Erwan Piriou

Early Age at Time of Primary Epstein–Barr Virus Infection Results in Poorly Controlled Viral Infection in Infants From Western Kenya: Clues to the Etiology of Endemic Burkitt Lymphoma

BACKGROUND Infection with Epstein-Barr virus (EBV) early in life and repeated malaria exposure have been proposed as risk factors for endemic Burkitt lymphoma (eBL). METHODS Infants were enrolled from 2 rural sites in Kenya: the Kisumu District, where malaria transmission is holoendemic and risk for eBL is high, and the Nandi District, where malaria transmission is limited and the risk for eBL is low. Blood samples were taken from infants through 2 years of age to measure EBV viral load, EBV antibodies, and malaria parasitemia. RESULTS We observed a significantly younger age at time of primary EBV infection in children from Kisumu compared with children from Nandi (mean age, 7.28 months [±0.33 SEM] in Kisumu vs 8.39 months [±0.26 SEM] in Nandi), with 35.3% of children in Kisumu infected before 6 months of age. To analyze how different predictors affected EBV viral load over time, we performed multilevel mixed modeling. This modeling revealed that residence in Kisumu and younger age at first EBV infection were significant predictors for having a higher EBV viral load throughout the period of observation. CONCLUSIONS Children from a region at high risk for eBL were infected very early in life with EBV, resulting in higher viral loads throughout infancy.

Altered EBV Viral Load Setpoint after HIV Seroconversion Is in Accordance with Lack of Predictive Value of EBV Load for the Occurrence of AIDS-Related Non-Hodgkin Lymphoma

In contrast to the situation in the post-transplant setting, in HIV-infected individuals an elevated EBV load is not predictive of EBV-related malignancies. To study whether a high EBV load is already a normal situation early in HIV infection and is not related to a decrease in immune function over time, we investigated EBV load and EBV-specific CD8+ T cells ∼1 year before and 1 year after HIV seroconversion. EBV load significantly increased after HIV seroconversion from 205 to 1002 copies/106 PBMC (p < 0.001), whereas no further increase in EBV load was observed between 1 and 5 years after HIV seroconversion (median, 1827–2478 copies/106 PBMC; p = 0.530). Interestingly, the absolute number of EBV lytic epitope, RAKFKQLL-specific CD8+ T cells increased over HIV seroconversion (4.78 to 9.54/μl; p = 0.011). Furthermore, the fraction of CD27-negative effector, RAK-specific CD8+ T cells tended to increase (from 12.2 to 17.31% CD27−; p = 0.051), in accordance with Ag-driven differentiation. In conclusion, both virological and immunological data support the idea that a new EBV viral setpoint is reached early in HIV infection, probably by EBV reactivation, as suggested by the preferential increase in EBV lytic epitope-specific CD8+ T cells. These data may thus help to explain the lack of predictive value of EBV load for the occurrence of AIDS-related lymphoma.

Serological evidence for long-term epstein–barr virus reactivation in children living in a holoendemic malaria region of Kenya†

To study the long term the effects of chronic exposure to P. falciparum malaria on Epstein–Barr virus (EBV) reactivation in children, EBV‐specific antibody levels were measured in a cross‐sectional survey of two groups of Kenyan children with divergent malaria exposure, varying in age from 1 to 14 years. A total of 169 children were analyzed within three age groups (1–4 years, 5–9 years and 10–14 years). Using a Luminex assay, elevated levels of IgG to EBV lytic and latent antigens were observed in children from the holoendemic malaria area; these remained elevated for each age group studied. In comparison, children from the sporadic malaria area had lower levels of EBV‐specific IgG antibodies and these levels declined across age groups. These data suggest that chronic exposure to malaria may lead to long‐term EBV reactivation. J. Med. Virol. 81:1088–1093, 2009.

Elevated anti-Zta IgG levels and EBV viral load are associated with site of tumor presentation in endemic Burkitt's lymphoma patients: a case control study.

BackgroundEndemic Burkitts lymphoma (BL) is an extranodal tumor appearing predominantly in the jaw in younger children while abdominal tumors predominate with increasing age. Previous studies have identified elevated levels of antibodies to Plasmodium falciparum schizont extracts and Epstein-Barr virus (EBV) viral capsid antigens (VCA) in endemic BL relative to malaria exposed controls. However, these studies have neither determined if there were any differences based on the site of clinical presentation of the tumor nor examined a broader panel of EBV and P. falciparum antigens.MethodsWe used a suspension bead Luminex assay to measure the IgG levels against EBV antigens, VCA, EAd, EBNA-1 and Zta as well as P. falciparum MSP-1, LSA-1, and AMA-1 antigens in children with BL (n = 32) and in population-based age-and sex-matched controls (n = 25) from a malaria endemic region in Western Kenya with high incidence of BL. EBV viral load in plasma was determined by quantitative PCR.ResultsRelative to healthy controls, BL patients had significantly increased anti-Zta (p = 0.0017) and VCA IgG levels (p < 0.0001) and plasma EBV viral loads (p < 0.0001). In contrast, comparable IgG levels to all P. falciparum antigens tested were observed in BL patients compared to controls. Interestingly, when we grouped BL patients into those presenting with abdominal tumors or with jaw tumors, we observed significantly higher levels of anti-Zta IgG levels (p < 0.0065) and plasma EBV viral loads (p < 0.033) in patients with abdominal tumors compared to patients with jaw tumors.ConclusionElevated antibodies to Zta and elevated plasma EBV viral load could be relevant biomarkers for BL and could also be used to confirm BL presenting in the abdominal region.

Epstein-Barr Virus Infects B and Non-B Lymphocytes in HIV-1–Infected Children and Adolescents

Epstein-Barr virus (EBV) is a widespread, persistent herpesvirus that can transform B cells and that is associated with malignant lymphomas. EBV dynamics and specific immunity in human immunodeficiency virus (HIV)-1-infected children are unknown. We found that, in 74% of EBV-seropositive, HIV-1-infected children, EBV DNA loads at the start of highly active antiretroviral therapy (HAART) were comparable with those in acutely EBV-infected, HIV-negative children. EBV DNA load remained elevated in most HIV-1-infected children for months to years of follow-up. Frequencies of interferon-gamma-producing EBV-specific CD8+ T cells were comparable with those in healthy control children, and antibodies to EBV nuclear antigen were detected in 73% of EBV-seropositive children. Detectable EBV DNA load was not correlated with HIV-1 RNA level or with CD4+ T cell count increase after the start of HAART. Because of its resemblance to chronic active EBV, we studied the cellular tropism of EBV in these patients. EBV DNA was found not only in the CD19+ B cell fraction but also--at stable levels--in the CD4+ and CD8+ T cell fractions. Although the reason for the aberrant T cell tropism of EBV remains unclear, these data may provide an explanation for the differential EBV dynamics in the presence of normal serological findings.

Suppression of circulating IgD+CD27+ memory B cells in infants living in a malaria-endemic region of Kenya

BackgroundPlasmodium falciparum infection leads to alterations in B cell subset distribution. During infancy, development of peripheral B cell subsets is also occurring. However, it is unknown if infants living a malaria endemic region have alterations in B cell subsets that is independent of an age effect.MethodsTo evaluate the impact of exposure to P. falciparum on B cell development in infants, flow cytometry was used to analyse the distribution and phenotypic characteristic of B cell subsets in infant cohorts prospectively followed at 12, 18 and 24 months from two geographically proximate regions in western Kenya with divergent malaria exposure i.e. Kisumu (malaria-endemic, n = 24) and Nandi (unstable malaria transmission, n = 21).ResultsThere was significantly higher frequency and absolute cell numbers of CD19+ B cells in Kisumu relative to Nandi at 12(p = 0.0440), 18(p = 0.0210) and 24 months (p = 0.0493). No differences were observed between the infants from the two sites in frequencies of naïve B cells (IgD+CD27-) or classical memory B cells (IgD-CD27+). However, immature transitional B cells (CD19+CD10+CD34-) were higher in Kisumu relative to Nandi at all three ages. In contrast, the levels of non-class switched memory B cells (CD19+IgD+CD27+) were significantly lower overall in Kisumu relative to Nandi at significantly at 12 (p = 0.0144), 18 (p = 0.0013) and 24 months (p = 0.0129).ConclusionsThese data suggest that infants living in malaria endemic regions have altered B cell subset distribution. Further studies are needed to understand the functional significance of these changes and long-term impact on ability of these infants to develop antibody responses to P. falciparum and heterologous infections.

Variation in Specificity of HIV Rapid Diagnostic Tests over Place and Time: An Analysis of Discordancy Data Using a Bayesian Approach

Background Recent trends to earlier access to anti-retroviral treatment underline the importance of accurate HIV diagnosis. The WHO HIV testing strategy recommends the use of two or three rapid diagnostic tests (RDTs) combined in an algorithm and assume a population is serologically stable over time. Yet RDTs are prone to cross reactivity which can lead to false positive or discordant results. This paper uses discordancy data from Médecins Sans Frontières (MSF) programmes to test the hypothesis that the specificity of RDTs change over place and time. Methods Data was drawn from all MSF test centres in 2007-8 using a parallel testing algorithm. A Bayesian approach was used to derive estimates of disease prevalence, and of test sensitivity and specificity using the software WinBUGS. A comparison of models with different levels of complexity was performed to assess the evidence for changes in test characteristics by location and over time. Results 106, 035 individuals were included from 51 centres in 10 countries using 7 different RDTs. Discordancy patterns were found to vary by location and time. Model fit statistics confirmed this, with improved fit to the data when test specificity and sensitivity were allowed to vary by centre and over time. Two examples show evidence of variation in specificity between different testing locations within a single country. Finally, within a single test centre, variation in specificity was seen over time with one test becoming more specific and the other less specific. Conclusion This analysis demonstrates the variable specificity of multiple HIV RDTs over geographic location and time. This variability suggests that cross reactivity is occurring and indicates a higher than previously appreciated risk of false positive HIV results using the current WHO testing guidelines. Given the significant consequences of false HIV diagnosis, we suggest that current testing and evaluation strategies be reviewed.

Impact of Plasmodium falciparum Coinfection on Longitudinal Epstein-Barr Virus Kinetics in Kenyan Children

Endemic Burkitt lymphoma is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum coinfection, although how P. falciparum exposure affects the dynamics of EBV infection is unclear. We have used a modeling approach to study EBV infection kinetics in a longitudinal cohort of children living in regions of high and low malaria transmission in Kenya. Residence in an area of high malaria transmission was associated with a higher rate of EBV expansion during primary EBV infection in infants and during subsequent episodes of EBV DNA detection, as well as with longer episodes of EBV DNA detection and shorter intervals between subsequent episodes of EBV DNA detection. In addition, we found that concurrent P. falciparum parasitemia also increases the likelihood of the first and subsequent peaks of EBV in peripheral blood. This suggests that P. falciparum infection is associated with increased EBV growth and contributes to endemic Burkitt lymphoma pathogenesis.

Tight Regulation of the Epstein-Barr Virus Setpoint: Interindividual Differences in Epstein-Barr Virus DNA Load Are Conserved after HIV Infection

Healthy individuals carry a constant number of Epstein-Barr virus-infected B cells in the peripheral blood over time. Here, we show that interindividual differences in Epstein-Barr virus DNA levels are maintained after HIV infection, providing evidence for the existence of an individual Epstein-Barr virus setpoint. Immune activation may contribute to the increase in this setpoint after human immunodeficiency virus seroconversion.

Feasibility of Xpert Ebola Assay in Médecins Sans Frontières Ebola Program, Guinea.

This assay provides results in less time than routine PCR and is equally sensitive.