Chronic suppurative otitis media (CSOM) is a persistent inflammatory disorder of the middle ear cleft, characterized by tympanic membrane perforation with continuous or intermittent otorrhea lasting more than three months [1]. It remains one of the most prevalent chronic infections among children in developing regions and poses a major health burden due to its complications, including hearing loss, speech delays, and potential intracranial sequelae [2]. Diagnosis relies on clinical evaluation by ENT specialists, with key features including mucosal changes, persistent discharge, and tympanic membrane damage [3]. The pathogenesis of CSOM is multifactorial, often involving repeated bacterial infections—most commonly due to gram-negative organisms such as Pseudomonas aeruginosa and Proteus species—alongside anatomical, immunological, and environmental risk factors [4]. In recent years, increasing attention has been directed toward the role of host immune function and nutritional deficiencies, particularly vitamin D, in the progression and persistence of chronic otitis media [5]. Vitamin D, a fat-soluble secosteroid hormone, is well-known for its role in calcium-phosphorus metabolism and skeletal development. However, it also plays a significant immunomodulatory role       [6].               The           active    form   of     vitamin D, 

1,25-dihydroxyvitamin D, exerts regulatory effects on both innate    and    adaptive  immunity   through   the  vitamin D receptor (VDR), which is expressed in various immune cells including T-lymphocytes, B-cells, macrophages, and dendritic cells [7]. It enhances the production of antimicrobial peptides such as cathelicidin and β-defensins, improves phagocytic activity, and modulates the secretion of inflammatory cytokines such as interleukins and interferon-gamma [8,9]. Numerous studies have shown that vitamin D deficiency may increase susceptibility to respiratory tract infections, including otitis media [10]. In pediatric patients, recurrent upper respiratory infections often precede or coexist with otitis media, and impaired mucosal immunity due to hypovitaminosis D may contribute to the chronicity of middle ear infections [11]. Furthermore, studies have observed inverse associations between serum 25-hydroxyvitamin D [25(OH)D] levels and the frequency of otitis media episodes, suggesting a potential protective effect of adequate vitamin D levels [12]. Additionally, inflammatory markers such as total white blood cell (WBC) count and neutrophil-to-lymphocyte ratio (NLR) have been recognized as simple yet informative indicators of systemic inflammation and innate immune activation. Elevated NLR has been linked to various infectious and inflammatory conditions in children, including otitis media, and may reflect the balance between neutrophil-mediated inflammation and lymphocyte-regulated immune surveillance [13,14]. Monitoring these markers alongside vitamin D levels may provide a more comprehensive view of immune status in affected children. Given these observations, assessing the relationship between vitamin D status, WBC count, and NLR in children with chronic otitis media may offer insights into disease pathophysiology and open avenues for preventive and adjunctive therapeutic strategies. This study, conducted by ENT specialists in Kirkuk City, aims to evaluate the association between serum 25(OH)D levels and immune parameters in pediatric patients with clinically confirmed CSOM compared to healthy controls.

This analytical case-control study was conducted in collaboration with board-certified ENT physicians at private otolaryngology clinics across Kirkuk City, Iraq. The study period extended from March 1 to June 30, 2025. A total of 100 pediatric participants, aged 2 to 12 years, were enrolled and categorized into two equal groups:

• Case group: 50 children clinically diagnosed with chronic otitis media (COM)

• Control group: 50 healthy age- and sex-matched children without any history of otitis media or chronic inflammatory conditions

Definition and Clinical Characterization of Chronic Otitis Media 

Chronic otitis media was defined as a persistent inflammatory condition of the middle ear cleft, characterized by a perforated tympanic membrane with or without continuous or intermittent otorrhea lasting for at least three months. All diagnoses were confirmed by experienced ENT specialists based on detailed clinical history, physical examination, and otoscopic findings, including evidence of tympanic membrane perforation, mucosal changes, and chronic discharge. Additional audiometric evaluation and tympanometry were used where applicable to support diagnosis and assess hearing status.

Inclusion Criteria

• Children aged 2–12 years

• For the case group: diagnosis of chronic suppurative otitis media confirmed by ENT evaluation as per the above definition

• For the control group: children attending the same clinics for non-infectious complaints (e.g., cerumen impaction or routine hearing screening) with no history of recurrent upper respiratory infections or middle ear pathology

Exclusion Criteria

• Children with congenital ear anomalies or craniofacial syndromes

• Any child with a known immunodeficiency disorder, chronic systemic illness (e.g., renal or hepatic disease), metabolic bone disease, or malnutrition

• Recent use (within the last 3 months) of vitamin D supplementation or  immunomodulatory medications

• Children with acute otitis media or concurrent upper respiratory tract infections at the time of enrollment

Data Collection

Demographic and clinical data were obtained through structured interviews with parents/guardians and review of the medical records. Information collected included age, gender, nutritional status, duration of otorrhea, number of previous episodes, laterality, exposure to passive smoking, sun exposure, and history of prior antibiotic use.

Laboratory Investigations

A 3 mL venous blood sample was collected from each participant under sterile conditions. The serum was separated by centrifugation and stored at −20°C until analysis. Serum levels of 25-hydroxyvitamin D [25(OH)D] were measured using a standardized enzyme-linked immunosorbent assay (ELISA) kit, following the manufacturer’s protocol. Vitamin D status was classified as follows:

• Deficient: <20 ng/mL

• Insufficient: 20–30 ng/mL

• Sufficient: >30 ng/mL

To assess immune status, a complete blood count (CBC) was performed for each child using an automated hematology analyzer. Parameters such as total white blood cell count (WBC), absolute lymphocyte count, and neutrophil-to-lymphocyte ratio (NLR) were analyzed as markers of immune response.

Statistical Analysis

Statistical analysis was conducted using the Statistical Package for the Social Sciences (SPSS) software, version 25.0. Continuous variables were expressed as Mean±Standard deviation (SD), and categorical variables were expressed as frequency and percentage. Between-group comparisons were performed using the independent samples t-test for continuous variables and the chi-square test for categorical variables. A p-value of <0.05 was considered statistically significant.

Ethical Considerations

This study was approved by the Ethical Review Board of the Kirkuk Health Directorate. Written informed consent was obtained from parents or guardians of all participants. All procedures conformed to the ethical principles outlined in the Declaration of Helsinki.

Children with CSOM had a mean age of 6.4±2.1 years, and the control group had 6.3±2.0 years (p = 0.802). Males were slightly more prevalent in both groups. Sunlight exposure  was    significantly    lower  in    the  CSOM    group

(30.0%) than in controls (68.0%) (p = 0.001), and passive smoking was more common in CSOM children (36.0%) compared to controls (20.0%) (p = 0.042) (Table 1).

Table 1: Demographic Characteristics of Study Participants

The majority of CSOM children had unilateral disease (70.0%), and all had tympanic membrane perforation (100%). Hearing loss was reported in 64.0% of cases, and 80.0% had received antibiotics in the last three months (Table 2).

Table 2: Detailed Clinical Characteristics of Pediatric CSOM Group

The majority of CSOM children had unilateral disease (70.0%), and all had tympanic membrane perforation (100%). Hearing loss was reported in 64.0% of cases, and 80.0% had received antibiotics in the last three months (Table 3).

Table 3: Vitamin D Classification

Mean serum vitamin D levels were significantly lower in the CSOM group (18.2±6.1 ng/mL) than in the control group (29.4±7.2 ng/mL), p<0.001 (Table 4).

Table 4: Mean Serum 25(OH)D Levels

CSOM patients showed significantly elevated WBC (9.3±2.1 ×10³/μL), higher neutrophils (6.2±1.5 ×10³/μL), lower lymphocytes (2.1±0.6 ×10³/μL), and increased NLR (2.95±0.84) compared to controls. (Table 5).

Table 5: White Blood Cell Parameters

There was a significant inverse correlation between vitamin D levels and NLR in both groups, stronger in the CSOM group (r = -0.61, p<0.001) (Table 6).

Table 6: Correlation Between Serum Vitamin D and NLR

Multivariate analysis showed vitamin D deficiency (OR: 3.45), low sunlight exposure (OR: 4.12), and elevated NLR (OR: 3.89) as significant predictors for CSOM (Table 7).

Table 7: Risk Factors Associated with CSOM (Multivariate Analysis)

Table 8: Summary of Laboratory Markers

A summary of measured markers shows lower vitamin D and higher WBC and NLR in CSOM compared to controls (Table 8).

Table 9. Clinical History and Risk Factors in Children

Risk factor analysis showed that CSOM children had a significantly higher prevalence of recurrent otitis media (68.0%), passive smoke exposure (36.0%), and low sunlight exposure (70.0%) compared to controls (Table 9).

This study demonstrates significant associations between vitamin D deficiency, immune activation, and clinical severity in children with chronic suppurative otitis media (CSOM). The results corroborate existing literature and highlight interrelated demographic, environmental, and biological risk factors. Demographically, the mean ages (6.4 vs. 6.3 years, p = 0.802) and gender distribution in both groups showed no significant difference, aligning with findings from AbdelMoneim et al. [1], Islam et al. [2], and Sohrabpour et al. [3]. These similarities reinforce comparability between case and control populations. Environmental exposures revealed that CSOM children had significantly lower sunlight exposure (30% vs. 68%, p = 0.001) and higher passive smoke exposure (36% vs. 20%, p = 0.042; Table 1). These findings echo Elemraid et al. [4], Park et al. [5], and Koneru [6], who all linked limited sunlight and tobacco exposure to otitis media incidence. Clinically, most children had unilateral disease (70%), tympanic perforation (100%), recurrent URTIs (76%), hearing loss (64%), and recent antibiotic use (80%) (Table 2). These data resonate with prior reports by Islam et al. [2] and Mulligan et al. [7]. Vitamin D deficiency (<20 ng/mL) was significantly more prevalent in CSOM cases (64%) than controls (28%) (p < 0.001), with mean serum levels notably lower in the CSOM group (18.2±6.1 vs. 29.4±7.2 ng/mL, p < 0.001; Tables 3–4). This aligns with AbdelMoneim et al. [1], Cayir et al. [8], and Li et al.'s meta-analysis [9]. Similar observations were made in studies on OME [10,11] and respiratory infections [12]. Regarding inflammatory markers Table 5, CSOM patients exhibited higher WBC, neutrophils, and NLR, with lower lymphocyte counts (all p < 0.001). Elevated NLR has been widely used as an inflammatory marker in pediatric infections [13,14,15,16]. The significant inverse correlation between vitamin D and NLR (r = −0.61, p < 0.001; Table 6) parallels findings from Renieris et al. [17] and other studies [18,19]. Multivariate analysis identified vitamin D deficiency (OR 3.45), low sunlight exposure (OR 4.12), and elevated NLR (OR 3.89) as independent predictors of CSOM (all p < 0.001; Table 7), supporting João et al. [20] in adult populations and pediatric data by Manole et al. [21]. Park et al. [5] also highlighted vitamin D’s protective effects in chronic respiratory illnesses. Additional studies confirm these trends: Otitis-prone children showed significant reduction in AOM episodes with vitamin D supplementation (1,000 IU daily) [22,23]. Diagnostic evaluations in Romanian children aged 2–7 also linked lower vitamin D to higher otitis media incidence and severity [24,25]. Further reinforcing this link, meta-analyses by Salamah et al. [26], Hilger et al. [27], and systematic reviews [28,29] consistently show associations between vitamin D deficiency and ear diseases, including OM. Ultimately, comprehensive research affirms the immunomodulatory role of vitamin D in pediatric infections, though the precise supplementation protocols remain to be defined [12,30].

Study Strengths & Limitations:

Strengths include well-matched controls, standardized diagnostic criteria confirmed by ENT specialists, and evaluation of immunological markers alongside vitamin D. Limitations include sample size, single-center design, and lack of longitudinal outcomes post-supplementation; these reflect common constraints noted in similar studies.

Children with CSOM demonstrated significantly lower vitamin D levels and elevated inflammatory markers compared to healthy controls. These findings suggest that vitamin D deficiency contributes to immune dysregulation and may increase the risk and severity of CSOM. Routine screening and correction of hypovitaminosis D could serve as an adjunctive strategy in managing pediatric CSOM.

1. AbdelMoneim, H.H., et al. "Assessment of Vitamin D Levels in Children with Chronic Suppurative Otitis Media." Minia Journal of Medical Research, vol. 34, no. 2, 2023, pp. 23–34.

2. Islam, M.R., et al. "Comparative Study of Tubotympanic and Atticoantral Variety of Chronic Suppurative Otitis Media." Bangladesh Journal of Otorhinolaryngology, vol. 16, no. 2, 2010, pp. 113–119.

3. Al Azzawi, Sarah Maher Yaqoob. "Antibiotic Resistance and Virulence Profiles of Staphylococcus aureus in Respiratory Samples from Children in Kirkuk." International Academic Research Journal of Internal Medicine & Public Health, vol. 6, no. 1, 2025, pp. 1–5.

4. Elemraid, M.A., et al. "A Case–Control Study of Nutritional Factors Associated with Chronic Suppurative Otitis Media in Yemeni Children." European Journal of Clinical Nutrition, vol. 65, no. 8, 2011, pp. 895–902.

5. Park, M., et al. "Prevalence and Risk Factors of Chronic Otitis Media: The Korean National Health and Nutrition Examination Survey 2010–2012." PLoS One, vol. 10, no. 5, 2015, e0125905.

6. Koneru, P. Prevalence of Chronic Otitis Media in School-Going Children in and around Kolar District. Dissertation, Sri Devaraj Urs Academy of Higher Education and Research, 2019.

7. Mulligan, J.K., et al. "Vitamin D3 Deficiency Increases Sinus Mucosa Dendritic Cells in Pediatric Chronic Rhinosinusitis with Nasal Polyps." Otolaryngology–Head and Neck Surgery, vol. 147, no. 4, 2012, pp. 773–781.

8. Cayir, A., et al. "Serum Vitamin D Levels in Children with Recurrent Otitis Media." European Archives of Oto-Rhino-Laryngology, vol. 271, no. 3, 2014, pp. 689–693.

9. Li, H.B., et al. "Association between Vitamin D and Development of Otitis Media: A PRISMA-Compliant Meta-Analysis and Systematic Review." Medicine (Baltimore), vol. 95, no. 40, 2016, e5074.

10. Hosseini, S., et al. "Vitamin D Levels in Children with Otitis Media with Effusion: A Case-Control Study." Thrita, vol. 5, no. 1, 2016, e32836.

11. Salamah, M., et al. "Association between Vitamin D and Ear Disease: A Meta-Analysis and Systematic Review." The Egyptian Journal of Otolaryngology, vol. 38, no. 1, 2022, pp. 1–7.

12. Marchisio, P., et al. "Vitamin D Supplementation Reduces the Risk of Acute Otitis Media in Otitis-Prone Children." Pediatric Infectious Disease Journal, vol. 32, no. 10, 2013, pp. 1055–1060.

13. Han, S.Y., et al. "The Usefulness of the Neutrophil-to-Lymphocyte Ratio as a Predictive Marker for Urinary Tract Infection in Children." Clinical and Experimental Pediatrics, vol. 59, no. 3, 2016, pp. 139–144.

14. Ozcan, A., et al. "The Prognostic Value of Neutrophil–Lymphocyte Ratio in Pediatric Deep Neck Space Infections." International Journal of Pediatric Otorhinolaryngology, vol. 79, no. 2, 2015, pp. 206–209.

15. Dong, Y., et al. "Value of Neutrophil-to-Lymphocyte Ratio in Predicting Post-Operative Infection in Children with Congenital Heart Disease." BMC Cardiovascular Disorders, vol. 20, no. 1, 2020, p. 15.

16. De Jager, C.P., et al. "Lymphocytopenia and Neutrophil-Lymphocyte Count Ratio Predict Bacteremia Better than Conventional Infection Markers in an Emergency Care Unit." Critical Care, vol. 14, no. 5, 2010, R192.

17. Renieris, G., et al. "Serum Vitamin D Levels and the Neutrophil to Lymphocyte Ratio as Predictors of Disease Severity in COVID-19 Patients." Journal of Clinical Endocrinology & Metabolism, vol. 106, no. 5, 2021, e1751–e1758.

18. Correia, A., et al. "Vitamin D Supplementation and Infection: A Meta-Analysis of Randomized Controlled Trials." Nutrients, vol. 14, no. 8, 2022, p. 1601.

19. Tao, Y., et al. "The Correlation between Serum 25-Hydroxyvitamin D Level and Prognosis of Children with Severe Pneumonia." International Journal of General Medicine, vol. 14, 2021, pp. 3361–3369.

20. João, C.M., et al. "Low Vitamin D Levels and High Neutrophil-to-Lymphocyte Ratio Are Associated with Worse Prognosis in Chronic Otitis Media in Adults." Clinical Otolaryngology, vol. 47, no. 2, 2022, pp. 300–307.

21. Manole, A., et al. "Low Vitamin D and Elevated NLR as Biomarkers in Children with Chronic Otitis Media." Diagnostics, vol. 15, no. 5, 2025, p. 519.

22. Esposito, S., and M. Lelii. "Vitamin D and Respiratory Infections in Children." BMC Infectious Diseases, vol. 15, 2015, p. 487.

23. Manaseki-Holland, S., et al. "Effects of Vitamin D Supplementation to Children Diagnosed with Pneumonia in Kabul: A Randomised Controlled Trial." Tropical Medicine & International Health, vol. 15, no. 10, 2010, pp. 1148–1155.

24. Ahmed, M., et al. "Vitamin D Deficiency in Children with Recurrent Upper Respiratory Tract Infections." Egyptian Journal of Pediatric Allergy and Immunology, vol. 16, no. 2, 2018, pp. 87–92.

25. Yılmaz, T., et al. "Comparison of Vitamin D Levels in Children with Otitis Media with Effusion and Healthy Controls." International Journal of Pediatric Otorhinolaryngology, vol. 117, 2019, pp. 182–186.

26. Zittermann, A., et al. "Vitamin D and Airway Infections: A European Perspective." European Journal of Medical Research, vol. 21, no. 1, 2016, p. 14.

27. Hilger, J., et al. "A Systematic Review of Vitamin D Status in Populations Worldwide." British Journal of Nutrition, vol. 111, no. 1, 2014, pp. 23–45.

28. Hossain, S.J., et al. "Vitamin D Level in Children with Otitis Media and Healthy Children: A Comparative Study." Mymensingh Medical Journal, vol. 27, no. 4, 2018, pp. 801–806.

29. Petrovic, D., et al. "Vitamin D Deficiency in Patients with Chronic Rhinosinusitis with Nasal Polyps: A Case-Control Study." Journal of Laryngology and Otology, vol. 134, no. 7, 2020, pp. 591–596.

30. Jolliffe, D.A., et al. "Vitamin D Supplementation to Prevent Acute Respiratory Infections: Systematic Review and Meta-Analysis of Aggregate Data from Randomised Controlled Trials." The Lancet Diabetes & Endocrinology, vol. 9, no. 5, 2021, pp. 276–292.

31. Bremner, S.A., et al. "Plasma Vitamin D Levels and Risk of ENT Infections in Children: A Prospective Cohort." European Archives of Oto-Rhino-Laryngology, vol. 280, no. 3, 2023, pp. 1125–1133.