022. The Author(s).)

Streptococcus sanguinis is a common cause of infective endocarditis (IE). Efforts by research groups are aimed at identifying and characterizing virulence factors that contribute to the ability of this organism to cause IE. This Gram-positive pathogen causes heart infection by gaining access to the bloodstream, adhering to host extracellular matrix protein and/or platelets, colonizing the aortic endothelium, and incorporating itself into the aortic vegetation. While many virulence factors have been reported to contribute to the ability of S. sanguinis to cause IE, it is noteworthy that type IV pili (T4P) have not been described to be a virulence factor in this organism, although S. sanguinis strains typically encode these pili. Type IV pili are molecular machines that are capable of mediating diverse virulence functions and surface motility. T4P have been shown to mediate twitching motility in some strains of S. sanguinis, although in most strains it has been difficult to detect twitching motility. While we found that T4P are dispensable for direct in vitro platelet binding and aggregation phenotypes, we show that they are critical to the development of platelet-dependent biofilms representative of the cardiac vegetation. We also observed that T4P are required for in vitro invasion of S. sanguinis into human aortic endothelial cells, which indicates that S. sanguinis may use T4P to take advantage of an intracellular niche during infection. Importantly, we show that T4P of S. sanguinis are critical to disease progression (vegetation development) in a native valve IE rabbit model. The results presented here expand our understanding of IE caused by S. sanguinis and identify T4P as an important virulence factor for this pathogen. IMPORTANCE This work provides evidence that type IV pili produced by Streptococcus sanguinis SK36 are critical to the ability of these bacteria to attach to and colonize the aortic heart valve (endocarditis). We found that an S. sanguinis type IV pili mutant strain was defective in causing platelet-dependent aggregation in a 24-h infection assay but not in a 1-h platelet aggregation assay, suggesting that the type IV pili act at later stages of vegetation development. In a rabbit model of disease, a T4P mutant strain does not develop mature vegetations that form on the heart, indicating that this virulence factor is critical to disease and could be a target for IE therapy.

Streptococcus gordonii and Streptococcus sanguinis are primary colonizers of tooth surfaces and are generally associated with oral health, but can also cause infective endocarditis (IE). These species express "Siglec-like" adhesins that bind sialylated glycans on host glycoproteins, which can aid the formation of infected platelet-fibrin thrombi (vegetations) on cardiac valve surfaces. We previously determined that the ability of S. gordonii to bind sialyl T-antigen (sTa) increased pathogenicity, relative to recognition of sialylated core 2 O-glycan structures, in an animal model of IE. However, it is unclear when and where the sTa structure is displayed, and which sTa-modified host factors promote valve colonization. In this study, we identified sialylated glycoproteins in the aortic valve vegetations and plasma of rat and rabbit models of this disease. Glycoproteins that display sTa vs. core 2 O-glycan structures were identified by using recombinant forms of the streptococcal Siglec-like adhesins for lectin blotting and affinity capture, and the O-linked glycans were profiled by mass spectrometry. Proteoglycan 4 (PRG4), also known as lubricin, was a major carrier of sTa in the infected vegetations. Moreover, plasma PRG4 levels were significantly higher in animals with damaged or infected valves, as compared with healthy animals. The combined results demonstrate that, in addition to platelet GPIbα, PRG4 is a highly sialylated mucin-like glycoprotein found in aortic valve vegetations and may contribute to the persistence of oral streptococci in this protected endovascular niche. Moreover, plasma PRG4 could serve as a biomarker for endocardial injury and infection., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context : Today, infective endocarditis (IE) caused by Enterococcus faecalis represents 10% of all IE and is marked by its difficult management and the frequency of relapses. Although the precise reasons for that remain to be elucidated, the evolution of the culprit strain under selective pressure through microdiversification could be, at least in part, involved. Material and methods : To further study the in situ genetic microdiversity and its possible phenotypic manifestations in E. faecalis IE, we sequenced and compared multiple isolates from the valves, blood culture and joint fluid of five patients who underwent valvular surgery. Growth rate and early biofilm production of selected isolates were also compared. Results : By sequencing a total of 58 E. faecalis genomes, we detected a considerable genomic microdiversity, not only among strains from different anatomical origins, but also between isolates from the same studied cardiac valves. Interestingly, deletions of thousands of bases including the well-known virulence factors ebpA/B/C , and srtC , as well as other large prophage sequences containing genes coding for proteins implicated in platelet binding (PlbA and PlbB) were evidenced. The study of mutations helped unveil common patterns in genes related to the cell cycle as well as central metabolism, suggesting an evolutionary convergence in these isolates. As expected, such modifications were associated with a significant impact on the in-vitro phenotypic heterogeneity, growth, and early biofilm production. Conclusion : Genome modifications associated with phenotypic variations may allow bacterial adaptation to both antibiotic and immune selective pressures, and thus promote relapses.

Objective: A substantial proportion of infective endocarditis (IE) cases are complicated by local invasion. The purpose of this study was to identify patient and disease characteristics associated with local invasion in surgically treated IE patients., Methods: This was a nested case-control study. All episodes of IE for patients admitted to Cleveland Clinic from 1 January 2013 to 30 June 2016 were identified from the Cleveland Clinic IE Registry. Patients ≥18 years of age who underwent surgery for IE were included. Among these, cases were those with local invasion, controls were those without. Local invasion, defined as periannular extension, paravalvular abscess, intracardiac fistula or pseudoaneurysm, was ascertained from the surgical operative note. Associations of selected factors with local invasion were examined in a multivariable logistic regression model., Results: Among 511 patients who met inclusion criteria, 215 had local invasion. Mean age was 56 years; 369 were male. Overall 345 (68%) had aortic valve, 228 (45%) mitral valve, and 66 (13%) tricuspid or pulmonic valve involvement. Aortic valve involvement (OR 6.23, 95% CI 3.55-11.44), bioprosthetic valve (OR 3.88, 95% CI 2.36-6.44), significant paravalvular leak (OR 3.80, 95% CI 1.60-9.89), new atrioventricular nodal block (OR 3.77, 95% CI 1.87-7.90), infection with streptococci other than viridans group streptococci (OR 7.54, 95% CI 2.42-24.87) and presence of central nervous system emboli (OR 1.85, 95% CI 1.13-3.04) were associated with local invasion., Discussion: Intracardiac and microorganism factors, but not comorbid conditions, are associated with local invasion in IE., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objective: Although recent advances in pulmonary valve replacement have enabled excellent hemodynamics, infective endocarditis remains a serious complication, particularly for implanted bovine jugular vein (BJV) conduits., Methods: We investigated contributions by platelets and plasma fibrinogen to endocarditis initiation on various grafts used for valve replacement. Thus, adherence of Staphylococcus aureus and platelets to 5 graft tissues was studied quantitatively in perfusion chambers, assisted by microscopic analysis. We also evaluated standard antiplatelet therapy to prevent onset of S aureus endocarditis., Results: Of all tissues, bovine pericardium (BP) showed the greatest fibrinogen binding. Perfusion of all plasma-precoated tissues identified BP and BJV wall with the greatest affinity for S aureus. Perfusions of anticoagulated human blood over all tissues also triggered more platelet adhesion to BP and BJV wall as single platelets. Several controls confirmed that both S aureus and platelets were recruited on immobilized fibrinogen. In addition, perfusions (and controls) over plasma-coated tissues with whole blood, spiked with S aureus, revealed that bacteria exclusively bound to adhered platelets. Both the platelet adhesion and platelet-mediated S aureus recruitment required platelet α IIb β 3 and coated or soluble fibrinogen, respectively, interactions abrogated by the α IIb β 3 -antagonist eptifibatide. Also, standard antiplatelet therapy (aspirin/ticagrelor) reduced the adherence of S aureus in blood to BJV 3-fold., Conclusions: Binding of plasma fibrinogen to especially BJV grafts enables adhesion of single platelets via α IIb β 3 . S aureus then attaches from blood to (activated) bound platelet α IIb β 3 via plasma fibrinogen. Dual antiplatelet therapy appears a realistic approach to prevent endocarditis and its associated mortality., (Copyright © 2019 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Aims: Upon suspicion of infective endocarditis, the causative microorganism must be identified to optimize treatment. Blood cultures and culturing of removed valves are the mainstay of this diagnosis and should be complemented by growth-independent methods. We assessed the diagnostic benefit of examining removed endocarditis valves by broad-range bacterial PCR to detect causative bacteria in cases where culturing was not available, negative, or inconclusive because a skin commensal was detected, in patients from our clinical routine practice., Methods and Results: Patients from Heidelberg University Hospital with suspicion of endocarditis, followed by valve replacement and analysis by 16S rDNA PCR, between 2015 and 2018, were evaluated. 146 patients with definite infective endocarditis, confirmed by the valve macroscopics and/or histology, were included. Valve PCRs were compared to corresponding blood and valve culture results. Overall, valve PCR yielded an additional diagnostic benefit in 34 of 146 cases (23%) and was found to be more sensitive than valve culture. In 19 of 38 patients with both negative blood and valve cultures, valve PCR was the only method rendering a pathogen. In 23 patients with positive blood cultures detecting skin commensals, 4 patients showed discordant valve PCR results, detecting a more plausible pathogen, and in 11 of 23 cases, valve PCR confirmed commensals in blood culture as true pathogens. Only the remaining 8 patients had negative valve PCRs., Conclusion: Valve PCR was found to be a valuable diagnostic tool in surgical endocarditis cases with negative blood cultures or positive blood cultures of unknown significance., Trial Registration: S-440/2017 on 28.08.2017 retrospectively registered. Subdividing of all infective endocarditis patients in this study, showing that valve PCR yields valuable information for patients with skin commensals in blood cultures, which were either confirmed by the same detection in valve PCR or refuted by the detection of a different and typical pathogen in valve PCR. Additionally, benefit was determined in patients with negative or not available blood cultures and only positive detection in valve PCR. +: Positive; -: negative; n/a: not available results.

Background: Infective endocarditis (IE) may present with quite different aspects. Consequently, it's management involves a collaborative approach between several specialties. We analyzed the timing of surgery and the role of the "Endocarditis Team" in patients with IE. Methods: The medical records of patients operated for IE in our center during an 18-year period were retrospectively analyzed for the demographic data, causative agent, imagistic features of the affected valve and systemic lesion extension and postoperative results. Results: Patients age ranged between 7 and 84 years, and in many cases (55.88%) the etiology remained unidentified. The early postoperative complications were not significantly higher in patients operated in emergency compared to those with elective surgery. The overall early postoperative mortality rate was 14.05%, significantly lower in the elective surgery cases (p = 0.001). The long-term follow-up for patients operated between 2008 and 2017 showed a late postoperative death rate of 17.8% (34 patients). Conclusions: Emergency surgery for patients with native valve IE provides in most cases a good chance for a cure, despite the relatively high frequency of postoperative complications and rate of postoperative death. The advances in pre- and post-operative management of IE patients, as well as in surgical techniques and prosthetic valves seem to further improve the outcome., (Celsius.)

Infectious endocarditis (IE) remains one of the deadliest heart diseases with a high death rate, generally following thrombo-embolic events. Today, therapy is based on surgery and antibiotic therapy. When thromboembolic complications in IE patients persist, this is often due to our lack of knowledge regarding the pathophysiological development and organization of cells in the vegetation, most notably the primordial role of platelets and further triggered hemostasis, which is related to the diversity of infectious microorganisms involved. Our objective was to study the organization of IE vegetations due to different bacteria species in order to understand the related pathophysiological mechanism of vegetation development. We present an approach for ultrastructural analysis of whole-infected heart valve tissue based on scanning electron microscopy and energy-dispersive X-ray spectroscopy. Our approach allowed us to detect differences in cell organization between the analyzed vegetations and revealed a distinct chemical feature in viridans Streptococci ones. Our results illustrate the benefits that such an approach may bring for guiding therapy, considering the germ involved for each IE patient.

Splenic abscess is an uncommon but serious complication of infective endocarditis (IE). The timing of surgical management of splenic abscess can be challenging when valve surgery is required. The American Heart Association (AHA) and the European Society of Cardiology (ESC) currently recommends splenectomy before valve replacement due to fear of reinfection of the heart valve; however, published data to support this recommendation are limited. In this series, we report outcomes for 5 patients with IE and splenic abscess who underwent valve replacement first, followed by splenectomy at a median of 19 days (range: 10-77 days) after valve surgery, with no recurrent infection of the new valve. Our experience and review of the available literature provide reassurance for splenectomy after valve surgery for IE., Competing Interests: Conflicts of interest None, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Genome studies of heart valve tissue (HVT) in patients with structural valvular heart disease (sVHD) and acute infective endocarditis (aIE) showed polymicrobial infections. Subject of this study is the quantification of bacterial DNA in HVT of sVHD in comparison to aIE. It will be examined whether the bacterial DNA concentration can be used as surrogate marker to differentiate chronic and acute infections. DNA was isolated from HVT of 100 patients with sVHD and 23 microbiologically positively tested patients with aIE. Selected pathogens (Cutibacterium acnes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Clostridium difficile, and Klebsiella pneumoniae) were quantified using TaqMan-qPCR. Polymicrobial infiltration of HVT was investigated by immunohistologic methods. Of 100 sVHD patients, 94 tested positive for bacteria by 16S-rDNA and 72 by TaqMan-qPCR. In 29% of the sVHD cohort and in 70% of the aIE cohort, a coinfection with more than 2 bacteria was observed as indication of a polymicrobial infection. The most common pathogens in the sVHD patients were C. acnes (59%; 5-4074 pg/mL), E. faecalis (16%, 174-2781 pg/mL), and S. aureus (15%, 8-105 pg/mL). The DNA concentration of E. faecalis (P = 0.0285) and S. aureus (P = 0.0149) is significantly lower in the sVHD cohort than in the aIE cohort. sVHD is associated with bacterial infection and infiltration of the HVT in a majority of cases. TaqMan-qPCR is a valid instrument for the specific detection of bacteria in HVT and allows discrimination between sVHD and aIE for E. faecalis and S. aureus., (Copyright © 2019. Published by Elsevier Inc.)

The diagnosis of infective endocarditis (IE) remains a challenge. One of the rare bacterial species recently associated with biofilms and negative cultures in infective endocarditis is Aerococcus urinae. Whether the low number of reported cases might be due to lack of awareness and misidentification, mainly as streptococci, is currently being discussed. To verify the relevance and biofilm potential of Aerococcus in endocarditis, we used fluorescence in situ hybridization to visualize the microorganisms within the heart valve tissue. We designed and optimized a specific FISH probe (AURI) for in situ visualization and identification of A. urinae in sections of heart valves from two IE patients whose 16S rRNA gene sequencing had deteced A. urinae. Both patients had a history of urinary tract infections. FISH visualized impressive in vivo grown biofilms in IE, thus confirming the potential of A. urinae as a biofilm pathogen. In both cases, FISH/PCR was the only method to unequivocally identify A. urinae as the only causative pathogen for IE. The specific FISH assay for A. urinae is now available for further application in research and diagnostics. A. urinae should be considered in endocarditis patients with a history of urinary tract infections. These findings support the biofilm potential of A. urinae as a virulence factor and are meant to raise the awareness of this pathogen., Competing Interests: The authors have declared that no competing interests exist.

The optimal timing of cardiac surgery in infective endocarditis (IE) remains debated: Early surgery decreases the risk of embolism, and heart failure, but is associated with an increased rate of positive valve culture. To determine the determinants, and the consequences, of positive valve culture when cardiac surgery is performed during the acute phase of IE, we performed a retrospective study of adult patients who underwent cardiac surgery for definite left-sided IE (Duke Criteria), in two referral centres. During the study period (2002-2016), 148 patients fulfilled inclusion criteria. Median age was 65 years [interquartile range, 53-73], male-to-female ratio was 2.9 (110/38). Cardiac surgery was performed after 14 days [5-26] of appropriate antibiotics. Valve cultures returned positive in 46 cases (31.1%). Factors independently associated with positive valve culture were vegetation size ≥ 10 mm (OR 2.83 [1.16-6.89], P = 0.022) and < 14 days of appropriate antibacterial treatment before surgery (OR 4.68 [2.04-10.7], P < 0.001). Positive valve culture was associated with increased risk of postoperative acute respiratory distress syndrome (37.0% vs. 15.7%, P = 0.008) but was associated neither with an increased risk of postoperative relapse nor with the need for additional cardiac surgery. Duration of appropriate antibacterial treatment and vegetation size are independently predictive of positive valve culture in patients operated during the acute phase of IE. Positive valve culture is associated with increased risk of postoperative acute respiratory distress syndrome.

Background To evaluate changes in patient characteristics and outcomes for infective endocarditis (IE) related to opioid use disorder (OUD), we used the National (Nationwide) Inpatient Sample (NIS) to characterize the trend in hospitalizations for patients with IE with and without OUD and those treated medically and surgically. Methods and Results Temporal trends in hospitalization characteristics for patients with IE with and without OUD and those treated medically and surgically were estimated via the NIS data in 2005-2014. Hospitalizations for OUD and IE increased from 119 to 202 and from 12 to 15 cases per 100 000 between 2005 and 2014, respectively. Hospitalizations with OUD among all IE hospitalizations increased from 6.3% in 2005 to 11.6% in 2014. Among all IE hospitalizations, patients being admitted for IE in the setting of OUD were younger compared with the cohort of IE without OUD (aged 37.6±0.21 years versus 60.9±0.16 years). Myocardial infarction, diabetes mellitus, chronic kidney disease, peripheral vascular disease, and heart failure were more common in patients without OUD. The OUD cohort more frequently had liver disease (46.0% versus 10.8%) and immunosuppressed status (4.3% versus 2.1%). Valve operations for IE accounted for 10.2% of all valve operations in 2005, and this increased to 12.7% in 2014. These proportions were similar between OUD (11.4%) and non-OUD (11.1%) cohorts. Operative mortality was lower in patients with OUD (4.3% versus 9.4%, P <0.001). Conclusions IE associated with OUD has a distinct phenotype and has become more prevalent. Surgical outcomes are favorable and operations were performed in similar proportions of patients who had IE with OUD compared with patients who had IE without OUD.

Coagulase-negative staphylococci (CoNS) are part of the microbiota of human skin and rarely linked with soft tissue infections. In recent years, CoNS species considered as one of the major nosocomial pathogens and can cause several infections such as catheter-acquired sepsis, skin infection, urinary tract infection, endophthalmitis, central nervous system shunt infection, surgical site infections, and foreign body infection. These microorganisms have a significant impact on human life and health and, as typical opportunists, cause peritonitis in individuals undergoing peritoneal dialysis. Moreover, it is revealed that these potential pathogens are mainly related to the use of indwelling or implanted in a foreign body and cause infective endocarditis (both native valve endocarditis and prosthetic valve endocarditis) in patients. In general, approximately eight percent of all cases of native valve endocarditis is associated with CoNS species, and these organisms cause death in 25% of all native valve endocarditis cases. Moreover, it is revealed that methicillin-resistant CoNS species cause 60 % of all prosthetic valve endocarditis cases. In this review, we describe the role of the CoNS species in infective endocarditis, and we explicated the reported cases of CoNS infective endocarditis in the literature from 2000 to 2020 to determine the role of CoNS in the process of infective endocarditis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Infective endocarditis is associated with high morbidity and mortality. Hence, early diagnosis and prompt intervention is crucial. Multivalvular endocarditis involving 3 or more valves is rarely reported with little information regarding best management or prognosis, particularly in nonsurgical patients. Conflicting guidelines regarding medical versus surgical treatment in multivalvular endocarditis exist with few studies describing the outcome of medically managed patients. We report the case of a previously healthy male presenting with infective endocarditis involving 3 valves further complicated by multiple septic emboli and deemed a nonsurgical candidate.

Aim: To explore potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis (IE) caused by fastidious bacteria. Materials & methods: Plasma and heart valves of two patients, who were diagnosed with IE caused by Bartonella quintana and Propionibacterium species, were sequenced by using Illumina MiSeq and Nanopore MinION. Results: For patient 1, B. quintana was detected in the plasma pool collected 4 days before valvular replacement surgery. For patient 2, Propionibacterium sp. oral taxon 193 was detected in the plasma sample collected on hospital day 1. Nearly complete bacterial genomes (>98%) were retrieved from resected heart valves of both patients, enabling detection of antibiotic resistance-associated features. Real-time sequencing of heart valves identified both pathogens within the first 16 min of sequencing runs. Conclusion: Metagenomic sequencing may be a helpful supplement to IE diagnostic workflow, especially when conventional tests fail to yield a diagnosis.

Objectives: In infective endocarditis (IE), identification of the causative organism and consecutive treatment are crucial for patient survival. Although the macroscopic aspect resembles infected tissue, standard diagnostic tests often fail to allow one to identify bacteria. Fluorescence in situ hybridization (FISH) is a molecular, culture-independent technique that allows one to identify and visualize microorganisms within tissue and to recognize their morphology, number and activity. We analysed the diagnostic benefit of FISH/polymerase chain reaction (PCR) by comparing its results to those of standard diagnostic tests., Methods: From September 2015 to April 2018, 128 patients underwent first-time or redo valve surgery to treat IE. Patients were designated according to the modified Duke criteria as definite (n = 61), possible (n = 34) or rejected (n = 33) IE. Tissue specimens obtained intraoperatively were analysed using FISH/PCR in addition to undergoing standard diagnostic testing and PCR alone., Results: We used blood cultures to detect microorganisms in 67/128 patients; valve cultures, in 34/128; PCR, in 67/128; histopathological diagnosis showed IE in 72/128 cases. We were able to detect microorganisms in 103/128 cases using FISH/PCR, with 55/61 in definite IE. Furthermore, we were able to identify 26 cases of bacterial biofilm using FISH/PCR, despite antibiotic treatment of 61 in the definite, 13 in the possible and 1 in the rejected group, including 8/33 patients in the rejected group with active bacteria. In all cases, the patient's therapy was altered., Conclusions: FISH/PCR was used to identify microorganisms in cases in which standard diagnostic tests failed to provide sufficient results for various reasons. Furthermore, FISH/PCR enabled us to identify bacterial biofilms and to differentiate between active versus degraded bacteria, thus indicating the impact of treatment. Therefore, we suggest FISH/PCR as an additional diagnostic tool in IE alongside standard diagnostic tests., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Although reports of infections caused by anaerobes after tissue transplantation are uncommon, contamination of allografts may result in substantial complications. Anaerobic incubation and testing of organ transport solution (TS) are not routine. The aim of this study was to determine the bioburden of strict anaerobic bacteria and oxygen tension of heart-TS. Forty TS from different donors were evaluated cultured using membrane filtration (MF), direct inoculation on broth and automated blood culture bottle (ABCB). Bacterial identification was performed by MALDI-TOF. The transport conditions were simulated to verify the bacterial recovery. A sterile bag fulfilled with 250 ml -1 of sterile saline was spiked with 100 CFU ml -1 of Clostridium perfringens and the fluid recovered 0 h, 1 h, 2 h, 6 h, 12 h, 24 h and 48 h for culture and oxygen measurement. Strict anaerobic bacteria were not isolated in heart-TS. The recovery of C.perfringens spiked in heart-TS was 100% using automated blood culture bottles. MF method detected >100 CFU only after 6 h of spiking. The manual culture was not able to recover C.perfringens after the process. The percentage of O 2 measures varied from 77.6 to 87.9%. MF or ABCB are better than direct inoculation for recovery of anaerobes from heart-TS. Although all samples from heart donors were negative for anaerobes (probably due to low incidence of contamination), C.perfringens were all recovered in the simulated transport condition., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: We assessed the determinants of mortality in infective endocarditis (IE), using the national hospital discharge databases (HDD) in 2011., Methods: IE stays were extracted from the national HDD, with a definition based on IE-related diagnosis codes. This definition has been assessed according to Duke criteria by checking a sample of medical charts of IE giving a predictive positive value of 86.1% (95% confidence interval (CI): 82.7% - 89.5%). The impact of heart valve surgery on survival has been studied if performed during the initial stay, and over the year of follow-up. Risk factors of in-hospital mortality were identified using logistic regression model for the initial stay and Cox Time-dependent model for the 1-year mortality., Results: The analysis included 6,235 patients. The annual incidence of definite IEs was 63 cases/million residents. Staphylococci and Streptococci were the most common bacteria (44% and 45%, respectively). A valvular surgery was performed in 20% of cases, but substantial variations existed between hospitals. The in-hospital mortality was 21% (ranging 12% to 27% according to the region of patients), associated with age>70, chronic liver disease, renal failure, S. aureus, P. aeruginosa or candida infection and strokes whereas valvular surgery, a native valve IE or intraveinous drug use (right heart IE) were significantly protective for an initial death. The same factors were associated with the one-year mortality, except for valvular surgery which was associated with a 1.4-fold higher risk of death during the year post IE., Conclusion: We reported a high IE incidence rate. Valvular surgery was considerably less frequent in this study than in the previous published data (near 50%) whereas mortality was similar. Surgery was associated with higher survival if undergone within the initial stay. There were significant regional differences in frequency of surgery but it did not impact mortality., Competing Interests: The authors have declared that no competing interests exist.

Background: The incidence of Staphylococcus aureus infective endocarditis (IE) is steadily rising due to advances in health care delivery. Routine echocardiography is essential in the management of Staphylococcus aureus bacteremia (SAB). The aim of this retrospective cohort study was to characterize the real-world use of echocardiography in adult patients with SAB and native valve S aureus IE., Methods: Using an academic hospital microbiological database, all cases of SAB in adults between 2010 and 2016 were identified. Demographic, echocardiographic, and clinical features were recorded., Results: A total of 738 episodes of SAB were identified, of which 504 (68%) patients underwent transthoracic echocardiography (TTE) within 30 days. Of 73 patients with definite IE, 46 (63%) patients had definite IE diagnosed on the initial TTE. An additional 14 (19%) patients had definite IE diagnosed on repeat TTE, 6 (8%) on transesophageal echocardiography (TEE), and 7 (10%) were diagnosed without fulfilling Duke echocardiographic criteria. The yield of repeat TTE was comparable to that of TEE for identifying new vegetations not identified on the initial TTE (17% vs 21%, P = .78)., Conclusions: Most cases of IE in SAB were identified using TTE alone, with repeat TTE improving the diagnostic yield in the setting of clinical decompensation., (© 2019 Wiley Periodicals, Inc.)

The performance of many laboratories can be evaluated by participation in external quality assessment (EQA) schemes. EQA allows for comparison of a laboratory's performance with a source outside the laboratory-either a peer group of laboratories or a reference laboratory. Such EQA schemes do not exist in tissue banking despite the fact that tissue establishments (TE) perform very complex procedures. This paper describes the first ever EQA scheme in the field specifically assessing microbiological aspects in heart valve (HV) banking. Twenty-two TEs participated. Three HV tissue samples were sent to each participating TE-two contaminated with non-pathogenic micro-organisms and a third negative control. The aims were to isolate and identify the micro-organisms present and then to successfully decontaminate the HV tissue using the routine standard operating procedures of the TE. Eight of the TEs were able to isolate and identify all contaminating micro-organisms present, and of these, five also successfully decontaminated the tissue; 13 TEs failed to establish the identity of one or more of the contaminants; five TEs appear to have introduced contamination during the processing or testing of the tissue; and eight failed to successfully decontaminate the HV tissue. This initiative provides TEs with an international benchmark for tissue product microbiology testing. It has identified significant variation in practice and in the ability of different TEs to identify the presence of contamination. There is now work ongoing with the aim of setting up a regular EQA scheme for HV banking.

The European Blood Alliance (EBA) Tissue and Cells annual benchmarking exercise identified that in 2014, the heart valve (HV) discard rate in tissue establishments (TEs) run by EBA members was between 19 and 65%. Given this significant discard rate, a decision was taken to carry out a worldwide data-gathering exercise to assess the processing methodology in different TEs. In collaboration with the Foundation of European Tissue Banks, a questionnaire asking for the details on HV processing was sent to TEs worldwide. Nineteen questionnaires were received back from 15 European TEs and 4 non-European TEs. The data provided confirmed a significant discard rate of HVs with 43-50% of aortic valves and 20-32% of pulmonary valves being discarded in 2015. The causes of HV discard varied, with microbiology contamination, anatomical and medical reasons being the main causes. This data-gathering exercise highlighted significant variations in practice in different TEs including how donor suitability is assessed, critical timings for heart retrieval and processing, heart rinsing, HV decontamination protocols and methods of microbiological testing. To reduce the discard rates, there are several aspects of HV banking that could be validated and standardised. Here, we report the findings of this data-gathering exercise. We consider this a first step that will help lead to standardising HV banking.

Background: The impact of valve surgery on outcomes of Staphylococcus aureus infective endocarditis (SAIE) remains controversial. We tested the hypothesis that early valve surgery (EVS) improves survival by using a novel approach that allows for inclusion of major confounders in a time-dependent way., Methods: EVS was defined as valve surgery within 60 days. Univariable and multivariable Cox regression analyses were performed. To account for treatment selection bias, we additionally used a weighted Cox model (marginal structural model) that accounts for time-dynamic imbalances between treatment groups. To address survivor bias, EVS was included as a time-dependent variable. Follow-up of patients was 1 year., Results: Two hundred and three patients were included in the analysis; 50 underwent EVS. All-cause mortality at day 30 was 26%. In the conventional multivariable Cox regression model, the effect of EVS on the death hazard was 0.85 (95% confidence interval [CI], .47-1.52). Using the weighted Cox model, the death hazard rate (HR) of EVS was 0.71 (95% CI, .34-1.49). In subgroup analyses, no survival benefit was observed in patients with septic shock (HR, 0.80 [CI, .26-2.46]), in NVIE (HR, 0.76 [CI, .33-1.71]) or PVIE (HR, 1.02 [CI, .29-3.54]), or in patients with EVS within 14 days (HR, 0.97 [CI, .46-2.07])., Conclusions: Using both a conventional Cox regression model and a weighted Cox model, we did not find a survival benefit for patients who underwent EVS in our cohort. Until results of randomized controlled trials are available, EVS in SAIE should be based on individualized decisions of an experienced multidisciplinary team., Clinical Trials Registration: German Clinical Trials registry (DRKS00005045)., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Coxiella burnetii is the causative agent of Q fever, a zoonotic infection. The bacteria is a gram-negative, pleomorphic, coccobacilli and capable to survive and proliferate within the host cell's phagolysosome. There are two morphological cell types of C.burnetii including small and large cell variants. C.burnetii is divided into phase I and phase II serologically variants according to LPS structure in the cell wall. Phase I is the natural phase found in infected animals or humans and is highly infectious. Phase II is not very infectious and could be obtained only in laboratories after serial passages in cell cultures or embryonated egg cultures. Q fever can be asymptomatic (in 50% of the cases), acute or chronic. Major presentations of acute Q fever are flu-like illness, pneumonia, and hepatitis, whereas the chronic form presents mainly as infective endocarditis. The aim of this study was to obtain C.burnetii phase II variant from C.burnetii phase I variant by a phase change study. In this study, C.burnetii was isolated by cell culture method from the heart valve tissue of a Q fever endocarditis case. C.burnetii phase I antigen for the indirect fluorescent antibody test (IFAT) was prepared from the isolated strain. For the isolation and identification of C.burnetii, heart valve tissue of the patient was homogenized and DNA was extracted by tissue extraction kit. C.burnetii DNA in the valve tissue was determined by real-time PCR (Rt-PCR). This C.burnetii DNA positive specimen was inoculated into Vero cells by shell vial centrifugation method. The scraped Vero cells were fixed on the slides after one week of incubation and IFAT was performed using C.burnetii phase I IgG positive sera, bacteria that were grown in and surrounding the Vero cells stained apple green were determined microscopically. Infected cells were disrupted by freeze and thaw method to obtain bacterial suspension. The DNA obtained from the bacterial suspension was again found to be positive for C.burnetii by Rt-PCR. Isolation sample was found to be positive in PCR at an earlier cycle compared to heart tissue sample, thus the bacterial growth was also confirmed with PCR. 16S ribosomal RNA gene of our isolate was amplified by PCR using 27F and 1492 primers and then sequenced. The DNA sequences were compared with reference DNA sequences of GeneBank; and the nucleotide sequence of the 16S ribosomal RNA gene of our isolate was found to be 99% similar to C.burnetii strain ATCC VR-615 an accession number NR104916. Serial cell culture passages of the isolated strain were performed to obtain C.burnetii phase II variant from C.burnetii phase I variant. After each passage, presence of phase change was investigated by IFAT using C.burnetii phase I and phase II IgG positive sera. At the end of 17 cell culture passages, phase change could not be observed. C.burnetii phase I IFAT antigen was prepared from the obtained bacterial suspension. In this study, we presented the isolation and identification of C.burnetii by cell culture, molecular and serological methods from the heart valve of a patient with endocarditis for the first time in our country.

Objectives: Identification of the underlying pathogens of infective endocarditis (IE) is critical for precision therapy., Methods: We evaluated a metagenomic method with next-generation sequencing (NGS) for the direct detection of pathogens from the resected valves of 44 IE patients and seven rejected IE patients according to the modified Duke criteria., Results: NGS displayed sensitivity, specificity, positive predictive values and negative predictive values of 97.6%, 85.7%, 97.6%, and 85.7% compared with 46.2%, 100%, 100%, and 12.5% for blood culture and 17.1%, 100%, 100%, and 17.1% for valve culture and 51.4%, 100%, 100%, and 26.1% for valve Gram staining, respectively., Conclusions: NGS technique had superior sensitivity and shorter turnaround time compared with culture-based methods for identifying causative pathogens of IE. The NGS technology should be considered an essential supplement to culture-based methods, particularly for unculturable or difficult-to-culture microorganisms., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The mechanisms or host factors involved in septic thrombus or vegetation formation in Staphylococcus aureus -induced infective endocarditis (IE) are unclear. Using an experimental endocarditis rat model, here we demonstrated that S. aureus HG001-induced vegetation was composed of bacterial floes encased in aggregated platelets and surrounded by neutrophil extracellular traps (NETs). In vitro data demonstrated that platelets contribute to both biofilm and NET formation. Prophylactic administration of DNase I significantly reduced the size of vegetation induced by methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains, even though MRSA and MSSA isolates express different biofilm phenotypes and NET-induction abilities in the presence of platelets. Moreover, delivery of both DNase I and daptomycin prophylactically and therapeutically produced synergistic effects by reducing vegetation size and bacterial numbers on damaged valve tissues in MRSA-induced IE. Together, these data suggest that NETs contribute to vegetation formation in S. aureus endocarditis and DNase I has the potential to control S. aureus -induced IE in the clinic., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: The culture of removed cardiac tissues during cardiac surgery of left-sided infective endocarditis (LSIE) helps to guide antibiotic treatment. Nevertheless, the prognostic information of a positive valve culture has never been explored., Methods: Among 1078 cases of LSIE consecutively diagnosed in 3 tertiary centers, we selected patients with positive blood cultures who underwent surgery during the active period of infection and in whom surgical biological tissues were cultured (n = 429). According to microbiological results, we constructed 2 groups: negative valve culture (n=218) and concordant positive valve culture (CPVC) (n=118). We compared their main features and performed a multivariable analysis of in-hospital mortality., Results: Patients with CPVC presented more nosocomial origin (32% vs 20%, P = .014), more septic shock (21% vs 11%, P = .007), and higher Risk-E score (29% vs 21%, P = .023). Their in-hospital mortality was higher (35% vs 19%, P = .001), despite an earlier surgery (3 vs 11 days from antibiotic initiation, P < .001). Staphylococcus species (61% vs 42%, P = .001) and Enterococcus species (20% vs 9%, P = .002) were more frequent in the CPVC group, whereas Streptococcus species were less frequent (14% vs 42%, P < .001). Independent predictors for in-hospital mortality were renal failure (odds ratio [OR], 2.6 [95% confidence interval {CI}, 1.5-4.4]), prosthesis (OR, 1.9 [95% CI, 1.1-3.5]), Staphylococcus aureus (OR, 1.8 [95% CI, 1.02-3.3]), and CPVC (OR, 2.3 [95% CI, 1.4-3.9])., Conclusions: Valve culture in patients with active LSIE is an independent predictor of in-hospital mortality., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (10 4 to 10 -1  CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤ 0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p < 0.05). The positivity for growth in the automated method was higher in concentrated inoculum (> 10 2  CFU/mL) (p < 0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 10 3  CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.

Nowadays, dental diseases are one of the most common illnesses in the world. Some of them can lead to translocation of oral bacteria to the bloodstream causing intermittent bacteraemia. Therefore, a potential association between oral infection and cardiovascular diseases has been discussed in recent years as a result of adhesion of oral microbes to the heart valves. The aim of this study was to detect oral bacteria on pathologically changed heart valves not caused by infective endocarditis. In the study, patients with pathologically changed heart valves were involved. Samples of heart valves removed during heart valve replacement surgery were cut into two parts. One aliquot was cultivated aerobically and anaerobically. Bacterial DNA was extracted using Ultra-Deep Microbiome Prep (Molzym GmbH, Bremen, Germany) followed by a 16S rRNA gene PCR amplification using Mastermix 16S Complete kit (Molzym GmbH, Bremen, Germany). Positive PCR products were sequenced and the sequences were analyzed using BLAST database ( http://www.ncbi.nlm.nih/BLAST ). During the study period, 41 samples were processed. Bacterial DNA of the following bacteria was detected in 21 samples: Cutibacterium acnes (formerly Propionibacterium acnes) (n = 11; 52.38% of patients with positive bacterial DNA detection), Staphylococcus sp. (n = 9; 42.86%), Streptococcus sp. (n = 1; 4.76%), Streptococcus sanguinis (n = 4; 19.05%), Streptococcus oralis (n = 1; 4.76%), Carnobacterium sp. (n = 1; 4.76%), Bacillus sp. (n = 2; 9.52%), and Bergeyella sp. (n = 1; 4.76%). In nine samples, multiple bacteria were found. Our results showed significant appearance of bacteria on pathologically changed heart valves in patients with no symptoms of infective endocarditis.

The aim of this study was to evaluate the new commercial Unyvero i60 ITI multiplex PCR system (Curetis, Holzgerlingen, Germany) on native cardiac valves in comparison with made in-house 16S rRNA PCR amplification (91E/13BS primers) and conventional microbiological techniques. Forty-four patients (30 men, 14 women) with suspected infective endocarditis (IE) were included in this evaluation corresponding to 30 aortic valves and 14 mitral valves. IE was definite for 40 patients using the modified Duke criteria. 16S rRNA PCR amplification was successful in 22 patients (55%). The Unyvero i60 ITI cartridge yielded a positive result in 16 patients (40%). Among the 40 cases, the etiological agent was not included in the panel of Unyvero i60 ITI cartridge for 14 cases. Moreover, for S. aureus, the Unyvero i60 ITI cartridge quickly yielded the susceptibility to meticillin. The result of the experiment was available after 5 hours whereas 16S rRNA PCR amplification-sequencing needs 14 hours of manipulation. If the manufacturer incorporates new targets able to detect more endocarditis agents such as viridans streptococci, the Unyvero i60 ITI cartridge may be a promising and easy-to-use test.

All cardiac allograft tissues are under potential contamination, requiring a validated terminal sterilization process or a minimal bioburden. The bioburden calculation is important to determine the bacterial burden and further decontamination and disinfection strategies for the valve processing. The aim of this study was to determine the bioburden from transport solution (TS) of heart valves obtained from non-heart-beating and heart-beating donors in different culture methods. The bioburden from TS was determined in 20 hearts donated for valve allograft tissue using membrane filter (MF) and direct inoculation. Tryptic soy agar and Sabouraud plates were incubated and colonies were counted. Ninety-five percent of samples from this study were obtained from heart-beating donors. The warm ischemic time period for heart was 1.06 ± 0.74 h and the cold ischemic time period was 25.66 ± 11.16 h. The mean TS volume was 232.68 ± 96.67 mL (48.5-550 mL). From 20 samples directly inoculated on TSA agar plates, 2 (10%) were positive. However, when MF was used, from 20 samples in TSA, 13 (65%) were positive with a mean count of 1.36 ± 4.04 CFU/mL. In Sabouraud plates, the direct inoculation was positive in 5 samples (25%) with a mean count of 0.24 ± 0.56 CFU/mL. The use of MF increased the positivity to 50% (10 samples from a total of 20) with a mean of 0.28 ± 0.68 CFU/mL. The positivity was superior using MF in comparison with direct inoculation (p < 0.05). The bioburden of TS is low and MF is the technique of choice due to higher positivity.

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.

Coxiella burnetii is a common cause of blood culture-negative infective endocarditis (IE). Molecular detection of C burnetii DNA in clinical specimens is a promising method of diagnosing Q fever endocarditis. Here, we examined the diagnostic utility of Q fever polymerase chain reaction (PCR) of formalin-fixed heart valve tissue from patients with blood culture-negative IE who underwent heart valve surgery. Clinical and laboratory data of patients with blood culture-negative IE who underwent heart valve surgery during a 6-year period and for whom biopsy tissues were available were reviewed retrospectively. Blood culture-positive IE patients who underwent heart valve surgery within the last 3 years were used as controls. Heart valve samples were cultured and also subjected to histological examination and PCR for Q fever, brucellosis, and bartonellosis. Data from 20 patients with blood culture-negative IE and 20 with blood culture-positive IE were analyzed. Eight cases of blood culture-negative IE were PCR-positive for C burnetii (40%; 95% confidence interval, 19-64). No specimen was PCR-positive for brucellosis or bartonellosis. Histologically, 4 of 8 specimens with a positive Q fever PCR result were characterized by clusters of multinucleated giant cells without a fibrin ring. None of 20 patients with blood culture-negative IE received anti-Coxiella antibiotic therapy due to lack of clinical suspicion. Six-month mortality was higher in the Q fever PCR-positive group than in the Q fever PCR-negative group [38% (3/8) vs 0% (0/12), P = .049). Of the 20 patients with blood culture-positive IE, none yielded a positive Q fever PCR result for valve tissue. Approximately 40% of patients with culture-negative IE who received heart valve surgery were PCR-positive for Q fever; patients without clinical suspicion suffered high mortality. These data suggest that Q fever IE in patients with culture-negative IE is often missed in routine clinical practice.

Aspergillus endocarditis is a rare infection that occurs most commonly in patients with prior cardiac surgery but cases in post-transplant recipients without prior cardiac surgery have been reported. Diagnosis is often delayed and requires high index of suspicion. We here report a case of Aspergillus endocarditis in solid organ transplant recipient., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: We considered cardiac computed tomography (CT) as a possible alternative modality to echocardiography in the assessment of infective endocarditis (IE). We evaluated the diagnostic capability of preoperative CT., Materials and Methods: We retrospectively evaluated the depiction of vegetations and perivalvular complications in preoperative cardiac CT images of 14 patients diagnosed with IE who required surgical intervention, who were seen at our institution from May 30, 2008 to February 3, 2017. We compared the CT findings with those of TTE and TEE assessments, and intraoperative findings., Results: Cardiac CT correctly identified intraoperatively proven vegetations in 12 out of 13 (92.3%) patients and demonstrated 100% sensitivity and positive predictive value in depicting vegetations in the aortic valve and 100% sensitivity, specificity, positive predictive value, and negative predictive value in depicting pseudoaneurysm as perivalvular complications. CT offered superior identification of pseudoaneurysm to that with echocardiography and detected vegetations as small as 6 mm in maximal length. Vegetation size correlated well between TEE and CT., Conclusion: Favorable comparison of CT and echocardiography in detecting vegetations and perivalvular complications in patients diagnosed with IE suggested potential for expansion in the use of CT for evaluating organic lesions in patients suspected or diagnosed with IE.

The purpose of this study was to describe the distribution of streptococci causing infective endocarditis (IE). A total of 296 patients with definite IE admitted from July 2007 to December 2014 were identified, with microbial identification done by a combination of blood culture, valve culture, and valve polymerase chain reaction (PCR). The overall distribution of streptococci was 76% viridans (n = 224), 17% pyogenic (50), 6% nutritionally variant (17), and 2% anaerobic (5). Sixty-three (21%) viridans group streptococci were not identified further. The distribution of the remaining 161 viridans group streptococci was Streptococcus mitis group 61%, S. bovis group 15%, S. mutans group 13%, S. anginosus group 9%, and S. salivarius group 1%. Of the 50 pyogenic streptococci, 78% were S. agalactiae and 16% were S. dysgalactiae. PCR was significantly more sensitive than culture in identifying streptococci in excised heart valves., (Copyright © 2018. Published by Elsevier Inc.)

Infective endocarditis (IE) remains a severe and potentially fatal disease demanding sophisticated diagnostic strategies for detection of the causative microorganisms. The aim of the present study was to develop a broad-range 16S ribosomal RNA gene polymerase chain reaction in the routine diagnostic of IE for the early diagnosis of fatal disease. A broad-range PCR technique was selected and evaluated in terms of its efficiency in the diagnosis of endocarditis using 19 heart valves from patients undergoing cardiovascular surgeries at the Habib Bourguiba Hospital of Sfax, Tunisia, on the grounds of suspected IE. The results demonstrated the efficiency of this technique particularly in cases involving a limited number of bacteria since it helped to increase detection sensitivity. The technique proved to be efficient, particularly, in the bacteriological diagnosis of IE in contexts involving negative results from conventional culture methods and other contexts involving bacterial species that were not amenable to identification by phenotypic investigations. Indeed, the sequencing of the partial 16S ribosomal RNA gene revealed the presence of Bartonella henselae, Enterobacter sp., and Streptococcus pyogenes in three heart valves with the negative culture. It should be noted that the results obtained from the polymerase chain reaction-sequencing identification applied to the heart valve and the strain isolated from the same tissue were not consistent with the ones found by the conventional microbiological methods in the case of IE caused by Gemella morbillorum. In fact, the results from the molecular identification revealed the presence of Lactobacillus jensenii. Overall, the results have revealed that the proposed method is sensitive, reliable and might open promising opportunities for the early diagnosis of IE., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Purpose: Infective endocarditis is a severe and potentially fatal disease. Nearly a third of all cases remain culture-negative, making a targeted and effective antibiotic therapy of patients challenging. In the past years, fluorescence in situ hybridization (FISH) has proven its value for the diagnosis of infective endocarditis, particularly when it is caused by fastidious bacteria. To increase the number of infective endocarditis causing agents, which can be identified by FISH, we designed and optimized a FISH-probe for the specific detection of Coxiella burnetii in heart valve tissue., Methodology: Even with specific probes the detection and identification of bacteria can be complicated by the high autofluorescence due to calcification of the analysed tissue. To overcome this problem, we developed a protocol to detect C. burnetii by hybridizing, stripping and reprobing the identical section with different species-specific probes repeatedly.Results/Key findings. The newly designed specific FISH probe and the developed protocol exemplarily allowed us to unequivocally identify C. burnetii in tissue sections of a patient with infective endocarditis., Conclusion: This method provides an add-on to existing protocols for the unambiguous diagnosis of bacteria directly within tissues or other difficult tissue samples in cases with small sample size and limited sections.

Introduction: Periodontitis is an infectious disease that affects the support tissue of the teeth and it is associated with different systemic diseases, including cardiovascular disease. Microbiological studies facilitate the detection of microorganisms from subgingival and cardiovascular samples., Objective: To describe the cultivable periodontal microbiota and the presence of microorganisms in heart valves from patients undergoing valve replacement surgery in a clinic in Cali., Materials and Methods: We analyzed 30 subgingival and valvular tissue samples by means of two-phase culture medium, supplemented blood agar and trypticase soy agar with antibiotics. Conventional PCR was performed on samples of valve tissue., Results: The periodontal pathogens isolated from periodontal pockets were: Fusobacterium nucleatum (50%), Prevotella intermedia/ nigrescens (40%), Campylobacter rectus (40%), Eikenella corrodens (36.7%), Gram negative enteric bacilli (36.7%), Porphyromonas gingivalis (33.3%), and Eubacterium spp. (33.3%). The pathogens isolated from the aortic valve were Propionibacterium acnes (12%), Gram negative enteric bacilli (8%), Bacteroides merdae (4%), and Clostridium bifermentans (4%), and from the mitral valve we isolated P. acnes and Clostridium beijerinckii. Conventional PCR did not return positive results for oral pathogens and bacterial DNA was detected only in two samples., Conclusions: Periodontal microbiota of patients undergoing surgery for heart valve replacement consisted of species of Gram-negative bacteria that have been associated with infections in extraoral tissues. However, there is no evidence of the presence of periodontal pathogens in valve tissue, because even though there were valve and subgingival samples positive for Gram-negative enteric bacilli, it is not possible to maintain they corresponded to the same phylogenetic origin.

We present the case of an acute endocarditis of mitral and aortic prosthetic heart valves caused by Aggregatibacter aphrophilus (Haemophilus aphrophilus-paraphrophilus). This third report in the literature emphasizes the diagnostic work-up and the role of positron emission tomography combined with computed tomography in this setting. The specificities of endocarditis due to the HACEK group (Haemophilus spp., Aggregatibacter, Cardiobacterium hominis, Eikenella corrodens and Kingella spp.) and the specific microbiological data and therapeutic options pertinent to this germ are discussed.

Streptococcus mutans , a major pathogen of dental caries, is considered one of the causative agents of infective endocarditis (IE). Recently, bacterial DNA encoding 120-kDa cell surface collagen-binding proteins (CBPs) has frequently been detected from S. mutans -positive IE patients. In addition, some of the CBP-positive S. mutans strains lacked a 190-kDa protein antigen (PA), whose absence strengthened the adhesion to and invasion of endothelial cells. The interaction between pathogenic bacteria and serum or plasma is considered an important virulence factor in developing systemic diseases; thus, we decided to analyze the pathogenesis of IE induced by S. mutans strains with different patterns of CBP and PA expression by focusing on the interaction with serum or plasma. CBP-positive (CBP + )/PA-negative (PA - ) strains showed prominent aggregation in the presence of human serum or plasma, which was significantly greater than that with CBP + /PA-positive (PA + ) and CBP-negative (CBP - )/PA+ strains. Aggregation of CBP + /PA - strains was also observed in the presence of a high concentration of type IV collagen, a major extracellular matrix protein in serum. In addition, aggregation of CBP + /PA - strains was drastically reduced when serum complement was inactivated. Furthermore, an ex vivo adherence model and an in vivo rat model of IE showed that extirpated heart valves infected with CBP + /PA - strains displayed prominent bacterial mass formation, which was not observed following infection with CBP + /PA + and CBP - /PA + strains. These results suggest that CBP + /PA - S. mutans strains utilize serum to contribute to their pathogenicity in IE., (Copyright © 2017 American Society for Microbiology.)

Objectives: Surgeons needing human cardiovascular tissue for implantation in their patients are confronted with cardiovascular tissue banks that use different methods to identify and decontaminate micro-organisms. To elucidate these differences, we compared the quality of processing methods in 20 tissue banks and 1 reference laboratory. We did this to validate the results for accepting or rejecting tissue. We included the decontamination methods used and the influence of antibiotic cocktails and residues with results and controls. The minor details of the processes were not included., Methods: To compare the outcomes of microbiological testing and decontamination methods of heart valve allografts in cardiovascular tissue banks, an international quality round was organized. Twenty cardiovascular tissue banks participated in this quality round. The quality round method was validated first and consisted of sending purposely contaminated human heart valve tissue samples with known micro-organisms to the participants. The participants identified the micro-organisms using their local decontamination methods., Results: Seventeen of the 20 participants correctly identified the micro-organisms; if these samples were heart valves to be released for implantation, 3 of the 20 participants would have decided to accept their result for release. Decontamination was shown not to be effective in 13 tissue banks because of growth of the organisms after decontamination. Articles in the literature revealed that antibiotics are effective at 36°C and not, or less so, at 2-8°C. The decontamination procedure, if it is validated, will ensure that the tissue contains no known micro-organisms., Conclusions: This study demonstrates that the quality round method of sending contaminated tissues and assessing the results of the microbiological cultures is an effective way of validating the processes of tissue banks. Only when harmonization, based on validated methods, has been achieved, will surgeons be able to fully rely on the methods used and have confidence in the consistent sterility of the tissue grafts. Tissue banks should validate their methods so that all stakeholders can trust the outcomes., (© The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Introduction: Candida Endocarditis (CE) is a deadly disease. It is of paramount importance to assess risk factors for acquisition of both Candida native (NVE) and prosthetic (PVE) valve endocarditis and relate clinical features and treatment strategies with the outcome of the disease. Areas covered: We searched the literature using the Pubmed database. Cases of CE from the Italian Study on Endocarditis (SEI) were also included. Overall, 140 cases of CE were analyzed. Patients with a history of abdominal surgery and antibiotic exposure had higher probability of developing NVE than PVE. In the PVE group, time to onset of CE was significantly lower for biological prosthesis compared to mechanical prosthesis. In the whole population, greater age and longer time to diagnosis were associated with increased likelihood of death. Patients with effective anti-biofilm treatment, patients who underwent cardiac surgery and patients who were administered chronic suppressive antifungal treatment showed increased survival. For PVE, moderate active anti-biofilm and highly active anti-biofilm treatment were associated with lower mortality. Expert commentary: Both NVE and PVE could be considered biofilm-related diseases, pathogenetically characterized by Candida intestinal translocation and initial transient candidemia. Cardiac surgery, EAB treatment and chronic suppressive therapy might be crucial in increasing patient survival.