Associations Between Salivary HHV-6 And Long COVID Severity in a Cross-Sectional, Case-Control Analysis

Background Reactivation of human herpesviruses (HHVs), particularly EBV, is associated with more severe acute SARS-CoV-2 infection and the development of Long COVID (LC). Reactivation of HHVs has been previously implicated in hormonal dysregulation, which has also been observed in LC, suggesting a potential link. This study aims to examine the relationship between LC symptoms and salivary HHV DNA, cortisol, testosterone, and oestradiol levels. Methods Our cross-sectional, case-control study enrolled 45 participants with LC, and 45 age-sex-matched controls. Surveys and validated health questionnaires were used to collect demographics, medical history, and symptom profiles. Saliva was self-collected at waking, 15, 30, and 45 minutes, and 8 and 16 hours after waking, across two consecutive days. Salivary cortisol, testosterone and oestradiol were measured, and extracted nucleic acid was tested for EBV, HSV 1/2, HCMV and HHV-6 A/B using multiplex qPCR, and for SARS-CoV-2 and RNaseP using RT-qPCR. Findings Detection of salivary EBV and HHV-6 DNA copies was highest early in the morning. There were no significant differences in salivary cortisol, testosterone, or oestradiol, or in EBV or HHV-6 DNA between the LC and control groups. However, salivary HHV-6 DNA levels were positively associated with a greater aggregated LC propensity score (r= 0.24, 95% CI= 0.02 to 0.44, p= 0.015, adj. p= 0.030), as well as anxiety ({beta}= 0.69, 95% CI= 0.02 to 1.36, p= 0.045) and depression ({beta}= 0.24, 95% CI= 0.05 to 0.42, p= 0.013) scores. Interpretation The observed correlation between salivary HHV-6 DNA and symptom severity suggests HHV-6 may contribute to post-acute disease, though mechanisms remain unclear. While we did not identify a relationship between salivary EBV DNA and LC, EBV may still play a role at earlier time points in the disease course, or in compartments not sampled in this study. These findings highlight the potential importance of HHV-6 in LC pathophysiology and underscore the need for longitudinal, multi-compartment studies of herpesvirus reactivation in LC.