Infective endocarditis (IE) is a bacterial infection of the heart valves, inserted material or surrounding struc-tures and is associated with a high morbidity and mortality. In patients with IE, anemia is considered to result from the underlying infection, prolonged sustained inflammatory response due to the often slow natural course of the disease, and coexisting comorbidities.

Previous studies have found that moderate to severe anemia is associated with a markedly higher risk of mortality in the 6-months recovery phase following treatment for IE. In many cardiac patients and patients with chronic inflammation, randomized trials have shown benefit of treatment of anemia with adjunctive therapy i.e., vitamins (vitamin B12/folic acid), intravenous iron, and erythropoiesis stimulating agents in alleviating anemia, without increased risk of infection. Despite these findings, anemia screening and management are not addressed in current endocarditis guidelines. Thus, in patients with IE and anemia, adjunctive treatment of the anemia might be beneficial for recovery and improve outcomes.

The aim of POET-IRON is to assess the efficacy of adjunctive anemia treatment in patients with IE, using intravenous iron supplementation, erythropoietin-stimulated erythropoiesis, and dietary optimization including vitamins if necessary, and its effect on hemoglobin levels compared to standard care.

The investigators hypothesize that this intervention is safe and will increase hemoglobin concentration, thereby alleviating symptoms of anemia and improving clinical outcomes through enhanced oxygen-carrying capacity, tissue oxygenation, and functional status.

BACKGROUND Infective endocarditis (IE) is a life-threatening condition with an almost 100% fatality rate if untreated and a one-year mortality of approximately 30% in treated patients. There are 600-700 annual cases in Denmark and there is a rising global incidence. Due to aging populations and increased use of invasive devices, IE remains a critical healthcare challenge. Beyond its direct complications such as heart failure, need for acute cardiac surgery, serious septic embolisms, and persistent systemic inflammation, IE is strongly associated with varying degrees of anemia, which is associated with a significant worsening of long-term outcomes. The Partial Oral versus Intravenous Antibiotic Treatment of Endocarditis study (POET) found that 85% of stable patients with IE had anemia, with 29% experiencing moderate to severe anemia, which was associated with a 5-fold higher mortality rate at 6-months follow-up

. Anemia of inflammation, often accompanied by iron deficiency anemia, results from disrupted iron distri-bution rather than depleted iron stores. Traditionally, infection related anemia has been attributed to a part of the innate immune response in relation to infection/inflammation. In this process, hepcidin, a hormone upregulated by inflammatory cytokines like interleukin-6 (IL-6), plays a pivotal role as it causes intracellular iron sequestration, primarily in macrophages, markedly reducing circulating levels of iron in the blood, and inhibits iron absorption from the gut. This protective mechanism is thought to be an evolutionary defense mechanism against bacteremia, as it restricts bacterial access to iron and thereby limits bacterial growth, with animal studies showing a higher risk of uncontrolled infection when intravenous (IV) iron was administered at time of infection. On this basis, iron treatment has been considered contraindicated in bacterial infections. Acute infection also triggers systemic inflammation, which increases metabolic turnover, redistributes trace elements, and accelerates urinary and gastrointestinal losses, which often results in in functional or absolute micronutrient deficits. The oft-cited "two-to threefold increase" in micronutrient requirements originates from critical care guidelines and is well-documented in severe infection, but remains less clearly defined in milder cases. Treatment of these patients support normalization, rather than high-dose supplementation, of vitamin C, vitamin D, zinc, and selenium. These vitamins and minerals support neutrophil and epithelial integrity, enhances antimicrobial peptide production, and maintains oxidative balance. For example, zinc supplementation has been shown to modestly reduce the incidence and shorten the duration of acute respiratory infections in adults, particularly among those who are deficient. Selenium is essential for selenoproteins that regulate redox balance and immune cell activity; supplementation in deficient patients may improve inflammatory control, though high-dose regimens in critical illness have shown inconsistent benefits.

In summary, targeted repletion of specific micronutrients such as vitamin C, vitamin D, zinc, and selenium can help restore immune competence and redox balance in deficient patients, though high-dose strategies in acute illness often yield inconsistent results. By contrast, the role of iron supplementation in the setting of infection related anemia remains more controversial. Yet, IV iron therapy (e.g., ferric derisomaltose) has been shown to reduce readmissions and improve physical capacity and quality of life in other cardiac patients and patients with chronic kidney failure, without increasing the risk of infection. In patients with pure inflammatory anemia i.e. rheumatic disease, IV iron therapy is a well-established treatment. Nevertheless, clinical studies investigating iron supplementation in patients with infective inflammation and anemia have shown mixed results with regard to infection risk.

A systematic review and meta-analysis of 154 randomized clinical trials (RCTs) found that IV iron therapy was associated with a modestly increased risk of bacterial infections (RR 1.16; 95% CI, 1.03-1.29). Notably, none of the RCTs reported data on positive microbiology cultures and only one study reported information on antibiotic treatment of infections. No association was found between IV iron therapy and mortality, or length of hospital stay. Thus, the authors highlighted considerable heterogeneity in the definitions and reporting of infections and concluded that there remains a critical need for well-designed studies using standardized infection endpoints to determine the clinical relevance of this association. In studies of iron supplementation in children with a high prevalence of anemia, an increased incidence of diarrhea and overgrowth of particularly gram-negative bacteria in the gastrointestinal tract was observed. However, no increase in hospital-requiring diarrhea was found and in a systematic review by the Cochrane Institute, which examined RCTs in this area, found no increased risk of death with oral iron supplementation.

Anemia in hemodialysis patients is a well-known issue, and these patients are treated with both erythro-poiesis-stimulating agents (ESA) and iron supplementation. As hemodialysis patients have a high risk of bacterial infections due to the nature of the dialysis procedure itself, the potentially increased risk of infec-tion associated with IV iron supplementation in these patients has been studied in several trials. Observational studies have found a weak association between iron supplementation and bacterial infec-tions. However, in the DRIVE study (Dialysis Patients' Response to IV Iron with Elevated Ferritin) (n=134), a randomized trial of IV iron gluconate supplementation in hemodialysis patients, no difference in infection incidence was found between the iron-treated and placebo groups during the 6-week study period. In an additional 6-week follow-up (DRIVE-II), a higher frequency of serious adverse events (SAEs) was observed in the control group, including 10 cases of infection compared to 4 cases in the IV iron group.

In another large (n=2141), randomized study involving hemodialysis patients (PIVOTAL - Intravenous Iron in Patients Undergoing Maintenance Hemodialysis), where patients were randomized to receive either high- or low-dose IV iron sucrose, no difference in infection rates was observed between the two groups. Although infection was not a primary outcome in any of these studies and thus the studies were not pow-ered to definitively detect differences in infection rates between the intervention and control groups, the findings from these trials are of relevance when evaluating the hypothetically increased risk of infection due to IV iron treatment. In 2015, a large retrospective cohort study analyzed 22,820 American hemodialysis patients who had recently received IV iron therapy and were hospitalized for bacterial infection. The study evaluated whether continued administration of IV iron from admission to discharge was associated with adverse outcomes, including 30-day mortality, all-cause mortality within the year, length of hospital stay, and risk of readmission or death within 30 days post-discharge. Administration of IV iron during hos-pitalization was not associated with higher mortality, longer hospital stays or increased short-term risk of infection-related readmission. These findings challenge existing recommendations to routinely with-hold IV iron in this setting and highlight the need for randomized controlled trials to establish definitive clin-ical guidance.

In patients with heart failure, IV iron therapy is already included in treatment recommendations as an adjunctive treatment. Randomized trials such as AFFIRM-AHF (n=1132) found significant benefits, including reduced mortality rates, improved functional capacity, and enhanced quality of life related to IV iron therapy. The IRONMAN trial (n=1137) found that correction of iron deficiency with ferric deriso-maltose did not increase the risk of infection related hospitalization or death. The findings led to the incorporation of routine screening and treatment of anemia into current European heart failure guidelines.

In general, European guidelines for patients with heart failure specifically endorse an IV iron approach when treating anemia of inflammation, as iron absorption from oral iron preparations is generally poor in patients with ongoing inflammation. This is due to the inflammation induced upregulation of the liver-derived hormone hepcidin, which downregulates the iron-transporting protein ferroportin in cellular membranes. This leads to significantly decreased iron uptake from the gut and resulting in slow and often inefficient iron repletion.

Furthermore, gastrointestinal side effects occur in up to 60% of patients, which may limit the tolerability of oral iron therapy. The European recommendations are supported by the IRONOUT-HF trial (n=225), where oral iron supplementation did not improve functional capacity. In contrast, when comparing with IV iron therapy, IV administration can quickly reach and maintain hemoglobin levels and reduce the need for further anemia management.

ANEMIA IN PATIENTS WITH IE Patients with IE are at high risk of developing anemia, as prolonged infection periods, long-term antibiotic therapy, malnutrition, frequent blood sampling, cardiac surgery, and impaired kidney function all contribute to lower hemoglobin levels. Additionally, infection elevates hepcidin levels and thereby inflammato-ry anemia. The current literature offers limited insight into anemia in patients with IE. In a POET substudy, the investigators found anemia in 85% of patients with medically managed IE after stabilization of infection, of which 29% was moderate to severe anemia. Moderate to severe anemia was independently associated with a 5-fold increase in mortality at 6-months follow-up.

In the study ANIE (Anemia in patients with Infective Endocarditis) (unpublished), a prospective observational study of anemia in patients with IE, the investigators sought to understand the natural course of anemia in patients with IE (n=100). The aim of the study was to gain insight and to support the design of a novel approach to treatment of inflammatory anemia in this population with minimal risk to patients, while still improving outcome of the disease.

Preliminary data from the ANIE study reaffirm that at least 90% of patients with IE are affected with some degree of anemia during the course of disease. The severity of anemia appears to worsen progressively with each week of antibiotic therapy, typically peaking during the final stages of the antibiotic treatment. Notably, the data also suggest that anemia often begins to develop gradually up to 30 days before the time of diagnosis. Following the termination of antibiotic treatment, hemoglobin levels are only gradually recovering in the following months, with normalization at 3 months post-treatment.

The evidence of the use of IV iron therapy in patients with bacterial infections remains limited, and there is a critical need to evaluate the safety and efficacy of anemia treatment in patients IE. A safety-oriented approach using IV iron therapy in combination with erythropoietin (EPO) and other means to increase the hemoglobin levels may improve both short- and long-term outcomes, including functional capacity.

To minimize the risk of exacerbating infection through iron supplementation, the timing of intervention is critical. The limited literature linking adjunctive anemia therapy, such as intravenous iron, with infection risk generally reports that most infections occur within the first 30 days after iron administration. In the POET trial, specific stabilization criteria were established to define when a patient's infection was considered sufficiently controlled to allow a safe switch from IV to oral antibiotic therapy. These criteria include 1) Absence of clinical signs of uncontrolled infection, 2) At least 10 days of IV antibiotic treatment (or 7 days postoperatively in cases involving valve surgery), 3) afebrile for at least 48 hours, c-reactive protein level <25 mg/L, or a clear downward trend, 4) Hemodynamic stability, 5) No signs of uncontrolled infection including persistent blood cultures and/or progression of vegetations or abscesses on echocardiography. At this time, the risk of relapse of infection within 6 months was under 3% in both the IV and oral treatment groups.

Also, the iron therapy will be initiated during ongoing antibiotic treatment, further reducing the risk. Additionally, clinically applied careful monitoring during and after the active treatment phase including infection parameters and blood cultures.

In the POET trial as well as the ANIE study, the investigators found that by the time of clinical stabilization more than one-third of patients with IE had moderate to severe anemia (unpublished for the ANIE trial), mainly characterized as anemia of inflammation. By choosing this timepoint to initiate the adjunctive treatment of anemia, the investigators believe that the treatment will be both effective, with minimal risk of exacerbating the existing infection.

If successful, the POET-IRON study has the potential to redefine IE management and support the integration of anemia treatment into future standard care guidelines, as well as change international guidelines on the treatment of patients with anemia caused by infections in general.

STUDY AIM The aim of this study is to explore adjunctive treatment of anemia in patients with IE after stabilization of the IE disease course according to predefined stabilization criteria. The investigators hypothesize, that

Adjunctive treatment of anemia in patients with IE through nutritional counselling, supplementing vitamins (i.e. vitamin B12 and folic acid), as well as IV iron, and erythropoietin infusion as a supportive supplement to the existing treatment regimen for patients with infective endocarditis and concurrent anemia, defined as hemoglobin ≤ 6.0 mmol/L, will safely and significantly faster increase functional capacity measured by 6 minute-walk-test (6MWT). This is expected also to improve the general clinical outcomes.

STUDY DESIGN This study is a prospective, open-label, randomized clinical trial with 3 months clinical follow-up and 10 years follow-up in medical records and National Health Registries. The total study duration is expected to be 10 years (2036), which will include long-term assessment. Randomization (1:1) will be con-ducted using a web-based system 1:1 to either anemia targeting management as adjunctive therapy to the existing treatment regimen (interventional group) or standard care only (control group). In this study, adjunctive treatment of anemia considers the multifactorial etiology of anemia in patients with IE and will start right after the randomization. The approach to anemia management is therefore multifaceted, including interventions such as dietary counseling, supplementation with vitamin B12 and/or folate, IV iron therapy, and EPO infusion as supportive treatment alongside the existing therapeutic regimen.

SUBGROUP ANALYSES

Subgroup analyses will be performed for each treatment category to assess whether there are differences between these groups in the effect of treatment strategy on the primary endpoint and for the following:

Age (analyses of patients ≤65.5 yr and >65.5) Sex (female vs. male) Diabetes Chronic kidney disease (CKD - normal-stage G3 vs stage G4 and below) Bacteria Streptococci Enterococcus faecalis Staphylococcus aureus Coagulase-negative staphylococci Culture-negative Surgical treatment Type of valve (prostethic heart valve vs. native heart valve) Involved valve (aortic valve vs. mitral valve) Double sided IE Right-sided IE Device-related IE

FOLLOW-UP REGIMEN Course of examinations and follow-up after intervention The follow-up strategy is visualized in table 1 with detailed explanation of all blood samples in the supple-mentary table 1. All participants in the study will also follow standard follow-up after IE. Additionally, specific to this study the participant will answer two questionnaires (Barthel-199 and EQ-5D-5L) as-sessing QOL, do a 6MWT and draw blood samples for metabolomics/proteomics. If a patient is unable to complete the follow-up assessment, the test will be omitted. The participants will also need to have additional blood samples at six weeks, and 3 months from time of randomization compared to standard follow-up regimen. The last physical follow-up visit per study protocol happens 3 months from the randomization date / date of intervention. Long-term follow-up includes 10 years through medi-cal records and national health registries.

ASSESMENT OF PHYSICAL CAPACITY The 6MWT will be conducted using a standardized, widely used, and validated protocol to quantify physiological/functional exercise capacity. The test will be performed on a flat, straight, indoor corridor (typically a 30-m course). Participants will be instructed to walk back and forth for 6 minutes at a self-selected pace, aiming to cover as much distance as possible, with the option to slow down, stop, and rest if needed, and to resume walking when able. Standardized encouragement will be provided at predefined time points. The total distance walked (meters) will be recorded as the primary outcome. Vital signs and perceived exertion (e.g., Borg scale) will assessed before and immediately after the test. The test will be discontinued if prede-fined safety criteria occur (e.g., chest pain, severe dyspnea, dizziness, syncope, or other clinically significant symptoms). The purpose of 6MWT is to capture an objective and clinically meaningful measure of physio-logical capacity and daily functional performance. In this study, it is used to assess functional status and to detect changes over time, including response to interventions.

STATISTICS In this study, power calculations were based on detecting a clinically meaningful difference in 6MWT distance. The expected difference was set at 28 meters, which is within the upper range of clinically important differences reported in chronic cardiac and post-infectious populations. A standard deviation of 40 meters was assumed, based on prior studies in heart failure, sepsis recovery, and older adults with anemia-related physical decline. Using a two-sided alpha of 0.05 and 80% power, the required sample size to detect a 28-meter difference in 6MWT was calculated to be 68 patients in total (34 per group). Taking an expected drop-out rate of 10% into account, the final sample size is 74 participants.

SAFETY In some studies, it has been reported, that elevated levels of free serum iron can be associated with a higher risk of exacerbation of bacterial infections. For patients randomized to adjunctive therapy, the intervention with IV iron therapy may pose a theoretical risk of prolonged treatment duration and reinfection. In rare cases, a reinfection can compose a risk of cardiac surgery, as the heart valve(s) is damaged by the infection. This condition can be life-threatening.

To reduce this risk most, clinical stability and eradication of intravascular bacterial flow is ensured by use of criteria, that are strongly inspired by the well-established POET criteria prior to study randomization. No patients will be randomized to treatment prior to fulfillment of these criteria. The POET criteria comprise a combination of clinical and paraclinical assessment parameters that ensure stability as well as clinical and paraclinical infection control (the criteria are elaborated in "Eligibility"). The POET criteria are also utilized in European guidelines when shifting from IV antibiotic therapy to oral therapy, precisely because it is essential in this process to ensure that patients are not put at risk of an inferior treatment.

The TREAT trial (n = 4,038) demonstrated that targeting higher hemoglobin levels with erythropoiesis-stimulating agents (ESA) was associated with an increased risk of stroke in patients with diabetes, chronic kidney disease, and moderate anemia. For patients in the intervention group, treatment with IV EPO will require close monitoring of hemoglobin levels. The risk of stroke is primarily associated with hemoglobin levels above 8.1 mmol/L, therefore, EPO will not be administered if hemoglobin exceeds this threshold. In this study, EPO dosing and administration will be conducted exclusively in close collaboration and consultation with specialists in hematology, who daily, use EPO as part of various treatment strategies, including in the management of advanced hematologic disorders.

By closely monitoring patients with extra clinical consultations, blood samples, and echocardiography after initiation of the intervention in hospital and after discharge, the investigators will be able to detect recurrences early, allowing for the prompt initiation of appropriate treatment.

SAFETY REPORTING

• Persistent bacteremia or relapse of the positive blood culture with the IE causing pathogen identi-fied during the present disease-course within 6 months.
• Infection related admission (hospitalization, minimum of one overnight stay) within 6 months
• All-cause admissions (hospitalization, minimum of one overnight stay) within 6 months
• Anaphylaxis or severe hypersensitivity reaction to adjunctive therapies i.e., locally to site of injec-tion within 6 months
• Iron overload (transferrin saturation (TSAT) >70% with ferritin rise, symptomatic manifestation) within 6 months.
• Hypokalemia or fluid shifts requiring hospitalization (in rare cases seen with rapid vitamin B12 cor-rection) within 30 days post randomization.
• Stroke (with symptomatic manifestation and/or verified by CT-cerebrum) within 6 months.
• All-cause mortality within 6 months
• Infection-related death within 6 months

A Data Safety Monitoring Board (DSMB) will be convened prior to study initiation.

ETHICAL CONSIDERATIONS The study will be conducted in compliance with the Helsinki II Declaration as adopted by the 18th World Medical Assembly in Helsinki, Finland, in 1964 and subsequent versions.

Each year, patients with IE account for nearly 20,000 hospital admission days in Denmark. The pro-longed course of admission increases the risk of functional decline for patients while placing a significant financial burden on the healthcare system. Functional decline due to prolonged hospitalization is well-documented across all age groups. Some studies also report that older patients are at high risk of poor functional outcomes, as they are less likely to recover function and more likely to develop new func-tional deficits during hospitalizations.

Currently, anemia in this high-risk population is an overlooked condition that is neither treated systemati-cally nor managed proactively. In patients with IE, anemia is independently linked to increased mortality. Other studies have already described how anemia is strongly linked to prolonged hospital length of stay, higher healthcare costs, and longer ICU duration. In other patient groups, adjunctive anemia treatment has been associated with clinical benefits, suggesting that in POET-IRON patients randomized to receive such treatment may similarly achieve these advantages.

Physiological, psychological, and societal benefits Treatment of anemia might reduce the symptoms of anemia including shortness of breath, fatigue, dizziness and worsening of symptoms from preexisting heart conditions. These effects are expected to lead to earlier mobilization, improved energy levels, fewer side-effects to prolonged infection, antibiotic treatment, and fewer cardiovascular complications i.e. faster recovery/rehabilitation. These benefits can in theory re-duce the length of hospital stay, lowering the risk of readmission, just as reducing societal and psychological burden of long hospital stays linked to IE.

There is no guarantee that the individual participant will achieve any benefits from participating in the trial. However, the clinical trial is performed with the expectation that the results will provide information that would help to improve not only anemia in patients with IE, but also other patient groups that share a similar underlying mechanism of anemia, such as those with prolonged bacterial or systemic infections. For these patients, POET-IRON has a significant potential to shed light on a serious and currently overlooked health challenge.

Finally, the results from POET-IRON may be definite in paving the way for the treatment of infection related anemia, for example by informing changes to clinical practice guidelines.

For patients randomized to standard care, participation in the study will not result in any substantial deviations from current clinical practice.

Inclusion Criteria:

• 18 years or older

• Confirmed bacterial endocarditis.

• Hb ≤6.0 mmol/l

• Stabilization criteria:

  • Stable condition (patients with satisfactory clinical response to initial treatment)
  • At least 10 days with IV administered antibiotic treatment and at least 7 days after surgery in cases of valve surgery.
  • Transesophageal echocardiography (TEE) performed prior to randomization with no signs of abscess formation or valve abnormalities, which would require surgery.
  • No fever for at least 48 hours prior to enrollment.No positive blood cultures within last 4 days prior to randomization.
  • CRP < 25 mg/L OR > 25% reduction from peak value.
  • Leucocytes <15 mia./L OR > 25% reduction from peak value.

Exclusion Criteria:

• Known or suspected immunocompromise (e.g., HIV infection, ongoing chemotherapy, systemic corti-costeroid treatment >20 mg prednisolone equivalent/day)
• Inability to provide informed consent for participation
• Relapsing infective endocarditis (endocarditis caused by the same microorganism within 6 months)
• Allergy / intolerance to EPO or iron therapy
• Inability to complete a 6MWT
• Hematological conditions, that contradicts use of IV iron therapy
• Death prior to clinical stabilization
• Failure to fulfill criteria of clinical stabilization

IPD sharing is currently undecided. Data may be shared upon reasonable request, subject to ethical approval and data protection regulations