Herpesvirus Screening in Childhood Hematopoietic Transplant Reveals High Systemic Inflammation in Episodes of Multiple Viral Detection and an EBV Association with Elevated IL-1β, IL-8 and Graft-Versus-Host Disease

Abstract

Infections remain a major cause of morbidity and mortality among hematopoietic stem cell transplant (HSCT) recipients. Unlike Epstein-Barr Virus (EBV) and Human Cytomegalovirus (HCMV), Human Herpesvirus (HHV) 6, HHV7 and HHV8 are not routinely monitored in many centers, especially in the pediatric population of low-medium income countries. We screened EBV, HCMV, HHV6, HHV7 and HHV8 in 412 leukocytes-plasma paired samples from 40 pediatric patients assisted in a tertiary hospital in Mexico. Thirty-two underwent allo-HSCT, whereas eight received auto-HSCT. Overall viral detection frequencies in allo- and auto-HSCT were: EBV = 43.7% and 30.0%, HCMV = 5.0% and 6.7%, HHV6 = 7.9% and 20.0% and HHV7 = 9.7% and 23.3%. HHV8 was not detected in any sample. Interestingly, HHV6 and HHV7 were more frequent in auto-HSCT, and HHV6 was observed in all episodes of multiple detection in auto-HSCT patients. We found EBV DNA in plasma samples, whereas HCMV, HHV6 and HHV7 DNA were predominantly observed in leukocytes, indicative of their expansion in cellular compartments. We also found that IL-1β, IL-2, IL-6 and IL-8 were significantly increased in episodes in which multiple viruses were simultaneously detected, and samples positive for EBV DNA and graft-versus-host disease had a further increase of IL-1β and IL-8. In conclusion, the EBV, HCMV, HHV6 and HHV7 burdens were frequently detected in allo- and auto-HSCT, and their presence associated with systemic inflammation.

Keywords: EBV; HCMV; HHV6; HHV7; graft-versus-host disease; hematopoietic stem cell transplant; herpesvirus; systemic inflammation.

Figure 1

Frequency of single and multiple detection of EBV, HCMV, HHV6 and HHV7 in childhood allogeneic (allo) and autologous (auto) hematopoietic stem cell transplant (HSCT). Comparison of detection status (a), overall detection (b) and multiple detection (c) episodes between allo- and auto-HSCT. In (b), frequencies were compared by the Test of Difference in proportions. (d) Magnitude of the viral load seen for each virus in allo-, auto-HSCT and healthy donors. Mann–Whitney’s test was used to compare allo- versus auto-HSCT.

Figure 2

EBV DNA is found mainly in plasma, whereas HCMV, HHV6 and HHV7 DNA are predominantly in leukocytes. (a) Example of the detection episodes in plasma and leukocytes in a patient with allo-HSCT during one year of follow-up. (b) Frequencies of EBV, HCMV, HHV6 and HHV7 detection in leukocytes, plasma or leukocytes and plasma simultaneously. (c) Paired analyses of the viral load in leukocytes and plasma for each virus. The mean value is up to the bars. Paired Mann–Whitney test. **** = p < 0.0001, ns = non-significant.

Figure 3

Samples in which multiple viruses were detected show high systemic levels of inflammatory cytokines. Plasma levels of IL-1β, IL-2, IL-6 and IL-8 in healthy donors, no viral detection, single detection ((a): any virus, (b): EBV) and multiple detection samples in patients with allogeneic transplant. Mann–Whitney test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 4

IL-1β and IL-8 increase in EBV-positive samples during GvHD. (a) Survival curve of patients with or without GvHD. Kaplan–Meier method, * = p < 0.05. (b) Plasma levels of the indicated cytokines in samples EBV-positive with or without GvHD. Mann–Whitney test., **** = p < 0.0001.