Procedures
INFECTIOUS DISEASES
Nasopharyngeal swab samples were collected at 9 months of age (on the same day as, but before, the booster dose was administered), 15 months of age (6 months after the booster), and 18 months of age (9 months after the booster), and were immediately placed in 1 mL skim milk-tryptone-glucose-glycerol (STGG) and transported at 4°C to the Vaccine and Infectious Diseases Analytics Research Unit at the University of the Witwatersrand (Wits VIDA) within 2 h of collection to be stored at −80°C (Total nucleic acid was automatically extracted from the transport media of the vortexed nasopharyngeal swab samples using the NucliSens easyMAG extraction system (BioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. As an initial step (pre-amplification of DNA) for the Biomark HD system (Standard BioTools [previously known as Fluidigm], San Francisco, CA, USA), specific target amplification (STA) was carried out on extracted DNA, as described elsewhere.All reactions included in the Standard BioTools qPCR were effective in amplifying their respective targets with the efficiency of the reactions ranging from 90% to 110%. Within the linear dynamic range, the correlation coefficients of the reactions were higher than 0·98, and all reactions showed high analytical sensitivities (lower limit of detection equivalent <10–100 copies per reaction). Both the inter-assay and intra-assay variations for all respective reactions were less than 0·1 SD, and the accuracy ratio was within 0·1. Standard curves were set up before sample screening and template controls for each reaction were included within each reaction plate. Reaction plates were grouped in batches of ten, and the Cq of controls within each plate was compared with the average cycle of quantification value within the batch and remained within at least 1 quantification cycle variation between standards of each run.Serotype-specific IgG concentrations were measured for all PCV13 capsular polysaccharides, as detailed previously.