Study design and population
As reported previously [24], this work was carried out within a rural African cohort, the General Population Cohort (GPC). The GPC is a community-based cohort of about 22,000 people in 25 adjacent villages in southwestern Uganda. It was established in 1989 to investigate the epidemiology of HIV; participants from the GPC have been followed ever since. The seroprevalence of KSHV is >90% in adults [26]. Between July 2017 and November 2017, we nested a cross-sectional study within the GPC enrolling 975 KSHV seropositive (tested previously [27]), HIV-negative individuals aged three to eighty-nine years. Participants were selected randomly after stratification for age and sex. Blood, stool and oral fluids samples were collected from these individuals. Socio-demographic data were collected using standard questionnaires. DNA was extracted from 2 million PBMCs collected and oral fluids pellets. This DNA was used to quantify both KSHV and EBV.
Peripheral blood mononuclear cells (PBMC) and plasma were obtained from the blood for immunological and virological analyses. Study participants were instructed to rinse with 5mL of Listerine mouthwash, and collect the resulting fluid in a polypropylene tube. Aliquots (of 1mL each) of oral fluids were spun at 13,000rcf for 10 minutes to form oral fluids pellets. Thereafter the supernatant was removed and the oral fluids pellet was stored at -80oC. A pellet of two million PBMCs and oral fluids pellets were processed for DNA extraction using a QIAamp blood kit (Qiagen, Valencia, CA), following the manufacturer's instructions.
EBV real-time PCR
Using DNA extracted previously [27], EBV DNA was quantified in PBMCs and oral fluids from 833 individuals with KSHV viral load data [24]. EBV viral load was quantified using real-time PCR. EBV DNA was amplified using primers (Balf5 EBV forward: 5' – CGG AAG CCC TCT GGA CTT C – 3', - Balf5 EBV reverse: 5' – CCC TGT TTA TCC GAT GGA ATG – 3') and probe ( Balf5 EBV Probe: 5' - /56-FAM/TGT ACA CGC ACG AGA AAT GCG CCT/3BHQ_1/ - 3') previously reported to be specific to the Balf5 gene [6, 28]. Additionally, B-Actin was amplified in the same sample as an internal positive control using primers (B-Actin forward: 5’ – TCA CCC ACA CTG TGC CCA TCT ACG A – 3’, B-Actin reverse: 5’ – CAG CGG AAC CGC TCA TTG CCA ATG G – 3’) and probe (B-Actin Probe: 5’ - /5HEX/ATG CCC TCC CCC ATG CCA TCC TGC GT/3BHQ_1/ - 3’) as previously reported [29].
KSHV and EBV serology
IgG antibody levels were quantified in plasma using ELISA and a multiplex bead-based assay as previously described [27, 30]. K8.1 and LANA/ORF73 recombinant proteins were used to quantify IgG by ELISA. Seropositivity was defined as reactivity to either K8.1 or ORF73 proteins. Each ELISA plate contained three positive and three negative control sera. The negative control sera were used to set a cut-off value on each plate as previously reported [31]. DNA was extracted from oral fluids and PBMCs of seropositive individuals. Twenty-five KSHV recombinant proteins including ORF73, K10.5, K5, K14, ORF6, ORF11, ORF55, ORF50, ORF60, K3, ORF38, ORF52, ORF59, ORF65, ORF61, ORF18, K11, K8.1, ORF19, ORF25, ORF26, ORF33, ORF37, ORF44 and ORF63 were included in the multiplex bead assay panel with three EBV proteins (EBNA-1, VCA and EA).
Statistical analysis
Statistical analysis was carried out using STATA version 13 (StataCorp, College Station, Texas USA). Graphs were drawn using STATA and GraphPad Prism version 8. Viral load levels were log10 transformed. First, risk factors associated with viral DNA detection (as a categorical outcome variable) in oral fluids and PBMCs, separately, were obtained using logistic regression modelling. Thereafter, risk factors associated with increasing levels of viral DNA (as a continuous outcome variable) in oral fluids and PBMCs, separately, were determined using linear regression modelling. Chi2 test, student T-test, Kruskal Wallis test and one-way ANOVA were used for crude analysis. The false discovery rate (FDR) was used to correct multiple comparisons of antibody data.