Latex agglutination

Cultured-enriched saliva samples in which a higher concentration of pneumococcus was detected (<30 Ct by qPCR) were re-visited in an attempt to isolate pure pneumococcus. Samples were serially diluted 10-fold over a range of 10⁻¹ to 10⁻⁵ in 1X PBS. The 10⁻⁵ and 10⁻⁶ serially diluted samples were plated (100 μL) onto plain blood agar plates. After overnight incubation, culture plates were visually screened for pneumococcal-like colonies. Each colony of pneumococcus-like morphology was streaked onto a plain blood agar plate and then inoculated into 50 μL of elution buffer (ThermoFisher Scientific) in a microcentrifuge tube. From each saliva sample, a total of 20 colonies were streaked onto one plain blood agar plate, with colonies pooled by 5 in each microcentrifuge tube. Pooled bacterial colonies were incubated for 10 min at 95°C on a heating block, then tested in qPCR for piaB and lytA to confirm the identity of the isolates. Streaked isolates from the pooled boilate samples that generated any signal <40 Ct for either piaB or lytA were individually tested for optochin susceptibility. Optochin susceptible colonies which tested positive for both piaB and lytA were then serotyped by latex agglutination (Statens Serum Institut) (39 (link)).

Isolates were cultured onto Columbia agar plates supplemented with horse blood (Oxoid Ltd., Hampshire, UK) and incubated aerobically at 37 °C for 24 h. Serological classification based on capsular polysaccharide types Ia, Ib and II to IX was performed using latex agglutination according to the manufacturer’s recommendations (Statens Serum Institute, Copenhagen, Denmark).

Standard laboratory methods for the identification of GBS on culture have been described [12 (link)]. Briefly, GBS from blood was isolated using the Bact/Alert microbial system (Organon Teknika, Durham, NC), plated on blood or chocolate agar. On CSF samples, gram-staining was done and samples plated onto blood or chocolate agar plates were inoculated into an enrichment broth (Brain Heart Infusion, Diagnostics Media Production; South Africa). A GBS antigen agglutination test (Wellcogen Bacterial Antigen Kit, Remel, UK) was also undertaken if the CSF culture was negative, but the CSF cyto-chemistry was suggestive of bacterial meningitis. Positive GBS isolates were archived at -70 degrees celsius at the Respiratory and Meningeal Pathogens Research Unit. Serotyping (Ia, Ib, II–IX) methods were consistently performed over the study period using latex agglutination (Statens Serum Institute, SSI, Sweden) [20 (link), 21 (link)]. Twelve (1.9%) non-typeable isolates were further characterized using single-plex PCR and primers as described (S1 Table) [22 (link), 23 (link)].