Development of a universal real-time RT-PCR assay for detection of pan-SARS-coronaviruses with an RNA-based internal control.

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for rapid diagnostic assays to prompt intensified virological monitoring both in human and wild animal populations. To date, there are no clinical validated assays for pan-SARS-coronavirus (pan-SARS-CoV) detection. Here, we suggest an innovative primer design strategy for the diagnosis of pan-SARS-CoVs targeting the envelope (E) gene using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, we developed a new primer-probe set targeting human ß2-microglobulin (B2M) as an RNA-based internal control for process efficacy. The universal RT-qPCR assay demonstrated no false-positive amplifications with other human coronaviruses or 20 common respiratory viruses, and its limit of detection (LOD) was 159.16 copies/ml at 95% detection probability. In clinical validation, the assay delivered 100% sensitive results in the detection of SARS-CoV-2-positive oropharyngeal samples (n = 120), including three variants of concern (Wuhan, Delta, and Omicron). Taken together, this universal RT-qPCR assay provides a highly sensitive, robust, and rapid detection of SARS-CoV-1, SARS-CoV-2, and animal-derived SARS-related CoVs.

Clinical features and viral etiology of acute respiratory infection in an outpatient fever clinic during COVID-19 pandemic in a tertiary hospital in Nanjing, China.

BACKGROUND: Clinical feature and viral etiology for acute respiratory infection (ARI) in the community was unknown during coronavirus disease 2019 (COVID-19) pandemic. OBJECTIVE: In a retrospective study, we aimed to characterize the clinical feature and etiology for the ARI patients admitted to the outpatient fever clinic in Nanjing Drum Tower Hospital between November 2020 and March 2021. METHODS: Fifteen common respiratory pathogens were tested using pharyngeal swabs by multiplex reverse transcriptase-polymerase chain reaction assays. RESULTS: Of the 242 patients, 56 (23%) were tested positive for at least one viral agent. The predominant viruses included human rhinovirus (HRV) (5.4%), parainfluenza virus type III (PIV-III) (5.0%), and human coronavirus-NL63 (HCoV-NL63) (3.7%). Cough, sputum, nasal obstruction, and rhinorrhea were the most prevalent symptoms in patients with viral infection. Elderly and the patients with underlying diseases were susceptible to pneumonia accompanied with sputum and chest oppression. Three (5.4%) patients in virus infection group, whereas 31 (16.7%) in non-viral infection group (p = 0.033), were empirically prescribed with antiviral agents. Among 149 patients who received antibiotic therapy, 30 (20.1%) patients were later identified with viral infection. CONCLUSION: Our study indicated the importance of accurate diagnosis of ARI, especially during the COVID-19 pandemic, which might facilitate appropriate clinical treatment.

The Third dose of CoronVac vaccination induces broad and potent adaptive immune responses that recognize SARS-CoV-2 Delta and Omicron variants.

The waning humoral immunity and emerging contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants resulted in the necessity of the booster vaccination of coronavirus disease 2019 (COVID-19). The inactivated vaccine, CoronaVac, is the most widely supplied COVID-19 vaccine globally. Whether the CoronaVac booster elicited adaptive responses that cross-recognize SARS-CoV-2 variants of concern (VoCs) among 77 healthy subjects receiving the third dose of CoronaVac were explored. After the boost, remarkable elevated spike-specific IgG and IgA responses, as well as boosted neutralization activities, were observed, despite 3.0-fold and 5.9-fold reduced neutralization activities against Delta and Omicron strains compared to that of the ancestral strain. Furthermore, the booster dose induced potent B cells and memory B cells that cross-bound receptor-binding domain (RBD) proteins derived from VoCs, while Delta and Omicron RBD-specific memory B cell recognitions were reduced by 2.7-fold and 4.2-fold compared to that of ancestral strain, respectively. Consistently, spike-specific circulating follicular helper T cells (cTfh) significantly increased and remained stable after the boost, with a predominant expansion towards cTfh17 subpopulations. Moreover, SARS-CoV-2-specific CD4+ and CD8+ T cells peaked and sustained after the booster. Notably, CD4+ and CD8+ T cell recognition of VoC spike was largely preserved compared to the ancestral strain. Individuals without generating Delta or Omicron neutralization activities had comparable levels of CD4+ and CD8+ T cells responses as those with detectable neutralizing activities. Our study demonstrated that the CoronaVac booster induced broad and potent adaptive immune responses that could be effective in controlling SARS-CoV-2 Delta and Omicron variants.

Knowledge and behaviour of community residents' face mask-wearing during the COVID-19 pandemic: a cross-sectional study in Shanghai, China.

OBJECTIVES: COVID-19 is an infectious disease spreading through respiratory droplets. Using a face mask correctly is one of the essential preventive measures. We launched a survey to discover the current face mask-wearing problems for better prevention and control of the pandemic. SETTING/PARTICIPANTS: A cross-sectional study was conducted with 1240 residents in Shanghai during 18-28 February 2020. Stratified random sampling was adopted in the urban area, urban-rural fringe area and rural area. A mobile self-designed questionnaire was used. MAIN OUTCOME MEASURES: Analysis of the demographic and sociological factors influencing residents' face mask type selection, disposable surgical mask-wearing behaviour, replacement time and the storage method for reuse of disposable surgical masks. RESULTS: The accuracy of residents' face mask type selection was rated 41.61% (513/1233), and that of urban-rural fringe residents, low-education residents, elderly residents, were low, respectively (p<0.001). 96.67% of residents always wore face masks in crowded public places. Multiple-factor analysis showed that region, gender and education level were the influencing factors for entirely fitting the disposable surgical mask to the face. Region was an influencing factor for not touching the mask's external surface while wearing or removing it. Education level was an influencing factor for recognising the external and inner mask surface, upper and lower edge of a disposable surgical mask. The accuracy of the mask replacement time was 25.63% (316/1223). It was low with elderly, low-education residents, respectively (p<0.001).The accuracy of residents' storage method for reuse of disposable surgical mask was 3.81% (47/1233), and region, age and education level were influencing factors. CONCLUSIONS: Shanghai residents recorded a high rate of wearing face masks. Selection of an appropriate type of face mask, disposable surgical mask-wearing behaviour, replacement time and storage method for reuse of disposable surgical masks should be particularly emphasised in future behaviour change interventions.

Human urinary kallidinogenase in acute ischemic stroke: A single-arm, multicenter, phase IV study (RESK study).

AIMS: Human urinary kallidinogenase (HUK) has shown favorable efficacies in acute ischemic stroke (AIS) treatment. We sought confirmation of the safety and efficacy of HUK for AIS in a large population. METHODS: RESK study enrolled patients with AIS of anterior circulation to receive HUK infusion. The primary endpoint was the incidence of treatment-emergent adverse events (AEs). Secondary endpoints assessed neurological and functional improvements and stroke recurrent rate. RESULTS: Of 1206 eligible patients, 1202 patients received at least one dose of HUK infusion and 983 (81.5%) completed the study. The incidence of treatment-emergent AEs and serious AEs were 55.99% and 2.41%, respectively. Pre-specified AEs of special interest occurred in 21.71% of patients, but the majority were mild and unrelated to therapy. Hypertension, age, treatment time, and drug combination were identified to be associated with drug-related blood pressure reduction. Neurological and functional evaluations revealed favorable outcomes from baseline to post-treatment assessment. The cumulative recurrence rate of stroke was 2.50% during the 90-day assessment. CONCLUSION: HUK had an acceptable safety and tolerability profile in AIS patients. Besides, HUK demonstrated the neurological and functional improvements in AIS, further confirming its clinical efficacy in a real-world large population.

Dynamic SARS-CoV-2-specific B-cell and T-cell responses following immunization with an inactivated COVID-19 vaccine.

OBJECTIVE: The dynamic adaptive immune responses elicited by the inactivated virus vaccine CoronaVac remain elusive. METHODS: In a prospective cohort of 100 healthcare professionals naïve for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who received two doses of CoronaVac, we analysed SARS-CoV-2-specific humoral and cellular responses at four different timepoints, including before vaccination (T1), 2 weeks after the first dose (T2), 2 weeks after the booster dose (T3), and 8-10 weeks after the booster dose (T4). SARS-CoV-2-specific antibodies, serum neutralizing activities, peripheral B cells, CD4+ and CD8+ T cells and their memory subsets were simultaneously measured in this cohort. RESULTS: SARS-CoV-2 spike-specific IgG responses reached a peak (geometric mean titre (GMT) 54827, 30969-97065) after two doses and rapidly declined (GMT 502, 212-1190) at T4, whereas suboptimal IgA responses were detected (GMT 5, 2-9). Spike-specific circulating B cells (0.60%, 0.46-0.73% of total B cells) and memory B cells (1.18%, 0.92-1.44% of total memory B cells) were effectively induced at T3 and sustained over time (0.33%, 0.23-0.43%; 0.87%, 0.05-1.67%, respectively). SARS-CoV-2-specific circulating CD4+ T cells (0.57%, 0.47-0.66%) and CD8+ T cells (1.29%, 1.04-1.54%) were detected at T3. At T4, 0.78% (0.43-1.20%) of memory CD4+ T cells and 0.68% (0.29-1.30%) of memory CD8+ T cells were identified as SARS-CoV-2-specific, while 0.62% (0.51-0.75%) of CD4+ T cells and 0.47% (0.38-0.58%) of CD8+ T cells were SARS-CoV-2-specific terminally differentiated effector memory cells. Furthermore, age and interval between doses affected the magnitude of CoronaVac-induced immune responses. SARS-CoV-2 memory CD4+ T cells were strongly associated with both receptor binding domain (RBD)-specific memory B cells (r 0.87, p <0.0001) and SARS-CoV-2-specific memory CD8+ T cells (r 0.48, p <0.0001). CONCLUSIONS: CoronaVac induced robust circulating and memory B cell and T cell responses. Our study offers new insight into the underlying immunobiology of inactivated virus vaccines in humans and may have implications for vaccine strategies in the future.