All laboratory methods were standardized across sites [18 ]. A flocked NP swab (flexible minitip; Copan) and a rayon oropharyngeal (OP) swab specimen were collected from each case and control and were placed into the same vial. The NP/OP specimen was tested for pneumococcus (lytA gene target) as part of a multiplex real-time polymerase chain reaction (PCR) assay (FTD Respiratory Pathogens 33; Fast-track Diagnostics) performed using an Applied Biosystems 7500 (ABI-7500) platform. Standard curves for quantification were generated on an approximately 3-monthly basis and were used to calculate pathogen density (in copies per milliliter) from the sample cycle threshold values. Densities <104 or >108 copies/mL were outside the linear range of the PCR assay, limiting precise density estimation.
A second NP specimen for S. pneumoniae culture was collected simultaneously with the first swab specimen; pneumococcal isolates were serotyped using Quellung reaction or latex agglutination, as described elsewhere [18 ]. Testing was performed at each site, and all sites participated in external quality assurance programs for both pneumococcal PCR and serotyping [18 ].
Cases, but not controls, had blood collected for culture. Some sites (Bangladesh, The Gambia, Mali, and South Africa) collected lung aspirates from children with consolidation on chest radiographs (CXRs) who met clinical and radiologic criteria for the procedure [19 (link)]. Pleural fluid was collected from cases when clinically indicated. Lung aspirate and pleural fluid specimens were tested for pneumococcus by means of culture and PCR; pleural fluid was also tested for pneumococcal antigen (Binax NOW; Alere).
DefinitionsAntibiotic pre-exposure was defined as either a positive serum bioassay result (cases and controls) or documentation of antibiotics administered at the referral or study hospital before specimen collection (cases only) [20 ]. Microbiologically confirmed pneumococcal pneumonia (MCPP) was defined, in PERCH cases, as detection of pneumococcus from a culture of blood, lung aspirate, or pleural fluid; by PCR of lung aspirate or pleural fluid; or by detection of pneumococcal antigen in pleural fluid. A control was considered to have a respiratory tract illness (RTI) if cough or runny nose were reported. RTI was also considered present if a child had (1) ear discharge, wheezing, or difficulty breathing and (2) either fever (temperature ≥38.0°C or reported fever in the past 48 hours) or sore throat.
CXRs were obtained at admission for cases, and each digital image was assessed by 2 members of a panel of 14 radiologists and pediatricians trained in the standardized interpretation of pediatric CXRs; films with discordant conclusions were adjudicated [21 (link), 22 ]. Clinical characteristics, including oxygen saturation, were assessed on the day of enrollment. Case mortality was assessed at hospital discharge and by contact 30 days after discharge.
A second NP specimen for S. pneumoniae culture was collected simultaneously with the first swab specimen; pneumococcal isolates were serotyped using Quellung reaction or latex agglutination, as described elsewhere [18 ]. Testing was performed at each site, and all sites participated in external quality assurance programs for both pneumococcal PCR and serotyping [18 ].
Cases, but not controls, had blood collected for culture. Some sites (Bangladesh, The Gambia, Mali, and South Africa) collected lung aspirates from children with consolidation on chest radiographs (CXRs) who met clinical and radiologic criteria for the procedure [19 (link)]. Pleural fluid was collected from cases when clinically indicated. Lung aspirate and pleural fluid specimens were tested for pneumococcus by means of culture and PCR; pleural fluid was also tested for pneumococcal antigen (Binax NOW; Alere).
DefinitionsAntibiotic pre-exposure was defined as either a positive serum bioassay result (cases and controls) or documentation of antibiotics administered at the referral or study hospital before specimen collection (cases only) [20 ]. Microbiologically confirmed pneumococcal pneumonia (MCPP) was defined, in PERCH cases, as detection of pneumococcus from a culture of blood, lung aspirate, or pleural fluid; by PCR of lung aspirate or pleural fluid; or by detection of pneumococcal antigen in pleural fluid. A control was considered to have a respiratory tract illness (RTI) if cough or runny nose were reported. RTI was also considered present if a child had (1) ear discharge, wheezing, or difficulty breathing and (2) either fever (temperature ≥38.0°C or reported fever in the past 48 hours) or sore throat.
CXRs were obtained at admission for cases, and each digital image was assessed by 2 members of a panel of 14 radiologists and pediatricians trained in the standardized interpretation of pediatric CXRs; films with discordant conclusions were adjudicated [21 (link), 22 ]. Clinical characteristics, including oxygen saturation, were assessed on the day of enrollment. Case mortality was assessed at hospital discharge and by contact 30 days after discharge.
