Effect of Plasmodium falciparum Malaria on Epstein Barr Virus (EBV) Reactivation during Pregnancy and Subsequent Shedding of EBV in Breast milk Postpartum

Abstract

Epstein–Barr virus (EBV) is an oncogenic virus that has been implicated in the etiology of endemic Burkitt’s lymphoma (eBL). Infection with EBV early in life and repeated Plasmodium falciparum malaria exposures have been linked to the etiology of eBL. Previous study reported that 35% of children in Kisumu were infected before 6 months of age. However, the underlying mechanism that predisposes infants to EBV infection before 6 months of age and the role of P. falciparum malaria in EBV transmission is a significant gap in the etiology of eBL. The objective of this study was to determine the effect of P. falciparum malaria infection on EBV reactivation during pregnancy, and subsequent EBV shedding in breast milk postpartum among pregnant women. The study enrolled 175 HIV negative pregnant women attending the antenatal clinic and followed them prospectively from antenatal to delivery through to postpartum. Data on demographic, obstetrics and socioeconomic status were collected. Blood and breast milk samples were collected at various visits. Malaria diagnosis and EBV load were measured by quantitative polymerase chain reaction assay (Q-PCR). EBV serology was measured using ELISA. DNAse I based assay was used to assess whether there was encapsidated virus in breast-milk. EBV DNA positive breast-milk supernatant was exposed to 106 cells/ml Peripheral Blood Mononuclear Cells (PBMC) and observed for evidence of transformation. Results show that pregnant women who had malaria during pregnancy were more likely to have a detectable EBV DNA than pregnant women who had no evidence of malaria during pregnancy (64% vs. 36%, p=0.01). EBV load, as quantified using area under the longitudinal observation curve (AUC), was significantly higher in women with P. falciparum malaria than in women without malaria (p =0.01). Increase in EBV load correlated with that of malaria load (p <0.0001). Independent of malaria infection, EBV load was significantly higher at third trimester (p =0.04) than first and second trimester of pregnancy. EBV DNA and EBV load in breast-milk was significantly higher at 6 weeks and decreased sequentially in subsequent visits (p < .0001). Virus in breast milk supernatant was found to be DNAse I resistant in 24/40 (60%) of samples, and showed evidence of lymphocyte transformation. Being infected with P. falciparum infection at delivery was significantly associated with increased EBV shedding in early breast milk (p = 0.02), whereas, levels of EBV load in maternal blood at delivery was positively correlated with that of EBV load at 6 weeks postpartum (p = 0.002). The findings suggest that malaria during pregnancy causes EBV reactivation leading to high EBV load in maternal circulation, which subsequently increases shedding of infectious EBV in breast milk, a possible conduit of EBV transmission in infants at an early age. This study recommends sustained long-term efforts in malaria control programs such as, use of insecticide treated bed nets, intermittent preventive therapy, and indoor vector control, with a view to reducing EBV reactivation during pregnancy and subsequent EBV shedding in breast milk, consequently stemming eBL.