Methods

The study was undertaken in a rural district of coastal Kenya that experiences a tropical climate with seasonal rains (roughly, during March—July and October—December). The population of predominantly subsistence farmers has a growth rate of 3.1% per annum, with 18% aged <5 years [14]. Malaria is endemic yet highly seasonal [15]. The community is served by a district hospital (Kilifi District Hospital [KDH]) based in the town of Kilifi (2006 population, 41,000). The Collaborative Research Programme between the Kenya Medical Research Institute and the Wellcome Trust, which provided the infrastructure for this study, runs the hospital pediatric wards and operates a Demographic Surveillance System within an area of ∼900 km² immediately surrounding KDH [16]. Mortality among infants and among children aged <5 years (per 1000 per year) in the Demographic Surveillance System is estimated to be 50 and 74, respectively (Kilifi Demographic Surveillance System, unpublished data). Ethical clearance for this study was obtained from the Kenyan National Ethics Review Board and Coventry Research Ethics Committee (Coventry, United Kingdom). The survey methods have been reported elsewhere [13] and are presented here in detail when relevant to the analysis.

Study participants were recruited in the maternity ward and at the maternal and child health clinic at KDH. Children were eligible if their home was located in the Demographic Surveillance System and within easy access to the hospital. Written informed consent was obtained for participation. Recruitment took place over 2 calendar years in 2 phases, each spanning approximately one‐half of a year and separated by 6 months. The birth cohort was monitored over 4 calendar years until each phase of recruits had experienced 3 epidemics of RSV infection. Surveillance was performed through active household visits by field workers that were time‐tabled each week during epidemics of RSV infection and, otherwise, monthly; surveillance was also performed at presentation (either passively or by referral at a home visit) to the research outpatient clinic at KDH or at admission to KDH. Children in their homes who had symptoms or signs predominantly reflecting lower respiratory tract involvement were referred to the clinic. Mothers were encouraged to bring their children to the research clinic if they identified any symptoms of respiratory infection. A blood smear for malaria was performed for children with an axillary temperature ⩾37.5°C or with a history of fever during the previous week and for all persons admitted to the hospital.

Nasal wash specimens [17] were collected at home or at clinic visits if, during the preceding week (inclusive of the day of the visit), the child had a history of or was observed to have a minimum of (1) difficulty in breathing, (2) a runny nose and/or nasal congestion, or (3) an acute cough. Within the KDH pediatric ward, a nasal specimen (nasal wash or nasal pharyngeal aspirate specimen) was collected if the child presented with LRTI or bronchiolitis or with severe or very severe pneumonia or if the child was hypoxic (pO₂, <90%), as determined by Oximeter (Nelcor). Samples were examined for RSV antigen using a direct immunofluorescence test (DFA; Chemicon). Details of collection, cold chain to the laboratory, and processing are reported elsewhere [13]. The severity of respiratory disease was ascribed following a clinical review that was uniform to the research clinic and the pediatric ward. “LRTI” was assigned to children with acute cough or difficulty in breathing in association with ⩾1 of the following: (1) increased respiratory rate for age, (2) intercostal indrawing, or (3) inability to feed, reduced conscious level, or hypoxia (O₂ saturation, <90%, by Oximetry); the latter group was required to have confirmation of LRTI by the clinician's own diagnosis of LRTI or bronchiolitis. “Severe LRTI” was assigned to a child with the second and/or third criteria. A “hospital admission” was assigned only if linked to a KDH inpatient record. For children not admitted to the hospital, “malaria” was ascribed using the criteria defined by Mwangi et al. [15] and, for admitted children, was assigned to those discharged from the hospital with a diagnosis that included malaria.

Data analysis was performed using Stata, version 8.2 (Stata). Observation time for each child included all days from date of recruitment to the last visit of the study or loss to follow‐up (table 1), excluding days absent from the district. A discrete episode of RSV infection was defined as a visit resulting in a specimen positive for RSV that occurred no less than 14 days after a previous episode of RSV infection. The severity of disease associated with an episode of RSV infection was the maximum identified over the 7‐day period beginning the day of nasal sampling. A concurrent diagnosis of malaria and RSV infection was exclusively assigned to RSV infection, whereas concurrent malaria and non—RSV‐associated LRTI was ascribed to malaria. Incidence of infection, defined as number of infections per 1000 child‐years of observation (CYO), was estimated by Poisson regression. Observation time was divided into epidemic and nonepidemic periods of RSV infection [13]. Incidence estimates are presented in crude form (i.e., number of cases per 1000 CYO) and adjusted to take into account bias in the ratio of observation time within and between epidemics arising from the discontinuous cohort recruitment and the censoring at the end of the study. For adjusted estimates, probability weights (W_ki; “pweights” in Stata) were applied to each observation in time period k (k=n,e; n=nonepidemic; e=epidemic) and in specified (6‐month) age class, i (1=1,m), as defined by which is the ratio of the expected total days at risk for age class i in time period k to observed total days at risk in age class i, time period k. Here, P_k is the average proportion of the year that is epidemic (k=e) or nonepidemic (k=n), and Y_ki is the number of person‐days at risk for age class i in time period k. Separate weights were calculated for observations used to estimate total, primary, and repeat RSV infection incidences. Weighted Poisson regression was undertaken with account taken of clustering of RSV infections within the individual.

Seasonality in total LRTIs is markedly less than that in RSV infections and is consequently ignored. Proportions were compared using Fisher's exact test (2‐tailed), with binomial exact confidence intervals, and assessment of trend in proportions across ordered groups was made using an extension to the Wilcoxon rank‐sum test (“nptrend” Stata command).

Results

Characteristics of recruitment, surveillance, and loss to follow‐up. A total of 635 children (51% male) were enrolled in the study at the KDH at or shortly after birth in 2 phases (January—May 2002 and December 2002—July 2003) (table 1). Five hundred thirty‐five (84%) of the children were recruited in the maternity ward at KDH. Forty‐seven percent of the cohort resided in Kilifi Township. Follow‐up over 3 epidemics for each phase realized a total of 1187 CYO (figure 1A and 1B). The mean age of a child at exit from the study was 22 months (median age, 25 months) (table 1). Of the recruits, 199 (31%) exited prior to the end of the study (a rate of 17% of the cohort per annum). For 16 (8%) of these children, the reason of study exit was death; 11 deaths occurred during the first year of life (562 CYO; mortality rate, 20 per 1000 infants).

The frequency of surveillance was 29 visits per CYO (1 visit every 13 days), with a mean duration between visits of 9 days within epidemic periods of RSV infection and 18 days between these periods. For each child there was a mean of 22 home visits and 6 clinic visits per year (these rates were highly homogeneous). The rate of clinical presentations within and between epidemics did not change (rate ratio, 1.02; 95% CI, 0.98–1.07; P=.324). There were 8716 visits fulfilling the criteria for nasal sampling, and nasal specimens were collected during 8492 (97%) of these visits (7 visits with a specimen collected per CYO). Blood smears were indicated at 5113 visits; during 570 (11.1%) of these visits, the blood smears tested parasite positive, and 490 (9.6%) blood smears accompanied a diagnosis of malaria, representing 1.4% of all visits. Laboratory immunofluorescent antibody test results were obtained for 8471 of the nasal samples, yielding 409 (4.8%) RSV antigen—positive samples constituting separate RSV episodes; 130 of these samples were obtained during active visits, 268 were obtained during clinic visits, and 11 were obtained at admission to KDH. Malaria was diagnosed concurrently in 86 (8.5%) of 1008 cases of LRTI that were negative for RSV, including 12 cases of severe LRTI and 10 cases for which the patient was hospitalized.

RSV infections in the birth cohort. Of the 409 separate episodes of RSV infection, 326 were the first observed in a particular child (hereafter, denoted as primary cases), and 83 were repeat infections. There were 4 distinct epidemics of RSV infection (figure 1A and 1B) occurring with approximate annual periodicity (intervals between the epidemics were 9, 14, and 10 months), with the majority of cases occurring during the first quarter of the year; these epidemic periods were not associated with any meteorological measures. Over the 4 calendar years of observation (2002–2005), 70 of 208 weeks (mean proportion of the year, 0.34) were defined as epidemic periods (figure 1). The proportion of the cohort observation period that was within defined epidemic periods was 0.44, indicating an oversampling from within epidemics.

Crude incidences for total infections, primary infections, and repeat infections were 345 cases per 1000 CYO, 394 cases per 1000 CYO, and 230 cases per 1000 CYO, respectively. The relevant observation periods for primary and repeat infections were 827 child years and 361 child years, respectively. Adjusted incidence estimates (weighted for epidemic oversampling) were 261 cases per 1000 CYO (95% CI, 236–287 cases per 1000 CYO), 298 cases per 1000 CYO (95% CI, 264–337 cases per 1000 CYO) and 169 cases per 1000 CYO (95% CI, 134–214 cases per 1000 CYO), respectively. There was no statistically significant difference in the incidence of RSV infections by age group (infants aged 0–11 months versus children aged 12–30 months) or by sex. The incidence of reinfection was approximately one‐half that for primary infections, regardless of age.

RSV‐associated disease in the cohort. Of the 409 episodes of RSV infection, 275 (67%) appeared to be confined to the upper respiratory tract, and 134 (33%) involved the lower respiratory tract; 66 (49%) of the RSV‐associated LRTIs were considered to be severe. Among all children with cases of RSV infection, 11 (3%) with severe LRTIs were admitted to KDH, none of whom had a cobacterial infection. No RSV infection coincided with the death of the child. A concurrent malaria diagnosis was received by 6 (1.5%) of 409 children with RSV infections; 2 of these patients had LRTIs, 1 of which was severe, and none were hospitalized.

The risks of LRTI, severe LRTI, and hospital admission were 35%, 18%, and 3%, respectively, following primary RSV infection and 24%, 8%, and 2%, respectively, following repeat RSV infection. Following primary infection, the peak disease risk tended to be in children aged <6 months, followed by a trend for decrease in risk with increasing age (table 2). For LRTI, this trend was gradual, with the risk never decreasing to <20% (i.e., ∼40% of the peak risk), even in children aged ⩾24 months. The risk of severe LRTI following RSV infection appears to decrease only for children 9–11 months of age onwards and most significantly for children 18–30 months of age. In relation to repeat infection, in children aged ⩾18 months, the data indicate a substantial risk of LRTI (risk, 25.4%; 95% CI, 15.3–37.9) and of severe LRTI (risk, 9.5%; 95% CI, 3.6–19.6), although the number of cases in the younger age groups was too low to identify any trend with age. Two of the children with RSV infection who were aged 18–30 months were admitted to the hospital.

Adjusted incidence estimates for RSV‐associated LRTI, severe LRTI, and hospitalization, by age group, are presented in table 3. For all disease categories, peak incidence occurred in infants aged <6 months and was significantly lower in the older age group (age, 6–30 months) for LRTI (incidence rate ratio, 0.494; 95% CI, 0.318–0.768; P=.002) and severe LRTI (incidence rate ratio, 0.347; 95% CI, 0.198–0.610; P<.001). For infants (age, 0–11 months), adjusted incidences of LRTI, severe LRTI, and hospital admission were 104 cases per 1000 CYO (95% CI, 79–137), 66 cases per 1000 CYO (95% CI, 47–91), and 13 cases per 1000 CYO (95% CI, 5–34), respectively. Corresponding estimates for children aged 12–30 months were 77 cases per 1000 CYO (95% CI, 39–151), 22 cases per 1000 CYO (95% CI, 9–56), and 7 cases per 1000 CYO (95% CI, 1–61), respectively. There was no relationship between incidence and sex of the child or low birth weight (<2.5kg).

RSV disease as a proportion of all‐cause LRTI and hospital admissions. The temporal occurrence of RSV‐associated LRTI and severe LRTI relative to all‐cause LRTI and severe LRTI is recorded in figure 1C, 1D, 1E, and 1F, respectively. RSV was identified in 13% of children with LRTI, 19% of children with severe LRTI, and 5% of children admitted to the hospital. Stratified by age (table 4), the data show a higher risk of RSV in cases of LRTI, cases of severe LRTI, and cases requiring hospitalization in infants, compared with older children, which is statistically significant only for LRTI.

The characteristics of repeat RSV infections. The distribution of the total number of clinical RSV infections per individual was 254 individuals with 1 infection, 64 with 2 infections, 6 with 3 infections, 1 with 4 infections, and 1 with 5 infections. Of the 72 children who experienced >1 episode of RSV infection, 19 (26%) had a repeat infection that involved the lower respiratory tract and 6 (8%) had severe LRTI. In 10 (14%) of the children, a repeat infection was more severe than the primary episode. Of the 83 repeat infections, 19 (23%) occurred during the same epidemic, with a median duration between reinfection of 24 days (range, 18–66 days). There were 5 children reinfected within the first 6 months of life (1 was reinfected twice). Of these 6 repeat infections, 4 resulted in LRTI (including 1 case of severe LRTI); 5 occurred during a single epidemic, with a duration between episodes of 19–63 days; and 1 occurred during the succeeding epidemic (113 days after the first infection). Of the 8 children with ⩾3 RSV infections, 4 were admitted to the hospital at least once; however, none had an RSV‐associated hospital admission, and none had inpatient reports that would indicate an immunocompromised condition.

Discussion

A birth cohort of 635 children from a rural Kenyan location was intensively monitored for a median of 25 months spanning 4 calendar years to identify RSV infections and disease outcomes. The estimated incidence of symptomatic episodes of RSV infection was 261 episodes per 1000 CYO; the estimated incidence of RSV‐associated LRTI, severe LRTI, and hospitalization at KDH was 90 cases per 1000 CYO, 43 cases per 1000 CYO, and 10 cases per 1000 CYO, respectively. RSV infection was identified in ∼13% of all LRTIs, ∼19% of all severe LRTIs, and ∼5% of all hospitalizations experienced by the cohort. Infants had the highest rates of RSV‐associated disease, with estimates of 104 cases per 1000 CYO, 66 cases per 1000 CYO, and 13 cases per 1000 CYO for LRTI, severe LRTI, and hospital admission, respectively, representing 16% of total LRTIs, 21% of severe LRTIs, and 6% of hospital admissions in this age group. If we extrapolate this disease burden to the total annual birth cohort in Kenya of 1.3 million [14], then RSV is expected to result annually in ∼135,000 cases of LRTI, ∼85,000 cases of severe LRTI, and >17,000 hospitalizations of infants.

The data provided by this study are unusual; a literature review reveals few community‐based studies of RSV infection incidence in resource‐poor settings [8, 9, 12], with estimates of RSV‐associated LRTI incidence in infants ranging widely, from ∼40 cases per 1000 CYO to >200 cases per 1000 CYO. Only 2 studies showed estimates for severe LRTI in infants—each showing ∼15 cases per 1000 CYO [8, 9]. Our study, which involved highly intensive surveillance, provides community incidences at the higher end of or, in the case of severe LRTI, well above those reported elsewhere. Furthermore, we can assume that these are underestimates, largely because case surveillance would have greater sensitivity using RT‐PCR methods and serologic examination [4, 13, 18, 19]. Not surprisingly, insensitivity decreases with increasing case severity, and our estimates of RSV‐associated hospital admissions are in line with those from the United States [4, 20] and other developing countries [12].

In our study, the rate of RSV‐associated severe LRTI was 4–5 times higher than the rate of RSV‐associated hospitalization. World Health Organization guidelines recommend all such cases for hospital admission [21]. A proportion (26%) of these children with RSV‐associated severe LRTI but with no record of admission to KDH had been referred for admission to KDH from the research clinic. The remaining three‐quarters were not deemed admissible by experienced clinical officers. The situation was the same for all‐cause severe LRTI. The study may have influenced the decision to refer, because the patients had relatively good hospital access and were able to return free of charge if the child's condition deteriorated. Nevertheless, only one‐half of the patients admitted to KDH were referred from the research clinic. It is clear from these data and data from the United States [22] that studies based on passive hospital surveillance overlook a significant community burden of severe RSV disease.

Although the estimated incidence of RSV‐associated LRTI, severe LRTI, and hospitalization was highest in the group aged <6 months, significant disease incidence was observed in older groups continuing into the third year of life. If we assume that children aged 6–30 months, relative to children aged <6 months, are ∼4 times greater in number and have an incidence that is one‐third to one‐half (table 3), then 55%–65% of significant disease due to RSV in the wider community occurs in ages outside the age range typically designated as the vaccine target. A delay in vaccination to an age when the child is more immunologically mature and interference from maternal antibody is less likely may have potential to prevent a majority of the community burden of RSV‐associated disease.

Although the highest risk of lower respiratory tract disease following primary symptomatic RSV infection was seen in children aged <6 months, a substantial risk remained up to 18 months of age. Even if scaled for underestimation of the true denominator of infection, these risks would remain substantial. We identify a significant risk of disease following reinfection in children during their second and third years of life, which suggests that potential benefits of delay in first exposure by use of maternal immunization [23] may be less than hoped for and that severe disease from reinfection should not be ignored when considering the merits of RSV vaccination. Furthermore, our data suggest that immunity to symptomatic reinfection (frequently involving the lower respiratory tract) is short‐lived in a substantial minority of children, including infected children aged <6 months. It follows that a candidate vaccine delivered in the first 3 months of life that provides no better protection than does natural infection might not prevent considerable early reinfection, with associated high risk of disease.

In summary, an intensively monitored birth cohort of >600 children in a rural location in a developing country has provided evidence of a substantial burden of RSV‐associated severe disease in the wider community that would not be identified through hospital‐based surveillance. The majority of such disease occurs in older infants and young children. We report a substantial risk of disease following primary infection beyond early infancy and on reinfection, as well as rapid loss of immunity to symptomatic reinfection. These findings have a bearing on the potential effectiveness of RSV vaccines. Consideration is required of the merits of broadening the target age range for a live attenuated vaccine in preventing a large community burden of severe RSV infection.