Standard antisera

Nasopharyngeal samples were collected, processed and analysed per WHO recommendations [22] as described previously [5] (link). A calcium alginate swab (Medical Wire & Equipment, Corsham, UK) was inserted into the posterior nasopharynx. The swab was transported in skim milk-tryptone-glucose-glycerol medium. Inoculated vials were stored at −20 °C within 6 h of collection, and were frozen at −80 °C within days until tested. Samples (30 µL) were cultured on gentamicin (5 µg/mL) sheep blood agar plates and incubated overnight at 37 °C with 5% carbon dioxide. Pneumococci were identified by morphology and sensitivity for optochin. A single colony was selected from the primary plate and a secondary plate was grown to obtain pure growth. Pneumococci from 2009 to 2011 were serogrouped using latex agglutination and serotyped by Quellung reaction. In 2014, latex agglutination was used for both steps. Both assays used standard antisera (Statens Serum Institute Denmark). The 13-valent latex serotyping kit was used to identify individual VT (1, 3, 4, 5, 6A, 6B, 7F, 9 V, 14, 18C, 19A, 19F, 23F) and provide some detail on NVT serogroup (e.g. 7A/7B/7C). Serotyping for NVT was not done due to budget constraints.

All surveillance and referred isolates were confirmed as forming alpha-haemolytic colonies on horse blood agar and being inhibited by a 5 µg optochin (ethylhydrocupreine hydrochloride) disc (Oxoid-Thermofisher, Basingstoke, UK). Isolates with atypical colonial morphology, or which could not be serotyped (below), were confirmed as being lysed within 30 min by 2% sodium deoxycholate, and being catalase-negative when tested with 3% hydrogen peroxide. For serotyping, isolates were grown overnight in Todd Hewitt broth at 35 °C with 5% CO₂, harvested by centrifugation at 453 g for 30 min, then re-suspended in a small residual volume of broth and subjected to slide agglutination tests with standard antisera (Statens Serum Institut, Copenhagen, Denmark) [26 ]. Agar dilution susceptibility tests were performed in accordance with BSAC guidelines [27 ], using IsoSensitest agar (Oxoid-Thermofisher) supplemented with 5% defibrinated horse blood and incubated at 35–37 °C in a 5% CO₂ atmosphere. ‘Triple resistance’ was defined as resistant to erythromycin (minimum inhibitory concentration (MIC) > 0.5 mg/L) and tetracycline (MIC > 2 mg/L), and non-susceptible to penicillin (MIC > 0.06 mg/L), based on EUCAST breakpoints [28 ])

Nasopharyngeal samples were collected and analyzed according to standard procedures (12 (link)). A calcium alginate swab (Medical Wire & Equipment, Corsham, United Kingdom) was inserted into the posterior nasopharynx. The swab was transported in a medium containing skim milk, tryptone, glucose, and glycerol. Inoculated vials were stored at −20°C within 6 hours of collection and were frozen at −80°C until tested. Samples were cultured on gentamicin (5 µg/mL) sheep blood agar plates and incubated overnight at 37°C with 5% carbon dioxide. Pneumococci were identified by morphology and sensitivity to optochin. One colony was isolated and cultured in Todd-Hewitt broth. Pneumococci were serogrouped using the latex agglutination method and serotyped using the Quellung method with standard antisera (Statens Serum Institut, Copenhagen, Denmark). Reagents were available to type 48 of the 92 possible serotypes, including the serotypes covered by the 13-valent pneumococcal conjugate vaccine (PCV13): 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.