The authors have declared that no competing interests exist.
Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract disease in early life and a target for vaccine prevention. Data on the ageprevalence of RSV specific antibodies will inform on optimizing vaccine delivery.
Archived plasma samples were randomly selected within age strata from 960 children less than 145 months of age admitted to Kilifi County Hospital pediatric wards between 2007 and 2010. Samples were tested for antibodies to RSV using crude virus IgG ELISA. Seroprevalence (and 95% confidence intervals) was estimated as the proportion of children with specific antibodies above a defined cutoff level. Nested catalytic models were used to explore different assumptions on antibody dynamics and estimate the rates of decay of RSV specific maternal antibody and acquisition of infection with age, and the average age of infection.
RSV specific antibody prevalence was 100% at age 0<1month, declining rapidly over the first 6 months of life, followed by an increase in the second half of the first year of life and beyond. Seroprevalence was lowest throughout the age range 5–11 months; all children were seropositive beyond 3 years of age. The best fit model to the data yielded estimates for the rate of infection of 0.78/person/year (95% CI 0.65–0.97) and 1.69/person/year (95% CI 1.27–2.04) for ages 0<1 year and 1<12 years, respectively. The rate of loss of maternal antibodies was estimated as 2.54/year (95% CI 2.30–2.90), i.e. mean duration 4.7 months. The mean age at primary infection was estimated at 15 months (95% CI 13–18).
The rate of decay of maternal antibody prevalence and subsequent ageacquisition of infection are rapid, and the average age at primary infection early. The vaccination window is narrow, and suggests optimal targeting of vaccine to infants 5 months and above to achieve high seroconversion.
Acute respiratory infection (ARI) is a leading cause of morbidity and mortality in children <5 years old worldwide [
To date there are no licensed vaccines for prevention of RSV disease in infants and young children. Some candidate vaccines have shown promising results[
The primary target for RSV vaccination is children under 6 months of age; a group highly susceptible to severe RSV disease[
Analysis of an age specific seroprevalence profile could inform on the age distribution at attack of the disease in a given population. Data from previous serological studies of RSV specific antibodies suggest early ageacquisition of RSV specific antibodies following decay in maternal antibody[
Catalytic models explain antibody dynamics as a function of age and use agestratified serological data to estimate the force of infection. The simple catalytic model can be modified to allow for varied structures for nonimmunizing infections[
In this study we present the agespecific prevalence of antibodies to RSV from a rural community at the Kenyan coast. We then develop three nested catalytic models that explore different assumptions on the RSV specific antibody dynamics. Samples were selected randomly from pediatric admissions to a County hospital in coastal Kenya, and screened for antibodies to RSV by ELISA. The data from this study provides basic understanding on natural response to RSV infection and has a bearing on the optimal age of RSV vaccine delivery.
We used data and archived plasma samples from children aged 1 day to less than 12 years (i.e. <145 months) who were admitted to Kilifi County Hospital (KCH) paediatric wards between 2007 and 2010 (inclusive). The County Hospital, previously known as Kilifi District Hospital (KDH), is located in a rural area along the Kenyan Coast. The Kenya Medical Research Institute (KEMRI)Wellcome Trust Research Programme provides clinical care on the hospital paediatric wards and runs the Kilifi Health and Demographic Surveillance System (KHDSS) covering an area of approximately 900km^{2} which is within 50km north, 50km south, and 30km west of the hospital, and includes a population (2010 enumeration) of approximately 260,000[
The study participants were selected at random from Kilifi Integrated Data Management System (KIDMS) admission register regardless of diagnosis and stratified by age into one month age groups up to 11 months, then 12<15m, 15<18m, 18<24m, 24<36m, 36<60m, 6084m, 84<108m and 108<145m. These children were linked to stored serum samples collected on admission, which were retrieved and an aliquot screened for the presence of antibodies to RSV. Being a resident of KHDSS, admitted to KCH between 2007 and 2010 and having a stored plasma sample at admission were inclusion criteria. All readmissions were excluded, i.e. the total number of samples in the present analysis is exactly the total number of study participants.
To reduce bias due to seasonal transmission and in order to provide an average seroprevalence in the presence of seasonal and longerterm variation in transmission, crosssectional sampling covered a period of four years. Each year was divided into quarters with each quarter contributing 60 samples such that each of the 20 age groups had 3 samples. This gave a total of 960 samples for the 4year period, 240 samples from each year. Of the 960 samples each of the 20 age groups had 48 samples, 12 collected each year. Sample size was estimated by standard techniques to provide seroprevalence precision estimates of 50% within a width of +/14%.
All parents and guardians gave written consent to have their children participate in the paediatric inpatient surveillance at KCH and for storage of blood samples for use in future research. The KEMRI Ethical Review Committee approved this study.
All admission plasma samples were tested for antibody concentration with an IgG based ELISA method using crude virus extract from lab adapted RSV A2 culture and specific antibody concentrations recorded as log arbitrary units as determined by a local standards procedure. The optimal dilutions for RSVA2 antigen and serum were determined by a checkerboard titration. The crude virus RSV lysate preparation was as previously described by Ochola et al [
The samples were run in duplicate to account for any variability in the assay operation such as caused by pipetting errors. Within every plate, both high and low controls were run alongside the samples. A graph was plotted over time to check for both the standards and coating antigen deterioration.
A pooled standard serum from adults, serially diluted in 2 fold dilutions from 1:50 to 1:2800 was run in each plate to generate a standard curve. The OD values of the standard sera dilutions were assigned arbitrary unit (AU) values. Standardized arbitrary units (and log_{10} transformed) of RSV specific IgG for samples were estimated by interpolation of a standard curve generated from the pooled adult serum (serum standard) tested in each ELISA run.
A frequency distribution of log_{10}AU values for all sera screened was made to determine a suitable cut off between seropositive and seronegative status. A cut off point of 1.5 as previously applied by Ochola et al, [
Top row: Scatter plots of antibody titer level by age groups. Bottom row: Histograms of antibody titers by age groups. Red lines show the 1.5 cutoff used to define seropositivity.
Data were analyzed using STATA version 11 (StataCorp, College Station, Texas, USA). The proportions of samples in each age class were derived with 95% confidence intervals.
We estimated the titerrelated risk of primary RSV infection and rate of loss of maternal antibodies concurrently, using a nested catalytic model built up from a simple model. The nested model allows for exploration of different assumptions on population level antibody dynamics. We assumed that antibodies were acquired through maternal transfer or exposure to infection.
A catalytic model is a population level model; individuals are grouped into different states (compartments) depending on assumptions in the model. The simple catalytic model had two compartments: (i) the proportion susceptible and seronegative at each age group,
We explored three main forms of the nested model structure (
The compartments in the model represent the following states of the population: M = Individuals with maternally acquired antibodies (split into 2, M_{1} and M_{2} to allow for improved fit), S_{0} = Seronegative after loss of maternally acquired antibodies, F_{0} = Permanent seropositive status after infection, F_{1} = Temporary seropositive status after primary infection, S_{1} = Seronegative after loss of infection acquired antibodies. When p = 0, the model reduces to MSF, when p = 1 and only 1 rate of infection is estimated
At age zero, all individuals are seropositive for maternally acquired RSV antibodies, the M compartment. They lose their maternally acquired seropositive status at a constant rate σ entering the susceptible compartment S_{0} in which they become infected at a constant rate λ_{0} and enter the F_{0} class of (ever) infected. In F_{0} they experience subsequent infections, at an unknown rate, while seropositive.
This is an extension of the MSF model where a proportion, p = 1, will get infected at a rate λ_{0} and enter the F_{1} compartment, but subsequently lose their seropositive status at a rate δ and join the S_{1} class of susceptibles. From S_{1} they get reinfected at a rate λ_{1} and move into the F_{0} class following reinfection and experience subsequent infections while seropositive.
An extension of the
The model structure is shown in (
Parameter  Description  Value 

Proportion that loses antibodies after primary infection.  0 or 1  
Rate of loss of antibodies post primary infection  4/person/year [ 

Rate of loss of maternal antibodies  Fitted  
Rate of primary infection (primary force of infection)  Fitted  
Rate of secondary infection (secondary force of infection)  Fitted 
Parameter fitting was done using maximum likelihood estimation (MLE)[
Using the parameters estimated, we calculated the average age at primary infection
There were 960 children selected from the admission register stratified into 20 age groups with each group having 48 children. Four hundred and one (41.8%) were female. Based on primary diagnosis at discharge, 298 (31.0%) were admitted with lower respiratory tract infection (LRTI), 181 (18.8%) with gastroenteritis, 174 (18.1%) with other conditions (cardiac problem, Burkitt’s lymphoma, epilepsy, hydrocephalus), 145 (15.1%) other infectious diseases, 57 (5.9%) malaria, 39 (4.1%) malnutrition, 37 (3.9%) congenital diseases, 17 (1.7%) anemia, while 12 (1.2%) had a missing diagnosis. Of those with a diagnosis of LRTI, 16% (5% of study participants) had an antigen confirmed RSV infection [
Overall seroprevalence was 65.7% (95% CI: 62.7–68.7). There was 100% seroprevalence in all children less than 1month old. Seroprevalence declined from 91.7% at 1 month to a low of 25% at 6 months. Thereafter, there was slow rise between 6 months to 9 months and then a rapid change in seroprevalence from 41.7% (11 to <12 months), 52.1% (12 to <15 months), 56.3% (15 to <18 months), 83.3% (18 to <24 months), 93.8% (24 to <36 months) and 100% (36 to <60months). All children above 3 years were seropositive. The proportion seropositive per age group is shown in
Age group in months  RSV IgG ELISA(%)  95% CI 

0<1  100  1 
1<2  91.67  83.56–99.78 
2<3  75  62.29–87.71 
3<4  64.58  59.88–85.96 
4<5  45.83  31.21–60.45 
5<6  27.08  14.04–40.12 
6<7  25.00  12.29–37.71 
7<8  39.58  25.23–53.93 
8<9  35.42  21.38–49.45 
9<10  43.75  29.19–58.31 
10<11  39.58  25.23–53.93 
11<12  41.47  27.2–56.13 
12<15  52.08  37.42–66.74 
15<18  56.25  41.69–70.81 
18<24  83.33  72.40–94.27 
24<36  93.75  86.65–1.01 
36<60  100  1 
60<84  100  1 
84<108  100  1 
108<145  100  1 
Each age group had a total of 48 children.
We explored different assumptions in three nested catalytic models and compared the fits to data; the comprehensive results are shown in S1 Table in
The MSF model fit the data best with an AIC_{C} value of 634.6, the MSFSF_{1} had an AIC_{C} of 635.6 while the MSFSF_{2} had 637.9
Parameters  LL  AIC_{C}  

Model  p 
δ 
σ  λ_{0}  λ_{1}  
0  NA  2.54  0.78, 1.69  NA  314.3  634.6  
1  4  2.71  1.80, 3.10  1.80, 3.10  314.8  635.6  
1  4  2.78  1.53  3.98  316.0  637.9 
p = proportion that loses antibodies post primary infection; δ = rate of loss of antibodies post primary infection; σ = rate of loss of maternal antibodies; λ_{0} = primary force of infection; λ_{1} = secondary force of infection; LL = the negative log likelihood value, the lower the value the better the model; AICc = the second order Akaike information criterion.
* These parameters are fixed (not estimated). All rates are per person per year.
The MSF model gave a rate of loss of maternal antibodies of 2.54/year (95% CI 2.30–2.90) and a force of infection of 0.78/person/year (95% CI 0.65–0.97) for ages 0–1 year and 1.69/person/year (95% CI 1.27–2.04) for ages 1–12 years. (
Main: Of the three models in the nested model structure, the MSF model gave the best fit to the data and is shown by the blue line. The parameters were reestimated to obtain the fits that gave the 95% confidence region by Bootstrapping method, grey lines. The red circles show the proportions seropositive by age group according to the data. Inset: A magnification for age range 0–3 years.
The dark blue bars show the proportion at different age groups that have maternally acquired antibodies while the pink bars show the proportion that have been infected and hence have infection acquired antibodies, as predicted by the MSF model. The dashed blue line shows the stepwise force of infection function.
The samples included in this analysis were from hospitalized children with different discharge diagnoses. Included in these were children diagnosed with LRTIs, a subset of which were RSV antigen positive. To check if the inclusion of these samples led to any bias, the MSF model with stepwise force of infection was refitted to 2 subsets of the data; excluding the LRTIs and the RSV antigen positives. The parameter estimates from these fits were not different from those obtained using all the data. This is shown in S2 Fig in
The model structure was modified to relax the assumption that the presence of maternal antibodies is refractive to infection. The force of infection acting on children prior to loss of maternal antibodies was estimated as 0.0/person/year, indicating that the data does not support this infection process (results not shown). The cutoff for seropositivity for ages 0–6 months was varied to try and account for differences between maternally acquired and infection acquired antibodies. We explored a range of cutoffs from 1.5 to 2, in steps of 0.25. Increasing the cutoff resulted in an increase in the rate of loss of maternal antibodies and a decrease in the force of infection. The estimate for the recommended optimal age of vaccination however, retained a relatively narrow interval of 4.7–7.1 months with different cutoffs, results in (S2 Table in
Vaccine intervention is likely to be key in controlling severe disease associated with RSV infection. Prior to vaccine introduction, epidemiological parameters such as the force of infection and average age at primary infection would be important baseline information. Analysis of age specific seroprevalence data can inform on the force of infection in a given population in the absence of vaccination. In this study we present the maternal antibody prevalence decay profile and subsequent age specific acquisition of antibodies to RSV among children in Kilifi and estimate the force of infection, average age at primary infection and the optimal age for RSV vaccine delivery.
The use of randomly selected inpatient plasma samples of children from 0–12 years, did not introduce bias to the estimation of seroprevalence in this study as might be speculated. Exclusion of all participants with LRTI or RSV positive antigen confirmed by IFAT/molecular method did not have a major change on the seroprevalence profiles. Interestingly, analysis of data on diagnosis at discharge showed that 31% of the admissions selected for this study were due to LRTI, with 16% of the admissions with LRTI caused by RSV. This clearly shows RSV as a major cause of lower respiratory tract disease in children in this setting, consistent with previous findings [
The serological prevalence for RSV varied with age and showed 100% seropositivity in samples from children in the first month of life, i.e. <1 month old. This percentage decreased to 25% in the 6–7 month age group before rising again to 100% in 3 year olds and remained so upwards to 12 years. Though the data does not extend to include adults, this profile is qualitatively similar to what would be observed for immunizing infections such as measles[
Interpretation of ageprevalence data for RSV can be complicated by several factors. Waning of antibodies post primary infection could mean a reversion of serostatus. There could also be age dependence in the rate of infection. We explored a variety of catalytic models based on various assumptions on the properties of RSV specific antibodies. Including agedependent force of infection provided a better fit to the data with or without assuming serostatus reversion following primary infection. An interpretation of this result is that while waning antibodies following primary infection have been observed, the reinfection rate is so rapid that the data are unable to distinguish a model with or without the process. If, however, vaccination were to reduce the rate of transmission of the virus, then the effect on the ageprevalence profile might be more pronounced and influence interpretation of such data.
The simplest model (MSF) gave estimates of the average duration of maternally acquired antibodies (D_{M}) of 4.7 months and the average age at primary infection (A) as 15.1 months. These were not very different from D_{M} = 4.42 months and A = 14.2 months obtained from the model that allows for waning of antibodies post primary infection (model MSFSF_{1}). In comparison, a seroepidemiological study by Cox et al [
Amaku et al [
An estimate of 0.0/person/year for the force of infection acting on children who still have maternally acquired antibodies is in line with studies that show that the infant IgG response following primary infection is very low [
The optimal time to vaccinate children to prevent infection would be at an age after they have lost maternally acquired antibodies (which might interfere with the vaccine) and before their first infection. Proportions positive for RSV antibodies begin to rise soon after 6 months of age due to infection, as such; delaying vaccination could result in missing out on a significant amount of preventable infection/disease. Looking at the ageseroprevalence profile suggests vaccination should be carried out at around 6 months when seropositivity is at its lowest. However using a more formal approach, catalytic models results suggest a period between 5 and 15 months. Given that the force of infection estimated for ages 1–12 years is double that for ages 0–1 year, implying greater infection risk after 12 months, it would thus suggest vaccination should occur between 5 and 12 months. The vaccination window established by our analysis overlaps with the vaccination window of between 5 and 10 months obtained by Kinyanjui et al using an agestructured deterministic compartmental model of RSV and data from the same location as our study[
The catalytic model used in the current analysis is relatively simple. Variations could be made to the structure to account for varied antibody dynamics. However, we found that there was no added benefit of including antibody loss post primary infection, or allowing for infection while seropositive for maternally acquired antibodies. Antibody dynamics, such as waning, that have been observed to occur at an individual level, were not supported by the model and data used in this analysis. For a better understanding of this, an analysis similar to one done by Kucharski et al [
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This paper is published with the permission of the Director of KEMRI. We thank all the study volunteers for the contribution of study samples. We acknowledge Field and Laboratory staff of the KEMRI Wellcome Trust Research Programme for collection and storage of the samples. We are also grateful to G.F. Medley for his comments on earlier versions of the manuscript.