Cytomegalovirus, Epstein-Barr virus and varicella zoster virus infection in the first two years of life: a cohort study in Bradford, UK

Cytomegalovirus (CMV), Epstein Barr virus (EBV) and varicella-zoster virus (VZV) are common herpesviruses frequently acquired in childhood. They establish persistent, latent infection and are likely to impact the developing immune system [1, 2]. In childhood, VZV causes chickenpox but CMV and EBV infections are usually asymptomatic. If acquired in adolescence, EBV can cause glandular fever. EBV has been associated with immune disorders such as multiple sclerosis and lymphoma [3, 4] and CMV seropositivity is associated with inflammation, atherosclerosis and immunosenescence [5, 6].

Chickenpox vaccination is not part of the UK routine childhood vaccination programme but zoster vaccine has been introduced for adults in their 70s to prevent shingles [7], while CMV and EBV vaccines are under development [8‐10]. Population-based data on the age-specific prevalence and incidence of these viruses is needed to inform the timing and target groups of potential vaccination programmes. Little is known about the epidemiology of CMV and EBV in contemporary paediatric UK populations, particularly in different ethnic groups. A recent US study identified differences in CMV seroprevalence by ethnic group among children but had limited data on other relevant variables such as childcare and breastfeeding [11].

The Allergy and Infection Study is a sub-study of the Born in Bradford birth cohort and aims to describe the maternal and paediatric epidemiology of CMV, EBV and VZV infection and to investigate the effect of age at infection on immune function and atopic allergy. The specific objectives of this paper are to estimate the incidence of CMV, EBV and VZV infection at age 1 and 2 years and to identify factors associated with infection, in particular to quantify any differences in age at infection by ethnic group.

Children enrolled in the Born in Bradford cohort who were born on or after 1 March 2008 with a maternal baseline questionnaire available were eligible for the Allergy and Infection Study (ALL IN). Details of the Born in Bradford (BiB) cohort are described elsewhere [12]. Briefly, BiB is a multi-ethnic birth cohort study aiming to examine the impact of environmental, psychological and genetic factors on maternal and child health. Bradford is a city in the North of England with high levels of socio-economic deprivation and ethnic diversity.

Mothers were invited to participate in ALL IN 1 month before their child’s first birthday. A questionnaire was completed by those who consented and a 5 ml venous blood sample was taken from the child, centrifuged and stored at -80 °C. This was repeated 1 year later to provide exposure data and serum at 12 and 24 months. See Additional file 1 for further details.

A sample of 1000 children was selected for serological testing (see Additional file 1 for sample size calculations); 700 based on having 4 or 5 aliquots of the 24-month sample and having received the relevant immunisations (for a separate analysis of response to routine vaccination) and 300 randomly selected from those with fewer than 4 aliquots.

At the Royal Free Virology laboratory, the 24-month samples were tested first for CMV-immunoglobulin G (IgG), EBV-IgG and VZV-IgG. If positive, the 12-month sample was tested in the same way. CMV-IgG was tested using the Abbott Architect assay (kit product no. 6c15-25) and EBV-IgG and VZV-IgG using the Diasorin Liaison assays (kit product numbers 310510 and 310850, respectively) [13]. For children who were CMV-IgG positive at 12 months, the cord blood sample was tested for CMV- deoxyribonucleic acid (DNA) using a quantitative real-time polymerase chain reaction method described in detail elsewhere [14]. To verify the expected loss of maternal antibody by 12 months so that IgG testing at this age can represent paediatric infection, a random sample of 100 children who were IgG negative at 24 months for each infection were tested for IgG at 12 months.

Of 7423 eligible children, the parents of 5891 were contacted and invited to take part in ALL IN and 3196 (54%) agreed. Of the 2695 parents who declined, 2013 gave a reason: 1075 (53%) not happy about having blood samples taken, 510 (25%) too busy and 428 (21%) gave other reasons. Figure 1 shows the number of children completing a 12- and 24-month visit. The children recruited were born between March 2008 and June 2011.

Of the 24-month samples, there were 5 equivocal results for VZV which were considered negative. One-third of children (323) were seronegative for all three infections and 677 seropositive for at least one. A 12-month sample was not available for 58 of 677 children so 12-month samples for 619 children were tested. 231 children were CMV-IgG positive at 12 months. Cord blood was available for 154 of these; one child had detectable CMV-DNA (52,268 CMV VL genomes/ml) while 153 were undetectable (<200 copies/ml). Testing to verify loss of maternal antibody gave the following results: For EBV none of the 101 children who were IgG negative at 24 months were IgG positive at 12 months. For CMV two of 103 children, and for VZV two of 101 children, who were IgG negative at 24 months were IgG positive at 12 months (with the same results on re-testing).

This analysis includes 1000 children with CMV, EBV and VZV infection status at 12 and 24 months. 391 (39%) children were born to White British women, 472 (47%) to Pakistani women and 137 (14%) to women of other ethnic groups (Table 1). Table 1 shows the characteristics of the children and their mothers by ethnic group (also see Additional file 2).

The 1000 children selected for this analysis were similar to the ALL IN children who were not included with respect to ethnic group, mother’s country of birth, sex, childcare, household size and number of children in the household but were more likely to have been breastfed (81% vs. 74%). Compared to the rest of the BiB cohort (12,538 children), the 1000 ALL IN children were similar in terms of most characteristics but their mothers were slightly more likely to be married and living with partner (72% vs. 65%) and to be in the least deprived quintiles of the Index of Multiple Deprivation.

There were marked differences in the incidence of CMV and EBV infection at 12 and 24 months between the White British and Pakistani children with the Pakistani children infected earlier (Fig. 2 and Additional file 3).

One-third of Pakistani children were CMV infected in the first year compared to 9% of White British children. By 2 years, half of Pakistani children were EBV infected compared to a quarter of White British children. The higher incidence of CMV and EBV infection among Pakistani children cannot be fully explained by differences in breastfeeding, family size, childcare and socio-economic indicators. VZV incidence from 12 to 24 months was lower among the Pakistani than White British children. Risk factors for CMV and EBV infection differed by ethnic group.

Our VZV estimates are similar to those from previous UK studies [18, 24]. In the Millennium Cohort Study, children from other ethnic groups were less likely to have had chickenpox by age 3 years than white children, based on maternal report, similar to our findings [18]. There is good evidence that VZV seroprevalence in the UK is lower among pregnant women born in Asia than those born in the UK [13, 25], due to later age at infection in many tropical countries compared to the UK. However, it is unclear why children born to Asian women in the UK are infected later, as VZV is predominantly transmitted between children rather than from contact with the mother. It has been suggested that infection with CMV affords some cross-protection against VZV [18, 26] which could explain the later VZV infection in Pakistani children in our study as they are infected with CMV earlier than the White British children.

Our CMV incidence results were similar to a cross-sectional study in the Netherlands [27]. CMV seroprevalence in 1- and 2-year-olds in a US study was lower than our overall results, although there were small numbers of children in each age group (<200) [11]. The only other UK study on CMV prevalence in young children was based in a London hospital 30 years ago and 20% of children were infected by 12 months [16]. Results from Australia and Sweden showed slightly higher seroprevalence at 2 years [28, 29]. Our CMV results on breastfeeding, childcare and socio-economic indicators are consistent with previous findings and we report novel results on birth order (Table 5).

Ethnicity was a key risk factor in several studies with consistent findings of higher CMV prevalence among children in non-white ethnic groups compared to white/European/Western groups [11, 27, 30]. This association was specifically investigated in relation to other factors in studies in New Zealand [31] and the Netherlands [32] in children aged 3.5 and 6 years, respectively. Differences by ethnic group could not be explained by differences in parity, day care, breastfeeding, and socio-economic indicators, similar to our findings.

The unexpected apparent protective effect of higher birth order on early CMV infection among Pakistani children is unlikely to be spurious for several reasons. First, the birth order effect commonly associated with childhood infections (i.e. children of higher birth order more likely to be infected earlier) was observed for VZV in our study. Second, estimates for the White British children are also less than one although the confidence intervals cross one. Third, Jansen et al. report a similar finding; multiparity was inversely associated with CMV seropositivity by age 6 years (OR 0.76, 95% CI 0.65–0.90) [32]. The ‘birth order effect’ is usually discussed in relation to the hygiene hypothesis and refers to the lower risk of atopic disease among children of higher birth order due to increased exposure to childhood infections [15]. An alternative or contributing explanation for this effect could be changes in the maternal immune system with increasing parity so that the effect is determined in the prenatal period [33]. Whether these changes could also affect the maternal immune response to CMV infection and thereby the risk of transmitting virus to her child (e.g. through breastmilk) warrants further investigation. Perhaps CMV infection in the first child acts like a vaccine to boost maternal immunity so she has lower viral load and takes longer to infect subsequent children. Any such effect might be manifest through acquiring immune responses to strains of CMV different from the initial maternal infection, because prototype CMV vaccines can boost the immunity of seropositive adults [8, 34, 35].

Our EBV incidence estimates are comparable to results from previous UK studies [36‐38]. Differences in paediatric EBV prevalence by ethnic group have been reported from two US studies [22, 39] and one in the Netherlands [32]. Socio-economic position and family size partly explained the ethnic differences observed in the Generation R study [32] but the lower prevalence in white children could not be explained by infection risk factors in the Minnesota study [39].

The unexpected association between attendance at mother/baby activities and later EBV infection among White British children may have arisen due to uncontrolled confounding by socio-economic factors; further investigation did not provide convincing evidence of a real effect as the association was only evident in a sub-group.

Ethnic differences in paediatric CMV and EBV incidence have not been reported before in the UK but our results are in line with findings in different populations. As these cannot be fully explained by factors influencing exposure to infection, genetic variation may play a role. For example, the prevalence of the NKG2C deletion is known to vary between populations and is important in NK cell responses to CMV infection such that individuals with the deletion are less able to control CMV viraemia so CMV may be more easily transmitted [40]. Genetic susceptibility may also be important in infectious mononucleosis following EBV infection [41].

A key strength of this study is having serology data at two time points early in childhood. Previous studies have been cross-sectional or with samples available for only one time point. Our results demonstrate that most of the CMV infections occurred in the first year and are generally due to contact with the mother, whereas most EBV and VZV infections are acquired in the second year through contact with siblings and other children. Pinpointing age at infection is important when examining associations with subsequent development of atopic disease [42, 43] as there is likely to be a critical window in immune development in the first 2 years during which infection with CMV and/or EBV may influence immune-related outcomes.

We collected detailed information on likely sources of exposure to infection, with few missing values due to interviewer-administered questionnaires with well-trained interviewers. This study had the statistical power to investigate differences in acquisition of infection between two large ethnic groups and the advantages of a prospective birth cohort design with rich characterisation and homogeneity within ethnic groups. There were few differences between the children in this analysis and the main BiB cohort and our results are generalisable to other multi-ethnic urban UK populations. Our results may not be generalisable to less deprived populations within the UK where the incidence of CMV and EBV infection by age 2 years is likely to be lower than in Bradford.

A limitation of our study is that maternal CMV serostatus was unavailable for most children. Adjustment for mother’s country of birth and maternal age would have partially accounted for maternal serostatus as South Asian women born abroad have higher seroprevalence than those born in the UK [13] and seroprevalence increases with age. The pattern of higher incidence at 12 months in Pakistani than White British children was still evident in the sub-group of CMV seropositive women.

Misclassification of age at CMV and VZV infection due to the occasional presence of maternal antibody at 12 months is likely to have had a minimal effect; the adjusted incidence figures rounded to the same percentage points.

This study provides current incidence data for three common herpesviruses in young children which are generalisable to urban UK populations. We have demonstrated large differences in incidence between White British and Pakistani children for the first time in the UK and have identified key risk factors for infection in each group, some novel, which suggest mechanisms of transmission to be investigated further.

Our data will inform the optimum schedule of potential CMV and EBV vaccination programmes. For CMV it has been proposed that toddlers as well as adolescents are vaccinated to prevent transmission to pregnant women and reduce the risk of congenital CMV infection [9]. Our results suggest that infants of Pakistani origin would need to be vaccinated before age one and the impact of vaccinating children with recent natural infection needs to be explored.