Comparison of next generation technologies and bioinformatics pipelines for capsular typing of Streptococcus pneumoniae.

Whole genome sequencing (WGS)-based approaches for pneumococcal capsular typing have become an alternative to serological methods. In silico serotyping from WGS has not yet been applied to long-read sequences produced by third-generation technologies. The objective of the study was to determine the capsular types of pneumococci causing invasive disease in Catalonia (Spain) using serological typing and WGS and to compare the performance of different bioinformatics pipelines using short- and long-read data from WGS. All invasive pneumococcal pediatric isolates collected in Hospital Sant Joan de Déu (Barcelona) from 2013 to 2019 were included. Isolates were assigned a capsular type by serological testing based on anticapsular antisera and by different WGS-based pipelines: Illumina sequencing followed by serotyping with PneumoCaT, SeroBA, and Pathogenwatch vs MinION-ONT sequencing coupled with serotyping by Pathogenwatch from pneumococcal assembled genomes. A total of 119 out of 121 pneumococcal isolates were available for sequencing. Twenty-nine different serotypes were identified by serological typing, with 24F (n = 17; 14.3%), 14 (n = 10; 8.4%), and 15B/C (n = 8; 6.7%) being the most common serotypes. WGS-based pipelines showed initial concordance with serological typing (>91% of accuracy). The main discrepant results were found at the serotype level within a serogroup: 6A/B, 6C/D, 9A/V, 11A/D, and 18B/C. Only one discrepancy at the serogroup level was observed: serotype 29 by serological testing and serotype 35B/D by all WGS-based pipelines. Thus, bioinformatics WGS-based pipelines, including those using third-generation sequencing, are useful for pneumococcal capsular assignment. Possible discrepancies between serological typing and WGS-based approaches should be considered in pneumococcal capsular-type surveillance studies.

Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection Among Children and Adults: A Seroprevalence Study of Family Households in the Barcelona Metropolitan Region, Spain.

BACKGROUND: Susceptibility of children and adults to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and persistence of antibody response to the virus after infection resolution remain poorly understood, despite their significant public health implications. METHODS: A prospective cross-sectional seroprevalence study with volunteer families that included at least 1 first-reported adult case positive by SARS-CoV-2 by polymerase chain reaction (PCR) and at least 1 child aged <15 years living in the same household under strict home confinement was conducted in the metropolitan Barcelona Health Region, Spain, during the pandemic period 28 April 2020-3 June 2020. All household members were tested at home using a rapid SARS-CoV-2 antibody assay with finger prick-obtained capillary blood. RESULTS: A total of 381 family households including 381 first-reported PCR-positive adult cases and 1084 contacts (672 children, 412 adults) were enrolled. SARS-CoV-2 seroprevalence rates were 17.6% (118 of 672) in children and 18.7% (77 of 335) in adult contacts (P = .64). Among first-reported cases, seropositivity rates varied from 84.0% in adults previously hospitalized and tested within 6 weeks since the first positive PCR result to 31.5% in those not hospitalized and tested after that lag time (P < .001). Nearly all (99.9%) positive children were asymptomatic or had mild symptoms. CONCLUSIONS: Children appear to have similar probability as adults to become infected by SARS-CoV-2 in quarantined family households but remain largely asymptomatic. Adult antibody protection against SARS-CoV-2 seems to be weak beyond 6 weeks post-infection confirmation, especially in cases that have experienced mild disease.

Carriage of multiple Streptococcus pneumoniae capsular types is frequent among children with invasive pneumococcal disease.

Streptococcus pneumoniae (pneumococcus) is a human pathogen that colonizes the nasopharynx. We investigated serotype distribution in paired invasive and nasopharyngeal samples obtained from 57 children during invasive pneumococcal disease. Of 39 nasopharyngeal samples positive for pneumococci, 46.2% contained a serotype different from the one causing disease. This study reports a high frequency of pneumococcal multiple serotype carriage in children with invasive pneumococcal disease. Whether multiple serotype carriage is important for the onset and progress to pneumococcal infection warrants further investigation.

Pneumococcal nasopharyngeal carriage among Bhutanese children hospitalized with clinical pneumonia: serotypes and viral co-infection.

BACKGROUND: Pneumococcal nasopharyngeal colonization (PNC) generally precedes pneumococcal disease. The purpose of this study was to determine the prevalence of PNC and to identify the pneumococcal serotypes circulating among Bhutanese children under five years of age admitted with clinical pneumonia, before the introduction of pneumococcal conjugate vaccine (PCV13) in the country. We also aimed to contribute to the understanding of the interplay between PNC and viral co-infection among this population. METHODS: This was a prospective study conducted at the Jigme Dorji Wangchuck National Referral Hospital in Bhutan over 12 consecutive months. Children aged 2 to 59 months admitted with WHO-defined clinical pneumonia were eligible for recruitment. We collected blood for bacterial culture and molecular identification of S. pneumoniae, and nasopharyngeal washing for screening of respiratory viruses, and for the detection and capsular typing of S. pneumoniae by real-time polymerase chain reaction (RT-PCR). RESULTS: Overall, 189 children were recruited, and PNC was tested in 121 of them (64.0%). PNC was found in 76/121 children (62.8%) and S. pneumoniae was identified in blood (both by culture and RT-PCR) in a single child. Respiratory viruses were detected in a similar proportion among children with (62/70; 88.6%) and without PNC (36/40; 90.0%; p = 1.000), but rhinovirus detection was less common among children with PNC (20/70; 28.6% versus 19/40; 47.5%; p = 0.046). Capsular typing identified 30 different serotypes. Thirty-nine children (51.3%) were colonised with two to five different serotypes. A third of the children presented with serotypes considered highly invasive. Over half of the children (44/76; 57.9%) were carrying at least one serotype included in PCV13. CONCLUSIONS: This study provides baseline information on the status of PNC among Bhutanese children admitted with clinical pneumonia prior to the introduction of PCV13, which is valuable to monitor its potential impact. PCV13 could theoretically have averted up to 58% of the pneumococcal infections among the children in this study, suggesting a future role for the vaccine to significantly reduce the burden associated with S. pneumoniae in Bhutan.

Validation and Implementation of a Diagnostic Algorithm for DNA Detection of Bordetella pertussis, B. parapertussis, and B. holmesii in a Pediatric Referral Hospital in Barcelona, Spain.

This study aimed to validate a comprehensive diagnostic protocol based on real-time PCR for the rapid detection and identification of Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii, as well as its implementation in the diagnostic routine of a reference children's hospital. The new algorithm included a triplex quantitative PCR (qPCR) targeting IS481 gene (in B. pertussis, B. holmesii, and some Bordetella bronchiseptica strains), pIS1001 (B. parapertussis-specific) and rnase P as the human internal control. Two confirmatory singleplex tests for B. pertussis (ptxA-Pr) and B. holmesii (hIS1001) were performed if IS481 was positive. Analytical validation included determination of linear range, linearity, efficiency, precision, sensitivity, and a reference panel with clinical samples. Once validated, the new algorithm was prospectively implemented in children with clinical suspicion of whooping cough presenting to Hospital Sant Joan de Deu (Barcelona, Spain) over 12 months. Lower limits of detection obtained were 4.4, 13.9, and 27.3 genomic equivalents/ml of sample for IS481 (on B. pertussis), pIS1001 and hIS1001, and 777.9 for ptxA-Pr. qPCR efficiencies ranged from 86.0% to 96.9%. Intra- and interassay variabilities were <3% and <5%, respectively. Among 566 samples analyzed, B. pertussis, B. holmesii, and B. parapertussis were detected in 11.1%, 0.9% (only in females >4 years old), and 0.2% of samples, respectively. The new algorithm proved to be a useful microbiological diagnostic tool for whooping cough, demonstrating a low rate of other non-pertussisBordetella species in our surveilled area.

Use of procalcitonin in the diagnosis of tuberculosis in infants and preschool children.

Normal procalcitonin (PCT) levels have been reported in adult pulmonary tuberculosis (TB) but have not been previously investigated in children. We aimed to assess PCT levels at diagnosis of TB in young children in a low-burden setting. In a cross-sectional observational study in a referral pediatric center in Barcelona (Spain), we assessed the value of PCT and other inflammatory markers (leucocyte counts, C-reactive protein, and erythrocyte sedimentation rate) in the diagnosis of TB in pre-school children (< 6 years at diagnosis, n = 45), as compared with two control groups (pneumococcal pneumonia, n = 25; and healthy controls, n = 49). Normal PCT levels were observed at diagnosis of TB in most cases, while C-reactive protein values and leucocyte counts were slightly elevated when compared to healthy controls. All three inflammatory biomarkers were significantly higher in children with pneumococcal pneumonia. CONCLUSIONS: In our study, PCT was not a useful diagnostic test for TB in young children. In a low-burden TB setting, PCT may be of some value in distinguishing pulmonary TB from pneumococcal pneumonia. What is Known: • Diagnosis of pediatric tuberculosis on clinical evidence is difficult, particularly in infants and small children. • Studies in adults with tuberculosis have mostly reported normal procalcitonin levels at diagnosis. What is New: • In pre-scholars with tuberculosis, erythrocyte sedimentation rate and white blood cell counts were higher than in healthy controls, but procalcitonin was not. • Procalcitonin may be useful in the differential diagnosis of intrathoracic tuberculosis and pneumococcal pneumonia.

Comparison of NxTAG Respiratory Pathogen Panel and Anyplex II RV16 Tests for Multiplex Detection of Respiratory Pathogens in Hospitalized Children.

Multiplex molecular techniques can detect a diversity of respiratory viruses and bacteria that cause childhood acute respiratory infection rapidly and conveniently. However, currently available techniques show high variation in performance. We sought to compare the diagnostic accuracy of the novel multiplex NxTAG respiratory pathogen panel (RPP) RUO test versus a routine multiplex Anyplex II RV16 assay in respiratory specimens collected from children <18 years of age hospitalized with nonspecific symptoms of acute lower respiratory infection. Parallel testing was performed on nasopharyngeal aspirates prospectively collected at referral Children's Hospital Sant Joan de Déu (Barcelona, Spain) between June and November 2015. Agreement values between the two tests and kappa coefficients were assessed. Bidirectional sequencing was performed for the resolution of discordant results. A total of 319 samples were analyzed by both techniques. A total of 268 (84.0%) of them yielded concordant results. Positive percent agreement values ranged from 83.3 to 100%, while the negative percent agreement was more than 99% for all targets except for enterovirus/rhinovirus (EV/RV; 94.4%). Kappa coefficients ranged from 0.83 to 1.00. Discrepancy analysis confirmed 66.0% of NxTAG RPP RUO results. A total of 260 viruses were detected, with EV/RV (n = 105, 40.4%) being the most prevalent target. Viral coinfections were found in 44 (14.2%) samples. In addition, NxTAG RPP RUO detected single bacterial and mixed viral-bacterial infections in seven samples. NxTAG RPP RUO showed high positive and negative agreement with Anyplex II RV16 for main viruses that cause acute respiratory infections in children, coupled with an additional capability to detect some respiratory bacteria.

Impact of the bacterial nasopharyngeal microbiota on the severity of genus enterovirus lower respiratory tract infection in children: A case-control study.

INTRODUCTION: Rhinoviruses (RV) and enteroviruses (EV) are among the main causative etiologies of lower respiratory tract infection (LRTI) in children. The clinical spectrum of RV/EV infection is wide, which could be explained by diverse environmental, pathogen-, and host-related factors. Little is known about the nasopharyngeal microbiota as a risk factor or disease modifier for RV/EV infection in pediatric patients. This study describes distinct nasopharyngeal microbiota profiles according to RV/EV LRTI status in children. METHODS: Cross-sectional case-control study, conducted at Hospital Sant de Déu (Barcelona, Spain) from 2017 to 2020. Three groups of children <5 years were included: healthy controls without viral detection (Group A), mild or asymptomatic controls with RV/EV infection (Group B), and cases with severe RV/EV infection admitted to the pediatric intensive care unit (PICU) (Group C). Nasopharyngeal samples were collected from participants for viral DNA/RNA detection by multiplex-polymerase chain reaction and bacterial microbiota characterization by 16S rRNA gene sequencing. RESULTS: A total of 104 subjects were recruited (A = 17, B = 34, C = 53). Children's nasopharyngeal microbiota composition varied according to their RV/EV infection status. Richness and diversity were decreased among children with severe infection. Nasopharyngeal microbiota profiles enriched in genus Dolosigranulum were related to respiratory health, while genus Haemophilus was specifically predominant in children with severe RV/EV LRTI. Children with mild or asymptomatic RV/EV infection showed an intermediate profile. CONCLUSIONS: These results suggest a close relationship between the nasopharyngeal microbiota and different clinical presentations of RV/EV infection.

Association between soluble angiotensin-converting enzyme 2 in saliva and SARS-CoV-2 infection: a cross-sectional study.

This study aimed to investigate the association between saliva soluble angiotensin-converting enzyme 2 (sACE2) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children and adults. We selected a convenience sample of adults with post-acute SARS-CoV-2 infection and their household children living in quarantined family households of the metropolitan Barcelona region (Spain) during the spring 2020 pandemic national lockdown. Participants were tested for saliva sACE2 quantification by western blot and nasopharyngeal SARS-CoV-2 RT-PCR detection. A total of 161 saliva samples [82 (50.9%) from children; 79 (49.1%) from females] yielded valid western blot and RT-PCR results. Saliva sACE2 was detected in 79 (96.3%) children and 76 (96.2%) convalescent adults. Twenty (24.4%) children and 20 (25.3%) convalescent adults were positive for SARS-CoV-2 in nasopharynx by RT-PCR. SARS-CoV-2 RT-PCR-negative children had a significantly higher mean proportional level of saliva sACE2 (0.540 × 10-3%) than RT-PCR-positive children (0.192 × 10-3%, p < 0.001) and convalescent adults (0.173 × 10-3%, p < 0.001). In conclusion, children negative for nasopharyngeal SARS-CoV-2 RT-PCR appear to exhibit a higher concentration of saliva sACE2 than SARS-CoV-2 RT-PCR-positive children and convalescent adults. Release of adequate levels of sACE2 in saliva could play a protective role against SARS-CoV-2.

Trends in invasive bacterial diseases during the first 2 years of the COVID-19 pandemic: analyses of prospective surveillance data from 30 countries and territories in the IRIS Consortium.

BACKGROUND: The Invasive Respiratory Infection Surveillance (IRIS) Consortium was established to assess the impact of the COVID-19 pandemic on invasive diseases caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus agalactiae. We aimed to analyse the incidence and distribution of these diseases during the first 2 years of the COVID-19 pandemic compared to the 2 years preceding the pandemic. METHODS: For this prospective analysis, laboratories in 30 countries and territories representing five continents submitted surveillance data from Jan 1, 2018, to Jan 2, 2022, to private projects within databases in PubMLST. The impact of COVID-19 containment measures on the overall number of cases was analysed, and changes in disease distributions by patient age and serotype or group were examined. Interrupted time-series analyses were done to quantify the impact of pandemic response measures and their relaxation on disease rates, and autoregressive integrated moving average models were used to estimate effect sizes and forecast counterfactual trends by hemisphere. FINDINGS: Overall, 116 841 cases were analysed: 76 481 in 2018-19, before the pandemic, and 40 360 in 2020-21, during the pandemic. During the pandemic there was a significant reduction in the risk of disease caused by S pneumoniae (risk ratio 0·47; 95% CI 0·40-0·55), H influenzae (0·51; 0·40-0·66) and N meningitidis (0·26; 0·21-0·31), while no significant changes were observed for S agalactiae (1·02; 0·75-1·40), which is not transmitted via the respiratory route. No major changes in the distribution of cases were observed when stratified by patient age or serotype or group. An estimated 36 289 (95% prediction interval 17 145-55 434) cases of invasive bacterial disease were averted during the first 2 years of the pandemic among IRIS-participating countries and territories. INTERPRETATION: COVID-19 containment measures were associated with a sustained decrease in the incidence of invasive disease caused by S pneumoniae, H influenzae, and N meningitidis during the first 2 years of the pandemic, but cases began to increase in some countries towards the end of 2021 as pandemic restrictions were lifted. These IRIS data provide a better understanding of microbial transmission, will inform vaccine development and implementation, and can contribute to health-care service planning and provision of policies. FUNDING: Wellcome Trust, NIHR Oxford Biomedical Research Centre, Spanish Ministry of Science and Innovation, Korea Disease Control and Prevention Agency, Torsten Söderberg Foundation, Stockholm County Council, Swedish Research Council, German Federal Ministry of Health, Robert Koch Institute, Pfizer, Merck, and the Greek National Public Health Organization.

Clinical impact of rapid viral respiratory panel testing on pediatric critical care of patients with acute lower respiratory infection.

BACKGROUND: We aimed to determine the impact of utilizing a rapid panel test of respiratory viral and atypical bacteria (FilmArray® Respiratory Panel, FA RP) on etiological diagnosis of acute lower respiratory infection (ALRI) and antimicrobial stewardship in critical care pediatric patients. METHODS: Prospective cohort study of patients aged<18 years with clinical diagnosis of ALRI that were admitted to the Pediatric Intensive Care Unit (PICU) of Hospital Sant Joan de Deu (Barcelona, Spain) during December 2015-February 2017. Patients were diagnosed by FA RP and by a bundle of routine microbiological assays. RESULTS: ALRI viral and bacterial etiology was confirmed by a composite reference standard of routine microbiological assays in 72 (55.4%) and 15 (11.5%) respiratory samples, respectively, that were collected from 130 children (median age, 3.5 months, IQR 1.1-14.8 months; 54.6% male). Comparatively, FA RP use increased etiological confirmation of ALRI in up to 123 (94.6%) samples (p<0.001) but only determined a bacterial origin in 2 (1.5%). Availability of diagnostic results before patient discharge from the PICU rose from 65.4 to 38.5% (p<0.001). Use of the new panel test directly influenced antimicrobial stewardship in 11 (8.4%) episodes, leading to discontinuation of antiviral drugs (n=5), administration of targeted antibiotics (n=3), antiviral therapy start (n=2) and both targeted antibiotic administration and discontinuation of antiviral drugs (n=1). CONCLUSION: FA RP contributed to improve etiological diagnosis of ALRI in a timely manner while enhancing a more rational use of antimicrobial drugs in critical care pediatric patients.

Characterization of the nasopharyngeal microbiome in patients with Kawasaki disease.

INTRODUCTION: The aetiology of Kawasaki disease (KD) remains unknown. Several studies have linked the human microbiome with some diseases. However, there are limited studies on the role of the respiratory microbiome in KD. The aim of our study was to make a more thorough analysis of the causes and processes that increase the susceptibility to KD. METHODS: Case-control study comparing the respiratory microbiome of KD patients with that of healthy children. The V3-V4 region of the 16S rRNA bacterial gene and 16 respiratory viruses were analysed by real-time polimerase-chain reaction. We used the Ribosomal Database Project (RDP) version 11.5 (taxonomic assignment). RESULTS: The initial sample included 11 cases and 11 controls matched for age, sex and seasonality. One of the cases was excluded to poor sample quality. The final analysis included 10 cases and 10 controls. In the case group, the analysis detected Haemophilus, Moraxella, Streptococcus and Corynebacterium species (27.62%, 19.71%, 25.28%, 11.86%, respectively). In the control group, it found Haemophilus, Streptococcus, Moraxella, and Dolosigranulum species (38.59%, 23.71%, 16.08, 8.93%, respectively). We found a higher relative abundance of Corynebacterium in patients with KD (11.86% vs. 1.55%; P = 0.004). CONCLUSIONS: To our knowledge, this is the first study that has found differences in the composition of the respiratory microbiome between patients with KD and healthy controls. The relative abundance of Corynebacterium spp. was greater in the KD group. This study shows differences in the microbiome between cases and controls, which suggests that the microbiome may play a role in facilitating the development of KD.

Concentrations of nitrogen compounds are related to severe rhinovirus infection in infants. A time-series analysis from the reference area of a pediatric university hospital in Barcelona.

BACKGROUND: There is scarce information focused on the effect of weather conditions and air pollution on specific acute viral respiratory infections, such as rhinovirus (RV), with a wide clinical spectrum of severity. OBJECTIVE: The aim of this study was to analyze the association between episodes of severe respiratory tract infection by RV and air pollutant concentrations (NOx and SO2 ) in the reference area of a pediatric university hospital. METHODS: An analysis of temporal series of daily values of NOx and SO2 , weather variables, circulating pollen and mold spores, and daily number of admissions in the pediatric intensive care unit (PICU) with severe respiratory RV infection (RVi) in children between 6 months and 18 years was performed. Lagged variables for 0-5 days were considered. The study spanned from 2010 to 2018. Patients with comorbidities were excluded. RESULTS: One hundred and fifty patients were admitted to the PICU. Median age was 19 months old (interquartile range [IQR]: 11-47). No relationship between RV-PICU admissions and temperature, relative humidity, cumulative rainfall, or wind speed was found. Several logistic regression models with one pollutant and two pollutants were constructed but the best model was that which included average daily NOx concentrations. Average daily NOx concentrations were related with the presence of PICU admissions 3 days later (odds ratio per IQR-unit increase: 1.64, 95% confidence interval: 1.20-2.25)). CONCLUSIONS: This study has shown a positive correlation between NOx concentrations at Lag 3 and children's PICU admissions with severe RV respiratory infection. Air pollutant data should be taken into consideration when we try to understand the severity of RVis.

Impact of COVID-19 Lockdown on the Nasopharyngeal Microbiota of Children and Adults Self-Confined at Home.

The increased incidence of COVID-19 cases and deaths in Spain in March 2020 led to the declaration by the Spanish government of a state of emergency imposing strict confinement measures on the population. The objective of this study was to characterize the nasopharyngeal microbiota of children and adults and its relation to SARS-CoV-2 infection and COVID-19 severity during the pandemic lockdown in Spain. This cross-sectional study included family households located in metropolitan Barcelona, Spain, with one adult with a previous confirmed COVID-19 episode and one or more exposed co-habiting child contacts. Nasopharyngeal swabs were used to determine SARS-CoV-2 infection status, characterize the nasopharyngeal microbiota and determine common respiratory DNA/RNA viral co-infections. A total of 173 adult cases and 470 exposed children were included. Overall, a predominance of Corynebacterium and Dolosigranulum and a limited abundance of common pathobionts including Haemophilus and Streptococcus were found both among adults and children. Children with current SARS-CoV-2 infection presented higher bacterial richness and increased Fusobacterium, Streptococcus and Prevotella abundance than non-infected children. Among adults, persistent SARS-CoV-2 RNA was associated with an increased abundance of an unclassified member of the Actinomycetales order. COVID-19 severity was associated with increased Staphylococcus and reduced Dolosigranulum abundance. The stringent COVID-19 lockdown in Spain had a significant impact on the nasopharyngeal microbiota of children, reflected in the limited abundance of common respiratory pathobionts and the predominance of Corynebacterium, regardless of SARS-CoV-2 detection. COVID-19 severity in adults was associated with decreased nasopharynx levels of healthy commensal bacteria.

Related Factors to Streptococcus pneumoniae Invasive Infection and Clinical Manifestations: The Potential Role of Nasopharyngeal Microbiome.

Infections of the lower respiratory tract, such as pneumonia, are one of the leading causes of death worldwide. Streptococcus pneumoniae might colonize the upper respiratory tract and is the main aetiological agent of community-acquired pneumonia (CAP). In the last decades, several factors related to the host, the microorganism and the antibiotic therapy have been investigated to identify risk factors associated with the development of invasive pneumococcal disease (IPD). Nevertheless, these factors themselves do not explain the risk of developing disease or its severity. Recently, some studies have focused on the importance of nasopharyngeal (NP) microbiome and its relation to respiratory health. This review presents existing evidence of the potential role of NP microbiome in the development of IPD.

Rapid Increase of Oral Bacteria in Nasopharyngeal Microbiota After Antibiotic Treatment in Children With Invasive Pneumococcal Disease.

Introduction: Antibiotics are commonly prescribed to young children for treating bacterial infections such as invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae. Despite the obvious benefits of antibiotics, little is known about their possible side effects on children's nasopharyngeal microbiota. In other ecological niches, antibiotics have been described to perturb the balanced microbiota with short- and long-term effects on children's health. The present study aims to evaluate and compare the nasopharyngeal microbiota of children with IPD and different degree of antibiotic exposure. Methods: We investigated differences in nasopharyngeal microbiota of two groups of children <18 years with IPD: children not exposed to antibiotics before sample collection (n=27) compared to children previously exposed (n=54). Epidemiological/clinical data were collected from subjects, and microbiota was characterized by Illumina sequencing of V3-V4 amplicons of the 16S rRNA gene. Results: Main epidemiological/clinical factors were similar across groups. Antibiotic-exposed patients were treated during a median of 4 days (IQR: 3-6) with at least one beta-lactam (100.0%). Higher bacterial richness and diversity were found in the group exposed to antibiotics. Different streptococcal amplicon sequence variants (ASVs) were differentially abundant across groups: antibiotic use was associated to lower relative abundances of Streptococcus ASV2 and Streptococcus ASV11 (phylogenetically close to S. pneumoniae), and higher relative abundances of Streptococcus ASV3 and Streptococcus ASV12 (phylogenetically close to viridans group streptococci). ASVs assigned to typical bacteria from the oral cavity, including Veillonella, Alloprevotella, Porphyromonas, Granulicatella, or Capnocytophaga, were associated to the antibiotic-exposed group. Common nosocomial genera such as Staphylococcus, Acinetobacter, and Pseudomonas were also enriched in the group exposed to antibiotics. Conclusion: Our results point toward a reduction of S. pneumoniae abundance on the nasopharynx of children with IPD after antibiotic treatment and a short-term repopulation of this altered niche by oral and nosocomial bacteria. Future research studies will have to evaluate the clinical implications of these findings and if these populations would benefit from the probiotic/prebiotic administration or even from the improvement on oral hygiene practices frequently neglected among hospitalized children.

Exploring the nasopharyngeal microbiota composition in infants with whooping cough: A test-negative case-control study.

PURPOSE: The purpose of this study was to characterize the nasopharyngeal microbiota of infants with possible and confirmed pertussis compared to healthy controls. METHODS: This prospective study included all infants <1 year with microbiologically confirmed diagnosis of pertussis attended at a University Hospital over a 12-month period. For each confirmed case, up to 2 consecutive patients within the same age range and meeting the clinical case definition of pertussis but testing PCR-negative were included as possible cases. A third group of asymptomatic infants (healthy controls) were also included. Nasopharyngeal microbiota was characterized by sequencing the V3-V4 region of the 16S rRNA gene. Common respiratory DNA/RNA viral co-infection was tested by multiplex PCR. RESULTS: Twelve confirmed cases, 21 possible cases and 9 healthy controls were included. Confirmed whooping cough was primarily driven by detection of Bordetella with no other major changes on nasopharyngeal microbiota. Possible cases had limited abundance or absence of Bordetella and a distinctive microbiota with lower bacterial richness and diversity and higher rates of viral co-infection than both confirmed cases and healthy controls. Bordetella reads determined by 16S rRNA gene sequencing were found in all 12 confirmed cases (100%), 3 out of the 21 possible cases (14.3%) but in any healthy control. CONCLUSION: This study supports the usefulness of 16S rRNA gene sequencing for improved sensitivity on pertussis diagnosis compared to real-time PCR and to understand other microbial changes occurring in the nasopharynx in children <1 year old with suspected whooping cough compared to healthy controls.

Lower Respiratory Tract Infection and Genus Enterovirus in Children Requiring Intensive Care: Clinical Manifestations and Impact of Viral Co-Infections.

Infection by rhinovirus (RV) and enterovirus (EV) in children ranges from asymptomatic infection to severe lower respiratory tract infection (LRTI). This cohort study evaluates the clinical impact of RV/EV species, alone or in codetection with other viruses, in young children with severe LRTI. Seventy-one patients aged less than 5 years and admitted to the Paediatric Intensive Care Unit (PICU) of a reference children's hospital with RV or EV (RV/EV) LRTI were prospectively included from 1/2018 to 3/2020. A commercial PCR assay for multiple respiratory pathogens was performed in respiratory specimens. In 22/71, RV/EV + respiratory syncytial virus (RSV) was found, and 18/71 had RV/EV + multiple viral detections. Patients with single RV/EV detection required invasive mechanical ventilation (IMV) as frequently as those with RSV codetection, whereas none of those with multiple viral codetections required IMV. Species were determined in 60 samples, 58 being RV. No EV-A, EV-C, or EV-D68 were detected. RV-B and EV-B were only found in patients with other respiratory virus codetections. There were not any associations between RV/EV species and severity outcomes. To conclude, RV/EV detection alone was observed in young children with severe disease, while multiple viral codetections may result in reduced clinical severity. Differences in pathogenicity between RV and EV species could not be drawn.

Differential nasopharyngeal microbiota composition in children according to respiratory health status.

Acute respiratory infections (ARIs) constitute one of the leading causes of antibiotic administration, hospitalization and death among children <5 years old. The upper respiratory tract microbiota has been suggested to explain differential susceptibility to ARIs and modulate ARI severity. The aim of the present study was to investigate the relation of nasopharyngeal microbiota and other microbiological parameters with respiratory health and disease, and to assess nasopharyngeal microbiota diagnostic utility for discriminating between different respiratory health statuses. We conducted a prospective case-control study at Hospital Sant Joan de Deu (Barcelona, Spain) from 2014 to 2018. This study included three groups of children <18 years with gradual decrease of ARI severity: cases with invasive pneumococcal disease (IPD) (representative of lower respiratory tract infections and systemic infections), symptomatic controls with mild viral upper respiratory tract infections (URTI), and healthy/asymptomatic controls according to an approximate case-control ratio 1:2. Nasopharyngeal samples were collected from participants for detection, quantification and serotyping of pneumococcal DNA, viral DNA/RNA detection and 16S rRNA gene sequencing. Microbiological parameters were included on case-control classification models. A total of 140 subjects were recruited (IPD=27, URTI=48, healthy/asymptomatic control=65). Children's nasopharyngeal microbiota composition varied according to respiratory health status and infection severity. The IPD group was characterized by overrepresentation of Streptococcus pneumoniae, higher frequency of invasive pneumococcal serotypes, increased rate of viral infection and underrepresentation of potential protective bacterial species such as Dolosigranulum pigrum and Moraxella lincolnii. Microbiota-based classification models differentiated cases from controls with moderately high accuracy. These results demonstrate the close relationship existing between a child's nasopharyngeal microbiota and respiratory health, and provide initial evidence of the potential of microbiota-based diagnostics for differential diagnosis of severe ARIs using non-invasive samples.

Low impact of SARS-CoV-2 infection among paediatric acute respiratory disease hospitalizations.

OBJECTIVE: This study describes the characteristics of children requiring admission with an acute lower-respiratory disease (ALRD) during the SARS-CoV-2 pandemics. METHODS: Epidemiological, clinical, and microbiological data from patients with ALRD (pneumonia, bronchiolitis, bronchospasm) admitted to a reference paediatric hospital in Spain during the pandemic peak (week 11-20/2020) were prospectively analysed. RESULTS: 110 patients were included. 7 were SARS-CoV-2(+) and they were older in comparison to SARS-CoV-2(-). Among SARS-CoV-2(+) patients, pneumonia was the main clinical diagnosis (6/7) and bronchospasm was absent. Only 1 of 29 infants diagnosed with bronchiolitis was SARS-CoV-2(+). Lower values of leucocytes, lymphocytes, neutrophils, and platelets and higher values of creatinine were found in SARS-CoV-2(+). Human-rhinovirus/enterovirus was the main detection (11/32). There were not differences in PICU admission rates between SARS-CoV-2(+) and (-). CONCLUSIONS: Most of the ALRD episodes identified during the pandemics were not related to SARS-CoV-2 infection. SARS-CoV-2 was mainly found causing pneumonia in older children.