Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage

Glennie, S; Gritzfeld, J F; Pennington, S H; Garner-Jones, M; Coombes, N; Hopkins, M J; Vadesilho, C F; Miyaji, E N; Wang, D; Wright, A D; Collins, A M; Gordon, S B; Ferreira, D M

Nasal wash samples were obtained 5 days before intranasal inoculation with S. pneumoniae serotype 6B and then on days 2, 7, and 14 after inoculation. Inoculation doses were between 50 Â 10 3 and 320 Â 10 3 colony-forming units (CFUs) per 100 ml. Bacterial preparation, inoculation of subjects with 6B pneumococcus, nasal wash sampling, carriage detection, and quantification of carriage density were done as described previously. 17 Detection and identification of URT virus. Oropharyngeal swabs were collected 5 days before intranasal inoculation and immediately put in viral transport medium (MWE, Corsham, UK). Viral multiplex PCR was performed as described elsewhere. 46, 47 Briefly, viral RNA was extracted from 200 ml of viral transport medium and eluted into 85 ml buffer using the standard Pathogen Complex 200 protocol on the QIAsymphony (QIAGEN, Crawley, UK) . The eluates were then analyzed using four multiplex PCR assays on the LC480 real-time PCR machine (Roche Diagnostics, Burgess Hill, UK). The assay panel covered the qualitative detection of influenza A and B, respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza viruses 1-4, and coronaviruses OC43, NL63, 229E, and HKU1.