76/176 children had at least 1 lacking sample Therefore, 39, 11, 5, 3 and 18 had 1, 2, 3, 4 and 5 lacking samples respectively.The 18 kids ARF3 with 5 consecutive missing samples were excluded through the analysis, leaving a complete of 158 kids for analysis. exchange and will occur in childhood in endemic regions [1]; KSHV prevalence increases with age [2]. The impact of age of infection with KSHV on the pathogenesis and control of KSHV has not been investigated. Early infection with Epstein-Barr virus (EBV), another gammaherpesvirus closely related to KSHV, is associated with higher subsequent viral load [3]. High viral capsid antigen (VCA) antibody titres and EBV viral load have been associated with risk of Burkitt lymphoma [4], the most common childhood malignancy in equatorial Africa, linked to both EBV and malaria [5]. High antibody titres to KSHV are an important predictor of risk of KS disease [6]; they are also a marker of KSHV reactivation [7]. This study was designed to determine the association between the age at which children from Uganda become KSHV seropositive (KSHV seroconversion age) and subsequent KSHV-specific IgG antibody values. Methods Study population Samples collected from children enrolled in the Entebbe Mother and Baby Study (EMaBS), were tested for KSHV IgG antibody responses retrospectively. Acebutolol HCl EMaBS was initiated as a randomised controlled trial, designed to investigate the impact of helminth treatment during pregnancy on childhood responses to vaccines and infectious diseases. The trial protocol and results have been described elsewhere [8]. A total of 2507 pregnant women from Entebbe, Uganda, a semi-urban area, were recruited and their children have been followed from birth. Blood and other samples have been collected annually and stored. Ethical approval This study was approved by the Uganda Virus Research Institute – Research and Ethics Committee (UVRI-REC), the Uganda National Council for Science and Technology (UNCST) and the London School of Hygiene & Tropical Medicine. Informed consent was obtained from study participants parents or guardians. KSHV serologic testing Plasma samples collected at age 6 years (annual 6) were tested for KSHV IgG antibodies to identify KSHV seropositive children. Acebutolol HCl KSHV sero-positivity was defined by sero-positivity to either ORF73 or K8.1 antigen. Any seropositive child was then tested at age 5, and so on retrospectively, until a seronegative specimen was identified. To determine if the effect of age at infection on subsequent antibody values is sustained for a longer time period, we then tested the available plasma samples at age 9 from the children who were seropositive at age 6 for IgG antibody responses to KSHV. The estimated age of KSHV seroconversion was defined as the midpoint between the last seronegative and the first seropositive specimen. Because of missing specimens, these samples were not always from consecutive years. An in-house multiplexed bead assay was used to measure KSHV-specific IgG antibody responses as previously described [9]. This assay has a wider dynamic range than an ELISA which is an important advantage when comparing antibody values. ORF73 and K8.1 recombinant proteins were coupled to fluorescent magnetic beads (Biorad, Hercules, CA) according to the manufacturers protocol. Coupled beads were mixed with plasma samples at a sample dilution of 1/200 and a bead concentration of 2,000 beads per well in assay/wash buffer (1% BSA/bovine serum albumin in 1XPBS/phosphate buffered saline), to make a total volume of 100 per well. The mixture was incubated for an hour under gentle agitation and washed with wash buffer thereafter. 100 of 0.5l/ml of detection antibody (goat F(ab)2 anti-human IgG R-PE conjugate) was added and incubated for 30 minutes under gentle agitation. After washing, 100 of assay buffer was Acebutolol HCl added per well, agitated for 2 minutes and the plate read using a Bioplex200 machine (Luminexcorp, USA) to obtain the Median Fluorescence Intensities (MFI). Each plate contained 3 negative and 3 positive control wells plus 2 blank wells. The cutoff Acebutolol HCl MFI values for OFR73 and K8.1 were 968 and 741 respectively plus the mean values of the negative control per plate. Statistical analysis Data analysis was performed using Stata-13 software (STATA 13.0, Statacorp, College Station, USA). Antibody values (measured as mean fluorescent intensities, MFI) were log10 transformed. Linear regression was used to examine the relationship between KSHV IgG antibody responses at ages 6.