Detection of HHV-6 and EBV and Cytokine Levels in Saliva From Children With Seizures: Results of a Multi-Center Cross-Sectional Study

Abstract

Background and Objective: One third of children with epilepsy are refractory to medications. Growing data support a role of common childhood infections with neurotropic viruses and inflammation in epileptogenesis. Our objective was to determine the frequency of Human Herpesvirus-6 (HHV-6) and Epstein-Barr Virus (EBV) infection and cytokine levels in saliva from children with seizures compared to healthy controls and to controls with a febrile illness without seizures. Methods: In this cross-sectional multi-center study, we collected saliva from 115 consecutive children with acute seizures (cases), 51 children with a fever and no seizures or underlying neurological disease (fever controls) and 46 healthy children (healthy controls). Specimens were analyzed by a novel droplet digital PCR for HHV-6 and EBV viral DNA and a bead-based immunoassay for neuroinflammatory cytokines. Results: Cases included febrile seizures (n = 30), acute seizures without (n = 53) and with fever (n = 4) in chronic epilepsy, new onset epilepsy (n = 13), febrile status epilepticus (n = 3), and first lifetime seizure (n = 12). HHV-6 DNA was found in 40% of cases vs. 37% fever controls and 35% healthy controls, with no statistically significant differences. EBV DNA was also detected with no differences in 17% cases, 16% fever controls, and 28% healthy controls. IL-8 and IL-1β were increased in saliva of 32 random samples from cases compared with 30 fever controls: IL-8 cases mean (SD): 1158.07 pg/mL (1427.41); controls 604.92 (754.04); p = 0.02. IL-1β 185.76 (230.57); controls 86.99 (187.39); p = 0.0002. IL-1β level correlated with HHV6 viral load (p = 0.007). Conclusion: Increase in inflammatory cytokines may play a role in the onset of acute seizures and saliva could represent an inexpensive and non-invasive method for detection of viral DNA and cytokines.

Keywords: Epstein-Barr Virus; epilepsy; herpesvirus 6; human; inflammation; interleukin-1; pediatric; saliva.

Figure 1

(A, top) Representative two-dimensional ddPCR plot from a 2 years old boy with febrile seizure who tested positive for HHV-6B (green dots, right lower quadrant) in saliva. The x-axis displays the amplitude of fluorescence, corresponding to HHV-6B, while the y-axis displays the fluorescence amplitude of HHV-6A. Different probes were used to distinguish between HHV-6A (FAM) and HHV-6B (VIC). (B, bottom left) Representative plot for the detection of EBV DNA (blue dots, left upper quadrant) in saliva from a 14 years-old young man with an acute seizure in the context of chronic epilepsy. (C, bottom right) Example of low-amplitude lmp1 mutation in a 8 years-old child with epilepsy and acute seizures. ddPCR is a novel technique that allows for the direct absolute quantification of a target gene in a given sample, providing a precise and reliable method for quantification of proviral load, particularly when applied to samples with low numbers of cells.

Figure 2

Higher levels of IL-8 (A, left; Wilcoxon rank-sum test, W = 641, p = 0.02) and IL-1β (B, right; Wilcoxon rank-sum test, W = 738, p = 0.0002) in children with seizures vs. controls with fever.

Figure 3

Lack of correlation between fever grade and IL-8 (A, left; Pearson's correlation, r = 0.03, 95% CI –0.2804–0.3498, p = 0.81) and IL-1β level (B, right; Pearson's correlation, r = 0.06, 95% CI –0.2611–0.3679, p = 0.72). Red dots = cases; black dots = fever controls.

Figure 4

Positive correlation between age and IL-8 (A, left; Pearson's correlation, t = 2.3, 95% CI 0.04–0.50, p = 0.02) and IL-1β (B, right; Pearson's correlation, t = 2.3, 95% CI 0.04–0.50, p = 0.002).

Figure 5

Positive correlation between HHV-6B viral load and IL-1β level (B, right; polyserial correlation, cor = 0.34, p = 0.007) and absence of correlation for IL-8 (A, left; polyserial correlation, cor = 0.21, p = 0.09). PVL = proviral load.