Juliana R. Oliveira

The role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country.

We report an investigation for 16 bacteria and viruses among 184 children hospitalized with pneumonia in Salvador, Brazil. Etiology was established in 144 (78%) cases. Viral, bacterial, and mixed infections were found in 110 (60%), 77 (42%), and 52 (28%) patients, respectively. Rhinovirus (21%) and Streptococcus pneumoniae (21%) were the most common pathogens. Our results demonstrate the importance of viral and pneumococcal infections among those patients.

Seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region

Abstract Community-acquired pneumonia (CAP) is a common cause of morbidity among children. Evidence on seasonality, especially on the frequency of viral and bacterial causative agents is scarce; such information may be useful in an era of changing climate conditions worldwide. To analyze the frequency of distinct infections, meteorological indicators and seasons in children hospitalized for CAP in Salvador, Brazil, nasopharyngeal aspirate and blood were collected from 184 patients aged <5 y over a 21-month period. Fourteen microbes were investigated and 144 (78%) cases had the aetiology established. Significant differences were found in air temperature between spring and summer (p = 0.02) or winter (p < 0.001), summer and fall (p = 0.007) or winter (p < 0.001), fall and winter (p = 0.002), and on precipitation between spring and fall (p = 0.01). Correlations were found between: overall viral infections and relative humidity (p = 0.006; r = 0.6) or precipitation (p = 0.03; r = 0.5), parainfluenza and precipitation (p = 0.02; r = −0.5), respiratory syncytial virus (RSV) and air temperature (p = 0.048; r = −0.4) or precipitation (p = 0.045; r = 0.4), adenovirus and precipitation (p = 0.02; r = 0.5), pneumococcus and air temperature (p = 0.04; r = −0.4), and Chlamydia trachomatis and relative humidity (p = 0.02; r = −0.5). The frequency of parainfluenza infection was highest during spring (32.1%; p = 0.005) and that of RSV infection was highest in the fall (36.4%; p < 0.001). Correlations at regular strength were found between several microbes and meteorological indicators. Parainfluenza and RSV presented marked seasonal patterns.

Community acquired pneumonia among pediatric outpatients in Salvador, Northeast Brazil, with emphasis on the role of pneumococcus

Pneumonia is one of the leading causes of hospitalization and death among children in developing countries, and mortality due to pneumonia has been associated with S. pneumoniae infection. This investigation was designed to describe the antimicrobial susceptibility and serotype patterns of pneumococcal strains recovered from the blood of children with community-acquired pneumonia (CAP) and to assess the clinical findings of pneumococcal bacteremic patients with pneumonia. In a 26 month prospective study, blood cultures were obtained as often as possible from children (<16 years of age) diagnosed with CAP in two emergency rooms. Antimicrobial drug susceptibility tests and serotyping were performed when pneumococcus was identified. We studied 3,431 cases and cultured blood samples from 65.5% of those. Pneumococcus was recovered from 0.8% of the blood samples. The differences in age, somnolence, wheezing and hospitalization among children with and without pneumococcal bacteremia were statistically significant. Pneumococcal bacteremia was age-related (mean 1.63 +/- 1.55; median 0.92) and associated with somnolence and hospitalization among children with CAP. One strain was recovered from pleural fluid. Penicillin resistance was detected in 21.0% (4/19) of the strains at an intermediate level, whereas 63.0% of the strains were resistant to trimethoprim-sulfamethoxazole. The most common serotypes were 14 and 6B, and these serotypes included the resistant strains. Eight of our 18 isolates from blood were of types included in the heptavalent conjugate pneumococcal vaccine, recently licensed in the USA.

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

Abstract 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 ≥6.25ng 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 (<10ng). 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.

Respiratory viral infections among children with community-acquired pneumonia and pleural effusion

Abstract Pleural effusion (PE), a complication of community-acquired pneumonia (CAP), is usually attributed to a bacterial infection. Nonetheless, viral infections have not been investigated routinely. We searched for bacterial and viral infections among 277 children hospitalized with CAP. Among these children 206 (74%) had radiographic confirmation, of whom 25 (12%) had PE. The aetiology was established in 18 (72%) PE cases: bacterial (n = 5; 28%), viral (n = 9; 50%), and viral–bacterial (n = 4; 22%) infections were found. Infection by rhinovirus (n = 3), enterovirus, Streptococcus pneumoniae (n = 2 each), Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, influenza A virus, and respiratory syncytial virus (RSV) (n = 1 each) were detected as probable sole infections. Parainfluenza virus 1/3 + influenza A virus and RSV + influenza A virus (n = 1 each) were identified as mixed viral–viral infections. Probable viral non-bacterial infection was identified in a third of the cases with CAP and PE. It is advisable to investigate viral as well as bacterial infections among children with CAP and PE.

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.

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.

Abstract 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.

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.