Trends in Immunotherapy

Article

The Effect of Vitamin D₃ Supplementation on Diarrhea Frequency, Duration, and Neutrophil-to-Lymphocyte Ratio (NLR) in Children with Diarrhea

Downloads

https://doi.org/10.54963/ti.v10i1.1493
Wibowo, S., Wulandari, K. T., & Amalia, N. (2026). The Effect of Vitamin D₃ Supplementation on Diarrhea Frequency, Duration, and Neutrophil-to-Lymphocyte Ratio (NLR) in Children with Diarrhea. Trends in Immunotherapy, 10(1), 130–143. https://doi.org/10.54963/ti.v10i1.1493
Volume 10, Issue 1 (2026)
Copyright (c) 2026 Satrio Wibowo, Kirnia Tri Wulandari, Nurlinah Amalia
Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Copyright

The authors shall retain the copyright of their work but allow the Publisher to publish, copy, distribute, and convey the work.

License

Trends in Immunotherapy (TI)  publishes accepted manuscripts under Creative Commons Attribution 4.0 International (CC BY 4.0). Authors who submit their papers for publication by TI agree to have the CC BY 4.0 license applied to their work, and that anyone is allowed to reuse the article or part of it free of charge for any purpose, including commercial use. As long as the author and original source are properly cited, anyone may copy, redistribute, reuse, and transform the content.

Authors

  • Satrio Wibowo

    Division of Gastroenterohepatology, Department of Pediatrics, Saiful Anwar General Hospital, Faculty of Medicine, Universitas Brawijaya, Malang 65122, Indonesia
  • Kirnia Tri Wulandari

    Division of Gastroenterohepatology, Department of Pediatrics, Saiful Anwar General Hospital, Faculty of Medicine, Universitas Brawijaya, Malang 65122, Indonesia
  • Nurlinah Amalia

    Faculty of Medicine, Universitas Brawijaya, Malang 65145, Indonesia
    Medical Research Center of Indonesia, Surabaya 61257, Indonesia

Received: 4 August 2025; Revised: 29 September 2025; Accepted: 22 October 2025; Published: 4 February 2026

Diarrhea remains a major cause of morbidity in children under five, and although current management is mainly supportive, Vitamin D₃ supplementation has emerged as a potential immunomodulatory approach whose clinical efficacy requires further clarification. This study aimed to evaluate the effect of Vitamin D₃ supplementation on clinical outcomes, specifically diarrhea frequency, duration, and neutrophil-to-lymphocyte ratio (NLR) in children with acute diarrhea. This single-blind, randomized controlled trial employed a pre-post test control group design involving 34 pediatric patients with diarrhea. Participants were allocated into two groups: one receiving standard therapy alone (control) and the other receiving standard therapy plus oral Vitamin D₃ supplementation for 14 days. Outcome measures included diarrhea frequency, duration, and neutrophil-to-lymphocyte ratio (NLR), evaluated pre-post intervention. Post-intervention analysis showed significant improvements in the intervention group compared to controls: shortened diarrhea duration (p = 0.002), reduced diarrhea frequency (p = 0.000), but no significant with NLR reduction (p = 0.124); however, the median delta NLR decreased more in the Vitamin D₃ group (−2.57) than in the control group (−0.13) (p = 0.031). No significant differences in dehydration status were observed between groups (p = 1.000). The intervention group also exhibited a marked increase in serum 25(OH)D levels post-supplementation (p = 0.000). However, after adjusting for baseline NLR using ANCOVA, the reduction was no longer directly attributable to the intervention (p = 0.09), though a strong trend favoring the Vitamin D group persisted. Vitamin D₃ supplementation may serve as an adjunctive immunotherapy, as it demonstrated beneficial effects on clinical outcomes in pediatric diarrhea, suggesting its potential through anti-inflammatory and immunomodulatory properties.

Keywords:

Vitamin D Diarrhea Children Neutrophil-to-Lymphocyte Ratio (NLR)

References

  1. Directorate General of Disease Prevention and Control (Ditjen P2P). Indonesian Nutrition Status Survey; Directorate General of Disease Prevention and Control: Jakarta, Indonesia, 2023.
  2. Bhutta, Z.A.; Saeed, M.A. Childhood Infectious Diseases: Overview. Int. Encycl. Public Health 2008, 620–640. DOI: https://doi.org/10.1016/B978-012373960-5.00568-2
  3. Shankar, S.; Durairaj, E. Diet and Management of Diarrhea. Indian J. Pediatr. 2024, 91, 590–597. DOI: https://doi.org/10.1007/s12098-023-04737-z
  4. Liu, J.; Ma, F.; Degen, A.; et al. The Effects of Zinc Supplementation on Growth, Diarrhea, Antioxidant Capacity, and Immune Function in Holstein Dairy Calves. Animals 2023, 13, 2493. DOI: https://doi.org/10.3390/ani13152493
  5. Abbott, M.; Ustoyev, Y. Cancer and the Immune System: The History and Background of Immunotherapy. Semin. Oncol. Nurs. 2019, 35, 150923. DOI: https://doi.org/10.1016/j.soncn.2019.08.002
  6. Legesse, B.T.; Wondie, W.T.; Gedefaw, G.D.; et al. Coutilisation of Oral Rehydration Solution and Zinc for Treating Diarrhoea and Its Associated Factors Among Under-Five Children in East Africa: A Multilevel Robust Poisson Regression. BMJ Open 2024, 14, e079618. DOI: https://doi.org/10.1136/bmjopen-2023-079618
  7. Kumar, A.R., Aeila, A.S. Sustained release matrix type drug delivery system: An overview. World J. Pharm. Pharm. Sci. 2019, 9, 470–480.
  8. Bauza, V.; Ye, W.; Liao, J.; et al. Interventions to improve sanitation for preventing diarrhoea. Cochrane Database Syst. Rev. 2023, 1, CD013328. DOI: https://doi.org/10.1002/14651858.CD013328.pub2
  9. Yazar, A.S.; Guven, S.; Dinleyici, E.C. Effects of Zinc or Synbiotic on the Duration of Diarrhea in Children with Acute Infectious Diarrhea. Turk. J. Gastroenterol. 2020, 27, 537–540. DOI: https://doi.org/10.5152/tjg.2016.16396
  10. Agarwal, G.; Agarwal, S.; Karar, P.K.; et al. Oral Sustained Release Tablets: An Overview with a Special Emphasis on Matrix Tablet. Am. J. Adv. Drug Deliv. 2017, 5, 64–76. DOI: https://doi.org/10.21767/2321-547X.1000013
  11. Dhingra, U.; Kisenge, R.; Sudfeld, C.R.; et al. Lower-Dose Zinc for Childhood Diarrhea — A Randomized, Multicenter Trial. N. Engl. J. Med. 2020, 383, 1231–1241. DOI: https://doi.org/10.1056/NEJMoa1915905
  12. Imdad, A.; Mayo-Wilson, E.; Herzer, K.; et al. Vitamin A Supplementation for Preventing Morbidity and Mortality in Children from Six Months to Five Years of Age. Cochrane Database Syst. Rev. 2017, 3, CD008524. DOI: https://doi.org/10.1002/14651858.CD008524.pub3
  13. Yurdak, K. Vitamin A Supplementation in Acute Diarrhea. J. Pediatr. Gastroenterol. Nutr. 2000, 31, 234–237.
  14. Khalighi Sikaroudi, M.; Mokhtare, M.; Janani, L.; et al. Vitamin D3 Supplementation in Diarrhea-Predominant Irritable Bowel Syndrome Patients: The Effects on Symptoms Improvement, Serum Corticotropin-Releasing Hormone, and Interleukin-6 – A Randomized Clinical Trial. Complement. Med. Res. 2020, 27, 302–309. DOI: https://doi.org/10.1159/000506149
  15. Cojic, M.; Kocic, R.; Klisic, A.; et al. The Effects of Vitamin D Supplementation on Metabolic and Oxidative Stress Markers in Patients With Type 2 Diabetes: A 6-Month Follow-Up Randomized Controlled Study. Front. Endocrinol. 2021, 12, 610893. DOI: https://doi.org/10.3389/fendo.2021.610893
  16. Platts-Mills, J.A.; Houpt, E.R.; Liu, J.; et al. Etiology and Incidence of Moderate-to-Severe Diarrhea in Young Children in Niger. J. Pediatric Infect. Dis. Soc. 2021, 10, 1062–1070. DOI: https://doi.org/10.1093/jpids/piab080
  17. Mileva, S.; Galunska, B.; Gospodinova, M.; et al. Vitamin D3 Status in Children with Acute Diarrhea. Integr. Food Nutr. Metab. 2014, 1, 98–99.
  18. Mahyar, A.; Ayazi, P.; Saffari Rad, M.; et al. The Correlation Between Vitamin D and Bacterial Diarrhea in Children. Arch. Pediatr. Infect. Dis. 2019, in press DOI: https://doi.org/10.5812/pedinfect.84382
  19. El-Desouky, A.I.; El Shaffi, M.M.; Sharaf, S.M.; et al. Association Between Vitamin D Deficiency and Recurrent Acute Diarrhea in Children. Egypt. J. Hosp. Med. 2020, 79, 538–543. DOI: https://doi.org/10.21608/ejhm.2020.85234
  20. Abd El-Azim, M.M.; Mousa, M.K.; Abdelmaaboud, R.M.; et al. Evaluation of the Role of Neutrophil-to-Lymphocyte Ratio (NLR), Platelet-to-Lymphocyte Ratio (PLR) and Mean Platelet Volume (MPV) Time Series as Predictors of Diagnosis and Prognosis of Hemotoxic Snakebite. Biomarkers 2023, 28, 652–662.
  21. Aluisio, A.R.; Maroof, Z.; Chandramohan, D.; et al. Vitamin D₃ Supplementation and Childhood Diarrhea: A Randomized Controlled Trial. Pediatrics 2013, 132, e832–e840. DOI: https://doi.org/10.1542/peds.2012-3986
  22. Dahlan, M.S. Determining the Sample Size Formula: Sample Size and Sampling Methods in Medical and Health Research 2016, 1st ed.; Epidemiologi Indonesia: Jakarta, Indonesia, 2016; p. 151.
  23. Workie, G.Y.; Akalu, T.Y.; Baraki, A.G. Environmental Factors Affecting Childhood Diarrheal Disease Among Under-Five Children in Jamma District, South Wello Zone, Northeast Ethiopia. BMC Infect. Dis. 2019, 19, 804. DOI: https://doi.org/10.1186/s12879-019-4445-x
  24. Hassam, I.; Kisenge, R.; Aboud, S.; et al. Association of Vitamin D and Diarrhoea in Children Aged Less Than Five Years at Muhimbili National Hospital, Dar es Salaam: An Unmatched Case Control Study. BMC Pediatr. 2019, 19, 237. DOI: https://doi.org/10.1186/s12887-019-1614-4
  25. Lazarus, G.; Putra, I.G.N.S.; Junaidi, M.C.; et al. The Relationship of Vitamin D Deficiency and Childhood Diarrhea: A Systematic Review and Meta-Analysis. BMC Pediatr. 2024, 24, 125. DOI: https://doi.org/10.1186/s12887-024-04599-0
  26. Holick, M.F.; Chen, T.C. Vitamin D deficiency: a worldwide problem with health consequences. Am. J. Clin. Nutr. 2008, 87, 1080S–1086S. DOI: https://doi.org/10.1093/ajcn/87.4.1080S
  27. Garg, M.; Rosella, O.; Rosella, G.; et al. Evaluation of a 12 week targeted vitamin D supplementation regimen in patients with active inflammatory bowel disease. Clin. Nutr. 2018, 37, 1375–1382. DOI: https://doi.org/10.1016/j.clnu.2017.06.011
  28. Misra, M.; Pacaud, D.; Petryk, A.; et al. Vitamin D Deficiency in Children and Its Management: Review of Current Knowledge and Recommendations. Pediatrics 2008, 122, 398–417. DOI: https://doi.org/10.1542/peds.2007-1894
  29. Ahlawat, R.; Weinstein, T.; Pettei, M.J. Vitamin D in pediatric gastrointestinal disease. Curr. Opin. Pediatr. 2017, 29, 122–127. DOI: https://doi.org/10.1097/MOP.0000000000000451
  30. Sun, J.; Zhang, Y.-G. Vitamin D Receptor Influences Intestinal Barriers in Health and Disease. Cells 2022, 11, 1129. DOI: https://doi.org/10.3390/cells11071129
  31. Singh, P.A.; Tushir, S.; Hazra, S.; et al. Novel approaches for the extraction and identification of phytoconstituents from herbs and spices. Nat. Prod. J. 2025, 15, E060624230784. DOI: https://doi.org/10.2174/0122103155293641240417072907
  32. Dlozi, P.N.; Ahmed, R.; Khoza, S.; et al. Vitamin D3 loaded polycaprolactone nanoparticles enhance the expression of the antimicrobial peptide cathelicidin in macrophages. Artif. Cells Nanomed. Biotechnol. 2025, 53, 207–219.
  33. Xiong, Y.; Sharma, H.P.; Goyal, R.K.; et al. Bioflavonoid combination attenuates diabetes induced nephropathy in rats via modulation of MMP 9/TIMP 1, TGF β, and GLUT 4 associated pathways. Heliyon 2024, 10, e33217. DOI: https://doi.org/10.1016/j.heliyon.2024.e33217
  34. Otayf, A.Y.; Agarwal, G.; Al Khairat, M.; et al. Fructose, the sweet culprit behind nonalcoholic fatty liver disease and type 2 diabetes. Curr. Diabetes Rev. 2025, 22, e15733998336827. DOI: https://doi.org/10.2174/0115733998336827250114071627
  35. Beard, J.A.; Bearden, A.; Striker, R. Vitamin D and the anti viral state. J. Clin. Virol. 2011, 50, 194–200. DOI: https://doi.org/10.1016/j.jcv.2010.12.006
  36. Dai, J.; Song, J.; Chen, X.; et al. 1,25(OH)₂D₃ treated mouse bone marrow derived dendritic cells alleviate autoimmune hepatitis in mice by improving TFR/TFH imbalance. Immunopharmacol. Immunotoxicol. 2025, 47, 59–67.
  37. Bishop, L.; Ismailova, A.; Dimeloe, S.; et al. Vitamin D and immune regulation: antibacterial, antiviral, anti-inflammatory. J. Bone Miner. Res. Plus 2021, 5, e10405. DOI: https://doi.org/10.1002/jbm4.10405
  38. Bui, L.; Zhu, Z.; Hawkins, S.; et al. Vitamin D regulation of the immune system and its implications for COVID 19: a mini review. SAGE Open Med. 2021, 9, 20503121211014073. DOI: https://doi.org/10.1177/20503121211014073
  39. Ragab, D.; Soliman, D.; Samaha, D.; et al. Vitamin D status and its modulatory effect on interferon gamma and interleukin 10 production by peripheral blood mononuclear cells in culture. Cytokine 2016, 85, 5–10. DOI: https://doi.org/10.1016/j.cyto.2016.05.024
  40. Yang, J.; Tian, G.; Chen, D.; et al. 1,25 Dihydroxyvitamin D₃ inhibits porcine epidemic diarrhea virus replication by regulating cell cycle resumption in IPEC J2 porcine epithelial cells. Microb. Pathog. 2021, 158, 105017. DOI: https://doi.org/10.1016/j.micpath.2021.105017
  41. Tantawy, A.A.G.; Adly, A.A.M.; Atif, H.M.; et al. Abdominal lymphadenopathy in children with Gaucher disease: relation to disease severity and glucosylsphingosine. Pediatr. Hematol. Oncol. 2022, 39, 304–317.
  42. Zhao, P.; Li, J.; Li, X.; et al. The NLRP3 inflammasome recognizes alpha 2 and alpha 7.3 giardins and decreases the pathogenicity of Giardia duodenalis in mice. Parasit. Vectors 2023, 16, 85. DOI: https://doi.org/10.1186/s13071-023-05688-2
  43. Zahorec, R. Neutrophil to lymphocyte ratio, past, present and future perspectives. Bratisl. Med. J. 2021, 122, 474–488. DOI: https://doi.org/10.4149/BLL_2021_078
  44. Yang, L.; Fang, Y.; Zheng, J.; et al. Correlation between serum vitamin D level and acute invasive enteritis in children. Immun. Inflamm. Dis. 2024, 12, e70024. DOI: https://doi.org/10.1002/iid3.70024
  45. Vagha, K.; Taksande, A.; Lohiya, S.; et al. Unlocking Vitality: a comprehensive review of vitamin D’s impact on clinical outcomes in critically ill children. Cureus 2024, 16, e60840. DOI: https://doi.org/10.7759/cureus.60840
  46. Aggeletopoulou, I.; Marangos, M.; Assimakopoulos, S.F.; et al. Vitamin D and microbiome: molecular interaction in inflammatory bowel disease pathogenesis. Am. J. Pathol. 2023, 193, 656–668. DOI: https://doi.org/10.1016/j.ajpath.2023.02.004
  47. Uwaezuoke, S.N.; Odimegwu, C.L.; Mbanefo, N.R.; et al. Vitamin D₃ supplementation as an adjunct in the management of childhood infectious diarrhea: a systematic review. BMC Infect. Dis. 2023, 23, 159. DOI: https://doi.org/10.1186/s12879-023-08077-3
  48. Sari, R.; Karakurt, Z.; Ay, M.; et al. Neutrophil to lymphocyte ratio as a predictor of treatment response and mortality in septic shock patients in the intensive care unit. Turk. J. Med. Sci. 2019, 49, 1336–1349. DOI: https://doi.org/10.3906/sag-1901-105
  49. Templeton, A.J.; McNamara, M.G.; Šeruga, B.; et al. Prognostic role of neutrophil to lymphocyte ratio in solid tumors: a systematic review and meta analysis. JNCI J. Natl. Cancer Inst. 2014, 106, dju124. DOI: https://doi.org/10.1093/jnci/dju124
  50. Jeyakumar, A.; Bhalekar, P.; Shambharkar, P. Effect of vitamin D supplementation on the immune response to respiratory tract infections and inflammatory conditions: a systematic review and meta analysis. Hum. Nutr. Metab. 2024, 37, 200272. DOI: https://doi.org/10.1016/j.hnm.2024.200272
  51. Tabatabaeizadeh, S.A.; Avan, A.; Bahrami, A.; et al. High dose supplementation of vitamin D affects measures of systemic inflammation: reductions in high sensitivity C reactive protein level and neutrophil to lymphocyte ratio (NLR) distribution. J. Cell. Biochem. 2017, 118, 4317–4322. DOI: https://doi.org/10.1002/jcb.26084
  52. Chowdhury, F.; Shahid, A.S.M.S.B.; Tabassum, M.; et al. Vitamin D supplementation among Bangladeshi children under five years of age hospitalised for severe pneumonia: a randomised placebo controlled trial. PLoS One 2021, 16, e0246460. DOI: https://doi.org/10.1371/journal.pone.0246460
  53. Posovszky, C.; Buderus, S.; Claßen, M.; et al. Acute Infectious Gastroenteritis in Infancy and Childhood. Dtsch. Ärztebl. Int. 2020, 117, 615–624. DOI: https://doi.org/10.3238/arztebl.2020.0615
  54. Black, R.E.; Perin, J.; Yeung, D.; et al. Estimated global and regional causes of deaths from diarrhoea in children younger than 5 years during 2000–21: a systematic review and Bayesian multinomial analysis. Lancet Glob. Health 2024, 12, e919–e928. DOI: https://doi.org/10.1016/S2214-109X(24)00078-0
  55. Shi, R.; Wang, B. Nutrient metabolism in regulating intestinal stem cell homeostasis. Cell Prolif. 2024, 57, e13602. DOI: https://doi.org/10.1111/cpr.13602