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nCounter is robust and sensitive for the simultaneous detection of viral (both RNA and DNA) and bacterial transcripts in NPA with low RNA input (<10 ng). This medium-throughput technique will increase our understanding of ARI pathogenesis and may provide an evidence-based approach for the targeted and rational use of antibiotics in pediatric ARI.

Acute respiratory infection (ARI) is the most frequent cause for hospitalization in infants and young children. Using multiplexed nCounter technology to digitally quantify 600 human mRNAs in parallel with 14 virus- and 5 bacterium-specific RNAs, we characterized viral and bacterial presence in nasopharyngeal aspirates (NPA) of 58 children with ARI and determined the corresponding in situ immune profiles. NPA contained different groups of organisms and these were classified into bacterial (n = 27), viral (n = 5), codetection [containing both viral and bacterial transcripts (n = 21), or indeterminate intermediate where microbial load is below threshold (n = 5)]. We then identified differentially expressed immune transcripts (DEITs) comparing NPAs from symptomatic children vs. healthy controls, and comparing children presenting NPAs with detectable microbial load vs. indeterminate. We observed a strong innate immune response in NPAs, due to the presence of evolutionarily conserved type I Interferon (IFN)-stimulated genes (ISG), which was correlated with total bacterial and/or viral load. In comparison with indeterminate NPAs, adaptive immunity transcripts discriminated among viral, bacterial, and codetected microbial profiles. In viral NPAs, B cell transcripts were significantly enriched among DEITs, while only type III IFN was correlated with viral load. In bacterial NPAs, myeloid cells and coinhibitory transcripts were enriched and significantly correlated with bacterial load. In conclusion, digital nCounter transcriptomics provide a microbial and immunological in situ “snapshot” of the nasopharyngeal interface in children with ARI. This enabled discrimination among viral, bacterial, codetection, and indeterminate transcripts in the samples using non-invasive sampling.

Respiratory syncytial virus (RSV) is one of the most common etiological agents of childhood respiratory infections globally. Information on seasonality of different antigenic groups is scarce. We aimed to describe the frequency, seasonality, and age of children infected by RSV antigenic groups A (RSVA) and B (RSVB) among children with ARI in a 4-year period.Children (6–23 months old) with respiratory infection for ≤7 days were enrolled in a prospective cross-sectional study, from September, 2009 to October, 2013, in Salvador, in a tropical region of Brazil. Upon recruitment, demographic, clinical data, and nasopharyngeal aspirates (NPA) were collected. A multiplex quantitative real-time polymerase chain reaction (RT-PCR) with a group-specific primer and probeset for RSVA and RSVB was used. Seasonal distribution of infection by RSV different antigenic groups was evaluated by Prais-Wisten regression.Of 560 cases, the mean age was 11.4 ± 4.5 months and there were 287 (51.3%) girls. Overall, RSV was detected in 139 (24.8%; 95% CI: 21.4%–28.5%) cases, RSVA in 74 (13.2%; 95% CI: 10.6%–16.2%) cases, and RSVB in 67 (12.0%; 95% CI: 9.5%–14.9%) cases. Two (0.4%; 95% CI: 0.06%–1.2%) cases had coinfection. RSVA frequency was 9.6%, 18.4%, 21.6%, and 3.1% in 2010, 2011, 2012, and 2013, respectively. RSVB frequency was 19.2%, 0.7%, 1.4%, and 35.4% in the same years. RSVA was more frequently found from August to January than February to July (18.2% vs. 6.4%, P < 0.001). RSVB was more frequently found (P < 0.001) between March and June (36.0%) than July to October (1.0%) or November to February (1.6%). RSVB infection showed seasonal distribution and positive association with humidity (P = 0.02) whereas RSVA did not. RSVA was more common among children ≥1-year-old (17.8% vs. 1.8%; P = 0.02), as opposed to RSVB (11.5% vs. 12.2%; P = 0.8).One quarter of patients had RSV infection. RSVA compromised more frequently children aged ≥1 year. RSVA predominated in 2011 and 2012 whereas RSVB predominated in 2010 and 2013. In regard to months, RSVA was more frequent from August to January whereas RSVB was more often detected between March and June. Markedly different monthly as well as yearly patterns for RSVA and RSVB reveal independent RSV antigenic groups’ epidemics.

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