Mucosal immunity, including secretory IgA (sIgA), plays an important role in early defenses against respiratory pathogens. Salivary testing, the most convenient way to measure sIgA, has been used to characterize mucosal immune responses to many viral infections including SARS, MERS, influenza, HIV, and RSV. However, its role has not yet been characterized in the COVID-19 pandemic. Here, we report development and validation of a rapid immunoassay for measuring salivary IgA against the SARS-CoV-2 virus, and report quantitative results in both pre-COVID-19 and muco-converted subjects.We developed and refined a specific test for salivary IgA against SARS-CoV-2 on the Brevitest platform, a rapid immunoassay system designed for point-of-care use. A qualitative test was validated as per FDA guidelines with saliva obtained from subjects prior to the emergence of COVID-19, and from PCR-confirmed COVID-19 patients. We also generated a quantitative measure of anti-SARS-CoV-2 salivary IgA. Time taken for saliva self-collection was measured and its ease-of-use assessed.We successfully validated a qualitative salivary assay for SARS-CoV-2 IgA antibodies, with positive and negative predictive values of 92% and 97%, respectively, and no observable cross-reactivity with any of seven potential confounders. Pre-COVID-19 saliva samples showed an 8-fold range of IgA concentrations, suggesting a broad continuum of natural antibody resistance against the novel virus, though at levels lower than that observed in COVID-19 PCR-confirmed subjects. Samples from muco-positive subjects also shown a ~9-fold variation in salivary IgA levels, with elevated salivary IgA observed beyond three months after onset of symptoms. We observed a correlation (r=0.4405) between salivary IgA levels and COVID-19 disease severity. In anecdotal observations, we observed individuals who exhibited antibodies early in the course of their disease, contemporaneously with a positive PCR test, as well as individuals who muco-converted despite no known direct exposure to a COVID-19 patient, no symptoms, and negative molecular and/or serum antibody tests. Salivary collection took 5–10 minutes, and was reported as being easy .Mucosal immunity, including secretory IgA, plays an important role in host defense against respiratory pathogens, and our early data suggest it may do so in COVID-19. Salivary IgA, an accessible marker of mucosal immunity, may be a useful indicator of several key parameters including individual and community immune response, disease severity, clinical risk, and herd immunity. The non-invasive nature and ease of saliva collection facilitates its potential use as a biomarker for ongoing patient assessment and management, as well as a community surveillance tool. By measuring mucosal immune responses directly and systemic immune responses indirectly, salivary IgA could be useful in developing and deploying a vaccine(s) against COVID-19. Quantitative IgA assessment could also potentially serve as a tool to segment the population into different risk categories and inform individual and collective decisions relating to appropriate activities and vaccine prioritization/delivery. These data reinforce the importance of further investigation into the role of mucosal immunity and IgA in host responses against COVID-19. COVID-19 is caused by a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the World Health Organization in February 20203. Coronaviruses have been responsible for several respiratory disease outbreaks over the previous two decades, including Severe Acute Respiratory Syndrome  which was primarily limited to Southeast Asia, and Middle East Respiratory Syndrome . Although the exact mechanisms behind the increased spread of SARS-CoV-2 remains to be discovered, one hypothesis suggests that SARS-CoV-2 spread is fueled by the infectivity of asymptomatic or pre-symptomatic carriers, making containment difficult and allowing the virus to spread worldwide through travel and community-based contacts5.The COVID-19 pandemic has been characterized by rapid global spread and has impacted the life of almost every person on the planet. First reported in the Wuhan province in China in December 2019, the COVID-19 disease reached pandemic status within six months and has spread to nearly every country. Although initially contained in several countries, COVID-19 has begun to resurface even as it continues to surge through other countries, such as the United States, Russia, India and Brazil, which have had less success with containment or are experiencing rapid increases in the number of cases6. Compounding the challenges of preventing transmission of the disease, symptoms can vary widely in severity; some patients remain largely asymptomatic or present with mild disease while others may develop a potentially fatal severe respiratory crisis7. Common symptoms include sore throat, fever, cough, muscle pain, headache, and a characteristic loss of taste or smell. Severe cases may result in progressive lung pathology beginning with difficulty breathing, and progressing to pneumonia or acute respiratory distress syndrome (ARDS), often requiring intubation and mechanical ventilation of the lungs8. ARDS is typically associated with a cytokine storm, and can result in organ damage and end stage failure in organs beyond the lung, including the brain, gastrointestinal tract, vasculature, kidney, liver and heart10.SARS-CoV-2 appears to be primarily spread via respiratory droplets which begin as mucosal secretions in infected individuals. These droplets become aerosolized by coughing, sneezing, or talking and can spread through the air or through contaminating surfaces. Respiratory droplets are particularly infectious when infected persons are in enclosed areas or in close contact with others14 targeted against the pathogen, along with the release of cytokines which attract neutrophils, natural killer cells, and lymphoid cells. In conjunction with mucociliary clearance, and, where appropriate, recruitment of additional responder cells , the mucosal innate immune system protects the respiratory tract and facilitates pathogen clearance.Outside of efforts to limit physical exposure, including wearing face coverings and limiting contact with others, the first line of defense against a viral respiratory infection is the mucosal immune system in the respiratory tract. The mucosal immune system plays critical roles in both innate and adaptive immunity. As part of the innate response, the mucosal immune system coats the lumen of the respiratory tract with a liquid lining containing surfactants, mucus, and periciliary fluid. A rapid response to a pathogen challenge to the respiratory tract is triggered by ‘sensor’ cells, which include epithelial cells, macrophages, dendritic cells, and mast cells. Upon detection of a pathogen, these cells trigger innate responses, which include the generation of reactive oxygen species and anti-microbial peptides17. Dendritic cells, play a critical role in activating an adaptive response.When the viral inoculum is large enough, it can trigger participation by the mucosal adaptive immune system, mediated in part through dendritic cells – protruding through the epithelium – to sample luminal contents18 primarily exists in the body in two forms: the predominant polymeric form is a dimer  found in mucosal secretions, while the less abundantly monomeric form is primarily found in serum. Plasma cells producing dimeric IgA localize along the lamina propria adjacent to mucosal membranes. Dimeric IgA is translocated across the epithelium via the polymeric immunoglobulin receptor18 and is secreted into the mucosa, where the covalent linkage confers protease resistance to the IgA molecule19. This secretory IgA (sIgA) has a number of essential functions within mucosal immunity. A major function is to prevent the cognate pathogens from infecting hos cells via immune exclusion, by competing for the host-cell ligands that trigger viral entry. sIgA further contributes to viral clearance via agglutination and shielding of microbial adhesins for later clearance via ciliary activity20. In the case of SARS-CoV-2, sIgA antibodies may prevent adhesion to target epithelial cells via neutralization of the coronavirus spike protein (and thus inhibiting its interaction with the host ACE-2 receptor21) or binding to the SARS-CoV-2 nucleocapsid protein. Beyond its role in immune exclusion, sIgA can initiate and regulate the process of myeloid immune responses through the FcaR receptor to the IgA FC region that is found on multiple immune and epithelial cells, resulting in a broad range of effector functions involving both humoral and cellular responses. The production of IgA, relative to other antibody classes is closely controlled by the classs-witching process.Part of this adaptive response is the generation of antibodies against the pathogen, including IgA which plays an important role in mucosal immunity. IgA, which is the antibody class produced in largest quantities by the body22, possibly as early as two days after onset of symptoms (versus five days for either IgM or IgG)23. This suggests that IgA may be the first antibody to appear in response to SARS-CoV-2 infection, but research into the role of IgA, especially mucosal IgA in COVID-19 has lagged.IgA responses have already been described as playing a role in immune responses to SARS-CoV-2. Serum IgA has been detected in COVID-19 patients and appears to be detectable earlier than IgM or IgG antibodies25Beyond its important role in mucosal immunity and its clear importance in regulating host response to respiratory infections, IgA is attractive for public health response for another reason: its localization to the mucosa. As a secreted antibody, sIgA is easily accessible in saliva, and compared with other sample types, multiple groups have recognized the attractiveness of salivary testing due to its ease of collectionHere we report results from over 250 tests IgA tests, including those from earlier versions of our assay. The IgA test was developed using the Brevitest platform, which is designed for rapid, quantitative, point-of-care ELISA testing. The test was validated for qualitative detection of IgA in saliva in accordance with FDA guidelines using PCR-confirmed subjects as the true positive group and saliva samples obtained before COVID-19 emergence as true negative. In addition, we share quantitative data on levels of salivary IgA against SARS-CoV-2 in both pre-COVID and muco-positive samples. These analyses demonstrate a broad range of IgA concentrations across subjects, suggest the persistence of anti-SARS-CoV-2 salivary IgA through at least 3 months after onset of symptoms and a potential correlation between salivary IgA levels and disease severity. We also present data on time and ease of use in self-collected saliva samples. Our findings suggest a potential role for salivary IgA testing in managing individual patients, understanding community infection rates, and supporting vaccine development and deployment.We utilized our Brevitest platform to develop and validate our salivary IgA assay, which we term BRAVO . Briefly, the Brevitest platform is a rapid, automated enzyme-linked immunosorbent assay (ELISA) designed for point-of-care (POC) use. As the platform is automated, minimal variation between loading, reagents, washing, and development times are expected. Additional details regarding the platform can be found in the Methods. We developed our COVID-19 salivary IgA assay to detect IgA antibodies against either the SARS-CoV-2 Spike S1 antigen (S1) or the SARS-CoV-2 Nucleocapsid Protein (NP) antigen. To validate the BRAVO assay and to ensure that we were detecting antibodies within a linear range, we spiked increasing concentrations of commercially available anti-S1 and anti-NP antibodies into simulated saliva. We observed a strong, linear correlation in our BRAVO assay  with a pAfter validating the linear range and parameters of the BRAVO assay, we next sought to determine a cut-off threshold for positive and negative sample types. We purchased true negative samples  to determine our negative mean. Interestingly, we observed significant variation (~9-fold) in salivary IgA levels among the true negative samples , potenti26 while IgA in serum is 70–400 mg/dl27), therefore, before testing, we conservatively diluted the serum samples 10-fold using true-negative patient saliva sample as the diluent. We tested multiple independent samples from each potentially cross-reactive anti-serum, all of which were negative , nonspecific human IgG, and rheumatoid factors. Although we would have preferred to utilize patient-derived saliva samples, we were unable to acquire cross-reactive saliva samples, therefore we purchased commercially available patient-derived serum samples. Antibody concentrations in serum are 10–30x higher than concentrations typically found in saliva  with symptoms consistent with COVID-19. Information on their relevant history was collected from the subjects including information on COVID-19-related symptoms and any additional serology testing they may have undergone. 48 of the 53 additional subjects (91%) tested muco-positive for salivary IgA. The data from these 83 muco-positive subjects (48+35) was analyzed further.As with the pre-COVID samples, we observed a broad range of salivary IgA concentrations , with an28.We then looked for correlations between IgA levels and other subject attributes. We examined the correlation between IgA levels and elapsed time between onset of symptoms and saliva collection . For the29. There was no apparent correlation between salivary IgA levels and duration of symptoms  or known exposure to COVID-19. This is again anecdotal, but we are sharing it since it raises some intriguing questions.To minimize risk to lab personnel of exposure to SARS-CoV-2, our clinical study was limited to salivary samples collected from individuals who were at least a month post-symptom onset, so we cannot report on when IgA levels first appear in saliva, though that work is currently underway. However, in limited anecdotal data collected since, we observe IgA levels arising early in the disease process, at the same time as with viral shedding. This early appearance of mucosal IgA is consistent with its role in protection against respiratory pathogens and is also consistent with reports that SARS-CoV-2 serum IgA appears earlier than IgG or IgM, possibly as early as two days after onset of symptoms.Finally, we sought to collect additional data regarding the utility of saliva collection for this, and future assays. Patients and sample donors in our optimization and clinical validation studies were asked to complete a questionnaire about sample collection, including an estimation of the overall time required and an assessment of the ease of use for the self-collection. Saliva collection in this study was by supervised self-collection. Time required to collect the sample, measured by the supervisor, was 5–10 minutes in each case . On a sc30 as well as effectors – primarily secretory immunoglobulins – that are generated by and integrated with adaptive immunity. Mucosal immunity has been shown to play a crucial role against a broad range of respiratory pathogens, although its role in COVID-19 has not yet been established.The classic model of human immunity comprises two interconnected, complementary systems: the innate immune system and the adaptive immune system. Lung mucosal immunity can act as a critical link between those systems with respiratory pathogens, playing an important, front-line defensive role. Lung mucosal immunity includes inducible innate resistance mediated by reactive oxygen species and antimicrobial peptides,Secretory IgA (sIgA) is a principal component of mucosal immunity, and can easily be measured in saliva. Brevitest has validated a simple assay for measuring salivary IgA against SARS-CoV-2, which has been submitted to FDA for an Emergency Use Authorization (EUA). To our knowledge, no salivary IgA tests for SARS-CoV-2 have been authorized to date, and authorization of the BRAVO test will provide a new tool to assess immune responses in COVID-19. The Brevitest platform, on which our BRAVO assay runs, is designed to be a rapid (<15-minute), point-of-care, quantitative immunoassay. Although we have not yet validated the test for point-of-care (PoC) use, that work is currently underway. Given the convenience of saliva collection, a PoC test could open the door to expanded testing in multiple settings.Here we demonstrate that quantitative data from true negative (pre-COVID) samples could be used to cleanly define a binary, qualitative threshold to assess muco-positivity. While the test we have submitted to FDA for an EUA is qualitative, the quantitative results we report here raise important questions about the host response to the SARS-CoV-2 virus, with potentially significant implications for the COVID-19 pandemic. These findings suggest that a more robust understanding of the role of mucosal immunity in COVID-19 could improve decision-making by patients, clinicians, and policymakers. We discuss some of these implications in three settings: individual immunity and clinical implications; community surveillance and herd immunity; and vaccine development.Our observations that (i) we see a large variation in salivary IgA titer, even in pre-COVID-19 samples; (ii) elevated IgA levels appear to persist for at least 2–3 months; and (iii) individuals may develop mucosal IgA without an overt SARS-CoV-2 infection, each raise intriguing questions.31 and can bind to a number of pathogenic antigens. Polyreactive IgA appears to be a carryover from a primordial immune system that co-exists in humans along with the more modern adaptive immune system, and acts as a front-line host response that prevents epithelial entry of large numbers of microorganisms.32 Polyreactive IgA may be artifacts of prior adaptive responses, since IgA production – like IgG – generates memory cells that are stored for future use and expansion. These “natural” antibodies may display variable affinity against novel pathogens, as those pathogens may have epitopes in common with prior pathogens. However, these antibodies can also bind with affinities comparable to those seen with monoreactive antibodies. Further, other group have recently reported the presence of both cellular33 and humoral35 immunity to SARS-CoV-2 in pre-COVID samples, which have been attributed to prior exposure to other coronaviruses. We do not know if the salivary IgA antibodies detected in the pre-COVID-19 samples are protective, or if protection is related to mucosal antibody concentrations. But if these natural antibodies are protective, or accelerate a full adaptive response by giving the immune system an effective “seed” for subsequent rounds of somatic hypermutation and clonal selection, they could contribute to the variable susceptibility to COVID-19 infection that has been observed.Our unexpected detection of SARS-CoV-2 IgA antibodies in pre-COVID-19 saliva  is supported by the literature. Polyreactive “natural” sIgA was first discovered in human saliva and colostrumIntriguingly, even among the 83 muco-positive subjects in our study, we observed an over 9-fold range in IgA levels. If mucosal IgA in COVID-19 behaves in ways similar to those described for other respiratory pathogens, salivary IgA levels at either end of the spectrum (very low or very high) may have a clinical significance.36 Several groups have looked at correlation between changes in salivary IgA and respiratory infections in individuals and found that decreases in IgA correlated with an increase in infections. These findings have been reported in a wide variety of subjects including from elite athletes,37 infants,38 healthy children41 and children with Down’s syndrome.42 These findings suggest that individuals that are muco-negative for salivary IgA against SARS-CoV-2 may be at an elevated risk for infection and/or disease severity.On the one hand, low levels of mucosal IgA have been associated with an increased incidence of respiratory infections. Byars et al. reviewed data from 1,189,061 individuals who had undergone tonsillectomy or adenoidectomy in childhood with a comparably sized control group, and found a significant increase in respiratory infections.44 and “near-miss” SIDS45 infants had hyperimmune responses with substantially elevated levels of mucosal antibody responses. Yu et al. also reported a correlation between IgA levels and APACHE-II score in critically ill patients with COVID-19 23 although they did not examine salivary IgA. COVID-19 is characterized by sudden respiratory deterioration in patients, and sIgA can induce an inflammatory response in the airway, including neutrophil activation,46 and recent pre-prints suggest that systemic neutrophilic activation appears to be a marker of severity in pandemic flu47 and COVID-19.49 Thus, it may be desirable to look for correlations between clinical outcome and excess salivary IgA levels. If such a correlation is established, increased salivary IgA could serve as a biomarker to identify patients at an elevated risk of clinical deterioration in COVID-19, and perhaps candidates for early intervention with steroids, which have been shown to be effective in some ventilated patients.50Conversely, there is also data suggesting that excessive levels of mucosal IgA can induce a pathologic inflammatory response. For example, both SIDSOne of our intriguing, though anecdotal observations was that individuals may develop mucosal IgA without an overt COVID-19 infection. We have conducted over 250 assays, including those with earlier versions of the test. Brevitest employees, including several of the authors on this paper, contributed multiple saliva samples over the course of two months as the assay was refined. Several individuals muco-converted over this period of time despite no known exposure to COVID-19, no symptoms, and no evidence of disease by PCR and/or serology.20 where the polymeric sIgA binds to the pathogen and prevents it from binding to its cell surface targets until it is eliminated by clearance or degradation. If successful, immune exclusion prevents the pathogen from penetrating the epithelial defenses and establishing an infection, while still potentially triggering a ramp-up of the body’s immune defenses through their interaction with the adaptive immune system. Part of IgA’s function is to engage with a broad range of host cells. The FcaR receptor to the IgA Fc region is found on multiple cells including both key immune mediators like dendritic cells, and epithelial cells resulting in a broad range of effector functions involving both humoral and cellular adaptive responses. Once triggered, the adaptive humoral response produces both plasma cells and memory cells, and an active class-switching process.51 We need deeper understanding of the role played by mucosal IgA and its interplay with other elements of the immune system in order to better understand the pathogenesis and epidemiology of COVID-19.An important role played by sIgA is to continually scan the mucosal surface and prevent pathogen entry through “immune exclusion”23 as early as two days after symptom onset are consistent with the early-response role played by IgA, as well as with our anecdotal observations that individuals can muco-convert to positive salivary IgA contemporaneously with viral detection by PCR. A major challenge to successful containment of COVID-19 has been the relatively high false negativity in PCR testing as well as lack of timely results that have impeded contact tracing. If the early emergence of SARS-CoV-2 IgA in saliva is validated, salivary IgA may serve as a useful POC early screening tool, far more attractive to patients and clinicians than a nasopharyngeal swab, especially with children, with viral PCR serving as a confirmatory test.Reports that systemic IgA may be detectable earlier than IgG or IgM,52 We did not track individual IgA responses over time (that work is currently underway), but experience with a number of other respiratory pathogens suggests that mucosal IgA defenses can persist for extended periods of time.15While we do not know whether sIgA antibodies protect against SARS-CoV-2 exposure, extensive experience with other respiratory pathogens suggests that they should provide at least some protection. Our observation that mucosal IgA antibodies against SARS-CoV-2 persist for months is especially intriguing considering the recently reported data that systemic IgG antibody titers can decay within a month.25 Given the simplicity of saliva collection, salivary IgA could be a particularly attractive tool for community surveillance including identifying and tracking vulnerable populations at increased risk. Sullivan et al. are currently validating home collection methods, including for salivary IgA.53Apart from playing a special role in respiratory infections, salivary IgA also interacts with the adaptive immune system. Hence it is not surprising that, as with other respiratory infections, salivary IgA appears to correlate with systemic antibody levels against SARS-CoV-2.Moreover, if sIgA reduces the percentage of SARS-CoV-2 challenges that become full-blown COVID-19 infections, mucosal immunity could be part of the reason why there appears to be an unusually large number of asymptomatic patients who have tested positive for the virus. The virus could be present and neutralized by sIgA in mucosal tissue, and thus detectable by RT-PCR, but unable to enter epithelial cells in sufficient quantity to trigger the classic systemic immune response precipitated by a viral infection. And finally, if our data on muco-positivity rates are at least somewhat representative of the true community rate, and mucosal IgA is protective, we may be closer to herd immunity than currently believed.57 and several inhalation and intranasal vaccines for COVID-19 are under development. Especially intriguing is our anecdotal finding that exposure to sub-infective doses of SARS-CoV-2 could induce a mucosal immune response as measured by salivary IgA. A speculative interpretation is that the threshold of viral exposure required to induce a mucosal IgA response may be below that required to induce and establish infection or generate a measurable IgG response. Could we already have an effective and scalable live attenuated vaccine, with attenuation done by dose-reduction to a non-infective titer?Understanding whether mucosal IgA plays a protective role can help determine the trade-offs between topical and parenteral vaccines (or whether we might need both). Live attenuated flu vaccines have demonstrated encouraging activity,62 Selecting a vaccine that induces both mucosal and systemic immunity in a Phase 1 clinical study might increase the probability of success in Phase 3. Conversely, high levels of salivary IgA have been associated with a blunted response to immunization,54 and over-representation of subjects with elevated sIgA in a placebo arm may mask the beneficial effect of a vaccine. Thus, it may be prudent to analyze or even stratify subjects in a vaccine clinical study by salivary IgA levels.Even with parenteral vaccines, tracking salivary IgA may be useful for several reasons. Not all mucosal vaccines lead to systemic immunity, but many do.In summary, the preliminary data on salivary IgA serology that we have generated with the Brevitest BRAVO assay is consistent with the extensive published data on the critical role played by mucosal immunity in protecting against respiratory viruses. This makes a strong case for the clinical, research, and public health communities to better understand sIgA’s role in COVID-19 and to evaluate the possibility of its use in patient management, community monitoring, and vaccine development.The Brevitest platform consists of an Analyzer and a single-use test Cartridge. The Analyzer is a miniaturized fully automated robotic ELISA device that can conduct a standard ELISA using 40 microliters of sample in less than fifteen minutes. BRAVO is an enzyme-Linked Immunosorbent Assay (ELISA) capable of quantitative detection of IgA antibodies to SARS-CoV-2 in human saliva though the assay has only been validated for qualitative IgA detection. The analyzer controls and automates the ELISA process. The automated mechanism controls the movement of magnetic microspheres across several compartments within the cartridge. In each compartment a separate reaction takes place on the surface of these microspheres. The microspheres are oscillated in each compartment using proprietary mixing patterns. The Analyzer incorporates a scanner that measures the optical signal at the end of the reaction and sends the data to the cloud for analysis. There are a number of controls and safeguards built into the platform to ensure that the cartridge is inserted properly, the correct test is performed, the cartridge is not past its expiry date, that the results are valid, and that patient identifiable information is not at risk.The cartridges are loaded with about 40μl of patient’s diluted saliva , and inserted into the analyzer. The analyzer is a cloud connected robotic device which moves the magnetic microspheres within the cartridge through the different compartments to perform the complete ELISA reaction. The color change at the end of the process is read by the analyzer’s on-board optical sensors. BRAVO uses the following reagents. Capture antigen: 1:1 ratio of SARS-CoV-2 Nucleocapsid antigen and SARS-CoV-2 Spike S1 antigen (Receptor Binding Domain), both purchased from Genscript. Secondary antibody: Goat Anti-Human IgA conjugated to HRP . COVID-19 antibody (Absolute Antibody) as our internal positive control.Each BRAVO assay utilizes a single-use cartridge, which in addition to the sample lane, contains separate positive- and negative-control lanes to ensure consistency. The optical density in the control lanes serve as a quality check on the assay/cartridge and are also used to inform the sample reading. The BRAVO result is determined by an algorithm that incorporates the results from the sample lane as well as the negative and positive controls, thereby controlling for cartridge to cartridge variation as well as any minor differences in the ambient environment.The BRAVO assay was validated as described in the Results section, using true negative pre-COVID saliva samples (Lee Biosolutions) and tested for cross-reactivity using serum samples for anti-influenza B IgG, anti-respiratory syncytial virus IgG, antinuclear antibodies, IgG, rheumatoid factors (Biomex ZMBH), HIV, and Hemophilus influenza antibodies (Cantor Bioconnect).For clinical sample collection, we enrolled participants who had presented themselves with symptoms consistent with COVID-19 infection to the Next Level Urgent Care Clinic  and subsequently underwent a PCR test using nasopharyngeal sample collection. Information on relevant history was collected from the subjects including information on COVID-19-related symptoms and any additional testing (PCR or serology) they may have undergone. Subjects were also asked to rate the ease of saliva sample collection on a scale of 1 (easy) to 10 (difficult). The details of the PCR test used for each subject were obtained from the clinic’s electronic health records. The clinical protocol for sample and data collection and the informed consent document were approved by IntegReview, an independent Institutional Review Board.For saliva collection, subjects were provided with a kit containing a salivary swab, a plastic syringe  and a 1.5 ml capacity tube . The swabs were FDA listed Class 1 saliva collection devices, safe for use in adults or children 6 years or older, and are qualified for recovery of analytes, comparable to saliva samples collected from passive drool. All three components were supplied individually wrapped by the manufacturer, and assembled in a single bag by Brevitest. Each bag contained a syringe, a collection tube and a salivary swab, together with instructions for saliva collection.For our clinical validation, subjects self-collected saliva supervised by a trained provider. The individuals supervising the procedure completed a survey as to the length of time taken for sample collection . Individuals also responded rating the procedure on a scale of 1 (easiest) to 10 (most difficult).The collection procedure is briefly described here. Specimens were collected in accordance with correct medical practices, observing all standard safety procedures for both patient and collector. The subjects were briefly verbally instructed on the procedure and a glove put on their dominant hand. They then opened the bag containing the swab package, syringe and collection tube, removed the contents, and released the swab from its packaging. The swab was placed under the tongue and kept there for 2 minutes. Meanwhile, the subject identifier code label was put on the tube, the syringe removed from its package and the plunger removed from the barrel. After collection, the swab was placed in the barrel, followed by the plunger. The tip of the syringe was inserted into the collection tube and the plunger slowly pushed down to squeeze the collected saliva into the tube. The tube was then closed and the user confirmed that the volume of saliva collected (excluding the bubbles/froth on top) reached the first mark on the tube (which ensures 250 μl of sample). If the sample volume was inadequate, the procedure was repeated, with the second sample added to the tube to make up the required volume. The closed tube was placed in a biosafety pouch by the healthcare provider and put in a freezer or container with dry ice for storage below −20°C and later transport. Samples were kept frozen until actual testing, for a minimum of 2 hours at −20°C.The frozen samples in collection tubes were thawed and warmed up to room temperature by removal from the freezer 10 to 15 minutes prior to testing. 400 μl of PBS  was placed in a new separate 1.5 ml tube. 100 μl of the saliva sample was added to this tube, and mixed with the PBS by gently inverting the tube 3–5 times. Care was taken to take the saliva sample from the bottom of the sample tube, avoiding any remaining bubbles or froth at the top.An appropriate cartridge for the BRAVO assay was taken from the refrigerator, removed from its pouch and placed on a clean, flat, dry surface with the barcode on the cartridge facing up. Before running the assay, the sample was electronically connected to the cartridge, to enable automated reading of results after the test. To electronically link the sample identification barcode (on the collection tube) to the cartridge code (on the cartridge), any web-enabled device (e.g. tablet or smartphone) with a camera and web browser could be used. The test operator logged into the appropriate website on the device and sequentially scanned the code on the cartridge, followed by the subject identifier code on the tube. Finally the linking (if done satisfactorily) was saved and the cartridge was ready to be used. 40 μl of the diluted sample was pipetted into the sample well in the cartridge using a calibrated pipette. The rest of the sample was saved until the test run was complete, in case of failures or the need to re-test.The PPA and NPA were calculated using standard procedures as explained in the Results section. The confidence limits were calculated for the population proportion using the Wilson method with standard statistical software. Inputs were the sample size and number of positive results, the desired level of confidence in the estimate (95%) and the number of decimal places required in the answer. Outputs were the estimated proportion plus upper and lower limits of the specified confidence interval.


Cytokines are key mediators in the regulation of the normal immune response. They are ambivalent molecules which can be either beneficial to the treatment of diseases, but can also be harmful and participate in pathogenesis. Indeed, despite regulatory controls at multiple levels, abnormal immune responses involving cytokines can occur and cause various pathologies, including autoimmunity and inflammation-induced cancer. For these reasons, it is crucial to continue efforts focused on understanding the different modes of action of cytokines with an eye toward the design of new selective drugs that modulate cytokine activities that direct beneficial immune responses.Deregulation of cytokine expression has a complex role in disease pathogenesis and novel therapeutic agents that neutralize cytokines have been successfully translated into clinical practice. For instance, the use of monoclonal anti-TNF antibodies have greatly improved the health of patients suffering from diseases like inflammatory bowel diseases, rheumatoid arthritis, spondyloarthritis, or psoriasis. Furthermore, additional cytokine blockers such as anti-IL-6R antibodies, IL-12/IL-23 p40 inhibitors and IL-23 p19 blockers have been approved for various immune-mediated diseases. The use of effector cytokines  either alone or in combination with other therapeutic reagents, such as checkpoint inhibitors and emerging immunocytokines, is accelerating in cancer immunotherapy. While, IL-2 was approved by the Food and Drug Administration for the treatment of metastatic kidney cancer in 1992 and for metastatic melanoma in 1998, researchers are still working to improve IL-2 efficacy and reduce toxicity. Given the broad range of biological activities of cytokines, the side effects of biologic therapies need to be carefully assessed and warrant the development of new therapeutics with improved specificity of action. Thus, fundamental discoveries on structural features of cytokines in interaction with their different receptor chains could lead to the identification of cytokines with reduced toxicity and increased specificity.In this Research Topic issue entitled “Modulating Cytokines as Treatment of Autoimmune Diseases and Cancer”, we have compiled 6 original research articles, 1 hypothesis and theory and 6 reviews. This collection is divided into four sections. The first section presents recent knowledge for a better understanding of the mode of action and the structural features of the interaction between cytokines and their receptors. The second section describes new targets for the treatment of autoimmune diseases. The third is devoted to the use of cytokines to induce tolerance and the last section presents different combinations between cytokines and other therapeutic agents for the treatment of cancer.Markovic and Savvides focused on the structure and the mode of signaling assemblies of two closely related cytokines that share the IL-7Rα chain, IL-7 and TSLP. The review of Metcalfe et al. is devoted to the IL-6 family with a focus on IL-11. The authors of both reviews present structural overviews of the two cytokine systems and their involvements in pathological conditions. They also provide an overview of the broad array of potential therapeutic agents in autoimmune diseases to thwart overexpression of the targeted cytokines including monoclonal antibodies, chemical compounds, soluble receptors, and muteins. Along this line, Holgado et al. focused on an original strategy for inhibiting the action of cytokines by generating cytokine-traps. Their approach is based on the generation of molecules consisting of the fusion of receptors chains to form soluble heterodimers capable of capturing cytokines before interacting with their membrane-bound receptors. They are able to efficiently modulate either IL-33 alone by the IL-33-Trap or two cytokines simultaneously using the dual IL-14/13-Trap to inhibit experimental airway inflammation.Increasing knowledge of the structural interactions between a cytokine and its receptor chains is fundamental for generating original selective reagents targeting cytokine’s action. In their review, Barnes and Somerville provide a new vision of the action of cytokines and lead us to bear in mind that the production of both cytokines and extracellular vesicles play an important role in pathology. These properties could be translated into therapy by engineering extracellular vesicles to deliver immune modulators such as cytokines in pathological conditions.Cytokines are primarily described as soluble factors, but they can also be packaged within extracellular vesicles. In their review, Brune et al. focused on IRF5, a key transcriptional regulatory factor of type-I interferon. Hyperactivation of IRF5 has been identified as key factor in several autoimmune diseases, including systemic lupus erythematosus. In their review, Brune et al. provide an original perspective on the complex role of IRF5 and focus on T cell functions and polarization.The research community is always on the lookout for new targets for the design of novel therapeutics. Thus, it is crucial to deepen our knowledge of relevant targets involved in pathology. Schulz-Kuhnt et al. focused on recent knowledge of the functions of human ILCs and provide a comprehensive view of the major regulators, including cytokines, that selectively support the three ILC subpopulations. A better understanding of the regulation of human ILC functions should help researchers use ILCs and modulate their action under inflammatory conditions in the future.Innate lymphoid cells (ILC) are unique cell populations that play important roles in immune defense in response to chronic inflammatory and autoimmune diseases. Hasseli et al. draw attention to visfatin and other adipokines as potentially interesting targets in the search for RA therapeutics.Adipokines are cytokines produced by adipocytes. Among them, visfatin appears to play an important role in the pathogenesis of rheumatoid arthritis (RA) by increasing the adhesion of RA synovial fibroblasts to endothelial cells. Lio et al. analyzed the literature that used dry eye disease models to find cells and cytokines that could be targeted in this pathology. They show the involvement of Th1 cells as well as IL-1β and TNFα proinflammatory cytokines. This meta-analysis also prompts precaution when using animal models that do not fully recapitulate human pathology.In generating new reagents, investigators need to assess their efficacy in relevant animal models. In their study, Cauwels and Tavernier proposed an original strategy to expand endogenous Tol-DC in vivo by the administration of AcTakine molecules. The latter consists of a targeting module (VHH is more commonly used) fused to a mutated cytokine with reduced affinity to its cognate receptor. They engineered a Tol-DC AcTaferon with IFN-I to induce tolerance in autoimmune diseases.Autoimmune diseases are characterized by the disruption of tolerance to self antigens. Different approaches have been designed by researchers to restore tolerance, including cell therapy by injecting tolerogenic dendritic cells (Tol-DC) or regulatory T (T-reg) cells. Another approach is to target tolerogenic cells directly in vivo. Ghelani et al. attempt to find the threshold required for IL-2 to selectively expand T-reg cells into effector cells. To this end, they generated a series of IL-2 muteins and found that minimal IL-2 receptor signaling is required to fully expand regulatory T cells and support their immunosuppressive functions.IL-2 is an important cytokine for the development of T-reg cells, which constitutively express IL-2Rα. The latter forms a trimeric receptor with IL-2Rβ and the common gamma chain and binds IL-2 with a high affinity allowing T-reg cells to respond to low dose of IL-2. In their study, Bézie et al. show that CD4 and CD8 FoxP3 regulatory T cells increase significantly when cultured in the presence of monocytes differentiated by IL-34. In addition, human CD8 regulatory T cells grown under these conditions suppress the immune response in a humanized model of acute GVHD by effectively increasing the survival of the graft after organ transplantation by acting on T-regs cells and monocytes. These results demonstrate that IL-34 should also be considered for therapy with regard to its property for promoting the development of regulatory T cells.IL-34 is another cytokine with tolerogenic properties. In their study, Waldmann et al. present assays that use IL-15 in combination with therapeutic monoclonal antibodies such as anti-CD40, anti-CD20, anti-CD52, anti-EGFR, anti-CTLA-4, anti-PD-1 and anti-PDL-1. Preliminary combination studies show better efficacy than individual agents alone and hold promise for the treatment of patients with metastatic malignancy.IL-15 is a cytokine that shares with IL-2 an important role in supporting the development and functions of effector cells, such as NK and CD8 T cells. Unlike IL-2, IL-15 does not support regulatory T cells. For these reasons, IL-15 was identified early as a potential candidate for use in cancer immunotherapy. However, IL-15 administrated as monotherapy did not show therapeutic efficacy despite a dramatic expansion of NK and CD8 T cells. In their review, Shen et al. fused anti-PD-1 antibody to IL-21 muteins. The advantages of such molecules are numerous. They improve both the half-life and bioavailability of cytokines. Targeting cytokine therapies to specific cells may better replicate the paracrine activities of physiologically delivered cytokines. This approach can thus enhance efficacy and limit off-target effects. To restrict IL-21 activity on targeted cells and slow down the clearance of the ICK, the investigators mutated the cytokine to decrease its affinity for its cognate receptor. Preliminary preclinical data reveal that the anti-PD1-attenuated-IL-21 immunocytokine shows promise for anti-cancer indications.Another way to combine cytokines and therapeutic monoclonal antibodies is to fuse them to generate immunocytokines (ICK). In their study, Collectively, the above studies highlight the potential of recombinant cytokines and their inhibitors to more effectively treat autoimmunity and cancer. These results open new avenues for research and may lead to improved therapeutic options in clinical therapy by precision editing of cytokine responses.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, none of the test exercises activated hamstring muscles more than an average of 60% of the maximal activation during top-speed sprinting. In conclusion, training programs aiming to be sprint-specific in terms of horizontal force production could include exercises such as the Upright-hip-extension and the Nordic hamstring exercise, in addition to maximal sprinting activity, which is the only exercise leading to high levels of hamstring muscle activity.To train hamstring muscle specifically to sprint, strengthening programs should target exercises associated with horizontal force production and high levels of hamstring activity. Therefore, the objectives of this study were to analyze the correlation between force production capacities during sprinting and hamstring strengthening exercises, and to compare hamstring muscle activity during sprinting and these exercises. Fourteen track and field regional level athletes performed two maximal 50-m sprints and six strengthening exercises: Nordic hamstring exercises without and with hip flexion, Upright-hip-extension in isometric and concentric modalities, Standing kick, and Slide-leg-bridge. The sprinting horizontal force production capacity at low (F0) and high (V0) speeds was computed from running velocity data. Hamstring muscle performances were assessed directly or indirectly during isolated exercises. Hamstring muscle electromyographic activity was recorded during all tasks. Our results demonstrate substantially large to very large correlations between V0 and performances in the Upright-hip-extension in isometric (r Sprinting acceleration is a key component in numerous sports aiming to cover a given distance in the shortest time  had their hamstring muscle peak activity at a similar hip and knee angle, none of the isolated exercises  that were tested induced hamstring electromyographic (EMG) activity >75% for the semimembranosus, 65% for the semitendinosus, and 40% for the biceps femoris of that measured during top-speed sprinting  to analyze the correlations between horizontal force production capacities during sprinting and the force production capacity during hamstring muscle-strengthening exercises, and (2) to compare hamstring muscle activity during sprinting and hamstring muscle-strengthening exercises. We first hypothesized that performance achieved during hamstring-dominant exercises executed in similar conditions to hamstring force production during sprinting would be correlated with the horizontal force production capacity during sprinting. Therefore, it could be expected that performance during exercises executed in full leg extension and high velocity would be correlated with horizontal force production at high running velocities (V0) and not at low running velocities (F0). On the opposite, performance in exercises executed at a low or null velocity, and without full leg extension, would be correlated with horizontal force production capacity at low running velocities (F0) and not at high running velocities (V0). Based on the results of van den Tillaar et al.  during tWe conducted an experimental cross-sectional study with adult male and female sprinters, who performed, during one session, two maximal sprints and six exercises selected for their ability to target hamstring muscles. Hamstring force production and muscle activity were assessed during the maximal sprints and the exercises. The study protocol was reviewed and approved by the Saint-Etienne University Hospital Ethics Committee .Participants were recruited from French athletics clubs and were included if they practiced sprinting, trained at least three times per week, were engaged in strength training on a weekly basis, and accepted to participate in this study. Participants with any recent history (<6 months) of HMI were excluded from the study, as were participants reporting pain or discomfort great enough to influence their abilities to sprint or to perform one of the exercises.*Power software, version 3.1.9.2, Germany) were determined to allow us (1) to analyze the correlations between horizontal force production capacities during sprinting and the force production capacity during hamstring muscle-strengthening exercises (to detect correlation of 0.7 between variables), and (2) to compare hamstring muscle activity during sprinting and hamstring muscle-strengthening exercises  and between V0 and the break-angle during both NHE0  and NHE90  (s = 0.60 (95% CI 0.494–0.705); p = 0.028] and between F0 and the break-point angle during the NHE0   between EMG activity during all exercises and the maximal EMG activity during the sprint (100%)  .Nordic-hamstring and the Upright-Hip-Extension in isometric (UHE-I) exercises were largely to very largely correlated with the force production during sprinting at both low (F0) and high (V0) velocity, and that (2) none of the exercises tested  induced hamstring EMG activity >60%  of the maximal hamstring EMG activity measured during maximal sprinting acceleration. This study is the first, to the authors' knowledge, to investigate the specificity of strengthening exercises in relation to determinants of over-ground sprinting mechanics in terms of force production functional capacity and surface EMG activity.The main findings of this study were that (1) performance during the Our results show that the ability to reach a greater break-point angle (indirectly estimating eccentric force production capacity) during both NHE0 and NHE90 exercises was correlated with greater levels of force production at low and high speed (F0 and V0). These results are partially in agreement with our hypothesis, since we expected low-speed exercises (NHE) to be correlated with force production at low velocity, but not with high velocity. This could be interpreted as the fact that the functional capacity of the hamstring muscles in the eccentric modality may be related to the ability to produce backward-oriented force during the entire sprint acceleration. This highlights a possible relationship between sprint performance and the eccentric contraction modality of the hamstring muscles, and offers indirect support to Chapman and Caldwell's  was strongly correlated with sprinting force production capacity at high (V0) and low velocity (F0). This suggests that hamstring force production at null velocity but maximal force is linked to horizontal force production capacities at both low- and high-speed running. As the UHE-I mimics the early stance phase, these results could support the findings of Clark and Weyand  of what it maximally sustained during a sprint. These results were similar to those reported by van den Tillaar et al.  from theUpright-Hip-Extension in isometric (UHE-I)] and stance [Upright-Hip-Extension in concentric (UHE-C)] phases of the sprint strikes. Similarly, Yu et al.  allowed for greater hamstring muscle activity, which was also reported by previous studies  did not facilitate greater hamstring muscle activity than the NHE0. To be more specific to sprint, Guex and Millet (Standing-Kick or Nordic-hamstring exercises) allowed for less muscle activity.Similarly to previous studies  to explain sprint specificity could be a reductionist approach. In practice, our results highlight the fact that sprinting targets hamstring muscle in an irreplicable manner and should be part of any hamstring conditioning or injury prevention program.Nordic-hamstring exercise, Sconce et al.  compared to sprinting is of great interest from a performance, primary prevention, and return-to-sport standpoint. The sprint-specificity of exercises assessed through force production and muscular activity revealed that force production ability during certain exercises such as the Nordic-hamstring or the Upright-Hip-Extension exercise were related to horizontal force production during sprinting. However, with none of these exercises was it possible to reach similar levels of EMG activity as the ones induced by sprinting activities. Maximum sprinting activities appears to be the only way to achieve high muscle activity. However, more investigation is needed to determine which exercise or combination of exercises could reduce hamstring injuries or increase sprint performance.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Saint-Etienne University Hospital Ethics Committee, Institutional Review Board: IORG0007394; IRBN322016/CHUSTE. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.CP collected the data and wrote manuscript. CP, PE, and PS designed the study and analyzed the data. CP, PE, PS, J-BM, JL, JM, and KG revised manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The leadless cardiac pacemaker was selected to provide empiric pacing support in this patient with a manifest mid-septal accessory pathway who had undergone a previous ablation resulting in injury to the compact atrioventricular node. Although this patient’s accessory pathway currently demonstrates stable antegrade conduction properties, diminished and complete resolution of manifest pre-excitation has been well described in patients with Wolff–Parkinson–White syndrome. Because of the patient’s young age, an increased risk is present for long-term complications inherent with traditional transvenous pacing. The Nanostim leadless pacemaker  was implanted into the right ventricular myocardium without complication. Pacing performance has remained stable, and the patient has been free of device-related adverse events at 19 months after implant. The patient reported that episodes of abrupt-onset tachycardia recurred 6 months after the initial procedure. More recently, the symptoms had increased in frequency and duration. She strongly preferred to avoid long-term pharmacologic therapy, and sought definitive ablation therapy.A healthy 46-year-old female with Wolff-Parkinson-White (WPW) syndrome was referred for catheter ablation to the Cleveland Clinic. She had undergone a reportedly successful accessory pathway (AP) ablation procedure at the age of 26 years at another institution, and has re-established the need for cardiovascular care only recently, when a health insurance examination documented ventricular pre-excitation , with an effective refractory period (ERP) of S1 600 ms, and S2 310 ms . An antegrade His-bundle potential was not identified during incremental pacing, or during programmed stimulation with single extrastimulus testing from the proximal coronary sinus or high right atrium. Programmed electrical stimulation from the right ventricular (RV) apex showed retrograde conduction via two distinct midline accessory pathways, but no evidence of ventriculoatrial activation via the His-Purkinje system and atrioventricular (AV) node  and 3B. Tachycardia was not inducible at baseline or with isoproterenol.A comprehensive electrophysiology study was performed, which verified the presence of a mid-septal AP, with the earliest ventricular activation observed at the His-bundle region. 1:1 antegrade conduction via the AP was maintained at a drive stimulus train of 290 ms  and 4B. Simultaneous loss of AV conduction was noted with block of the accessory pathway within 3.5 s from onset of radiofrequency ablation (RFA), with a maximum of 15 watts of delivered energy . With termination of RF energy, accessory pathway conduction recovered immediately and remained durable thereafter.Because of reported tachycardia and observed robust antegrade conduction properties, the AP was targeted for ablation. The earliest ventricular activation was mapped to the mid-septum, and preceded the onset of the delta wave by 25 ms Figure 6 should have alerted us to the possibility of severe intrinsic AV node dysfunction. Whether explained as an AV node echo beat  or a bundle branch reentry beat , we speculate severely impaired baseline antegrade AV node function to be present.In the present case, complete heart block concurrent with block in the septal pathway during RFA occurred without preceding accelerated junctional rhythm; the latter is typically seen when heating the AV junction. We initially surmised that AV conduction was intact based on reproducibly observing a visible His-bundle electrogram with programmed stimulation when double extrastimuli were delivered from the RV apex, which was inferred to be inscribed in the antegrade direction. However, in retrospect, we conclude that the sequence shown in The mid-septal AP was left to remain intact, and its ante-grade conduction properties persisted unaltered. Yet, knowing the unreliable state of this patient’s AV node, the benefit of empirical pacing support was entertained.4 Given this, empiric pacing support was contemplated, and a single chamber ventricular pacing system was ultimately felt to be sufficient.It has been well described that antegrade conduction via an AP diminishes over time and in some cases disappears. The rate of spontaneous resolution of ventricular pre-excitation has been reported to be between 10% and 79% in cohort studies, with the mean longitudinal follow-up periods of 3–40 years.8 A self-contained leadless cardiac pacemaker implanted directly within the right ventricle was felt to be ideal, as it avoids repetitive lead stress and the potential of lead malfunction.Of note, this patient’s younger age increases the risk for the development of long-term complications, such as venous thrombosis, lead malfunction, and device infection inherent with the use of a traditional transvenous pacemaker system. In such an event, device-assisted lead extraction may be necessary. Fatal cardiovascular injury is the most serious complication, and risk of such increases with the age and type of lead, lead-related calcification, and female gender.In addition, eliminating both the device pocket and the transvenous lead prevents the risk of long-term complications like surgical-related infection, hematoma formation, vascular occlusion, or tricuspid regurgitation. and 7D using previously reported techniques.9 Briefly, a delivery catheter pre-loaded with the device was gently deflected and advanced into the right ventricle within a protective sleeve that served to guard the helix while allowing for small contrast injections (approximately 1 cc to 2 cc each) to judge tip proximity to the RV endocardium. Once located approximately 1 cm from the endocardial surface, the device was then extended beyond the simultaneously withdrawn protective sleeve, and the helix fixated into the preferred implant site, which involved the apical RV septum by application of clockwise rotation movement . The device was then released from the catheter while remaining fixed to the docking button by two small cables (tethered mode) for electrical testing. With normal function confirmed, the device was then released completely by bringing the anchoring cables slightly out of alignment to trigger a full release from the docking button. Appropriate fixation was then confirmed by evaluating device motion with the cardiac silhouette in right and left anterior oblique views by fluoroscopy. At this time, if necessary, the device can be percutaneously retrieved by ensnaring the docking button and re-engaging the catheter to the device, using a specially designed retrieval catheter to disengage the helix from the tissue through counter-clockwise rotation.In this case, the Nanostim leadless cardiac pacemaker  was implanted into the RV myocardium without complication With the use of this device, pacing performance has remained stable in the current case, and the patient has been free of device-related adverse events to date at 19 months’ post-implantation.


In the recent SARS-CoV-2 pandemic, public health experts have emphasized testing, tracking infected people, and tracing their contacts as an effective strategy to reduce the spread of the virus. Several diagnostic methods are reported for detecting the coronavirus in clinical, research, and public health laboratories. Some tests detect the infection directly by detecting the viral RNA and other tests detect the infection indirectly by detecting the host antibodies. A diagnostic test during the pandemic should help make an appropriate clinical decision in a short period of time. Recently reported diagnostic methods for SARS-CoV-2 have varying throughput, batching capacity, requirement of infrastructure setting, analytical performance, and turnaround times ranging from a few minutes to several hours. These factors should be considered while selecting a reliable and rapid diagnostic method to help make an appropriate decision and prompt public health interventions. This paper reviews recent SARS-CoV-2 diagnostic methods published in journals and reports released by regulatory agencies. We compared the analytical efficiency including limit of detection, sensitivity, specificity, and throughput. In addition, we also looked into ease of use, affordability, and availability of accessories. Finally, we discuss the limitations of the methods and provide our perspectives on priorities for future test development. The coronavirus disease 2019 (COVID-19) follows multiple past epidemics caused by highly transmissible respiratory viral infections. COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that was first reported in Wuhan, China [The SARS-CoV-2 is a positive-sense single-stranded RNA β family coronavirus that is genetically similar to SARS coronavirus and bat SARS-like coronaviruses . Recent Several diagnostic methods have been used to detect the coronavirus in clinical, research, and public health laboratories. Direct tests detect the infection directly by detecting the viral RNA, while indirect tests measure antibodies against the virus in a host that has been exposed. A diagnostic test method should have sufficient sensitivity and accuracy to make appropriate clinical decisions rapidly during a pandemic .Nucleic acid amplification using the reverse transcription polymerase chain reaction (RT-PCR) is the most widely used method for direct SARS-CoV-2 diagnosis . ImmunoaIn this paper, we review recently published SARS-CoV-2 diagnosis methods and compare their analytical efficiency in terms of limit of detection (LOD), sensitivity, specificity, and throughput. In addition, we also evaluated ease of use, affordability, and availability of accessories. Note that other tests from a wide variety of companies are being used for COVID-19 diagnostics, with additional products entering the market each week; however, in the rush to get the tests operational, the information on these critical parameters and the company-unique information are often not yet published. Finally, we discuss the limitations of the methods and provide our perspective on priorities for future test development. The intended audience for this paper are students and researchers working on method development and clinical testing of viruses including SARS-CoV-2. People who make decision on which test method should be recommended to a clinical laboratory for the diagnosis of COVID-19 will also benefit from this review paper.Two types of sample specimens are being primarily used for the diagnosis of COVID-19. Respiratory specimens are used for direct detection of virus and serum samples are used for identification of antiviral antibodies . Direct The virus samples should be processed and tested as soon as possible. If immediate testing is not possible, the sample can be stored up to 72 h at 2–8 °C. However, for more than 72 h storage, the specimens should be frozen at -70 °C as soon as possible after collection . It is rThe right type of sample, appropriate collection procedure, and reliable transportation must be in place to minimize the risk of inaccurate results. Based on the stage of infection on a person and the purpose of test, an appropriate sample should be collected. Higher viral loads of SARS-CoV-2 have been reported from specimens collected from nose than from throat . SimilarThe molecular detection methods involve the analysis of nucleic acids present in the sample to identify the virus. The most commonly used laboratory detection method for the clinical diagnosis of COVID-19 is real-time reverse transcriptase polymerase chain reaction (RT-PCR). The same technique has been used in the diagnosis and surveillance of various other viral diseases including SARS-CoV and MERS-CoV , 37, 38.The RT-PCR tests take less than an hour to a couple of days to give results, depending on the version of the PCR. The RT-PCR assay can be carried out in one- or two-step approaches. One-step approach is faster in which both RT and DNA polymerase are combined together to carry out their respective reaction in the same reaction tube and is a preferred approach for the detection of SARS-CoV-2 . The twoThe RT-PCR test result relies on sample collection, primers and probes used, analysis of fluorescence curves, use of suitable controls, and reliability of the temperature control. A negative control is used to check sample cross-contamination, and the positive control is used to assess the chemical integrity of the reagents, primers, and probes. In addition to these controls, the US CDC recommends the use of a human specimen control (HSC)  to ensurRespiratory specimens may contain different genera of coronaviruses along with other major viral pathogens. In the last six decades, before SARS-CoV-2, the human population was already infected with six other members  of the CoV family . False pThe RT-PCR assays in centralized laboratories are generally performed in 96-well plates for signal reading in batches. The high-throughput 384-well assay system using lower volume was reported recently with detection sensitivity down to 5 copies of viral genome per microliter . The higThe US CDC real-time RT-PCR diagnostic panel under EUA targets two different loci of the N gene . The FDA−4 to 2 × 103 TCID50/reaction) was obtained using RNA extracted from cells infected by SARS coronavirus as a positive control using one-step RT-PCR assays to detect two different regions (ORF1b and N) of the viral genome [Primer sets (IP2 and IP4) designed by Pasteur Institute, when used individually in an assay, can detect about 100 copies of RNA genome equivalent per reaction at 95% detection probability. A lower LOD of 10 copies was obtained with a multiplex assay using these primer sets . Chu et l genome .Further improvement in the sensitivity was found in the Charité protocol . They coThe PCR assays developed by Pasteur Institute have been claimed to not cross-react with respiratory viruses like influenza A H1N1, H3N2), enterovirus, adenovirus, human coronaviruses , and MERS-CoV, indicating 100% specificity. Assays from HKU are specific to only subgenus Sarbecoviruses . This as, enterovMultiple studies have shown that the clinical sensitivity of a RT-PCR assay is under the influence of specimen type, amount of virus in a swab and the specimen collection time in relation to the onset of symptoms. In one study with 205 patients, RT-PCR sensitivity was 93% for BLF, 72% for sputum, 63% for nasal swabs, and only 32% for throat swabs . The preThe point-of-care (POC) methods are less complex to perform, give results within several minutes, and can be performed on site. They may provide alternative diagnostic methods more suitable for widespread population testing.A preliminary study reported a POC system capable of detecting genes coding for the N protein of SARS-CoV-2 in respiratory samples . MoleculIn an effort to enhance the sensitivity of the LAMP method, a two-stage isothermal amplification (COVID-19 Penn-RAMP) method has been described, producing 10 to 100 fold better sensitivity than conventional LAMP and RT-PCR tests. This test is carried out in a closed tube and employs fluorescence or colorimetric detection. This POC method has been proposed as a rapid, highly sensitive molecular test amenable for use at home, in the clinic and at point of entry by minimally trained individuals and with minimal instrumentation. This method as reported, however, was not yet tested in patient samples .Two of the first POC platforms receiving EUA from FDA are produced by Abbott and Cepheid. The Abbott ID NOW COVID-19 system gives positive results in 5 min and negative results in 13 min after sample preparation. The Xpert® Xpress SARS-CoV-2 system can give results in ~ 30 min with less than a minute sample preparation. It is interesting to note that the Abbott technology missed one-third of samples detected positive by Cepheid Xpert Xpress .The ePlex® SARS-CoV-2 is a qualitative automated nucleic acid multiplex platform and sample-to-answer system capable of automating extraction, amplification, and detection in a single-use cartridge. A simple visual readout similar to a pregnancy test has been described. This POC system is based on SHERLOCK technology and has been called STOPCovid (SHERLOCK Testing in One Pot) . It usesWhile having the advantage of accommodating large batches of samples, the conventional RT-PCR test and the facility it requires, including the PCR machines, are too expensive. When transport to a competent laboratory is included, the turnaround time is measured in days. Additionally, sample preparation and assay procedures require well-trained manpower. These shortcomings limit the wider use of the technology during viral pandemics. In addition, high demand during the pandemic creates a shortage of swabs, personal protective equipment, PCR reagents, and equipment such as thermocyclers and biosafety level-2 cabinets, further delaying the diagnosis. Since RT-PCR assays amplify specific target loci, the assays will report a negative result if the particular target locus is not present in the sample. Collective genetic data indicate changes in the viral genome due to insertion or deletion, recombination, and interchange are frequent among CoVs , 61. If Apart from conventional RT-PCR, additional molecular diagnostic tools are emerging for SARS-CoV-2. Whole-genome sequencing of SARS-CoV-2 has the potential to overcome the limitations of RT-PCR. Genomic sequencing can detect fragments even when a complete genome is not present in the sample. Notably, the SARS-CoV-2 genome is free of repeats, making it susceptible to complete characterization using short sequence reads. The genome sequencing involves the construction of RNA library, next-generation sequencing (NGS) of the RNA construct, de novo assembly of the quality-trimmed reads to generate a contig map, and phylogenetic analysis . The genAnother emerging method is CRISPR-based technologies. A rapid (< 40 min), easy-to-implement, and accurate CRISPR–Cas12-based lateral flow assay for the detection of SARS-CoV-2 from respiratory swab RNA extracts has been recently reported. The CRISPR-based DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) has a visual read for results. This assay performed simultaneous RT and LAMP steps, followed by Cas12 detection of predefined coronavirus sequences, after which cleavage of a reporter molecule confirms detection of the virus. The LOD of this method was 10 copies per microliter reaction. Clinical sensitivity and specificity were 95% and 100%, respectively, for the detection of the coronavirus in 83 total respiratory swab samples . A new pDroplet-based digital PCR (dPCR) methods have also been sought as a more sensitive method to test for the RNA of SARS-CoV-2. This method, when tested in 77 patient samples, showed an improved sensitivity from 44 to 94% and the same specificity when compared with the RT-PCR method . The LODA dual-functional plasmonic biosensor that combined the plasmonic photothermal effect and localized surface plasmon resonance sensing transduction was reported as an alternative for the clinical diagnosis of COVID-19 . Two-dimThough relatively slow and expensive, computed tomography (CT) of the chest has also been explored in the diagnosis of COVID-19 as a complementary tool to molecular techniques when combined with medical history and clinical observations . In a reImmunoassays are one of the widely used bioanalytical methods to detect and/or quantify an analyte using an antigen-antibody interaction. While there are a few reports using antibodies in a biosensor to detect virus directly , the affEnzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs) are the most widely practiced techniques to detect the antibodies against SARS-CoV-2 . A two-sRecently, a peptide-based magnetic chemiluminescence enzyme immunoassay was developed for the diagnosis of COVID-19 . The tesThe sensitivity of the immunoassays for the presence of antibodies in human samples may depend on the viral titer and time of sample collected after viral infection; both factors impact circulating antibody concentration . ELISA aThe immunoassay-based rapid diagnostic test (RDT) kits, depending on the vendor type, are reported to have sensitivity in the range of 60–80% and selectivity of 85–100% at a confidence interval of 95% . The RDTThe Single Molecule Array (SIMOA) multiplexed method developed by Quanterix targets three immunoglobulin responses  to four viral proteins  in a single sample, thereby enabling the quantification of 12 antibody isotype-viral protein interactions. It gives a high-resolution profile of immune response of SARS-CoV-19 when compared with a traditional ELISA where only a single interaction can be interrogated. The SIMOA method demonstrated a sensitivity of 86% and a specificity of 100% during the first week of infection, and 100% sensitivity and specificity thereafter .RDTs employing serological immunoassays are less complex, cost less than the molecular tests, and can give results in a short time period. Immunoassays are good tools to track and study past infections, especially in asymptomatic cases. Serological assays can be used to determine the infection rate and to estimate the population extent and prevalence of infection. Results from a serological survey can also be used to project mortality rates in a community. Furthermore, they are useful to characterize the immune response to the virus. A serological assay is critical for identifying potential plasma donors and for developing vaccines. These tests are relatively quick, are easy to use, and require no highly trained personnel and sophisticated equipment. By early August 2020, a quick scan of the internet showed over 20 rapid tests under commercialization with the European CE Mark approval.Immunoassays are not as specific as the tests recognizing RNA sequences in the virus. In the past, molecules like interferon, rheumatoid factor, and non-specific IgM have been shown to cause problems in immunoassays , and levImmunoassays to detect the antibody response to virus may produce false negative results during the early stage of the infection. The sensitivity may be low when tested in local populations with asymptomatic or mildly symptomatic individuals who may generate only low-titer antibodies. Sensitivity may be even lower if there are many such cases. Results may also be biased due to prior COVID-like illnesses that confound the specificity of the antibody response. One of the difficulties in validating an assay for antiviral antibodies is the availability of appropriate negative and positive controls. Negative controls are easier to come by and can be serum pools taken before 2019. Positive controls can be convalescent samples from COVID-19 patients or monoclonal antibodies like CR3022 . DespiteTo add to the complexity of interpreting results from diagnostics for antiviral antibodies, the antibody response in a patient depends on age, nutritional status, and existing medical conditions and medications , 87. TheIt has been suggested to detect multiple antibodies to avoid false results. Antisera raised against N proteins lack specificity and need to be used in combination with antibodies to other proteins; e.g., antisera against N proteins and S proteins might be used to develop a more reliable serum-based quick diagnostic test kits , 92, 93.Among various methods available for the detection of COVID-19, the RT-PCR is the most reliable and widely used method. During pandemic emergencies, shortage of resources such as PCR kits is common. It is therefore important to have multiple options for the diagnostic methods. Alternative testing platforms and accessories that could be locally manufactured, even in a small scale, are equally important. Such platforms would be appropriate in resource-limited settings as well. The currently practiced RT-PCR methods are costly, and therefore many countries, especially the low-income ones, cannot afford enough number of COVID-19 tests to screen larger population. Important gaps remain in screening asymptomatic persons in the incubation phase. Accurate determination of live viral shedding among patients in the convalescence phase to inform de-isolation decisions is also challenging.Quality assurance and regulatory frameworks surrounding testing remain a challenge. There are several instances of COVID-19 test kits being recalled in several countries due to suspicious quality. The lack of an established reference standard, the use of differing sample collection and preparation methods, and an incomplete understanding of viral dynamics across the time course of infection hamper the rigorous assessment of the diagnostic accuracy of the many newly introduced SARS-CoV-2 assays .New technologies such as CRISPR-based diagnostics and implementation of practical approaches for POC applications such as RT-LAMP will provide rapid, simple, low-cost, portable, temperature-stable assay systems that are appropriate resource-limited settings, not only for doctor’s offices but also for airports, border crossings, and remote locations. One of the research areas to be explored for diagnosis of COVID-19 is to perform the serological assays in microfluidic POC systems. Microfluidic systems offer advantages of small sample volume, miniaturization, portability, multiplexed analyses, and rapid detection and can increase the sensitivity of analyte detection using signal amplification strategies . The app


We investigated the impact of regionally imposed social and healthcare restrictions due to coronavirus disease 2019 (COVID-19) to the time metrics in the management of acute ischemic stroke patients admitted at the regional stroke referral site for Central South Ontario, Canada.We compared relevant time metrics between patients with acute ischemic stroke receiving intravenous tissue plasminogen activator (tPA) and/or endovascular thrombectomy (EVT) before and after the declared restrictions and state of emergency imposed in our region .p = 0.008) and/or EVT  after the start of social and healthcare restrictions in our region compared to the previous 12 months. For patients receiving intravenous tPA treatment, we also found a significant increase (p = 0.005) in the median door-to-needle time . No delays in the time from symptom onset to hospital presentation were uncovered for patients receiving tPA and/or endovascular reperfusion treatments in the first 1.5 months after the establishment of regional and institutional restrictions due to the COVID-19 pandemic.We identified a significant increase in the median door-to-CT times for patients receiving intravenous tPA : 14–27 min vs. 13 min, IQR: 9–17 min, We detected an increase in our institutional time to treatment metrics for acute ischemic stroke patients receiving tPA and/or endovascular reperfusion therapies, related to delays from hospital presentation to the acquisition of cranial CT imaging for both tPA- and EVT-treated patients, and an added delay to treatment with tPA. Stroke experts and international organizations have highlighted the need to preserve the best standards and comprehensiveness of care at all stages during the COVID-19 pandemic outbreak.3 Reports of declining stroke admission volumes and delays in hospital presentation, which have resulted in decreased systematic and endovascular reperfusion treatments, due to presumed patient fears, as well as the social and logistical barriers imposed by community and healthcare preventive measures, are accumulating.7The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), originally emerging in Wuhan, has quickly spread worldwide and coronavirus disease 2019 (COVID-19) was declared as a pandemic outbreak on March 11, 2020.In the present report, we investigate the impact of regionally imposed social and healthcare restrictions to the quality of care time metrics in the hyperacute management of patients presenting with acute ischemic strokes to a large comprehensive stroke center in Ontario, Canada.8We performed a retrospective review on the time metrics of consecutive stroke patients admitted or transferred for acute stroke treatment to the Hamilton General Hospital, Hamilton Health Sciences between March 1, 2019 and April 30, 2020. The Hamilton General Hospital is the regional referral comprehensive stroke center for a population of 2.2 million people in Central South Ontario, Canada and cares for over 1500 patients with stroke and threatened stroke annually.For the aforementioned time period, we retrospectively searched hospital databases and records to obtain the total numbers of acute ischemic stroke patients receiving systemic and/or endovascular reperfusion therapies with the relevant time metrics. Additional informations for patients receiving endovascular thrombectomy (EVT) were obtained from an established national EVT registry (OPTIMISE) that focuses on improving the quality of management of patients receiving EVT for acute ischemic stroke. The primary outcome of interest was the time from hospital admission to treatment initiation, using the door-to-needle and door-to-groin puncture times for patients receiving treatment with intravenous tissue plasminogen activator (tPA) and/ or EVT, respectively.For acute ischemic stroke patients receiving intravenous tPA treatment, we extracted additional data on the time from symptom onset to hospital presentation (onset-to-door time), time spent in triage at the emergency room (ER triage), time from the presentation at our institution to the first neuroimaging acquisition (door-to-CT time), time from first neuroimaging acquisition to the start of the tPA bolus (CT-to-needle time), and the total time from symptom onset to tPA bolus administration (onset-to-treatment time). For patients receiving EVT, we extracted data on the time from arrival at our institution to the initiation of EVT (door-to-groin puncture), the time from angiography suite arrival to groin puncture time, and the time from stroke symptoms onset to groin puncture (onset-to-groin puncture time). Finally, we also reported the time from hospital arrival to the last angiographic run establishing any degree of reperfusion . For patients transferred for EVT to our center from a primary stroke care center, we additionally obtained data on the time that the patient stayed in the place of the first arrival until the initiation of transfer to our institution (door-in-to-door-out time).For the primary analyzes, we compared all relevant time metrics between patients with acute ischemic stroke receiving intravenous tPA and/or EVT prior to the declared lockdown restrictions and state of emergency imposed in Ontario  and acute ischemic stroke patients receiving intravenous tPA and/or EVT between March 1, 2019 and March 16, 2020. Following the declaration of government-imposed social distancing on March 17, 2020, our institution instantly activated staff redeployment, physical distancing, screening at all hospital entrances, and the default use of personal protective equipment for all new admissions to the emergency department.As sensitivity analyzes, we compared the aforementioned time metrics between acute ischemic stroke patients presenting between March 17, 2020 and April 30, 2020 and ischemic stroke patients treated during the respective time period the previous year . Additionally, using box plots, we present a biweekly temporal overview of door-to-CT time, which is a common metric for both tPA- and EVT-treated patients, from December, 2019 to April, 2020. Dichotomous variables were presented as percentages, while continuous variables were summarized using median values and corresponding interquartile ranges (IQRs). Dichotomous variables were analyzed using Pearson’s chi-square test and continuous variables with the Mann–Whitney U test. Analyzes were performed with the Stata Statistical Software Release 13 for Windows .p = 0.005) in the median door-to-needle time  compared to patients receiving tPA treatment the previous 12 months . No significant differences in onset-to-door (p = 0.480), ER triage (p = 0.888), and onset-to-treatment times (p = 0.394) were found between patients receiving tPA treatment before and after the start of social distancing measures  following the official implementation of social and healthcare restrictions in Ontario. Interestingly, the median time from symptom onset to admission in our institution (onset-to-door time) was shorter for patients presenting after March 17, 2020 . Although the median door-to-recanalization time was found to be prolonged , this difference did not reach statistical significance (p = 0.348). When restricting the comparison cohort to patients with acute ischemic stroke receiving EVT during the corresponding time interval a year ago  only a significant difference in door-to-CT times was uncovered  volumes during COVID-19 measures could have contributed to these observations. Additionally, social restrictions urged family members to spend significantly more time together at home, which might lead to timely recognition of stroke symptoms and prompt EMS notification. Therefore, according to our findings, we suggest that institutional activated restrictions and preventive measures had a significant impact on the in-hospital acute stroke management, while we were not able to uncover any relevant impact of social distancing and overall public apprehension on the acute stroke prehospital pathway. Our data derive from a single regional comprehensive stroke center in Central South Ontario and thus might not be relevant to other institutions or regions. The disparity of our findings with previous reports highlights the need for intensive quality monitoring of stroke care delivery during the COVID-19 pandemic.Our report is in line with a recent publication from a Spanish regional stroke care system also highlighting an increased door-to-needle time for tPA patients in the COVID-19 era.13Our single-center experience suggests that healthcare institutional restrictions imposed in response to the COVID-19 pandemic have had a negative impact on acute ischemic stroke care time metrics known to predict stroke-related clinical outcomes. To preserve quality in acute stroke care patient access and outcomes, while decreasing potential COVID-19 exposure to patients and healthcare providers, institutions should consider simulation training programs to prepare their medical teams for protected code strokes during the COVID-19 pandemic.