This retrospective study was conducted from November 2021 to October 2022 at Montgomery Allergy and Asthma in Montgomery, Alabama, and included a total of 195 patients aged 18 years and older. To be included in the selection, patients had to be diagnosed clinically with Chronic Spontaneous Urticaria and were currently being treated for this disorder. The testing for comorbidities was both routine and based on their current symptoms. Researchers reviewed medical records for demographic data—including age, sex, and race—along with documentation of comorbid conditions and relevant serological testing. Each patient was assigned an anonymous unique study identifier to maintain confidentiality. The presence of HSV-2 was tested by an IgG Serological Blood test. Vitamin D was measured using the 25 Hydroxyvitamin D Blood Test and a value <30 ng/ml was considered deficient in Vitamin D.

This study was reviewed and approved by the Institutional Review Board (IRB) at Huntingdon College. All patients had agreed to the release of their medical records for this study. This methodology aligns with the Declaration of Helsinki, which outlines ethical principles for medical research involving human subjects.

A significant portion of our CSU cohort tested positive for HSV-2, at a prevalence of 52.27% among those evaluated. This figure markedly exceeds the US national average of 11.9% [12] . Additionally, 90.7% of those who tested positive were women, echoing national trends that show a higher HSV-2 prevalence among females (25%) compared to males (17%) [12] . Due to the mucosal environment of the female reproductive track, a male-to female transmission is easier. However, the higher percentage we observe here may be due to the greater likelihood that women get tested more for HSV-2.

While our findings do not confirm causality, existing research suggests that HSV-2 infection can intensify certain immune pathways [13] . Specifically, HSV-2 infection of human genital epithelial cells is linked to increased Toll-like receptor 9 (TLR9) expression through the SP1/JNK signaling pathway—a key modulator of inflammatory processes [14] . The correlation between HSV-2 and CSU observed here may reflect an overlapping mechanism of heightened immune and inflammatory activity, though additional studies are needed to define the exact nature of this relationship.

Observed Among the 116 patients tested for Vitamin D, 84 (72.41%) were found to have deficient serum levels. These data are consistent with prior research showing that patients with Urticaria (acute and chronic) usually have lower Vitamin D levels compared to control groups [15] - [17] . This association suggests a possible connection between Vitamin D levels and inflammatory states associated with CSU, but it does not establish a direct causal relationship. We might expect Urticaria patients with positive HSV-2 test to have lower levels of Vitamin D. We tested this on our population. A total of 88 individuals were assessed for vitamin D deficiency and HSV-2 positivity. Of those who were vitamin D deficient, 23 (56.1%) tested positive for HSV-2, while among those who were not deficient, 23 (48.9%) were HSV-2 positive. The test yielded a χ² (n = 88) = 0.44 and a p = 0.50 indicating that there is no statistically significant association between Vitamin D deficiency and HSV-2 positivity in the cohort study. Vitamin D deficiency may be both a product of, and a contributor to, systemic inflammation, a hypothesis that warrants more in-depth evaluation through mechanistic and longitudinal studies.

Despite providing valuable insights, this study has limitations that should be addressed in future research. First, the single-institution, retrospective design limits the generalizability of our findings. Larger, multicenter studies could offer broader demographic representation and enhance statistical power, enabling more definitive conclusions about the associations observed. Moreover, while correlations were identified, causative relationships cannot be established without longitudinal or interventional approaches that can clarify temporal and mechanistic links between HSV-2, Vitamin D deficiency, and CSU.