Seasonality of distinct respiratory viruses in a tropical city: implications for prophylaxis.

OBJECTIVE: The frequency and seasonality of viruses in tropical regions are scarcely reported. We estimated the frequency of seven respiratory viruses and assessed seasonality of respiratory syncytial virus (RSV) and influenza viruses in a tropical city. METHODS: Children (age ≤ 18 years) with acute respiratory infection were investigated in Salvador, Brazil, between July 2014 and June 2017. Respiratory viruses were searched by direct immunofluorescence and real-time polymerase chain reaction for detection of RSV, influenza A virus, influenza B virus, adenovirus (ADV) and parainfluenza viruses (PIV) 1, 2 and 3. Seasonal distribution was evaluated by Prais-Winsten regression. Due to similar distribution, influenza A and influenza B viruses were grouped to analyse seasonality. RESULTS: The study group comprised 387 cases whose median (IQR) age was 26.4 (10.5-50.1) months. Respiratory viruses were detected in 106 (27.4%) cases. RSV (n = 76; 19.6%), influenza A virus (n = 11; 2.8%), influenza B virus (n = 7; 1.8%), ADV (n = 5; 1.3%), PIV 1 (n = 5; 1.3%), PIV 3 (n = 3; 0.8%) and PIV 2 (n = 1; 0.3%) were identified. Monthly count of RSV cases demonstrated seasonal distribution (b3 = 0.626; P = 0.003). More than half (42/76 [55.3%]) of all RSV cases were detected from April to June. Monthly count of influenza cases also showed seasonal distribution (b3 = -0.264; P = 0.032). Influenza cases peaked from November to January with 44.4% (8/18) of all influenza cases. CONCLUSIONS: RSV was the most frequently detected virus. RSV and influenza viruses showed seasonal distribution. These data may be useful to plan the best time to carry out prophylaxis and to increase the number of hospital beds.

In situ Immune Signatures and Microbial Load at the Nasopharyngeal Interface in Children With Acute Respiratory Infection.

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.

Diagnostic accuracy of digital RNA quantification versus real-time PCR for the detection of respiratory syncytial virus in nasopharyngeal aspirates from children with acute respiratory infection.

BACKGROUND: Virus-specific molecular assays such as real-time polymerase chain reaction (RT-PCR) are regularly used as the gold standard to diagnose viral respiratory tract infections, but simultaneous detection of multiple different pathogens is often challenging. A multiplex digital method of RNA quantification, nCounter (NanoString Technologies), can overcome this disadvantage and identify, in a single reaction, the presence of different respiratory viruses. OBJECTIVES: To evaluate the accuracy of nCounter to identify and quantify RSV-A and RSV-B in nasopharyngeal aspirates (NPA) of children (6-23-months-old) with acute respiratory infection. STUDY DESIGN: NPA was collected at enrolment in a prospective cross-sectional study conducted in Salvador, Brazil. A quantitative RT-PCR with a subgroup-specific primer and probeset for RSV-A and RSV-B was performed in parallel with a customized nCounter probeset containing viral targets in NPA. RESULTS: Of 559 NPA tested, RSV was detected by RT-PCR in 139 (24.9%), by nCounter in 122 (21.8%) and by any method in 158 (28.3%) cases. Compared to the gold standard of qRT-PCR, sensitivity of nCounter was 74.3% (95%CI:63.3%-82.9% RSV-A) and 77.6% (95%CI:66.3%-85.9% RSV-B); specificity was 98.4% (95%CI:96.8%-99.2% RSV-A) and 97.8% (95%CI:96.0%-98.8% RSV-B); positive predictive value was 87.3% (95%CI:76.9%-93.4% RSV-A) and 82.5% (95%CI:71.4%-90.0% RSV-B) and negative predictive value was 96.1% (95%CI:94.1%-97.5% RSV-A), and 96.9% (95%CI:95.1%-98.2% RSV-B). Accuracy was 95.2% (95%CI:93.1%-96.7%) for RSV-A and 95.3% (95%CI:93.3%-96.9%) for RSV-B, while both methods significantly correlated for RSV-A (r = 0.44, p = 8 × 10-5) and RSV-B (r = 0.73, p = 3 × 10-12) quantification. CONCLUSIONS: nCounter is highly accurate in detecting RSV-A/B in NPA. Robustness and high-throughput multiplexing indicate its use in large-scale epidemiological studies.

Antibody responses against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in children with acute respiratory infection with or without nasopharyngeal bacterial carriage.

BACKGROUND: We studied Immunoglobulin G (IgG) antibody responses against Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in young children with acute viral type respiratory infection and analyzed the findings in a multivariate model including age, nasopharyngeal carriage of the tested bacteria and pneumococcal vaccination. METHODS: We included 227 children aged 6-23 months with acute respiratory infection. Nasopharyngeal aspirates were tested for bacterial carriage through detection of messenger RNA (mRNA) transcript with nCounter analysis. Acute and convalescent serum samples were tested for IgG antibody response against eight pneumococcal proteins, three proteins from H. influenzae and five proteins from M. catarrhalis in a fluorescent multiplex immunoassay. RESULTS: A two-fold or greater increase in antibodies to S. pneumoniae, H. influenzae and M. catarrhalis was detected in 27.8, 9.7 and 14.1%, respectively. Nasopharyngeal carriage of each of the studied bacteria was not associated with antibody response detection against each respective bacterium. Furthermore, neither age nor pneumococcal vaccination were independently associated to detection of antibody response against the studied bacteria. Children who carried H. influenzae had higher frequency of colonization by M. catarrhalis (175 [80.3%] vs. 2 [22.2%]; p < .001) than those without H. influenzae. Also, children with acute otitis media tended to have higher frequency of antibody response to S. pneumoniae. CONCLUSION: Nasopharyngeal colonization by S. pneumoniae, H. influenzae and M. catarrhalis did not induce significant increases in antibody levels to these bacteria. Carriage of pathogenic bacteria in the nasopharynx is not able to elicit antibody responses to protein antigens similar to those caused by symptomatic infections.

10-valent pneumococcal conjugate vaccine (PCV10) decreases metabolic activity but not nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae.

BACKGROUND: The effect of pneumococcal vaccination is widely variable when measured by nasopharyngeal carriage of vaccine and non-vaccine targets. The aim of this study was to compare the carriage rates and metabolic activity of Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Moraxella catarrhalis among children who were or were not vaccinated with PCV10. METHODS: We included children with acute respiratory infection aged 6-23months from a cross-sectional study (CHIADO-IVAS). Nasopharyngeal aspirates were collected and respiratory pathogens were quantified by nCounter digital transcriptomics (Nanostring) and metagenomic sequencing of 16S ribosomal RNA (Illumina). The metabolic rate was calculated by the ratio between RNA transcripts and 16S DNA reads. RESULTS: Out of the 80 patients in this study, 53 were vaccinated with PCV10 and 27 were unvaccinated. There was no difference in nasopharyngeal carriage rates of S. pneumoniae, S. aureus, H. influenzae or M. catarrhalis by either transcriptomic analysis or 16S metagenomics. However, unvaccinated children presented a higher metabolic rate for S. pneumoniae compared to PCV10-vaccinated children (Median [25-75th percentiles]: 126 [22.75-218.41] vs. 0[0-47.83], p=0.004). Furthermore, unvaccinated children presented a positive correlation between mRNA counts and 16S DNA reads for S. pneumoniae (r=0.707; p<0.001) and H. influenzae (r=0.525; p=0.005), in contrast to vaccinated children. No such effect was observed for S. aureus and M. catarrhalis. CONCLUSIONS: Vaccination by PCV10 exerts a pathogen-specific effect on pneumococcal metabolic rate. Pathogen RNA/DNA ratio might represent a more sensitive readout for vaccine follow-up, as compared to nasopharyngeal carriage.

Respiratory syncytial virus a and b display different temporal patterns in a 4-year prospective cross-sectional study among children with acute respiratory infection in a tropical city.

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 ±â€Š4.5 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.

Frequency of complications and the effects of pneumococcal vaccination in young children with acute respiratory tract infection.

BACKGROUND: Acute respiratory infection (ARI) is the most frequent reason for children being seen by doctors worldwide. We aimed to estimate the frequency of complications in children aged 6-23 months during ARI episode and to evaluate risk factors present on recruitment associated with complications after the universal implementation of pneumococcal vaccine (PCV10) in our region. METHODS: This prospective cohort enrolled children who had shown ARI for up to 7 days and who were subsequently followed up 14-21 days after, in Salvador, Brazil. Data on recruitment were registered. The vaccine card was personally checked. Complication was defined when hospitalization, pneumonia or acute otitis media (AOM) were informed during the follow-up visit. Pneumonia and AOM were diagnosed by a doctor. Multiple logistic regression analysis was performed. RESULTS: Of 576 children, 422 (73%) returned and 79 (19%; 95%CI: 15-23%) had complications. The mean interval between admission and follow-up was 23±13 days. Pneumonia (n=47; 11%), hospitalization (n=28; 7%), and AOM (n=17; 4%) were reported. Most of the patients presented one complication (n=66; 84%) followed by two (n=13; 16%). Report of fever (92% versus 79%; OR [95%CI]: 2.90 [1.18-7.14]), bird at home (24% versus 14%; OR [95%CI]: 2.13 [1.07-4.26]), ronchi (48% versus 36%; OR [95%CI]: 2.06 [1.16-3.67]) or crackles (17% versus 7%; OR [95%CI]: 2.36 [1.04-5.38]) on auscultation were directly associated with complications whereas PCV10 (59% versus 75%; OR [95%CI]: 0.46 [0.26-0.82]) was inversely associated. Bird at home (OR [95%CI]: 5.80 [1.73-19.38]) and ronchi (OR [95%CI]: 6.39 [1.96-20.85]) were associated with AOM; PCV10 was inversely associated with AOM (OR [95%CI]: 0.16 [0.05-0.52]). Crackles were associated with pneumonia (OR [95%CI]: 2.55 [1.01-6.40]). CONCLUSIONS: One fifth of the children presented complications. PCV10 was independently associated with lower odds of development of AOM. Bird at home and ronchi are risk factors of otitis. Crackles are associated with pneumonia.

Pathogen transcriptional profile in nasopharyngeal aspirates of children with acute respiratory tract infection.

BACKGROUND: Acute respiratory tract infections (ARI) present a significant morbidity and pose a global health burden. Patients are frequently treated with antibiotics although ARI are most commonly caused by virus, strengthening the need for improved diagnostic methods. OBJECTIVES: Detect viral and bacterial RNA in nasopharyngeal aspirates (NPA) from children aged 6-23 months with ARI using nCounter. STUDY DESIGN: A custom-designed nCounter probeset containing viral and bacterial targets was tested in NPA of ARI patients. RESULTS: Initially, spiked control viral RNAs were detectable in ≥6.25 ng input RNA, indicating absence of inhibitors in NPA. nCounter applied to a larger NPA sample (n=61) enabled the multiplex detection of different pathogens: RNA viruses Parainfluenza virus (PIV 1-3) and RSV A-B in 21%, Human metapneumovirus (hMPV) in 5%, Bocavirus (BoV), CoV, Influenza virus (IV) A in 3% and, Rhinovirus (RV) in 2% of samples, respectively. RSV A-B was confirmed by Real Time PCR (86.2-96.9% agreement). DNA virus (AV) was detected at RNA level, reflecting viral replication, in 10% of samples. Bacterial transcripts from Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae and Chlamydophila pneumoniae were detected in 77, 69, 26, 8, 3 and 2% of samples, respectively. CONCLUSION: 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.