Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B.

The coronavirus disease 2019 (COVID-19) pandemic caused extensive loss of life worldwide. Further, the COVID-19 and influenza mix-infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real-time reverse-transcription PCR (RT-PCR) assay for early diagnosis of COVID-19 was developed, but detection was time-consuming (4-6 h). To improve the diagnosis of COVID-19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS-CoV-2 were selected, and specific primers were designed. We validated our method using SARS-CoV-2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID-19 and Influenza A/B (RT-PCR-verified) positive patients. The method could detect SARS-CoV-2 plasmid standard DNA quantitatively between 102 and 105 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID-19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS-CoV-2, H1N1, H3N2 and influenza B, and adapted for point-of-care (POC) detection of a broad range of infectious pathogens in resource-limited settings.

Polylactic acid face masks: Are these the sustainable solutions in times of COVID-19 pandemic?

The global massive consumption of disposable face masks driven by the ongoing COVID-19 pandemic has emerged as a blooming disaster to both the land and marine environment that might last for generations. Growing public concerns have been raised over the management and control of this new form of plastic pollution, and one of the proposed sustainable solution is to use renewable and/or biodegradable resources to develop mask materials in order to minimize their environmental impacts. As a representative biodegradable polymer, polylactic acid (PLA) has been proposed as a promising candidate to produce non-woven face masks instead of those fossil-based polymers. To further explore the feasibility of this alternative mask material, the present work aims to study both the hydrolytic and bio-degradation behaviors of pure PLA-derived 3-ply disposable face masks at ambient temperature. Hydrolytic degradability was investigated at different pH conditions of 2, 7 and 13 with the whole piece of face mask soaked for regular timed intervals up to 8 weeks. Weight loss study showed neutral and acidic conditions had minimal effect on PLA masks, but rapid degradation occurred under basic conditions in the first week with a sharp 25% decrease in weight that slowly tapered off, coupled with solution pH dropping from 13 to 9.6. This trend was supported by mechanical property, bacterial filtration efficiency (BFE) and particulate filtration efficiency (PFE) studies. Masks soaked in basic conditions had their modulus and tensile strength dropped by more than 50% after 8 weeks where the middle layer reached 68% and 90% respectively just after 48 h, and BFE and PFE decreased by 14% and 43% respectively after 4 weeks, which was much more significant than those in neutral and acidic conditions. Base degradation was also supported by nuclear magnetic resonance (NMR) and fourier transform infrared (FTIR), which disclosed that only the middle layer undergo major degradation with random chain scission and cleavage of enol or enolate chain ends, while outer and inner layers were much less affected. Scanning electron microscopy (SEM) attributed this observation to thinner PLA fibers for the middle layer of 3-7 µm diameter, which on average is 3 times smaller. This degradation was further supported by gel permeation chromatography (GPC) which saw an increase in lower molecular weight fragment Mw ~ 800 Da with soaking duration. The biodegradation behavior was studied under OECD 301F specification in sewage sludge environment. Similarly, degradation to the middle meltblown layer was more extensive, where the average weight loss and carbon loss was 25.8% and 25.7% respectively, double that of outer/inner spunbond layer. The results showed that the face masks did not completely disintegrate after 8 weeks, but small solubilized fragments of PLA formed in the biodegradation process can be completely mineralized into carbon dioxide without generation of secondary microplastic pollution in the environment. PLA masks are therefore a slightly greener option to consider in times of a pandemic that the world was caught unprepared; however future research on masks could be geared towards a higher degradability material that fully breaks down into non-harmful components while maintaining durability, filtration and protection properties for users.

Identification of potential therapeutic targets and mechanisms of COVID-19 through network analysis and screening of chemicals and herbal ingredients.

After experiencing the COVID-19 pandemic, it is widely acknowledged that a rapid drug repurposing method is highly needed. A series of useful drug repurposing tools have been developed based on data-driven modeling and network pharmacology. Based on the disease module, we identified several hub proteins that play important roles in the onset and development of the COVID-19, which are potential targets for repositioning approved drugs. Moreover, different network distance metrics were applied to quantify the relationship between drug targets and COVID-19 disease targets in the protein-protein-interaction (PPI) network and predict COVID-19 therapeutic effects of bioactive herbal ingredients and chemicals. Furthermore, the tentative mechanisms of candidates were illustrated through molecular docking and gene enrichment analysis. We obtained 15 chemical and 15 herbal ingredient candidates and found that different drugs may play different roles in the process of virus invasion and the onset and development of the COVID-19 disease. Given pandemic outbreaks, our method has an undeniable immense advantage in the feasibility analysis of drug repurposing or drug screening, especially in the analysis of herbal ingredients.

Association between vaccinations and clinical manifestations in children with COVID-19.

BACKGROUND: The infection rate of Coronavirus Disease 2019 (COVID-19) in children was less than that in adults. However, the underlining reason is not well known. METHODS: Children with COVID-19 were recruited from two Children's Hospitals in Wuhan and Shanghai in this case-control study. The associations of initial symptoms with age, vaccinations of Bacillus Calmette Guerin (BCG), and influenza and pathogens were determined by Chi-square t-test. RESULTS: We evaluated 248 confirmed cases, and 56 suspected cases with COVID-19. The median age was 6.82 years old, and 118 cases (38.82%) were girls. Furthermore, 30.26% of all patients were asymptomatic cases. The percentage of asymptomatic cases vaccinated with BCG was not significantly higher than that without BCG vaccination [86/280 (30.71%) vs. 6/13 (46.15%), P=0.203], and initial symptoms were not related with immunized influenza vaccine (P=0.267). Compared to parameters in pediatric patients with normal body temperatures, patients with fever had higher C reactive protein (CRP) (P<0.001). CONCLUSIONS: Pediatric COVID-19 patients with BCG vaccinations exhibit similar clinical manifestations compared to those without BCG vaccinations, and the severity of symptoms in pediatric patients may be related to the maturity of immune function.

Clinical and Immune Features of Hospitalized Pediatric Patients With Coronavirus Disease 2019 (COVID-19) in Wuhan, China.

Importance: The epidemiologic and clinical characteristics of pediatric patients with coronavirus disease 2019 (COVID-19) have been reported, but information on immune features associated with disease severity is scarce. Objective: To delineate and compare the immunologic features of mild and moderate COVID-19 in pediatric patients. Design, Setting, and Participants: This single-center case series included 157 pediatric patients admitted to Wuhan Children's Hospital with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data were collected from January 25 to April 18, 2020. Exposures: Documented SARS-CoV-2 infection. Main Outcomes and Measures: Clinical and immunologic characteristics were collected and analyzed. Outcomes were observed until April 18, 2020. Results: Of the 157 pediatric patients with COVID-19, 60 (38.2%) had mild clinical type with pneumonia, 88 (56.1%) had moderate cases, 6 (3.8%) had severe cases, and 3 (1.9%) were critically ill. The 148 children with mild or moderate disease had a median (interquartile range [IQR]) age of 84 (18-123) months, and 88 (59.5%) were girls. The most common laboratory abnormalities were increased levels of alanine aminotransferase (ALT) (median [IQR], 16.0 [12.0-26.0] U/L), aspartate aminotransferase (AST) (median [IQR], 30.0 [23.0-41.8] U/L), creatine kinase MB (CK-MB) activity (median [IQR], 24.0 [18.0-34.0] U/L), and lactate dehydrogenase (LDH) (median [IQR], 243.0 [203.0-297.0] U/L), which are associated with liver and myocardial injury. Compared with mild cases, levels of inflammatory cytokines including interleukin 6, tumor necrosis factor α, and interferon γ were unchanged, whereas the level of immune suppressive interleukin 10 was markedly increased in moderate cases compared with mild cases (median [IQR], 3.96 [3.34-5.29] pg/mL vs 3.58 [3.10-4.36] pg/mL; P = .048). There was no statistically significant difference in absolute number of lymphocytes (including T cells and B cells) between mild and moderate cases, but moderate cases were associated with a decrease in neutrophil levels compared with mild cases (median [IQR], 2310/µL [1680/µL-3510/µL] vs 3120/µL [2040/µL-4170/µL]; P = .01). Immunoglobin G and the neutrophil to lymphocyte ratio were negatively associated with biochemical indices related to liver and myocardial injury (immunoglobulin G, ALT: r, -0.3579; AST: r, -0.5280; CK-MB activity: r, -0.4786; LDH: r, -0.4984; and neutrophil to lymphocyte ratio, ALT: r, -0.1893; AST: r, -0.3912; CK-MB activity: r, -0.3428; LDH: r, -0.3234), while counts of lymphocytes, CD4+ T cells, and interleukin 10 showed positive associations (lymphocytes, ALT: r, 0.2055; AST: r, 0.3615; CK-MB activity: r, 0.338; LDH: r, 0.3309; CD4+ T cells, AST: r, 0.4701; CK-MB activity: r, 0.4151; LDH: r, 0.4418; interleukin 10, ALT: r, 0.2595; AST: r, 0.3386; CK-MB activity: r, 0.3948; LDH: r, 0.3794). Conclusions and Relevance: In this case series, systemic inflammation rarely occurred in pediatric patients with COVID-19, in contrast with the lymphopenia and aggravated inflammatory responses frequently observed in adults with COVID-19. Gaining a deeper understanding of the role of neutrophils, CD4+ T cells, and B cells in the pathogenesis of SARS-CoV-2 infection could be important for the clinical management of COVID-19.

SARS-CoV-2 infection in children - Understanding the immune responses and controlling the pandemic.

In December 2019, a cluster of patients with severe pneumonia caused by a novel coronavirus (SARS-CoV-2) emerged in the city of Wuhan, China. The disease is now termed coronavirus disease 2019 (COVID-19). In the early reports, the patients were mainly middle-aged and elderly men, and children appeared to be less susceptible to this infection. With modern and efficient transportation, the disease quickly spread to almost all corners of the world and the mortality far exceeds that caused by severe acute respiratory syndrome (SARS) coronavirus or Middle East respiratory syndrome (MERS) coronavirus. As the number of children with COVID-19 gradually increases, the disease has been documented in premature babies, infants, children, and adolescents. Severe and fatal cases in children are relatively rare. The burden of disease in children has been relatively low, but the high proportions of asymptomatic or mildly symptomatic infections in children deserve careful attention. A clear understanding of the immune responses to the virus in children and the transmission potential of asymptomatic children is of paramount importance for the development of specific treatments and vaccine in order to effectively control the ongoing pandemic.