We did a cross-sectional survey of nasopharyngeal carriage prevalence among healthy subjects of all ages during the dry season and repeated it three months later in the rainy season in the same individuals. The sampling frame for the study was the population register of the Kilifi Demographic Surveillance System (DSS). The DSS began in 2000 with a population census of all households within a predefined area of 891 km2 surrounding Kilifi District Hospital. Vital status was subsequently updated in the population register by questionnaires administered at household visits conducted approximately twice a year. In 2005 Kilifi DSS was admitted to the INDEPTH network (www.indepth-network.net ) a network of DSS sites with common data reporting standards. In January 2003 the population register numbered 220 000 individuals and we used this register to generate lists of randomly selected individuals within 64 age-sex-location strata aiming to recruit 480 individuals, 7-8 from each stratum. The strata were composites of two sexes, 4 locations and 8 age groups (<1, 1-2, 3-4, 5-9, 10-19, 20-39, 40-49, ≥50 years.) For logistic reasons we restricted selection to two semi-urban locations from township settings (Kilifi, Roka) and two rural locations (Marere, Pinglikani). In Kilifi District Hib vaccine was introduced into the routine childhood immunization program in 2001. At 12 months of age 87% of children have received three doses of the vaccine [14 (link)]. Pneumococcal carriage prevalence is higher among HIV-infected than HIV-uninfected individuals [15 (link)], however, we chose not to test study subjects for HIV status because the prevalence of HIV in Coastal Kenya is low at 4.8% in men and 6.6% in women [16 ].
In Kilifi there is a long rainy season in May-July and a short rainy season in October-November. The climate between January and March is very dry. To select dates for our rainy and dry season surveys we aggregated daily rainfall data from a meteorologic recording station at Kilifi Agricultural Research Institute in Kilifi town into weekly rainfall means for the preceding 10 years, 1994-2003. The dates of sampling selected were 2-24 March 2004 and 2 June-3 July 2004. The survey in the rainy season took longer to conduct because the rains impeded transport throughout the study area.
A nasopharyngeal specimen was collected from each consenting individual by trained field workers according to the WHO guidelines [17 (link)]. A rayon-tipped flexible aluminium-shaft (Medical Wire and Equipment Company, Town, UK) was passed via the anterior nares to the posterior nasopharynx to a depth predefined by the external distance from the tip of the nose to the external auditory canal. It was left in place for approximately 2 seconds and rotated through 180 degrees before removal. Swabs were immediately placed in STGG transport medium and transported to the laboratory within 8 hours. Putative risk factors for nasopharyngeal colonization, including cigarette smoking, coryza in the last two weeks, use of antibiotics or folate synthesis inhibitors (sulfadoxine/pyrimethamine or sulfamethoxazole/trimethoprim) in the preceding two weeks and number of children aged <5 years resident in the house, were ascertained by questionnaire at the same time. Those who had taken medications were asked to remember the name, describe the package and, if possible, produce the package for verification.
STGG swab specimens were processed at the Wellcome Trust/Kenya Medical Research Institute microbiology laboratories according to the WHO guidelines [17 (link)] either immediately (fresh) or after a period of freezing at -80°C for up to two months. Fresh or thoroughly thawed specimens were vortexed for 10 seconds and 10μl was inoculated directly onto 7% horse blood agar with gentamicin 2.5 μg/ml and 7% chocolate agar. Media were incubated overnight at 37°C in 5% CO2. S. pneumoniae was identified by colony morphology, α-haemolysis, optochin susceptibility, bile solubility and serotyping. Four morphologically distinct colonies were sub-cultured for typing from each primary plate. Pneumococci were serogrouped by latex agglutination and serotyped by the Quellung reaction using polyclonal rabbit antisera (Statens Serum Institute, Copenhagen, Denmark). Haemophilus species were identified by growth on chocolate agar alone, colony morphology, X and V factor dependency and serotype. Swabs from children aged <5 years were also cultured on media containing Hib antiserum to increase the sensitivity of detection of Hib carriage by identification of precipitation haloes [18 (link)]. Serotype results for H. influenzae isolates were confirmed in England by polymerase chain reaction-based capsular genotyping using primers designed to amplify the type-specific regions of the cap loci in each of the 6 (a-f) capsular types [Falla 1994]. [19 ]
STATA (version 8.2) was used for statistical analyses. The prevalence of nasopharyngeal carriage of S. pneumoniae and H. influenzae was presented as proportions of individuals in different age, sex and location strata. The effect of season on carriage was calculated as a matched odds ratio and tested with McNemar’s χ2 tests. Logistic regression was used to analyze risk factors for carriage entering subject identity as a random effect to take account of the correlation of response variables from the same individual in the two surveys. The contribution of each variable to the model was determined by Likelihood Ratio Tests and only those with a p value <0.05 were retained in the final model except for age, in 8 strata, which was retained as an a priori confounder.
The study was approved by the Kenya Medical Research Institute/National Ethical Review Committee and The Oxford Tropical Research Ethics Committee and written informed consent was obtained for all participants.
In Kilifi there is a long rainy season in May-July and a short rainy season in October-November. The climate between January and March is very dry. To select dates for our rainy and dry season surveys we aggregated daily rainfall data from a meteorologic recording station at Kilifi Agricultural Research Institute in Kilifi town into weekly rainfall means for the preceding 10 years, 1994-2003. The dates of sampling selected were 2-24 March 2004 and 2 June-3 July 2004. The survey in the rainy season took longer to conduct because the rains impeded transport throughout the study area.
A nasopharyngeal specimen was collected from each consenting individual by trained field workers according to the WHO guidelines [17 (link)]. A rayon-tipped flexible aluminium-shaft (Medical Wire and Equipment Company, Town, UK) was passed via the anterior nares to the posterior nasopharynx to a depth predefined by the external distance from the tip of the nose to the external auditory canal. It was left in place for approximately 2 seconds and rotated through 180 degrees before removal. Swabs were immediately placed in STGG transport medium and transported to the laboratory within 8 hours. Putative risk factors for nasopharyngeal colonization, including cigarette smoking, coryza in the last two weeks, use of antibiotics or folate synthesis inhibitors (sulfadoxine/pyrimethamine or sulfamethoxazole/trimethoprim) in the preceding two weeks and number of children aged <5 years resident in the house, were ascertained by questionnaire at the same time. Those who had taken medications were asked to remember the name, describe the package and, if possible, produce the package for verification.
STGG swab specimens were processed at the Wellcome Trust/Kenya Medical Research Institute microbiology laboratories according to the WHO guidelines [17 (link)] either immediately (fresh) or after a period of freezing at -80°C for up to two months. Fresh or thoroughly thawed specimens were vortexed for 10 seconds and 10μl was inoculated directly onto 7% horse blood agar with gentamicin 2.5 μg/ml and 7% chocolate agar. Media were incubated overnight at 37°C in 5% CO2. S. pneumoniae was identified by colony morphology, α-haemolysis, optochin susceptibility, bile solubility and serotyping. Four morphologically distinct colonies were sub-cultured for typing from each primary plate. Pneumococci were serogrouped by latex agglutination and serotyped by the Quellung reaction using polyclonal rabbit antisera (Statens Serum Institute, Copenhagen, Denmark). Haemophilus species were identified by growth on chocolate agar alone, colony morphology, X and V factor dependency and serotype. Swabs from children aged <5 years were also cultured on media containing Hib antiserum to increase the sensitivity of detection of Hib carriage by identification of precipitation haloes [18 (link)]. Serotype results for H. influenzae isolates were confirmed in England by polymerase chain reaction-based capsular genotyping using primers designed to amplify the type-specific regions of the cap loci in each of the 6 (a-f) capsular types [Falla 1994]. [19 ]
STATA (version 8.2) was used for statistical analyses. The prevalence of nasopharyngeal carriage of S. pneumoniae and H. influenzae was presented as proportions of individuals in different age, sex and location strata. The effect of season on carriage was calculated as a matched odds ratio and tested with McNemar’s χ2 tests. Logistic regression was used to analyze risk factors for carriage entering subject identity as a random effect to take account of the correlation of response variables from the same individual in the two surveys. The contribution of each variable to the model was determined by Likelihood Ratio Tests and only those with a p value <0.05 were retained in the final model except for age, in 8 strata, which was retained as an a priori confounder.
The study was approved by the Kenya Medical Research Institute/National Ethical Review Committee and The Oxford Tropical Research Ethics Committee and written informed consent was obtained for all participants.
