Identification of Drugs Blocking SARS-CoV-2 Infection using Human Pluripotent Stem Cell-derived Colonic Organoids

Abstract: The current COVID-19 pandemic is caused by SARS-coronavirus 2 (SARS-CoV-2). There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with worse COVID-19 outcomes1. Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple hESC-derived colonic cell types, but highly enriched in enterocytes. Multiple cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. We used hPSC-derived COs in a high throughput platform to screen 1280 FDA-approved drugs against viral infection. Mycophenolic acid and quinacrine dihydrochloride were found to block the infection of SARS-CoV-2 pseudo-entry virus in COs both in vitro and in vivo, and confirmed to block infection of SARS-CoV-2 virus. This study established both in vitro and in vivo organoid models to investigate infection of SARS-CoV-2 disease-relevant human colonic cell types and identified drugs that blocks SARS-CoV-2 infection, suitable for rapid clinical testing.


Identification of potent and safe antiviral therapeutic candidates against SARS-CoV-2

Abstract: COVID-19 pandemic has infected millions of people with mortality exceeding 300,000. There is an urgent need to find therapeutic agents that can help clear the virus to prevent the severe disease and death. Identifying effective and safer drugs can provide with more options to treat the COVID-19 infections either alone or in combination. Here we performed a high throughput screen of approximately 1700 US FDA approved compounds to identify novel therapeutic agents that can effectively inhibit replication of coronaviruses including SARS-CoV-2. Our two-step screen first used a human coronavirus strain OC43 to identify compounds with anti-coronaviral activities. The effective compounds were then screened for their effectiveness in inhibiting SARS-CoV-2. These screens have identified 24 anti-SARS-CoV-2 drugs including previously reported compounds such as hydroxychloroquine, amlodipine, arbidol hydrochloride, tilorone 2HCl, dronedarone hydrochloride, and merfloquine hydrochloride. Five of the newly identified drugs had a safety index (cytotoxic/effective concentration) of >600, indicating wide therapeutic window compared to hydroxychloroquine which had safety index of 22 in similar experiments. Mechanistically, five of the effective compounds were found to block SARS-CoV-2 S protein-mediated cell fusion. These FDA approved compounds can provide much needed therapeutic options that we urgently need in the midst of the pandemic.


Antiviral activity of Glucosylceramide synthase inhibitors against SARS-CoV-2 and other RNA virus infections

Abstract: The need for antiviral drugs is real and relevant. Broad spectrum antiviral drugs have a particular advantage when dealing with rapid disease outbreaks, such as the current COVID-19 pandemic. Since viruses are completely dependent on internal cell mechanisms, they must cross cell membranes during their lifecycle, creating a dependence on processes involving membrane dynamics. Thus, in this study we examined whether the synthesis of glycosphingolipids, biologically active components of cell membranes, can serve as an antiviral therapeutic target. We examined the antiviral effect of two specific inhibitors of GlucosylCeramide synthase (GCS); (i) Genz-123346, an analogue of the FDA-approved drug Cerdelga®, (ii) GENZ-667161, an analogue of venglustat which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit the replication of four different enveloped RNA viruses of different genus, organ-target and transmission route: (i) Neuroinvasive Sindbis virus (SVNI), (ii) West Nile virus (WNV), (iii) Influenza A virus, and (iv) SARS-CoV-2. Moreover, GCS inhibitors significantly increase the survival rate of SVNI-infected mice. Our data suggest that GCS inhibitors can potentially serve as a broad-spectrum antiviral therapy and should be further examined in preclinical and clinical trial. Analogues of the specific compounds tested have already been studied clinically, implying they can be fast-tracked for public use. With the current COVID-19 pandemic, this may be particularly relevant to SARS-CoV-2 infection. One Sentence Summary An analogue of Cerdelga®, an FDA-approved drug, is effective against a broad range of RNA-viruses including the newly emerging SARS-CoV-2.


Several FDA-approved drugs effectively inhibit SARS-CoV-2 infection in vitro

Abstract: To identify drugs that are potentially used for the treatment of COVID-19, the potency of 1403 FDA-approved drugs were evaluated using a robust pseudovirus assay and the candidates were further confirmed by authentic SARS-CoV-2 assay. Four compounds, Clomiphene (citrate), Vortioxetine, Vortioxetine (hydrobromide) and Asenapine (hydrochloride), showed potent inhibitory effects in both pseudovirus and authentic virus assay. The combination of Clomiphene (citrate), Vortioxetine and Asenapine (hydrochloride) is much more potent than used alone, with IC50 of 0.34 μM.


Synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of SARS-associated coronavirus.

Abstract: A novel severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. We previously isolated and characterized SARS-CoV and SARS-CoV-like viruses from human and animals, respectively, suggesting that SARS could be transmitted from wild/farmed animals to humans. Comparison of the viral genomes indicated that sequence variation between animal and human isolates existed mainly in the spike (S) gene. We hypothesized that these variations may underlie a change of binding specificity of the S protein to the host cells, permitting viral transmission from animals to humans. Here we report that four 20-mer synthetic peptides (S protein fragments), designed to span these sequence variation hotspots, exhibited significant antiviral activities in a cell line. SARS-CoV infectivity was reduced over 10 000-fold through pre-incubation with two of these peptides, while it was completely inhibited in the presence of three peptides. Molecular modelling of the SARS-CoV peplomer suggests that three of these antiviral peptides map to the interfaces between the three monomers of the trimeric peplomer rather than the heptad repeat region from which short peptides are known to inhibit viral entry. Our results revealed novel regions in the spike protein that can be targeted to inhibit viral infection. The peptides identified in this study could be further developed into antiviral drugs.


Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor.

Abstract: A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.


Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design.

Abstract: Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M(pro)), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) M(pro) and a severe acute respiratory syndrome CoV (SARS-CoV) M(pro) mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M(pro). A monomeric form of IBV M(pro) was identified for the first time in CoV M(pro) structures. A comparison of these two structures to other available M(pro) structures provides new insights for the design of substrate-based inhibitors targeting CoV M(pro)s. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M(pro) and was found to demonstrate in vitro inactivation of IBV M(pro) and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M(pro).


Inactivated SARS-CoV vaccine prepared from whole virus induces a high level of neutralizing antibodies in BALB/c mice.

Abstract: We tested the ability of inactivated SARS-CoV vaccine to induce neutralizing antibodies in BALB/c mice. The inactivated vaccine was prepared by SARS-CoV virus propagation in Vero cells, with subsequent beta-propiolactone inactivation and Sepharose 4FF column chromatography purification. One hundred forty BALB/c female mice were divided into seven groups of 20 mice each. Of the seven groups, three groups were inoculated with 0.1, 1, and 3 microg of the vaccine without adjuvant while three other groups were inoculated at the same three dosages of vaccine with aluminum hydroxide as adjuvant, respectively. The remaining group was set up as a blank control. Each mouse was inoculated twice at an interval of 3 weeks. One week after the second immunization, mice sera were collected to detect serum neutralizing antibodies. An assay for determining neutralizing antibody titers was developed. The results can be summarized as follows: (1) higher dosages of vaccine induced higher levels of neutralizing antibody titer; (2) the level of neutralizing antibodies induced by the inoculation with aluminum hydroxide adjuvant was slightly higher than that without adjuvant, but the difference was not statistically significant.


Protection from infection with severe acute respiratory syndrome coronavirus in a Chinese hamster model by equine neutralizing F(ab')2.

Abstract: To warrant potential clinical testing, the equine anti-severe acute respiratory syndrome coronavirus (SARS-CoV) F(ab')(2) requires evaluation in as many animal models as possible. In this study, we established a new animal model, the Chinese hamster, susceptible to SARS-CoV infection. SARS-CoV could propagate effectively and sustain high levels for 1 wk in animal lungs. All animals were protected from SARS-CoV infection in preventive settings. Further, when used therapeutically this antibody led to an approximately 4-log(10) decrease in viral burden in infected animal lungs. The pathological changes in lungs correlated closely with the dose of antibody administered. The excellent preventive and therapeutic roles of equine anti-SARS-CoV F(ab')(2) in several animal models, including the novel Chinese hamster model described in this study, have provided exciting data concerning its potential clinical study.


Baculovirus surface display of SARS coronavirus (SARS-CoV) spike protein and immunogenicity of the displayed protein in mice models.

Abstract: The baculovirus surface display technique has provided an ideal tool to display foreign proteins with natural conformation, functions, and immunogenicity. In this work, we explored the application of this technique on SARS-associated coronavirus (SARS-CoV) spike (S) protein, and further analyzed the immunogenicity of displayed S protein. The entire ectodomain of S protein was fused between the gp64 signal peptide and the VSV-G membrane anchor and successfully displayed on the baculovirus surface. Subcutaneous injection with purified S-displayed baculoviruses without adjuvant elicited highly effective production of specific and neutralizing antibodies against S protein in mice. These results confirmed a successful surface display of S protein on baculoviruse, and suggested a potential role of S-displayed baculoviruses as a novel live virus-based vaccine candidate for SARS-CoV.


The aetiology of SARS: Koch's postulates fulfilled.

Abstract: Proof that a newly identified coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV) is the primary cause of severe acute respiratory syndrome (SARS) came from a series of studies on experimentally infected cynomolgus macaques (Macaca fascicularis). SARS-CoV-infected macaques developed a disease comparable to SARS in humans; the virus was re-isolated from these animals and they developed SARS-CoV-specific antibodies. This completed the fulfilment of Koch's postulates, as modified by Rivers for viral diseases, for SARS-CoV as the aetiological agent of SARS. Besides the macaque model, a ferret and a cat model for SARS-CoV were also developed. These animal models allow comparative pathogenesis studies for SARS-CoV infections and testing of different intervention strategies. The first of these studies has shown that pegylated interferon-alpha, a drug approved for human use, limits SARS-CoV replication and lung damage in experimentally infected macaques. Finally, we argue that, given the worldwide nature of the socio-economic changes that have predisposed for the emergence of SARS and avian influenza in Southeast Asia, such changes herald the beginning of a global trend for which we are ill prepared.


Small molecules targeting severe acute respiratory syndrome human coronavirus.

Abstract: Severe acute respiratory syndrome (SARS) is an infectious disease caused by a novel human coronavirus. Currently, no effective antiviral agents exist against this type of virus. A cell-based assay, with SARS virus and Vero E6 cells, was developed to screen existing drugs, natural products, and synthetic compounds to identify effective anti-SARS agents. Of >10,000 agents tested, approximately 50 compounds were found active at 10 microM; among these compounds, two are existing drugs (Reserpine 13 and Aescin 5) and several are in clinical development. These 50 active compounds were tested again, and compounds 2-6, 10, and 13 showed active at 3 microM. The 50% inhibitory concentrations for the inhibition of viral replication (EC(50)) and host growth (CC(50)) were then measured and the selectivity index (SI = CC(50)/EC(50)) was determined. The EC(50), based on ELISA, and SI for Reserpine, Aescim, and Valinomycin are 3.4 microM (SI = 7.3), 6.0 microM (SI = 2.5), and 0.85 microM (SI = 80), respectively. Additional studies were carried out to further understand the mode of action of some active compounds, including ELISA, Western blot analysis, immunofluorescence and flow cytometry assays, and inhibition against the 3CL protease and viral entry. Of particular interest are the two anti-HIV agents, one as an entry blocker and the other as a 3CL protease inhibitor (K(i) = 0.6 microM).


The FDA-approved gold drug auranofin inhibits novel coronavirus (SARS-COV-2) replication and attenuates inflammation in human cells

Abstract: SARS-COV-2 has recently emerged as a new public health threat. Herein, we report that the FDA-approved drug, auranofin, inhibits SARS-COV-2 replication in human cells at low micro molar concentration. Treatment of cells with auranofin resulted in a 95% reduction in the viral RNA at 48 h after infection. Auranofin treatment dramatically reduced the expression of SARS-COV-2-induced cytokines in human cells. These data indicate that auranofin could be a useful drug to limit SARS-CoV-2 infection and associated lung injury due to its antiviral, anti-inflammatory and anti-reactive oxygen species (ROS) properties. Further animal studies are warranted to evaluate the safety and efficacy of auranofin for the management of SARS-COV-2 associated disease.


Development of multi-specific humanized llama antibodies blocking SARS-CoV-2/ACE2 interaction with high affinity and avidity

Abstract: Coronaviruses cause severe human viral diseases including SARS, MERS and COVID-19. Most recently SARS-CoV-2 virus (causing COVID-19) has led to a pandemic with no successful therapeutics. The SARS-CoV-2 infection relies on trimeric spike (S) proteins to facilitate virus entry into host cells by binding to ACE2 receptor on host cell membranes. Therefore, blocking this interaction with antibodies are promising agents against SARS-CoV-2. Here we describe using humanized llama antibody VHHs against SARS-CoV-2 that would overcome the limitations associated with polyclonal and monoclonal combination therapies. From two llama VHH libraries, unique humanized VHHs that bind to S protein and block the S/ACE2 interaction were identified. Furthermore, pairwise combination of VHHs showed synergistic blocking. Multi-specific antibodies with enhanced affinity and avidity, and improved S/ACE2 blocking are currently being developed using an in-silico approach that also fuses VHHs to Fc domains. Importantly, our current bi-specific antibody shows potent S/ACE2 blocking (KD - 0.25 nM, IC100 â¼ 36.7 nM, IC95 â¼ 12.2 nM, IC50 â¼ 1 nM) which is significantly better than individual monoclonal VHH-Fcs. Overall, this design would equip the VHH-Fcs multiple mechanisms of actions against SARS-CoV-2. Thus, we aim to contribute to the battle against COVID-19 by developing therapeutic antibodies as well as diagnostics.


Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection

Abstract: The recent emergence of the novel pathogenic SARS-coronavirus 2 (SARS-CoV-2) is responsible for a worldwide pandemic. Given the global health emergency, drug repositioning is the most reliable option to design an efficient therapy for infected patients without delay. The first step of the viral replication cycle [i.e. attachment to the surface of respiratory cells, mediated by the spike (S) viral protein] offers several potential therapeutic targets. The S protein uses the angiotension-converting enzyme-2 (ACE-2) receptor for entry, but also sialic acids linked to host cell surface gangliosides. Using a combination of structural and molecular modelling approaches, this study showed that chloroquine (CLQ), one of the drugs currently under investigation for SARS-CoV-2 treatment, binds sialic acids and gangliosides with high affinity. A new type of ganglioside-binding domain at the tip of the N-terminal domain of the SARS-CoV-2 S protein was identified. This domain (111-158), which is fully conserved among clinical isolates worldwide, may improve attachment of the virus to lipid rafts and facilitate contact with the ACE-2 receptor. This study showed that, in the presence of CLQ [or its more active derivative, hydroxychloroquine (CLQ-OH)], the viral S protein is no longer able to bind gangliosides. The identification of this new mechanism of action of CLQ and CLQ-OH supports the use of these repositioned drugs to cure patients infected with SARS-CoV-2. The in-silico approaches used in this study might also be used to assess the efficiency of a broad range of repositioned and/or innovative drug candidates before clinical evaluation.


The anti-HIV drug nelfinavir mesylate (Viracept) is a potent inhibitor of cell fusion caused by the SARSCoV-2 spike (S) glycoprotein warranting further evaluation as an antiviral against COVID-19 infections

Abstract: Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is the causative agent of the coronavirus disease-2019 (COVID-19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV-2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID-19. These results suggest that SARS CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA-approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n- and S-o-mediated cell fusion with complete inhibition at a 10-µM concentration. In-silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n- and S-o-mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV-2 symptoms appear.


Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

Abstract: Countermeasures to prevent and treat COVID-19 are a global health priority. We enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen over 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. We showed that passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters, as revealed by maintained weight and low lung viral titers in treated animals. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.


Discovery of the FDA-approved drugs bexarotene, cetilistat, diiodohydroxyquinoline, and abiraterone as potential COVID-19 treatments with a robust two-tier screening system

Abstract: Coronavirus Disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a crude case fatality rate of about 0.5-10 % depending on locality. A few clinically approved drugs, such as remdesivir, chloroquine, hydroxychloroquine, nafamostat, camostat, and ivermectin, exhibited anti-SARS-CoV-2 activity in vitro and/or in a small number of patients. However, their clinical use may be limited by anti-SARS-CoV-2 50 % maximal effective concentrations (EC50) that exceeded their achievable peak serum concentrations (Cmax), side effects, and/or availability. To find more immediately available COVID-19 antivirals, we established a two-tier drug screening system that combines SARS-CoV-2 enzyme-linked immunosorbent assay and cell viability assay, and applied it to screen a library consisting 1528 FDA-approved drugs. Cetilistat (anti-pancreatic lipase), diiodohydroxyquinoline (anti-parasitic), abiraterone acetate (synthetic androstane steroid), and bexarotene (antineoplastic retinoid) exhibited potent in vitro anti-SARS-CoV-2 activity (EC50 1.13-2.01 µM). Bexarotene demonstrated the highest Cmax:EC50 ratio (1.69) which was higher than those of chloroquine, hydroxychloroquine, and ivermectin. These results demonstrated the efficacy of the two-tier screening system and identified potential COVID-19 treatments which can achieve effective levels if given by inhalation or systemically depending on their pharmacokinetics.


In Vitro and Animal Models for SARS-CoV-2 research

Abstract: Basic research on SARS-CoV-2 is essential to understand its detailed pathophysiology and identify best drug targets. Models that can faithfully reproduce the viral life cycle and reproduce the pathology of COVID-19 are required. Here, we briefly review the cell lines, organoids, and animal models that are currently being used in COVID-19 research.


Antiviral activities of mycophenolic acid and IMD-0354 against SARS-CoV-2

Abstract: In this study, anti-SARS-CoV-2 activity of mycophenolic acid (MPA) and IMD-0354 was analyzed, these compounds were chosen based on their antiviral activities against other coronaviruses. Since they also inhibit Dengue virus (DENV) infection, other anti-DENV compounds/drugs were also assessed. Using SARS-CoV-2-infected VeroE6/TMPRSS2 cells, MPA and IMD-0354, but not other anti-DENV compounds/drugs, showed significant anti-SARS-CoV-2 activity. Although MPA reduced the viral RNA level by only ~100-fold, its EC50 was as low as 0.87µM, which is easily achievable at therapeutic doses of mycophenolate mofetil. MPA targets coronaviral papain-like protease and its study would be useful in the development of novel anti-SARS-CoV-2 drugs. This article is protected by copyright. All rights reserved.


Animal models of mechanisms of SARS-CoV-2 infection and COVID-19 pathology

Abstract: The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 infections has led to substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS-CoV-2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID-19. At the time of writing, in vivo studies of SARS-CoV-2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partially model the mechanisms implicated in human severe COVID-19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID-19. These models need to recapitulate key mechanisms of COVID-19 severe acute respiratory distress syndrome and reproduce the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS-CoV-2 infection and COVID-19-related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID-19 pathogenesis and for assessing potential treatments.


The search for a COVID-19 animal model


A Potently Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for millions of infections and hundreds of thousands of deaths globally. There are no widely available licensed therapeutics against SARS-CoV-2, highlighting an urgent need for effective interventions. The virus enters host cells through binding of a receptor-binding domain within its trimeric spike glycoprotein to human angiotensin-converting enzyme 2. In this article, we describe the generation and characterization of a panel of murine mAbs directed against the receptor-binding domain. One mAb, 2B04, neutralized wild-type SARS-CoV-2 in vitro with remarkable potency (half-maximal inhibitory concentration of <2 ng/ml). In a murine model of SARS-CoV-2 infection, 2B04 protected challenged animals from weight loss, reduced lung viral load, and blocked systemic dissemination. Thus, 2B04 is a promising candidate for an effective antiviral that can be used to prevent SARS-CoV-2 infection.


Identification of Antiviral Drug Candidates against SARS-CoV-2 from FDA-Approved Drugs

Abstract: Drug repositioning is the only feasible option to immediately address the COVID-19 global challenge. We screened a panel of 48 FDA-approved drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which were preselected by an assay of SARS-CoV. We identified 24 potential antiviral drug candidates against SARS-CoV-2 infection. Some drug candidates showed very low 50% inhibitory concentrations (IC50s), and in particular, two FDA-approved drugs-niclosamide and ciclesonide-were notable in some respects.


Computational screening of antagonists against the SARS-CoV-2 (COVID-19) coronavirus by molecular docking

Abstract: In the current spread of novel coronavirus (SARS-CoV-2), antiviral drug discovery is of great importance. AutoDock Vina was used to screen potential drugs by molecular docking with the structural protein and non-structural protein sites of new coronavirus. Ribavirin, a common antiviral drug, remdesivir, chloroquine and luteolin were studied. Honeysuckle is generally believed to have antiviral effects in traditional Chinese medicine. In this study, luteolin (the main flavonoid in honeysuckle) was found to bind with a high affinity to the same sites of the main protease of SARS-CoV-2 as the control molecule. Chloroquine has been proved clinically effective and can bind to the main protease; this may be the antiviral mechanism of this drug. The study was restricted to molecular docking without validation by molecular dynamics simulations. Interactions with the main protease may play a key role in fighting against viruses. Luteolin is a potential antiviral molecule worthy of attention.


The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner

Abstract: Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.


Ribavirin and interferon-β synergistically inhibit SARS-associated coronavirus replication in animal and human cell lines

Abstract: Abstract Initial in vitro investigations demonstrated type I interferons (IFNs: IFN-α, IFN-β) to inhibit replication of SARS coronavirus (SARS-CoV), but found the nucleoside analogue ribavirin ineffective in Vero cells. In this report, ribavirin was shown to inhibit SARS-CoV replication in five different cell types of animal or human origin at therapeutically achievable concentrations. Since clinical anti-SARS-CoV activity of type I interferons or ribavirin is limited, we investigated the combination of IFN-β and ribavirin. Determination of the virus yield indicated highly synergistic anti-SARS-CoV action of the combination suggesting the consideration of ribavirin plus IFN-β for the treatment of SARS.


Antiviral activities of type I interferons to SARS-CoV-2 infection

Abstract: There is an urgent need to identify antivirals to curtail the COVID-19 pandemic. Herein, we report the sensitivity of SARS-CoV-2 to recombinant human interferons α and β (IFNα/β). Treatment with IFN-α or IFN-β at a concentration of 50 international units (IU) per milliliter reduces viral titers by 3.4 log or over 4 log, respectively, in Vero cells. The EC(50) of IFN-α and IFN-β treatment is 1.35 IU/ml and 0.76 IU/ml, respectively, in Vero cells. These results suggest that SARS-CoV-2 is more sensitive than many other human pathogenic viruses, including SARS-CoV. Overall, our results demonstrate the potential efficacy of human Type I IFN in suppressing SARS-CoV-2 infection, a finding which could inform future treatment options for COVID-19.


Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel human coronavirus that causes coronavirus disease 2019 (COVID-19), was first discovered in December 2019 as the cause of an outbreak of pneumonia in the city of Wuhan, Hubei province, China. The clinical presentation of COVID-19 is fairly non-specific, and symptoms overlap with other seasonal respiratory infections concurrently circulating in the population. Furthermore, it is estimated that up to 80% of infected individuals experience mild symptoms or are asymptomatic, confounding efforts to reliably diagnose COVID-19 empirically. To support infection control measures, there is an urgent need for rapid and accurate molecular diagnostics to identify COVID-19 positive patients. In the present study, we have evaluated the analytical sensitivity and clinical performance of four SARS-CoV-2 molecular diagnostic assays granted Emergency Use Authorization by the FDA using nasopharyngeal swabs from symptomatic patients: the New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel (Modified CDC), the Simplexa COVID-19 Direct (Diasorin Molecular), GenMark ePlex SARS-CoV-2 assay (GenMark) and the Hologic Panther Fusion® SARS-CoV-2 assay (Hologic). This information is crucial for both laboratories and clinical teams, as decisions on which testing platform to implement are made.


Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection.

Abstract: The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid based tests currently offer the most sensitive and early detection of COVID-19. However, the "gold standard" test pioneered by the United States Center for Disease Control & Prevention, takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the "gold-standard" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own labs. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.


Comparison of Commercially Available and Laboratory Developed Assays for in vitro Detection of SARS-CoV-2 in Clinical Laboratories.

Abstract: Multiple laboratory developed tests and commercially available assays have emerged to meet diagnostic needs related to the SARS-CoV-2 pandemic. To date, there is limited comparison data for these different testing platforms. We compared the analytical performance of a laboratory developed test (LDT) developed in our clinical laboratory based on CDC primer sets and four commercially available, FDA emergency use authorized assays for SARS-CoV-2 (Cepheid, DiaSorin, Hologic Panther, and Roche Cobas) on a total of 169 nasopharyngeal swabs. The LDT and Cepheid Xpert Xpress SARS-CoV-2 assays were the most sensitive assays for SARS-CoV-2 with 100% agreement across specimens. The Hologic Panther Fusion, DiaSorin Simplexa, and Roche Cobas 6800 only failed to detect positive specimens near the limit of detection of our CDC-based LDT assay. All assays were 100% specific, using our CDC-based LDT as the gold standard. Our results provide initial test performance characteristics for SARS-CoV-2 RT-PCR and highlight the importance of having multiple viral detection testing platforms available in a public health emergency.


Covid-19: What is the UK's testing strategy?


Current practice and potential strategy in diagnosing COVID-19.

Abstract: OBJECTIVE To summarize the current practice and potential strategy in diagnosing coronavirus disease 2019 (COVID-19). MATERIALS AND METHODS PubMed, Web of Science were systematically searched using terms including "COVID-19", "SARS-CoV-2" and "2019-nCoV". After removing duplicates, we then identified articles, letters and commentaries regarding diagnosing COVID-19. RESULTS Here we summarized relatively mature diagnostic methods like nuclear acid test and computed tomography. Besides, new aspects regarding these detection methods like suitable specimens for nuclear acid test, possible use of 18F-FDG PET/CT were also reported. Especially, we also presented several novel techniques for diagnosing COVID-19 like lung ultrasound. CONCLUSIONS Chinese Clinical Guidance for COVID-19 Pneumonia Diagnosis and Treatment (7th edition) by National Health Commission is recommended to follow as it provides detailed diagnostic procedures using currently available tools. We suggest clinicians further explore the saliva's utility as a specimen for nuclear acid test and the use of lung ultrasound.


A SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Towards the Development of Point of Care Tests Using Commercially Available Reagents.

Abstract: The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, based on previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this paper we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/ mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.


Performance of Abbott ID NOW COVID-19 rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a New York City academic institution.

Abstract: The recent emergence of the SARS-CoV-2 pandemic has posed formidable challenges for clinical laboratories seeking reliable laboratory diagnostic confirmation. The swift advance of the crisis in the United States has led to Emergency Use Authorization (EUA) facilitating the availability of molecular diagnostic assays without the more rigorous examination to which tests are normally subjected prior to FDA approval. Our laboratory currently uses two real time RT-PCR platforms, the Roche Cobas SARS-CoV2 and the Cepheid Xpert Xpress SARS-CoV-2. Both platforms demonstrate comparable performance; however, the run times for each assay are 3.5 hours and 45 minutes, respectively. In search for a platform with shorter turnaround time, we sought to evaluate the recently released Abbott ID NOW COVID-19 assay which is capable of producing positive results in as little as 5 minutes. We present here the results of comparisons between Abbott ID NOW COVID-19 and Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs transported in viral transport media and comparisons between Abbott ID NOW COVID-19 and Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs transported in viral transport media for Cepheid and dry nasal swabs for Abbott ID NOW. Regardless of method of collection and sample type, Abbott ID NOW COVID-19 had negative results in a third of the samples that tested positive by Cepheid Xpert Xpress when using nasopharyngeal swabs in viral transport media and 45% when using dry nasal swabs.


Covid-19 mass testing facilities could end the epidemic rapidly.


A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry.

Abstract: The 2019 novel coronavirus (COVID-19) is a newly emerged strain that has never been found in humans before. At present, the laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the main method to confirm COVID-19 infection. The intensification of the COVID-19 epidemic overwhelms limited clinical resources in particular, but not only, in developing countries, resulting in many patients not being tested for the infection and in large queues of potentially infected individuals waiting to be tested while providing a breeding ground for the disease. We describe here a rapid, highly sensitive, point-of-care, molecular test amenable for use at home, in the clinic, and at points of entry by minimally trained individuals and with minimal instrumentation. Our test is based on loop mediated isothermal amplification (COVID-19 LAMP) and for higher sensitivity on nested nucleic acid, two stage isothermal amplification (COVID-19 Penn-RAMP). Both tests can be carried out in closed tubes with either fluorescence or colorimetric (e.g., leuco crystal violet LCV) detection. COVID-19 LAMP performs on par with COVID-19 RT-PCR. COVID-19 RAMP has 10 fold better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing purified targets and 100 times better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing rapidly prepared sample mimics. Due to fortunate scarcity of COVID-19 infections in the USA, we were not able to test our assays and methods with patient samples. We hope that such tests will be carried out by colleagues in impacted countries. Our Closed-Tube Penn-RAMP has the potential to significantly reduce false negatives while being amenable to use with minimal instrumentation and training.


Evaluation of eleven rapid tests for detection of antibodies against SARS-CoV-2.

Abstract: Objectives SARS-CoV-2, causing COVID-19, has emerged to cause a human pandemic. Detection of SARS-CoV-2 in respiratory samples by using PCR is the standard laboratory diagnostic tool. Our aim was to perform a limited evaluation of the diagnostic performance and user-friendliness of eleven rapid tests for detection of antibodies against SARS-CoV-2. Methods All participants were tested with PCR against SARS-CoV-2 at a clinical microbiology laboratory. Comparing with results from PCR tests, we evaluated the rapid tests' performances in three arms; 1) 20 hospitalized patients with PCR-confirmed COVID-19, 2) 23 recovered outpatients with former PCR-confirmed COVID-19, and 3) 49 participants with suspected COVID-19 presenting at a primary care emergency room. Results All eleven tests detected antibodies in hospitalized COVID-19 patients, though with varying sensitivities. In former outpatients recovered from COVID-19, there were differences between tests in the immunoglobulin type G (IgG) sensitivity, with five tests having a sensitivity below 65%. In participants with suspected COVID-19 infection, the rapid tests had very low sensitivities. Most rapid tests were easy to perform and interpret. Conclusions Rapid tests were not suited as stand-alone tests to detect present infection in a Norwegian primary care emergency room population. All the rapid tests were able to detect SARS-CoV-2 antibodies, although sensitivities varied and were generally higher in the study arm of more severely affected participants. Rapid tests with high IgG sensitivity (and specificity) may be useful for confirmation of past infection. An independent evaluation should be performed in the intended population before introducing a rapid test.


Clinical Evaluation of Three Sample-To-Answer Platforms for the Detection of SARS-CoV-2.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread across the globe. As part of the worldwide response, many molecular diagnostic platforms have been granted Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) to identify SARS-CoV-2 positive patients. Our objective was to evaluate three sample-to-answer molecular diagnostic platforms (Cepheid Xpert® Xpress SARS-CoV-2 [Xpert Xpress], Abbott ID NOW™ COVID-19 [ID NOW], GenMark ePlex® SARS-CoV-2 Test [ePlex]) to determine analytical sensitivity, clinical performance, and workflow for the detection of SARS-CoV-2 in nasopharyngeal swabs from 108 symptomatic patients. We found that the Xpert Xpress had the lowest limit of detection (100% detection at 100 copies/mL), followed by the ePlex (100% detection at 1,000 copies/mL), and the ID NOW (20,000 copies/mL). The Xpert Xpress also had highest positive percent agreement (PPA) when compared to our reference standard (98.3%) followed by the ePlex (91.4%) and ID now (87.7%). All three assays showed 100% negative percent agreement (NPA). In the workflow analysis, the ID NOW produced the most rapid time to result per specimen (∼17 minutes) as compared to the Xpert Xpress (∼46 minutes) and the ePlex (∼1.5 hours), but what the ID NOW gained in rapid results, it lost in analytical and clinical performance. The ePlex had the longest time to results and showed a slight improvement in PPA over the ID NOW. Information about the clinical and analytical performance of these assays, as well as workflow, will be critical in making informed and timely decisions on testing platform.


INSIGHT: a scalable isothermal NASBA-based platform for COVID-19 diagnosis

Abstract: We present here INSIGHT (Isothermal NASBA-Sequencing based hIGH-througput Test): a two-stage COVID-19 testing strategy, using a combination of an isothermal NASBA reaction and next generation sequencing. From commercially acquired human saliva with spiked-in viral RNA as input, the first stage employs isothermal amplification of viral RNA to give a rapid result in one to two hours, using either fluorescence detection or a dipstick readout, whilst simultaneously incorporating sample-specific barcodes into the amplification product. In the first stage, fluorescent viral RNA detection can be consistently achieved at 10-100 copies per 20 µl reaction. The second stage pools post-amplification barcoded products from multiple samples for scalable sequencing that could be centralised, to further improve the accuracy of the test in a massively parallel way. Our two-stage testing strategy is suitable for further development into a home-based or point-of-care assay, and is potentially scalable to population level. IMPORTANT This protocol has not been validated on patient samples and should not be used for clinical diagnosis without further validation and certification. ***This is ongoing research to develop a testing strategy for the SARS-CoV-2 virus. The Wellcome Sanger Institute is not in a position to develop this into a commercial product, but would be open to discussions with third parties about how they could develop this further to achieve the societal benefits of this work.***


Pooling of Nasopharyngeal Swab Specimens for SARS-CoV-2 detection by RT-PCR

Abstract: Systematic testing of large population groups by RT-PCR is mandatory to Covid-19 case identification and contact tracing in order to minimize the likelyhood of resurgence in contagion. Sample pooling for RT-PCR has been effectively used to detect community transmission of SARS CoV-2. Nevertheless, this procedure may decrease the sensitivity of RT-PCR assays due to specimen dilution. We evaluated the efficacy of this strategy for diagnosis of Covid-19 using a sensitive commercially-available RT-PCR targeting SARS CoV-2 E and RdRp genes in a single reaction. A total of 20 mini-pools containing either 5 (n=10) or 10 (n=10) nasopharyngeal exudates collected in universal transport medium were made, each of which including a unique positive NP specimen. Positive specimens yielding CT <32 for the E gene (6 out of 10) or <35.2 for the RdRP gene (7 out of 10) were detected in mini-pools of both sizes. In contrast, most NP samples displaying CTs > 35.8 for the E gene or 35.7 for the RdRP gene remained undetected in mini-pools of 5 specimens (3/4 and 2/3, respectively) or in mini-pools of 10 samples (4/4 and 3/3, respectively.


Immunochromatographic test for the detection of SARS-CoV-2 in saliva

Abstract: We evaluated the rapid immunochromatographic test for SARS-CoV-2 antigen detection using 16 saliva specimens collected from 6 COVID-19 hospitalized patients, and detected N-antigen in 4 of 7 RT-PCR positive specimens. The POCT antigen test using saliva is highly considered to be a game-changer for COVID-19 diagnosis.


Performance of Abbott ID NOW COVID-19 rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a New York City academic institution


Massive Multiplexing Can Deliver a $1 Test for COVID-19

Abstract: The severe acute respiratory syndrome virus, SARS-CoV-2 (hereafter COVID-19), rapidly achieved global pandemic status, provoking large-scale screening programs in many nations. Their activation makes it imperative to identify methods that can deliver a diagnostic result at low cost. This paper describes an approach which employs sequence variation in the gene coding for its envelope protein as the basis for a scalable, inexpensive test for COVID-19. It achieves this by coupling a simple RNA extraction protocol with low-volume RT-PCR, followed by E-Gel screening and sequencing on high-throughput platforms to analyze 10,000 samples in a run. Slight modifications to the protocol could support screening programs for other known viruses and for viral discovery. Just as the $1,000 genome is transforming medicine, a $1 diagnostic test for viral and bacterial pathogens would represent a major advance for public health.


High-throughput extraction of SARS-CoV-2 RNA from nasopharyngeal swabs using solid-phase reverse immobilization beads

Abstract: The ongoing pandemic of the novel coronavirus, SARS-CoV-2, has led to a global surge in laboratory testing for the virus. The gold standard approach to detecting an active viral infection is the use of RT-qPCR. This approach requires the isolation of viral RNA from respiratory specimens, such as nasopharyngeal swabs. We developed a method using a widely available lysis buffer coupled with solid-phase reverse immobilization (SPRI) beads to extract viral RNA from swabs collected in viral transport medium (VTM) which can be performed manually or on a Hamilton STAR liquid-handling robot. Using a WHO recommended, laboratory-developed RT-qPCR for SARS-CoV-2, we validated this method in a CAP-accredited laboratory, against the IVD-labelled bioMérieux NucliSENS easyMAG automated extraction platform. Our method demonstrates a comparable sensitivity and specificity, making it suitable for large-scale testing and monitoring of suspected COVID-19 cases and healthcare workers. This is especially important as the world faces critical shortages of viral RNA extraction reagents for the existing commercial extraction systems.


SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification

Abstract: As the current SARS-CoV-2 pandemic spreads, the need for more diagnostic capabilities is great. In order to address this need, we have developed a highly sensitive RT-LAMP assay compatible with current reagents, that utilizes a colorimetric readout in as little as 30 minutes. In addition to this, we have developed an inexpensive pipeline to further increase sensitivity without requiring highly specialized equipment. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was also developed to increase sensitivity and sample stability. This protocol, combined with our RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with our RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. We hope that this inactivation and purification pipeline, which costs approximately $0.07 per sample and which uses readily available reagents, will increase the availability of SARS-CoV-2 testing, as well as expand the settings in which this testing can be performed.


AI based Chest X-Ray (CXR) Scan Texture Analysis Algorithm for Digital Test of COVID-19 Patients

Abstract: Chest Imaging in COVID-19 patient management is becoming an essential tool for controlling the pandemic that is gripping the international community. It is already indicated in patients with COVID-19 and worsening respiratory status. The rapid spread of the pandemic to all continents, albeit with a nonuniform community transmission, necessitates chest imaging for medical triage of patients presenting moderate-severe clinical COVID-19 features. This paper reports the development of innovative machine learning schemes for the analysis of Chest X-Ray (CXR) scan images of COVID-19 patients in almost real-time, demonstrating significantly high accuracy in identifying COVID-19 infection. The performance testing was conducted on a combined dataset comprising CXRs of positive COVID-19 patients, patients with various viral and bacterial infections, as well as persons with a clear chest. The test resulted in successfully distinguishing CXR COVID-19 infection from the other cases with an average accuracy of 94.43%, sensitivity 95% and specificity 93.86%.


A rapid systematic review and case study on test, contact tracing, testing, and isolation policies for Covid-19 prevention and control

Abstract: Objectives To conduct a rapid review on the efficacy and policy of contact tracing, testing, and isolation (TTI) in Covid-19 prevention and control, including a case study for their delivery. Method Research articles and reviews on the use of contact tracing, testing, self-isolation and quarantine for Covid-19 management published in English within 1 year (2019 to 28th May, 2020) were eligible to the review. We searched MEDLINE (PubMed), Cochrane Library, SCOPUS and JSTOR with search terms included "contact tracing" or "testing" or "self-isolation" or "quarantine" in the title in combination with "Covid-19" or "COVID-19" or "coronavirus" in the title or abstract. Studies not associated with TTI or Covid-19 or being solely commentary were excluded. A narrative synthesis with a tabulation system was used to analyse studies for their diverse research designs, methods, and implications. Information for the case study was obtained from the Centers for Disease Control Taiwan. Results Among the 160 initial publications, 30 eligible studies are included in the review. Included studies applied various designs: experiments, clinical studies, Government Documents, systematic reviews, observational studies, surveys, practice guidelines, technical reports. A case study on TTI delivery is summarised based on policy and procedures in Taiwan. Conclusions The information included in the review may inform the TTI program in the UK.


Self-Collected Oral Fluid and Nasal Swabs Demonstrate Comparable Sensitivity to Clinician Collected Nasopharyngeal Swabs for Covid-19 Detection

Abstract: Background: Currently, there is a pandemic caused by the 2019 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes Covid-19. We wanted to compare specimen types and collection methods to explore if a simpler to collect specimen type could expand access to testing. Methods: We recruited individuals recently tested for SARS-CoV-2 infection through a testing program (drive-through). In homes of participants, we assessed the performance of self-collected oral fluid swab specimens with and without clinician supervision, clinician-supervised self-collected mid-turbinate (nasal) swab specimens, and clinician-collected nasopharyngeal swab specimens. We tested specimens with a validated reverse transcription-quantitative polymerase chain reaction assay for the detection of SARS-CoV-2 and measured cycle threshold values. Symptom status and date of onset of symptoms was also recorded for each participant. Results: We recruited 45 participants. The median age of study participant was 42 years old (Interquartile range, 31 to 52 years). Of the participants, 29 had at least one specimen test positive for SARS-CoV-2. Of those, 21 (73%) of 29 reported active symptoms. By specimen type and home-based collection method, clinician-supervised self-collected oral fluid swab specimens detected 26 (90%) of 29 infected individuals, clinician-supervised self-collected nasal swab specimens detected 23 (85%) of 27, clinician-collected posterior nasopharyngeal swab specimens detected 23 (79%) of 29, and unmonitored self-collected oral fluid swab specimens detected 19 (66%) of 29. Despite nasopharyngeal swabs being considered the gold standard, 4 participants tested negative by clinician-collected nasopharyngeal swab and positive by the 3 other specimen types. Additionally, false negative results by each sample type were seen to generally not overlap. Conclusions: Supervised self-collected oral fluid and nasal swab specimens performed similarly to, if not better than clinician-collected nasopharyngeal swab specimens for the detection of SARS-CoV-2 infection. No sample type captured all SARS-CoV-2 infections, suggesting potential heterogeneity in the distribution of viral load in different parts of the respiratory tract between individuals. Supervised self-collection performed comparably to clinician collection and would allow for rapid expansion of testing capacity in the United States by reducing the need for trained healthcare workers, reducing exposure of healthcare workers, and reducing the amount of PPE (personal protective equipment) being used for testing during a critical shortage.


Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP

Abstract: The ability to detect an infectious agent in a widespread epidemic is crucial to the success of quarantine efforts in addition to sensitive and accurate screening of potential cases of infection from patients in a clinical setting. Enabling testing outside of sophisticated laboratories broadens the scope of control and surveillance efforts, but also requires robust and simple methods that can be used without expensive instrumentation. Here we report a method to identify SARS-CoV-2 (COVID-19) virus RNA from purified RNA or cell lysis using loop-mediated isothermal amplification (LAMP) using a visual, colorimetric detection. This test was additionally verified using RNA samples purified from respiratory swabs collected from COVID-19 patients in Wuhan, China with equivalent performance to a commercial RT-qPCR test while requiring only heating and visual inspection. This simple and sensitive method provides an opportunity to facilitate virus detection in the field without a requirement for complex diagnostic infrastructure.


Diagnostic accuracy of a host response point-of-care test for identifying COVID-19

Abstract: Rationale Management of the COVID-19 pandemic is hampered by long delays associated with centralised laboratory PCR testing. In hospitals this leads to poor patient flow and nosocomial transmission and rapid, accurate diagnostic tests are urgently required. The FebriDx is a point-of-care test that detects an antiviral host response protein in finger prick blood within 10 minutes, but its accuracy for the detection of COVID-19 is unknown. Objectives To evaluate the diagnostic accuracy of FebriDx in hospitalised patients during the first wave of the pandemic. Methods Measures of diagnostic accuracy were calculated based on FebriDx results compared to the reference standard of PCR, and stratified by duration of symptoms. A multivariable predictive model was developed and underwent internal validation. Results FebriDx was performed on 251 patients and gave a valid result in 248. 118 of 248 (48%) were PCR positive for COVID-19. Sensitivity of FebriDx for the identification of COVID-19 was 93% (110/118; 95% CI 87 to 97%) and specificity was 86% (112/130; 95%CI 79 to 92%). Positive and negative likelihood ratios were 6.73 (95%CI 4.37 to 10.37) and 0.08 (95%CI 0.04 to 0.15) respectively. In the multivariate model diagnosis of COVID-19 was not significantly influenced by clinical symptoms and signs, and FebriDx accuracy was not improved by restricting testing to those with duration of symptoms of less than seven days. Conclusions During the first wave of the pandemic, FebriDx had high sensitivity for the identification of COVID-19 in hospitalised adults and could be deployed as a front door triage tool.


Sensitivity and specificity of a rapid test for assessment of exposure to SARS-CoV-2 in a community-based setting in Brazil

Abstract: Background: While the recommended laboratory diagnosis of COVID-19 is a molecular based assay, population-based studies to determine the prevalence of COVID-19 usually use serological assays. Objective: To evaluate the sensitivity and specificity of a rapid diagnostic test for COVID-19 compared to quantitative reverse transcription polymerase chain reaction (qRT-PCR). Methods: We evaluated the sensitivity using a panel of finger prick blood samples from participants >18 years of age that had been tested for COVID-19 by qRT-PCR. For assessing specificity, we used serum samples from the 1982 Pelotas (Brazil) Birth Cohort participants collected in 2012 with no exposure to SARS-CoV-2. Results: The sensitivity of the test was 77.1% (95% CI 66.6 - 85.6), based upon 83 subjects who had tested positive for qRT-PCR at least 10 days before the rapid diagnostic test (RDT). Based upon 100 sera samples, specificity was 98.0% (95% CI 92.9 - 99.8). There was substantial agreement (Kappa score 0.76) between the qRT-PCR results and the RDT. Interpretation. The validation results are well in line with previous assessments of the test, and confirm that it is sufficiently precise for epidemiological studies aimed at monitoring levels and trends of the COVID-19 pandemic.


Saliva Sample as a Non-Invasive Specimen for the Diagnosis of Coronavirus Disease-2019 (COVID-19): a Cross-Sectional Study

Abstract: Objectives. Amid the increasing number of global pandemic coronavirus disease 2019 (COVID-19) cases, there is a need for a quick and easy method to obtain a non-invasive sample for the detection of this novel coronavirus 2019 (SARS-CoV-2). We aimed to investigate the potential use of saliva samples as a non-invasive tool for the diagnosis of COVID-19. Methods. From 27 March to 4 April, 2020, we prospectively collected saliva samples and a standard nasopharyngeal and throat swab in persons seeking care at an acute respiratory infection clinic in a university hospital during the outbreak of COVID-19. Real-time polymerase chain reaction (RT-PCR) was performed, and the results of the two specimens were compared. Results. Two-hundred pairs of the samples were collected. Sixty-nine (34.5%) patients were male, and the median (interquartile) age was 36 (28-48) years. Using nasopharyngeal and throat swab RT-PCR as the reference standard, the prevalence of COVID-19 diagnosed by nasopharyngeal and throat swab RT-PCR was 9.5%. The sensitivity and specificity of the saliva sample RT-PCR were 84.2% [95% confidence interval (CI) 79.2%-89.3%], and 98.9% (95% CI 97.5-100.3%), respectively. An analysis of the agreement between the two specimens demonstrated 97.5% observed agreement (kappa coefficient 0.851, 95% CI 0.723-0.979; p <0.001). Conclusions. Saliva specimens can be used for the diagnosis of COVID-19. The collection method is non-invasive, and non-aerosol generating. Using a saliva sample as a specimen for the detection of SARS-CoV-2 could facilitate the diagnosis of the disease, which is one of the strategies that helps in controlling the epidemic.


Combination of Antibody based rapid diagnostic tests used in an algorithm may improve their performance in SARS CoV-2 diagnosis.

Abstract: Background: Globally response to the SARS-CoV-2 pandemic is highly limited by diagnostic methods. Currently, World Health Organization (WHO) recommends the use of molecular assays for confirmation of SARS-CoV-2 infection which are highly expensive and require specialized laboratory equipment. This is a limitation in mass testing and in low resource settings. SARS CoV-2 IgG/IgM antibody tests have had poor diagnostic performance that do not guarantee their use in diagnostics. In this study we demonstrate a concept of using a combination of RDTs in an algorithm to improve their performance for diagnostics. Method: Eighty six (86) EDTA whole blood samples were collected from SARS-CoV-2 positive cases admitted at Masaka and Mbarara Regional Referral Hospitals in Uganda. These were categorized from day when confirmed positive as follows; category A (0-3 days, 10 samples), category B (4-7 days, 20 samples), Category C (8-17 days, 11 samples) and Category D (18-28 days, 20 samples). Plasma was prepared, transported to the testing laboratory and stored at -200C prior to testing. A total of 13 RDTS were tested following manufacturers instructions. Data was entered in Microsoft Excel exported to STATA for computation of sensitivity and specificity. We computed for all possible combinations of 2 of the 13 RDTS (13C2) that were evaluated in parallel algorithm. Results: The individual sensitives of the RDTs ranged between 74% and 18% and there was a general increasing trend across the categories with days since PCR confirmation. A total of 78 possible combinations of the RDTs to be used in parallel was computated. The combinations of the 2 RDTS improved the sensitivities to 90%. Discussion: We demonstrate that use of RDTs in combinations can improve their overall sensitivity. This approach when used on a wider range of combination of RDTs may yield combinations that can give sensitivities that are of diagnostics relevance in mass testing and low resource setting.


Recommendations for sample pooling on the Cepheid GeneXpert® system using the Cepheid Xpert® Xpress SARS-CoV-2 assay

Abstract: The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the Xpert® Xpress SARS-CoV-2 assay limit of detection was comparable to central laboratory reverse-transcription quantitative PCR tests with observed SARS-CoV-2 detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were diluted into six sample pools. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted by population public health needs. The suggested number of samples per pool, or pooling depth, is unique for each point-of-care test site and should be determined by assessing positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.


Evaluation of performance of two SARS-CoV-2 Rapid whole-blood finger-stick IgM-IgGCombined Antibody Tests

Abstract: Background The SARS-CoV-2 virus is responsible for the infectious respiratory disease called COVID-19 (COronaVIrus Disease). In response to the growing COVID-19 pandemic, Rapid Diagnostic Tests (RDTs) have been developed to detect specific antibodies, IgG and IgM, to SARS-CoV-2 virus in human whole blood. We conducted a real-life study to evaluate the performance of two RDTs, COVID-PRESTO and COVID-DUO, compared to the gold standard, RT-PCR. Methods RT-PCR testing of SARS-Cov-2 was performed from nasopharyngeal swab specimens collected in adult patients visiting the infectious disease department at the hospital (Orleans, France). Fingertip whole blood samples taken at different time points after onset of the disease were tested with RDTs. The specificity and sensitivity of the rapid test kits compared to test of reference (RT-PCR) were calculated. Results Among 381 patients with symptoms of COVID-19 who went to the hospital for a diagnostic, 143 patients were RT-PCR negative. Results of test with RDTs were all negative for these patients, indicating a specificity of 100% for both RDTs. In the RT-PCR positive subgroup (n=238), 133 patients were tested with COVID-PRESTO and 129 patients were tested with COVID-DUO (24 patients tested with both). The further the onset of symptoms was from the date of collection, the greater the sensitivity. The sensitivity of COVID-PRESTO test ranged from 10.00% for patients having experienced their 1st symptoms from 0 to 5 days ago to 100% in patients where symptoms had occurred more than 15 days before the date of tests. For COVID-DUO test, the sensitivity ranged from 35.71% [0-5 days] to 100% (> 15 days). Conclusion COVID-PRESTO and DUO RDTs turned out to be very specific (none false positive) and to be sensitive enough after 15 days from onset of symptom. These easy to use IgG/IgM combined test kits are the first ones allowing a screening with capillary blood sample, by typing from a finger prick. These rapid tests are particularly interesting for screening in low resource settings.


Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: a reliable, faster and economical method.

Abstract: Background: Corona virus disease 2019 (COVID-19) which initially started as a cluster of pneumonia cases in the Wuhan city of China has now become a full blown pandemic. Timely diagnosis of COVID-19 is the key in containing the pandemic and breaking the chain of In low and middle income countries availability of testing kits has become the major bottle neck in testing. Novel methods like pooling of samples are the need of the hour. Method: Extracted RNA samples were randomly placed in pools of 8 on a 96 well plate. Both individual RNA (ID) and pooled RNA RT-qPCR for the screening E gene were done in the same plate and the positivity for the E gene was seen. Results: The present study demonstrated that pool testing with 8 RNA samples can easily detect even up to a single positive sample with Ct value as high as 38. The present study also showed that the results of pool testing is not affected by number of positive samples in a pool. Conclusion: Pooling of 8 RNA samples can reduce the time and expense by one eighth, and can help expand diagnostic capabilities, especially during constrained supply of reagents and PCR kits for the diagnosis of SARS-CoV-2 infection.


Comparison of Cepheid Xpert Xpress and Abbott ID Now to Roche cobas for the Rapid Detection of SARS-CoV-2

Abstract: The SARS-CoV-2 pandemic has created an urgent and unprecedented need for rapid large-scale diagnostic testing to inform timely patient management. This study compared two recently-authorized rapid tests, Cepheid Xpert Xpress SARS-CoV-2 and Abbott ID Now SARS-CoV-2 to the Roche cobas SARS-CoV-2 assay. A total of 113 nasopharyngeal swabs were tested, including 88 positives spanning the full range of observed Ct values on the cobas assay. Compared to cobas, the overall positive agreement was 73.9% with ID Now and 98.9% with Xpert. Negative agreement was 100% and 92.0% for ID Now and Xpert, respectively. Both ID Now and Xpert showed 100% positive agreement for medium and high viral concentrations (Ct value <30). However, for Ct values >30, positive agreement was 34.3% for ID Now and 97.1% for Xpert. These findings highlight an important limitation of ID Now for specimens collected in viral or universal transport media with low viral concentrations. Further studies are needed to evaluate the performance of ID Now for direct swabs.


iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2

Abstract: The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.


Noisy Pooled PCR for Virus Testing

Abstract: Fast testing can help mitigate the coronavirus disease 2019 (COVID-19) pandemic. Despite their accuracy for single sample analysis, infectious diseases diagnostic tools, like RT-PCR, require substantial resources to test large populations. We develop a scalable approach for determining the viral status of pooled patient samples. Our approach converts group testing to a linear inverse problem, where false positives and negatives are interpreted as generated by a noisy communication channel, and a message passing algorithm estimates the illness status of patients. Numerical results reveal that our approach estimates patient illness using fewer pooled measurements than existing noisy group testing algorithms. Our approach can easily be extended to various applications, including where false negatives must be minimized. Finally, in a Utopian world we would have collaborated with RT-PCR experts; it is difficult to form such connections during a pandemic. We welcome new collaborators to reach out and help improve this work!


Evaluation on the diagnostic efficiency of different methods in detecting COVID-19.

Abstract: Objective: To evaluate the diagnostic efficiency of different methods in detecting COVID-19 to provide preliminary evidence on choosing favourable method for COVID-19 detection. Methods: PubMed, Web of Science and Embase databases were searched for identifing eligible articles. All data were calculated utilizing Meta Disc 1.4, Revman 5.3.2 and Stata 12. The diagnostic efficiency was assessed via these indicators including summary sensitivity and specificity, positive likelihood ratio (PLR), negative LR (NLR), diagnostic odds ratio (DOR), summary receiver operating characteristic curve (sROC) and calculate the AUC. Results: 18 articles (3648 cases) were included. The results showed no significant threshold exist. EPlex: pooled sensitivity was 0.94; specificity was 1.0; PLR was 90.91; NLR was 0.07; DOR was 1409.49; AUC=0.9979, Q*=0.9840. Panther Fusion: pooled sensitivity was 0.99; specificity was 0.98; PLR was 42.46; NLR was 0.02; DOR was 2300.38; AUC=0.9970, Q*=0.9799. Simplexa: pooled sensitivity was 1.0; specificity was 0.97; PLR was 26.67; NLR was 0.01; DOR was 3100.93; AUC=0.9970, Q*=0.9800. Cobas: pooled sensitivity was 0.99; specificity was 0.96; PLR was 37.82; NLR was 0.02; DOR was 3754.05; AUC=0.9973, Q*=0.9810. RT-LAMP: pooled sensitivity was 0.98; specificity was 0.99; PLR was 36.22; NLR was 0.04; DOR was 751.24; AUC=0.9905, Q*=0.9596. Xpert Xpress: pooled sensitivity was 0.99; specificity was 0.97; PLR was 27.44; NLR was 0.01; DOR was 3488.15; AUC=0.9977, Q*=0.9829. Conclusions: These methods (ePlex, Panther Fusion, Simplexa, Cobas, RT-LAMP and Xpert Xpress) bear higher sensitivity and specificity, and might be efficient methods complement to the gold standard.


A new role for Biofoundries in rapid prototyping, development, and validation of automated clinical diagnostic tests for SARS-CoV-2

Abstract: The SARS-CoV-2 pandemic has shown how the rapid rise in demand for patient and community sample testing, required for tracing and containing a highly infectious disease, has quickly overwhelmed testing capability globally. With most diagnostic infrastructure dependent on specialised instruments, their exclusive reagent supplies quickly become bottlenecks in times of peak demand, creating an urgent need for novel approaches to boost testing capacity. We address this challenge by refocusing the full synthetic biology stack available at the London Biofoundry onto the development of alternative patient sample testing pipelines. We present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled, and that accepts a diverse range of reagents. Using an in-house-generated, open-source, MS2-virus-like-particle-SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two novel detection assays based on CRISPR-Cas and Loop-mediated isothermal Amplification (LAMP) approaches. In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and benchmark SARS-CoV-2 detection in patient samples via RT-qPCR, CRISPR-Cas, and LAMP against clinical test sets. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs and now contributes to increased patient sample processing in the UK while we continue to refine and develop novel high-throughput diagnostic methods. Finally, our workflows and protocols can be quickly implemented and adapted by members of the Global Biofoundry Alliance and the wider scientific and medical diagnostics community.


A SIMPLE COLORIMETRIC MOLECULAR DETECTION OF NOVEL CORONAVIRUS (COVID-19), AN ESSENTIAL DIAGNOSTIC TOOL FOR PANDEMIC SCREENING

Abstract: The recent outbreak of the newly emerged novel coronavirus (SARS-CoV-2) presents a big challenge for public health laboratories as virus isolates are not available while there is an increasing evidence that the epidemic is more widespread than initially thought, as well as spreading internationally across borders through travellers does already happen warranting a methodology for the rapid detection of the infection to control SARS-CoV-2. Aim: We intended to develop and deploy a robust and rapid diagnostic methodology using LAMP assay for use in point of care settings to detect SARS-COV-2 infection. Methodology: In the present study, we have developed a validated rapid diagnostic procedure to detect SARS-CoV-2 using LAMP assay, its design relying on isothermal amplification of the nucleic acids of the SARS-CoV-2. Results: The LAMP assay developed detects SARS-CoV-2 infection rapidly with high sensitivity and reliability. The data generated by LAMP assay were comparable and at par with the data generated by real-time PCR method. Conclusion: The present study demonstrates that the LAMP assay developed was a rapid, reliable, sensitive and cost effective method to detect SARS-CoV-2 infection in a point of care as well as in laboratory settings.


Head-to-head comparison of four antigen-based rapid detection tests for the diagnosis of SARS-CoV-2 in respiratory samples

Abstract: In the context of the Covid-19 pandemic, the development and validation of rapid and easy-to-perform diagnostic methods are of high priority. We compared the performance of four rapid antigen detection tests for SARS-CoV-2 in respiratory samples. Immunochromatographic SARS-CoV-2 assays from RapiGEN, Liming bio, Savant, and Bioeasy were evaluated using universal transport medium containing naso-oropharyngeal swabs from suspected Covid-19 cases. The diagnostic accuracy was determined in comparison to SARS-CoV-2 RT-PCR. A total of 111 samples were included; 80 were RT-PCR positive. Median patients’ age was 40 years, 55% were female, and 88% presented within the first week after symptom onset. The evaluation of the Liming bio assay was discontinued due to insufficient performance. The overall sensitivity values of RapiGEN, Liming bio, and Bioeasy tests were 62.0% (CI95% 51.0–71.9), 16.7% (CI95% 10.0–26.5), and 85.0% (CI95% 75.6–91.2), respectively, with specificities of 100%. Sensitivity was significantly higher in samples with high viral loads (RapiGEN, 84.9%; Bioeasy, 100%). The study highlighted the significant heterogeneity of test performance among evaluated assays, which might have been influenced by the use of a non-validated sample material. The high sensitivity of some tests demonstrated that rapid antigen detection has the potential to serve as an alternative diagnostic method, especially in patients presenting with high viral loads in early phases of infection. This is particularly important in situations with limited access to RT-PCR or prolonged turnaround time. Further comparative evaluations are necessary to select products with high performance among the growing market of diagnostic tests for SARS-CoV-2.


Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens

Abstract: The novel human coronavirus SARS-CoV-2 was first discovered in the city of Wuhan, Hubei province, China, causing an outbreak of pneumonia in January 2020. As of April 10, 2020, the virus has rapidly disseminated to over 200 countries and territories, causing more than 1.6 million confirmed cases of COVID-19 and 97,000 deaths worldwide. The clinical presentation of COVID-19 is fairly non-specific, and symptoms overlap with other seasonal respiratory infections concurrently circulating in the population. Further, it is estimated that up to 80% of infected individuals experience mild symptoms or are asymptomatic, confounding efforts to reliably diagnose COVID-19 empirically. To support infection control measures, there is an urgent need for rapid and accurate molecular diagnostics to identify COVID-19 positive patients. In the present study, we have evaluated the analytical sensitivity and clinical performance of four SARS-CoV-2 molecular diagnostic assays granted Emergency Use Authorization by the FDA using nasopharyngeal swabs from symptomatic patients. This information is crucial for both laboratories and clinical teams, as decisions on which testing platform to implement are made.


Rapid, sensitive, full genome sequencing of Severe Acute Respiratory Syndrome Virus Coronavirus 2 (SARS-CoV-2)

Abstract: SARS-CoV-2 recently emerged, resulting a global pandemic. Rapid genomic information is critical to understanding transmission and pathogenesis. Here, we describe validated protocols for generating high-quality full-length genomes from primary samples. The first employs multiplex RT-PCR followed by MinION or MiSeq sequencing. The second uses singleplex, nested RT-PCR and Sanger sequencing.


Laboratory capability for molecular detection and confirmation of novel coronavirus in Europe, November 2012.

Abstract: A rapid survey by the European Centre for Disease Prevention and Control (ECDC) and the World Health Organization (WHO) Regional Office for Europe ascertained the availability of national reference laboratory testing for a recently detected novel coronavirus as of 28 November 2012. Screening by internal quality controlled upE-RT-PCR assay was available in 23/46 of responding countries in the WHO European Region, of which 19/30 in European Union (EU) and European Economic Area (EEA) countries. Confirmation of positive screened samples by either ORF1b - RT-PCR, or other target RT-PCR assays with sequence analysis or whole-genome sequence analysis was available in 22/46 responding countries of which 18/30 in EU/EEA countries.


The Appropriate Use of Testing for COVID-19

Abstract: Many public officials are calling for increased testing for the 2019 novel coronavirus disease (COVID-19), and some governments have taken extraordinary measures to increase the availability of testing. However, little has been published about the sensitivity and specificity of the reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs that are commonly used for testing. This narrative review evaluates the literature regarding the accuracy of these tests, and makes recommendations based on this literature. In brief, a negative RT-PCR nasopharyngeal swab test is insufficient to rule out COVID-19. Thus, over-reliance on the results of the test may be dangerous, and the push for widespread testing may be overstated.


Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19

Abstract: Recently, a novel coronavirus (SARS-CoV-2; coronavirus disease 2019, COVID-19) has emerged, rapidly spreading and severely straining the capacity of the global health community. Many nations are employing combinations of containment and mitigation strategies, where early diagnosis of COVID-19 is vital in controlling illness progression and limiting viral spread within the population. Thus, rapid and accurate methods of early detection are vital to contain COVID-19 and prevent further spread and predicted subsequent infectious waves of viral recurrence in future. Immediately after its initial characterization, Chinese and American Centers for Disease Control and Prevention (CDCs) rapidly employed molecular assays for detection of COVID-19, mostly employing real-time polymerase chain reaction (RT-PCR) methods. However, such methods require specific expensive items of equipment and highly trained analysts, requiring upwards of 4-8 h to process. These requirements coupled with associated financial pressures may prevent effective deployment of such diagnostic tests. Loop mediated isothermal amplification(LAMP) is method of nucleic acid amplification which exhibits increased sensitivity and specificity are significantly rapid, and do not require expensive reagents or instruments, which aids in cost reduction for coronavirus detection. Studies have shown the successful application of LAMP assays in various forms to detect coronavirus RNA in patient samples, demonstrating that 1-10 copies of viral RNA template per reaction are sufficient for successful detection, ~100-fold more sensitive than conventional RT-PCR methods. Importantly, studies have also now demonstrated the effectiveness of LAMP methodology in the detection of SARS-CoV-2 RNA at significantly low levels, particularly following numerous improvements to LAMP assay protocols. We hypothesise that recent advancements in enhanced LAMP protocols assay perhaps represent the best chance for a rapid and robust assay for field diagnosis of COVID-19, without the requirement of specialized equipment and highly trained professionals to interpret results. Herein, we present our arguments with a view to disseminate such findings, to assist the combat of this virus that is proving so devastating. We hope that this strategy could be applied rapidly, and confirmed for viability with clinical samples, before being rolled out for mass-diagnostic testing in these current times.


Combining PCR and CT testing for COVID

Abstract: We analyze the effect of using a screening CT-scan for evaluation of potential COVID-19 infections in order to isolate and perform contact tracing based upon a viral pneumonia diagnosis. RT-PCR is then used for continued isolation based upon a COVID diagnosis. Both the low false negative rates and rapid results of CT-scans lead to dramatically reduced transmission. The reduction in cases after 60 days with widespread use of CT-scan screening compared to PCR by itself is as high as $50\times$, and the reduction of effective reproduction rate $R(t)$ is $0.20$. Our results imply that much more rapid extinction of COVID is possible by combining social distancing with CT-scans and contact tracing.


Diagnostic testing for SARS-CoV-2


Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2

Abstract: Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.


Trends and Innovations in Biosensors for COVID-19 Mass Testing

Abstract: Fast and widespread diagnosis is crucial to fighting against the outbreak of COVID-19. This work surveys the landscape of available and emerging biosensor technologies for COVID-19 testing. Molecular diagnostic assays based on quantitative reverse transcription polymerase chain reaction are used in most clinical laboratories. However, the COVID-19 pandemic has overwhelmed testing capacity and motivated the development of fast point-of-care tests and the adoption of isothermal DNA amplification. Antigenic and serological rapid tests based on lateral-flow immunoassays suffer from low sensitivity. Advanced digital systems enhance performance at the expense of speed and the need for large equipment. Emerging technologies, including CRISPR gene-editing tools, benefit from high sensitivity and specificity of molecular diagnostics and the easy use of lateral-flow assays. DNA sequencing and sample pooling strategies are highlighted to bring out the full capacity of the available biosensor technologies and accelerate mass testing.


Laboratory information system requirements to manage the COVID-19 pandemic: a report from the Belgian national reference testing center

Abstract: OBJECTIVE: To describe the development, implementation and requirements of laboratory information system (LIS) functionality to manage test ordering, registration, sample flow, and result reporting during the COVID-19 pandemic. CONTEXT AND SETTING: Our large (>12,000,000 tests/year) academic hospital laboratory is the Belgian National Reference Center (NRC) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We performed a moving total of > 25,000 SARS-CoV-2 PCR tests in parallel to standard routine testing since the start of the outbreak. A LIS implementation team dedicated to develop tools to remove the bottlenecks, primarily situated in the pre- and post-analytical phase, was established early in the crisis. RESULTS: We outline the design, implementation and requirements of LIS functionality related to managing increased test demand during the COVID-19 crisis, including tools for test ordering, standardized order sets integrated into a computerized provider order entry module, notifications on shipping requirements, automated triaging based on digital metadata forms, and the establishment of databases with contact details of other laboratories and primary care physicians to enable automated reporting. We also describe our approach to data mining and reporting of actionable daily summary statistics to governing bodies and other policymakers. DISCUSSION: Rapidly developed, agile extendable LIS functionality and its meaningful use alleviates the administrative burden on laboratory personnel and improves turn-around-time of SARS-CoV-2 testing. It will be important to maintain an environment that is conducive for the rapid adoption of meaningful LIS tools post-COVID crisis.


Development of a rapid test kit for SARS-CoV-2: an example of product design

Abstract: We present an example of applying 'need-driven' product design principle to the development of a rapid test kit to detect SARS-COV-2 (COVID-19). The tests are intended for use in the field and, longer term, for home use. They detect whether a subject is currently infected with the virus and is infectious. The urgent need for large numbers of tests in field setting imposes constraints such as short test time and lack of access to specialist equipment, laboratories and skilled technicians to perform the test and interpret results. To meet these needs, an antigen test based on RT-LAMP with colorimetric readout was chosen. Direct use of swab sample with no RNA extraction was explored. After extensive experimental study (reported elsewhere), a rapid test kit has been fabricated to satisfy all design criteria.