Nasopharyngeal Aspirate Samples Research Articles

ATP-P2X7R pathway activation limits the Tfh cell compartment during pediatric RSV infection.

Follicular helper T cells (Tfh) are pivotal in B cell responses. Activation of the purinergic receptor P2X7 on Tfh cells regulates their activity. We investigated the ATP-P2X7R axis in circulating Tfh (cTfh) cells during Respiratory Syncytial Virus (RSV) infection. We analyzed two cohorts: children with RSV infection (moderate, n=30; severe, n=21) and healthy children (n=23). We utilized ELISA to quantify the levels of PreF RSV protein-specific IgG antibodies, IL-21 cytokine, and soluble P2X7R (sP2X7R) in both plasma and nasopharyngeal aspirates (NPA). Additionally, luminometry was employed to determine ATP levels in plasma, NPA and supernatant culture. The frequency of cTfh cells, P2X7R expression, and plasmablasts were assessed by flow cytometry. To evaluate apoptosis, proliferation, and IL-21 production by cTfh cells, we cultured PBMCs in the presence of Bz-ATP and/or P2X7R antagonist (KN-62) and a flow cytometry analysis was performed. In children with severe RSV disease, we observed diminished titers of neutralizing anti-PreF IgG antibodies. Additionally, severe infections, compared to moderate cases, were associated with fewer cTfh cells and reduced plasma levels of IL-21. Our investigation revealed dysregulation in the ATP-P2X7R pathway during RSV infection. This was characterized by elevated ATP levels in both plasma and NPA samples, increased expression of P2X7R on cTfh cells, lower levels of sP2X7R, and heightened ATP release from PBMCs upon stimulation, particularly evident in severe cases. Importantly, ATP exposure decreased cTfh proliferative response and IL-21 production, while promoting their apoptosis. The P2X7R antagonist KN-62 mitigated these effects. Furthermore, disease severity positively correlated with ATP levels in plasma and NPA samples and inversely correlated with cTfh frequency. Our findings indicate that activation of the ATP-P2X7R pathway during RSV infection may contribute to limiting the cTfh cell compartment by promoting cell death and dysfunction, ultimately leading to increased disease severity.

Circulation patterns and molecular characteristics of respiratory syncytial virus among hospitalized children in Tianjin, China, before and during the COVID-19 pandemic (2017–2022)

Respiratory syncytial virus (RSV) is the main pathogen that causes hospitalization for acute lower respiratory tract infections (ALRIs) in children. With the reopening of communities and schools, the resurgence of RSV in the COVID-19 post-pandemic era has become a major concern. To understand the circulation patterns and genotype variability of RSV in Tianjin before and during the COVID-19 pandemic, a total of 19,531 nasopharyngeal aspirate samples from hospitalized children in Tianjin from July 2017 to June 2022 were evaluated. Direct immunofluorescence and polymerase chain reaction (PCR) were used for screening RSV-positive samples and subtyping, respectively. Further analysis of mutations in the second hypervariable region (HVR2) of the G gene was performed through Sanger sequencing. Our results showed that 16.46% (3,215/19,531) samples were RSV positive and a delayed increase in the RSV infection rates occurred in the winter season from December 2020 to February 2021, with the average RSV-positive rate of 35.77% (519/1,451). The ON1, with H258Q and H266L substitutions, and the BA9, with T290I and T312I substitutions, are dominant strains that alternately circulate every 1–2 years in Tianjin, China, from July 2017 to June 2022. In addition, novel substitutions, such as N296Y, K221T, N230K, V251A in the BA9 genotype, and L226I in the ON1 genotype, emerged during the COVID-19 pandemic. Analysis of clinical characteristics indicated no significant differences between RSV-A and RSV-B groups. This study provides a theoretical basis for clinical prevention and treatment. However, further studies are needed to explore the regulatory mechanism of host immune responses to different lineages of ON1 and BA9 in the future.

Torque Teno Virus Monitoring in Pediatric Hematopoietic Stem Cell Transplantation

Background Immune reconstitution (IR) after allogeneic hematopoietic stem cell transplantation (HSCT) is crucial to avoid infection, relapse, and graft versus host disease (GvHD). Torque Teno Virus (TTV) is a highly prevalent and ubiquitous small DNA virus which constitutes the main component of human virome. In the transplant setting, TTV viral load (VL) seems to correlate with immunodeficiency status, but most studies have focused on solid organ transplant recipients. Aims To study the kinetics of TTV VL in blood (log10 copies/ml), and its presence in nasopharyngeal aspirate (NPA) by qualitative PCR in children undergoing HSCT, and to study their correlation with HSCT outcomes and other markers of IR in the first year after the procedure. Methods Prospective single center study including patients <18 years old who underwent HSCT in 2020-2022, excluding those with lung disease. Blood and NPA samples were collected pre-HSCT and at days 0, +15, +30, +90, +180, +270 and +360 post-HSCT. Immune cell typing including T/B/NK, and NK phenotyping and cytotoxicity (%CD107a+) were performed in blood, and T/NK cells by flow cytometry and respiratory viruses by multiplex PCR were tested in NPA. TTV in blood was analyzed with TTV R GENE® kits (Biomérieux, France), whereas for NPA an in house PCR developed at the National Centre for Microbiology was used. Results We included 30 patients, whose main characteristics are summarized in Table 1. The most frequent indication for HSCT was malignant disease (76.7%). Donor was mainly HLA mismatch (67%). All patients had engraftment (median 10 days, IQR 10-13.5), but one experienced secondary graft failure. Acute GvHD occurred in 73% of patients (63% grades II-IV), and chronic in 37% (10% severe). Almost all patients (93%) presented at least one episode of infection (median 3, IQR 1-6). Endothelial injury complications occurred in 17%. During the first year post-HSCT, 2/21 patients relapsed. Overall survival was 67% (median follow up 601 days, IQR 424.25-880). No significant differences were found in pre HSCT TTV VL regarding patient age, sex, underlying disease, nor CMV status. TTV VL (Graphic 1) decreased after conditioning to minimum at +15 (mean log10 3.149, SD 1.8), increased from +30 being highest at +270 (mean log10 6.778, SD 1.7), and at +360 was still higher than pre-HSCT (mean log10 6.358, SD 1.9). At day +15, each increase in log10 TTV VL increased the risk of dying 39% (p=0.027). At day +30, TTV VL was higher when total lymphoid irradiation (TLI) was used vs serotherapy (p=0.01). No differences were found regarding GvHD development or infections episodes. TTV could be detected in NPA, although its prevalence seems lower than in blood: 14/44 NPA samples resulted positive (32%), most at day +15 (36%); while 50/182 (27.5%) were positive for respiratory virus, the majority at +270 (28%), without identifying any association with TTV positivity. We found several associations between TTV VL and some parameters of IR: there was an inverse correlation with leukocytes, total, and B lymphocytes at +90 (p=0.001, 0.013, and 0.035 respectively), and with leukocytes at +270 (p=0.042). There was also an inverse correlation at +90 with %CD4+CD45RA+ (p=0.043), and direct with %CD4+CD45RO+ (p=0.038). Finally, TTV VL was direclty associated at pre-HSCT with %CD3+HLADR+ (p=0.035). In our series, associations between TTV VL and NK were positive, the stronger with %CD16+ at +270 (p=0.023). Nevertheless, an inverse correlation was found with %CD107a+ at baseline (p=0.04) and at +15 (p=0.013). There was a lack of a consistent pattern of expression of NK receptors along our study. Conclusions 1. TTV is usually detected in blood in children undergoing HSCT, whereas its detection in NPA is less frequent. 2. TTV could be a potential early biomarker of survival after pediatric HSCT. 3. TTV correlates with less NK citotoxicity before and early after HSCT. 4. TTV VL correlates with slower IR at day 90 after HSCT.

The diagnostic yield of nasopharyngeal aspirate for pediatric pulmonary tuberculosis: a systematic review and meta-analysis

BackgroundTuberculosis (TB) is a leading cause of death in children, but many cases are never diagnosed. Microbiological diagnosis of pulmonary TB is challenging in young children who cannot spontaneously expectorate sputum. Nasopharyngeal aspirates (NPA) may be more easily collected than gastric aspirate and induced sputum and can be obtained on demand, unlike stool. However, further information on its diagnostic yield is needed.MethodsWe systematically reviewed and meta-analyzed the diagnostic yield of one NPA for testing by either culture or nucleic acid amplification testing (NAAT) to detect Mycobacterium tuberculosis from children. We searched three bibliographic databases and two trial registers up to 24th November 2022. Studies that reported the proportion of children diagnosed by NPA compared to a microbiological reference standard (MRS) were eligible. Culture and/or WHO-endorsed NAAT on at least one respiratory specimen served as the MRS. We also estimated the incremental yield of two NPA samples compared to one and summarized operational aspects of NPA collection and processing. Univariate random-effect meta-analyses were performed to calculate pooled diagnostic yield estimates.ResultsFrom 1483 citations, 54 were selected for full-text review, and nine were included. Based on six studies including 256 children with microbiologically confirmed TB, the diagnostic yield of NAAT on one NPA ranged from 31 to 60% (summary estimate 44%, 95% CI 36–51%). From seven studies including 242 children with confirmed TB, the diagnostic yield of culture was 17–88% (summary estimate 58%, 95% CI 42–73%). Testing a second NPA increased the yield by 8–19% for NAAT and 4–35% for culture. NPA collection procedures varied between studies, although most children had NPA successfully obtained (96–100%), with a low rate of indeterminate results (< 5%). Data on NPA acceptability and specifically for children under 5 years were limited.ConclusionsNPA is a suitable and feasible specimen for diagnosing pediatric TB. The high rates of successful collection across different levels of healthcare improve access to microbiological testing, supporting its inclusion in diagnostic algorithms for TB, especially if sampling is repeated. Future research into the acceptability of NPA and how to standardize collection to optimize diagnostic yield is needed.

Epidemiology of acute respiratory viral infections of children with cancer before the COVID-19 pandemic

Children with cancer became a major high-risk group during the COVID-19 pandemic. It has become difficult for patients to reach practical and safe care and treatment all over the world. The late diagnosis of many childhood cancers was driven by limited access to healthcare due to the pandemic and fears of COVID-19 that prevented parents from making an early medical assessment of their child's symptoms. Acute respiratory diseases are an important cause of morbidity and mortality in children with cancer, regardless of the COVID-19 pandemic and were seen less frequently in the pandemic during the lockdown and distant education period in comparison to the time before the pandemic in children with cancer. Acute respiratory viral infections frequently cause febrile neutropenia attacks in children under five years of age. A three year retrospective cross-sectional study was performed in a tertiary care university hospital. Children with cancer who presented to the Department of Pediatric Hematology and Oncology with the diagnosis of acute respiratory viral infection were included in the study if they had available results of multiplex polymerase chain reaction (PCR) of nasopharyngeal aspirate samples. The qualitative detection of 18 respiratory viruses and four bacterias were detected by the real-time multiplex polymerase chain reaction. Sixty-six patients with 93 acute respiratory viral infection were included in this study. Seventy of 93 (75%) samples were positive for at least one pathogen. The most common three viruses were HRV, Influenza H1N1, and Influenza H1N3, respectively. Other than COVID-19, the risk of severe acute respiratory viral infections are always important in children with cancer and during the pandemic, hygiene measures and social restrictions caused a reduction in the number of acute respiratory viral infection. This study is critical because it shows the distribution of agents in children with cancer who had acute viral upper respiratory tract infections in the near term before the pandemic.

Co-Infection Rates between SARS-CoV-2 and RSV in Oropharyngeal, Nasopharyngeal Aspirate and Saliva Samples of COVID-19 Patients, Shiraz, South of Iran.

Determining the prevalence of respiratory viruses' coinfection with coronavirus disease 2019 (COVID-19) is essential to defining its true clinical influence. This study aimed to evaluate co-infection rates between severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) in infected patients in Shiraz, south of Iran. In a cross-sectional descriptive study, oropharyngeal, nasopharyngeal aspirate (NPA), and saliva samples of 50 COVID-19 patients who were referred to Ali-Asghar hospital (Shiraz, Iran) from March to August 2020, were collected. A control group consisted of age and sex-matched healthy participants. The nasopharyngeal and oropharyngeal aspirates were collected by sterile swabs. All cases were hospitalized, and all SARS-CoV-2 patients had a fever and respiratory symptoms. The samples were packed in a vial with 1 mL of transport medium and transported to the Valfagre specialty laboratory, where they were tested for RSV using a real-time polymerase chain reaction (PCR). 100 nasopharyngeal/oropharyngeal aspirates and saliva samples including 50 healthy controls (24 females, 26 males) and 50 COVID-19 patients' samples (27 males and 23 females) were studied. There was no significant difference regarding age as well as gender between both groups (p> 0.05). None of the healthy subjects was infected with RSV; however, 5(10%) patients from COVID-19 group were infected with the RSV virus. Chi-square test did not show a significant difference between RSV infection in COVID-19 patients and healthy subjects. The outcome of present research showed that concurrent RSV with COVID 19 infection might be seen in hospitalized patients in Shiraz Southwest of Iran. For more reliable findings, further research on bigger populations, including more pathogens in several places around the country, and considering the severity of symptoms is required.

Molecular Profiling of Inflammatory Mediators in Human Respiratory Syncytial Virus and Human Bocavirus Infection.

Infections due to human respiratory syncytial virus (HRSV) and human bocavirus (HBoV) can mediate the release of several pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α, which are usually associated with disease severity in children. In this study, the change in the expression profile of cytokines and chemokines were determined during HRSV, HBoV, and HRSV coinfection with HBoV in 75 nasopharyngeal aspirates (NPAs) samples, positive real-time reverse transcriptase PCR Assay (rRT-PCR) for HRSV (n = 36), HBoV (n = 23) infection alone or HRSV coinfection with HBoV (n = 16). The samples were collected from hospitalized children. qPCR-based detection revealed that the levels of IL-6, IL-8, IL-10, IL-13, IL-33, and G-CSF were significantly (p < 0.05) greater in patients than in controls. IL-4, IL-17, GM-CSF, and CCL-5 were significantly elevated in children with HRSV coinfection with HBoV than in other groups (p < 0.05). TNF-α, IL-6, IL-8, IL-10, IL-13, and IL-33 in children with HRSV were significantly increased in severe infections compared to mild infections. Whereas, IL-10, IL-13, and IL-33 were significantly increased in severe infection in compared a mild infection in children with HBoV. Further large-scale investigations involving isolates are needed to enhance our knowledge of the association between viral infections and cytokine expression patterns during the different stages of HRSV and HBoV infection.

Modulation ofAutophagy and mTOR signalling pathway genes in respiratory epithelium by respiratory syncytial virus (RSV) in children suffering from acute lower respiratory tract infections.

The study was planned to carry out the molecular characterization of the Respiratory Syncytial Virus (RSV) circulating strains and to elucidate the gene expression of autophagy and mTOR signalling pathways in children with acute lower respiratory tract infection (ALRTI). Nasopharyngeal aspirate (NPA) samples (n=145) from children suffering from ALRTI were subjected to the detection of RSV. Of them, 31 RSV positive strains were subjected for sequencing. Semi-quantitative gene expression analysis for mTOR signalling and autophagy pathway genes was performed in respiratory tract epithelial cells using 25 RSV positive cases, and 10 age and sex matched healthy control subjects. Five representative genes were selected for each pathway and subjected to SYBR green real-time polymerase chain reaction (PCR). RSV was positive in 69 (47.6%) samples and the representative (n=31) RSV strains belonged to RSV-A. Thirty-one strains of RSV-A on phylogenetic analysis clustered with the novel ON1 genotype having 72bp nucleotide duplicationby targeting the ecto-domain portion of the G gene. Further, the stains belonged to lineage 1 (51.6%), followed by lineage 3 (29%) and lineage 2 (19.4%). Autophagy gene expression analysis revealed significant upregulation in NPC1 and ATG3 autophagy genes. mTOR, AKT1, and TSC1 genes of the mTOR pathway were significantly down-regulated in RSV positive patients. Thus, RSV infection inducing autophagy pathway genes (NPC1 and ATG3) and suppressing mTOR signalling pathway genes (AKT1, mTOR, and TSC1) to possibly evade the host immune system through dysregulating these pathways for its way of survival within the host. This article is protected by copyright. All rights reserved.

New, fast, and precise method of COVID-19 detection in nasopharyngeal and tracheal aspirate samples combining optical spectroscopy and machine learning.

Fast, precise, and low-cost diagnostic testing to identify persons infected with SARS-CoV-2 virus is pivotal to control the global pandemic of COVID-19 that began in late 2019. The gold standard method of diagnostic recommended is the RT-qPCR test. However, this method is not universally available, and is time-consuming and requires specialized personnel, as well as sophisticated laboratories. Currently, machine learning is a useful predictive tool for biomedical applications, being able to classify data from diverse nature. Relying on the artificial intelligence learning process, spectroscopic data from nasopharyngeal swab and tracheal aspirate samples can be used to leverage characteristic patterns and nuances in healthy and infected body fluids, which allows to identify infection regardless of symptoms or any other clinical or laboratorial tests. Hence, when new measurements are performed on samples of unknown status and the corresponding data is submitted to such an algorithm, it will be possible to predict whether the source individual is infected or not. This work presents a new methodology for rapid and precise label-free diagnosing of SARS-CoV-2 infection in clinical samples, which combines spectroscopic data acquisition and analysis via artificial intelligence algorithms. Our results show an accuracy of 85% for detection of SARS-CoV-2 in nasopharyngeal swab samples collected from asymptomatic patients or with mild symptoms, as well as an accuracy of 97% in tracheal aspirate samples collected from critically ill COVID-19 patients under mechanical ventilation. Moreover, the acquisition and processing of the information is fast, simple, and cheaper than traditional approaches, suggesting this methodology as a promising tool for biomedical diagnosis vis-à-vis the emerging and re-emerging viral SARS-CoV-2 variant threats in the future.

Naso-pharyngeal carriage and antimicrobial susceptibility of Streptococcus pneumoniae in community-acquired pneumonia in children

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia (CAP) in children. Recently, the rapid emergence of antibiotic-resistant pneumococci has posed enormous challenges for CAP treatment and public health. This study aims to provide clinicians with updated data about the antimicrobial susceptibility of S. pneumoniae and improve the treatment guidelines in CAP in children. This cross-sectional and in-vitro study was conducted at three hospitals in Quang Nam province and Da Nang city in Vietnam. Pneumococcal strains were isolated from nasopharyngeal aspirate samples of 360 CAP in-patients under five years of age. The susceptibility of clinically used antibiotics was investigated using the disk diffusion test and the E-test for identifying the MIC. Multi-drug resistant (MDR) S. pneumoniae isolates were also determined. 21.9% of CAP children patients were pneumococcal carriage. The susceptibility testing demonstrated that all 56 tested pneumococcal isolates were resistant to erythromycin and azithromycin, whereas none of the isolates developed the resistance to levofloxacin, vancomycin, and linezolid. The clindamycin- and cotrimoxazole-resistant pneumococci rates were 96.4% and 87.5%, respectively. For β-lactam antibiotics, the resistance proportions of pneumococcal isolates to penicillin G, co-amoxiclav, amoxicillin, cefotaxime, and ceftriaxone were 3.6%,1.8%, 1.8%, 1.8%, and 3.6%, respectively. 83.9% of the tested isolates were MDR S. pneumoniae. Our data support using penicillin G, amoxicillin, co-amoxiclav, or cefotaxime as the first-line therapy for uncomplicated-pneumococci-induced CAP in Quang Nam – Da Nang, while vancomycin, linezolid, and levofloxacin should be used as alternatives or in MDR cases.

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.

Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization. Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples. The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness. Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

122. Streptococcus pneumoniae Nasopharyngeal Colonization in a Colombian Cohort of Patients with Comorbid Conditions

Abstract Background Community-acquired pneumonia (CAP) remains the first cause of infectious death and morbidity worldwide. Nevertheless, its etiological pathogen is only isolated in almost 30% of cases, and its most representative bacteria is S. pneumoniae. Some researchers have proposed that the nasopharyngeal colonization by S. pneumoniae could risk developing CAP. However, the prevalence of nasopharyngeal colonization in patients with chronic comorbid conditions in Latin America is unknown, where vaccination rates are low. This study aims to determine the rate of S. pneumoniae colonization in a Colombian cohort. Figure 1.Pneumococcal vaccination and colonization in the whole cohort. Methods This is a prospective cohort study in 3 centers in Colombia carried out between 2020 and 2022. According to the world health organization guidelines, the nasopharyngeal aspirate (NPA) samples from each participant were obtained. Samples were incubated with 5% CO2 in blood agar, and MALDI-TOFF identified the colonies obtained. We compared the number of S. pneumoniae isolated and stratified by anti-pneumococcal vaccination status. Results The cohort was composed of 810 patients. The median [IQR] age was 63 [53-62] and the most common comorbidities were 52.2% [423/810] arterial hypertension, 21.7% [176/810] coronary disease, 19.0% [154/810] congestive heart failure, and 17.4% [141/810] chronic kidney disease. Nasopharyngeal colonization by S. pneumonie was only documented in the patients' 2.1% [15/723] (Figure 1). Moreover, 10.7% [87/810] patients from the cohort were vaccinated against S. pneumoniae, and none were colonized. Conclusion Nasopharyngeal colonization by S. pneumoniae in our cohort of adults with chronic comorbidities was low. Notably, all the colonized patients were not vaccinated, at higher risk of developing the invasive pneumococcal disease. More robust vaccination policies should be implemented for adults in Colombia. Disclosures All Authors: No reported disclosures.

Effect of systematic tuberculosis detection on mortality in young children with severe pneumonia in countries with high incidence of tuberculosis: a stepped-wedge cluster-randomised trial

Tuberculosis diagnosis might be delayed or missed in children with severe pneumonia because this diagnosis is usually only considered in cases of prolonged symptoms or antibiotic failure. Systematic tuberculosis detection at hospital admission could increase case detection and reduce mortality. We did a stepped-wedge cluster-randomised trial in 16 hospitals from six countries (Cambodia, Cameroon, Côte d'Ivoire, Mozambique, Uganda, and Zambia) with high incidence of tuberculosis. Children younger than 5 years with WHO-defined severe pneumonia received either the standard of care (control group) or standard of care plus Xpert MTB/RIF Ultra (Xpert Ultra; Cepheid, Sunnyvale, CA, USA) on nasopharyngeal aspirate and stool samples (intervention group). Clusters (hospitals) were progressively switched from control to intervention at 5-week intervals, using a computer-generated random sequence, stratified on incidence rate of tuberculosis at country level, and masked to teams until 5 weeks before switch. We assessed the effect of the intervention on primary (12-week all-cause mortality) and secondary (including tuberculosis diagnosis) outcomes, using generalised linear mixed models. The primary analysis was by intention to treat. We described outcomes in children with severe acute malnutrition in a post hoc analysis. This study is registered with ClinicalTrials.gov (NCT03831906) and the Pan African Clinical Trial Registry (PACTR202101615120643). From March 21, 2019, to March 30, 2021, we enrolled 1401 children in the control group and 1169 children in the intervention group. In the intervention group, 1140 (97·5%) children had nasopharyngeal aspirates and 942 (80·6%) had their stool collected; 24 (2·1%) had positive Xpert Ultra. At 12 weeks, 110 (7·9%) children in the control group and 91 (7·8%) children in the intervention group had died (adjusted odds ratio [OR] 0·986, 95% CI 0·597-1·630, p=0·957), and 74 (5·3%) children in the control group and 88 (7·5%) children in the intervention group had tuberculosis diagnosed (adjusted OR 1·238, 95% CI 0·696-2·202, p=0·467). In children with severe acute malnutrition, 57 (23·8%) of 240 children in the control group and 53 (17·8%) of 297 children in the intervention group died, and 36 (15·0%) of 240 children in the control group and 56 (18·9%) of 297 children in the intervention group were diagnosed with tuberculosis. The main adverse events associated with nasopharyngeal aspirates were samples with blood in 312 (27·3%) of 1147 children with nasopharyngeal aspirates attempted, dyspnoea or SpO2 less than 95% in 134 (11·4%) of children, and transient respiratory distress or SpO2 less than 90% in 59 (5·2%) children. There was no serious adverse event related to nasopharyngeal aspirates reported during the trial. Systematic molecular tuberculosis detection at hospital admission did not reduce mortality in children with severe pneumonia. High treatment and microbiological confirmation rates support more systematic use of Xpert Ultra in this group, notably in children with severe acute malnutrition. Unitaid and L'Initiative. For the French translation of the abstract see Supplementary Materials section.

Prevalence, pattern of distribution and characterization of respiratory syncytial virus associated acute respiratory tract infections in hospitalized children less than 5 years in a general hospital in Sri Lanka from 2016–2018

Respiratory Syncytial Virus (RSV) is one of the most common respiratory viruses causing acute respiratory tract infections (ARTI) in children. Detailed data on RSV infections including the RSV types circulating in Sri Lanka are not available. This study aimed to determine the prevalence, patterns and characterization of RSV associated ARTI in hospitalized children less than 5 years in a general hospital in Sri Lanka. We tested 500 nasopharyngeal aspirate (NPA) samples collected from children with suspected viral ARTI from May 2016 to July 2018 from Kegalle General Hospital, Sri Lanka for RSV using antigen detection by an immunofluorescence assay (IFA). RSV positive samples were further characterized using the real time RT-PCR. RSV was the predominant virus associated with ARTI with a prevalence of 28% (140/500) in the study sample. RSV in was also detected in more co-infections with other respiratory viruses. RSV was detected throughout the year with peak periods from June to August 2016, March to July 2017 and May to July 2018. Of the 140 RSV positive children tested, 72.14% had RSV-B, while 27.86% had RSV-A infection. Both RSV subtypes were detected throughout the study period with overlapping patterns. A few co-infections between RSV-A and RSV-B were detected during the co-circulation. RSV was the most prevalent virus and RSV-B was the predominant subgroup associated with ARTI in the children <5 years in Sri Lanka from May 2016 to July 2018. RSV was detected throughout the study period with peaks in certain months in the study area.

Clinical characteristics of hospitalized term and preterm infants with community-acquired viral pneumonia

BackgroundPneumonia is a serious problem that threatens the health of newborns. This study aimed to investigate the clinical characteristics of hospitalized term and preterm infants with community-acquired viral pneumonia.MethodsThis was a retrospective analysis of cases of community-acquired viral pneumonia in the Neonatal Department. Nasopharyngeal aspirate (NPA) samples were collected for pathogen detection, and clinical data were collected. We analysed pathogenic species and clinical characteristics among these infants.ResultsRSV is the main virus in term infants, and parainfluenza virus (PIV) 3 is the main virus in preterm infants. Patients infected with PIV3 were more susceptible to coinfection with bacteria than those with respiratory syncytial virus (RSV) infection (p < 0.05). Preterm infants infected with PIV3 were more likely to be coinfected with bacteria than term infants (p < 0.05), mainly gram-negative bacteria (especially Klebsiella pneumonia). Term infants with bacterial infection were more prone to fever, cyanosis, moist rales, three concave signs, elevated C-reactive protein (CRP) levels, respiratory failure and the need for higher level of oxygen support and mechanical ventilation than those with simple viral infection (p < 0.05). The incidence of hyponatremia in neonatal community-acquired pneumonia (CAP) was high.ConclusionsRSV and PIV3 were the leading causes of neonatal viral CAP. PIV3 infection is the main cause of viral CAP in preterm infants, and these individuals are more likely to be coinfected with bacteria than term infants, mainly gram-negative bacteria. Term infants with CAP coinfected with bacteria were more likely to have greater disease severity than those with single viral infections.

Simian virus 40 DNA in immunocompetent children with respiratory disease.

Evidence of Simian virus 40 (SV40) DNA sequences or gene products has been reported in a variety of organ systems in humans. However, the route of transmission and the significance of SV40 polyomavirus infection in human are unknown. The aim of study was to characterize the frequency of SV40 infection in immunocompetent and immunocompromised patients with respiratory diseases. Respiratory specimens from patients with respiratory tract illness obtained from nasopharyngeal aspirates (n = 280) were screened for SV40 polyomavirus using real-time PCR; coinfection with other viruses was examined. Positive results were confirmed with sequencing. Of the 280 samples analysed, 2 (0.71%) were positive for SV40. SV40 was identified in nasopharyngeal aspirate samples from children aged 8 and 14 months who were immunocompetent. Both patients had upper or lower respiratory tract infection. Coinfections with other viruses were found in 50% of the SV40 positive samples. The data suggest that SV40 can infect respiratory tract, that respiratory tract may represent a route of transmission or a site for virus persistence, and that with the high rate of co-infection, SV40 may not involved in respiratory diseases.

Respiratory Viral Infections in Pediatric Acute Leukemia Patients Presenting with Febrile Neutropenia in a Tertiary Hospital in Ankara, Turkey

Background: The prevalence and roles of respiratory viral pathogens in pediatric acute leukemia patients with febrile neutropenia is not well understood and laboratory tests to detect viral agents are not a routine practice in the investigation of these patients.Patients and methods: The medical records of 50 neutropenic episodes in pediatric acute leukemia patients in the Hacettepe University Children’s hospital, Ankara-Turkey, were reviewed. Blood samples were obtained for blood culture, aspergillus antigen and other routine tests. Nasopharyngeal aspirate samples were collected and transferred to the laboratory in a viral transport medium. Therapy for febrile neutropenia was initiated according to our institution’s protocols.Results: Fifty (50) consecutive febrile neutropenic episodes in 44 pediatric ALL and AML patients were included in the study between 1st October 2009 and 31st August 2010. Microbiologically documented infections were found in 36% of the episodes, clinically documented infections in 16% of the episodes and 48% of the episodes were accepted as Fever of Unknown Origin. Twenty-two percent (22%) of the microbiologically documented infections were due to viral agents, 56 % were due to Gram positive bacteria, 21% were due to Gram negative bacteria (E. coli) and only one episode of fungemia was documented.Conclusions: Fever of Unknown Origin constituted nearly 50% of the febrile neutropenic episodes in this study despite the availability of advanced laboratory diagnostic methods. Among the episodes with microbiologically documented infections, bacterial pathogens were especially common. Presenting complaints like cough and rhinorrhea are not specific to a viral etiology and care should be taken not to miss potentially threatening bacterial pathogens in such episodes.

Empirical treatment against cytomegalovirus and tuberculosis in HIV-infected infants with severe pneumonia: study protocol for a multicenter, open-label randomized controlled clinical trial

BackgroundPneumonia is the primary cause of death among HIV-infected children in Africa, with mortality rates as high as 35–40% in infants hospitalized with severe pneumonia. Bacterial pathogens and Pneumocystis jirovecii are well known causes of pneumonia-related death, but other important causes such as cytomegalovirus (CMV) and tuberculosis (TB) remain under-recognized and undertreated.The immune response elicited by CMV may be associated with the risk of developing TB and TB disease progression, and CMV may accelerate disease caused both by HIV and TB. Minimally invasive autopsies confirm that CMV and TB are unrecognized causes of death in children with HIV. CMV and TB may also co-infect the same child.The aim of this study is to compare the impact on 15-day and 1-year mortality of empirical treatment against TB and CMV plus standard of care (SoC) versus SoC in HIV-infected infants with severe pneumonia.MethodsThis is a Phase II-III, open-label randomized factorial (2 × 2) clinical trial, conducted in six African countries. The trial has four arms. Infants from 28 to 365 days of age HIV-infected and hospitalized with severe pneumonia will be randomized (1:1:1:1) to (i) SoC, (ii) valganciclovir, (iii) TB-T, and (iv) TB-T plus valganciclovir. The primary endpoint of the study is all-cause mortality, focusing on the short-term (up to 15 days) and long-term (up to 1 year) mortality. Secondary endpoints include repeat hospitalization, duration of oxygen therapy during initial admission, severe and notable adverse events, adverse reactions, CMV and TB prevalence at enrolment, TB incidence, CMV viral load reduction, and evaluation of diagnostic tests such as GeneXpert Ultra on fecal and nasopharyngeal aspirate samples and urine TB-LAM.DiscussionGiven the challenges in diagnosing CMV and TB in children and results from previous autopsy studies that show high rates of poly-infection in HIV-infected infants with respiratory disease, this study aims to evaluate a new approach including empirical treatment of CMV and TB for this patient population. The potential downsides of empirical treatment of these conditions include toxicity and medication interactions, which will be evaluated with pharmacokinetics sub-studies.Trial registrationClinicalTrials.gov, NCT03915366, Universal Trial Number U111-1231-4736, Pan African Clinical Trial Registry PACTR201994797961340.

Herpesvirus infections in adenoids in patients with chronic adenotonsillar disease.

Adenoids and tonsils have gained interest as a new in vivo model to study local immune functions and virus reservoirs. Especially herpesviruses are interesting because their prevalence and persistence in local lymphoid tissue are incompletely known. Our aim was to study herpesvirus and common respiratory virus infections in nonacutely ill adenotonsillar surgery patients. Adenoid and/or palatine tonsil tissue and nasopharyngeal aspirate (NPA) samples were collected from elective adenoidectomy (n = 45) and adenotonsillectomy (n = 44) patients (median age: 5, range: 1–20). Real‐time polymerase chain reaction was used to detect 22 distinct viruses from collected samples. The overall prevalence of herpesviruses was 89% and respiratory viruses 94%. Human herpesviruses 6 (HHV6), 7 (HHV7), and Epstein–Barr virus (EBV) were found, respectively, in adenoids (33%, 26%, 25%), tonsils (45%, 52%, 23%), and NPA (46%, 38%, 25%). Copy numbers of the HHV6 and HHV7 genome were significantly higher in tonsils than in adenoids. Patients with intra‐adenoid HHV6 were younger than those without. Detection rates of EBV and HHV7 showed agreement between corresponding sample types. This study shows that adenoid and tonsil tissues commonly harbor human herpes‐ and respiratory viruses, and it shows the differences in virus findings between sample types.

Decreased Pediatric Viral Burden and Increased Rhinovirus Infection During The COVID-19 Pandemic

Objective: Primary preventive nonpharmaceutical interventions were introduced to reduce viral transmission and disease spread at the beginning of the COVID-19 pandemic. Therefore, herein, we aimed to determine and assess the impact of the nonpharmaceutical interventions on bronchiolitis and varicella infection rates in the pediatric population during the pandemic compared to the previous four years. We also aimed to evaluate which viruses cause viral respiratory tract infections during the pandemic period. Material and Methods: Diagnosis and laboratory data of the patients who were one month to 18 years of age were retrospectively retrieved from hospital records. The distribution of the number of patients with bronchiolitis and chickenpox diagnoses was shown monthly between January 2016 and December 2020. Viral agents detected by polymerase chain reaction (PCR) in the nasopharyngeal aspirate samples obtained at the first application of the patients during the pandemic period were investigated. Results: The data of 2.254.877 pediatric patients admitted to our hospital from January 2016 to December 2020 were examined. There were 38.458 bronchiolitis and 954 chickenpox cases reported both as inpatients and outpatients. There was a 85.6% decrease in the rate of bronchiolitis compared to previous years, and chickenpox peak was not observed in the pandemic period. Rhinovirus was found to be the most common etiologic agent of bronchiolitis during the pandemic period and Respiratory Syncytial Virus (RSV) came second. A significant decrease in the frequency of influenza was also observed. Conclusion: Our study reveals that the measures which curtail social life and prioritize social distancing prevent the spread of viral infections. It has also shown that there is an increase in the frequency of Rhinovirus infection during the pandemic period.