Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus with high nucleotide identity to SARS-CoV and to SARS-related coronaviruses that have been detected in horseshoe bats, has spread across the world and had a global effect on healthcare systems and economies1,2. A suitable small animal model is needed to support the development of vaccines and therapies. Here we report the pathogenesis and transmissibility of SARS-CoV-2 in golden (Syrian) hamsters (Mesocricetus auratus). Immunohistochemistry assay demonstrated the presence of viral antigens in nasal mucosa, bronchial epithelial cells and areas of lung consolidation on days 2 and 5 after inoculation with SARS-CoV-2, followed by rapid viral clearance and pneumocyte hyperplasia at 7 days after inoculation. We also found viral antigens in epithelial cells of the duodenum, and detected viral RNA in faeces. Notably, SARS-CoV-2 was transmitted efficiently from inoculated hamsters to naive hamsters by direct contact and via aerosols. Transmission via fomites in soiled cages was not as efficient. Although viral RNA was continuously detected in the nasal washes of inoculated hamsters for 14 days, the communicable period was short and correlated with the detection of infectious virus but not viral RNA. Inoculated and naturally infected hamsters showed apparent weight loss on days 6-7 post-inoculation or post-contact; all hamsters returned to their original weight within 14 days and developed neutralizing antibodies. Our results suggest that features associated with SARS-CoV-2 infection in golden hamsters resemble those found in humans with mild SARS-CoV-2 infections.

Antigen identification and high-throughput interaction mapping by reprogramming viral entry.

Deciphering immune recognition is critical for understanding a broad range of diseases and for the development of effective vaccines and immunotherapies. Efforts to do so are limited by a lack of technologies capable of simultaneously capturing the complexity of adaptive immunoreceptor repertoires and the landscape of potential antigens. To address this, we present receptor-antigen pairing by targeted retroviruses, which combines viral pseudotyping and molecular engineering approaches to enable one-pot library-on-library interaction screens by displaying antigens on the surface of lentiviruses and encoding their identity in the viral genome. Antigen-specific viral infection of cell lines expressing human T or B cell receptors allows readout of both antigen and receptor identities via single-cell sequencing. The resulting system is modular, scalable and compatible with any cell type. These techniques provide a suite of tools for targeted viral entry, molecular engineering and interaction screens with broad potential applications.

Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay.

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.

Diagnostics for COVID-19: moving from pandemic response to control.

Diagnostics have proven to be crucial to the COVID-19 pandemic response. There are three major methods for the detection of SARS-CoV-2 infection and their role has evolved during the course of the pandemic. Molecular tests such as PCR are highly sensitive and specific at detecting viral RNA, and are recommended by WHO for confirming diagnosis in individuals who are symptomatic and for activating public health measures. Antigen rapid detection tests detect viral proteins and, although they are less sensitive than molecular tests, have the advantages of being easier to do, giving a faster time to result, of being lower cost, and able to detect infection in those who are most likely to be at risk of transmitting the virus to others. Antigen rapid detection tests can be used as a public health tool for screening individuals at enhanced risk of infection, to protect people who are clinically vulnerable, to ensure safe travel and the resumption of schooling and social activities, and to enable economic recovery. With vaccine roll-out, antibody tests (which detect the host's response to infection or vaccination) can be useful surveillance tools to inform public policy, but should not be used to provide proof of immunity, as the correlates of protection remain unclear. All three types of COVID-19 test continue to have a crucial role in the transition from pandemic response to pandemic control.

Sensitive Immunodetection of Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern 501Y.V2 and 501Y.V1.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may influence the effectiveness of existing laboratory diagnostics. In the current study we determined whether the British (20I/501Y.V1) and South African (20H/501Y.V2) SARS-CoV-2 variants of concern are detected with an in-house S1-based antigen detection assay, analyzing spiked pools of quantitative reverse-transcription polymerase chain reaction-negative nasopharyngeal swab specimens. The assay, combining 4 monoclonal antibodies, allowed sensitive detection of both the wild type and the variants of concern, despite accumulation of several mutations in the variants' S1 region-results suggesting that this combination, targeting distinct epitopes, enables both specificity and the universality.

Performance Characteristics of a Rapid Severe Acute Respiratory Syndrome Coronavirus 2 Antigen Detection Assay at a Public Plaza Testing Site in San Francisco.

We evaluated the performance of the Abbott BinaxNOW rapid antigen test for coronavirus disease 2019 (Binax-CoV2) to detect virus among persons, regardless of symptoms, at a public plaza site of ongoing community transmission. Titration with cultured severe acute respiratory syndrome coronavirus 2 yielded a human observable threshold between 1.6 × 104-4.3 × 104 viral RNA copies (cycle threshold [Ct], 30.3-28.8). Among 878 subjects tested, 3% (26 of 878) were positive by reverse-transcription polymerase chain reaction, of whom 15 of 26 had a Ct <30, indicating high viral load; of these, 40% (6 of 15) were asymptomatic. Using this Ct threshold (<30) for Binax-CoV2 evaluation, the sensitivity of Binax-CoV2 was 93.3% (95% confidence interval, 68.1%-99.8%) (14 of 15) and the specificity was 99.9% (99.4%-99.9%) (855 of 856).

Performance and Implementation Evaluation of the Abbott BinaxNOW Rapid Antigen Test in a High-Throughput Drive-Through Community Testing Site in Massachusetts.

Rapid diagnostic tests (RDTs) for SARS-CoV-2 antigens (Ag) that can be performed at point of care (POC) can supplement molecular testing and help mitigate the COVID-19 pandemic. Deployment of an Ag RDT requires an understanding of its operational and performance characteristics under real-world conditions and in relevant subpopulations. We evaluated the Abbott BinaxNOW COVID-19 Ag card in a high-throughput, drive-through, free community testing site in Massachusetts using anterior nasal (AN) swab reverse transcriptase PCR (RT-PCR) for clinical testing. Individuals presenting for molecular testing in two of seven lanes were offered the opportunity to also receive BinaxNOW testing. Dual AN swabs were collected from symptomatic and asymptomatic children (≤18 years of age) and adults. BinaxNOW testing was performed in a testing pod with temperature/humidity monitoring. One individual performed testing and official result reporting for each test, but most tests had a second independent reading to assess interoperator agreement. Positive BinaxNOW results were scored as faint, medium, or strong. Positive BinaxNOW results were reported to patients by phone, and they were instructed to isolate pending RT-PCR results. The paired RT-PCR result was the reference for sensitivity and specificity calculations. Of 2,482 participants, 1,380 adults and 928 children had paired RT-PCR/BinaxNOW results and complete symptom data. In this study, 974/1,380 (71%) adults and 829/928 (89%) children were asymptomatic. BinaxNOW had 96.5% (95% confidence interval [CI], 90.0 to 99.3) sensitivity and 100% (95% CI, 98.6 to 100.0) specificity in adults within 7 days of symptoms and 84.6% (95% CI, 65.1 to 95.6) sensitivity and 100% (95% CI, 94.5 to 100.0) specificity in children within 7 days of symptoms. Sensitivity and specificity in asymptomatic adults were 70.2% (95% CI, 56.6 to 81.6) and 99.6% (95% CI, 98.9 to 99.9), respectively, and in asymptomatic children, they were 65.4% (95% CI, 55.6 to 74.4) and 99.0% (95% CI, 98.0 to 99.6), respectively. By cycle threshold (CT ) value cutoff, sensitivity in all subgroups combined (n = 292 RT-PCR-positive individuals) was 99.3% with CT values of ≤25, 95.8% with CT values of ≤30, and 81.2% with CT values of ≤35. Twelve false-positive BinaxNOW results (out of 2,308 tests) were observed; in all 12, the test bands were faint but otherwise normal and were noted by both readers. One invalid BinaxNOW result was identified. Interoperator agreement (positive versus negative BinaxNOW result) was 100% (n = 2,230/2,230 double reads). Each operator was able to process 20 RDTs per hour. In a separate set of 30 specimens (from individuals with symptoms ≤7 days) run at temperatures below the manufacturer's recommended range (46 to 58.5°F), sensitivity was 66.7% and specificity 95.2%. BinaxNOW had very high specificity in both adults and children and very high sensitivity in newly symptomatic adults. Overall, 95.8% sensitivity was observed with CT values of ≤30. These data support public health recommendations for use of the BinaxNOW test in adults with symptoms for ≤7 days without RT-PCR confirmation. Excellent interoperator agreement indicates that an individual can perform and read the BinaxNOW test alone. A skilled laboratorian can perform and read 20 tests per hour. Careful attention to temperature is critical.

High performance of a novel antigen detection test on nasopharyngeal specimens for diagnosing SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has become a major public health issue worldwide. Developing and evaluating rapid and easy-to-perform diagnostic tests is a high priority. The current study was designed to assess the diagnostic performance of an antigen-based rapid detection test (COVID-VIRO®) in a real-life setting. Two nasopharyngeal specimens of symptomatic or asymptomatic adult patients hospitalized in the Infectious Diseases Department or voluntarily accessing the COVID-19 Screening Department of the Regional Hospital of Orléans, France, were concurrently collected. The diagnostic specificity and sensitivity of COVID VIRO® results were compared to those of real-time reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) results. A subset of patients underwent an additional oropharyngeal and/or saliva swab for rapid testing. A total of 121 patients confirmed to be infected and 127 patients having no evidence of recent or ongoing infection were enrolled for a total of 248 nasopharyngeal swab specimens. Overall, the COVID-VIRO® sensitivity was 96.7% (CI, 93.5%-99.9%). In asymptomatic patients, symptomatic patients having symptoms for more than 4 days and those with an RT-qPCR cycle threshold value ≥ 32, the sensitivities were 100%, 95.8%, and 91.9%, respectively. The concordance between RT-qPCR and COVID VIRO® rapid test results was 100% for the 127 patients with no SARS-CoV-2 infection. The COVID-VIRO® test had 100% specificity and sensitivity greater than 95%, which are better than the recommendations set forth by the WHO (specificity ≥ 97%-100%, sensitivity ≥ 80%). These rapid tests may be particularly useful for large-scale screening in emergency departments, low-resource settings, and airports.

Placental SARS-CoV-2 in a pregnant woman with mild COVID-19 disease.

The full impact of coronavirus disease 2019 (COVID-19) on pregnancy remains uncharacterized. Current literature suggests minimal maternal, fetal, and neonatal morbidity and mortality. COVID-19 manifestations appear similar between pregnant and nonpregnant women. We present a case of placental severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a woman with mild COVID-19 disease, then review the literature. Reverse transcriptase polymerase chain reaction was performed to detect SARS-CoV-2. Immunohistochemistry staining was performed with specific monoclonal antibodies to detect SARS-CoV-2 antigen or to identify trophoblasts. A 29-year-old multigravida presented at 40-4/7 weeks for labor induction. With myalgias 2 days prior, she tested positive for SARS-CoV-2. We demonstrate maternal vascular malperfusion, with no fetal vascular malperfusion, as well as SARS-CoV-2 virus in chorionic villi endothelial cells, and also rarely in trophoblasts. To our knowledge, this is the first report of placental SARS-CoV-2 despite mild COVID-19 disease (no symptoms of COVID-19 aside from myalgias); patient had no fever, cough, or shortness of breath, but only myalgias and sick contacts. Despite her mild COVID-19 disease in pregnancy, we demonstrate placental vasculopathy and presence of SARS-CoV-2 virus across the placenta. Evidence of placental COVID-19 raises concern for placental vasculopathy (potentially leading to fetal growth restriction and other pregnancy complications) and possible vertical transmission-especially for pregnant women who may be exposed to COVID-19 in early pregnancy. This raises important questions of whether future pregnancy guidance should include stricter pandemic precautions, such as screening for a wider array of COVID-19 symptoms, increased antenatal surveillance, and possibly routine COVID-19 testing throughout pregnancy.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

Activation of the Liver X Receptor Pathway Inhibits HBV Replication in Primary Human Hepatocytes.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is ranked among the top health priorities worldwide. Accumulating evidence suggests that HBV infection and replication are closely associated with liver metabolism. The liver X receptors (LXRs), which belong to the superfamily of nuclear hormone receptors, are important physiological regulators of lipid and cholesterol metabolism. However, the association between the LXR pathway and HBV infection remains largely unclear. APPROACH AND RESULTS: In this study, the antiviral activity of LXR agonists was investigated using multiple HBV cellular models. We observed that in HBV-infected primary human hepatocytes (PHHs), synthetic LXR agonists (T0901317, GW3965, and LXR-623), but not an LXR antagonist (SR9238), potently inhibited HBV replication and gene expression, as demonstrated by substantial reductions in viral RNA, DNA, and antigen production following agonist treatment. However, covalently closed circular DNA (cccDNA) levels were not significantly reduced by the agonists. In addition, no rebound in viral replication was observed after treatment withdrawal, indicating a long-lasting inhibitory effect. These results suggest that LXR agonists decrease the transcriptional activity of cccDNA. In contrast, no significant anti-HBV effect was observed in HepG2-derived cell lines. Interestingly, LXR agonist treatment strongly reduced cholesterol 7α-hydroxylase 1 (CYP7A1) mRNA levels. Knockdown of CYP7A1 gene expression with small interfering RNA inhibited HBV activity in PHHs, suggesting CYP7A1 as a potential factor contributing to the antiviral effects of LXR agonists. CONCLUSIONS: We found that activation of the LXR pathway with synthetic LXR agonists could elicit potent anti-HBV activity in PHHs, possibly through sustained suppression of cccDNA transcription. Our work highlights the therapeutic potential of targeting the LXR pathway for the treatment of chronic HBV infection.

Evaluation of SARS-CoV-2 antigen-based rapid diagnostic kits in Pakistan: formulation of COVID-19 national testing strategy.

Rapid diagnosis of SARS-CoV-2 during pandemic enables timely treatment and prevention of COVID-19. Evaluating the accuracy and reliability of rapid diagnostic testing kits is crucial for surveillance and diagnosis of SARS-CoV-2 infections in general population, injection drug users, multi-transfused populations, healthcare workers, prisoners, barbers and other high risk populations. The aim of this study was to evaluate performance and effectiveness of nasopharyngeal swab (NSP) and saliva based rapid antigen detection testing kits in comparison with USFDA approved triple target gold standard real-time polymerase chain reaction. A cross-sectional study was conducted on 33,000 COVID-19 suspected patients. From RT-PCR positive patients, nasopharyngeal swab (NSP) and saliva samples were obtained for evaluation of rapid COVID-19 testing kits (RDT). 100/33,000 (0.3%) of specimens were RT-PCR positive for SARS-CoV-2. Among RT-PCR positive, 62% were males, 34% were females, and 4% were children. The NSP-RDT (Lepu Medical China) analysis revealed 53% reactivity among males, 58% reactivity among females, and 25% reactivity among children. However saliva based RDT (Lepu Medical China) analysis showed 21% reactivity among males and 23% among females, and no reactivity in children. False negative results were significantly more pronounced in saliva based RDT as compared to NSP-RDT. The sensitivity of these NSP-RDT and saliva based RDT were 52% and 21% respectively. The RDTs evaluated in this study showed limited sensitivities in comparison to gold standard RT-PCR, indicating that there is a dire need in Pakistan for development of suitable testing to improve accurate COVID-19 diagnosis in line with national demands.

Full-length G glycoprotein directly extracted from rabies virus with detergent and then stabilized by amphipols in liquid and freeze-dried forms.

Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly targeted by the host immune response. Obtaining these MPs in a soluble and stable form constitutes a real challenge, regardless of the application purposes (e.g. quantification/characterization assays, diagnosis, and preventive and curative strategies). A rapid process to obtain a native-like antigen by solubilization of a full-length MP directly from a pathogen is reported herein. Rabies virus (RABV) was used as a model for this demonstration and its full-length G glycoprotein (RABV-G) was stabilized with amphipathic polymers, named amphipols (APols). The stability of RABV-G trapped in APol A8-35 (RABV-G/A8-35) was evaluated under different stress conditions (temperature, agitation, and light exposure). RABV-G/A8-35 in liquid form exhibited higher unfolding temperature (+6°C) than in detergent and was demonstrated to be antigenically stable over 1 month at 5°C and 25°C. Kinetic modeling of antigenicity data predicted antigenic stability of RABV-G/A8-35 in a solution of up to 1 year at 5°C. The RABV-G/A8-35 complex formulated in an optimized buffer composition and subsequently freeze-dried displayed long-term stability for 2-years at 5, 25, and 37°C. This study reports for the first time that a natural full-length MP extracted from a virus, complexed to APols and subsequently freeze-dried, displayed long-term antigenic stability, without requiring storage under refrigerated conditions.

Expression of HCV genotype-4 core antigen in prokaryotic E. coli system for diagnosis of HCV infection in Egypt.

BACKGROUND: Egypt has a high prevalence of hepatitis C virus (HCV) infection with 92.5% of genotype-4. AIM: This study aimed to clone and express the core gene of HCV genotype-4 for using it to develop a highly sensitive, specific, and cost-effective diagnostic assay for detecting HCV infection. METHODS: Using synthetic HCV genotype-4 core gene, pET15b as E. coli expression vector, and 1 mM lactose as inducer, the HCV core protein (MW 17 kDa) was expressed in the form of inclusion bodies (IBs) that was purified and solubilized using 8 M guanidinium HCl. The recombinant core protein was in vitro refolded by a rapid dilution method for further purification using weak cation exchange liquid chromatography. The immunogenicity of the purified protein was tested by ELISA using 129 serum samples. RESULTS: The recombinant core protein was successfully expressed and purified. The results also showed that the in-house anti-HCV core assay is accurate, specific (~96.6%), and highly sensitive (~100%) in accordance with the commercial ELISA kit. CONCLUSION: The sensitivity, specificity, and reproducibility of the developed assay were high and promising to be used as a screening assay for detecting HCV infection.

Detection of diarrhoea associated rotavirus and co-infection with diarrhoeagenic pathogens in the Littoral region of Cameroon using ELISA, RT-PCR and Luminex xTAG GPP assays.

BACKGROUND: Despite the global roll-out of rotavirus vaccines (RotaTeq/Rotarix / ROTAVAC/Rotasiil), mortality and morbidity due to group A rotavirus (RVA) remains high in sub-Saharan Africa, causing 104,000 deaths and 600,000 hospitalizations yearly. In Cameroon, Rotarix™ was introduced in March 2014, but, routine laboratory diagnosis of rotavirus infection is not yet a common practice, and vaccine effectiveness studies to determine the impact of vaccine introduction have not been done. Thus, studies examining RVA prevalence post vaccine introduction are needed. The study aim was to determine RVA prevalence in severe diarrhoea cases in Littoral region, Cameroon and investigate the role of other diarrheagenic pathogens in RVA-positive cases. METHODS: We carried out a study among hospitalized children < 5 years of age, presenting with acute gastroenteritis in selected hospitals of the Littoral region of Cameroon, from May 2015 to April 2016. Diarrheic stool samples and socio-demographic data including immunization and breastfeeding status were collected from these participating children. Samples were screened by ELISA (ProSpecT™ Rotavirus) for detection of RVA antigen and by gel-based RT-PCR for detection of the VP6 gene. Co-infection was assessed by multiplexed molecular detection of diarrheal pathogens using the Luminex xTAG GPP assay. RESULTS: The ELISA assay detected RVA antigen in 54.6% (71/130) of specimens, with 45, positive by VP6 RT-PCR and 54, positive using Luminex xTAG GPP. Luminex GPP was able to detect all 45 VP6 RT-PCR positive samples. Co-infections were found in 63.0% (34/54) of Luminex positive RVA infections, with Shigella (35.3%; 12/34) and ETEC (29.4%; 10/34) detected frequently. Of the 71 ELISA positive RVA cases, 57.8% (41/71) were fully vaccinated, receiving two doses of Rotarix. CONCLUSION: This study provides insight on RVA prevalence in Cameroon, which could be useful for post-vaccine epidemiological studies, highlights higher than expected RVA prevalence in vaccinated children hospitalized for diarrhoea and provides the trend of RVA co-infection with other enteric pathogens. RVA genotyping is needed to determine circulating rotavirus genotypes in Cameroon, including those causing disease in vaccinated children.

Simple workflow to repurpose SARS-CoV-2 swab/serum samples for the isolation of cost-effective antibody/antigens for proteotyping applications and diagnosis.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.

The evaluation of a newly developed antigen test (QuickNavi™-COVID19 Ag) for SARS-CoV-2: A prospective observational study in Japan.

INTRODUCTION: Several antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed worldwide, but their clinical utility has not been well established. In this study, we evaluated the analytical and clinical performance of QuickNavi™-COVID19 Ag, a newly developed antigen test in Japan. METHODS: This prospective observational study was conducted at a PCR center between October 7 and December 5, 2020. The included patients were referred from a local public health center and 89 primary care facilities. We simultaneously obtained two nasopharyngeal samples with flocked swabs; one was used for the antigen test and the other for real-time reverse transcription PCR (RT-PCR). Using the results of real-time RT-PCR as a reference, the performance of the antigen test was evaluated. RESULTS: A total of 1186 patients were included in this study, and the real-time RT-PCR detected SARS-CoV-2 in 105 (8.9%). Of these 105 patients, 33 (31.4%) were asymptomatic. The antigen test provided a 98.8% (95% confidence interval [CI]: 98.0%-99.4%) concordance rate with real-time RT-PCR, along with a sensitivity of 86.7% (95% CI: 78.6%-92.5%) and a specificity of 100% (95% CI: 99.7%-100%). False-negatives were observed in 14 patients, 8 of whom were asymptomatic and had a low viral load (cycle threshold (Ct) > 30). In symptomatic patients, the sensitivity was 91.7% (95% CI: 82.7%-96.9%). CONCLUSION: QuickNavi™-COVID19 Ag showed high specificity and sufficient sensitivity for the detection of SARS-CoV-2. This test is a promising potential diagnostic modality especially in symptomatic patients.

Immunochromatography and chemiluminescent enzyme immunoassay for COVID-19 diagnosis.

INTRODUCTION: The rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required to prevent the spread of COVID-19. This study evaluated the utility of two SARS-CoV-2 antigen detection methods. METHODS: We evaluated two types of antigen detection methods using immunochromatography (Espline) and quantitative chemiluminescent enzyme immunoassay (Lumipulse). RT-PCR was performed as a standard procedure for COVID-19 diagnosis. Lumipulse and RT-PCR were performed for all 486 nasopharyngeal swabs and 136 saliva samples, and the Espline test was performed for 271 nasopharyngeal swabs and 93 saliva samples. RESULTS: The sensitivity and specificity of the Espline test were 10/11 and 260/260 (100%), respectively for the nasopharyngeal swabs and 3/9 and 84/84 (100%), respectively for the saliva samples. High sensitivities for both saliva (8/9) and nasopharyngeal swabs (22/24) were observed in the Lumipulse test. The specificities of the Lumipulse test for nasopharyngeal swabs and saliva samples were 460/462 (99.6%) and 123/127 (96.9%), respectively. CONCLUSION: The Espline test is not effective for saliva samples but is useful for simple and rapid COVID-19 tests using nasopharyngeal swabs because it does not require special devices. The Lumipulse test is a powerful high-throughput tool for COVID-19 diagnosis because it has high detection performance for nasopharyngeal swabs and saliva samples.

Large scale production and characterization of SARS-CoV-2 whole antigen for serological test development.

BACKGROUND: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID-19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS-CoV-2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory. METHODS: An enzyme-linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings. RESULTS: Preliminary data from 10 sera (5 patients with COVID-19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS-CoV-2-positive individuals. CONCLUSION: This ELISA appears to be a specific and reliable method for detecting COVID-19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus.

Rotavirus VP6 as a potential vaccine candidate.

By the age of 5 years, virtually all children have been infected by group A rotavirus (RVA), which is responsible for around half million mortality annually prior to vaccination. Relatively high rate of the morbidity and mortality highlights the necessity of applying preventive procedures particularly in developing countries. Two live attenuated RVA vaccines (Rotarix and RotaTeq) are licensed and now being used in many countries worldwide. Although these vaccines are shown to reduce the mortality up to 50%, several key questions yet remained to answer. Indeed, the licensed RV vaccines were found to be less effective in countries of sub-Saharan Africa and Southeast Asia. Therefore, developing next generation RVA vaccines is warranted. VP6 is highly abundant and conserved protein that forms the middle layer of RV particles and was shown to be both antigenic and immunogenic. Although it does not induce neutralizing antibodies, different VP6 preparations were found to induce homologous and cross-reactive immune responses with partial protection from RVA replication. Although the molecular mechanisms are not fully elucidated, VP6-based RVA vaccine candidates are worthy of further consideration. This review aims to focus on different aspects of VP6 protein and its potentiality for an alternative RV vaccine against RV disease.