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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.)

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.

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.

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.

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.

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.

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.

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.)

Aims: In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE., Material and Methods: Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM)., Results: Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified., Conclusion: The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may result in improved outcomes.

Background  A considerable proportion of patients with acute infective endocarditis require surgical treatment. The aim of this study was to collate our short- and medium-term results of surgical treatment of infective endocarditis and to compare the results in native and prosthetic endocarditis. Method  Total 254 operations for infective endocarditis from January 2008 to May 2015 were included in this retrospective study. There were 182 operations for native valve endocarditis and 72 for prosthetic valve endocarditis. Patient characteristics, operative details, complications, and mortality were registered. Results  The endocarditis was left sided in 247 operations (146 aortic, 78 mitral, and 23 double-valve) and right-sided in 7 (5 tricuspid and 2 pulmonary). Twenty-two patients (8.7%) died within 30 days (7.7% with native valve endocarditis and 11.1% with prosthetic valve endocarditis, p  = 0.31). Severe perioperative complications occurred in 99 of 254 operations (39%). Overall cumulative survival at 1 and 5 years was 86% and 75%, respectively, and it was not significantly different for native and prosthetic endocarditis ( p  = 0.31). Eighteen patients (8%) had one ( n  = 16) or two ( n  = 2) recurrent episodes of endocarditis requiring surgery. Conclusion  Surgery for infective endocarditis is still associated with a high early mortality rate and a considerable complication rate. Long-term outcome is acceptable. In our study population, morbidity and mortality were not significantly different in native and prosthetic endocarditis., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Bacterial endocarditis is a recognised disease in humans and animals. In humans, infection with Coxiella burnetii can cause endocarditis, but this has not been investigated thoroughly in animals. Endocarditis in cattle is a common post-mortem finding in abattoirs and studies have identified Trueperella pyogenes as a major cause. Despite exposure of cattle to C. burnetii, the significance of this particular bacterium for development and progression of endocarditis has not been studied in detail. Cardiac valves of cattle affected with endocarditis (n = 100) were examined by histology, fluorescence in situ hybridization (FISH) and real time quantitative polymerase chain reaction (PCR). Serum was examined for anti-C. burnetii antibodies by enzyme-linked immunosorbent assay (ELISA)., Results: Serology revealed that 70% of the cattle were positive for antibodies to C. burnetii, while PCR analysis identified 25% of endocarditis valve samples as being positive. C. burnetii was not detected by FISH, probably due to the low infection levels. Most cattle had chronic valvular vegetative endocarditis with lesions being characterised by a core of fibrous tissue covered by significant amounts of fibrin, sometimes with areas of liquefaction, and with a coagulum covering the surface. In a few cases, including the case with the highest infection level, lesions were characterized by extensive fibrosis and calcification. Histologically, bacteria other than C. burnetii were observed in most cases., Conclusions: The presence of C. burnetii DNA is relatively common in cattle affected with valvular endocarditis. The role of C. burnetii remains however unknown as lesions did not differ between C. burnetii infected and non-infected cattle and because T. pyogenes-like bacteria were present in the inflamed valves; a bacterium able to induce the observed lesions. Heart valves of normal cattle should be investigated to assess if C. burnetii may be present without preexisting lesions.

Introduction: The aim of this study was to evaluate the outcomes of operations for endocarditis in Iceland, but such results have not been reported before., Materials and Methods: Retrospective nation-wide study of pa--tients that underwent open-heart surgery for infective endocarditis at Landspitali University Hospital in 1997-2013. Variables were collected from hospital charts. Long-term survival was analysed using Kaplan- Meier methods. Mean follow-up time was 7.2 years., Results: Out of 179 patients diagnosed with endocarditis, 38 (21%) -underwent open heart surgery. Two patients were excluded due to missing information leaving 36 patients for analysis. The number of operations steadily increased, or from 8 to 21 during the first and last 5-years of the study period (OR: 1.12, 95% CI: 1.05-1.21, p=0.002). The most common pathogen was S. aureus and 81% (29/36) of the patients had positive blood cultures. Three patients had history of previous cardiac surgery and five had history of intravenous drug abuse. The aortic valve was most often infected (72%), followed by the mitral valve (28%). The infected valve was replaced in 35 cases 14 with a mechanical prosthesis and 20 with a bioprosthesis. In addition two mitral valves were repaired. Postoperative complications included perioperative myocardial infarction (35%), respiratory failure (44%) and reoperation for bleeding (25%). Thirty-day mortality was 11% (4 patients) with 5- and 10-year survival of 59% and 49%, respectively., Conclusion: One out of five patients with endocarditis underwent surgery, most commonly aortic or mitral valve replacement. Outcomes were comparable to other studies. In comparison to elective valve replace-ment surgery the rate of post-operative complications and 30-day mortality were higher and long-term survival was less favorable. Key words: Endocarditis, surgical treatment, valve replacement, complications, outcome. Correspondence: Arnar Geirsson, arnargeirsson@yahoo.com.

Cardiovascular allografts are usually disinfected using antibiotics, but protocols vary significantly between tissue banks. It is likely that different disinfection protocols will not have the same level of efficacy; they may also have varying effects on the structural integrity of the tissue, which could lead to significant differences in terms of clinical outcome in recipients. Ideally, a disinfection protocol should achieve the greatest bioburden reduction with the lowest possible impact on tissue integrity. We conducted a systematic review of methods applied to disinfect cardiovascular tissues. The use of multiple broad spectrum antibiotics in conjunction with an antifungal agent resulted in the greatest reduction in bioburden. Antibiotic incubation periods were limited to less than 24 h, and most protocols incubated tissues at 4 °C, however one study demonstrated a greater reduction of microbial load at 37 °C. None of the reviewed studies looked at the impact of these disinfection protocols on the risk of infection or any other clinical outcome in recipients., Competing Interests: Production of this report has been made possible through a financial contribution from Health Canada, and the Provincial and Territorial governments and has been facilitated by Canadian Blood Services. The views expressed herein do not necessarily represent the views of the Federal, Provincial, Territorial governments or Canadian Blood Services. Funds were provided by Canadian Blood Services to authors from McMaster University for their assistance in performing and writing the systematic review. No conflicts-of-interest were identified on disclosure forms completed by members of the Committee.

Identification of the causative pathogen of infective endocarditis (IE) is crucial for adequate management and therapy. A broad-range PCR-electrospray ionization mass spectrometry (PCR-ESI-MS) technique was compared with broad-spectrum 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) for the detection of bacterial pathogens in 40 heart valves obtained from 34 definite infective endocarditis patients according to the modified Duke criteria and six nonendocarditis patients. Concordance between the two molecular techniques was 98% for being positive or negative, 97% for concordant identification up to the genus level, and 77% for concordant identification up to the species level. Sensitivity for detecting the causative pathogen (up to the genus level) in excised heart valves was 88% for 16S rRNA PCR and 85% for PCR-ESI-MS; the specificity was 83% for both methods. The two molecular techniques were significantly more sensitive than valve culture (18%) and accurately identified bacteria in excised heart valves. In eight patients with culture-negative IE, the following results were obtained: concordant detection of Coxiella burnetii (n = 2), Streptococcus gallolyticus (n = 1), Propionibacterium acnes (n = 1), and viridans group streptococci (n = 1) by both molecular tests, detection of P. acnes by PCR-ESI-MS whereas the 16S rRNA PCR was negative (n = 1), and a false-negative result by both molecular techniques (n = 2). In one case of IE caused by viridans streptococci, PCR-ESI-MS was positive for Enterococcus spp. The advantages of PCR-ESI-MS compared to 16S rRNA PCR are its automated workflow and shorter turnaround times., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background and Aim of the Study: Infective endocarditis (IE) is a major cause of morbidity and mortality in patients with end-stage renal disease (ESRD). The study aim was to evaluate the demographic, clinical, microbiological and inpatient mortality data of IE in hemodialysis patients., Methods: Data were analyzed for all IE patients admitted to the authors' 800-bed tertiary care hospital between January 2001 and December 2014. These data included demographics, comorbidities, clinical and microbiological characteristics, echocardiographic findings, complications, outcomes, and in-patient mortality., Results: A total of 296 patients, including 52 on hemodialysis, was admitted with a diagnosis of IE. The median age of patients with ESRD (28 females, 24 males) was 55.9 ± 15.47 years. The prevalences of comorbidities such as hypertension (80%) and diabetes mellitus (46%) were significantly higher in ESRD patients, whereas other comorbidities were similarly distributed in both groups. The mitral valve was the most commonly involved (55.8%), followed by aortic (21.7%), tricuspid (21.2%) and pulmonary (1.9%) valves. Staphylococcus aureus was the most common organism (40%), followed by Enterococcus sp. (13.7%), Gram-negative staphylococci (13.7%), coagulase-negative staphylococci (11.5%), and Streptococcus sp. (5.7%). Polymicrobes were found in 11.5% of patients and cultures were negative in 19%. The mean ejection fraction in these patients was 42 ± 4.19% and the mean area of vegetation was 63.5 ± 40 mm2. The in-hospital course of 11 patients was complicated by embolic events, while three patients had acute heart failure and one patient had heart block secondary to IE. A total of four patients (7.7%) died during the index hospitalization., Conclusions: IE in patients receiving chronic hemodialysis is a very frequent occurrence. Its diagnosis is complex and its presence should be considered in all hemodialysis patients with bacteremia. In the present study the etiology was shown to be multifactorial, with the mitral valve being the most commonly involved and S. aureus the most common organism.

Background and Aim of the Study: Q fever, caused by the rickettsia Coxiella burnetii, is a worldwide zoonotic disease with both acute and chronic manifestations. Endocarditis is the principal chronic manifestation. Q fever can easily be mistaken for degenerative valve disease due to its indolent presentation, the fastidious nature of the organism (routine cultures are negative), and the absence of a typical echocardiographic and macroscopic appearance for endocarditis. Prosthetic valve failure, with associated morbidity and mortality, have been described following unrecognized infections., Methods: Previous studies have documented the value of screening strategies in areas of high prevalence. Hence, a pilot study was conducted in a low-prevalence setting, in which 139 patients at two tertiary cardiac centers attending for elective valve replacement for degenerative valvular disease underwent testing for chronic Q fever infection by serological and molecular methods on blood and valve tissue., Results: Five patients (3.7%) had serological evidence of past exposure to Q fever (consistent with rates in the literature). None had evidence of chronic Q fever endocarditis. The cost of adopting a universal screening strategy is around £40,000 per case (if serology is used to screen patients prior to surgery)., Conclusions: Alternative and more cost-effective methods for identifying clinically quiet cases of chronic Q fever endocarditis are required.

Background: Staphylococcus lugdunensis is an emerging cause of endocarditis. To cause endovascular infections, S. lugdunensis requires mechanisms to overcome shear stress. We investigated whether platelets and von Willebrand factor (VWF) mediate bacterial adhesion to the vessel wall and the cardiac valves under flow., Methods: S. lugdunensis binding to VWF, collagen, and endothelial cells was studied in a parallel flow chamber in the absence and presence of platelets. In vivo adhesion of S. lugdunensis was evaluated in a mouse microvasculature perfusion model and a new mouse model of endocarditis., Results: Contrary to other coagulase-negative staphylococci, S. lugdunensis bound to VWF under flow, thus enabling its adhesion to endothelial cells and to the subendothelial matrix. In inflamed vessels of the mesenteric circulation, VWF recruited S. lugdunensis to the vessel wall. In a novel endocarditis mouse model, local inflammation and the resulting release of VWF enabled S. lugdunensis to bind and colonize the heart valves., Conclusions: S. lugdunensis binds directly to VWF, which proved to be vital for withstanding shear forces and for its adhesion to the vessel wall and cardiac valves. This mechanism explains why S. lugdunensis causes more-aggressive infections, including endocarditis, compared with other coagulase-negative staphylococci., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background and Aim of the Study: Infective endocarditis (IE) is diagnosed by blood and/or resected valve cultivation and echocardiographic findings, as defined by the Duke criteria. Unfortunately, cultures may be negative due to prior antibiotic therapy or fastidious or slow-growing microorganisms. The study aim was to investigate the value of the broad-range polymerase chain reaction (PCR) in addition to blood and valve culture for the detection of causative microorganisms., Methods: Between February 2012 and March 2015, valve samples from 36 patients undergoing cardiac surgery were analyzed; of these patients, 26 had a preoperative diagnosis of IE and 10 served as controls. Multiple blood cultures were obtained from 34 patients before antibiotic therapy was commenced. Valve samples were inoculated on bacteriological media and underwent analysis using broad-range PCR (16S rDNA)., Results: IE was confirmed microbiologically in 21 of the 26 patients (80.7%); in 20 cases (76.9%) this was by positive blood cultures and in 16 (61.5%) by positive valves. Valves were positive in 15 blood culturepositive patients, and in one blood-culture negative patient. Broad-range PCR detected a microorganism in valves significantly more frequently (n = 14; 53.8%) compared to valve culture (n = 8; 30.7%) (chisquare 11.5, p <0.001). The predominant microorganisms were Staphylococcus aureus, Streptococcus of the viridans group, coagulasenegative staphylococci and Enterococcus faecalis. Blood, valve cultures and broad-range PCR were negative in five patients (19.3%) with IE, and in all 10 subjects of the control group., Conclusions: Broad-range PCR on valves was more sensitive than valve culture. However, blood culture, if taken before the start of antibiotic therapy, was the best method for detecting IE.

Despite recent advances in diagnostics and treatments, infective endocarditis is still associated with substantial morbidity and mortality. Even prolonged courses of broad-spectrum antimicrobials often fail to eradicate the infection, making surgical intervention necessary in many cases. In this review, we present recent advances in molecular microbiology techniques that have uncovered a plausible explanation for this resistance to treatment: the recently discovered social behavior of some microbes, in which colonies form a nearly impenetrable barrier around themselves called a biofilm. These biofilm structures isolate the colony from the body׳s immune response and antimicrobial drugs. We also present current thinking about possible ways biofilms can be destroyed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Structural deficiencies and functional abnormalities of heart valves represent an important cause of cardiovascular morbidity and mortality, and a number of diseases, such as aortic stenosis, have been recently associated with infectious agents. This study aimed to analyze oral bacteria in dental plaque, saliva, and cardiac valves of patients with cardiovascular disease. Samples of supragingival plaque, subgingival plaque, saliva, and cardiac valve tissue were collected from 42 patients with heart valve disease. Molecular analysis of Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis, and Treponema denticola was performed through real-time PCR. The micro-organism most frequently detected in heart valve samples was the S. mutans (89.3%), followed by P. intermedia (19.1%), P. gingivalis (4.2%), and T. denticola (2.1%). The mean decayed, missing, filled teeth (DMFT) was 26.4 ± 6.9 (mean ± SD), and according to the highest score of periodontal disease observed for each patient, periodontal pockets > 4 mm and dental calculus were detected in 43.4% and 34.7% of patients, respectively. In conclusion, oral bacteria, especially S. mutans, were found in the cardiac valve samples of patients with a high rate of caries and gingivitis/periodontitis., Competing Interests: The authors declare no potential conflicts of interest with respect to authorship and/or publication of this article.

Iranian Tissue Bank established in 1994 provides soft tissues for implantation in Iran. This study was designed to evaluate the efficacy of decontamination process of cardiac and soft tissues in Iranian Tissue Bank. In this bank after initial assessments, the tissues were incubated in a 5-antibiotic cocktail at room temperature for 24 h and then at 4 °C for 14 days. Contamination status was compared before and after antibiotic cocktail incubation. Of 3,315 assessed tissues, 1,057 were pericardia, 1,051 were fascia and 1,207 were other soft tissues including tibialis and aorta. The initial contamination rate was 36.86%. Pericardia showed the highest contamination rate. Klebsiella species was the most prevalent organism causing contamination. Decontamination rate after antibiotic incubation was 86.91% with the highest successful decontamination rate for fascia tissue. Klebsiella species was the major source of contamination in tissues that remained contaminated after antibiotic incubation. This may be due to resistance of this organism to applied antibiotics in the decontamination cocktail possibly due to a negative drug interaction between aminoglycoside and penicillin derivatives in this antibiotic cocktail. In conclusion collected data shows comparable efficacy of the decontamination process that is used in ITB compared with homograft banks of other countries.

Infective endocarditis (IE) has been increasingly diagnosed in patients without previously detected predisposing heart disease, but its clinical features have yet to be fully determined. A recent single-center study including echocardiographic images and surgical findings investigated the incidence of undiagnosed, clinically silent valvular or congenital heart diseases and healthcare-associated infective endocarditis (HAIE). The study confirmed that a large proportion of patients with IE have no previous history of heart disease. Analysis of underlying disease in these patients showed that undetected mitral valve prolapse was the most common disease, followed by an apparently structurally normal valve. The patients who developed IE of apparently structurally normal valves had different clinical characteristics and worse outcomes. IE involving a structurally normal valve was associated with both nosocomial and non-nosocomial HAIE, whereas community-acquired IE was more frequent than HAIE. The pathophysiologic mechanism involving the development of non-HAIE or community-acquired IE due to predominantly staphylococcal infection in an apparently structurally normal valve is not yet clearly understood. Structurally normal valves are not necessarily free of regurgitation or abnormal turbulence and, given the dynamic nature and fluctuating hemodynamic effects of conditions such as poorly controlled hypertension, end-stage renal disease, and sleep apnea, further investigation is necessary to evaluate the potential role of these diseases in the development of IE. An apparently normal-looking valve is associated with IE development in patients without previously recognized predisposing heart disease, warranting repartition of at-risk groups to achieve better clinical outcomes.

Eustachian valve endocarditis caused by Actinomyces species is extremely rare. A literature review revealed only one reported case-caused by Actinomyces israelii in an intravenous drug abuser. Our patient, a 30-year-old woman who at first appeared to be in good health, presented with fever, a large mobile mass on the eustachian valve, and extensive intra-abdominal and pelvic masses that looked malignant. Histopathologic examination of tissue found in association with an intrauterine contraceptive device revealed filamentous, branching microorganisms consistent with Actinomyces turicensis. This patient was treated successfully with antibiotic agents. In addition to presenting a new case of a rare condition, we discuss cardiac actinomycotic infections in general and eustachian valve endocarditis in particular: its predisposing factors, clinical course, sequelae, and our approaches to its management.

Background: Testing excised valves in surgically treated infective endocarditis (IE) patients provides an opportunity to identify the microbial etiology of IE. Microbial sequencing (universal bacterial, mycobacterial, or fungal polymerase chain reaction followed by DNA sequencing) of valves can identify microorganisms accurately, but the value it adds beyond information provided by blood and valve cultures has not been adequately explored., Methods: Three hundred fifty-six patients who underwent surgery for active IE from January 1, 2010, to January 1, 2013, were identified from our cardiovascular information registry and outpatient parenteral antibiotic therapy registry. Their records were reviewed to identify 174 patients whose valves were sent for sequencing. The microbial etiology of IE was defined using comprehensive clinical, pathologic, and microbiological criteria. Blood culture, valve culture, and valve sequencing were examined to determine how frequently they identified the definitive cause of IE., Results: Of the 174 patients, 162 (93%) had acute inflammation on histopathologic examination of their valves. Valve sequencing was significantly more sensitive than valve culture in identifying the causative pathogen (90% versus 31%, p < 0.001), and yielded fewer false positive results (3% versus 33%, p <0.001). The pathogen would not have been identified in 25 patients (15%) had it not been for valve sequencing. All the value provided by sequencing was attributable to bacterial DNA sequencing; mycobacterial and fungal sequencing provided no additional information beyond that provided by blood culture, histopathology, and valve culture., Conclusions: Valve sequencing, not valve culture, should be considered the primary test for identifying bacteria in excised cardiac valves., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: In infective endocarditis (IE), blood cultures are negative in 2.5-31% of cases because of a previously prescribed antimicrobial treatment. Molecular methods may represent an alternative to conventional microbiological techniques to identify the causative agent. The aim of this prospective study was to evaluate the performance of a new primer pair (341F/785R) for 16S rDNA amplification in heart valves compared with primers 91E/13BS already used for the diagnosis of IE. The primer pair 341F/785R was previously selected in silico to allow 16S rDNA amplification for a large coverage of bacterial species., Results: A total of 74 patients suspected of having IE were included in this study. IE was diagnosed in 55 of these patients using the modified Duke criteria, which was the gold standard here. 91E/13BS primers were more sensitive than 341F/785R primers: 38/55 (69.1%) samples were positive using 91E/13BS primers against 28/55 (50.9%) with 341F/785R (p = 0.013). When at least one of the two molecular methods was positive, the sensitivity and specificity of 16S rDNA amplification was 72.7% and 94.7%, respectively., Conclusion: Even if the new primer pair 341F/785R seemed promising in silico, it was less sensitive for 16S rDNA amplification in heart valves than the 91E/13BS pair already used. This study underlines a lack of standardization for 16S rDNA amplification for clinical samples.