Research articlePrimary gamma-herpesviral infection in Zambian children

Background: HHV-8 is closely related to Epstein-Barr virus (EBV), but the clinical presentations of these two infections in early childhood are not well understood. Also, it is not known whether infection by one virus correlates with another. Here, we compare the natural history of infection by these two viruses along with the clinical manifestations and risk factors that are associated with early childhood infection in Zambia, which is an endemic area for HHV-8. Methods: This study was conducted in a cohort of 12 month old Zambian children (N = 677). Data on socio-economic status and a wide range of clinical manifestations were collected. Logistic regression was used to test for significant associations between the collected variables and HHV-8 or EBV serostatus at 12 months of age. Results: We observed a significantly higher seroprevalence for EBV (58.9%) as compared to HHV-8 (13.4%). HIV-1 infected children had at a significantly higher risk of being infected with HHV-8 (Odds ratio [OR] 3.69, 95% confidence interval [CI] 1.64 8.32). HIV-1 infection of the mothers was a significant risk factor for increased acquisition of EBV but not HHV-8 by children (OR 1.86, 05% CI 1.20 2.87). Self reported rash was marginally associated with primary infection for HHV-8 and EBV. Conclusions: These results suggest that there is no correlation between EBV and HHV-8 infections. Infection by one does not increase the susceptibility for the second virus. Primary HHV-8 and EBV infection in early childhood may clinically present as rash but remains largely asymptomatic and may remain undetected in this population. HIV infection in the mother or child are important risk factors that contribute to EBV or HHV-8 infection. Background Human herpesvirus-8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-herpesvirus reported to be associated with all forms of Kaposi's sarcoma (KS) [1,2]. HHV-8 is closely related to Epstein-Barr virus (EBV), the only other known human gamma-herpesvirus with both having a latent and lytic phase of replication. EBV is the most commonly identified virus associated with AIDS-related non-Hodgkin lymphomas, CNS lymphomas, African Burkitt's Lymphoma, and Nasopharyngeal Carcinoma [3,4]. HIV-associated immunosuppression has led to an increase in the incidence of childhood Kaposi's sarcoma and non-Hodgkin lymphomas including African Burkitt's lymphoma in sub-Saharan Africa [5]. Little is known about the acquisition of HHV-8 and EBV in early childhood in endemic African countries like Zambia especially after the onset of the HIV-1 epidemic in this region. Whether infection by one virus enhances the susceptibility to infection by the other in the context of HIV-1 infection and immune-suppression is not known. Most studies that documented the prevalence of HHV-8 have sampled a cross-section of the population. These studies have reported various factors that are associated with increased risk for HHV-8 prevalence but not on primary HHV-8 infection and the associated clinical symptoms. In addition, it is still not clear whether primary infection with HHV-8 produces an acute clinical syndrome and what factors increase the risk of acquisition of HHV-8 and EBV. Only one study conducted on immunocompetent children in Egypt has reported that primary HHV-8 infection may present itself as a febrile illness and the infected children developed a maculopapular rash [6]. Whether similar clinical symptoms are observed in endemic areas such as Zambia and whether * Correspondence: cwood@unlnotes.unl.edu 1 Nebraska Center for Virology and School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 USA Full list of author information is available at the end of the article Minhas et al. BMC Infectious Diseases 2010, 10:115 http://www.biomedcentral.com/1471-2334/10/115 Page 2 of 11 symptoms are similar for primary HHV-8 and EBV infections is still not known. As a part of our previous cohort studies in Zambia, we recruited newborn infants and followed them for up to 48 months of age. We have found that approximately 40% of children became infected with HHV-8 by 48 months of age [7]. Our cohort provided us an opportunity to further investigate early childhood infection in children less than 12 months of age. We anticipate that children of this age group will most likely undergo primary infection rather than reactivation of latent infection. Due to the prospective design and data collection during follow-up, our study provides a unique opportunity to examine potential risks with these two herpesviruses in the same population. The objective of this study was to determine HHV-8 and EBV seroprevalence in the children, the effect of HIV-1 infection, the specific clinical manifestations and the risk factors involved. Here, we report the prevalence of HHV-8 and/or EBV serostatus in 12 month old Zambian children where primary infection event had occurred. We also report the clinical symptoms during the primary infection, the association of primary infection with the HIV-1 status of the child and analyze the association of a positive serostatus at 12 months of age with markers of social and economic status. Methods Study participants Enrollment and follow-upAs described previously [7], study participants were recruited as part of a prospective cohort of mother/child pairs at the University Teaching Hospital (UTH) in Lusaka, Zambia between October 1998 and April 2004. Women in the early stages of labor and their newborn children were enrolled in a prospective cohort study after the mothers were counseled, educated about the study, and had given written informed consent. The study was approved by the Institutional Review Boards of the University of Zambia, University of Nebraska, and University of Miami. A total of 1,424 mother-child pairs who returned for at least one postpartum visit constituted our longitudinal cohort (Figure 1). Inclusion and exclusion criteria Among the cohort of 684 children of 12 months of age, adequate amount of plasma for reliable testing for HHV-8 and EBV was not available for 7 children. Therefore, the present analysis includes 677 children who survived and were followed up till at least 24 months of age in order to establish reliable HIV-1 status. Children who did not return for a follow-up visit at age 24 months (n = 740) were excluded from this analysis. Other reasons for exclusion included early mortality, early withdrawal, and loss to follow-up before HIV-1 serostatus could be reliably established. Diagnostic PCR testing for HIV-1 in children below 18 months of age was not available in Zambia at the time of this study. Therefore, HIV-1 serology was performed between 18 24 months of age. By 24 months of age, 6 percent (41/677) of the children tested positive for HIV-1. Serological testing Blood specimens collected from children were coded by a unique identification number and analyzed without knowledge of the personal identity of the study participants. To rule out detection of transplacental maternal HHV-8 antibodies, child plasma was not screened for HHV-8 infection until the age of 12 months and for HIV1 infection until the age of 18 months. HHV-8 serology All plasma samples were tested by monoclonal-enhanced immunofluorescence assay (mIFAs) as described previously [8]. Briefly, BC3 mIFAs were conducted by stimulating BC-3 cells with tetradecanoyl phorbol acetate (TPA), which were then fixed with 4% paraformaldehyde and permeabilized after 72 hours. To reduce subjectivity in observing specific fluorescence, slides were read independently by two laboratory workers. All plasma determined to be positive by BC-3 mIFA was further confirmed using Sf9 mIFA. Recombinant baculoviruses expressing glutathione S-transferase tagged lytic proteins, ORF65 and K8.1A, and latent protein, ORF73 were used to develop Sf9 mIFAs. Baculovirus-infected Sf9 cells expressing GST alone were used as a negative control to detect background and nonspecific fluorescence. All infections were initiated separately, to maximize the level of proteins produced, harvested at 72 hpi and fixed using the BC3 cell method. The procedure of Sf9 mIFA was the same as that for BC3 mIFA. A sample was considered seropositive for HHV-8 only if it was positive at a standard serum dilution of 1:40 for both BC3 and Sf9 mIFA (with at least one antigen). To rule out the detection of residual maternal antibodies, plasma of all HHV-8-seropositive children at 12 months who were born to HHV-8seropositive mothers was titered at birth, at 6 months, and at 12 months. Primary infection event occurred when previously seronegative children became seropositive at 12 months of age. EBV serology IgG antibodies against EBV viral capsid antigen (VCA) were detected using a standard Enzyme Linked Immunosorbent Assay kit. (ELISA) (Diagnostic Automation, California). This commercially available kit is based on recombinant purified protein of the VCA complex and has a specificity and sensitivity of 100% as reported by the manufacturer. Plasma was diluted 1:21 as per the manufacturer's instructions, and three calibrators and control sera were tested in each test run. The optical density was read on a microplate reader (Biotek instruments) at 450 Minhas et al. BMC Infectious Diseases 2010, 10:115 http://www.biomedcentral.com/1471-2334/10/115 Page 3 of 11 nm. Results were accepted when all the quality control criteria were met as per the manufacturer's recommendation. HIV-1 Test All plasma was screened by a standard HIV-1 test kit (Capillus HIV-1/2 Agglutination test kit, Trinity Biotech.) and confirmed for HIV-1 antibodies by another standard kit (Abbott Determine HIV-1/2 EIA, Abbott Laboratories). Statistical Analysis Data was analyzed using the statistical software package, SPSS version 17 (SPSS, Chicago, IL). Logistic regression was used to test for significant associations between the collected variables and serostatus at 12 months