RAPID IDENTIFICATION OF ETIOLOGICAL AGENTS OF RESPIRATORY TRACT INFECTIONS DURING THE COVID-19 PANDEMIC USING THE MULTIPLEX-PCR METHOD.

Objective. Most respiratory infections have similar symptoms, so it is clinically difficult to determine their etiology. This study aimed to show the importance of molecular diagnostics in identifying the etiological agent of respiratory infections, especially during the coronavirus disease 2019 (COVID-19) pandemic. Methods. A total of 849 samples from patients hospitalized at the University Clinical Center Kragujevac (from January 1 to August 1, 2022) were examined using automated multiplex-polymerase chain reaction (PCR) tests. The BioFire-FilmArray-Respiratory Panel 2.1 test was used for 742 nasopharyngeal swabs [identification of 19 viruses (including SARS-CoV-2) and four bacteria], while the BioFire-FilmArray-Pneumonia Panel was used [identification of 18 bacteria and nine viruses] (BioMerieux, Marcy l'Etoile, France) for 107 tracheal aspirates. The tests were performed according to the manufacturer's instructions, and the results were available within an hour. Results. In 582 (78.4%) samples, the BioFire-FilmArray-Respiratory Panel 2.1 plus test identified at least one pathogen. The rhinovirus (20.6%), SARS-CoV-2 (17.7%), influenza A (17.5%), respiratory syncytial virus (12.4%), and parainfluenza 3 (10.1%) were the most common. Other viruses were found less frequently, and Bordetella parapertussis was detected in one sample. In 85 (79.4%) samples, the BioFire-FilmArray-Pneumonia Panel test identified at least one bacterium or virus. The most prevalent bacteria were Staphylococcus aureus (42.4%), Haemophilus influenzae (41.2%), Streptococcus pneumoniae (36.5%), Moraxella catarrhalis (22.3%), and Legionella pneumophila (2.4%). Among viruses, rhinovirus (36.5%), adenovirus (23.5%), influenza A (11.8%), and the genus Coronavirus (4.7%), were detected. Conclusion. Multiplex-PCR tests improved the implementation of therapeutic and epidemiological measures, preventing the spread of the COVID-19 infection and Legionnaires' disease.Copyright © 2022, Serbian Medical Society. All rights reserved.

Analysis of pathogen detection results of upper respiratory tract specimens from fever clinic patients

Objective: To explore the pathogen composition and distribution characteristics of pathogens in respiratory samples from patients with fever of unknown origin. Methods: A total of 96 respiratory samples of patients with unknown cause fever with respiratory symptoms were collected from four hospitals above grade II in Shijiazhuang area (Hebei Provincial Hospital of Traditional Chinese Medicine, Luancheng District People's Hospital, Luquan District People's Hospital, Shenze County Hospital) from January to April 2020, and multiplex-fluorescent polymerase chain reaction(PCR)was used to detect influenza A virus, influenza B virus, enterovirus, parainfluenza virus I/II/III/IV, respiratory adenovirus, human metapneumovirus, respiratory syncytial virus, human rhinovirus, human bocavirus, COVID-19, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae, Group A streptococcus, Haemophilus influenzae, Staphylococcus aureus nucleic acid detection, the results were analyzed for chi-square. Results: A total of 8 pathogens were detected in the upper respiratory tract samples of 96 fever patients, including 1 kind of virus, 6 kinds of bacterias, and Mycoplasma pneumoniae. There were 12 viruses including influenza virus and parainfluenza virus, Legionella pneumophila and Chlamydia pneumoniae were not detected. The pathogen detection rates in descending order were Streptococcus pneumoniae (58/96, 60.42%), Haemophilus influenzae(38/96, 39.58%), Klebsiella pneumoniae (14/96, 14.58%), Staphylococcus aureus (10/96, 10.42%), Mycoplasma pneumoniae (8/96, 8.33%), Pseudomonas aeruginosa (6/96, 6.25%), Group A streptococcus (4/96, 4.17%) and human rhinovirus (2/96, 2.08%). The proportions of single-pathogen infection and multi-pathogen mixed infection in fever clinic patients were similar, 41.67% (40/96) and 45.83% (44/96), respectively, and 12.50% (12/96)of the cases had no pathogens detected. The infection rate of Mycoplasma pneumoniae in female patients with fever (21.43%) was higher than that in male patients with fever (2.94%) (P < 0.05). There was no statistical difference between the distribution of of other pathogens and gender and age(P > 0.05). Conclusions: The upper respiratory tract pathogens were mainly bacterial infections, and occasional human rhinovirus and Mycoplasma pneumonia infections. In clinical diagnosis and treatment, comprehensive consideration should be given to the pathogen detection.

The influence of COVID-19 prevention and control measures on the transmission and epidemic of influenza in Chongqing

Objective: To evaluate the influence of coronavirus disease 2019 (COVID-19) prevention and control measures on the transmission and epidemic of influenza in Chongqing, so as to provide references for formulating targeted influenza prevention and control strategies. Methods: The influenza surveillance data, during the year 2018 to 2020, were collected through the "China Influenza Surveillance Information System", and the seasonal characteristics of influenza epidemic were analyzed. The percentage of influenza like cases (ILI%) and influenza virus positive rate between 2020 and 2018-2019 were compared, so as to evaluate the impact of COVID-19 prevention and control measures on influenza epidemic characteristics. Results: The annual proportions of ILI cases in Chongqing were respectively 3.53%, 2.23% and 1.2% from 2018 to 2020, while the positive rates of influenza virus were respectively 13.97%, 23.81% and 2.65%. The distribution trend of ILI% from 2018 to 2019 fluctuated were similar, but it continued to drop and remain at a low level since February 2020. The positive rate of influenza virus showed an epidemic peak from December to March in 2018-2019, also peaked from November 2019 to January 2020, but decreased to 0 in March. ILI% was positively correlated with the positive rate of influenza virus (r=0.404 8, P < 0.05). In 2020, compared with the same period of 2018-2019, the growth rate of ILI% was -66.09% and -46.32%, respectively. The positive rate of influenza virus in 2020 decreased by 81.03% and 88.87% compared with the same period of 2018-2019, respectively. The growth rates of influenza virus positive rate in January 2020 were decreased with a small rate of about 39.87%, and with a significantly decline of more than 93.65% from February. No influenza epidemic was found after March. Conclusions: Since COVID-19 prevention and control measures were implemented in January 2020 in Chongqing, the ILI% and the positive rate of influenza virus in sentinel hospitals decreased significantly. In the season of high incidence of respiratory infectious diseases, personal protection and other measures can effectively reduce influenza virus infection.

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.

Strategic construction of mRNA vaccine derived from conserved and experimentally validated epitopes of avian influenza type A virus: a reverse vaccinology approach.

Purpose: The development of vaccines that confer protection against multiple avian influenza A (AIA) virus strains is necessary to prevent the emergence of highly infectious strains that may result in more severe outbreaks. Thus, this study applied reverse vaccinology approach in strategically constructing messenger RNA (mRNA) vaccine construct against avian influenza A (mVAIA) to induce cross-protection while targeting diverse AIA virulence factors. Materials and Methods: Immunoinformatics tools and databases were utilized to identify conserved experimentally validated AIA epitopes. CD8+ epitopes were docked with dominant chicken major histocompatibility complexes (MHCs) to evaluate complex formation. Conserved epitopes were adjoined in the optimized mVAIA sequence for efficient expression in Gallus gallus. Signal sequence for targeted secretory expression was included. Physicochemical properties, antigenicity, toxicity, and potential cross-reactivity were assessed. The tertiary structure of its protein sequence was modeled and validated in silico to investigate the accessibility of adjoined B-cell epitope. Potential immune responses were also simulated in C-ImmSim. Results: Eighteen experimentally validated epitopes were found conserved (Shannon index <2.0) in the study. These include one B-cell (SLLTEVETPIRNEWGCR) and 17 CD8+ epitopes, adjoined in a single mRNA construct. The CD8+ epitopes docked favorably with MHC peptide-binding groove, which were further supported by the acceptable ΔGbind (-28.45 to -40.59 kJ/mol) and Kd (<1.00) values. The incorporated Sec/SPI (secretory/signal peptidase I) cleavage site was also recognized with a high probability (0.964814). Adjoined B-cell epitope was found within the disordered and accessible regions of the vaccine. Immune simulation results projected cytokine production, lymphocyte activation, and memory cell generation after the 1st dose of mVAIA. Conclusion: Results suggest that mVAIA possesses stability, safety, and immunogenicity. In vitro and in vivo confirmation in subsequent studies are anticipated.

Influenza A (H1N1) virus induced long-term remission in a refractory acute myeloid leukaemia.

There have been reports of haematological cancer patients achieving spontaneous remission after being infected with the influenza A or SARS-COV-2 virus. Here, we present the first case of long-term complete remission (CR) induced by influenza A (IAV, H1N1 subtype) in a refractory AML patient and have functionally validated this finding in two different animal disease models. We observed a significant increase in the proportion of helper T cells in the patient after IAV infection. The levels of cytokines, including IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α, were higher in IAV-infected patients compared with control groups. These findings indicate that the anti-tumour effects induced by IAV are closely related to the modification of the immune response. Our study provides new evidence of the anti-tumour effects of IAV from a clinical practice perspective.

Identification of small molecules capable of enhancing viral membrane fusion.

Several approaches have been developed to analyze the entry of highly pathogenic viruses. In this study, we report the implementation of a Bimolecular Multicellular Complementation (BiMuC) assay to safely and efficiently monitor SARS-CoV-2 S-mediated membrane fusion without the need for microscopy-based equipment. Using BiMuC, we screened a library of approved drugs and identified compounds that enhance S protein-mediated cell-cell membrane fusion. Among them, ethynylestradiol promotes the growth of SARS-CoV-2 and Influenza A virus in vitro. Our findings demonstrate the potential of BiMuC for identifying small molecules that modulate the life cycle of enveloped viruses, including SARS-CoV-2.

Impact of Influenza in South-East Asia

Influenza infection is asymptomatic in up to 75% of cases, but outbreaks result in significant morbidity. Reports found that severe influenza complications tend to occur among the very young (<5 years) and very old (>65 years), especially those with underlying co-morbidities like diabetes mellitus and heart disease. Even with no co-morbidity, some older persons with severe influenza may require hospitalisation or intensive care, with increased risk of myocardial infarction and stroke. In South-East Asia, influenza was often seen as a mild problem and was not deemed notifiable until the appearance of the Influenza A(H1N1) pandemic in 2009. For decades the data made available were based on extrapolated estimates collected mainly from paediatric populations, resulting in inconsistent findings. Following expanded surveillance across the region using national surveillance systems for influenza-like illness (ILI) and severe acute respiratory illness (SARI), and better diagnostic methods, improved estimates of disease burden was achieved in South-East Asia. However, two studies conducted in 2008-2010 reported findings ranging from 2-3% to 11%. With regards to increased risk of complications, the estimated global annual attack rates for influenza were 5-10% in adults and 20-30% in children, resulting in 3-5 million cases of severe illness and 290,000-650,000 deaths. A study In Singapore reported that influenza is associated with annual excess mortality rates (EMR) of 11-14.8 per 100 000 person-years, especially affecting the elderly;these rates are comparable to that of the USA. As for hospitalisation rates of children under 5 years with seasonal influenza, the USA estimated a rate of 1.4 per 100,000. Comparable rates were reported in Singapore (0.7-0.9), Thailand (2.4), Viet Nam (3.9-4.7), and the Philippines (4.7). In 2018, an updated study reported a mean annual influenza-associated respiratory EMR of 4.0-8.8 per 100 000 individuals, with South-East Asia showing a high mortality rate of 3.5-9.2 per 100,000 individuals. It was already estimated in Thailand in 2004 that influenza resulted in USD23-63 million in economic costs, with the main contribution from lost productivity due to missed workdays. Thus, comparable to countries in temperate climate, the clinical and socioeconomic impact of influenza in South-East Asia appear to be just as substantial. With the emergence of the COVID-19 pandemic in 2020, global influenza incidence dropped dramatically. In South-East Asia, the trend in influenza detections was similar to the rest of the world, with numbers slightly higher than average in early 2020, followed by a quick drop-off by the end of April 2020. After April 2020, the detection rate remained low until late July 2020, when Influenza A(H3N2) predominated in Cambodia, Malaysia, the Philippines, Singapore, Thailand and Timor-Leste;influenza B in Lao People's Democratic Republic but with an upsurge in A(H3N2) activity. Following a two-year hiatus, influenza outbreaks began to re-emerge significantly since early 2022. From February through August 2022, influenza activity in the southern hemisphere remained lower than in pre-COVID-19 pandemic years, but was at the highest level compared to similar periods since the start of the COVID-19 pandemic. Reasons for the reduction during the COVID-19 pandemic include non-pharmaceutical interventions (NPIs), reduced population mixing and reduced travel, and possibly viral interference between SARS-CoV-2 and influenza virus in the same host. In general, the reduction in influenza detections however does not appear to be associated with lack of testing. The World Health Organisation (WHO) continues to recommend that vaccination is the most effective way to prevent infection and severe outcomes caused by influenza viruses. Although influenza vaccine is not commonly used in most countries in South-East Asia, its burden is similar in other parts of the world where influenza vaccine is now routinely used. Currently, the countries in South-East Asia that are providing free influenza vacc na ion for those at high risk include Thailand, Singapore, the Philippines and Lao People's Democratic Republic.Copyright © 2023

PARNAS: Objectively Selecting the Most Representative Taxa on a Phylogeny.

The use of next-generation sequencing technology has enabled phylogenetic studies with hundreds of thousands of taxa. Such large-scale phylogenies have become a critical component in genomic epidemiology in pathogens such as SARS-CoV-2 and influenza A virus. However, detailed phenotypic characterization of pathogens or generating a computationally tractable dataset for detailed phylogenetic analyses requires objective subsampling of taxa. To address this need, we propose parnas, an objective and flexible algorithm to sample and select taxa that best represent observed diversity by solving a generalized k-medoids problem on a phylogenetic tree. parnas solves this problem efficiently and exactly by novel optimizations and adapting algorithms from operations research. For more nuanced selections, taxa can be weighted with metadata or genetic sequence parameters, and the pool of potential representatives can be user-constrained. Motivated by influenza A virus genomic surveillance and vaccine design, parnas can be applied to identify representative taxa that optimally cover the diversity in a phylogeny within a specified distance radius. We demonstrated that parnas is more efficient and flexible than existing approaches. To demonstrate its utility, we applied parnas to (i) quantify SARS-CoV-2 genetic diversity over time, (ii) select representative influenza A virus in swine genes derived from over 5 years of genomic surveillance data, and (iii) identify gaps in H3N2 human influenza A virus vaccine coverage. We suggest that our method, through the objective selection of representatives in a phylogeny, provides criteria for quantifying genetic diversity that has application in the the rational design of multivalent vaccines and genomic epidemiology. PARNAS is available at https://github.com/flu-crew/parnas.

Acute hemorrhagic pancreatitis following influenza infection: a case report.

BACKGROUND: Acute hemorrhagic pancreatitis is a life-threatening condition leading to shock and multiorgan failure. Although prevalent in the general population, the incidence during pregnancy is low, with a high maternal and fetal mortality rate. The highest incidence is in the third trimester/early postpartum period. Infectious etiology for acute hemorrhagic pancreatitis is rare with only a handful of cases following influenza infection being documented in the literature. CASE PRESENTATION: A 29-year-old Sinhalese pregnant lady in the third trimester presented with an upper respiratory tract infection and abdominal pain, for which she was managed with oral antibiotics. An elective caesarean section was done at 37 weeks gestation due to a past section. On postoperative day 3 she developed a fever with difficulty in breathing. Despite treatment, she succumbed to death on the sixth postoperative day. The autopsy revealed extensive fat necrosis with saponification. The pancreas was necrosed and hemorrhagic. The lungs showed features of adult respiratory distress syndrome and necrosis was observed in the liver and kidneys. Polymerase chain reaction of lungs detected influenza A virus (subtype H3). CONCLUSION: Although rare, acute hemorrhagic pancreatitis from an infectious etiology carries risk of morbidity and mortality. Therefore, a high level of clinical suspicion must be upheld among clinicians to minimize adverse outcomes.

Single center retrospective study on the distribution of pathogens in elderly patients with acute exacerbation of chronic obstructive pulmonary disease

Objective: To explore the distribution and correlation of pathogens in the elderly patients with AECOPD, so as to guide the rational use of antibiotics and hormones in clinic. Methods: A total of 111 patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) admitted to Nanjing First Hospital from January 2019 to January 2020 were retrospectively analyzed. The basic data such as eosinophil, neutrophil and lymphocyte count, the levels of C-reactive protein(CRP) and erythrocyte sedimentation rate (ESR)in blood routine examination were collected. Further, the pathogens were qualified by sputum fluorescence quantitative polymerase chain reaction, and the pathogens distribution was analyzed. Results: The level of ESR and the ratio of cardiovascular diseases showed significant differences between the pathogen-positive group and pathogen-negative group. In this study, the top five pathogens in AECOPD patients were EB virus (21.6%), Haemophilus influenzae (19.8%), Streptococcus pneumoniae (17.1%), herpes simplex virus(14.4%), influenza A virus(14.4%). The detection rate of influenza A virus was correlated with influenza B virus and Aspergillus (P < 0.05);The detection rate of respiratory syncytial virus was correlated with Candida, Moraxella catarrholis, Streptococcus pneumoniae and Haemophilus influenzae (P < 0.05);The detection rate of Escherichia coli was correlated with rhinovirus, adenovirus, Klebsiella pneumoniae and Acinetobacter baumannii (P < 0.05);The detection rate of Candida was correlated with that of Moraxella catarrholis and Pseudomonas aeruginosa(P<0.05);The detection rate of human coronavirus was correlated with Haemophilus influenzae, herpes simplex virus and Streptococcus pneumoniae(P < 0.05). Conclusions: AECOPD are mostly induced by different pathogens, especially mixed infection of bacteria and virus. It is helpful to guide the rational use of antibiotics by analyzing the etiological characteristics in the elderly patients with AECOPD.

Outbreaks of different viral etiologies amidst COVID-19 pandemic

The COVID-19 pandemic has severely affected public health system and surveillance of other communicable diseases across the globe. The lockdown, travel constraints and COVID phobia turned down the number of people with illness visiting to the clinics or hospitals. Besides this, the heavy workload of SARS-CoV-2 diagnosis has led to the reduction in differential diagnosis of other diseases. Consequently, it added to the underlying burden of many diseases which remained under-diagnosed. Amidst the pandemic, the rise of emerging and re-emerging infectious diseases was observed worldwide and reported to the World Health Organization i.e., Crimean Congo Hemorrhagic Fever (2022, Iraq;2021 India), Nipah virus (2021, India), Zika virus (2021, India), and H5N1 influenza (2021, India), Monkeypox (2022, multicountry outbreak), Ebola virus disease (2022, DRC, Uganda;2021, DRC, Guinea;2020, DRC), Marburg (2022, Ghana;2021, Guinea), Yellow fever (2022, Uganda, Kenya, West and Central Africa;2021, Ghana, Venezuela, Nigeria;2020, Senegal, Guinea, Nigeria, Gabon;2020, Ethiopia, Sudan, Uganda), Dengue (2022, Nepal, Pakistan, Sao Tome, Temor-Leste;2021, Pakistan), Middle east respiratory syndrome coronavirus (2022, Oman, Qatar;2021, Saudi Arabia, UAE;2020, Saudi Arabia, UAE), Rift valley fever (2021, Kenya;2020, Mauritania), wild poliovirus type 1 (2022, Mozambique), Lassa fever (2022, Guinea, Togo, Nigeria;2020, Nigeria), Avian Influenza (H3N8) (2022, China), Avian Influenza (H5N1) (2022, USA), H10N3 influenza (2021, China), Hepatitis E virus (2022, Sudan), Measles (2022, Malawi, Afghanistan;2020, Burundi, Mexico), Mayaro virus disease (2020, French Guiana), Oropouche virus disease (2020, French Guiana). All these diseases were associated with high morbidity and burdened the public health system during the COVID-19 pandemic. During this critical public health menace, majority of the laboratory workforce was mobilized to the SARS-CoV-2 diagnosis. This has limited the surveillance efforts that likely led to under diagnosis and under-detection of many infectious pathogens. Lockdowns and travel limitations also put a hold on human and animal surveillance studies to assess the prevalence of these zoonotic viruses. In addition, lack of supplies and laboratory personnel and an overburdened workforce negatively impacted differential diagnosis of the diseases. This is especially critical given the common symptoms between COVID-19 and other pathogens causing respiratory illnesses. Additionally, the vaccination programs against various vaccine preventable diseases were also hampered which might have added to the disease burden. Despite these challenges, the world is better prepared to detect and respond to emerging/re-emerging pathogens. India now has more than 3000 COVID-19 diagnostic laboratories and an enhanced hospital infrastructure. In addition, mobile BSL-3 facilities are being validated for onsite sampling and testing in remote areas during outbreak situations and surveillance activities. This will undoubtedly be valuable as the COVID-19 pandemic evolves as well as during future outbreaks and epidemics. In conclusion, an increase in the emergence and re-emergence of viruses demonstrates that other infectious diseases have been neglected during the COVID-19 pandemic. Lessons learned from the infrastructure strengthening, collaborations with multiple stakeholders, increased laboratory and manufacturing capacity, large-scale COVID-19 surveillance, extensive network for laboratory diagnosis, and intervention strategies can be implemented to provide quick, concerted responses against the future threats associated with other zoonotic pathogens.

Respiratory Syncytial Virus (RSV) infections in children: recent concepts in immunopathogenesis and diagnostic dilemma

Respiratory viral infections are important cause of morbidity and mortality in early life. The relative influence of host and viral factors possibly contribute to the disease pathogenesis. Predisposing conditions like prematurity, Low birth weight and congenital heart diseases etc. have been incriminated in the disease progression. The development of cough, wheezing, and tachypnea, usually peaking on days 4 to 5, go parallel with host cytokine responses and viral load. Various host cytokines, chemokines and molecules involved in the immune response against RSV infection might be responsible for the outcome of the disease process. Nasopharyngeal aspirates (NPAs) from children (n = 349) between 2013-2017 were subjected for IL-17A, IFN-gamma, TNF-alpha, IL-10, IL-6 levels by CBA and MMP-9 and TIMP-1 levels by ELISA. The viral load in RSV positive samples and cytokine levels were correlated with the WHO criteria for acute lower respiratory tract illness (ALRTI). RSV viral load, Pro-inflammatory cytokine (TNF-alpha) levels in severe ALRTI patients were significantly higher than the ALRTI patients [p<0.001]. Whereas Th17 cytokine (IL-17) was found to be significantly higher (p<0.05) in ALRTI patients than severe patients. MMP-9 is secreted in higher levels in severe ALRTI patients (n = 77) in comparison to Acute LRTI patients (n = 35) with an increase of thirty seven fold (p<0.001). Thus, the study highlights the role of TNF -alpha, IL-17 and Th2 cytokine biasness in the pathogenesis of RSV disease with the possible contribution of higher MMP-9/TIMP-1 ratio as a bad prognostic marker towards disease severity. To study the gene expression of autophagy and mTOR signalling pathways in RSV infected children with ALRTI. Nasopharyngeal aspirate (NPA) samples (n = 145) from children suffering from ALRTI were subjected for detection of RSV (Oct 2019 to March 2020). Semi-quantitative gene expression analysis for 5 representative genes each of mTOR signalling and autophagy pathway were performed in respiratory tract epithelial cells using 25 RSV positive cases and 10 healthy controls subjects. Autophagy gene expression analysis revealed significant upregulation in NPC1 and ATG3 autophagy genes. mTOR, AKT1 and TSC1 genes of mTOR pathway were significantly down-regulated in RSV positive patients except RICTOR gene which was significantly upregulated. Thus, survival of RSV within autophagosome might have been facilitated by upregulation of autophagy and downregulation of mTOR signalling genes. To assess the impact of SARS-CoV2 pandemic on RSV, samples were collected from children with ALRTIs admitted to emergency, PICU and indoor admissions during pre-pandemic period (October 2019 to February 2020;n = 166) and during COVID-19 Pandemic (July 2021 to July 2022;n = 189, SARS-CoV2 negative). These NP swabs were analyzed for pdm InfA H1N1, InfA H3N2, Inf B, RSV, hMPV, hBoV, hRV, PIV-2 and PIV-3 by PCR. Higher proportion of children with ALRTIs have had virus/es isolated during pre-pandemic period than during pandemic period (p<0.001). During pre-pandemic period, significantly higher proportion of children had RSV positivity (p<0.001);and significantly lower positivity for hRV (p<0.05), hMPV (p<0.05), and hBoV (p <= 0.005). The occurrence of COVID-19 pandemic has significantly impacted the frequency and pattern of detection of RSV among hospitalized children with LRTIs. RSV Fusion protein plays a critical role in the entry of the virus into the host cell by initiating the fusion of host and viral membranes. It happens to be a target of neutralizing antibodies paving the way as a vaccine candidate. Hence effort was made to introduce point mutation in hRSV fusion protein which can confer stability in its prefusion form. In-silico a stable structure of RSV fusion protein was generated making it a potential vaccine candidate. The timely diagnosis of RSV infection in this population is important for initiating therapy and instituting appropriate infection prevention measures. Serological testing is not widely used for the diagnosis of RSV. C ll Cultures including shell vial culture were used for RSV diagnosis. However, culture approaches lack sensitivity, often quite significantly, compared to nucleic acid amplification assays for the diagnosis of RSV infections. Molecular multiplex assays now offer increased sensitivity for a more accurate diagnosis. However issues with the use of these types of commercial panel assays include the requirement for substantial training, quality systems, and infrastructure to maintain and run these assays and many a times identification of viruses where the true pathogenic potential of those multiple viruses are debatable. Studies are available with laboratory- developed nucleic acid amplification test systems for the detection of RSVA and RSVB in clinical specimens either by PCRbased technologies or RT-LAMP. Gene targets of laboratory-developed molecular assays point towards M gene and the N gene in RSVA and -B with the benefits of flexibility to modify assays when targets are under evolutionary pressure to change, as well as a perceived initial low cost to carry out testing.

Establishment of multi-target rapid detection method of upper repiratory susceptible viruses based on digital microfluidic technology

Objective: Provide a digital microfluidic RT-qPCR chip for rapid detection of several upper respiratory diseases. Methods: Several specific primer-probe sets were designed according to the conserved sequences of 2019 novel corona virus(2019-n COV), influenza A virus(Flu A), influenza B virus(Flu B), severe acute respiratory syndrome corona virus(SARS-Co V), Middle East respiratory syndrome corona virus(MERS-Co V), and then packaged into a digital microfluidic chip which allowed simultaneous detection of five upper respiratory tract pathogens with the help of reverse transcription quantitative PCR(RT-q PCR)technology. In the meanwhile, the detection limit, specificity and sensitivity of this digital microfluidic chip were evaluated base on the clinical specimens, plasmids and unrelated pathogens. Results: The established digital microfluidic RT-q PCR chip for 2019-n COV, Flu A, Flu B,SARS-Co V,MERS-Co V had a detection limit of 12 copies/reaction, while the detection limit of the RT-q PCR method without digital microfluidics was 15 copy/reaction;the detection limit of the two methods was basically the same. For nucleic acid samples extracted from clinical samples, the detection results of digital microfluidic RT-q PCR chips were all negative without non-specific amplification. At the same time, the RT-qPCR method and the digital microfluidic RT-qPCR chip method were used to carry out clinical comparative tests of 5 items in 20 clinical samples, total 100 tests. The results showed that the sensitivity of the digital microfluidic RT-q PCR chip reached 94%, the specificity was 100%. SPSS was used to analyze the consistency of the two methods, and the results showed that the two methods had a high degree of consistency(Kappa=0.962, P<0.05). Conclusion: Based on digital microfluidic RT-q PCR chip technology,a multi-target rapid detection method of upper respiratory tract susceptible virus was established, which could provide a new detection method for early clinical identification of respiratory pathogens.

Influence of novel coronavirus prevention and control measures on common upper respiratory tract pathogen infection in children in Guangzhou

Objective: To understand the epidemiological changes of children infected with 8 respiratory viral pathogens under the protection strategy of the new coronavirus epidemic in Guangzhou. Methods: A total of 13 606 children diagnosed with upper respiratory tract infection from January 2019 to December 2020 were retrospectively analyzed. Children were divided into four groups: infant group, toddler group, preschool group and school age group. Indirect immunofluorescence assay (IFA) was used to detect eight major respiratory virus pathogens, including: adenovirus (ADV), respiratory syncytial virus(RSV), influenza A virus (IV-A), influenza B virus (IV-B), parainfluenza virus (PIV), Chlamydia pneumonia(CP), Legionella pneumophila (LP) and Mycoplasma pneumonia (MP). Results: Sinc ethe outbreak of COVID-19, the positive rates of eight respiratory pathogens had decreased, especially influenza A and B. The positive rate of IV-A showed significant decrease from 30.126% to 12.930%, and the positive rate of IV-B from 25.597% to 19.268%, the defferences were statistically significant (X2=52.849, 369.778, all P < 0.05). Among different age groups, the positive rates of IV-A and ADV infection in each group decreased significantly, especially in infant group and toddler group. The positive rate of IV-A showed significant decrease from 4.588% to 1.979%, and the positive rate of ADV from 43.007% to 21.240%, the defferences were statistically significant(X2=18.910, 197.714, all P < 0.05). Before and after the outbreak of COVID-19, the differences in ADV, IV-A, IV-B, MP and PIV between the male and female groups were statistically significant (P < 0.05). Conclusion: Novel coronavirus protective measures were effective in preventing some common respiratory diseases in children.

Immunogenetic studies on SARS-CoV-2: Our experience at SKIMS

Background: The pathophysiology of viral-infections is highly complex and involves host immunocompetence, host genetics, and gene-environment interactions. We hypothesized that polymorphic variants in host genes, blood group and previous vaccination status against H1N1 may affect the clinical course of covid-19 infection. Method(s): A total of 202 subjects who were RT-PCR negative after Covid-19 infection were recruited. We investigated association between Covid-19 infection (Severity and recovery period) and multiple factors including ABO and Rh blood groups, H1N1 vaccination, polymorphism in Viral susceptibility genes (ACE2 G8790A), and polymorphism in host response genes (ACE I/D rs4646994, IL6- 174G/C, GSTT1/GSTM1 I/D and GSTP1 Ile 105 Val). Result(s): B-ve and O-ve ABO and Rh blood groups had significantly higher Covid-19 recovery period applied on one-vs.-all in a nonparametric t-test (p<0.05). Subjects who had vaccinated themselves against H1N1 presented with a lower recovery-period (p<0.05). Both variables (blood group and H1N1 vaccination) were not however associated with Covid-19 severity. Out of the studied polymorphisms, ACE2 G8790A and GSTT1/GSTM1 were significantly associated with covid-19 infection. Our results indicated that G/G genotype of ACE2 G8790A (OR 3.52, P 0.007) and GSTT1/ GSTM1 null (M1 - / - OR = 3.98, P = 0.0004;T1 - / - OR 3.84, P = 0.004) and double null (M1 - / - /T1 - / - OR = 9.66, P = 0.001) are likely to be associated with an increased risk for severe-critical outcomes in individuals with COVID-19. Other polymorphisms analyzed in this study were found to have no significant association with Covid-19 outcome. Conclusion(s): This study suggests that outcome of Covid-19 infection is affected by both clinical and genetic factors. Thus it seems plausible to utilize these factors as prediction and susceptibility markers in the prognosis of COVID-19, which may help to personalize the treatment.

Influenza surveillance in Ezhou city, Hubei Province, 2016-2021

ObjectiveTo analyze the influenza surveillance data in Ezhou City, Hubei Province from 2016 to 2021, determine the epidemiological characteristics and etiological trend of influenza like illness (ILI), and to provide scientific evidence for influenza prevention and control. MethodsThe ILI surveillance data were reported by Ezhou influenza sentinel hospitals and etiological examination results were collected by network laboratory. Influenza surveillance data from 2016 to 2021 were analyzed. ResultsFrom 2016 to 2021, the percentage of ILI visits (ILI%) in Ezhou city was 2.81% and increased over years. Majority (55.55%) of ILI cases were 0-4 years. A total of 7 716 ILI samples were examined from 2016 to 2021, of which 1 467 tested positive with a positive rate of 19.01%. Influenza A H1N1 was mainly concentrated in January-April, A H3N2 mainly in August-December, B Victoria mainly in April-July and December-March, and B Yamagata mainly in December-February. Influenza network laboratory isolated influenza virus from the 1 467 positive samples by using MDCK cells and SPF chicken embryos. The overall isolation rate was 32.78%, which was 26.93% by MDCK cells and 5.86% by SPF chicken embryos. From 2016 to 2021, a total of 13 ILI outbreaks were reported in Ezhou City. Temporally, the outbreaks mainly occurred in winter and spring. Spatially, they were mainly in primary schools, middle schools and kindergartens. ConclusionThe winter and spring are the key time period of influenza prevention and control in Ezhou City, as they are susceptible to influenza outbreaks. Children aged 0-14 are the key population of prevention and control. Diverse subtypes of influenza virus alternate by years, which warrants continually strengthening monitoring. Additionally, certain countermeasures against COVID-19 may be recommended in the prevention and control of influenza.

Extracellular vesicles as delivery vectors to enhance killing of bacteria and viruses by airway-recruited human neutrophils

Background: Polymorphonuclear neutrophils (PMNs) recruited to the airway lumen in cystic fibrosis (CF) undergo a rapid transcriptional program, resulting in exocytosis of granules and inhibition of bacterial killing. As a result, chronic infection, feed-forward inflammation, and structural tissue damage occur. Because CF airway PMNs are also highly pinocytic, we hypothesized that we could deliver protein- and ribonucleic acid (RNA)-based therapies to modulate their function to benefit patients. We elected to use extracellular vesicles (EVs) as a delivery vector because they are highly customizable, and airway PMNs have previously been shown by our group to process and use their cargo efficiently [1]. Furthermore, our prior work on CF airway PMNs [2] led to identification of the long noncoding RNA MALAT1, the transcription factor Ehf, and the histone deacetylase/long-chain fatty deacylase HDAC11 as potential targets to modulate CF airway PMN dysfunction. Method(s): H441 human club epithelial cells were chosen for EV production because they efficiently communicate with lung-recruited primary human PMNs [1]. Relevant constructs were cloned into an expression plasmid downstream of a constitutive cytomegalovirus or U6 promoter with an additional puromycin selection cassette. EVs were generated in serumdepleted media and purified by differential centrifugation. Quality and concentration of EVs was determined by electron microscopy and nanoparticle tracking analysis and cargo content by western blot (protein) or qualitative reverse transcription polymerase chain reaction (RNA). Enhanced green fluorescent protein and messenger ribonucleic acid (mRNA) were used as controls. To test delivery to primary human PMNs, generated EVs were applied in the apical fluid of an airway transmigration model [2]. PMN activation was assessed by flow cytometry, and bacterial (PA01 and Staphylococcus aureus 8325-4) killing and viral (influenza Avirus [IAV] H1N1/PR/8/34;SARS-CoV-2/Washington) clearance assays were conducted. Result(s): To package protein, we used EV-loading motifs such as the tetraspanin CD63, Basp1 amino acids 1-9, and the palmitoylation signal of Lyn kinase. To load mRNA, a C'D box motif recognized by the RNA-binding protein L7Ae was included in the 3' untranslated region of the expressed RNA, and CD63-L7Ae was co-expressed. Airway-recruited PMNs treated with EVs containing small interfering RNAs against MALAT1 or HDAC11 showed greater ability to clear bacteria. Conversely, PMNs treated with constructs encasing MALAT1 or HDAC11 efficiently cleared IAV and SARSCoV- 2. PMNs expressing Ehf showed greater clearance of bacteria and viruses. Conclusion(s): Our findings suggest mutually exclusive roles of MALAT-1 and HDAC11 in regulating bacterial and viral clearance by airway-recruited PMNs. Expression of Ehf in airway PMNs may be a pathogen-agnostic approach to enhancing clearance by airway-recruited PMNs. Overall, our study brings proof-of-concept data for therapeutic RNA/protein transfer to airway-recruited PMNs in CF and other lung diseases and for use of EVs as a promising method for cargo delivery to these cells. It is our expectation that, by treating the immune compartment of CF airway disease, pathogentherapies, such as antibiotics will be more effective, and epithelial-targeted therapies, such as CFTR modulators, will have greater penetrance into the cell types of interest.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Rapid Virucidal Activity of Japanese Saxifraga Species-Derived Condensed Tannins against SARS-CoV-2, Influenza A Virus, and Human Norovirus Surrogate Viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus are global threats to human health. The application of effective virucidal agents, which contribute to the inactivation of viruses on hands and environmental surfaces, is important to facilitate robust virus infection control measures. Naturally derived virucidal disinfectants have attracted attention owing to their safety and eco-friendly properties. In this study, we showed that multiple Japanese Saxifraga species-derived fractions demonstrated rapid, potent virucidal activity against the SARS-CoV-2 ancestral strain and multiple variant strains, IAV, and two human norovirus surrogates: feline calicivirus (FCV) and murine norovirus (MNV). Condensed tannins were identified as active chemical constituents that play a central role in the virucidal activities of these fractions. At a concentration of 25 µg/mL, the purified condensed tannin fraction Sst-2R induced significant reductions in the viral titers of the SARS-CoV-2 ancestral strain, IAV, and FCV (reductions of ≥3.13, ≥3.00, and 2.50 log10 50% tissue culture infective doses [TCID50]/mL, respectively) within 10 s of reaction time. Furthermore, at a concentration of 100 µg/mL, Sst-2R induced a reduction of 1.75 log10 TCID50/mL in the viral titers of MNV within 1 min. Western blotting and transmission electron microscopy analyses revealed that Sst-2R produced structural abnormalities in viral structural proteins and envelopes, resulting in the destruction of viral particles. Furthermore, Saxifraga species-derived fraction-containing cream showed virucidal activity against multiple viruses within 10 min. Our findings indicate that Saxifraga species-derived fractions containing condensed tannins can be used as disinfectants against multiple viruses on hands and environmental surfaces. IMPORTANCE SARS-CoV-2, IAV, and norovirus are highly contagious pathogens. The use of naturally derived components as novel virucidal/antiviral agents is currently attracting attention. We showed that fractions from extracts of Saxifraga species, in the form of a solution as well as a cream, exerted potent, rapid virucidal activities against SARS-CoV-2, IAV, and surrogates of human norovirus. Condensed tannins were found to play a central role in this activity. The in vitro cytotoxicity of the purified condensed tannin fraction at a concentration that exhibited some extent of virucidal activity was lower than that of 70% ethanol or 2,000 ppm sodium hypochlorite solution, which are popular virucidal disinfectants. Our study suggests that Saxifraga species-derived fractions containing condensed tannins can be used on hands and environmental surfaces as safe virucidal agents against multiple viruses.