Viral Nucleic Acid Research Articles

Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43–25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

ЦИРКОВИРУС-МИКОПЛАЗМЕННАЯ ИНФЕКЦИЯ ПОРОСЯТ В ЭКСПЕРИМЕНТЕ

The aim of the study is to investigate the manifestation of circovirus-mycoplasma infection in piglets both as a mixed infection and as a monoinfection in the experiment. Objectives: to study the interaction of circovirus type 2 with Mycoplasma hyopneumoniae in the body of weaned piglets, as well as the manifestation of monoinfection in piglets during experimental infection; to assess the pathogenicity of PCV2d/M.hyo mixed infection, PCV2d monoinfection and M. hyo monoinfection; to identify the clinical and pathomorphological manifestation of mixed and single circovirus and mycoplasma infection in the experiment. Four groups of 23-day-old piglets (5 heads each) were formed and tested for the absence of pathogens. Experimental group I – mixed circovirus-mycoplasma infection, experimental group II – PCV2d monoinfection, experimental group III – M.hyo monoinfection, group IV – control. After infection, rectal temperature was measured daily and clinical manifestations were observed, nasal swabs were taken to determine the number of copies of M. hyo nucleic acid, blood was taken from each piglet weekly to detect viral nucleic acid and antibodies. All experimental and control piglets were euthanized on the 35th day from the beginning of the experiment. Pathomorphological and pathohistological studies were carried out. The experimental study reveals the synergistic pathogenicity of PCV2d and M. hyo in mixed infection. In experimental infection of weaning piglets with both M. hyo monoinfection and M. hyo/PCV2d mixed infection, it was found that M. hyo nucleic acid was detected in nasal smears in 80 % of animals and in lung tissue in 100 %. The number of copies of the PCV2d circovirus genome in the inguinal lymph node tissue of piglets in the mixed infection group was higher than in the monoinfection group. Circovirus type 2 in piglets can proceed latently, and circovirus-mycoplasma infection can be a complex respiratory disease.

Novel immunomodulatory properties of adenosine analogs promote their antiviral activity against SARS-CoV-2

The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics.

Structure-optimized iridium complex coordination-functionalized ZIF-8-based nanoemitters with HEPES as novel efficient and eco-friendly ECL system for multiplex assays of HSV and VZV

Both herpes simplex virus (HSV) and varicella-zoster virus (VZV) belong to herpesviruses family, and the cutaneous infections with HSV or VZV often present similar clinical symptoms, making it challenging in differentiation. Hence, simultaneous detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) holds great significance for protecting the public health safety. Herein, simultaneous detection of HSV and VZV was innovatively realized based on electrochemiluminescence (ECL) biosensor. Structure-optimized iridium(III) complex coordinated in ZIF-8 nanoemitters (Irbt-ZIF-8-NH2) were synthesized as anodic emitters. Notably, Irbt-ZIF-8-NH2 showed remarkable ECL performance with N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) as the novel efficient co-reactant over the traditional TPrA. The proposed biosensor in this work demonstrated outstanding performance within the linear range of 10 fM to 1 nM, with the detection limit of 1.5 fM for HSV and 2.2 fM for VZV. Importantly, it also exhibited satisfactory recoveries with RSDs in the analysis of viral nucleic acids samples extracted from HSV or VZV infected cells. This work inevitably provides a promising alternative assay for simultaneously detecting HSV and VZV, which would promote to the advancement of efficient and sensitive techniques to tackle significant global health concerns caused by herpesviruses outbreak in the future.

Rapid discrimination of African swine fever virus nucleic acid and virions using BenzoNuclease

African swine fever (ASF) is an acute and severe infectious disease caused by the African Swine Fever Virus (ASFV). ASFV exhibits significant resistance and stability in the environment, which, coupled with its double-stranded DNA and large genome, predisposes it to contaminate laboratory samples. This characteristic can lead to false-positive results in swine farm settings even days after disinfection, as detectable through polymerase chain reaction (PCR) or real-time fluorescent quantitative PCR (qPCR) assays. To meet the demand for efficient clinical methods capable of discriminating between ASFV nucleic acid and ASFV virions, this study aims to ascertain the efficacy of the nuclease “BenzoNuclease” in distinguishing ASFV nucleic acid (ASFV-DNA) from ASFV virions. BenzoNuclease is a versatile nucleic acid enzyme with the capacity to degrade nearly all forms of DNA and RNA. Initially, this research established a highly sensitive general PCR detection method for ASFV. Subsequently, a positive control was constructed using the M13 bacteriophage to substitute for active ASFV, facilitating the development of an improved qPCR method. It is important to note that common disinfectants have the potential to deactivate BenzoNuclease. However, in an environment simulating actual production applications, residual disinfectants do not interfere with the enzymatic efficacy of BenzoNuclease, thus not affecting the detection capabilities of this method. Positive clinical samples from pig farms, upon testing with the improved method, revealed that three samples were positive, indicating the presence of viral particles, while the remaining samples were negative, indicating the presence of nucleic acids. This provides an additional new option for sample testing in pig farms.

Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

Mechanisms of action of microbicides commonly used in infection prevention and control.

SUMMARYUnderstanding how commonly used chemical microbicides affect pathogenic microorganisms is important for formulation of microbicides. This review focuses on the mechanism(s) of action of chemical microbicides commonly used in infection prevention and control. Contrary to the typical site-specific mode of action of antibiotics, microbicides often act via multiple targets, causing rapid and irreversible damage to microbes. In the case of viruses, the envelope or protein capsid is usually the primary structural target, resulting in loss of envelope integrity or denaturation of proteins in the capsid, causing loss of the receptor-binding domain for host cell receptors, and/or breakdown of other viral proteins or nucleic acids. However, for certain virucidal microbicides, the nucleic acid may be a significant site of action. The region of primary damage to the protein or nucleic acid is site-specific and may vary with the virus type. Due to their greater complexity and metabolism, bacteria and fungi offer more targets. The rapid and irreversible damage to microbes may result from solubilization of lipid components and denaturation of enzymes involved in the transport of nutrients. Formulation of microbicidal actives that attack multiple sites on microbes, or control of the pH, addition of preservatives or potentiators, and so on, can increase the spectrum of action against pathogens and reduce both the concentrations and times needed to achieve microbicidal activity against the target pathogens.

Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

Evaluation of dried blood spots for Epstein–Barr virus nucleic acid testing

Epstein–Barr virus (EBV) is a ubiquitous and oncogenic virus that is associated with various malignancies and non-malignant diseases and EBV DNA detection is widely used for the diagnosis and prognosis prediction for these diseases. The dried blood spots (DBS) sampling method holds great potential as an alternative to venous blood samples in geographically remote areas, for individuals with disabilities, or for newborn blood collection. Therefore, the objective of this study was to assess the viability of detecting EBV DNA load from DBS. Matched whole blood and DBS samples were collected for EBV DNA extraction and quantification detection. EBV DNA detection in DBS presented a specificity of 100 %. At different EBV DNA viral load in whole blood, the sensitivity of EBV DNA detection in DBS was 38.78 % (≥1 copies/mL), 43.18 % (≥500 copies/mL), 58.63 % (≥1000 copies/mL), 71.43 % (≥2000 copies/mL), 82.35 % (≥4000 copies/mL), and 92.86 % (≥5000 copies/mL), respectively. These results indicated that the sensitivity of EBV DNA detection in DBS increased with elevating viral load. Moreover, there was good correlation between EBV DNA levels measured in whole blood and DBS, and on average, the viral load measured in whole blood was about 6-fold higher than in DBS. Our research firstly demonstrated the feasibility of using DBS for qualitative and semi-quantitative detection of EBV DNA for diagnosis and surveillance of EBV-related diseases.

Bovine Rhinitis B Virus Variant as the Putative Cause of Bronchitis in Goat Kids.

A diagnostic investigation into an outbreak of fatal respiratory disease among young goats in Iowa, USA revealed bronchitis lesions of unknown etiology and secondary bacterial bronchopneumonia. Hypothesis-free metagenomics identified a previously unreported picornavirus (USA/IA26017/2023), and further phylogenetic analysis classified USA/IA26017/2023 as an aphthovirus related to bovine rhinitis B virus. Viral nucleic acid was localized to lesions of bronchitis using in situ hybridization. This marks the first report of a picornavirus putatively causing respiratory disease in goats and highlights the potential for cross-species transmission of aphthoviruses.

Hepatitis B transmission/reactivation associated with Hepatitis B core antibody and Hepatitis C nucleic acid testing positive organs: A report from the Organ Procurement and Transplantation Network Disease Transmission Advisory Committee.

Better access to direct-acting antiviral (DAA) therapy has broadened the utilization of hepatitis C virus (HCV) nucleic acid testing (NAT) positive organs with excellent outcomes. However, DAA therapy has been associated with hepatitis B virus (HBV) reactivation. To determine the risk of HBV transmission or reactivation with utilization of HBV core antibody positive (HBcAb+) and HCV NAT positive (HCV+) organs, which presumably required DAA therapy. The number of HBcAb+ donors with delineated HCV NAT status was obtained from the Organ Procurement and Transplantation Network (OPTN) database. The number of unexpected HBV infections from transplanted organs adjudicated as "proven" or "probable" transmission was obtained from the OPTN Ad Hoc Disease Transmission Advisory Committee database. A chart review of the donors of "proven" or "probable" cases was conducted. From January 1, 2016, to December 31, 2021, 7735 organs were procured from 3767 HBcAb+ donors and transplanted into 7469 recipients; 545 (14.5%) donors were also HCV+. HBV transmission or reactivation occurred in seven recipients. The rate is not significantly different between recipients of HCV+ (0.18%, 2/1115) and the HCV NAT negative (HCV-) organs (0.08%, 5/6354) (p = 0.28) or between recipients of HCV+ and HCV- livers as well as non-liver organs. HBV transmission or reactivation occurred within a median of 319 (range, 41-1117) days post-transplant in the setting of missing, inadequate, or truncated prophylaxis. HBV reactivation associated with DAA therapy for HBcAb+ HCV+ organs is less frequent than reported in the non-transplant population, possibly due to the common use of HBV prophylaxis in the at-risk transplant population.

Orthomolecular Therapy for Management & Prevention of SARS-CoV-2 Disease

The renowned coronavirus spread throughout the entire world within a short duration of time and in the process it destroyed everything it encountered in its path, like an invisible fire. This article is to review some of the key features of the coronavirus pandemic and also the virus that causes it. That is, to review the covid-19 virus, which is also known as SARS-CoV-2 (Severe Acute Respiratory Syndrome – Corona Virus – 2). Several vaccines have been developed worldwide for the prevention and treatment of this virus, but due to the limitation of their distribution and manufacturing capacities, the targeted global “herd immunity” has not been achieved. Meanwhile, SARS-CoV-2 is constantly mutating and that unfortunately limits the effectiveness of various vaccination campaigns, notwithstanding the speed of the campaigns. Meanwhile, the current antiviral drugs being used including ivermectin, hydroxychloroquine, ritonavir and lopinavir have not resulted into any conclusive benefits to those infected. Therefore, more effective treatments that are affordable, safe and available globally are urgently needed for targeting the SARS-CoV-2 infections. Covid-19 infections affects the upper respiratory tract and also the lower respiratory tract. However, about 80% of the patients with the disease will not require any hospitalization because their symptoms are relatively mild. But, for the remaining 20% patients, the disease deteriorates to the lower respiratory tract and results into pneumonia. Unfortunately, about 6% of the covid-19 patients with pneumonia (out of the 20% patients with lower respiratory tract involvement) eventually develop respiratory failure which usually require urgent admission to an Intensive Care Unit (ICU) for adequate support. The most frequent and commonly reported symptoms of covid-19 infections are cough, shortness of breath, and fever. The clinical diagnosis for covid-19 infections can be made using the symptoms, chest imaging, and exposures history. In addition to that, an individual that is suspected to have covid-19 can be evaluated and thereafter confirmed for SARS-CoV-2 based on quantitative Real Time – Polymerase Chain Reaction (qRT-PCR), which is a viral nucleic acid test. So far, covid-19 has proven to be a serious global health threat and it has strained the global healthcare capacity. Therefore, it is very important and necessary to really study it and develop more effective pharmacological treatments which are effective for the treatment and prevention of the disease. In this article, we will review an anti-viral therapy made up of quercetin, zinc and ascorbic acid (vitamin C) combination. This combination has proven to be quite effective and safe. Also, various clinical studies and anecdotal evidence supports its efficacy in the management and prevention of covid-19 infections. The evidence includes a study done at Liaquat University of Medical and Health Sciences (LUMHS), Jamshoro, Pakistan and another study at the Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, China.

RNA extraction-free reduced graphene oxide-based RT-LAMP fluorescence assay for highly sensitive SARS-CoV-2 detection

Infectious diseases have always been a seriously endanger for human life and health. A rapid, accurate and ultra-sensitive virus nucleic acid detection is still a challenge to deal with infectious diseases. Here, a RNA extraction-free reduced graphene oxide-based reverse transcription-loop-mediated isothermal amplification (EF-G-RT-LAMP) fluorescence assay was developed to achieve high-throughput, rapid and ultra-sensitive SARS-CoV-2 RNA detection. The whole detection process only took ∼36 min. The EF-G-RT-LAMP assay achieves a detection limit of 0.6 copies μL−1 with a wide dynamic range of aM-pM. A large number (up to 384) of samples can be detected simultaneously. Simulated detection of the COVID-19 pseudovirus and clinical samples in nasopharyngeal swabs demonstrated a high-throughput, rapid and ultra-sensitive practical detection capability of the EF-G-RT-LAMP assay. The results proved that the assay would be used as a rapid, easy-to-implement approach for epidemiologic diagnosis and could be extended to other nucleic acid detections.

Outcome of RT-PCR in the Diagnosis of Patients with COVID-19 Symptoms: A Study in a COVID-19 Referral Hospital

<i>Introduction: </i>Corona virus (SARS-CoV-2) (COVID-19) outbreak was first reported in China rapidly spread around the world within short period causing global public health emergency. COVID 19 is a highly infectious disease caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). The initial symptoms were fever, cough, dyspnea, myalgia or fatigue, headache, hemoptysis, diarrhea and acute respiratory distress syndrome (ARDS). Hence in diagnosis of COVID-19 RT-PCR of viral nucleic acid is regarded as the reference standard to detect the COVID-19 positive cases. <i>Objective: </i>The aim of this study was to determine the outcome of RT-PCR of the patients with COVID-19 symptoms. <i>Methodology: </i>This was a cross-sectional prospective study carried out in the department of Radiology and imaging in collaboration with the department of Virology and COVID unit in IbnSina Hospital, Dhaka, Bangladesh during January, 2022 to June, 2022. A total of 239 OPD patients with COVID-19 symptoms aged above 18 years were enrolled in this study. The collected data were cleaned, edited and entered into computer for analysis. The data were analyzed using Statistical Package for Social Sciences (SPSS) software, version 23.0. The ethical clearance of this study was obtained from the Ethics Committee of School of Public Health & Life Science, University of South Asia, and Dhaka, Bangladesh. <i>Results: </i>In this study a total of (n=239) patients with COVID-19 symptoms were enrolled. According to age distribution, the majority., 142 (59.42%) patients belonged to the age group >52 years, which was the highest and followed by 44 (18.42%) age group (43-52) years, 36 (15.06%) (33-42) years, 14 (5.85%) (22-32) years and 3 (1.25%) <22 years. According to sex distribution, the majority, 167 (69.85%) were male and 72 (30.12%) were female. Distribution by symptoms, all the patients239 (100%) had general weakness and followed by sneezing 221 (92.46%), fever 236 (98.74%), headache 228 (95.39%), cough 232 (97.07%), respiratory distress, 225 (94.14%), cough and SOB, 237 (99.16%), loss of smell, 212 (88.74%), lac of apatite, 224 (73.72%), asthma 218 (91.21%) and pneumonia, 118 (49.37%). In RT_PCR lab test results, the majority 195 (81.58%) were observed COVID-19 Negative (-) and 44 (18.41%) were observed COVID-19 Positive (+). Among the positive cases, the majority 26 (60%) were male and 18 (40%) were female. <i>Conclusion: </i>This study prevailed around 20 % of the patients with COVID-19 symptoms got COVID-19 Positive in RT-PCR lab test results. Therefore, the patients with COVID-19 symptoms must conduct RT-PCR lab test for their being sure of their COVID-19 affection. At the same time, the male are more vulnerable to COVID-19 than their counter part female.

DNA Polymerase-Endonuclease Efficiently Synthesizes DNA to Prepare DNA Materials and Develop Novel Signal Amplification System.

DNA biosynthesis, a focus of fundamental and applied research, typically involves DNA polymerases by using templates, primers, and dNTPs. Some polymerases can polymerize dNTPs for DNA de novo synthesis, although this is generally to occur randomly. This novel synthesis method has garnered our attention and practical use. Herein, we observed that the addition of endonuclease significantly enhances the efficiency of the de novo synthesis reaction catalyzed by the DNA polymerase. We further investigated the reaction conditions that influence this efficiency. Building on the optimal reaction conditions, we developed a rapid and efficient strategy for preparing DNA hydrogel. Further, coupled with the CRISPR-Cas system, we developed a nucleic acid signal amplification system characterized by versatility, sensitivity, specificity, and no risk of aerosol contamination. We successfully detected viral nucleic acids in clinical samples. In summary, our study demonstrates the significant potential of DNA polymerase- and endonuclease-catalyzed DNA de novo synthesis in diverse applications.

Repeated vaccination does not appear to significantly weaken the protective effect of influenza vaccine in the elderly: A test-negative case-control study in China

BackgroundThe impact of repeated influenza vaccination on vaccine effectiveness has been a topic of debate. Conducting more multinational, multicenter studies in different influenza seasons is crucial for a better understanding of this issue. There is a lack of comprehensive related research reports in China. MethodsUsing the Regional Health Information Platform, we conducted a test-negative case-control study to evaluate the impact of repeated vaccination on the prevention of laboratory-confirmed influenza in individuals aged 60 and above in Ningbo during four influenza seasons from 2018–19 to 2021–22. Influenza-positive cases and negative controls were matched in a 1:1 ratio based on the visiting hospital and the date of influenza testing. Propensity score adjustment and multivariable logistic regression were used to estimate risk and address confounding effects. ResultsDuring the study period, a total of 30,630 elderly patients underwent influenza virus nucleic acid or antigen testing. After exclusions, we included 1976 cases of influenza-positive and 1976 cases of influenza-negative controls. Multivariable logistic regression analysis revealed that individuals receiving the vaccine in two consecutive seasons did not exhibit a significantly increased risk of influenza illness compared to those receiving the vaccine only in the current season (adjusted odds ratio: 1.22, 95% confidence interval: 0.94–1.58). However, the risk of influenza illness was found to be elevated in individuals who received the vaccine only in the previous season (adjusted odds ratio: 1.56, 95% confidence interval: 1.15–2.10) and even further elevated in those who had not received the vaccine in either of the consecutive two seasons (adjusted odds ratio: 3.39, 95% confidence interval: 2.80–4.09). ConclusionsRegardless of the vaccination history in the previous season, receiving the current season influenza vaccine is the best choice for the elderly population. Our study supports the initiative to vaccinate elderly individuals against influenza annually.

A novel syndrome of silent rhinovirus-associated bronchoalveolitis in children with recurrent wheeze

BackgroundRhinovirus (RV) infections trigger wheeze episodes in children. Thus, understanding of the lung inflammatory response to RV in children with wheeze is important. ObjectiveTo examine the associations of RV on lung lavage (BAL) granulocyte patterns and biomarkers of inflammation with age in children with treatment-refractory, recurrent wheeze (n=616). MethodsChildren underwent BAL to examine viral nucleic acid sequences, bacterial cultures, granulocyte counts, and phlebotomy for both general and type-2 inflammatory markers. ResultsDespite the absence of cold symptoms, RV was the most common pathogen detected (30%), and when present, was accompanied by BAL granulocytosis in 75% of children. Compared to children with no BAL pathogens (n=341), those with RV alone (n=127) had greater (p < 0.05) isolated neutrophilia (43% versus 16%), mixed eosinophils and neutrophils (26% versus 11%), and less pauci-granulocytic (27% versus 61%) BAL. Children with RV alone furthermore had biomarkers of active infection with higher total blood neutrophils and serum c-reactive protein (CRP), but no differences in blood eosinophils or total IgE. With advancing age, the log odds of BAL RV alone were lower, 0.82 [0.76-0.88, p < 0.001], but higher, 1.58 [1.01-2.51, p = 0.04], with high-dose daily corticosteroid treatment. ConclusionsChildren with severe recurrent wheeze often (22%) have a silent syndrome of lung RV infection with granulocytic bronchoalveolitis and elevated systemic markers of inflammation. The syndrome is less prevalent by school age, and not informed by markers of type-2 inflammation. We speculate that dysregulated mucosal innate antiviral immunity is a responsible mechanism.

Analytical validation of a semi-automated methodology for quantitative measurement of SARS-CoV-2 RNA in wastewater collected in northern New England.

Wastewater-based epidemiology (WBE) can be used to monitor the community presence of infectious disease pathogens of public health concern such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral nucleic acid has been detected in the stool of SARS-CoV-2-infected individuals. Asymptomatic SARS-CoV-2 infections make community monitoring difficult without extensive and continuous population screening. In this study, we validated a procedure that includes manual pre-processing, automated SARS-CoV-2 RNA extraction and detection workflows using both reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR). Genomic RNA and calibration materials were used to create known concentrations of viral material to determine the linearity, accuracy, and precision of the wastewater extraction and SARS-CoV-2 RNA detection. Both RT-qPCR and RT-ddPCR perform similarly in all the validation experiments, with a limit of detection of 50 copies/mL. A wastewater sample from a care facility with a known outbreak was assessed for viral content in replicate, and we showed consistent results across both assays. Finally, in a 2-week survey of two New Hampshire cities, we assessed the suitability of our methods for daily surveillance. This paper describes the technical validation of a molecular assay that can be used for long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.IMPORTANCEThis paper describes the technical validation of a molecular assay that can be used for the long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.

Logistic regression analysis of the value of biomarkers, clinical symptoms, and imaging examinations in COVID-19 for SARS-CoV-2 nucleic acid detection.

The detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) nucleic acid detection provides a direct basis for diagnosing Coronavirus Disease 2019. However, nucleic acid test false-negative results are common in practice and may lead to missed diagnosis. Certain biomarkers, clinical symptoms, and imaging examinations are related to SARS-CoV-2 nucleic acid detection and potential predictors. We examined nucleic acid test results, biomarkers, clinical symptoms, and imaging examination data for 116 confirmed cases and asymptomatic infections in Zhuhai, China. Patients were divided into nucleic acid-positive and -false-negative groups. Predictive values of biomarkers, symptoms, and imaging for the nucleic acid-positive rate were calculated by Least Absolute Shrinkage and Selection Operators regression analysis and binary logistic regression analysis, and areas under the curve of these indicators were calculated. Hemoglobin (OR = 1.018, 95% CI: 1.006-1.030; P = .004) was higher in the respiratory tract-positive group than the nucleic acid-negative group, but platelets (OR = 0.996, 95% CI: 0.993-0.999; P = .021) and eosinophils (OR = 0.013, 95% CI: 0.001-0.253; P = .004) were lower; areas under the curve were 0.563, 0.614, and 0.642, respectively. Some biomarkers can predict SARS-CoV-2 viral nucleic acid detection rates in Coronavirus Disease 2019 and are potential auxiliary diagnostic tests.

A Virus-Inspired Inhalable Liponanogel Induces Potent Antitumor Immunity and Regression in Metastatic Lung Tumors.

Pulmonary delivery of immunostimulatory agents such as poly(I:C) to activate double-stranded RNA sensors MDA5 and RIG-I within lung-resident antigen-presenting cells is a potential strategy to enhance antitumor immunity by promoting type I interferon secretion. Nevertheless, following pulmonary delivery, poly(I:C) suffers from rapid degradation and poor endosomal escape, thus limiting its potency. Inspired by the structure of a virus that utilizes internal viral proteins to tune the loading and cytosolic delivery of viral nucleic acids, we developed a liponanogel (LNG)-based platform to overcome the delivery challenges of poly(I:C). The LNG comprised an anionic polymer hyaluronic acid-based nanogel core coated by a lipid shell, which served as a protective layer to stabilize the nanogel core in the lungs. The nanogel core was protonated within acidic endosomes to enhance the endosomal membrane permeability and cytosolic delivery of poly(I:C). After pulmonary delivery, LNG-poly(I:C) induced 13.7-fold more IFNβ than poly(I:C) alone and two-fold more than poly(I:C) loaded in the state-of-art lipid nanoparticles [LNP-poly(I:C)]. Additionally, LNG-poly(I:C) induced more potent CD8+ T-cell immunity and stronger therapeutic effects than LNP-poly(I:C). The combination of LNG-poly(I:C) and PD-L1 targeting led to regression of established lung metastases. Due to the ease of manufacturing and the high biocompatibility of LNG, pulmonary delivery of LNG may be broadly applicable to the treatment of different lung tumors and may spur the development of innovative strategies for cancer immunotherapy. Significance: Pulmonary delivery of poly(I:C) with a virus-inspired inhalable liponanogel strongly activates cytosolic MDA5 and RIG-I and stimulates antitumor immunity, representing a promising strategy for safe and effective treatment of metastatic lung tumors.