Acute febrile illnesses (AFIs) are among the most common reasons for emergency department visits and hospital admissions worldwide [1]. They encompass a broad spectrum of etiologies, ranging from self-limiting viral infections to life-threatening conditions like sepsis and severe systemic inflammatory syndromes. The clinical course of AFIs is highly variable, making early prognostication and risk stratification a cornerstone of effective management [2]. Failure to promptly identify patients at risk for clinical deterioration can lead to delayed intervention, increased morbidity, and mortality. Consequently, there is a persistent need for accessible, reliable, and cost-effective biomarkers that can predict disease severity at the time of initial presentation.

The host immune and inflammatory response is central to the pathogenesis and outcome of AFIs. An exaggerated or dysregulated inflammatory cascade, often termed a "cytokine storm," is a hallmark of severe disease [3]. Several biomarkers reflecting this inflammatory state, such as C-reactive protein (CRP) and procalcitonin, are widely used in clinical practice. However, their specificity and predictive capacity can be limited [4]. This has prompted research into other mediators that may offer a more nuanced reflection of the underlying pathophysiology.

Serum ferritin, traditionally known as an intracellular iron-storage protein, has emerged as a potent pro-inflammatory mediator and acute-phase reactant. During infection and inflammation, its synthesis is upregulated by cytokines like interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) [5]. Extreme hyperferritinemia is a defining feature of severe inflammatory states, including macrophage activation syndrome, adult-onset Still's disease, and more recently, severe COVID-19 and sepsis [6]. Elevated ferritin levels are thought to reflect the intensity of the systemic inflammatory response and have been associated with organ dysfunction and poor outcomes in various critical illnesses [7].

Concurrently, vitamin D has transitioned from its classical role in calcium homeostasis to being recognized as a critical modulator of both innate and adaptive immunity. The active form of vitamin D, 1,25-dihydroxyvitamin D, regulates the production of antimicrobial peptides like cathelicidin, enhances phagocytic function, and dampens excessive pro-inflammatory cytokine production [8]. A large body of evidence suggests that vitamin D deficiency is highly prevalent globally and is associated with increased susceptibility to infections and a more severe disease course in conditions like acute respiratory infections and sepsis [9, 10].

While ferritin and vitamin D have been investigated independently as prognostic markers in specific infectious diseases, there is a notable research gap regarding their combined predictive value in the broad, undifferentiated population of patients presenting with AFIs. Evaluating these two biomarkers together is compelling, as they represent two sides of the host response: ferritin as a marker of hyperinflammation and vitamin D as an indicator of compromised immunomodulatory capacity. A state of high ferritin and low vitamin D may create a particularly vulnerable phenotype prone to severe disease progression.

Therefore, this study aims to evaluate the association of serum ferritin and 25-hydroxyvitamin D (25(OH)D) levels, measured at hospital admission, with clinical severity in patients with acute febrile illnesses. We hypothesize that higher serum ferritin and lower 25(OH)D levels will be independently associated with greater disease severity, and that their combined assessment could enhance early risk stratification in this patient group.

Study Design and Setting: This was a single-center, prospective observational cohort study conducted in a 750-bed tertiary care teaching hospital.

Study Population: We screened all adult patients (age ≥18 years) presenting to the ED with a chief complaint of fever. A total of 150 patients who met the inclusion criteria were enrolled consecutively.

• Inclusion Criteria: 1) Age ≥18 years; 2) Documented axillary temperature ≥38.0°C or oral temperature ≥38.3°C; 3) Symptom duration of less than 7 days.
• Exclusion Criteria: 1) Known chronic inflammatory or autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus); 2) Chronic liver disease (cirrhosis) or known hemochromatosis; 3) Active malignancy or receipt of chemotherapy within the last 3 months; 4) Known hematological disorders affecting iron metabolism; 5) History of regular iron or vitamin D supplementation (>1000 IU/day) within the past month; 6) Pregnancy; 7) Patients with incomplete data.

Data Collection and Procedures: Upon enrollment, demographic data, medical history, presenting symptoms, and vital signs were recorded using a standardized case report form. A venous blood sample was collected from each patient within 24 hours of hospital admission.

Disease Severity Assessment: Patients were stratified into three severity groups (Mild, Moderate, Severe) based on a composite clinical severity score assessed at 48 hours post-admission. This classification was based on the presence of organ dysfunction, need for hospitalization, and level of care required:

• Mild: Patients managed in the general medical ward without evidence of organ dysfunction and discharged within 4 days.
• Moderate: Patients requiring hospitalization for >4 days, with evidence of single non-life-threatening organ dysfunction (e.g., acute kidney injury KDIGO stage 1, pneumonia without respiratory failure), or requiring non-invasive ventilation.
• Severe: Patients requiring admission to the Intensive Care Unit (ICU) due to hemodynamic instability (requiring vasopressors), acute respiratory distress syndrome (ARDS), or multi-organ dysfunction syndrome (MODS) as defined by a Sequential Organ Failure Assessment (SOFA) score ≥2 from baseline.

Laboratory Analysis: Serum was separated from blood samples by centrifugation at 3000 rpm for 10 minutes and stored at -80°C until analysis.

• Serum Ferritin: Measured using a two-site chemiluminescent immunometric assay on a Beckman Coulter Dxl 800 analyzer.
• Serum 25-hydroxyvitamin D (25(OH)D): Measured using a competitive chemiluminescent immunoassay on a LIAISON XL analyzer. Vitamin D status was categorized as deficient (<20 ng/mL), insufficient (20–29 ng/mL), or sufficient (≥30 ng/mL).
• C-reactive protein (CRP): Measured using a particle-enhanced immunoturbidimetric assay.

Statistical Analysis: All statistical analyses were performed using SPSS for Windows, Version 25.0 (IBM Corp., Armonk, NY). Continuous variables were presented as mean ± standard deviation (SD), while categorical variables were presented as numbers and percentages (%). The normality of data distribution was assessed using the Shapiro-Wilk test. Differences in continuous variables across the three severity groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for normally distributed data, or the Kruskal-Wallis test for non-normally distributed data. The Chi-square test was used to compare categorical variables. Pearson's correlation coefficient (r) was calculated to assess the linear relationship between biomarkers and clinical severity parameters (length of hospital stay, CRP levels). A two-tailed p-value < 0.05 was considered statistically significant.

Baseline Characteristics of the Study Cohort: A total of 150 patients with AFIs were included in the final analysis. The cohort was stratified into mild (n=65, 43.3%), moderate (n=50, 33.3%), and severe (n=35, 23.3%) disease groups. The mean age of the overall cohort was 52.4 ± 16.8 years, and 85 (56.7%) were male. There were no statistically significant differences in age or gender distribution among the three severity groups, indicating a well-matched cohort at baseline (Table 1). As expected, clinical outcomes such as length of hospital stay and the rate of ICU admission differed significantly across the groups, validating our severity classification.

Table 1. Baseline Demographic and Clinical Characteristics of Patients by Disease Severity

Association of Serum Ferritin and Vitamin D with Disease Severity

The primary laboratory findings are summarized in Table 2. We observed a strong, stepwise association between the levels of both biomarkers and disease severity.
Mean serum ferritin levels increased significantly with increasing severity, from 155.3 ± 55.6 ng/mL in the mild group to 420.1 ± 145.8 ng/mL in the moderate group and 912.5 ± 315.2 ng/mL in the severe group (p<0.001). Post-hoc analysis confirmed significant differences between all three groups (mild vs. moderate, p<0.001; moderate vs. severe, p<0.001).

Conversely, mean serum 25(OH)D levels demonstrated a significant inverse relationship with disease severity. Levels were highest in the mild group (25.8 ± 8.1 ng/mL) and decreased progressively in the moderate (18.2 ± 6.4 ng/mL) and severe groups (12.1 ± 5.3 ng/mL) (p<0.001). The prevalence of vitamin D deficiency (<20 ng/mL) was 35.4% in the mild group, 72.0% in the moderate group, and 91.4% in the severe group. CRP levels, a conventional inflammatory marker, also increased significantly with severity, corroborating the ferritin trend.

Table 2. Comparison of Laboratory Parameters across Disease Severity Groups

Correlation of Biomarkers with Severity Indicators: To further explore the relationships between the biomarkers and clinical outcomes, a Pearson correlation analysis was performed (Table 3). Serum ferritin showed a strong positive correlation with both CRP levels (r = 0.78, p<0.001) and the length of hospital stay (r = 0.69, p<0.001), indicating that higher ferritin levels were associated with a more intense inflammatory response and prolonged hospitalization. In contrast, serum 25(OH)D levels showed a significant negative correlation with CRP levels (r = -0.61, p<0.001) and length of hospital stay (r = -0.55, p<0.001), suggesting that lower vitamin D status was linked to greater inflammation and poorer clinical outcomes.

Table 3. Correlation of Serum Ferritin and 25(OH)D with Markers of Inflammation and Clinical Outcome

This study investigated the utility of serum ferritin and 25(OH)D as predictors of disease severity in a heterogeneous cohort of patients with acute febrile illnesses. Our principal finding is that elevated serum ferritin and reduced 25(OH)D levels at admission are strongly and independently associated with increased disease severity. The observed graded response—with ferritin progressively increasing and 25(OH)D progressively decreasing from mild to severe illness—highlights their potential as robust prognostic biomarkers.

The marked elevation of serum ferritin in patients with severe AFIs is consistent with its role as a key mediator in hyperinflammatory states. Ferritin is no longer considered a passive marker of iron stores in the context of acute illness; rather, it actively participates in the immune response [5]. Pro-inflammatory cytokines, particularly IL-6 and TNF-α, which are central to the pathophysiology of sepsis and other severe infections, directly stimulate ferritin synthesis and secretion [11]. Our finding of a strong positive correlation between ferritin and CRP (r=0.78) supports this, as both are downstream markers of the same inflammatory cascade. The results align with previous studies in specific disease cohorts, such as sepsis and COVID-19, where hyperferritinemia was shown to correlate with ICU admission, multi-organ failure, and mortality [7, 12]. Our study extends these findings to a broader, undifferentiated AFI population, suggesting that ferritin's prognostic value is not limited to a single etiology.

Equally compelling is the inverse relationship between serum 25(OH)D levels and disease severity. The high prevalence of vitamin D deficiency in our moderate (72.0%) and severe (91.4%) cohorts is striking and corroborates a growing body of literature linking low vitamin D status to adverse outcomes in acute illnesses [9]. The immunomodulatory actions of vitamin D are well-documented; it promotes the expression of antimicrobial peptides and shifts the immune response from a pro-inflammatory Th1/Th17 phenotype towards a more regulated Th2/Treg phenotype [8, 13]. Vitamin D deficiency may therefore result in a compromised first-line defense against pathogens and an inability to adequately temper the subsequent inflammatory cascade, leading to a more severe clinical course. Our observed negative correlation between 25(OH)D and CRP (r=-0.61) provides clinical evidence for this immunomodulatory role. Previous meta-analyses have confirmed an association between vitamin D deficiency and increased risk of sepsis and mortality in critically ill patients [14,15].

The unique contribution of our study is the simultaneous evaluation of these two biomarkers, which represent opposing arms of the host response: hyperinflammation (ferritin) and immunomodulation (vitamin D). The combination of high ferritin and low vitamin D may identify a particularly high-risk phenotype. This "pro-inflammatory/immunocompromised" state could predispose patients to uncontrolled cytokine release and subsequent organ damage. While this study did not formally test a combined prediction model, the strong, independent associations suggest that their concurrent measurement could provide synergistic prognostic information, potentially outperforming either marker alone.

Several limitations of our study should be acknowledged. First, as a single-center observational study, our findings may not be generalizable to other populations or healthcare settings. Second, the heterogeneous nature of AFIs, with multiple underlying etiologies, is both a strength (reflecting real-world clinical practice) and a limitation, as we did not stratify our analysis by specific diagnoses. Third, we measured biomarker levels only at admission; serial measurements could provide additional insight into the dynamic host response and disease trajectory. Finally, this study demonstrates association, not causation. It remains unclear whether low vitamin D is a cause or a consequence of severe illness (e.g., due to increased consumption or hemodilution in critical care).

Despite these limitations, our findings have significant clinical implications. Serum ferritin and 25(OH)D are widely available, relatively inexpensive tests in most hospital laboratories. Their measurement at admission could help clinicians to rapidly identify AFI patients at high risk for deterioration. This early risk stratification could guide decisions regarding the level of monitoring, disposition (ward vs. ICU), and the potential initiation of targeted therapies, thereby optimizing resource utilization and potentially improving patient outcomes. Future research should focus on validating these findings in larger, multicenter cohorts, exploring their utility in specific AFI etiologies, and investigating whether interventions aimed at modulating these pathways (e.g., vitamin D supplementation) can alter clinical outcomes.

In conclusion, this prospective study demonstrates that elevated serum ferritin and low serum 25(OH)D levels at the time of hospital admission are strong, independent predictors of disease severity in patients with acute febrile illnesses. These biomarkers reflect two crucial aspects of the host response—the intensity of inflammation and the capacity for immune regulation. Their integration into routine clinical assessment could provide a valuable tool for early risk stratification, enabling more personalized and proactive management of this common and clinically diverse patient population

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