The major strengths of the present study were its size, its prospective nature, and the availability of HHV-8 and HIV-1 coinfection data from mother-infant pairs collected at multiple time points from birth to 48 months of follow-up. These strengths enabled us to provide the first documentation of annual HHV-8 incidence rates in early childhood in an African endemic area. Our results indicate that the HIV-1 status of the child is a strong predictor of HHV-8 seroconversion. Incidence rates were generally high among these children between birth and 48 months of age. In addition, fluctuations in detectable HHV-8 titers leading to seroreversions among HHV-8-seroconverted children may produce frequent underestimation of childhood HHV-8 seroprevalence in cross-sectional studies.
The observed HHV-8 seroprevalence in Zambian children is generally comparable to prevalences reported in cross-sectional studies from other parts of sub-Saharan Africa (17
). While the prospective, longitudinal design of this study makes it difficult to compare its results directly with those of published cross-sectional studies conducted in the region, HHV-8 seroprevalence of 20–60 percent has been reported among young children (22
). Kaplan-Meier analysis (data not shown) estimating the probability of HHV-8 seroconversion in this longitudinally followed cohort revealed that more than 40 percent of the children seroconverted for HHV-8 by age 48 months, with a clear increase in HHV-8 seroprevalence with age. These results show that children become infected at a young age in Zambia and that adult seroprevalence levels may be reached relatively early in life. These results indicate that HIV-1-infected children are more likely to become HHV-8-infected, but it remains unclear whether increased risk is due to HIV-1-infected children's having 1) a higher likelihood of being exposed to HHV-8, 2) a higher likelihood of becoming infected when exposed, or 3) a higher likelihood of antibody detection when infected. In addition, the specific risk factors associated with each of these possibilities have yet to be determined.
Potential routes of horizontal HHV-8 transmission are poorly understood, but salivary contact may be the major route of transmission in early childhood. Our laboratory and others have previously found that HHV-8 DNA can be readily detected in the saliva of infected persons and is detected more frequently in persons with higher antibody titers (16
). While Zambian children are usually breast-fed up to the age of 18–24 months, HHV-8 cannot be easily detected in breast milk, suggesting that it is an unlikely source of infection (16
). We frequently observed HHV-8 seroconversion in children born to mothers who were HHV-8-seronegative at delivery, indicating that horizontal transmission is possible in early childhood. These results are consistent with Mantina et al.'s finding that in utero infection of infants is infrequent (18
). However, we cannot rule out the possibility that some children who are HHV-8-seropositive at 12 months have HHV-8 that is due to perinatal transmission. Incidence in this cohort could still be underestimated if the perinatally infected children seroreverted before age 12 months. Determining serostatus in children below age 12 months is difficult because of the presence of maternal antibodies.
In this cohort, maternal HHV-8 infection was not an independent risk factor associated with transmission of HHV-8 to children—a finding which was somewhat unexpected, because in the early phase of life the mother is usually the primary caregiver and has close contact with the child. This suggests that mothers may not be the only source of transmission to children and that other members of the household or nonfamilial contacts could be responsible for horizontal transmission to young children. Some reports from sub-Saharan Africa have shown a positive correlation between the HHV-8 status of the mother and that of the child (24
). However, other studies reported only a marginal-to-weak correlation (25
), and a recent study demonstrated that young infants' risk of acquiring HHV-8 infection in South Africa was not dependent on maternal serostatus (41
). Molecular evidence for this has come from Uganda, where a mother and child were demonstrated to have different HHV-8 subtypes (42
). The strength of this association could depend on locally common child-care practices, such as kissing, premastication of food, or sharing of food and utensils (43
). The impact of such practices has not been explored fully and may be different in Zambia than in other HHV-8-endemic countries.
Brayfield et al. (16
) previously reported risk factors associated with HHV-8 infection in a much smaller group of infants at 12 months while active follow-up of mother-infant pairs was still ongoing. A commercially available enzyme-linked immunosorbent assay was used to validate BC-3 mIFA results, and we observed that it underestimated HHV-8 seroprevalence by missing patients with distinct punctate nuclear staining, which led us to develop the Sf9 mIFA. We believe that patients with specific punctate staining could have low-titer antibodies that were missed by enzyme-linked immunosorbent assay because of the higher optical density cutoff values. Employing two assays as part of an algorithm provided us with a reliable, highly specific and conservative method. Sf9 mIFAs have matched negative controls that are lacking for BC-3 mIFAs, thus contributing to a low number of false-positive results. Analysis of a panel of positive and negative control serum samples revealed a high concordance between the two mIFAs (κ = 0.75; unpublished data). Zhu et al. (44
) have suggested that confirmation of HHV-8 serostatus should not be based on a single antigen, since infected persons may demonstrate variable reactivity against different antigens. This variability may explain the fluctuations in the anti-HHV-8 antibody titers seen in this study and may explain why certain children have undetectable antibody titers during follow-up. Such variation has been observed in adults, and seroreversion has been reported for other herpesviruses and for hepatitis C virus (45
). HHV-8 DNA has been detected in certain HHV-8-seronegative patients, and biopsy-proven Kaposi's sarcoma patients in full remission have been reported to undergo HHV-8 seroreversion (50
Seroreversion may be partial or complete, resulting from a loss of detectable titer to one or more antigens or a total loss of all specific antibodies. We believe that the observed seroreversions in our cohort could be both partial and complete seroreversions. It is unlikely that the HHV-8 seropositivity of children who tested positive at 12 months and subsequently tested negative was due to residual maternal HHV-8 antibodies, because all children born to HHV-8-seropositive mothers were titered at birth and at age 6 months. Eight of 10 children who were seropositive at 12 months and were seronegative at all later time points were born to HHV-8-seronegative mothers and were themselves seronegative at birth. It is possible that the observed seroreversions in our cohort were due to antibody titers that were below the limit of detection of our assays or due to the stringent detection criteria used. However, seroreversion has also been reported in studies using enzyme-linked immunosorbent assay (45
). The lack of a gold-standard assay with established 100 percent accuracy makes it difficult to confirm these results. Performance studies of type-specific commercial assays designed to distinguish between herpes simplex virus types 1 and 2 have also reported that seroreversions were introduced because of poor assay sensitivity (52
). An absence of antigenic stimulation after establishment of latency or viral clearance could lead to seroreversion. In addition, immunocompetent children may be able to efficiently control further reactivation, and thus the antibody titers drop below detection levels. It has been proposed that HHV-8-specific antibodies might be more readily detectable due to broadening of epitope recognition over time or due to subsequent reactivation after primary HHV-8 infection (45
). HIV-1-related immunosuppression has also been proposed to be a factor responsible for HHV-8 seroreversion, especially in the Zambian population, which is experiencing a generalized HIV-1 epidemic. We observed that only four out of 41 HIV-1-infected children underwent seroreversion at one or more time points, but this number was too small for us to draw any conclusion, and a much larger cohort of HIV-1-infected children will be needed in order to understand this phenomenon.
Our study did have some important limitations. Although the number of HIV-1-infected children followed was limited, we found significantly higher HHV-8 incidence among HIV-1-infected children than among uninfected children. The high rate of attrition in HIV-1-infected children probably led to an underestimate of the true HHV-8 incidence and of the impact of child HIV-1 infection status on the risk of HHV-8 acquisition during early childhood. The number of children born to HIV-1-positive mothers was higher in the group that was not included in the analysis. To be eligible for inclusion in the analysis, a child had to survive and return at 24 months of age for reliable detection of HIV-1 antibodies. In addition, neither degree of clinical immunosuppression nor HIV-1 viral load could be assessed, because methods for determining CD4 cell count and viral load were not yet available in Zambia. Transmission from siblings could not be examined because siblings were not recruited. Attrition due to mortality was high in children, especially those who were HIV-1-infected, because of lack of availability of antiretroviral therapy at the time of the study (53
). None of the HIV-1-infected children returned after 36 months, probably because of high mortality in this group, which was without access to antiretroviral agents during the time period of this study. Pediatric antiretroviral therapy has since been implemented at primary health-care clinics throughout Lusaka (54
). Common reasons for withdrawal from the study were religious beliefs, disapproval of a spouse or family, or lack of interest. Some families were untraceable because both the mother and the child died, relocated, or provided a wrong address. Most members of the study population were of lower socioeconomic status, which contributed to their being highly mobile.
In conclusion, horizontal transmission appears to be the major route of HHV-8 transmission in early childhood, and HIV-1 infection of the child is an important risk factor for HHV-8 acquisition in an area that is highly endemic for both viruses. The frequent seroreversions observed demonstrate that cross-sectional serologic screening for HHV-8 underestimates true rates of HHV-8 infection and may not provide a true representation of HHV-8 prevalence and incidence in a population.