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Determinants of Gammaherpesvirus Shedding in Saliva Among Ugandan Children and Their Mothers

Determinants of Gammaherpesvirus Shedding in Saliva Among Ugandan Children and Their Mothers Abstract Background Epstein-Barr virus (EBV) and Kaposi sarcoma–associated herpesvirus (KSHV) are transmitted via saliva, but factors associated with salivary shedding are unknown. Methods We measured the DNA load of both viruses in saliva specimens collected from approximately 500 Ugandan mothers and their 6-year-old children, testing all participants for EBV and KSHV-seropositive individuals for KSHV. Results EBV and KSHV were shed by 72% and 22% of mothers, respectively, and by 85% and 40% of children, respectively; boys were more likely than girls to shed KSHV (48% vs 30%) but were equally likely to shed EBV. Children shed more KSHV and EBV than mothers, but salivary loads of EBV and KSHV were similar. KSHV shedding increased with increasing anti-KSHV (K8.1) antibodies in mothers and with decreasing antimalarial antibodies both in mothers and children. Among mothers, 40% of KSHV shedders also shed EBV, compared with 75% of KSHV nonshedders; among children, EBV was shed by 65% and 83%, respectively. Conclusions In summary, in this population, individuals were more likely to shed EBV than KSHV in saliva. We identified several factors, including child’s sex, that influence KSHV shedding, and we detected an inverse relationship between EBV and KSHV shedding, suggesting a direct or indirect interaction between the two viruses. EBV, KSHV, saliva, shedding, Uganda Kaposi sarcoma–associated herpesvirus (KSHV) is a necessary cause of Kaposi sarcoma [1, 2]. Unlike other human herpesviruses, KSHV is not ubiquitous across human populations; rather, the prevalence of infection shows considerable geographical variation, largely mirroring the variations seen in the incidence of Kaposi sarcoma. KSHV prevalence is relatively high in sub-Saharan Africa, lower in some Mediterranean countries, and lowest in most northern European and Asian populations [3], suggesting, in part, differences in transmission across regions. The factors that sustain higher rates of transmission in sub-Saharan Africa as compared to most other parts of the world are unclear. In Africa, primary infection begins in childhood, and prevalence increases with age. KSHV is shed in saliva [1, 4, 5], and this is the primary mode of transmission[1]. Conversely, infection by the related gammaherpesvirus Epstein-Barr virus (EBV), which also causes a number of human malignancies, is highly prevalent in all human populations [6]. Like KSHV, EBV is transmitted via saliva, but, in low-income settings, infection generally occurs much earlier in childhood, compared with high-income settings [7]. To investigate possible explanations for the differing epidemiology of these viruses, we compare here the prevalence and determinants of shedding of KSHV and EBV in saliva specimens collected from apparently healthy people in a Ugandan population with a high KSHV seroprevalence, on the assumption that viral shedding is an essential step in transmission [8]. We examined sociodemographic, clinical, and serological factors, including exposure to helminths and malaria, because, in previous work within this cohort, we have found these factors to be associated with KSHV seroprevalence [9, 10]. METHODS This was a cross-sectional study performed within the context of a clinical trial, the Entebbe Mother and Baby study (EMaBS; clinical trials registration ISRCTN32849447). The EMaBS is an ongoing birth cohort that originated as a double-blinded, randomized, placebo-controlled trial designed to determine the impact of helminth infections and their treatment on vaccine responses and infectious diseases outcomes; the details have been reported elsewhere [3, 4, 11, 12]. A total of 2507 pregnant women from Entebbe, Uganda, who consented, were enrolled into EMaBS and they and their children continue to be followed. In 2010, we systematically recruited into this substudy consenting human immunodeficiency virus (HIV)–negative mothers and their children, seen sequentially in the follow-up clinic. Additional plasma samples, together with a saliva sample, were collected from both mother and child and stored. Three mothers with HIV seroconversion after enrollment in the original study were excluded. Stored plasma samples from mothers and their 6-year old children were tested for the presence of KSHV antibodies by means of 2 enzyme-linked immunosorbent assays (ELISAs) using KSHV recombinant proteins: a lytic structural glycoprotein, K8.1, and the main latent nuclear protein, latency-associated nuclear antigen, encoded by ORF73. Results are reported as ODs. Each plate contained 3 positive and 3 negative controls; each assay cutoff was calculated on the basis of the performance of the negative controls. This procedure has been reported elsewhere [5, 13]. Individuals with positive results of either assay were considered KSHV seropositive. The same plasma samples were tested for malaria parasite antibodies, using 2 Plasmodium falciparum antigens: merozoite surface protein 1 (MSP-1) and apical membrane antigen 1 (AMA-1) [6, 14]. A pool of P. falciparum–positive plasma samples from patients known to be infected with malarial parasites was used to make standard dilutions. This pool was diluted serially 5 times, starting from 1:50 for MSP-1 and 1:100 for AMA-1, to make 6 standards with a 4-fold dilution increment. Blank wells were used to subtract background absorbance from the standards and the samples. The observed ODs were then exported into Microsoft Excel, and antibody titers for each sample and each antigen were derived from the standard curve of ODs. This procedure has been reported elsewhere [7, 15]. Saliva specimens were collected by having participants rinse their mouth with alcohol-based mouthwash and subsequently discharge this fluid into a 50-mL conical tube. DNA was extracted using a Qiagen blood and body fluids kit according to the manufacturer’s instructions. The KSHV load was measured using a quantitative real-time polymerase chain reaction (PCR) assay targeting the K6 gene [9, 10, 16, 17]. Similarly, the EBV load was determined using a quantitative real-time PCR specific for the EBNA1 gene [11, 18]. Another real-time assay for detection of the human endogenous retrovirus 3 (ERV-3), present in 2 copies in each diploid cell, was used to quantify cellular DNA [12, 19]. For each real-time PCR assay, each sample was assayed in triplicate, and an average of the 3 individual reactions was used to estimate the number of copies of the target gene. The viral DNA load was then calculated as the number of viral copies (genome equivalents) per million cells. Samples were designated as qualitatively positive if they could not be reliably quantitated (ie, if the amplification level for all 3 replicates exceeded the threshold value, but the average viral copy number was <3 in the KHSV assay or <10 in the EBV assay or if they failed to amplify in 1 or 2 of the 3 triplicate reactions). Qualitatively positive samples were retested, and if the results were confirmed, they were assigned the arbitrary value of 1 viral copy in further analyses. All mothers and children were tested for EBV in saliva, while KSHV-seropositive individuals were also tested for KSHV. Data analyses involved generating binary outcome variables for KSHV shedding; creating binary and categorical variables for demographic, socioeconomic, and illness information; and creating serological variables, including malarial antibody titers and KSHV antibody levels. For quantitative analyses, viral loads were log10 transformed. Variables considered to be possible risk factors or confounders for shedding in children were sex of child, maternal age group (categorized as 14–19, 20–24, 25–29, 30–34, or ≥35 years), maternal education level (none, primary, secondary, or tertiary), parity (1, 2–4, or ≥5 pregnancies), household socioeconomic status, maternal virus shedding, helminthiasis, anemia, anti-KSHV antibody levels (anti-K8.1 antigen and anti-ORF73 antigen, categorized as tertiles), and anti–P. falciparum antibody levels (categorized as tertiles). For shedding in mothers, possible risk factors considered were age group, education level, household socioeconomic status, HIV infection, anemia, helminthiasis, anti-KSHV antibody level, and anti–P. falciparum antibody levels. Because analysis on KSHV shedding is subset on KSHV-seropositive individuals, anti-KSHV antibody tertiles include only values predefined as positive, whereas anti–P. falciparum antibody levels include the entire range encountered in the sample. Initial analysis involved generating descriptive statistics by cross-tabulating viral shedding outcome variables and demographic, socioeconomic, illness, and immunologic variables that were considered to be possible risk factors for shedding. Logistic and linear regression models were fitted to examine variables predictive of shedding and viral load, respectively; nested modeling was used when twin children were included. Both prior knowledge from previous studies and a P value of <.05 in univariate analyses were used to select factors to be included in multivariable analyses. Likelihood ratio tests were used to determine adjusted P values. Analyses were conducted using Stata, version 13.1 (StataCorp, College Station, TX). Ethics Statement This study was approved by the Science and Ethics Committee of the Uganda Virus Research Institute, Uganda National Council for Science and Technology, and by the London School of Hygiene and Tropical Medicine Research Ethics Committee. Written and verbal information was provided in English and the vernacular; informed consent was obtained according to the Declaration of Helsinki and was recorded by signature or, in case of a participant’s inability to provide a signature, by thumbprint, as approved by the study’s institutional review boards. In Uganda, minors who are married, pregnant or have children are considered emancipated, and do not require parent/guardian consent to participate in research, therefore all participating mothers provided consent autonomously. Consent for participating children was provided by their mothers, fathers, or guardians. RESULTS Among participants in the EMaBS [11], we accrued for the present investigation 560 HIV-negative mothers and their 567 (including twins) 6-year-old children with KSHV serological data. Sociodemographic and other characteristics of the mothers at enrollment are shown in Table 1. Compared with the entire EMaBS cohort, mothers participating in this study were more likely to have higher education and income levels and less likely to have anemia. Table 1. Characteristics of Participating Mothers Characteristics . No. (%)a . Age group, y  14–19 112 (20.0)  20–24 222 (39.7)  25–29 136 (24.3)  30–34 62 (11.1)  ≥35 27 (4.8) Education level  None/primary 264 (47.2)  Secondary 232 (41.5)  Tertiary 63 (11.3) Marital status  Single/divorced/separated/widower 70 (12.5)  Married 488 (87.5) Monthly income, UGS  <30000 428 (80.6)  ≥30000 103 (19.4) Household socioeconomic status  Lower 234 (42.6)  Higher 314 (57.4) Pregnancies, no.  1 139 (24.9)  2–4 324 (58.0)  ≥5 97 (17.2) Characteristics . No. (%)a . Age group, y  14–19 112 (20.0)  20–24 222 (39.7)  25–29 136 (24.3)  30–34 62 (11.1)  ≥35 27 (4.8) Education level  None/primary 264 (47.2)  Secondary 232 (41.5)  Tertiary 63 (11.3) Marital status  Single/divorced/separated/widower 70 (12.5)  Married 488 (87.5) Monthly income, UGS  <30000 428 (80.6)  ≥30000 103 (19.4) Household socioeconomic status  Lower 234 (42.6)  Higher 314 (57.4) Pregnancies, no.  1 139 (24.9)  2–4 324 (58.0)  ≥5 97 (17.2) aValues might not sum to 560 because of missing data. Percentages were calculated using 560 as the denominator. bA total of 30000 Ugandan shillings (UGS) corresponds to the median national income. Open in new tab Table 1. Characteristics of Participating Mothers Characteristics . No. (%)a . Age group, y  14–19 112 (20.0)  20–24 222 (39.7)  25–29 136 (24.3)  30–34 62 (11.1)  ≥35 27 (4.8) Education level  None/primary 264 (47.2)  Secondary 232 (41.5)  Tertiary 63 (11.3) Marital status  Single/divorced/separated/widower 70 (12.5)  Married 488 (87.5) Monthly income, UGS  <30000 428 (80.6)  ≥30000 103 (19.4) Household socioeconomic status  Lower 234 (42.6)  Higher 314 (57.4) Pregnancies, no.  1 139 (24.9)  2–4 324 (58.0)  ≥5 97 (17.2) Characteristics . No. (%)a . Age group, y  14–19 112 (20.0)  20–24 222 (39.7)  25–29 136 (24.3)  30–34 62 (11.1)  ≥35 27 (4.8) Education level  None/primary 264 (47.2)  Secondary 232 (41.5)  Tertiary 63 (11.3) Marital status  Single/divorced/separated/widower 70 (12.5)  Married 488 (87.5) Monthly income, UGS  <30000 428 (80.6)  ≥30000 103 (19.4) Household socioeconomic status  Lower 234 (42.6)  Higher 314 (57.4) Pregnancies, no.  1 139 (24.9)  2–4 324 (58.0)  ≥5 97 (17.2) aValues might not sum to 560 because of missing data. Percentages were calculated using 560 as the denominator. bA total of 30000 Ugandan shillings (UGS) corresponds to the median national income. Open in new tab Mothers were generally young (median age, 23 years; range, 14–40 years), half had no education above primary school, and about a quarter had enrolled at their first pregnancy. Mothers were roughly distributed between lower (42.6%) and higher (57.4%) household socioeconomic status. Of mothers and children whose serological data were available, 299 (53%) and 102 (22%), respectively, were KSHV seropositive. KSHV DNA was detected in saliva twice as frequently in seropositive children (40% [40 of 99 tested]) as in seropositive mothers (21.5% [64 of 297]); however, while 27 of 56 boys (48%) had a detectable KSHV load, only 13 of 43 girls (30%) did (Figure 1). Among KSHV shedders, the median KSHV load was 3.6 log copies (interquartile range [IQR], 2.3–4.6 log copies) in mothers, versus 4.4 log copies (IQR, 3.5–4.7 log copies) in children (P = .03); there was no significant difference by sex of the child (median, 4.5 log copies [IQR, 3.5–4.9 log copies] in boys vs 4.4 log copies [IQR, 3.1–4.6 log copies] in girls). Figure 1. Open in new tabDownload slide Salivary shedding in mothers and children. Unadjusted proportion of mothers and children of either sex who are shedding Kaposi sarcoma–associated virus (KSHV) are estimated on KSHV-seropositive individuals only, whereas the prevalence of Epstein-Barr virus (EBV) shedding is estimated on the entire sample because all are assumed to be EBV seropositive. Figure 1. Open in new tabDownload slide Salivary shedding in mothers and children. Unadjusted proportion of mothers and children of either sex who are shedding Kaposi sarcoma–associated virus (KSHV) are estimated on KSHV-seropositive individuals only, whereas the prevalence of Epstein-Barr virus (EBV) shedding is estimated on the entire sample because all are assumed to be EBV seropositive. EBV DNA was prevalent in saliva specimens from both mothers (72% [402 of 559 tested]) and their children (85% [474 of 560]); the prevalence was similar in boys (84% [232 of 277]) and girls (86% [238 of 277]; Figure 1). However, the median viral load among EBV shedders was significantly higher in children than in their mothers (median, 4.7 log copies [IQR, 3.6–5.4 log copies] vs 3.9 log copies [IQR, 2.4–4.7 log copies]; P < .001) and did not differ significantly with respect to the sex of the child (4.6 log copies [IQR, 3.6–5.4 log copies] in boys vs 4.8 log copies [IQR, 3.6–5.4 log copies] in girls). In multivariate analysis, among mothers (Table 2), a detectable KSHV load was not associated with any demographic or clinical variable. KSHV shedding was directly associated with increasing levels of antibodies against KSHV K8.1 (odds ratio [OR] for medium and high OD tertiles to The low OD tertile, 80; 95% confidence interval [CI], 11–560) and inversely associated with antibodies against P. falciparum AMA-1 (OR, 0.25; 95% CI, .07–.86), but there was no association with level of antibodies against KSHV ORF73 nor with anti–P. falciparum MSP-1 antibodies. Detection of EBV DNA in mothers was not associated with any sociodemographic, clinical, or serological factor. Table 2. Factors Associated With Shedding of Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) in Saliva Among Mothers Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . aORb (95% CI) . Pc . Proportion (%) . aORb (95% CI) . Pc . Age group, y  14–19 14/73 (19.2) Reference NS 80/112 (71.4) Reference NS  20–24 24/116 (20.3) 1.09 (.52–2.29) 162/221 (73.3) 1.10 (.66–1.85)  25–29 16/65 (24.6) 1.42 (.62–3.26) 93/136 (68.4) 0.89 (.50–1.57)  30–34 8/29 (26.7) 1.47 (.53–4.06) 45/62 (72.6) 1.05 (.52–2.14)  ≥35 2/14 (14.3) 0.72 (.14–3.65) 21/27 (77.8) 1.67 (.57–4.87) Education level  Primary/none 36/147 (24.2) Reference NS 191/264 (72.0) Reference NS  Secondary 22/123 (17.7) 0.69 (.38–1.27) 167/231 (72.3) 1.00 (.66–1.49)  Tertiary 6/27 (22.2) 0.91 (.33–2.51) 44/63 (69.8) 0.89(.47–1.69) Household SES  Lower 29/154 (18.8) Reference 168/234 (71.8) Reference NS  Higher 35/140 (24.5) 0.70 (.40–1.24) NS 228/314 (72.6) 1.05 (.71–1.55) Anemia  No 35/188 (18.6) Reference 269/377 (71.4) Reference NS  Yes 29/109 (25.9) 1.60 (.90–2.83) NS 132/181 (72.9) 1.13 (.75–1.70) Anti-KSHV antibody OD  ORF73   Low 22/99 (22.0) Reference NS 34/99 (34.3) Reference NS   Medium 16/99 (15.0) 0.65 (.31–1.34) 27/99 (27.3) 1.00 (.53–1.90)   High 26/99 (27.0) 1.20 (.61–2.34) 33/99 (33.3) 0.71 (.38–1.31)  K8.1   Low 5/98 (5.0) Reference <.001 29/99 (29.6) Reference NS   Medium 25/98 (25.0) 7.22 (2.58–20.24) 30/100 (30.0) 1.36 (.73–2.53)   High 34/98 (34.0) 10.61 (3.87–29.12) 35/99 (35.4) 1.16 (.63–2.14) Anti–P. falciparum antibody titer  PfAMA1   Low 23/81 (28.4) Reference .02 136/183 (74.3) Reference NS   Medium 21/104 (20.2) 0.58 (.29–1.17) 132/184 (71.7) 0.87 (.54–1.39)   High 17/110 (15.5) 0.39 (.19–0.82) 129/186 (69.7) 0.83 (.52–1.33)  PfMSP1   Low 20/79 (25.3) Reference NS 136/181 (75.1) Reference NS   Medium 20/105 (19.0) 0.60 (.29–1.25) 140/185 (75.7) 1.04 (.64–1.70)   High 21/111 (18.9) 0.58 (.28–1.19) 121/186 (65.1) 0.60 (.38–0.96) Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . aORb (95% CI) . Pc . Proportion (%) . aORb (95% CI) . Pc . Age group, y  14–19 14/73 (19.2) Reference NS 80/112 (71.4) Reference NS  20–24 24/116 (20.3) 1.09 (.52–2.29) 162/221 (73.3) 1.10 (.66–1.85)  25–29 16/65 (24.6) 1.42 (.62–3.26) 93/136 (68.4) 0.89 (.50–1.57)  30–34 8/29 (26.7) 1.47 (.53–4.06) 45/62 (72.6) 1.05 (.52–2.14)  ≥35 2/14 (14.3) 0.72 (.14–3.65) 21/27 (77.8) 1.67 (.57–4.87) Education level  Primary/none 36/147 (24.2) Reference NS 191/264 (72.0) Reference NS  Secondary 22/123 (17.7) 0.69 (.38–1.27) 167/231 (72.3) 1.00 (.66–1.49)  Tertiary 6/27 (22.2) 0.91 (.33–2.51) 44/63 (69.8) 0.89(.47–1.69) Household SES  Lower 29/154 (18.8) Reference 168/234 (71.8) Reference NS  Higher 35/140 (24.5) 0.70 (.40–1.24) NS 228/314 (72.6) 1.05 (.71–1.55) Anemia  No 35/188 (18.6) Reference 269/377 (71.4) Reference NS  Yes 29/109 (25.9) 1.60 (.90–2.83) NS 132/181 (72.9) 1.13 (.75–1.70) Anti-KSHV antibody OD  ORF73   Low 22/99 (22.0) Reference NS 34/99 (34.3) Reference NS   Medium 16/99 (15.0) 0.65 (.31–1.34) 27/99 (27.3) 1.00 (.53–1.90)   High 26/99 (27.0) 1.20 (.61–2.34) 33/99 (33.3) 0.71 (.38–1.31)  K8.1   Low 5/98 (5.0) Reference <.001 29/99 (29.6) Reference NS   Medium 25/98 (25.0) 7.22 (2.58–20.24) 30/100 (30.0) 1.36 (.73–2.53)   High 34/98 (34.0) 10.61 (3.87–29.12) 35/99 (35.4) 1.16 (.63–2.14) Anti–P. falciparum antibody titer  PfAMA1   Low 23/81 (28.4) Reference .02 136/183 (74.3) Reference NS   Medium 21/104 (20.2) 0.58 (.29–1.17) 132/184 (71.7) 0.87 (.54–1.39)   High 17/110 (15.5) 0.39 (.19–0.82) 129/186 (69.7) 0.83 (.52–1.33)  PfMSP1   Low 20/79 (25.3) Reference NS 136/181 (75.1) Reference NS   Medium 20/105 (19.0) 0.60 (.29–1.25) 140/185 (75.7) 1.04 (.64–1.70)   High 21/111 (18.9) 0.58 (.28–1.19) 121/186 (65.1) 0.60 (.38–0.96) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum; SES, socioeconomic status. aData are for KSHV-seropositive individuals only. bAdjusted odds ratios (aORs) are estimated from multivariate logistic regression models that include age group, education level, and household SES, with the first 3 covariates adjusted for the other 2. cCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab Table 2. Factors Associated With Shedding of Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) in Saliva Among Mothers Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . aORb (95% CI) . Pc . Proportion (%) . aORb (95% CI) . Pc . Age group, y  14–19 14/73 (19.2) Reference NS 80/112 (71.4) Reference NS  20–24 24/116 (20.3) 1.09 (.52–2.29) 162/221 (73.3) 1.10 (.66–1.85)  25–29 16/65 (24.6) 1.42 (.62–3.26) 93/136 (68.4) 0.89 (.50–1.57)  30–34 8/29 (26.7) 1.47 (.53–4.06) 45/62 (72.6) 1.05 (.52–2.14)  ≥35 2/14 (14.3) 0.72 (.14–3.65) 21/27 (77.8) 1.67 (.57–4.87) Education level  Primary/none 36/147 (24.2) Reference NS 191/264 (72.0) Reference NS  Secondary 22/123 (17.7) 0.69 (.38–1.27) 167/231 (72.3) 1.00 (.66–1.49)  Tertiary 6/27 (22.2) 0.91 (.33–2.51) 44/63 (69.8) 0.89(.47–1.69) Household SES  Lower 29/154 (18.8) Reference 168/234 (71.8) Reference NS  Higher 35/140 (24.5) 0.70 (.40–1.24) NS 228/314 (72.6) 1.05 (.71–1.55) Anemia  No 35/188 (18.6) Reference 269/377 (71.4) Reference NS  Yes 29/109 (25.9) 1.60 (.90–2.83) NS 132/181 (72.9) 1.13 (.75–1.70) Anti-KSHV antibody OD  ORF73   Low 22/99 (22.0) Reference NS 34/99 (34.3) Reference NS   Medium 16/99 (15.0) 0.65 (.31–1.34) 27/99 (27.3) 1.00 (.53–1.90)   High 26/99 (27.0) 1.20 (.61–2.34) 33/99 (33.3) 0.71 (.38–1.31)  K8.1   Low 5/98 (5.0) Reference <.001 29/99 (29.6) Reference NS   Medium 25/98 (25.0) 7.22 (2.58–20.24) 30/100 (30.0) 1.36 (.73–2.53)   High 34/98 (34.0) 10.61 (3.87–29.12) 35/99 (35.4) 1.16 (.63–2.14) Anti–P. falciparum antibody titer  PfAMA1   Low 23/81 (28.4) Reference .02 136/183 (74.3) Reference NS   Medium 21/104 (20.2) 0.58 (.29–1.17) 132/184 (71.7) 0.87 (.54–1.39)   High 17/110 (15.5) 0.39 (.19–0.82) 129/186 (69.7) 0.83 (.52–1.33)  PfMSP1   Low 20/79 (25.3) Reference NS 136/181 (75.1) Reference NS   Medium 20/105 (19.0) 0.60 (.29–1.25) 140/185 (75.7) 1.04 (.64–1.70)   High 21/111 (18.9) 0.58 (.28–1.19) 121/186 (65.1) 0.60 (.38–0.96) Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . aORb (95% CI) . Pc . Proportion (%) . aORb (95% CI) . Pc . Age group, y  14–19 14/73 (19.2) Reference NS 80/112 (71.4) Reference NS  20–24 24/116 (20.3) 1.09 (.52–2.29) 162/221 (73.3) 1.10 (.66–1.85)  25–29 16/65 (24.6) 1.42 (.62–3.26) 93/136 (68.4) 0.89 (.50–1.57)  30–34 8/29 (26.7) 1.47 (.53–4.06) 45/62 (72.6) 1.05 (.52–2.14)  ≥35 2/14 (14.3) 0.72 (.14–3.65) 21/27 (77.8) 1.67 (.57–4.87) Education level  Primary/none 36/147 (24.2) Reference NS 191/264 (72.0) Reference NS  Secondary 22/123 (17.7) 0.69 (.38–1.27) 167/231 (72.3) 1.00 (.66–1.49)  Tertiary 6/27 (22.2) 0.91 (.33–2.51) 44/63 (69.8) 0.89(.47–1.69) Household SES  Lower 29/154 (18.8) Reference 168/234 (71.8) Reference NS  Higher 35/140 (24.5) 0.70 (.40–1.24) NS 228/314 (72.6) 1.05 (.71–1.55) Anemia  No 35/188 (18.6) Reference 269/377 (71.4) Reference NS  Yes 29/109 (25.9) 1.60 (.90–2.83) NS 132/181 (72.9) 1.13 (.75–1.70) Anti-KSHV antibody OD  ORF73   Low 22/99 (22.0) Reference NS 34/99 (34.3) Reference NS   Medium 16/99 (15.0) 0.65 (.31–1.34) 27/99 (27.3) 1.00 (.53–1.90)   High 26/99 (27.0) 1.20 (.61–2.34) 33/99 (33.3) 0.71 (.38–1.31)  K8.1   Low 5/98 (5.0) Reference <.001 29/99 (29.6) Reference NS   Medium 25/98 (25.0) 7.22 (2.58–20.24) 30/100 (30.0) 1.36 (.73–2.53)   High 34/98 (34.0) 10.61 (3.87–29.12) 35/99 (35.4) 1.16 (.63–2.14) Anti–P. falciparum antibody titer  PfAMA1   Low 23/81 (28.4) Reference .02 136/183 (74.3) Reference NS   Medium 21/104 (20.2) 0.58 (.29–1.17) 132/184 (71.7) 0.87 (.54–1.39)   High 17/110 (15.5) 0.39 (.19–0.82) 129/186 (69.7) 0.83 (.52–1.33)  PfMSP1   Low 20/79 (25.3) Reference NS 136/181 (75.1) Reference NS   Medium 20/105 (19.0) 0.60 (.29–1.25) 140/185 (75.7) 1.04 (.64–1.70)   High 21/111 (18.9) 0.58 (.28–1.19) 121/186 (65.1) 0.60 (.38–0.96) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum; SES, socioeconomic status. aData are for KSHV-seropositive individuals only. bAdjusted odds ratios (aORs) are estimated from multivariate logistic regression models that include age group, education level, and household SES, with the first 3 covariates adjusted for the other 2. cCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab Amongst seropositive mothers shedding KSHV (Table 3), no factor was associated with KSHV load. Similarly, no factors were associated with EBV load in EBV shedders. Table 3. Factors Associated With Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) Loads in Saliva Specimens From Shedding Mothers Factor . KSHV Load (n = 64) . EBV Load (n = 404) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Age group, y  14–19 Reference NS Reference NS  20–24 −0.36 (−1.61–0.90) −0.17 (−0.69–0.34)  25–29 0.14 (−1.24–1.52) −0.39 (−0.98–0.19)  30–34 −0.71 (−2.36–.94) −0.20 (−.90–.50)  ≥35 0.71 (−2.17–3.59) −0.13 (−1.05–.79) Education level  Primary/none Reference NS Reference NS  Secondary 0.78 (−.23–1.79) 0.24 (−.17–.64)  Tertiary 1.35 (−.31–3.01) 0.14 (−.51–.79) Household SES −0.03 (−.98–.93) NS −0.22 (−.60–.17) NS Anemia −0.23 (−1.22–.76) NS −0.01 (−.41–.40) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium 0.83 (−1.05–2.71) …   High 0.94 (−.89–2.77) …  ORF73   Low Reference NS …   Medium −0.81 (−2.08–.47) …   High 0.46 (−.70–1.62) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.19 (−.98–1.36) 0.26 (−.20–.72)   High 0.19 (−1.08–1.46) 0.28 (−.18–.73)  PfMSP1   Low Reference NS Reference NS   Medium −0.09 (−1.35–1.16) 0.14 (−.31–.59)   High 0.06 (−1.17–1.29) 0.36 (−.10–.83) Factor . KSHV Load (n = 64) . EBV Load (n = 404) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Age group, y  14–19 Reference NS Reference NS  20–24 −0.36 (−1.61–0.90) −0.17 (−0.69–0.34)  25–29 0.14 (−1.24–1.52) −0.39 (−0.98–0.19)  30–34 −0.71 (−2.36–.94) −0.20 (−.90–.50)  ≥35 0.71 (−2.17–3.59) −0.13 (−1.05–.79) Education level  Primary/none Reference NS Reference NS  Secondary 0.78 (−.23–1.79) 0.24 (−.17–.64)  Tertiary 1.35 (−.31–3.01) 0.14 (−.51–.79) Household SES −0.03 (−.98–.93) NS −0.22 (−.60–.17) NS Anemia −0.23 (−1.22–.76) NS −0.01 (−.41–.40) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium 0.83 (−1.05–2.71) …   High 0.94 (−.89–2.77) …  ORF73   Low Reference NS …   Medium −0.81 (−2.08–.47) …   High 0.46 (−.70–1.62) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.19 (−.98–1.36) 0.26 (−.20–.72)   High 0.19 (−1.08–1.46) 0.28 (−.18–.73)  PfMSP1   Low Reference NS Reference NS   Medium −0.09 (−1.35–1.16) 0.14 (−.31–.59)   High 0.06 (−1.17–1.29) 0.36 (−.10–.83) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum; SES, socioeconomic status. aCoefficients expressing variation in log10 genome equivalents/million cells are estimated in shedding individuals only, using multivariate models that include age group, education level, and household SES, with the first 3 covariates adjusted for the other 2. bCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab Table 3. Factors Associated With Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) Loads in Saliva Specimens From Shedding Mothers Factor . KSHV Load (n = 64) . EBV Load (n = 404) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Age group, y  14–19 Reference NS Reference NS  20–24 −0.36 (−1.61–0.90) −0.17 (−0.69–0.34)  25–29 0.14 (−1.24–1.52) −0.39 (−0.98–0.19)  30–34 −0.71 (−2.36–.94) −0.20 (−.90–.50)  ≥35 0.71 (−2.17–3.59) −0.13 (−1.05–.79) Education level  Primary/none Reference NS Reference NS  Secondary 0.78 (−.23–1.79) 0.24 (−.17–.64)  Tertiary 1.35 (−.31–3.01) 0.14 (−.51–.79) Household SES −0.03 (−.98–.93) NS −0.22 (−.60–.17) NS Anemia −0.23 (−1.22–.76) NS −0.01 (−.41–.40) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium 0.83 (−1.05–2.71) …   High 0.94 (−.89–2.77) …  ORF73   Low Reference NS …   Medium −0.81 (−2.08–.47) …   High 0.46 (−.70–1.62) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.19 (−.98–1.36) 0.26 (−.20–.72)   High 0.19 (−1.08–1.46) 0.28 (−.18–.73)  PfMSP1   Low Reference NS Reference NS   Medium −0.09 (−1.35–1.16) 0.14 (−.31–.59)   High 0.06 (−1.17–1.29) 0.36 (−.10–.83) Factor . KSHV Load (n = 64) . EBV Load (n = 404) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Age group, y  14–19 Reference NS Reference NS  20–24 −0.36 (−1.61–0.90) −0.17 (−0.69–0.34)  25–29 0.14 (−1.24–1.52) −0.39 (−0.98–0.19)  30–34 −0.71 (−2.36–.94) −0.20 (−.90–.50)  ≥35 0.71 (−2.17–3.59) −0.13 (−1.05–.79) Education level  Primary/none Reference NS Reference NS  Secondary 0.78 (−.23–1.79) 0.24 (−.17–.64)  Tertiary 1.35 (−.31–3.01) 0.14 (−.51–.79) Household SES −0.03 (−.98–.93) NS −0.22 (−.60–.17) NS Anemia −0.23 (−1.22–.76) NS −0.01 (−.41–.40) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium 0.83 (−1.05–2.71) …   High 0.94 (−.89–2.77) …  ORF73   Low Reference NS …   Medium −0.81 (−2.08–.47) …   High 0.46 (−.70–1.62) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.19 (−.98–1.36) 0.26 (−.20–.72)   High 0.19 (−1.08–1.46) 0.28 (−.18–.73)  PfMSP1   Low Reference NS Reference NS   Medium −0.09 (−1.35–1.16) 0.14 (−.31–.59)   High 0.06 (−1.17–1.29) 0.36 (−.10–.83) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum; SES, socioeconomic status. aCoefficients expressing variation in log10 genome equivalents/million cells are estimated in shedding individuals only, using multivariate models that include age group, education level, and household SES, with the first 3 covariates adjusted for the other 2. bCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab The children of mothers shedding KSHV were not more likely to be KSHV seropositive (crude odds ratio [OR], 1.63 [95% CI, .9–2.93]; OR adjusted for maternal age group, education level, socioeconomic status, and sex of the child, 1.63 [95% CI, .9–2.99]). In multivariate analysis, among children (Table 4), the odds of shedding KSHV were higher in boys versus girls (OR, 2.22; 95% CI, .97–5.10) and in children with helminthiasis as compared to those without (OR, 7.63; 95% CI, .63–83.45), although the differences did not reach statistical significance, and the latter finding was based on only 4 children. The odds of shedding KSHV were inversely associated with antibodies against P. falciparum AMA-1 (OR for medium and high titers vs low titers, 0.11; 95% CI, .01–.77) and tended to increase with higher anti-KSHV antibody levels, although there was no statistically significant association. Table 4. Factors Associated With Shedding of Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) in Saliva Among Children Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . ORb (95% CI) . Pc . Proportion (%) . ORb (95% CI) . Pc . Sex  Female 13/43 (30.2) Reference NS 238/277 (85.9) Reference NS  Male 27/56 (48.2) 2.15 (.93–4.96) 232/277 (83.8) 0.72 (.30–1.73) Maternal age group, y  14–19 6/14 (42.9) Reference NS 100/110 (90.9) Reference .05  20–24 15/36 (41.7) 0.89 (.25–3.18) 186/224 (83.0) 0.28 (.07–1.09)  25–29 10/29 (34.5) 0.72 (.19–2.73) 111/131 (84.7) 0.36 (.08–1.52)  30–34 6/11 (54.5) 1.36 (.27–6.92) 54/63 (85.7) 0.42 (.07–2.34)  ≥35 3/9 (33.3) 0.54 (.09–3.19) 20/27 (74.1) 0.10 (.01–0.98) Parity  1 12/22 (54.5) Reference NS 114/135 (84.4) Reference NS  2–4 16/55 (29.6) 0.37 (.13–1.06) 279/324 (86.1) 1.27 (.45–3.62)  ≥5 12/23 (52.2) 0.90 (.27–2.94) 78/96 (81.3) 0.66 (.17–2.52) Maternal virus shedding  No 29/68 (42.6) Reference NS 124/150 (82.7) NS  Yes 4/17 (23.5) 0.39 (.11–1.33) 338/396 (85.4) 1.23 (.74–2.04) Anemia  No 29/77 (37.6) Reference NS 367/430 (85.3) Reference NS  Yes 11/22 (50.0) 1.51 (.57–3.98) 107/130 (82.3) 0.63 (.23–1.76) Helminthiasis  No 15/41 (36.5) Reference NS 251/287 (87.6) Reference NS  Yes 3/4 (75.0) 7.23 (.63–83.45) 14/18 (77.8) 0.18 (.01–5.52) Anti-KSHV antibody OD  ORF73   Low 11/33 (33.3) Reference NS 9/33 (27.3) Reference NS   Medium 12/33 (36.4) 1.28 (.45–3.63) 9/33 (27.3) 0.86 (.28–2.65)   High 17/33 (51.5) 2.60 (.92–7.38) 6/33 (18.2) 1.39 (.41–4.66)  K8.1   Low 12/33 (36.4) Reference NS 4/33 (12.1) Reference NS   Medium 9/33 (27.3) 0.55 (.19–1.61) 11/33 (33.3) 0.33 (.09–1.23)   High 19/33 (57.6) 2.06 (.75–5.70) 9/33 (27.3) 0.45 (.12–1.71) Anti–P. falciparum antibody titer  PfAMA1   Low 11/24 (45.8) Reference NS 157/184 (85.3) Reference NS   Medium 11/25 (44.0) 1.02 (.32–3.23) 152/186 (81.7) 0.58 (.20–1.69)   High 17/48 (35.4) 0.67 (.24–1.86) 157/182 (86.3) 1.06 (.37–3.05)  PfMSP1   Low 14/23 (60.9) Reference .03 157/186 (84.4) Reference NS   Medium 12/36 (33.3) 0.32 (.11–0.97) 161/185 (87.0) 1.46 (.50–4.27)   High 13/38 (34.2) 0.33 (.11–1.00) 148/181 (81.8) 0.65 (.23–1.85) Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . ORb (95% CI) . Pc . Proportion (%) . ORb (95% CI) . Pc . Sex  Female 13/43 (30.2) Reference NS 238/277 (85.9) Reference NS  Male 27/56 (48.2) 2.15 (.93–4.96) 232/277 (83.8) 0.72 (.30–1.73) Maternal age group, y  14–19 6/14 (42.9) Reference NS 100/110 (90.9) Reference .05  20–24 15/36 (41.7) 0.89 (.25–3.18) 186/224 (83.0) 0.28 (.07–1.09)  25–29 10/29 (34.5) 0.72 (.19–2.73) 111/131 (84.7) 0.36 (.08–1.52)  30–34 6/11 (54.5) 1.36 (.27–6.92) 54/63 (85.7) 0.42 (.07–2.34)  ≥35 3/9 (33.3) 0.54 (.09–3.19) 20/27 (74.1) 0.10 (.01–0.98) Parity  1 12/22 (54.5) Reference NS 114/135 (84.4) Reference NS  2–4 16/55 (29.6) 0.37 (.13–1.06) 279/324 (86.1) 1.27 (.45–3.62)  ≥5 12/23 (52.2) 0.90 (.27–2.94) 78/96 (81.3) 0.66 (.17–2.52) Maternal virus shedding  No 29/68 (42.6) Reference NS 124/150 (82.7) NS  Yes 4/17 (23.5) 0.39 (.11–1.33) 338/396 (85.4) 1.23 (.74–2.04) Anemia  No 29/77 (37.6) Reference NS 367/430 (85.3) Reference NS  Yes 11/22 (50.0) 1.51 (.57–3.98) 107/130 (82.3) 0.63 (.23–1.76) Helminthiasis  No 15/41 (36.5) Reference NS 251/287 (87.6) Reference NS  Yes 3/4 (75.0) 7.23 (.63–83.45) 14/18 (77.8) 0.18 (.01–5.52) Anti-KSHV antibody OD  ORF73   Low 11/33 (33.3) Reference NS 9/33 (27.3) Reference NS   Medium 12/33 (36.4) 1.28 (.45–3.63) 9/33 (27.3) 0.86 (.28–2.65)   High 17/33 (51.5) 2.60 (.92–7.38) 6/33 (18.2) 1.39 (.41–4.66)  K8.1   Low 12/33 (36.4) Reference NS 4/33 (12.1) Reference NS   Medium 9/33 (27.3) 0.55 (.19–1.61) 11/33 (33.3) 0.33 (.09–1.23)   High 19/33 (57.6) 2.06 (.75–5.70) 9/33 (27.3) 0.45 (.12–1.71) Anti–P. falciparum antibody titer  PfAMA1   Low 11/24 (45.8) Reference NS 157/184 (85.3) Reference NS   Medium 11/25 (44.0) 1.02 (.32–3.23) 152/186 (81.7) 0.58 (.20–1.69)   High 17/48 (35.4) 0.67 (.24–1.86) 157/182 (86.3) 1.06 (.37–3.05)  PfMSP1   Low 14/23 (60.9) Reference .03 157/186 (84.4) Reference NS   Medium 12/36 (33.3) 0.32 (.11–0.97) 161/185 (87.0) 1.46 (.50–4.27)   High 13/38 (34.2) 0.33 (.11–1.00) 148/181 (81.8) 0.65 (.23–1.85) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum. aData are for KSHV-seropositive individuals only. bOdds ratios (ORs) are adjusted for sex except when sex is the factor of interest. cCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab Table 4. Factors Associated With Shedding of Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) in Saliva Among Children Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . ORb (95% CI) . Pc . Proportion (%) . ORb (95% CI) . Pc . Sex  Female 13/43 (30.2) Reference NS 238/277 (85.9) Reference NS  Male 27/56 (48.2) 2.15 (.93–4.96) 232/277 (83.8) 0.72 (.30–1.73) Maternal age group, y  14–19 6/14 (42.9) Reference NS 100/110 (90.9) Reference .05  20–24 15/36 (41.7) 0.89 (.25–3.18) 186/224 (83.0) 0.28 (.07–1.09)  25–29 10/29 (34.5) 0.72 (.19–2.73) 111/131 (84.7) 0.36 (.08–1.52)  30–34 6/11 (54.5) 1.36 (.27–6.92) 54/63 (85.7) 0.42 (.07–2.34)  ≥35 3/9 (33.3) 0.54 (.09–3.19) 20/27 (74.1) 0.10 (.01–0.98) Parity  1 12/22 (54.5) Reference NS 114/135 (84.4) Reference NS  2–4 16/55 (29.6) 0.37 (.13–1.06) 279/324 (86.1) 1.27 (.45–3.62)  ≥5 12/23 (52.2) 0.90 (.27–2.94) 78/96 (81.3) 0.66 (.17–2.52) Maternal virus shedding  No 29/68 (42.6) Reference NS 124/150 (82.7) NS  Yes 4/17 (23.5) 0.39 (.11–1.33) 338/396 (85.4) 1.23 (.74–2.04) Anemia  No 29/77 (37.6) Reference NS 367/430 (85.3) Reference NS  Yes 11/22 (50.0) 1.51 (.57–3.98) 107/130 (82.3) 0.63 (.23–1.76) Helminthiasis  No 15/41 (36.5) Reference NS 251/287 (87.6) Reference NS  Yes 3/4 (75.0) 7.23 (.63–83.45) 14/18 (77.8) 0.18 (.01–5.52) Anti-KSHV antibody OD  ORF73   Low 11/33 (33.3) Reference NS 9/33 (27.3) Reference NS   Medium 12/33 (36.4) 1.28 (.45–3.63) 9/33 (27.3) 0.86 (.28–2.65)   High 17/33 (51.5) 2.60 (.92–7.38) 6/33 (18.2) 1.39 (.41–4.66)  K8.1   Low 12/33 (36.4) Reference NS 4/33 (12.1) Reference NS   Medium 9/33 (27.3) 0.55 (.19–1.61) 11/33 (33.3) 0.33 (.09–1.23)   High 19/33 (57.6) 2.06 (.75–5.70) 9/33 (27.3) 0.45 (.12–1.71) Anti–P. falciparum antibody titer  PfAMA1   Low 11/24 (45.8) Reference NS 157/184 (85.3) Reference NS   Medium 11/25 (44.0) 1.02 (.32–3.23) 152/186 (81.7) 0.58 (.20–1.69)   High 17/48 (35.4) 0.67 (.24–1.86) 157/182 (86.3) 1.06 (.37–3.05)  PfMSP1   Low 14/23 (60.9) Reference .03 157/186 (84.4) Reference NS   Medium 12/36 (33.3) 0.32 (.11–0.97) 161/185 (87.0) 1.46 (.50–4.27)   High 13/38 (34.2) 0.33 (.11–1.00) 148/181 (81.8) 0.65 (.23–1.85) Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . ORb (95% CI) . Pc . Proportion (%) . ORb (95% CI) . Pc . Sex  Female 13/43 (30.2) Reference NS 238/277 (85.9) Reference NS  Male 27/56 (48.2) 2.15 (.93–4.96) 232/277 (83.8) 0.72 (.30–1.73) Maternal age group, y  14–19 6/14 (42.9) Reference NS 100/110 (90.9) Reference .05  20–24 15/36 (41.7) 0.89 (.25–3.18) 186/224 (83.0) 0.28 (.07–1.09)  25–29 10/29 (34.5) 0.72 (.19–2.73) 111/131 (84.7) 0.36 (.08–1.52)  30–34 6/11 (54.5) 1.36 (.27–6.92) 54/63 (85.7) 0.42 (.07–2.34)  ≥35 3/9 (33.3) 0.54 (.09–3.19) 20/27 (74.1) 0.10 (.01–0.98) Parity  1 12/22 (54.5) Reference NS 114/135 (84.4) Reference NS  2–4 16/55 (29.6) 0.37 (.13–1.06) 279/324 (86.1) 1.27 (.45–3.62)  ≥5 12/23 (52.2) 0.90 (.27–2.94) 78/96 (81.3) 0.66 (.17–2.52) Maternal virus shedding  No 29/68 (42.6) Reference NS 124/150 (82.7) NS  Yes 4/17 (23.5) 0.39 (.11–1.33) 338/396 (85.4) 1.23 (.74–2.04) Anemia  No 29/77 (37.6) Reference NS 367/430 (85.3) Reference NS  Yes 11/22 (50.0) 1.51 (.57–3.98) 107/130 (82.3) 0.63 (.23–1.76) Helminthiasis  No 15/41 (36.5) Reference NS 251/287 (87.6) Reference NS  Yes 3/4 (75.0) 7.23 (.63–83.45) 14/18 (77.8) 0.18 (.01–5.52) Anti-KSHV antibody OD  ORF73   Low 11/33 (33.3) Reference NS 9/33 (27.3) Reference NS   Medium 12/33 (36.4) 1.28 (.45–3.63) 9/33 (27.3) 0.86 (.28–2.65)   High 17/33 (51.5) 2.60 (.92–7.38) 6/33 (18.2) 1.39 (.41–4.66)  K8.1   Low 12/33 (36.4) Reference NS 4/33 (12.1) Reference NS   Medium 9/33 (27.3) 0.55 (.19–1.61) 11/33 (33.3) 0.33 (.09–1.23)   High 19/33 (57.6) 2.06 (.75–5.70) 9/33 (27.3) 0.45 (.12–1.71) Anti–P. falciparum antibody titer  PfAMA1   Low 11/24 (45.8) Reference NS 157/184 (85.3) Reference NS   Medium 11/25 (44.0) 1.02 (.32–3.23) 152/186 (81.7) 0.58 (.20–1.69)   High 17/48 (35.4) 0.67 (.24–1.86) 157/182 (86.3) 1.06 (.37–3.05)  PfMSP1   Low 14/23 (60.9) Reference .03 157/186 (84.4) Reference NS   Medium 12/36 (33.3) 0.32 (.11–0.97) 161/185 (87.0) 1.46 (.50–4.27)   High 13/38 (34.2) 0.33 (.11–1.00) 148/181 (81.8) 0.65 (.23–1.85) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum. aData are for KSHV-seropositive individuals only. bOdds ratios (ORs) are adjusted for sex except when sex is the factor of interest. cCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab The odds of shedding EBV tended to be lower in children born to mothers aged ≥20 years (OR, 0.3; 95% CI, .09–1.05) but were not otherwise associated with any sociodemographic, clinical, or serological factor. Among children shedding KSHV (Table 5), the viral load was higher in those who had helminths (2.1 log copies; 95% CI, .37–3.83) but was not associated with any other factor, whether demographic, clinical, or serological. Table 5. Factors Associated With Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) Loads in Saliva Specimens From Shedding Children Factor . KSHV Load (n = 40) . EBV Load (n = 477) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Male sex −0.10 (−1.19–.99) NS 0.20 (−.14–.56) NS Maternal age group, y  14–19 Reference NS Reference NS  20–24 1.03 (−.53–2.60) −0.05 (−.52–.42)  25–29 1.22 (−.46–2.89) −0.17 (−.69–.35)  30–34 0.34 (−1.52–2.21) −0.34 (−.98–.30)  ≥35 2.05 (−.29–4.39) −0.23 (−1.16–.70) Parity Reference .05 Reference NS  2–4 1.29 (.09–2.50) −0.25 (−.67–.17)  ≥5 0.91 (−.36–2.19) −0.20 (−.76–.36) Maternal viral loadc −3.83 (−16.54–8.89) NS 0.15 (.04–.25) .007 Anemia 0.09 (−1.07–1.25) NS −0.21 (−.62–.21) NS Helminthiasis 2.10 (.37–3.83) .02 −0.16 (−1.26–.93) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium −0.14 (−1.60–1.32) …   High 0.63 (−.67–1.93) …  ORF73   Low Reference NS …   Medium 0.54 (−.83–1.90) …   High 0.09 (−1.22–1.39) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.63 (−.79–2.05) −0.19 (−.62–.25)   High 0.11 (−1.22–1.45) −0.08 (−.51–.35)  PfMSP1   Low Reference NS Reference .01   Medium −0.35 (−1.78–1.07) −0.37 (−.75–.02)   High −0.12 (−1.47–1.24) −0.55 (−.97–−0.12) Factor . KSHV Load (n = 40) . EBV Load (n = 477) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Male sex −0.10 (−1.19–.99) NS 0.20 (−.14–.56) NS Maternal age group, y  14–19 Reference NS Reference NS  20–24 1.03 (−.53–2.60) −0.05 (−.52–.42)  25–29 1.22 (−.46–2.89) −0.17 (−.69–.35)  30–34 0.34 (−1.52–2.21) −0.34 (−.98–.30)  ≥35 2.05 (−.29–4.39) −0.23 (−1.16–.70) Parity Reference .05 Reference NS  2–4 1.29 (.09–2.50) −0.25 (−.67–.17)  ≥5 0.91 (−.36–2.19) −0.20 (−.76–.36) Maternal viral loadc −3.83 (−16.54–8.89) NS 0.15 (.04–.25) .007 Anemia 0.09 (−1.07–1.25) NS −0.21 (−.62–.21) NS Helminthiasis 2.10 (.37–3.83) .02 −0.16 (−1.26–.93) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium −0.14 (−1.60–1.32) …   High 0.63 (−.67–1.93) …  ORF73   Low Reference NS …   Medium 0.54 (−.83–1.90) …   High 0.09 (−1.22–1.39) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.63 (−.79–2.05) −0.19 (−.62–.25)   High 0.11 (−1.22–1.45) −0.08 (−.51–.35)  PfMSP1   Low Reference NS Reference .01   Medium −0.35 (−1.78–1.07) −0.37 (−.75–.02)   High −0.12 (−1.47–1.24) −0.55 (−.97–−0.12) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum. aCoefficients expressing variation in log10 genome equivalents/million cells are estimated in shedding individuals only and are adjusted for sex except when sex is the factor of interest. bCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. cScaled in increments of log10 copies. Open in new tab Table 5. Factors Associated With Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) Loads in Saliva Specimens From Shedding Children Factor . KSHV Load (n = 40) . EBV Load (n = 477) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Male sex −0.10 (−1.19–.99) NS 0.20 (−.14–.56) NS Maternal age group, y  14–19 Reference NS Reference NS  20–24 1.03 (−.53–2.60) −0.05 (−.52–.42)  25–29 1.22 (−.46–2.89) −0.17 (−.69–.35)  30–34 0.34 (−1.52–2.21) −0.34 (−.98–.30)  ≥35 2.05 (−.29–4.39) −0.23 (−1.16–.70) Parity Reference .05 Reference NS  2–4 1.29 (.09–2.50) −0.25 (−.67–.17)  ≥5 0.91 (−.36–2.19) −0.20 (−.76–.36) Maternal viral loadc −3.83 (−16.54–8.89) NS 0.15 (.04–.25) .007 Anemia 0.09 (−1.07–1.25) NS −0.21 (−.62–.21) NS Helminthiasis 2.10 (.37–3.83) .02 −0.16 (−1.26–.93) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium −0.14 (−1.60–1.32) …   High 0.63 (−.67–1.93) …  ORF73   Low Reference NS …   Medium 0.54 (−.83–1.90) …   High 0.09 (−1.22–1.39) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.63 (−.79–2.05) −0.19 (−.62–.25)   High 0.11 (−1.22–1.45) −0.08 (−.51–.35)  PfMSP1   Low Reference NS Reference .01   Medium −0.35 (−1.78–1.07) −0.37 (−.75–.02)   High −0.12 (−1.47–1.24) −0.55 (−.97–−0.12) Factor . KSHV Load (n = 40) . EBV Load (n = 477) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Male sex −0.10 (−1.19–.99) NS 0.20 (−.14–.56) NS Maternal age group, y  14–19 Reference NS Reference NS  20–24 1.03 (−.53–2.60) −0.05 (−.52–.42)  25–29 1.22 (−.46–2.89) −0.17 (−.69–.35)  30–34 0.34 (−1.52–2.21) −0.34 (−.98–.30)  ≥35 2.05 (−.29–4.39) −0.23 (−1.16–.70) Parity Reference .05 Reference NS  2–4 1.29 (.09–2.50) −0.25 (−.67–.17)  ≥5 0.91 (−.36–2.19) −0.20 (−.76–.36) Maternal viral loadc −3.83 (−16.54–8.89) NS 0.15 (.04–.25) .007 Anemia 0.09 (−1.07–1.25) NS −0.21 (−.62–.21) NS Helminthiasis 2.10 (.37–3.83) .02 −0.16 (−1.26–.93) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium −0.14 (−1.60–1.32) …   High 0.63 (−.67–1.93) …  ORF73   Low Reference NS …   Medium 0.54 (−.83–1.90) …   High 0.09 (−1.22–1.39) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.63 (−.79–2.05) −0.19 (−.62–.25)   High 0.11 (−1.22–1.45) −0.08 (−.51–.35)  PfMSP1   Low Reference NS Reference .01   Medium −0.35 (−1.78–1.07) −0.37 (−.75–.02)   High −0.12 (−1.47–1.24) −0.55 (−.97–−0.12) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum. aCoefficients expressing variation in log10 genome equivalents/million cells are estimated in shedding individuals only and are adjusted for sex except when sex is the factor of interest. bCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. cScaled in increments of log10 copies. Open in new tab The EBV load was significantly higher in children of mothers with a higher EBV load (0.15 log copies; 95% CI, .04–.26). Furthermore, the EBV load was lower in children with medium or high P. falciparum MSP-1 antibody levels (−1.05 log copies; 95% CI, −1.77 to −.33). Among KSHV-seropositive participants tested for both viruses, 57% shed only EBV, 13% only KSHV, 13% shed both viruses, and 17% did not shed either virus. Among mothers, 40% of KSHV shedders also shed EBV, compared with 75% of those who did not shed KSHV; for children, values were 65% and 83%, respectively (Supplementary Table 1). When we examined the relationship between KSHV load and EBV load, restriction of the analysis to KSHV-seropositive individuals revealed a negative correlation (ρ = −0.22; P < .0001). The correlation strengthened among KSHV shedders (ρ = −0.30; P < .001). However, when the analysis was restricted to EBV shedders, a modest positive correlation was observed (ρ = 0.15; P = .01). Stratification of mothers and children yielded similar results (except that the variance was larger in the latter case because of the smaller number of KSHV-seropositive children). DISCUSSION To our knowledge, this is the first study of factors associated with shedding of KSHV and EBV in saliva—a mechanism for viral transmission—in a sample of apparently healthy, HIV-uninfected people from a population in which both viruses are very prevalent and in which the tumors they cause, Kaposi sarcoma and Burkitt lymphoma, are endemic [20]. The prevalence of KSHV shedding was similar between mothers and girls, but it tended to be higher in boys. Examination of the KSHV load in individuals who shed revealed that it was higher in children than in mothers but was similar in boys and girls. The reasons for the greater prevalence and, presumably, frequency of shedding among boys, compared with that among mothers and girls, are not clear. In 2 other studies of adults and children from Uganda that examined the prevalence of KSHV in blood, Mbulaiteye and colleagues showed that males were twice as likely to have detectable virus in blood than females [18, 21, 22]. One of these studies [21] found no difference in the prevalence of detectable virus in saliva between boys and girls. However, in combination with the fact that, among people without HIV infection, Kaposi sarcoma shows a marked excess incidence among men as compared to women [23, 24], this might suggest that males are less able to control KSHV replication than females. Our results further suggest that such differential control may be established at an early age, suggesting that nonreproductive factors might be at play, whether genetic or environmental, due to early sex- or gender-specific exposures/behaviors. That many more children than mothers shed also suggests that children may be an important source of KSHV transmission both within the family and in the wider community. Further studies of KSHV transmission between children are warranted. In mothers, we detected strong associations between KSHV shedding and anti–KSHV K8.1 antibody levels; this is consistent with the hypothesis that antibodies against the K8.1 antigen reflect lytic viral replication [25]. Such an association in children was not significant. While children shed more frequently, it is conceivable that the shorter time since KSHV acquisition did not yet result in levels of KSHV-specific antibodies that were greater than those in nonshedders; alternatively, or additionally, in the course of recent infection in children, the antibody response profile might be different than during established infection in adults. The viral load in peripheral blood mononuclear cells (PBMCs) is more likely to be relevant to KSHV pathogenesis than the viral load in saliva, and the correlation between the load in PBMCs and anti-KSHV antibody levels will need to be investigated directly. However, we can now confirm a previously observed association between anti-K8.1 antibody levels and KSHV reactivation, resulting in salivary shedding [8]. This further validates our previous observations on the possible role of the anti-K8.1 antibody level as a prognostic marker in the natural history of infection [25]. In previous work within this cohort, we identified associations between KSHV prevalence and malaria parasitemia in both mothers and children [9, 10]. More recently, we found that the KSHV prevalence was also associated with antibodies against P. falciparum malaria (both AMA-1 and MSP-1) in both mothers and children [26]. In this study, we found an association between AMA-1 levels and KSHV shedding in mothers and between MSP-1 levels and KSHV shedding in children, but we found no association between these anti–P. falciparum antibodies and the KSHV load in shedders, either mothers or children. Further prospective research on the role of malaria in the natural history of KSHV infection is justified. In keeping with our previous findings, we also observed a sizable association between fecal detection of helminths in children and both KSHV shedding and viral load, although for the former finding, the small number of affected children did not allow the association to reach significance. This also deserves further study in larger cohorts, especially in light of the recent observations that murine gammaherpesvirus 68 undergoes reactivation in latently infected mice with acute with intestinal helminth infection [27]. Consistent with earlier reports, a large majority of participants (adults and children of both sexes) were shedding EBV [28]. Such a high proportion of shedders explains to some extent why, contrary to KSHV, EBV is ubiquitous in this population from a very early age. A child presumably had a higher likelihood of exposure to EBV than to KSHV at any given time, even though we detected similar virus loads in shedders: in children, the median KSHV load was 4.4 log copies (IQR, 3.5–4.7 log copies), and the EBV load was 4.7 log copies (IQR, 3.6–5.4 log copies); in mothers, viral loads were 3.6 log copies (IQR, 2.3–4.6 log copies) and 3.9 log copies (IQR, 2.4–4.7 log copies), respectively. Susceptibility to acquisition of either viral infection might also differ in the same individuals, in part because of the different role of exposure cofactors. In mothers, no factor was associated with EBV shedding or viral load. In children, however, some maternal factors were significant. Children of older mothers tended to shed less, emphasizing the possible role of environmental exposures unmeasured in our study. Likewise, children of mothers with a high EBV load tended to shed more themselves, again suggesting a common exposure or perhaps a genetic factor, also worthy of further examination. We have recently performed a genome-wide association study in a population-based rural Ugandan cohort, and we have identified an association between 5 novel loci and anti-EBV antibody levels [29]. Finally, we have identified negative associations between anti–MSP-1 (but not anti–AMA-1) antibody levels and EBV load in shedding children (but not mothers). The relationship between EBV and malaria has been investigated since the discovery of the virus [30], and very recent data provide interesting insight on the role of malaria in EBV-associated lymphomagenesis [31]. However, as with KSHV, the contribution of malaria to EBV pathogenesis and natural history must be further investigated throughout the life-span. For the first time, we also present data on the relationship between KSHV and EBV shedding in saliva. It is notable that individuals shedding KSHV tend to shed less EBV, while individuals shedding EBV tend to shed more KSHV. This suggests that there may be direct or indirect interaction between EBV and KSHV oropharyngeal replication, but the mechanisms controlling replication and shedding in saliva have not yet been investigated. Sparse in vitro data are available on dually infected primary effusion lymphoma lines, showing KSHV inhibition by EBV [32], mutual inhibition by the 2 viruses [33], differential transactivation of the 2 viruses by host immune factors [34], and even synergistic in vivo tumorigenicity in animal models [35]. Further investigation of the interactions between the 2 human gammaherpesviruses and their host is warranted. In conclusion, this study investigated salivary shedding of KSHV and EBV in apparently healthy mothers and their children and is the first to identify several factors that influence shedding or salivary viral load during infection with either virus. Our findings contribute to knowledge about the transmission, epidemiology, and natural history of EBV and KSHV infection in an East African population, in which the 2 viruses and associated malignancies are endemic. Notes Disclaimer.   The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Financial support. This work was supported by the Intramural Program of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services (contract HHSN261200800001E to D. W.), by the Wellcome Trust (grants 064693, 079110, 95778 to A. M. E., and 090132 to K. W.), and by the UK Medical Research Council (MRC), and the UK Department for International Development (DfID), under the MRC/DfID concordat (to the Entebbe Mother and Baby Study). Potential conflicts of interest.  All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: 18th International Workshop on KSHV and Related Agents, Miami, Florida, 30 June–3 July 2015; 4th Workshop on Emerging Issues in Oncogenic Virus Research, Manduria, Italy, 15–19 June 2016; 16th International Conference on Malignancies in HIV/AIDS, Bethesda, Maryland, 23–24 October 2017. References 1. Proceedings of the IARC Working Group on the Evaluation of Carcinogenic Risks to Humans . Epstein-Barr virus and Kaposi’s sarcoma herpesvirus/human herpesvirus 8 . 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N. is a Senior Visiting Scientist at the International Agency for Research on Cancer, Lyon, France. A. M. E. is the Principal Investigator of EMaBS, in which this project is based. Published by Oxford University Press for the Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. This Open Access article contains public sector information licensed under the Open Government Licence v2.0 (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/2/). Published by Oxford University Press for the Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Determinants of Gammaherpesvirus Shedding in Saliva Among Ugandan Children and Their Mothers

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Oxford University Press
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Copyright © 2022 Infectious Diseases Society of America
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0022-1899
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1537-6613
DOI
10.1093/infdis/jiy262
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Abstract

Abstract Background Epstein-Barr virus (EBV) and Kaposi sarcoma–associated herpesvirus (KSHV) are transmitted via saliva, but factors associated with salivary shedding are unknown. Methods We measured the DNA load of both viruses in saliva specimens collected from approximately 500 Ugandan mothers and their 6-year-old children, testing all participants for EBV and KSHV-seropositive individuals for KSHV. Results EBV and KSHV were shed by 72% and 22% of mothers, respectively, and by 85% and 40% of children, respectively; boys were more likely than girls to shed KSHV (48% vs 30%) but were equally likely to shed EBV. Children shed more KSHV and EBV than mothers, but salivary loads of EBV and KSHV were similar. KSHV shedding increased with increasing anti-KSHV (K8.1) antibodies in mothers and with decreasing antimalarial antibodies both in mothers and children. Among mothers, 40% of KSHV shedders also shed EBV, compared with 75% of KSHV nonshedders; among children, EBV was shed by 65% and 83%, respectively. Conclusions In summary, in this population, individuals were more likely to shed EBV than KSHV in saliva. We identified several factors, including child’s sex, that influence KSHV shedding, and we detected an inverse relationship between EBV and KSHV shedding, suggesting a direct or indirect interaction between the two viruses. EBV, KSHV, saliva, shedding, Uganda Kaposi sarcoma–associated herpesvirus (KSHV) is a necessary cause of Kaposi sarcoma [1, 2]. Unlike other human herpesviruses, KSHV is not ubiquitous across human populations; rather, the prevalence of infection shows considerable geographical variation, largely mirroring the variations seen in the incidence of Kaposi sarcoma. KSHV prevalence is relatively high in sub-Saharan Africa, lower in some Mediterranean countries, and lowest in most northern European and Asian populations [3], suggesting, in part, differences in transmission across regions. The factors that sustain higher rates of transmission in sub-Saharan Africa as compared to most other parts of the world are unclear. In Africa, primary infection begins in childhood, and prevalence increases with age. KSHV is shed in saliva [1, 4, 5], and this is the primary mode of transmission[1]. Conversely, infection by the related gammaherpesvirus Epstein-Barr virus (EBV), which also causes a number of human malignancies, is highly prevalent in all human populations [6]. Like KSHV, EBV is transmitted via saliva, but, in low-income settings, infection generally occurs much earlier in childhood, compared with high-income settings [7]. To investigate possible explanations for the differing epidemiology of these viruses, we compare here the prevalence and determinants of shedding of KSHV and EBV in saliva specimens collected from apparently healthy people in a Ugandan population with a high KSHV seroprevalence, on the assumption that viral shedding is an essential step in transmission [8]. We examined sociodemographic, clinical, and serological factors, including exposure to helminths and malaria, because, in previous work within this cohort, we have found these factors to be associated with KSHV seroprevalence [9, 10]. METHODS This was a cross-sectional study performed within the context of a clinical trial, the Entebbe Mother and Baby study (EMaBS; clinical trials registration ISRCTN32849447). The EMaBS is an ongoing birth cohort that originated as a double-blinded, randomized, placebo-controlled trial designed to determine the impact of helminth infections and their treatment on vaccine responses and infectious diseases outcomes; the details have been reported elsewhere [3, 4, 11, 12]. A total of 2507 pregnant women from Entebbe, Uganda, who consented, were enrolled into EMaBS and they and their children continue to be followed. In 2010, we systematically recruited into this substudy consenting human immunodeficiency virus (HIV)–negative mothers and their children, seen sequentially in the follow-up clinic. Additional plasma samples, together with a saliva sample, were collected from both mother and child and stored. Three mothers with HIV seroconversion after enrollment in the original study were excluded. Stored plasma samples from mothers and their 6-year old children were tested for the presence of KSHV antibodies by means of 2 enzyme-linked immunosorbent assays (ELISAs) using KSHV recombinant proteins: a lytic structural glycoprotein, K8.1, and the main latent nuclear protein, latency-associated nuclear antigen, encoded by ORF73. Results are reported as ODs. Each plate contained 3 positive and 3 negative controls; each assay cutoff was calculated on the basis of the performance of the negative controls. This procedure has been reported elsewhere [5, 13]. Individuals with positive results of either assay were considered KSHV seropositive. The same plasma samples were tested for malaria parasite antibodies, using 2 Plasmodium falciparum antigens: merozoite surface protein 1 (MSP-1) and apical membrane antigen 1 (AMA-1) [6, 14]. A pool of P. falciparum–positive plasma samples from patients known to be infected with malarial parasites was used to make standard dilutions. This pool was diluted serially 5 times, starting from 1:50 for MSP-1 and 1:100 for AMA-1, to make 6 standards with a 4-fold dilution increment. Blank wells were used to subtract background absorbance from the standards and the samples. The observed ODs were then exported into Microsoft Excel, and antibody titers for each sample and each antigen were derived from the standard curve of ODs. This procedure has been reported elsewhere [7, 15]. Saliva specimens were collected by having participants rinse their mouth with alcohol-based mouthwash and subsequently discharge this fluid into a 50-mL conical tube. DNA was extracted using a Qiagen blood and body fluids kit according to the manufacturer’s instructions. The KSHV load was measured using a quantitative real-time polymerase chain reaction (PCR) assay targeting the K6 gene [9, 10, 16, 17]. Similarly, the EBV load was determined using a quantitative real-time PCR specific for the EBNA1 gene [11, 18]. Another real-time assay for detection of the human endogenous retrovirus 3 (ERV-3), present in 2 copies in each diploid cell, was used to quantify cellular DNA [12, 19]. For each real-time PCR assay, each sample was assayed in triplicate, and an average of the 3 individual reactions was used to estimate the number of copies of the target gene. The viral DNA load was then calculated as the number of viral copies (genome equivalents) per million cells. Samples were designated as qualitatively positive if they could not be reliably quantitated (ie, if the amplification level for all 3 replicates exceeded the threshold value, but the average viral copy number was <3 in the KHSV assay or <10 in the EBV assay or if they failed to amplify in 1 or 2 of the 3 triplicate reactions). Qualitatively positive samples were retested, and if the results were confirmed, they were assigned the arbitrary value of 1 viral copy in further analyses. All mothers and children were tested for EBV in saliva, while KSHV-seropositive individuals were also tested for KSHV. Data analyses involved generating binary outcome variables for KSHV shedding; creating binary and categorical variables for demographic, socioeconomic, and illness information; and creating serological variables, including malarial antibody titers and KSHV antibody levels. For quantitative analyses, viral loads were log10 transformed. Variables considered to be possible risk factors or confounders for shedding in children were sex of child, maternal age group (categorized as 14–19, 20–24, 25–29, 30–34, or ≥35 years), maternal education level (none, primary, secondary, or tertiary), parity (1, 2–4, or ≥5 pregnancies), household socioeconomic status, maternal virus shedding, helminthiasis, anemia, anti-KSHV antibody levels (anti-K8.1 antigen and anti-ORF73 antigen, categorized as tertiles), and anti–P. falciparum antibody levels (categorized as tertiles). For shedding in mothers, possible risk factors considered were age group, education level, household socioeconomic status, HIV infection, anemia, helminthiasis, anti-KSHV antibody level, and anti–P. falciparum antibody levels. Because analysis on KSHV shedding is subset on KSHV-seropositive individuals, anti-KSHV antibody tertiles include only values predefined as positive, whereas anti–P. falciparum antibody levels include the entire range encountered in the sample. Initial analysis involved generating descriptive statistics by cross-tabulating viral shedding outcome variables and demographic, socioeconomic, illness, and immunologic variables that were considered to be possible risk factors for shedding. Logistic and linear regression models were fitted to examine variables predictive of shedding and viral load, respectively; nested modeling was used when twin children were included. Both prior knowledge from previous studies and a P value of <.05 in univariate analyses were used to select factors to be included in multivariable analyses. Likelihood ratio tests were used to determine adjusted P values. Analyses were conducted using Stata, version 13.1 (StataCorp, College Station, TX). Ethics Statement This study was approved by the Science and Ethics Committee of the Uganda Virus Research Institute, Uganda National Council for Science and Technology, and by the London School of Hygiene and Tropical Medicine Research Ethics Committee. Written and verbal information was provided in English and the vernacular; informed consent was obtained according to the Declaration of Helsinki and was recorded by signature or, in case of a participant’s inability to provide a signature, by thumbprint, as approved by the study’s institutional review boards. In Uganda, minors who are married, pregnant or have children are considered emancipated, and do not require parent/guardian consent to participate in research, therefore all participating mothers provided consent autonomously. Consent for participating children was provided by their mothers, fathers, or guardians. RESULTS Among participants in the EMaBS [11], we accrued for the present investigation 560 HIV-negative mothers and their 567 (including twins) 6-year-old children with KSHV serological data. Sociodemographic and other characteristics of the mothers at enrollment are shown in Table 1. Compared with the entire EMaBS cohort, mothers participating in this study were more likely to have higher education and income levels and less likely to have anemia. Table 1. Characteristics of Participating Mothers Characteristics . No. (%)a . Age group, y  14–19 112 (20.0)  20–24 222 (39.7)  25–29 136 (24.3)  30–34 62 (11.1)  ≥35 27 (4.8) Education level  None/primary 264 (47.2)  Secondary 232 (41.5)  Tertiary 63 (11.3) Marital status  Single/divorced/separated/widower 70 (12.5)  Married 488 (87.5) Monthly income, UGS  <30000 428 (80.6)  ≥30000 103 (19.4) Household socioeconomic status  Lower 234 (42.6)  Higher 314 (57.4) Pregnancies, no.  1 139 (24.9)  2–4 324 (58.0)  ≥5 97 (17.2) Characteristics . No. (%)a . Age group, y  14–19 112 (20.0)  20–24 222 (39.7)  25–29 136 (24.3)  30–34 62 (11.1)  ≥35 27 (4.8) Education level  None/primary 264 (47.2)  Secondary 232 (41.5)  Tertiary 63 (11.3) Marital status  Single/divorced/separated/widower 70 (12.5)  Married 488 (87.5) Monthly income, UGS  <30000 428 (80.6)  ≥30000 103 (19.4) Household socioeconomic status  Lower 234 (42.6)  Higher 314 (57.4) Pregnancies, no.  1 139 (24.9)  2–4 324 (58.0)  ≥5 97 (17.2) aValues might not sum to 560 because of missing data. Percentages were calculated using 560 as the denominator. bA total of 30000 Ugandan shillings (UGS) corresponds to the median national income. Open in new tab Table 1. Characteristics of Participating Mothers Characteristics . No. (%)a . Age group, y  14–19 112 (20.0)  20–24 222 (39.7)  25–29 136 (24.3)  30–34 62 (11.1)  ≥35 27 (4.8) Education level  None/primary 264 (47.2)  Secondary 232 (41.5)  Tertiary 63 (11.3) Marital status  Single/divorced/separated/widower 70 (12.5)  Married 488 (87.5) Monthly income, UGS  <30000 428 (80.6)  ≥30000 103 (19.4) Household socioeconomic status  Lower 234 (42.6)  Higher 314 (57.4) Pregnancies, no.  1 139 (24.9)  2–4 324 (58.0)  ≥5 97 (17.2) Characteristics . No. (%)a . Age group, y  14–19 112 (20.0)  20–24 222 (39.7)  25–29 136 (24.3)  30–34 62 (11.1)  ≥35 27 (4.8) Education level  None/primary 264 (47.2)  Secondary 232 (41.5)  Tertiary 63 (11.3) Marital status  Single/divorced/separated/widower 70 (12.5)  Married 488 (87.5) Monthly income, UGS  <30000 428 (80.6)  ≥30000 103 (19.4) Household socioeconomic status  Lower 234 (42.6)  Higher 314 (57.4) Pregnancies, no.  1 139 (24.9)  2–4 324 (58.0)  ≥5 97 (17.2) aValues might not sum to 560 because of missing data. Percentages were calculated using 560 as the denominator. bA total of 30000 Ugandan shillings (UGS) corresponds to the median national income. Open in new tab Mothers were generally young (median age, 23 years; range, 14–40 years), half had no education above primary school, and about a quarter had enrolled at their first pregnancy. Mothers were roughly distributed between lower (42.6%) and higher (57.4%) household socioeconomic status. Of mothers and children whose serological data were available, 299 (53%) and 102 (22%), respectively, were KSHV seropositive. KSHV DNA was detected in saliva twice as frequently in seropositive children (40% [40 of 99 tested]) as in seropositive mothers (21.5% [64 of 297]); however, while 27 of 56 boys (48%) had a detectable KSHV load, only 13 of 43 girls (30%) did (Figure 1). Among KSHV shedders, the median KSHV load was 3.6 log copies (interquartile range [IQR], 2.3–4.6 log copies) in mothers, versus 4.4 log copies (IQR, 3.5–4.7 log copies) in children (P = .03); there was no significant difference by sex of the child (median, 4.5 log copies [IQR, 3.5–4.9 log copies] in boys vs 4.4 log copies [IQR, 3.1–4.6 log copies] in girls). Figure 1. Open in new tabDownload slide Salivary shedding in mothers and children. Unadjusted proportion of mothers and children of either sex who are shedding Kaposi sarcoma–associated virus (KSHV) are estimated on KSHV-seropositive individuals only, whereas the prevalence of Epstein-Barr virus (EBV) shedding is estimated on the entire sample because all are assumed to be EBV seropositive. Figure 1. Open in new tabDownload slide Salivary shedding in mothers and children. Unadjusted proportion of mothers and children of either sex who are shedding Kaposi sarcoma–associated virus (KSHV) are estimated on KSHV-seropositive individuals only, whereas the prevalence of Epstein-Barr virus (EBV) shedding is estimated on the entire sample because all are assumed to be EBV seropositive. EBV DNA was prevalent in saliva specimens from both mothers (72% [402 of 559 tested]) and their children (85% [474 of 560]); the prevalence was similar in boys (84% [232 of 277]) and girls (86% [238 of 277]; Figure 1). However, the median viral load among EBV shedders was significantly higher in children than in their mothers (median, 4.7 log copies [IQR, 3.6–5.4 log copies] vs 3.9 log copies [IQR, 2.4–4.7 log copies]; P < .001) and did not differ significantly with respect to the sex of the child (4.6 log copies [IQR, 3.6–5.4 log copies] in boys vs 4.8 log copies [IQR, 3.6–5.4 log copies] in girls). In multivariate analysis, among mothers (Table 2), a detectable KSHV load was not associated with any demographic or clinical variable. KSHV shedding was directly associated with increasing levels of antibodies against KSHV K8.1 (odds ratio [OR] for medium and high OD tertiles to The low OD tertile, 80; 95% confidence interval [CI], 11–560) and inversely associated with antibodies against P. falciparum AMA-1 (OR, 0.25; 95% CI, .07–.86), but there was no association with level of antibodies against KSHV ORF73 nor with anti–P. falciparum MSP-1 antibodies. Detection of EBV DNA in mothers was not associated with any sociodemographic, clinical, or serological factor. Table 2. Factors Associated With Shedding of Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) in Saliva Among Mothers Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . aORb (95% CI) . Pc . Proportion (%) . aORb (95% CI) . Pc . Age group, y  14–19 14/73 (19.2) Reference NS 80/112 (71.4) Reference NS  20–24 24/116 (20.3) 1.09 (.52–2.29) 162/221 (73.3) 1.10 (.66–1.85)  25–29 16/65 (24.6) 1.42 (.62–3.26) 93/136 (68.4) 0.89 (.50–1.57)  30–34 8/29 (26.7) 1.47 (.53–4.06) 45/62 (72.6) 1.05 (.52–2.14)  ≥35 2/14 (14.3) 0.72 (.14–3.65) 21/27 (77.8) 1.67 (.57–4.87) Education level  Primary/none 36/147 (24.2) Reference NS 191/264 (72.0) Reference NS  Secondary 22/123 (17.7) 0.69 (.38–1.27) 167/231 (72.3) 1.00 (.66–1.49)  Tertiary 6/27 (22.2) 0.91 (.33–2.51) 44/63 (69.8) 0.89(.47–1.69) Household SES  Lower 29/154 (18.8) Reference 168/234 (71.8) Reference NS  Higher 35/140 (24.5) 0.70 (.40–1.24) NS 228/314 (72.6) 1.05 (.71–1.55) Anemia  No 35/188 (18.6) Reference 269/377 (71.4) Reference NS  Yes 29/109 (25.9) 1.60 (.90–2.83) NS 132/181 (72.9) 1.13 (.75–1.70) Anti-KSHV antibody OD  ORF73   Low 22/99 (22.0) Reference NS 34/99 (34.3) Reference NS   Medium 16/99 (15.0) 0.65 (.31–1.34) 27/99 (27.3) 1.00 (.53–1.90)   High 26/99 (27.0) 1.20 (.61–2.34) 33/99 (33.3) 0.71 (.38–1.31)  K8.1   Low 5/98 (5.0) Reference <.001 29/99 (29.6) Reference NS   Medium 25/98 (25.0) 7.22 (2.58–20.24) 30/100 (30.0) 1.36 (.73–2.53)   High 34/98 (34.0) 10.61 (3.87–29.12) 35/99 (35.4) 1.16 (.63–2.14) Anti–P. falciparum antibody titer  PfAMA1   Low 23/81 (28.4) Reference .02 136/183 (74.3) Reference NS   Medium 21/104 (20.2) 0.58 (.29–1.17) 132/184 (71.7) 0.87 (.54–1.39)   High 17/110 (15.5) 0.39 (.19–0.82) 129/186 (69.7) 0.83 (.52–1.33)  PfMSP1   Low 20/79 (25.3) Reference NS 136/181 (75.1) Reference NS   Medium 20/105 (19.0) 0.60 (.29–1.25) 140/185 (75.7) 1.04 (.64–1.70)   High 21/111 (18.9) 0.58 (.28–1.19) 121/186 (65.1) 0.60 (.38–0.96) Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . aORb (95% CI) . Pc . Proportion (%) . aORb (95% CI) . Pc . Age group, y  14–19 14/73 (19.2) Reference NS 80/112 (71.4) Reference NS  20–24 24/116 (20.3) 1.09 (.52–2.29) 162/221 (73.3) 1.10 (.66–1.85)  25–29 16/65 (24.6) 1.42 (.62–3.26) 93/136 (68.4) 0.89 (.50–1.57)  30–34 8/29 (26.7) 1.47 (.53–4.06) 45/62 (72.6) 1.05 (.52–2.14)  ≥35 2/14 (14.3) 0.72 (.14–3.65) 21/27 (77.8) 1.67 (.57–4.87) Education level  Primary/none 36/147 (24.2) Reference NS 191/264 (72.0) Reference NS  Secondary 22/123 (17.7) 0.69 (.38–1.27) 167/231 (72.3) 1.00 (.66–1.49)  Tertiary 6/27 (22.2) 0.91 (.33–2.51) 44/63 (69.8) 0.89(.47–1.69) Household SES  Lower 29/154 (18.8) Reference 168/234 (71.8) Reference NS  Higher 35/140 (24.5) 0.70 (.40–1.24) NS 228/314 (72.6) 1.05 (.71–1.55) Anemia  No 35/188 (18.6) Reference 269/377 (71.4) Reference NS  Yes 29/109 (25.9) 1.60 (.90–2.83) NS 132/181 (72.9) 1.13 (.75–1.70) Anti-KSHV antibody OD  ORF73   Low 22/99 (22.0) Reference NS 34/99 (34.3) Reference NS   Medium 16/99 (15.0) 0.65 (.31–1.34) 27/99 (27.3) 1.00 (.53–1.90)   High 26/99 (27.0) 1.20 (.61–2.34) 33/99 (33.3) 0.71 (.38–1.31)  K8.1   Low 5/98 (5.0) Reference <.001 29/99 (29.6) Reference NS   Medium 25/98 (25.0) 7.22 (2.58–20.24) 30/100 (30.0) 1.36 (.73–2.53)   High 34/98 (34.0) 10.61 (3.87–29.12) 35/99 (35.4) 1.16 (.63–2.14) Anti–P. falciparum antibody titer  PfAMA1   Low 23/81 (28.4) Reference .02 136/183 (74.3) Reference NS   Medium 21/104 (20.2) 0.58 (.29–1.17) 132/184 (71.7) 0.87 (.54–1.39)   High 17/110 (15.5) 0.39 (.19–0.82) 129/186 (69.7) 0.83 (.52–1.33)  PfMSP1   Low 20/79 (25.3) Reference NS 136/181 (75.1) Reference NS   Medium 20/105 (19.0) 0.60 (.29–1.25) 140/185 (75.7) 1.04 (.64–1.70)   High 21/111 (18.9) 0.58 (.28–1.19) 121/186 (65.1) 0.60 (.38–0.96) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum; SES, socioeconomic status. aData are for KSHV-seropositive individuals only. bAdjusted odds ratios (aORs) are estimated from multivariate logistic regression models that include age group, education level, and household SES, with the first 3 covariates adjusted for the other 2. cCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab Table 2. Factors Associated With Shedding of Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) in Saliva Among Mothers Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . aORb (95% CI) . Pc . Proportion (%) . aORb (95% CI) . Pc . Age group, y  14–19 14/73 (19.2) Reference NS 80/112 (71.4) Reference NS  20–24 24/116 (20.3) 1.09 (.52–2.29) 162/221 (73.3) 1.10 (.66–1.85)  25–29 16/65 (24.6) 1.42 (.62–3.26) 93/136 (68.4) 0.89 (.50–1.57)  30–34 8/29 (26.7) 1.47 (.53–4.06) 45/62 (72.6) 1.05 (.52–2.14)  ≥35 2/14 (14.3) 0.72 (.14–3.65) 21/27 (77.8) 1.67 (.57–4.87) Education level  Primary/none 36/147 (24.2) Reference NS 191/264 (72.0) Reference NS  Secondary 22/123 (17.7) 0.69 (.38–1.27) 167/231 (72.3) 1.00 (.66–1.49)  Tertiary 6/27 (22.2) 0.91 (.33–2.51) 44/63 (69.8) 0.89(.47–1.69) Household SES  Lower 29/154 (18.8) Reference 168/234 (71.8) Reference NS  Higher 35/140 (24.5) 0.70 (.40–1.24) NS 228/314 (72.6) 1.05 (.71–1.55) Anemia  No 35/188 (18.6) Reference 269/377 (71.4) Reference NS  Yes 29/109 (25.9) 1.60 (.90–2.83) NS 132/181 (72.9) 1.13 (.75–1.70) Anti-KSHV antibody OD  ORF73   Low 22/99 (22.0) Reference NS 34/99 (34.3) Reference NS   Medium 16/99 (15.0) 0.65 (.31–1.34) 27/99 (27.3) 1.00 (.53–1.90)   High 26/99 (27.0) 1.20 (.61–2.34) 33/99 (33.3) 0.71 (.38–1.31)  K8.1   Low 5/98 (5.0) Reference <.001 29/99 (29.6) Reference NS   Medium 25/98 (25.0) 7.22 (2.58–20.24) 30/100 (30.0) 1.36 (.73–2.53)   High 34/98 (34.0) 10.61 (3.87–29.12) 35/99 (35.4) 1.16 (.63–2.14) Anti–P. falciparum antibody titer  PfAMA1   Low 23/81 (28.4) Reference .02 136/183 (74.3) Reference NS   Medium 21/104 (20.2) 0.58 (.29–1.17) 132/184 (71.7) 0.87 (.54–1.39)   High 17/110 (15.5) 0.39 (.19–0.82) 129/186 (69.7) 0.83 (.52–1.33)  PfMSP1   Low 20/79 (25.3) Reference NS 136/181 (75.1) Reference NS   Medium 20/105 (19.0) 0.60 (.29–1.25) 140/185 (75.7) 1.04 (.64–1.70)   High 21/111 (18.9) 0.58 (.28–1.19) 121/186 (65.1) 0.60 (.38–0.96) Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . aORb (95% CI) . Pc . Proportion (%) . aORb (95% CI) . Pc . Age group, y  14–19 14/73 (19.2) Reference NS 80/112 (71.4) Reference NS  20–24 24/116 (20.3) 1.09 (.52–2.29) 162/221 (73.3) 1.10 (.66–1.85)  25–29 16/65 (24.6) 1.42 (.62–3.26) 93/136 (68.4) 0.89 (.50–1.57)  30–34 8/29 (26.7) 1.47 (.53–4.06) 45/62 (72.6) 1.05 (.52–2.14)  ≥35 2/14 (14.3) 0.72 (.14–3.65) 21/27 (77.8) 1.67 (.57–4.87) Education level  Primary/none 36/147 (24.2) Reference NS 191/264 (72.0) Reference NS  Secondary 22/123 (17.7) 0.69 (.38–1.27) 167/231 (72.3) 1.00 (.66–1.49)  Tertiary 6/27 (22.2) 0.91 (.33–2.51) 44/63 (69.8) 0.89(.47–1.69) Household SES  Lower 29/154 (18.8) Reference 168/234 (71.8) Reference NS  Higher 35/140 (24.5) 0.70 (.40–1.24) NS 228/314 (72.6) 1.05 (.71–1.55) Anemia  No 35/188 (18.6) Reference 269/377 (71.4) Reference NS  Yes 29/109 (25.9) 1.60 (.90–2.83) NS 132/181 (72.9) 1.13 (.75–1.70) Anti-KSHV antibody OD  ORF73   Low 22/99 (22.0) Reference NS 34/99 (34.3) Reference NS   Medium 16/99 (15.0) 0.65 (.31–1.34) 27/99 (27.3) 1.00 (.53–1.90)   High 26/99 (27.0) 1.20 (.61–2.34) 33/99 (33.3) 0.71 (.38–1.31)  K8.1   Low 5/98 (5.0) Reference <.001 29/99 (29.6) Reference NS   Medium 25/98 (25.0) 7.22 (2.58–20.24) 30/100 (30.0) 1.36 (.73–2.53)   High 34/98 (34.0) 10.61 (3.87–29.12) 35/99 (35.4) 1.16 (.63–2.14) Anti–P. falciparum antibody titer  PfAMA1   Low 23/81 (28.4) Reference .02 136/183 (74.3) Reference NS   Medium 21/104 (20.2) 0.58 (.29–1.17) 132/184 (71.7) 0.87 (.54–1.39)   High 17/110 (15.5) 0.39 (.19–0.82) 129/186 (69.7) 0.83 (.52–1.33)  PfMSP1   Low 20/79 (25.3) Reference NS 136/181 (75.1) Reference NS   Medium 20/105 (19.0) 0.60 (.29–1.25) 140/185 (75.7) 1.04 (.64–1.70)   High 21/111 (18.9) 0.58 (.28–1.19) 121/186 (65.1) 0.60 (.38–0.96) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum; SES, socioeconomic status. aData are for KSHV-seropositive individuals only. bAdjusted odds ratios (aORs) are estimated from multivariate logistic regression models that include age group, education level, and household SES, with the first 3 covariates adjusted for the other 2. cCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab Amongst seropositive mothers shedding KSHV (Table 3), no factor was associated with KSHV load. Similarly, no factors were associated with EBV load in EBV shedders. Table 3. Factors Associated With Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) Loads in Saliva Specimens From Shedding Mothers Factor . KSHV Load (n = 64) . EBV Load (n = 404) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Age group, y  14–19 Reference NS Reference NS  20–24 −0.36 (−1.61–0.90) −0.17 (−0.69–0.34)  25–29 0.14 (−1.24–1.52) −0.39 (−0.98–0.19)  30–34 −0.71 (−2.36–.94) −0.20 (−.90–.50)  ≥35 0.71 (−2.17–3.59) −0.13 (−1.05–.79) Education level  Primary/none Reference NS Reference NS  Secondary 0.78 (−.23–1.79) 0.24 (−.17–.64)  Tertiary 1.35 (−.31–3.01) 0.14 (−.51–.79) Household SES −0.03 (−.98–.93) NS −0.22 (−.60–.17) NS Anemia −0.23 (−1.22–.76) NS −0.01 (−.41–.40) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium 0.83 (−1.05–2.71) …   High 0.94 (−.89–2.77) …  ORF73   Low Reference NS …   Medium −0.81 (−2.08–.47) …   High 0.46 (−.70–1.62) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.19 (−.98–1.36) 0.26 (−.20–.72)   High 0.19 (−1.08–1.46) 0.28 (−.18–.73)  PfMSP1   Low Reference NS Reference NS   Medium −0.09 (−1.35–1.16) 0.14 (−.31–.59)   High 0.06 (−1.17–1.29) 0.36 (−.10–.83) Factor . KSHV Load (n = 64) . EBV Load (n = 404) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Age group, y  14–19 Reference NS Reference NS  20–24 −0.36 (−1.61–0.90) −0.17 (−0.69–0.34)  25–29 0.14 (−1.24–1.52) −0.39 (−0.98–0.19)  30–34 −0.71 (−2.36–.94) −0.20 (−.90–.50)  ≥35 0.71 (−2.17–3.59) −0.13 (−1.05–.79) Education level  Primary/none Reference NS Reference NS  Secondary 0.78 (−.23–1.79) 0.24 (−.17–.64)  Tertiary 1.35 (−.31–3.01) 0.14 (−.51–.79) Household SES −0.03 (−.98–.93) NS −0.22 (−.60–.17) NS Anemia −0.23 (−1.22–.76) NS −0.01 (−.41–.40) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium 0.83 (−1.05–2.71) …   High 0.94 (−.89–2.77) …  ORF73   Low Reference NS …   Medium −0.81 (−2.08–.47) …   High 0.46 (−.70–1.62) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.19 (−.98–1.36) 0.26 (−.20–.72)   High 0.19 (−1.08–1.46) 0.28 (−.18–.73)  PfMSP1   Low Reference NS Reference NS   Medium −0.09 (−1.35–1.16) 0.14 (−.31–.59)   High 0.06 (−1.17–1.29) 0.36 (−.10–.83) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum; SES, socioeconomic status. aCoefficients expressing variation in log10 genome equivalents/million cells are estimated in shedding individuals only, using multivariate models that include age group, education level, and household SES, with the first 3 covariates adjusted for the other 2. bCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab Table 3. Factors Associated With Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) Loads in Saliva Specimens From Shedding Mothers Factor . KSHV Load (n = 64) . EBV Load (n = 404) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Age group, y  14–19 Reference NS Reference NS  20–24 −0.36 (−1.61–0.90) −0.17 (−0.69–0.34)  25–29 0.14 (−1.24–1.52) −0.39 (−0.98–0.19)  30–34 −0.71 (−2.36–.94) −0.20 (−.90–.50)  ≥35 0.71 (−2.17–3.59) −0.13 (−1.05–.79) Education level  Primary/none Reference NS Reference NS  Secondary 0.78 (−.23–1.79) 0.24 (−.17–.64)  Tertiary 1.35 (−.31–3.01) 0.14 (−.51–.79) Household SES −0.03 (−.98–.93) NS −0.22 (−.60–.17) NS Anemia −0.23 (−1.22–.76) NS −0.01 (−.41–.40) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium 0.83 (−1.05–2.71) …   High 0.94 (−.89–2.77) …  ORF73   Low Reference NS …   Medium −0.81 (−2.08–.47) …   High 0.46 (−.70–1.62) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.19 (−.98–1.36) 0.26 (−.20–.72)   High 0.19 (−1.08–1.46) 0.28 (−.18–.73)  PfMSP1   Low Reference NS Reference NS   Medium −0.09 (−1.35–1.16) 0.14 (−.31–.59)   High 0.06 (−1.17–1.29) 0.36 (−.10–.83) Factor . KSHV Load (n = 64) . EBV Load (n = 404) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Age group, y  14–19 Reference NS Reference NS  20–24 −0.36 (−1.61–0.90) −0.17 (−0.69–0.34)  25–29 0.14 (−1.24–1.52) −0.39 (−0.98–0.19)  30–34 −0.71 (−2.36–.94) −0.20 (−.90–.50)  ≥35 0.71 (−2.17–3.59) −0.13 (−1.05–.79) Education level  Primary/none Reference NS Reference NS  Secondary 0.78 (−.23–1.79) 0.24 (−.17–.64)  Tertiary 1.35 (−.31–3.01) 0.14 (−.51–.79) Household SES −0.03 (−.98–.93) NS −0.22 (−.60–.17) NS Anemia −0.23 (−1.22–.76) NS −0.01 (−.41–.40) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium 0.83 (−1.05–2.71) …   High 0.94 (−.89–2.77) …  ORF73   Low Reference NS …   Medium −0.81 (−2.08–.47) …   High 0.46 (−.70–1.62) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.19 (−.98–1.36) 0.26 (−.20–.72)   High 0.19 (−1.08–1.46) 0.28 (−.18–.73)  PfMSP1   Low Reference NS Reference NS   Medium −0.09 (−1.35–1.16) 0.14 (−.31–.59)   High 0.06 (−1.17–1.29) 0.36 (−.10–.83) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum; SES, socioeconomic status. aCoefficients expressing variation in log10 genome equivalents/million cells are estimated in shedding individuals only, using multivariate models that include age group, education level, and household SES, with the first 3 covariates adjusted for the other 2. bCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab The children of mothers shedding KSHV were not more likely to be KSHV seropositive (crude odds ratio [OR], 1.63 [95% CI, .9–2.93]; OR adjusted for maternal age group, education level, socioeconomic status, and sex of the child, 1.63 [95% CI, .9–2.99]). In multivariate analysis, among children (Table 4), the odds of shedding KSHV were higher in boys versus girls (OR, 2.22; 95% CI, .97–5.10) and in children with helminthiasis as compared to those without (OR, 7.63; 95% CI, .63–83.45), although the differences did not reach statistical significance, and the latter finding was based on only 4 children. The odds of shedding KSHV were inversely associated with antibodies against P. falciparum AMA-1 (OR for medium and high titers vs low titers, 0.11; 95% CI, .01–.77) and tended to increase with higher anti-KSHV antibody levels, although there was no statistically significant association. Table 4. Factors Associated With Shedding of Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) in Saliva Among Children Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . ORb (95% CI) . Pc . Proportion (%) . ORb (95% CI) . Pc . Sex  Female 13/43 (30.2) Reference NS 238/277 (85.9) Reference NS  Male 27/56 (48.2) 2.15 (.93–4.96) 232/277 (83.8) 0.72 (.30–1.73) Maternal age group, y  14–19 6/14 (42.9) Reference NS 100/110 (90.9) Reference .05  20–24 15/36 (41.7) 0.89 (.25–3.18) 186/224 (83.0) 0.28 (.07–1.09)  25–29 10/29 (34.5) 0.72 (.19–2.73) 111/131 (84.7) 0.36 (.08–1.52)  30–34 6/11 (54.5) 1.36 (.27–6.92) 54/63 (85.7) 0.42 (.07–2.34)  ≥35 3/9 (33.3) 0.54 (.09–3.19) 20/27 (74.1) 0.10 (.01–0.98) Parity  1 12/22 (54.5) Reference NS 114/135 (84.4) Reference NS  2–4 16/55 (29.6) 0.37 (.13–1.06) 279/324 (86.1) 1.27 (.45–3.62)  ≥5 12/23 (52.2) 0.90 (.27–2.94) 78/96 (81.3) 0.66 (.17–2.52) Maternal virus shedding  No 29/68 (42.6) Reference NS 124/150 (82.7) NS  Yes 4/17 (23.5) 0.39 (.11–1.33) 338/396 (85.4) 1.23 (.74–2.04) Anemia  No 29/77 (37.6) Reference NS 367/430 (85.3) Reference NS  Yes 11/22 (50.0) 1.51 (.57–3.98) 107/130 (82.3) 0.63 (.23–1.76) Helminthiasis  No 15/41 (36.5) Reference NS 251/287 (87.6) Reference NS  Yes 3/4 (75.0) 7.23 (.63–83.45) 14/18 (77.8) 0.18 (.01–5.52) Anti-KSHV antibody OD  ORF73   Low 11/33 (33.3) Reference NS 9/33 (27.3) Reference NS   Medium 12/33 (36.4) 1.28 (.45–3.63) 9/33 (27.3) 0.86 (.28–2.65)   High 17/33 (51.5) 2.60 (.92–7.38) 6/33 (18.2) 1.39 (.41–4.66)  K8.1   Low 12/33 (36.4) Reference NS 4/33 (12.1) Reference NS   Medium 9/33 (27.3) 0.55 (.19–1.61) 11/33 (33.3) 0.33 (.09–1.23)   High 19/33 (57.6) 2.06 (.75–5.70) 9/33 (27.3) 0.45 (.12–1.71) Anti–P. falciparum antibody titer  PfAMA1   Low 11/24 (45.8) Reference NS 157/184 (85.3) Reference NS   Medium 11/25 (44.0) 1.02 (.32–3.23) 152/186 (81.7) 0.58 (.20–1.69)   High 17/48 (35.4) 0.67 (.24–1.86) 157/182 (86.3) 1.06 (.37–3.05)  PfMSP1   Low 14/23 (60.9) Reference .03 157/186 (84.4) Reference NS   Medium 12/36 (33.3) 0.32 (.11–0.97) 161/185 (87.0) 1.46 (.50–4.27)   High 13/38 (34.2) 0.33 (.11–1.00) 148/181 (81.8) 0.65 (.23–1.85) Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . ORb (95% CI) . Pc . Proportion (%) . ORb (95% CI) . Pc . Sex  Female 13/43 (30.2) Reference NS 238/277 (85.9) Reference NS  Male 27/56 (48.2) 2.15 (.93–4.96) 232/277 (83.8) 0.72 (.30–1.73) Maternal age group, y  14–19 6/14 (42.9) Reference NS 100/110 (90.9) Reference .05  20–24 15/36 (41.7) 0.89 (.25–3.18) 186/224 (83.0) 0.28 (.07–1.09)  25–29 10/29 (34.5) 0.72 (.19–2.73) 111/131 (84.7) 0.36 (.08–1.52)  30–34 6/11 (54.5) 1.36 (.27–6.92) 54/63 (85.7) 0.42 (.07–2.34)  ≥35 3/9 (33.3) 0.54 (.09–3.19) 20/27 (74.1) 0.10 (.01–0.98) Parity  1 12/22 (54.5) Reference NS 114/135 (84.4) Reference NS  2–4 16/55 (29.6) 0.37 (.13–1.06) 279/324 (86.1) 1.27 (.45–3.62)  ≥5 12/23 (52.2) 0.90 (.27–2.94) 78/96 (81.3) 0.66 (.17–2.52) Maternal virus shedding  No 29/68 (42.6) Reference NS 124/150 (82.7) NS  Yes 4/17 (23.5) 0.39 (.11–1.33) 338/396 (85.4) 1.23 (.74–2.04) Anemia  No 29/77 (37.6) Reference NS 367/430 (85.3) Reference NS  Yes 11/22 (50.0) 1.51 (.57–3.98) 107/130 (82.3) 0.63 (.23–1.76) Helminthiasis  No 15/41 (36.5) Reference NS 251/287 (87.6) Reference NS  Yes 3/4 (75.0) 7.23 (.63–83.45) 14/18 (77.8) 0.18 (.01–5.52) Anti-KSHV antibody OD  ORF73   Low 11/33 (33.3) Reference NS 9/33 (27.3) Reference NS   Medium 12/33 (36.4) 1.28 (.45–3.63) 9/33 (27.3) 0.86 (.28–2.65)   High 17/33 (51.5) 2.60 (.92–7.38) 6/33 (18.2) 1.39 (.41–4.66)  K8.1   Low 12/33 (36.4) Reference NS 4/33 (12.1) Reference NS   Medium 9/33 (27.3) 0.55 (.19–1.61) 11/33 (33.3) 0.33 (.09–1.23)   High 19/33 (57.6) 2.06 (.75–5.70) 9/33 (27.3) 0.45 (.12–1.71) Anti–P. falciparum antibody titer  PfAMA1   Low 11/24 (45.8) Reference NS 157/184 (85.3) Reference NS   Medium 11/25 (44.0) 1.02 (.32–3.23) 152/186 (81.7) 0.58 (.20–1.69)   High 17/48 (35.4) 0.67 (.24–1.86) 157/182 (86.3) 1.06 (.37–3.05)  PfMSP1   Low 14/23 (60.9) Reference .03 157/186 (84.4) Reference NS   Medium 12/36 (33.3) 0.32 (.11–0.97) 161/185 (87.0) 1.46 (.50–4.27)   High 13/38 (34.2) 0.33 (.11–1.00) 148/181 (81.8) 0.65 (.23–1.85) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum. aData are for KSHV-seropositive individuals only. bOdds ratios (ORs) are adjusted for sex except when sex is the factor of interest. cCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab Table 4. Factors Associated With Shedding of Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) in Saliva Among Children Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . ORb (95% CI) . Pc . Proportion (%) . ORb (95% CI) . Pc . Sex  Female 13/43 (30.2) Reference NS 238/277 (85.9) Reference NS  Male 27/56 (48.2) 2.15 (.93–4.96) 232/277 (83.8) 0.72 (.30–1.73) Maternal age group, y  14–19 6/14 (42.9) Reference NS 100/110 (90.9) Reference .05  20–24 15/36 (41.7) 0.89 (.25–3.18) 186/224 (83.0) 0.28 (.07–1.09)  25–29 10/29 (34.5) 0.72 (.19–2.73) 111/131 (84.7) 0.36 (.08–1.52)  30–34 6/11 (54.5) 1.36 (.27–6.92) 54/63 (85.7) 0.42 (.07–2.34)  ≥35 3/9 (33.3) 0.54 (.09–3.19) 20/27 (74.1) 0.10 (.01–0.98) Parity  1 12/22 (54.5) Reference NS 114/135 (84.4) Reference NS  2–4 16/55 (29.6) 0.37 (.13–1.06) 279/324 (86.1) 1.27 (.45–3.62)  ≥5 12/23 (52.2) 0.90 (.27–2.94) 78/96 (81.3) 0.66 (.17–2.52) Maternal virus shedding  No 29/68 (42.6) Reference NS 124/150 (82.7) NS  Yes 4/17 (23.5) 0.39 (.11–1.33) 338/396 (85.4) 1.23 (.74–2.04) Anemia  No 29/77 (37.6) Reference NS 367/430 (85.3) Reference NS  Yes 11/22 (50.0) 1.51 (.57–3.98) 107/130 (82.3) 0.63 (.23–1.76) Helminthiasis  No 15/41 (36.5) Reference NS 251/287 (87.6) Reference NS  Yes 3/4 (75.0) 7.23 (.63–83.45) 14/18 (77.8) 0.18 (.01–5.52) Anti-KSHV antibody OD  ORF73   Low 11/33 (33.3) Reference NS 9/33 (27.3) Reference NS   Medium 12/33 (36.4) 1.28 (.45–3.63) 9/33 (27.3) 0.86 (.28–2.65)   High 17/33 (51.5) 2.60 (.92–7.38) 6/33 (18.2) 1.39 (.41–4.66)  K8.1   Low 12/33 (36.4) Reference NS 4/33 (12.1) Reference NS   Medium 9/33 (27.3) 0.55 (.19–1.61) 11/33 (33.3) 0.33 (.09–1.23)   High 19/33 (57.6) 2.06 (.75–5.70) 9/33 (27.3) 0.45 (.12–1.71) Anti–P. falciparum antibody titer  PfAMA1   Low 11/24 (45.8) Reference NS 157/184 (85.3) Reference NS   Medium 11/25 (44.0) 1.02 (.32–3.23) 152/186 (81.7) 0.58 (.20–1.69)   High 17/48 (35.4) 0.67 (.24–1.86) 157/182 (86.3) 1.06 (.37–3.05)  PfMSP1   Low 14/23 (60.9) Reference .03 157/186 (84.4) Reference NS   Medium 12/36 (33.3) 0.32 (.11–0.97) 161/185 (87.0) 1.46 (.50–4.27)   High 13/38 (34.2) 0.33 (.11–1.00) 148/181 (81.8) 0.65 (.23–1.85) Factor . KSHV Shedding . EBV Shedding . Proportiona (%) . ORb (95% CI) . Pc . Proportion (%) . ORb (95% CI) . Pc . Sex  Female 13/43 (30.2) Reference NS 238/277 (85.9) Reference NS  Male 27/56 (48.2) 2.15 (.93–4.96) 232/277 (83.8) 0.72 (.30–1.73) Maternal age group, y  14–19 6/14 (42.9) Reference NS 100/110 (90.9) Reference .05  20–24 15/36 (41.7) 0.89 (.25–3.18) 186/224 (83.0) 0.28 (.07–1.09)  25–29 10/29 (34.5) 0.72 (.19–2.73) 111/131 (84.7) 0.36 (.08–1.52)  30–34 6/11 (54.5) 1.36 (.27–6.92) 54/63 (85.7) 0.42 (.07–2.34)  ≥35 3/9 (33.3) 0.54 (.09–3.19) 20/27 (74.1) 0.10 (.01–0.98) Parity  1 12/22 (54.5) Reference NS 114/135 (84.4) Reference NS  2–4 16/55 (29.6) 0.37 (.13–1.06) 279/324 (86.1) 1.27 (.45–3.62)  ≥5 12/23 (52.2) 0.90 (.27–2.94) 78/96 (81.3) 0.66 (.17–2.52) Maternal virus shedding  No 29/68 (42.6) Reference NS 124/150 (82.7) NS  Yes 4/17 (23.5) 0.39 (.11–1.33) 338/396 (85.4) 1.23 (.74–2.04) Anemia  No 29/77 (37.6) Reference NS 367/430 (85.3) Reference NS  Yes 11/22 (50.0) 1.51 (.57–3.98) 107/130 (82.3) 0.63 (.23–1.76) Helminthiasis  No 15/41 (36.5) Reference NS 251/287 (87.6) Reference NS  Yes 3/4 (75.0) 7.23 (.63–83.45) 14/18 (77.8) 0.18 (.01–5.52) Anti-KSHV antibody OD  ORF73   Low 11/33 (33.3) Reference NS 9/33 (27.3) Reference NS   Medium 12/33 (36.4) 1.28 (.45–3.63) 9/33 (27.3) 0.86 (.28–2.65)   High 17/33 (51.5) 2.60 (.92–7.38) 6/33 (18.2) 1.39 (.41–4.66)  K8.1   Low 12/33 (36.4) Reference NS 4/33 (12.1) Reference NS   Medium 9/33 (27.3) 0.55 (.19–1.61) 11/33 (33.3) 0.33 (.09–1.23)   High 19/33 (57.6) 2.06 (.75–5.70) 9/33 (27.3) 0.45 (.12–1.71) Anti–P. falciparum antibody titer  PfAMA1   Low 11/24 (45.8) Reference NS 157/184 (85.3) Reference NS   Medium 11/25 (44.0) 1.02 (.32–3.23) 152/186 (81.7) 0.58 (.20–1.69)   High 17/48 (35.4) 0.67 (.24–1.86) 157/182 (86.3) 1.06 (.37–3.05)  PfMSP1   Low 14/23 (60.9) Reference .03 157/186 (84.4) Reference NS   Medium 12/36 (33.3) 0.32 (.11–0.97) 161/185 (87.0) 1.46 (.50–4.27)   High 13/38 (34.2) 0.33 (.11–1.00) 148/181 (81.8) 0.65 (.23–1.85) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum. aData are for KSHV-seropositive individuals only. bOdds ratios (ORs) are adjusted for sex except when sex is the factor of interest. cCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. Open in new tab The odds of shedding EBV tended to be lower in children born to mothers aged ≥20 years (OR, 0.3; 95% CI, .09–1.05) but were not otherwise associated with any sociodemographic, clinical, or serological factor. Among children shedding KSHV (Table 5), the viral load was higher in those who had helminths (2.1 log copies; 95% CI, .37–3.83) but was not associated with any other factor, whether demographic, clinical, or serological. Table 5. Factors Associated With Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) Loads in Saliva Specimens From Shedding Children Factor . KSHV Load (n = 40) . EBV Load (n = 477) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Male sex −0.10 (−1.19–.99) NS 0.20 (−.14–.56) NS Maternal age group, y  14–19 Reference NS Reference NS  20–24 1.03 (−.53–2.60) −0.05 (−.52–.42)  25–29 1.22 (−.46–2.89) −0.17 (−.69–.35)  30–34 0.34 (−1.52–2.21) −0.34 (−.98–.30)  ≥35 2.05 (−.29–4.39) −0.23 (−1.16–.70) Parity Reference .05 Reference NS  2–4 1.29 (.09–2.50) −0.25 (−.67–.17)  ≥5 0.91 (−.36–2.19) −0.20 (−.76–.36) Maternal viral loadc −3.83 (−16.54–8.89) NS 0.15 (.04–.25) .007 Anemia 0.09 (−1.07–1.25) NS −0.21 (−.62–.21) NS Helminthiasis 2.10 (.37–3.83) .02 −0.16 (−1.26–.93) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium −0.14 (−1.60–1.32) …   High 0.63 (−.67–1.93) …  ORF73   Low Reference NS …   Medium 0.54 (−.83–1.90) …   High 0.09 (−1.22–1.39) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.63 (−.79–2.05) −0.19 (−.62–.25)   High 0.11 (−1.22–1.45) −0.08 (−.51–.35)  PfMSP1   Low Reference NS Reference .01   Medium −0.35 (−1.78–1.07) −0.37 (−.75–.02)   High −0.12 (−1.47–1.24) −0.55 (−.97–−0.12) Factor . KSHV Load (n = 40) . EBV Load (n = 477) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Male sex −0.10 (−1.19–.99) NS 0.20 (−.14–.56) NS Maternal age group, y  14–19 Reference NS Reference NS  20–24 1.03 (−.53–2.60) −0.05 (−.52–.42)  25–29 1.22 (−.46–2.89) −0.17 (−.69–.35)  30–34 0.34 (−1.52–2.21) −0.34 (−.98–.30)  ≥35 2.05 (−.29–4.39) −0.23 (−1.16–.70) Parity Reference .05 Reference NS  2–4 1.29 (.09–2.50) −0.25 (−.67–.17)  ≥5 0.91 (−.36–2.19) −0.20 (−.76–.36) Maternal viral loadc −3.83 (−16.54–8.89) NS 0.15 (.04–.25) .007 Anemia 0.09 (−1.07–1.25) NS −0.21 (−.62–.21) NS Helminthiasis 2.10 (.37–3.83) .02 −0.16 (−1.26–.93) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium −0.14 (−1.60–1.32) …   High 0.63 (−.67–1.93) …  ORF73   Low Reference NS …   Medium 0.54 (−.83–1.90) …   High 0.09 (−1.22–1.39) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.63 (−.79–2.05) −0.19 (−.62–.25)   High 0.11 (−1.22–1.45) −0.08 (−.51–.35)  PfMSP1   Low Reference NS Reference .01   Medium −0.35 (−1.78–1.07) −0.37 (−.75–.02)   High −0.12 (−1.47–1.24) −0.55 (−.97–−0.12) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum. aCoefficients expressing variation in log10 genome equivalents/million cells are estimated in shedding individuals only and are adjusted for sex except when sex is the factor of interest. bCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. cScaled in increments of log10 copies. Open in new tab Table 5. Factors Associated With Kaposi Sarcoma–Associated Herpesvirus (KSHV) and Epstein-Barr Virus (EBV) Loads in Saliva Specimens From Shedding Children Factor . KSHV Load (n = 40) . EBV Load (n = 477) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Male sex −0.10 (−1.19–.99) NS 0.20 (−.14–.56) NS Maternal age group, y  14–19 Reference NS Reference NS  20–24 1.03 (−.53–2.60) −0.05 (−.52–.42)  25–29 1.22 (−.46–2.89) −0.17 (−.69–.35)  30–34 0.34 (−1.52–2.21) −0.34 (−.98–.30)  ≥35 2.05 (−.29–4.39) −0.23 (−1.16–.70) Parity Reference .05 Reference NS  2–4 1.29 (.09–2.50) −0.25 (−.67–.17)  ≥5 0.91 (−.36–2.19) −0.20 (−.76–.36) Maternal viral loadc −3.83 (−16.54–8.89) NS 0.15 (.04–.25) .007 Anemia 0.09 (−1.07–1.25) NS −0.21 (−.62–.21) NS Helminthiasis 2.10 (.37–3.83) .02 −0.16 (−1.26–.93) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium −0.14 (−1.60–1.32) …   High 0.63 (−.67–1.93) …  ORF73   Low Reference NS …   Medium 0.54 (−.83–1.90) …   High 0.09 (−1.22–1.39) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.63 (−.79–2.05) −0.19 (−.62–.25)   High 0.11 (−1.22–1.45) −0.08 (−.51–.35)  PfMSP1   Low Reference NS Reference .01   Medium −0.35 (−1.78–1.07) −0.37 (−.75–.02)   High −0.12 (−1.47–1.24) −0.55 (−.97–−0.12) Factor . KSHV Load (n = 40) . EBV Load (n = 477) . Coefficienta (95% CI) . Pb . Coefficienta (95% CI) . Pb . Male sex −0.10 (−1.19–.99) NS 0.20 (−.14–.56) NS Maternal age group, y  14–19 Reference NS Reference NS  20–24 1.03 (−.53–2.60) −0.05 (−.52–.42)  25–29 1.22 (−.46–2.89) −0.17 (−.69–.35)  30–34 0.34 (−1.52–2.21) −0.34 (−.98–.30)  ≥35 2.05 (−.29–4.39) −0.23 (−1.16–.70) Parity Reference .05 Reference NS  2–4 1.29 (.09–2.50) −0.25 (−.67–.17)  ≥5 0.91 (−.36–2.19) −0.20 (−.76–.36) Maternal viral loadc −3.83 (−16.54–8.89) NS 0.15 (.04–.25) .007 Anemia 0.09 (−1.07–1.25) NS −0.21 (−.62–.21) NS Helminthiasis 2.10 (.37–3.83) .02 −0.16 (−1.26–.93) NS Anti-KSHV antibody OD  K8.1   Low Reference NS …   Medium −0.14 (−1.60–1.32) …   High 0.63 (−.67–1.93) …  ORF73   Low Reference NS …   Medium 0.54 (−.83–1.90) …   High 0.09 (−1.22–1.39) … Anti–P. falciparum antibody titer  PfAMA1   Low Reference NS Reference NS   Medium 0.63 (−.79–2.05) −0.19 (−.62–.25)   High 0.11 (−1.22–1.45) −0.08 (−.51–.35)  PfMSP1   Low Reference NS Reference .01   Medium −0.35 (−1.78–1.07) −0.37 (−.75–.02)   High −0.12 (−1.47–1.24) −0.55 (−.97–−0.12) Abbreviations: CI, confidence interval; NS, not significant; P. falciparum, Plasmodium falciparum. aCoefficients expressing variation in log10 genome equivalents/million cells are estimated in shedding individuals only and are adjusted for sex except when sex is the factor of interest. bCalculated by means of likelihood ratio tests. Values of <.05 are considered statistically significant. cScaled in increments of log10 copies. Open in new tab The EBV load was significantly higher in children of mothers with a higher EBV load (0.15 log copies; 95% CI, .04–.26). Furthermore, the EBV load was lower in children with medium or high P. falciparum MSP-1 antibody levels (−1.05 log copies; 95% CI, −1.77 to −.33). Among KSHV-seropositive participants tested for both viruses, 57% shed only EBV, 13% only KSHV, 13% shed both viruses, and 17% did not shed either virus. Among mothers, 40% of KSHV shedders also shed EBV, compared with 75% of those who did not shed KSHV; for children, values were 65% and 83%, respectively (Supplementary Table 1). When we examined the relationship between KSHV load and EBV load, restriction of the analysis to KSHV-seropositive individuals revealed a negative correlation (ρ = −0.22; P < .0001). The correlation strengthened among KSHV shedders (ρ = −0.30; P < .001). However, when the analysis was restricted to EBV shedders, a modest positive correlation was observed (ρ = 0.15; P = .01). Stratification of mothers and children yielded similar results (except that the variance was larger in the latter case because of the smaller number of KSHV-seropositive children). DISCUSSION To our knowledge, this is the first study of factors associated with shedding of KSHV and EBV in saliva—a mechanism for viral transmission—in a sample of apparently healthy, HIV-uninfected people from a population in which both viruses are very prevalent and in which the tumors they cause, Kaposi sarcoma and Burkitt lymphoma, are endemic [20]. The prevalence of KSHV shedding was similar between mothers and girls, but it tended to be higher in boys. Examination of the KSHV load in individuals who shed revealed that it was higher in children than in mothers but was similar in boys and girls. The reasons for the greater prevalence and, presumably, frequency of shedding among boys, compared with that among mothers and girls, are not clear. In 2 other studies of adults and children from Uganda that examined the prevalence of KSHV in blood, Mbulaiteye and colleagues showed that males were twice as likely to have detectable virus in blood than females [18, 21, 22]. One of these studies [21] found no difference in the prevalence of detectable virus in saliva between boys and girls. However, in combination with the fact that, among people without HIV infection, Kaposi sarcoma shows a marked excess incidence among men as compared to women [23, 24], this might suggest that males are less able to control KSHV replication than females. Our results further suggest that such differential control may be established at an early age, suggesting that nonreproductive factors might be at play, whether genetic or environmental, due to early sex- or gender-specific exposures/behaviors. That many more children than mothers shed also suggests that children may be an important source of KSHV transmission both within the family and in the wider community. Further studies of KSHV transmission between children are warranted. In mothers, we detected strong associations between KSHV shedding and anti–KSHV K8.1 antibody levels; this is consistent with the hypothesis that antibodies against the K8.1 antigen reflect lytic viral replication [25]. Such an association in children was not significant. While children shed more frequently, it is conceivable that the shorter time since KSHV acquisition did not yet result in levels of KSHV-specific antibodies that were greater than those in nonshedders; alternatively, or additionally, in the course of recent infection in children, the antibody response profile might be different than during established infection in adults. The viral load in peripheral blood mononuclear cells (PBMCs) is more likely to be relevant to KSHV pathogenesis than the viral load in saliva, and the correlation between the load in PBMCs and anti-KSHV antibody levels will need to be investigated directly. However, we can now confirm a previously observed association between anti-K8.1 antibody levels and KSHV reactivation, resulting in salivary shedding [8]. This further validates our previous observations on the possible role of the anti-K8.1 antibody level as a prognostic marker in the natural history of infection [25]. In previous work within this cohort, we identified associations between KSHV prevalence and malaria parasitemia in both mothers and children [9, 10]. More recently, we found that the KSHV prevalence was also associated with antibodies against P. falciparum malaria (both AMA-1 and MSP-1) in both mothers and children [26]. In this study, we found an association between AMA-1 levels and KSHV shedding in mothers and between MSP-1 levels and KSHV shedding in children, but we found no association between these anti–P. falciparum antibodies and the KSHV load in shedders, either mothers or children. Further prospective research on the role of malaria in the natural history of KSHV infection is justified. In keeping with our previous findings, we also observed a sizable association between fecal detection of helminths in children and both KSHV shedding and viral load, although for the former finding, the small number of affected children did not allow the association to reach significance. This also deserves further study in larger cohorts, especially in light of the recent observations that murine gammaherpesvirus 68 undergoes reactivation in latently infected mice with acute with intestinal helminth infection [27]. Consistent with earlier reports, a large majority of participants (adults and children of both sexes) were shedding EBV [28]. Such a high proportion of shedders explains to some extent why, contrary to KSHV, EBV is ubiquitous in this population from a very early age. A child presumably had a higher likelihood of exposure to EBV than to KSHV at any given time, even though we detected similar virus loads in shedders: in children, the median KSHV load was 4.4 log copies (IQR, 3.5–4.7 log copies), and the EBV load was 4.7 log copies (IQR, 3.6–5.4 log copies); in mothers, viral loads were 3.6 log copies (IQR, 2.3–4.6 log copies) and 3.9 log copies (IQR, 2.4–4.7 log copies), respectively. Susceptibility to acquisition of either viral infection might also differ in the same individuals, in part because of the different role of exposure cofactors. In mothers, no factor was associated with EBV shedding or viral load. In children, however, some maternal factors were significant. Children of older mothers tended to shed less, emphasizing the possible role of environmental exposures unmeasured in our study. Likewise, children of mothers with a high EBV load tended to shed more themselves, again suggesting a common exposure or perhaps a genetic factor, also worthy of further examination. We have recently performed a genome-wide association study in a population-based rural Ugandan cohort, and we have identified an association between 5 novel loci and anti-EBV antibody levels [29]. Finally, we have identified negative associations between anti–MSP-1 (but not anti–AMA-1) antibody levels and EBV load in shedding children (but not mothers). The relationship between EBV and malaria has been investigated since the discovery of the virus [30], and very recent data provide interesting insight on the role of malaria in EBV-associated lymphomagenesis [31]. However, as with KSHV, the contribution of malaria to EBV pathogenesis and natural history must be further investigated throughout the life-span. For the first time, we also present data on the relationship between KSHV and EBV shedding in saliva. It is notable that individuals shedding KSHV tend to shed less EBV, while individuals shedding EBV tend to shed more KSHV. This suggests that there may be direct or indirect interaction between EBV and KSHV oropharyngeal replication, but the mechanisms controlling replication and shedding in saliva have not yet been investigated. Sparse in vitro data are available on dually infected primary effusion lymphoma lines, showing KSHV inhibition by EBV [32], mutual inhibition by the 2 viruses [33], differential transactivation of the 2 viruses by host immune factors [34], and even synergistic in vivo tumorigenicity in animal models [35]. Further investigation of the interactions between the 2 human gammaherpesviruses and their host is warranted. In conclusion, this study investigated salivary shedding of KSHV and EBV in apparently healthy mothers and their children and is the first to identify several factors that influence shedding or salivary viral load during infection with either virus. Our findings contribute to knowledge about the transmission, epidemiology, and natural history of EBV and KSHV infection in an East African population, in which the 2 viruses and associated malignancies are endemic. Notes Disclaimer.   The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Financial support. This work was supported by the Intramural Program of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services (contract HHSN261200800001E to D. W.), by the Wellcome Trust (grants 064693, 079110, 95778 to A. M. E., and 090132 to K. 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N. is a Senior Visiting Scientist at the International Agency for Research on Cancer, Lyon, France. A. M. E. is the Principal Investigator of EMaBS, in which this project is based. Published by Oxford University Press for the Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. This Open Access article contains public sector information licensed under the Open Government Licence v2.0 (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/2/). Published by Oxford University Press for the Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Journal

The Journal of Infectious Diseases – Oxford University Press

Published: Aug 14, 2018

Keywords: herpesvirus 4, human; child; human herpesvirus 8; mothers; saliva; viruses; antibodies; gammaherpesvirus

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