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Atopic dermatitis (AD) is a common skin disorder resulting in malfunctioning of the skin barrier and immune system that facilitate the growth of various microorganisms. Several studies reported that there was a significant reduction of vitamin D and cathelicidin levels in AD patients. It was also reported that the administration of vitamin D in AD patients sharply increased cathelicidin level that can be advantageous as a skin defense against microbial infection. In this study the supplementation of vitamin D3 was administered to AD pediatric patients and the ability of Vitamin D3 to reduce infections in AD patients was measured by observing Staphylococcus aureus colonization. A double-blind randomized experimental study was carried out after appropriate sampling on 20 AD patients who came for treatment in Dermatopediatric Division of Dr. Soetomo General Hospital Surabaya and met the criteria for inclusion. A supplementation of vitamin D3 syrup was used in the experimental group, and a placebo syrup was used in the control group. Lesional skin swabs were taken before and after 28 days supplementation of vitamin D3. There was significant reduction of the Staphylococcus aureus colonization after vitamin D3 administration while there was no significant difference in the number of Staphylococcus aureus colonies after placebo administration. The statistic analysis of the two groups confirmed that there was a significant difference in the percentage of Staphylococcus aureus colonization in the vitamin D3 group compared to placebo. The supplementation of vitamin D3 decreased Staphylococcus aureus colonization in AD children, without significant side effects.