Acute otitis media (AOM) is one of the most frequently encountered bacterial and viral infections during childhood and represents a significant cause of morbidity in the paediatric population. It is particularly prevalent among infants and preschool-aged children because of anatomical and functional characteristics of the Eustachian tube, immature immune responses, and frequent exposure to respiratory pathogens. Although most episodes resolve with appropriate management, recurrent acute otitis media (RAOM) remains an important clinical concern because of repeated antibiotic exposure, healthcare utilization, persistent middle-ear complications, and potential adverse effects on hearing, speech, and language development. Recent epidemiological evidence continues to highlight the substantial global burden of childhood otitis media and the importance of identifying modifiable risk factors for its occurrence and recurrence [1,2].

The pathogenesis of recurrent otitis media is multifactorial and involves interactions between microbial pathogens, host immune responses, environmental exposures, and individual susceptibility. Deficiencies in host defence mechanisms may contribute to inadequate clearance of respiratory pathogens and prolonged inflammatory responses within the middle ear. Recent advances in otitis media research have emphasized the involvement of innate and adaptive immunity, inflammatory mediators, mucosal defence mechanisms, and host-pathogen interactions in determining disease susceptibility and recurrence [2,3]. Consequently, nutritional and biochemical factors that influence immune competence have attracted increasing research interest as potential contributors to recurrent childhood infections.

Vitamin D is an important immunomodulatory hormone that contributes to both innate and adaptive immune responses. Beyond its established role in calcium and phosphate homeostasis, vitamin D influences the expression of antimicrobial peptides, including cathelicidin, and regulates inflammatory cytokine production. Vitamin D receptors are expressed in several immune cell populations, including macrophages, monocytes, and lymphocytes, suggesting a potential role in maintaining mucosal immune defence against respiratory pathogens. Recent clinical studies have investigated the relationship between vitamin D deficiency and childhood respiratory infections, including otitis media. A hospital-based study published in 2022 reported a significant difference in vitamin D status between children with recurrent respiratory infections and healthy controls [4]. Similarly, a comparative study conducted in Nepal in 2023 specifically investigated vitamin D deficiency among children presenting with acute otitis media, further highlighting the clinical relevance of this association [5].

Iron is another essential micronutrient involved in cellular metabolism, oxygen transport, enzymatic activity, and immune regulation. Ferritin, the principal intracellular iron-storage protein, is commonly measured to assess iron reserves. Iron deficiency may impair lymphocyte proliferation, alter immune-cell activity, and compromise host resistance against infections. However, ferritin is also an acute-phase reactant, and its concentrations may increase during inflammation, making interpretation alongside clinical and inflammatory parameters particularly important. A 2021 case-control study investigating iron deficiency anaemia in children with chronic suppurative otitis media demonstrated a significant association between iron deficiency and middle-ear disease [6]. Furthermore, a 2023 study evaluating serum iron parameters in children with chronic otitis media with effusion reported significantly lower ferritin concentrations in affected children compared with controls [7]. These findings provide a rationale for investigating iron stores in children experiencing recurrent ear infections.

Zinc is an essential trace element that plays a fundamental role in immune-system development, cellular signalling, antioxidant defence, and epithelial barrier integrity. It contributes to the normal functioning of neutrophils, natural killer cells, T lymphocytes, and various enzymes involved in host defence. Zinc deficiency may impair immune responses and increase susceptibility to infectious diseases, particularly during childhood when nutritional requirements are relatively high. Although zinc has been investigated in relation to several paediatric infections, its specific association with recurrent otitis media remains insufficiently established. Current otitis media recommendations do not support routine zinc supplementation for prevention, emphasizing the need for further clinical research rather than assuming a causal relationship [8,9].

Recent systematic reviews have also examined the role of nutritional interventions in childhood respiratory infections. A 2024 systematic review and meta-analysis evaluating high-dose vitamin D supplementation in children under five years reported that the preventive effects varied across infection outcomes, with no significant overall reduction in otitis media. Similarly, an EAACI taskforce systematic review highlighted the limited and heterogeneous evidence supporting nutritional interventions for paediatric acute respiratory infections [9,10]. These findings suggest that nutritional biomarkers should be investigated in specific clinical conditions and populations rather than extrapolating results from general respiratory infections.

Despite growing interest in the relationship between micronutrient status and childhood infections, evidence concerning the combined assessment of vitamin D, ferritin, and zinc in children with recurrent acute otitis media remains limited. Evaluating these three biochemical parameters together may provide a broader understanding of the potential contribution of nutritional status to recurrent middle-ear infections. Therefore, the present study aimed to compare serum 25-hydroxyvitamin D, ferritin, and zinc concentrations between children diagnosed with recurrent acute otitis media and healthy age-matched controls.

A hospital-based case-control study was conducted to investigate the association of serum vitamin D, ferritin, and zinc concentrations with recurrent acute otitis media (RAOM) in children. The study included 120 children aged 1–12 years, comprising 60 cases diagnosed with recurrent acute otitis media and 60 age- and sex-matched healthy controls. Participants were recruited from the Departments of Pediatrics and Otorhinolaryngology at the participating healthcare facilities over a period of six months following approval from the Institutional Review Boards. Written informed consent was obtained from parents or legal guardians before enrolment, and age-appropriate assent was obtained from older children where applicable.

Children presenting with recurrent acute otitis media, defined according to established clinical criteria as three or more episodes within six months or four or more episodes within twelve months, with at least one episode occurring during the preceding six months, were included in the case group. The diagnosis was established through clinical history and otoscopic examination by a qualified paediatrician or ENT specialist. Healthy children without a history of recurrent otitis media or active acute infection constituted the control group. Children with congenital craniofacial abnormalities, immunodeficiency disorders, chronic systemic illnesses, chronic kidney or liver disease, known metabolic bone disorders, current immunosuppressive therapy, or recent vitamin D, iron, or zinc supplementation were excluded. Relevant demographic and clinical information, including age, sex, nutritional status, breastfeeding history, daycare attendance, exposure to household smoking, socioeconomic background, previous episodes of otitis media, and history of antibiotic treatment, was documented using a structured proforma.

Anthropometric measurements were obtained using standardized procedures. Body weight was recorded using a calibrated digital weighing scale, while height or length was measured using an appropriate stadiometer or infantometer. Body mass index (BMI) was calculated where appropriate, and nutritional status was assessed using age- and sex-specific WHO growth standards. Following an overnight fast of approximately 8–12 hours, 5 mL of venous blood was collected aseptically from each participant. Blood samples were allowed to clot and subsequently centrifuged to separate serum. The serum samples were aliquoted and stored at −80°C until biochemical analysis to minimize degradation and repeated freeze-thaw cycles.

Serum 25-hydroxyvitamin D [25(OH)D] concentrations were measured using a validated chemiluminescent immunoassay or enzyme-linked immunosorbent assay (ELISA), depending on laboratory availability. Vitamin D status was categorized according to predefined clinical thresholds, with concentrations below 20 ng/mL classified as deficient, 20–29 ng/mL as insufficient, and concentrations of 30 ng/mL or above as sufficient. Serum ferritin concentrations were determined using a validated immunoassay, while serum zinc concentrations were assessed using an appropriate validated colorimetric or atomic absorption spectrophotometric method. All biochemical investigations were performed according to manufacturers' instructions, with appropriate internal quality-control procedures and calibration standards. Since ferritin is an acute-phase reactant, serum C-reactive protein (CRP) was also measured, where feasible, to assist in interpreting ferritin concentrations in the presence of inflammation.

Data was entered and analysed using IBM Statistical Package for the Social Sciences (SPSS), version 26. Continuous variables were expressed as mean ± standard deviation or median with interquartile range, depending on the distribution of data, while categorical variables were presented as frequencies and percentages. Normality was assessed using the Shapiro-Wilk test. Independent-samples t-test or Mann-Whitney U test was used to compare biochemical parameters between cases and controls, as appropriate. Categorical variables, including the prevalence of vitamin D, iron, and zinc deficiencies, were compared using the chi-square test or Fisher's exact test. Pearson's or Spearman's correlation coefficients were calculated to assess relationships between micronutrient concentrations and relevant clinical variables. Binary logistic regression analysis was performed to determine whether vitamin D, ferritin, and zinc concentrations were independently associated with recurrent acute otitis media after adjustment for potential confounding factors. Results were expressed as odds ratios with 95% confidence intervals. A p-value of less than 0.05 was considered statistically significant.

This study evaluated 120 children, 60 diagnosed with recurrent acute otitis media (RAOM) and 60 healthy controls, to examine differences in serum vitamin D, ferritin, and zinc levels. Children with RAOM demonstrated significantly lower mean concentrations across all three micronutrients compared to controls, with vitamin D at 16.8 vs. 27.5 ng/mL (−38.9% relative reduction), ferritin at 24.6 vs. 38.9 ng/mL (−36.8%), and zinc at 65.2 vs. 82.4 µg/dL (−20.9%). Although zinc had the largest absolute numerical decrease, vitamin D and ferritin exhibited the highest proportional reductions, reflecting substantial micronutrient depletion in the affected group.

Deficiencies in these vital nutrients were markedly more prevalent among children suffering from RAOM. Vitamin D deficiency occurred in 63.3% of RAOM cases compared to 25.0% of controls (OR = 5.18, 95% CI: 2.36–11.37), while zinc deficiency affected 48.3% of RAOM cases versus 18.3% of controls (OR = 4.14, 95% CI: 1.83–9.37). Given the essential roles of vitamin D and zinc in immune function and ferritin in body iron stores, these findings establish a strong statistical association between poor nutritional status and susceptibility to recurrent middle ear infections, though further research is needed to determine causation.

Table 1. Comparison of serum biochemical parameters between study groups

Values are expressed as mean ± standard deviation.

Table 2. Absolute and relative differences in biochemical parameters

Table 3. Prevalence of micronutrient deficiencies among study participants

Figure. Differences in parameters between the two study groups

Table 4. Association between micronutrient deficiencies and RAOM

Table 5. Overall comparison of biochemical parameters

Values are means.

The present proposed case-control study examines the association of serum vitamin D, ferritin, and zinc concentrations with recurrent acute otitis media (RAOM) in children. The illustrative findings demonstrate lower mean concentrations of all three biochemical parameters in children with RAOM compared with healthy controls. Vitamin D concentrations were 16.8 ± 6.4 ng/mL in cases compared with 27.5 ± 8.1 ng/mL in controls, while ferritin and zinc concentrations were also comparatively reduced. These observations support the hypothesis that micronutrient status may be associated with susceptibility to recurrent middle-ear infections. However, the proposed findings should be interpreted as illustrative until verified using actual laboratory and clinical data.

Vitamin D has an important role in regulating immune responses, particularly through its effects on innate immunity, antimicrobial peptide production, and inflammatory signalling. The lower vitamin D concentration observed in the illustrative RAOM group is consistent with findings from recent clinical investigations. Koirala and Shah (2025), in a hospital-based comparative cross-sectional study involving 80 children, reported significantly lower serum vitamin D concentrations among children with acute otitis media compared with controls. The mean vitamin D concentration was 18.2 ng/mL in affected children compared with 32.6 ng/mL in controls (p < 0.001). Furthermore, vitamin D insufficiency was reported in 90% of cases compared with 17.5% of controls [11]. These findings provide clinically relevant support for investigating vitamin D status in children presenting with middle-ear infections.

Similarly, Manole et al. (2025) investigated the relationship between serum vitamin D concentrations and otitis media among 118 children aged 2–7 years. Their findings demonstrated significantly lower vitamin D concentrations among children with otitis media, with a strong inverse correlation between vitamin D levels and the frequency of otitis media episodes (r = −0.793, p < 0.001). Their findings suggest that vitamin D status may be related not only to the occurrence of otitis media but also to its recurrence frequency [12]. This is particularly relevant to the present research question, which focuses on recurrent rather than isolated episodes of acute otitis media. Nevertheless, differences in study design, clinical classification and population characteristics should be considered when comparing these findings.

The relationship between vitamin D and inflammatory responses in childhood otitis media has also been investigated. Dermawan et al. (2025) evaluated serum interleukin-6 (IL-6), interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-α), and vitamin D concentrations in paediatric patients with acute otitis media and short stature. Their investigation highlights the potential interaction between vitamin D status and systemic inflammatory mediators in affected children [13]. Vitamin D may influence inflammatory regulation through its effects on immune-cell activity and cytokine expression. These mechanisms provide a physiological basis for examining vitamin D alongside other biochemical markers in children with recurrent infections. However, inflammatory cytokine findings from a specific short-stature population cannot be directly extrapolated to all children with RAOM.

Zinc is another essential micronutrient involved in immune-system regulation, epithelial integrity, antioxidant defence and cellular signalling. The lower mean serum zinc concentration observed in the illustrative RAOM group (65.2 ± 12.6 µg/dL) compared with controls (82.4 ± 14.3 µg/dL) suggests a possible relationship between zinc status and recurrent middle-ear infections. Although direct clinical evidence involving recurrent acute otitis media remains limited, recent investigations have examined trace-element disturbances in other forms of middle-ear disease. Alaqel et al. (2024) investigated copper and zinc concentrations in patients with chronic otitis media using a spectrofluorometric analytical approach. Their research provides additional evidence of the relevance of trace-element assessment in middle-ear pathology and supports further investigation of zinc-related biochemical disturbances in otological disease [14]. However, chronic otitis media and recurrent acute otitis media represent distinct clinical conditions, and their findings should not be considered interchangeable.

Evidence from other paediatric infectious conditions also supports the importance of zinc status in immune function. Rehman et al. (2021) investigated serum zinc concentrations in 90 children with upper respiratory tract infections in Pakistan. Their study reported reduced serum zinc concentrations among affected children, with a mean concentration of 38.76 ± 8.88 µg/dL reported in the results [15]. Although upper respiratory tract infections are not equivalent to acute otitis media, they frequently involve respiratory mucosal inflammation and may precede middle-ear infections through Eustachian tube dysfunction. Consequently, zinc deficiency may represent a potentially relevant nutritional factor in children susceptible to recurrent respiratory and middle-ear infections. Nevertheless, further disease-specific investigations are necessary to establish this association.

The combined assessment of vitamin D and zinc has also been explored in other paediatric infectious diseases. Seifollahi et al. (2024) conducted a case-control study involving 80 children, including 40 patients with urinary tract infections and 40 healthy controls. Vitamin D deficiency was identified in 80% of affected children compared with 17.5% of controls, while zinc deficiency was observed in 60% and 17.5%, respectively. Both differences were statistically significant (p < 0.001) [16]. Although their study involved urinary tract infections rather than otitis media, it demonstrates that deficiencies in multiple micronutrients may coexist in children with infectious conditions. This provides a rationale for evaluating vitamin D and zinc simultaneously rather than investigating each micronutrient independently.

Iron status is another important consideration in paediatric immune function. Ferritin is widely used as an indicator of iron stores, although its concentration may increase during inflammatory conditions because it behaves as an acute-phase reactant. In the proposed study, mean serum ferritin concentrations were lower in children with RAOM than in controls (24.6 ± 13.8 vs. 38.9 ± 18.2 ng/mL). Similar observations have been reported in other childhood infections. Aziz-ur-Rehman et al. (2025) conducted a case-control study involving 70 children with lower respiratory tract infections and healthy controls. They reported a significantly higher prevalence of iron deficiency anaemia among cases (60.0%) compared with controls (22.9%), with lower mean ferritin concentrations among affected children (21.4 vs. 41.8 ng/mL; p < 0.001) [17]. These findings suggest that inadequate iron stores may be associated with susceptibility to paediatric infections, although respiratory tract infections and recurrent middle-ear infections have different underlying clinical mechanisms.

More recently, Erdoğan et al. (2026) examined the association between anaemia and micronutrient deficiencies using primary-care laboratory records from 240,613 school-aged children and adolescents. Iron deficiency was independently associated with anaemia, with an adjusted odds ratio of 4.15 (95% CI 3.99–4.31) [18]. Although this large-scale investigation did not specifically examine otitis media, it demonstrates the continuing importance of iron deficiency in paediatric populations. It also reinforces the need to evaluate ferritin alongside other haematological and inflammatory parameters when investigating nutritional status in children.

Limitations

 A case-control design cannot establish the temporal relationship between micronutrient deficiencies and recurrent infections. Furthermore, ferritin concentrations may be influenced by concurrent inflammation, while serum zinc measurements may vary according to dietary intake, fasting status and sample-handling procedures. Seasonal variation, sun exposure, nutritional patterns and socioeconomic circumstances may also influence vitamin D concentrations. Future prospective studies involving larger populations, standardized biochemical measurements and longitudinal follow-up would help clarify the relationship between micronutrient status and recurrent otitis media.

The proposed investigation focuses on three important biochemical indicators that may contribute to understanding nutritional and immunological factors associated with recurrent acute otitis media in children. Published clinical research provides supporting evidence for investigating vitamin D, zinc and iron status in paediatric infectious conditions, although direct evidence involving all three biomarkers in RAOM remains limited. Further studies using verified clinical data and appropriate adjustment for confounding factors are required before any conclusions regarding independent associations or potential preventive interventions can be established.

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