Design of a cost-effective diagnosis tool for SARSCov-2 variant detection through a next generation sequencing (NGS) based strategy

Background/Objectives: Current pandemic situation, together with the continuous emergence of new SARS-CoV-2 variants reveal the need to develop a more versatile tool than PCR-based methods that allows both high throughput COVID-19 diagnostic and specific variant detection at reduced cost and fast turnaround times. Thus, with the aim of overcoming current test limitations and providing a strategy with these characteristics arises our novel next generation sequencing based approach. Method(s): The developed strategy works with RNA samples obtained from nasopharyngeal swabs. RNA samples are processed with our custom laboratory protocol and can be sequenced with any Illumina platform to generate results within a 24h timeframe. A tailored bioinformatic pipeline analyzes the data and generates a clinical-level report. Result(s): Clinical validation results have shown that the designed solution, sensitively and specifically identifies negative and positive samples that display a broad range in viral loads and readily identifies the following major SARS-CoV-2 variants of concern (VoC): Alpha, Beta, Gamma, Delta, Lambda and Omicron (BA.1 and BA.2). Conclusion(s): The versatility of our solution allows the capability of identifying the presence of other common respiratory viruses as well as identifying patients at risk through the identification of susceptibility human variants in the host. This, together with the possibility of easily adding new VoC as they emerge, will make VoC monitoring in entire populations feasible, providing a new perspective on the application of NGS methods in the field of clinical microbiology.

Burden of respiratory coinfections in haematology patients with COVID-19

Introduction: Following the lifting of generalised restrictions and universal masking, severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2)- infected patients, especially the clinically extremely vulnerable (CEV) haematology patients, are at an increased risk for other respiratory viral coinfections;therefore, physicians need to be cognizant about excluding other treatable respiratory pathogens. Here, we report coinfection with SARS-CoV- 2 and other respiratory pathogens in patients with haematological cancers presenting to a large tertiary care hospital. Method(s): From July 2022-December 2022, patients with haematological disorders were screened for SARS-CoV- 2 and other 10 common respiratory pathogens by PCR. We performed a retrospective analysis of patients with concurrent respiratory viruses and will prospectively evaluate the same from Jan 2023 to March 2023. Result(s): During this period a total of 322 inpatients had routine screening and additional 6213 swabs were done in the outpatient/ambulatory setting, of which 294 were positive in 221 patients. We excluded all patients who had a single positive PCR swab result and specifically analysed only patients with coinfections. We identified 30 patients (14%) who had respiratory coinfections with 73 viral infections/reactivations over 6 months period, which represented 25% of all positive swabs: 25 inpatients (19 symptomatic/6 asymptomatic) and 48 in outpatients (32 symptomatic/16 asymptomatic). The median age of the cohort was 47.3 years (21-77). Patients were post allograft (n = 15), autograft (n = 7), post CART (n = 5) and postchemotherapy (n = 4). Of the 30 cases, 13 patients had concurrent infections: 5 SARS-CoV2, 10 Respiratory syncytial virus (RSV), 7 Rhino and 4 Influenza A, with all patients having dual viral infection. The remaining 17 patients had multiple viral infections but separated by a median of 54 days (range 27-137 days): 16 SARS-CoV2, 5 RSV, 6 Rhino, 2 Parainfluenza, 2 Adeno and one each of Influenza A, Influenza B, and metapneumovirus. Of the treatable infections (n = 46), 22% were detected on routine asymptomatic swabbing, with 50% of SARS-CoV2 detected on routine swabs. All 8 patients with Influenza were treated with oseltamivir, of 16 RSV cases one was treated with oral ribavirin and of the 22 SARS-CoV2 patients, 5 were treated (4 Paxlovid and 1 Remdesivir). No patients needed intensive care support and no deaths were reported. Conclusion(s): The burden of respiratory coinfections in CEV cohort has a significant impact on respiratory isolation and management, including appropriate & timely initiation of therapy for treatable viral infections. Although mortality was not increased secondary to respiratory coinfections and none needed intensive care, larger prospective cohorts are needed to assess the exact impact.

Unexpected Severe Bocavirus Infections among Hospitalized Children during the COVID-19 Pandemic

Objective: Acute respiratory tract infections are one of the leading causes of morbidity and mortality in children. Although human bocavirus (HBoV) infections are not as common as other seasonal respiratory viruses, children who are infected with HBoV are more likely to suffer from a variety of respiratory conditions, including the common cold, acute otitis media, asthma exacerbations, bronchiolitis pneumonia, some of the affected children require pediatric intensive care unit stay. Here, we aimed to evaluate pediatric bocavirus (HBoV) cases presenting with severe respiratory tract symptoms during the coronavirus disease 2019 (COVID-19) pandemic. Method(s): This retrospective study evaluated the medical records of children diagnosed with respiratory infections, followed up at the Faculty of Medicine, Eskisehir Osmangazi University between September 2021 and March 2022. In this study, patients with HBoV identified using nasopharyngeal polymerase chain reaction (PCR) were considered positive. Cases were analyzed retrospectively for their clinical characteristics. Result(s): This study included 54 children (29 girls and 25 boys) with HBoV in nasopharyngeal PCR samples. The cases ranged in age from 1 month to 72 months (median 25 months). At the time of presentation, cough, fever, and respiratory distress were the most prevalent symptoms. Hyperinflation (48%), pneumonic consolidation (42%), and pneumothorax-pneumomediastinum (7%) were observed on the chest X-ray;54% of the children required intensive care unit stay. The median length of hospitalization was 6 days. Bacterial coinfection was detected in 7 (17%) children, while HBoV and other viruses were present in 20 (37%) children;57% of children received supplemental oxygen by mask, 24% high-flow nasal oxygen, 7% continuous positive airway pressure, and 9% invasive mechanical ventilation support. Antibiotics were given to 34 (63%) cases, and systemic steroid treatment was given to 41 (76%) cases. Chest tubes were inserted in three out of the four cases with pneumothorax-pneumomediastinum. All patients were recovered and were discharged from the hospital. Conclusion(s): The COVID-19 pandemic changed the epidemiology of seasonal respiratory viruses and the clinical course of the diseases. Although it usually causes mild symptoms, severe respiratory symptoms can lead to life-threatening illnesses requiring intensive care admission.Copyright © 2023. The Author(s).

Pulmonary influenza infection promotes the awakening of dormant metastatic breast cancer cells

Breast cancer is the most common form of cancer and the second cancer-causing death in females. Although remission rates are high if detected early, survival rates drop substantially when breast cancer becomes metastatic. The most common sites of metastatic breast cancer are bone, liver and lung. Respiratory viral infections inflict illnesses on countless people. The latest pandemic caused by the respiratory virus, SARS-CoV-2, has infected more than 600 million worldwide, with documented COVID-related death upward of 1 million in the United States alone. Respiratory viral infections result in increased inflammation with immune cell influx and expansion to facilitate viral clearance. Prior studies have shown that inflammation, including through neutrophils, can contribute to dormant cancer cells reawakening and outgrowth. Moreover, inhibition of IL6 has been shown to decrease breast cancer lung metastasis in mouse models. However, how respiratory viral infections contribute to breast cancer lung metastasis remains to be unraveled. Using MMTV/PyMT and MMTV/NEU mouse models of breast cancer lung metastasis and influenza A virus as a model respiratory virus, we demonstrated that acute influenza infection and the accompanying inflammation and immune cell influx awakens and dramatically increased proliferation and expansion of dormant disseminated cancer cells (DCC) in the lungs. Acute influenza infection leads to immune influx and expansion, including neutrophils and macrophages, with increased proportion of MHCII+ macrophages in early time points, and a sustained decrease in CD206+ macrophages starting 6 days post-infection until 28 days after the initial infection. Additionally, we observed a sustained accumulation of CD4+ T cells around expanding tumor cells for as long as 28 days after the infection. Notably, neutrophil depletion or IL6 knockout reversed the flu-induced dormant cell expansion in the lung. Finally, awakened DCC exhibited downregulation of vimentin immunoreactivity, suggesting a role for phenotypic plasticity in DCC outgrowth following viral infection. In conclusion, we show that respiratory viral infections awaken and increase proliferation of dormant breast cancer cells in the lung, and that depletion of neutrophils or blocking IL6 reverses influenza-induced dormant cell awakening and proliferation.

Validating saliva as a biological sample for cost-effective, rapid and routine screening for SARS-CoV-2

Purpose: Compared to nasopharyngeal/oropharyngeal swabs (N/OPS-VTM), non-invasive saliva samples have enormous potential for scalability and routine population screening of SARS-CoV-2. In this study, we investigate the efficacy of saliva samples relative to N/OPS-VTM for use as a direct source for RT-PCR based SARS-CoV-2 detection. Method(s): We collected paired nasopharyngeal/oropharyngeal swabs and saliva samples from suspected positive SARS-CoV-2 patients and tested using RT-PCR. We used generalized linear models to investigate factors that explain result agreement. Further, we used simulations to evaluate the effectiveness of saliva-based screening in restricting the spread of infection in a large campus such as an educational institution. Result(s): We observed a 75.4% agreement between saliva and N/OPS-VTM, that increased drastically to 83% in samples stored for less than three days. Such samples processed within two days of collection showed 74.5% test sensitivity. Our simulations suggest that a test with 75% sensitivity, but high daily capacity can be very effective in limiting the size of infection clusters in a workspace. Guided by these results, we successfully implemented a saliva-based screening in the Bangalore Life Sciences Cluster (BLiSC) campus. Conclusion(s): These results suggest that saliva may be a viable alternate source for SARS-CoV-2 surveillance if samples are processed immediately. Although saliva shows slightly lower sensitivity levels when compared to N/OPS-VTM, saliva collection is logistically advantageous. We strongly recommend the implementation of saliva-based screening strategies for large workplaces and in schools, as well as for population-level screening and routine surveillance as we learn to live with the SARS-CoV-2 virus.Copyright © 2023 Indian Association of Medical Microbiologists

Respiratory syncytial virus outbreak DURING THE COVID-19 PANDEMIC. How has it changed?

INTRODUCTION: The epidemiology of respiratory syncytial virus (RSV) infection has changed during the COVID-19 pandemic. Our objectives were to describe the RSV epidemic in 2021 and compare it with the previous years to the pandemic. METHODS: Retrospective study performed in Madrid (Spain) in a large paediatric hospital comparing the epidemiology and clinical data of RSV admissions during 2021 and the two previous seasons. RESULTS: 899 children were admitted for RSV infection during the study period. During 2021, the outbreak peaked in June and the last cases were identified in July. Previous seasons were detected in autumn-winter. The number of admissions in 2021 was significantly lower than in previous seasons. There were no differences between seasons regarding age, sex or disease severity. CONCLUSION: RSV hospitalizations during 2021 in Spain moved to summer with no cases in autumn and winter 2020-2021. Unlike other countries, clinical data were similar between epidemics.

Comprehensive surveillance of acute respiratory infections during the COVID-19 pandemic: a methodological approach using sentinel networks, Castilla y León, Spain, January 2020 to May 2022.

BackgroundSince 1996, epidemiological surveillance of acute respiratory infections (ARI) in Spain has been limited to seasonal influenza, respiratory syncytial virus (RSV) and potential pandemic viruses. The COVID-19 pandemic provides opportunities to adapt existing systems for extended surveillance to capture a broader range of ARI.AimTo describe how the Influenza Sentinel Surveillance System of Castilla y León, Spain was rapidly adapted in 2020 to comprehensive sentinel surveillance for ARI, including influenza and COVID-19.MethodsUsing principles and methods of the health sentinel network, we integrated electronic medical record data from 68 basic surveillance units, covering 2.6% of the regional population between January 2020 to May 2022. We tested sentinel and non-sentinel samples sent weekly to the laboratory network for SARS-CoV-2, influenza viruses and other respiratory pathogens. The moving epidemic method (MEM) was used to calculate epidemic thresholds.ResultsARI incidence was estimated at 18,942 cases per 100,000 in 2020/21 and 45,223 in 2021/22, with similar seasonal fold increases by type of respiratory disease. Incidence of influenza-like illness was negligible in 2020/21 but a 5-week epidemic was detected by MEM in 2021/22. Epidemic thresholds for ARI and COVID-19 were estimated at 459.4 and 191.3 cases per 100,000 population, respectively. More than 5,000 samples were tested against a panel of respiratory viruses in 2021/22.ConclusionExtracting data from electronic medical records reported by trained professionals, combined with a standardised microbiological information system, is a feasible and useful method to adapt influenza sentinel reports to comprehensive ARI surveillance in the post-COVID-19 era.

Viral infection in chronic otitis media with effusion in children.

Background: The role of respiratory viruses in chronic otitis media with effusion (COME) in children is not clearly defined. In our study we aimed to investigate the detection of respiratory viruses in middle ear effusions (MEE) as well as the association with local bacteria, respiratory viruses in the nasopharynx and cellular immune response of children with COME. Methods: This 2017-2019 cross-sectional study included 69 children aged 2-6 undergoing myringotomy for COME. MEE and nasopharyngeal swabs were analyzed via PCR and CT-values for the genome and loads of typical respiratory viruses. Immune cell populations and exhaustion markers in MEE related to respiratory virus detection were studied via FACS. Clinical data including the BMI was correlated. Results: Respiratory viruses were detected in MEE of 44 children (64%). Rhinovirus (43%), Parainfluenzavirus (26%) and Bocavirus (10%) were detected most frequently. Average Ct values were 33.6 and 33.5 in MEE and nasopharynx, respectively. Higher detection rates correlated with elevated BMI. Monocytes were elevated in MEE (9.5 ± 7.3%/blood leucocytes). Exhaustion markers were elevated on CD4+ and CD8+ T cells and monocytes in MEE. Conclusion: Respiratory viruses are associated with pediatric COME. Elevated BMI was associated with increased rates of virus associated COME. Changes in cell proportions of innate immunity and expression of exhaustion markers may be related to chronic viral infection.

Comparative Analysis of Antimicrobial Antibodies between Mild and Severe COVID-19.

Patients with 2019 coronavirus disease (COVID-19) exhibit a broad spectrum of clinical presentations. A person's antimicrobial antibody profile, as partially shaped by past infection or vaccination, can reflect the immune system health that is critical to control and resolve the infection. We performed an explorative immunoproteomics study using microbial protein arrays displaying 318 full-length antigens from 77 viruses and 3 bacteria. We compared antimicrobial antibody profiles between 135 patients with mild COVID-19 disease and 215 patients with severe disease in 3 independent cohorts from Mexico and Italy. Severe disease patients were older with higher prevalence of comorbidities. We confirmed that severe disease patients elicited a stronger anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) response. We showed that antibodies against HCoV-229E and HcoV-NL63 but not against HcoV-HKU1 and HcoV-OC43 were also higher in those who had severe disease. We revealed that for a set of IgG and IgA antibodies targeting coronaviruses, herpesviruses, and other respiratory viruses, a subgroup of patients with the highest reactivity levels had a greater incidence of severe disease compared to those with mild disease across all three cohorts. On the contrary, fewer antibodies showed consistent greater prevalence in mild disease in all 3 cohorts. IMPORTANCE The clinical presentations of COVID-19 range from asymptomatic to critical illness that may lead to intensive care or even death. The health of the immune system, as partially shaped by past infections or vaccinations, is critical to control and resolve the infection. Using an innovative protein array platform, we surveyed antibodies against hundreds of full-length microbial antigens from 80 different viruses and bacteria in COVID-19 patients from different geographic regions with mild or severe disease. We not only confirmed the association of severe COVID-19 disease with higher reactivity of antibody responses to SARS-CoV-2 but also uncovered known and novel associations with antibody responses against herpesviruses and other respiratory viruses. Our study represents a significant step forward in understanding the factors contributing to COVID-19 disease severity. We also demonstrate the power of comprehensive antimicrobial antibody profiling in deciphering risk factors for severe COVID-19. We anticipate that our approach will have broad applications in infectious diseases.

The Effect of Respiratory Viral Infections on Breakthrough Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis and thrombosis and is associated with significant morbidity and mortality. Although complement inhibitors have significantly changed the outcomes in PNH patients, breakthrough hemolysis (BTH) may still occur as a response to stress factors such as pregnancy, surgery, and infections. Despite the well-described association between bacterial infections and hemolysis in PNH patients, little is known about the effect of respiratory viruses on triggering hemolytic episodes. This is the first study, to our knowledge, addressing this question. We retrospectively analyzed 34 patients with PNH disease between 2016 and 2018, who were on eculizumab treatment and who presented with respiratory symptoms and were subsequently tested for 10 respiratory viruses (influenza A, influenza B, parainfluenza, respiratory syncytial virus, adenovirus, rhinovirus, and human metapneumovirus). NTS+ patients had higher inflammatory markers, with the majority requiring antibiotics. Acute hemolysis, along with a significant drop in hemoglobin, was noted in the NTS+ group, with three of them requiring a top-up transfusion and two requiring an extra dose of eculizumab. Furthermore, the time from the last eculizumab dose was longer in the NTS+ patients who had BTH, than those who did not. Our data indicate that respiratory virus infections pose a significant risk for BTH in PNH patients on complement inhibitor treatment, underlining the need for regular screening and close monitoring of patients with respiratory symptoms. Furthermore, it implies a higher risk for patients who are not established on complement inhibitors, suggesting the necessity for greater vigilance in these patients.

pH-dependent endocytosis mechanisms for influenza A and SARS-coronavirus.

The ongoing SARS-CoV-2 pandemic and the influenza epidemics have revived the interest in understanding how these highly contagious enveloped viruses respond to alterations in the physicochemical properties of their microenvironment. By understanding the mechanisms and conditions by which viruses exploit the pH environment of the host cell during endocytosis, we can gain a better understanding of how they respond to pH-regulated anti-viral therapies but also pH-induced changes in extracellular environments. This review provides a detailed explanation of the pH-dependent viral structural changes preceding and initiating viral disassembly during endocytosis for influenza A (IAV) and SARS coronaviruses. Drawing upon extensive literature from the last few decades and latest research, I analyze and compare the circumstances in which IAV and SARS-coronavirus can undertake endocytotic pathways that are pH-dependent. While there are similarities in the pH-regulated patterns leading to fusion, the mechanisms and pH activation differ. In terms of fusion activity, the measured activation pH values for IAV, across all subtypes and species, vary between approximately 5.0 to 6.0, while SARS-coronavirus necessitates a lower pH of 6.0 or less. The main difference between the pH-dependent endocytic pathways is that the SARS-coronavirus, unlike IAV, require the presence of specific pH-sensitive enzymes (cathepsin L) during endosomal transport. Conversely, the conformational changes in the IAV virus under acidic conditions in endosomes occur due to the specific envelope glycoprotein residues and envelope protein ion channels (viroporins) getting protonated by H+ ions. Despite extensive research over several decades, comprehending the pH-triggered conformational alterations of viruses still poses a significant challenge. The precise mechanisms of protonation mechanisms of certain during endosomal transport for both viruses remain incompletely understood. In absence of evidence, further research is needed.

Respiratory syncytial virus outbreak during the COVID-19 pandemic. How has it changed?

INTRODUCTION: The epidemiology of respiratory syncytial virus (RSV) infection has changed during the COVID-19 pandemic. Our objectives were to describe the RSV epidemic in 2021 and compare it with the previous years to the pandemic. METHODS: Retrospective study performed in Madrid (Spain) in a large pediatric hospital comparing the epidemiology and clinical data of RSV admissions during 2021 and the two previous seasons. RESULTS: 899 children were admitted for RSV infection during the study period. During 2021, the outbreak peaked in June and the last cases were identified in July. Previous seasons were detected in autumn-winter. The number of admissions in 2021 was significantly lower than in previous seasons. There were no differences between seasons regarding age, sex or disease severity. CONCLUSION: RSV hospitalizations during 2021 in Spain moved to summer with no cases in autumn and winter 2020-2021. Unlike other countries, clinical data were similar between epidemics.

Pan-Respiratory Disease Surveillance in Southeast Asia: Past, Present and Future

The Global Influenza Surveillance and Response System (GISRS) was established by WHO in 1952 to conduct surveillance for influenza to inform strain selection for seasonal vaccines and to monitor for influenza pandemics. In 2016 WHO initiated a pilot project to add RSV to influenza surveillance platforms;this was disrupted by the SARS CoV-2 pandemic, and SARS CoV-2 was the first pathogen to be incorporated into influenza sentinel surveillance on a wide scale. This resulted in a "GISRS-plus" surveillance network for influenza and SARS CoV-2 that is now being standardized by WHO. In the wake of the SARS CoV-2 pandemic, there is global interest and funding to support pan-respiratory disease surveillance, which could result in expanding influenza/SARS CoV-2 surveillance platforms to include other pathogens and enhancing event- and indicator-based surveillance. Challenges with expanding sentinel surveillance include overburdening sentinel surveillance systems, reduced number of samples collected and loss of data quality for influenza and SARS CoV-2;thus, other types of surveillance for respiratory diseases might also be considered. This talk describes CDC-supported influenza surveillance platforms in Southeast Asia and recent successes and challenges in adding SARS CoV-2 to this surveillance. It discusses potential risks and benefits to GISRS-plus surveillance created by including other pathogens. Finally, it discusses decision-making steps on which methods to use for collecting data on respiratory viruses.Copyright © 2023

Examining the Association of Air Quality on Covid-19 Patient Outcomes Using Area Level Data from the Electronic Health Record

Background: Within the coronavirus 2019 (COVID-19) pandemic, literature has found worsened patient outcomes and increased virus transmissibility associated with reduced air quality. This factor, a structural social determinant of health (SDOH), has shown great promise as a link between air quality and patient outcomes during the COVID-19 pandemic. Researching SDOH within our patient populations is often difficult and limited by poor documentation or extensive questionnaires or surveys. The use of demographic data derived from the electronic health record (EHR) to more accurately represent SDOH holds great promise. The use of area-level determinants of health outcomes has been shown to serve as a good surrogate for individual exposures. We posit that an area level measure of air quality, the county-level Air Quality Index (AQI), will be associated with disease worsening in intensive care unit (ICU) patients being treated for COVID-19. Method(s): We will calculate AQI using a combination of open-source records available via the United States Environmental Protection Agency (EPA) and manual calculations using geospatial informatics systems (GIS) methods. Subjects will be identified as adult (> 18 years) patients admitted to Vanderbilt University Medical Center's ICUs between January 1, 2020, and March 31, 2022 with a positive SARS-CoV-2 laboratory analysis result. We will exclude patients without a home address listed. Patient demographic and hospital data from ICU admission to 28 days following admission will include: age, sex, home address, race, insurance type, primary language, employment status, highest level of education, and hospital course data. Together these will be collated to produce our primary outcome variable of WHO Clinical Progression Scale score. These validated scores range from 0 (uninfected) to 10 (dead) to track clinically meaningful progression of COVID-19 infected patients. Our AQI variable will be obtained from the EPA available county-level monitoring station spatial data combined with open-source state/county center point spatial data. These data contain historic cataloguing to determine air quality at both specific time points and averages over time. Where a county's average yearly AQI is not available due to lack of a monitoring station, we will use spatial data tools to calculate an average based on data from nearby stations. We will utilize yearly averages of AQI in the year prior to COVID-19 diagnosis to describe overall impact of air quality on patients' respiratory outcomes as opposed to single day exposures. Linkage of patient data to AQI database will be performed using patient addresses. Discussion(s): By combining area level data with electronic health record (EHR) data, we will be positioned to understand the contribution of environmental and social determinants of health on patient outcomes. Our long-term goal is to elucidate which social and environmental determinants of health are associated with worse outcomes from COVID-19 and other respiratory viruses, using data extracted from the EHR.

A novel anterior nasal swab to detect respiratory viruses: a prospective study of diagnostic accuracy.

Detection of respiratory viruses requires testing of the upper respiratory tract to obtain specimens for analysis. However, nasal and throat swabs can cause discomfort and procedural anxiety in children. Respiratory sampling methods which are accurate and less invasive are needed. We aim to determine the positive and negative percentage agreement of a novel anterior nasal swab (ANS) compared with the combined throat and anterior nasal swab (CTN), the reference standard, for detection of respiratory viruses. Children 5 - 18 years of age presenting to a tertiary paediatric hospital with respiratory symptoms were tested with both swabs in randomised order. Respiratory samples were tested on a multiplex RT-PCR panel. Viral detections, RT-PCR cycle-threshold values and child/parent/clinician experience of the swab were recorded. There were 157 viral detections from 249 participant CTN swabs. In comparison with the CTN, the overall positive and negative percentage agreement of ANS for detection of respiratory viruses was 96.2% (95% CI, 91.8-98.3%) and 99.8% (95% CI, 99.6-99.9%), respectively. The ANS was "extremely comfortable", or only a "little uncomfortable" for 90% of children compared with 48% for CTN. 202 children (84%) rated the ANS as the preferred swab, and 208 (87%) indicated they would prefer ANS for future testing. The ANS required additional laboratory handling processes compared to the CTN. The ANS has high positive percentage agreement and is comparable to the current standard of care. The high acceptability from the less invasive ANS provides a more comfortable method for respiratory virus testing in children.Trial registrationClinicalTrials.gov ID NCT05043623.

Impact of the COVID-19 pandemic on antiviral drug development for other community-acquired respiratory viruses' infections.

The coronavirus disease 2019 (COVID-19) pandemic indirectly resulted in missed therapeutic opportunities for many diseases. Here we focus on community-acquired respiratory viruses other than severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) [respiratory syncytial virus, parainfluenza and influenza A], and highlight the pandemics impact on clinical trials to develop novel therapies for other severe respiratory viral infections. We retrospectively reviewed inclusion rates within respiratory antiviral clinical trials in comparison with all other clinical trials in our clinical investigations center, before and during the COVID-19 pandemic. As opposed to the remaining clinical trials developed within our unit, respiratory antiviral trials inclusion rates did not recover after the initial recruitment decrease observed across all trials during the first pandemic wave. These results were discussed in the context of non-COVID-19 respiratory viral infection rates within our center, showing a general decline in seasonal respiratory viruses spread since the COVID-19 pandemic onset. Virus epidemiology changes upon the wide SARS-CoV-2 expansion as well as the lifestyle changes globally adopted to prevent SARS-CoV-2 transmission could have therefore contributed to the negative impact of the COVID-19 pandemic on antiviral drug development. Our study highlights the peculiarity of respiratory antiviral drug development during the COVID-19 pandemic era and describes potential explanations for such drug development halting.

Comparison of the reactogenicity and immunogenicity between two-dose mRNA COVID-19 vaccine and inactivated COVID-19 vaccine followed by an mRNA vaccine in children aged 5-11 years.

To compare the reactogenicity and immunogenicity between the two-dose mRNA COVID-19 vaccine regimen and one or two doses of inactivated vaccine followed by an mRNA vaccine regimen in healthy children between 5 and 11 years of age, a prospective cohort study was performed at King Chulalongkorn Memorial Hospital in Thailand between March to June 2022. Healthy children between 5 and 11 years of age were enrolled and received the two-dose mRNA COVID-19 vaccine (BNT162b2) regimen or the inactivated (CoronaVac) vaccine followed by the BNT162b2 vaccine regimen. In addition, healthy children who received two doses of BBIBP-CorV between 1 and 3 months prior were enrolled to receive a heterologous BNT162b2 as a third dose (booster). Reactogenicity was assessed by a self-reported online questionnaire. Immunogenicity analysis was performed to determine binding antibodies to wild-type SARS-CoV-2. Neutralizing antibodies to Omicron variants (BA.2 and BA.5) were tested using the focus reduction neutralization test. Overall, 166 eligible children were enrolled. Local and systemic adverse events which occurred within 7 days after vaccination were mild to moderate and well-tolerated. The two-dose BNT162b2, CoronaVac followed by BNT162b2, and two-dose BBIBP-CorV followed by BNT162b2 groups elicited similar levels of anti-receptor-binding domain (RBD) IgG. However, the two-dose BNT162b2 and two-dose BBIBP-CorV followed by BNT162b2 groups elicited higher neutralizing activities against the Omicron BA.2 and BA.5 variant than the CoronaVac followed by BNT162b2 group. The CoronaVac followed by BNT162b2 group elicited low neutralizing activities against the Omicron BA.2 and BA.5 variant. A third dose (booster) mRNA vaccine should be prioritized for this group.

Role of antiviral therapy in respiratory infections in children: Analysis of clinical and laboratory observations.

Objective. To evaluate the efficacy of therapy for acute respiratory viral infections (ARVIs) in children with antiviral medications: inosine pranobex (Groprinosin, Gedeon Richter) and Kagocel (Kagocel, Niarmedic Pharma LLC) in comparison with symptomatic treatment without etiotropic agents based on clinical and laboratory parameters. Patients and methods. The clinical and laboratory observation was conducted in an outpatient setting in the pre-COVID-19 period between 2018 and 2020. Acute respiratory infections were diagnosed using licensed testing systems by multiplex polymerase chain reaction (PCR) with detection of nucleic acid viral genomes: influenza, rhinovirus, respiratory syncytial virus, metapneumovirus, parainfluenza, seasonal coronaviruses, adenoviruses, and bocavirus). A total of 151 children aged 3 to 15 years were examined and monitored in dynamics, with 78.7% of positive and 21.3% of negative results detected by PCR in the nasopharyngeal and oropharyngeal swabs. The patients were randomized into three groups depending on the antiviral medication prescribed: group 1 (53 children) received Groprinosin;group 2 (52 children) received Kagocel;group 3 (control, 46 children) received only symptomatic therapy without antiviral agents. Results. The study demonstrated a significant positive effect in patients in group 1 treated with Groprinosin (n = 53). At the end of therapy for both mono- and mixed infections, there were 95.8% of negative results (according to PCR diagnosis, that is, the absence of viral genome). In children in group 2 (n = 52) treated with Kagocel, the absence of viral nucleic acids (NAs) was observed less frequently (in 77.3% of cases). In children in group 3 (n = 46) who did not receive etiotropic antiviral therapy, there were only 40.3% of negative results after the end of treatment, and viral NAs were detected in 59.7% of patients. In this case, a 5-day course of Groprinosin was prescribed, after which the PCR results became negative in all patients. Therefore, children with recurrent respiratory infections, mixed infections, and herpesvirus infections require longer therapy. Additionally, a high frequency of ARVI complications was noted in group 3 (5 (10.9%) patients, where otitis was observed in 1 case, sinusitis - in 2 cases, bronchitis - in 2 cases), whereas 1 (1.8%) patient taking Groprinosin had otitis, and 1 (1.9%) patient taking Kagocel had pneumonia. Conclusion. This study was the first to investigate antibody titers to respiratory viruses in dynamics at 3, 6 and 12 months after the onset of ARVI. It showed that the development of antibodies to respiratory viruses is very unstable and does not occur in all patients. Antibodies almost disappeared by the third month after ARVI and were no longer detectable by the sixth month. After 12 months, patients suffered a new ARVI and developed the corresponding antibodies. This information will be especially relevant in conditions of the rise in the incidence of ARVIs, as well as the COVID-19 pandemic observed in recent years.Copyright © 2022, Voprosy Prakticheskoi Pediatrii. All rights reserved.

Molecular diagnostics for clinical respiratory virus on a total integrated centrifugal microsystem using reverse transcription-loop-mediated isothermal amplification

Respiratory viruses are highly contagious agents that can cause endemic and epidemic infections in humans. Early detection of these viruses is crucial in preventing economic damage and reducing mortality rates. In this study, we present a total integrated genetic analyzer to perform a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of 7 respiratory viruses (Influenza A H1N1 and H3N2, Influenza B, Respiratory syncytial virus A and B, Adenovirus, and COVID-19). The primer sets for the RT-LAMP assay were designed and evaluated in comparison with the RT-PCR assay using clinical samples, confirming high specificity and efficiency. The entire process of viral RNA extraction, reagent mixing, gene amplification, and detection was completed on the device in 1hr 20min. The constructed portable diagnostic instrument is equipped with a rotary motor, two sets of peltier heaters, a fluorescence detector, and a touch screen for inputting experimental parameters and displaying result. The proposed point-of-care (POC) diagnostic platform correctly analyzed a total of 21 clinical samples (3 for each of the 7 viruses). The limit-of-detection (LOD) for Influenza A subtype H3N2 was 101 pfu/mL, which demonstrates the high performance of our proposed centrifugal microsystem for on-site molecular diagnostics in medical centers.