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Lateral flow assay (LFA) is a handful diagnostic technology that can identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other common respiratory viruses in one strip, which can be tested at the point-of-care without the need for equipment or skilled personnel outside the laboratory. Although its simplicity and practicality make it an appealing solution, it remains a grand challenge to substantially enhance the colorimetric LFA sensitivity. In this work, we present a straightforward approach to enhance the sensitivity of LFA by imposing the flow constraints in nitrocellulose (NC) membranes via a number of vertical femtosecond laser micromachined microchannels which is important for prolonged specific binding interactions. Porous NC membrane surfaces were structured with different widths and densities µ-channels employing a second harmonic of the Yb:KGW femtosecond laser and sample XYZ translation over a microscope objective-focused laser beam. The influence of the microchannel parameters on the vertical wicking speed was evaluated from the video recordings. The obtained results indicated that µ-channel length, width, and density in NC membranes controllably increased the immunological reaction time between the analyte and the labeled antibody by 950%. Image analysis of the colorimetric indicators confirmed that the flow rate delaying strategy enhanced the signal sensitives by 40% compared with pristine NC LFA., (© 2024. The Author(s).)

Different types of pathogenic viruses that have common transmission path can be co-infected, inducing distinct disease procession in comparison to that infection of one. Also, in the post COVID-19 time, more types of respiratory infectious virus are becoming prevalent and are concurrent. Those bring an urgent need for detection of co-existing viruses. Here, we propose a visualized lateral flow assay for logic determination of co-existing viral RNA fragments. In the presence of specific viral RNA inputs, DNAzyme is de-blocked according to defined logic, and catalyzes the hydrolysis of hairpin-structural substrate. One of cleaved substrates contains DNAzyme domain to realize dual signal amplification, which obtains copious of the other cleaved substrates. The cleaved substrates act as linking strands for bridging DNA-modified gold nanoparticles onto lateral flow strip to induce coloration on test line. "AND", "OR" and "INHIBIT" controlled lateral flow assays are respectively demonstrated for co-existing viral RNA detection, and the visual results can be obtained by the same kind of prepared strip, without need of re-fabricating strips according to logic systems. The work provides a flexible, convenient, visual and logic-processing strategy for simultaneous analysis of co-existing viruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We assessed the performance of three different multiplex lateral flow assays manufactured by SureScreen, Microprofit and Goldsite which provide results for influenza, respiratory syncytial virus (RSV) and SARS-CoV-2. Between 4 April and 20 October 2023, 1646 patients 6 months and older presenting to an outpatient department of a hospital in Hong Kong with ≥2 symptoms or signs of an acute respiratory illness were enrolled. The point estimates for all three multiplex tests had sensitivity >80% for influenza A and SARS-CoV-2 compared to PCR, and the tests manufactured by Microprofit and Goldsite had sensitivity >84% to detect RSV. Specificity was >97% for all three tests except for the SureScreen test which had specificity 86.2% (95% CI: 83.9% to 88.3%) for influenza A. Sensitivity was lower than reported by the manufacturers, resulting in a higher risk of false negatives. The three multiplex tests performed better in patients with high viral loads., Competing Interests: Declaration of competing interest BJC consults for AstraZeneca, Fosun Pharma, GSK, Haleon, Moderna, Novavax, Pfizer, Roche, and Sanofi Pasteur. The authors report no other potential conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An enhanced lateral flow assay (LFA) is presented for rapid and highly sensitive detection of acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens with gold nanoflowers (Au NFs) as signaling markers and gold enhancement to amplify the signal intensities. First, the effect of the morphology of gold nanomaterials on the sensitivity of LFA detection was investigated. The results showed that Au NFs prepared by the seed growth method showed a 5-fold higher detection sensitivity than gold nanoparticles (Au NPs) of the same particle size, which may benefit from the higher extinction coefficient and larger specific surface area of Au NFs. Under the optimized experimental conditions, the Au NFs-based LFA exhibited a detection limit (LOD) of 25 pg mL -1 for N protein using 135 nm Au NFs as the signaling probes. The signal was further amplified by using a gold enhancement strategy, and the LOD for the detection of N protein achieved was 5 pg mL -1 . The established LFA also exhibited good repeatability and stability and showed applicability in the diagnosis of SARS-CoV-2 infection., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Lateral flow assays (LFAs) are currently the most popular point-of-care diagnostics, rapidly transforming disease diagnosis from expensive doctor checkups and laboratory-based tests to potential on-the-shelf commodities. Yet, their sensitive element, a monoclonal antibody, is expensive to formulate, and their long-term storage depends on refrigeration technology that cannot be met in resource-limited areas. In this work, LCB1 affibodies (antibody mimetic miniproteins) were conjugated to bovine serum albumin (BSA) to afford a high-avidity synthetic capture (LCB1-BSA) capable of detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and virus like particles (VLPs). Substituting the monoclonal antibody 2B04 for LCB1-BSA (stable up to 60 °C) significantly improved the thermal stability, shelf life, and affordability of plasmonic-fluor-based LFAs (p-LFAs). Furthermore, this substitution significantly improved the sensitivity of p-LFAs toward the spike protein and VLPs with precise quantitative ability over 2 and 3 orders of magnitude, respectively. LCB1-BSA sensors could detect VLPs at 100-fold lower concentrations, and this improvement, combined with their robust nature, enabled us to develop an aerosol sampling technology to detect aerosolized viral particles. Synthetic captures like LCB1-BSA can increase the ultrasensitivity, availability, sustainability, and long-term accuracy of LFAs while also decreasing their manufacturing costs.

Objective: Neutralizing antibodies against SARS-CoV-2 have shown to be an effective tool for the analysis of the immunity generated against COVID-19. Numerous seroprevalence studies carried out in different groups have made it possible to draw a global map of vaccination coverage through the use of rapid lateral flow immunochromatography serological tests for clinical and epidemiological purposes. The objective of our study was to determine the degree of immunity against SARS-CoV-2 associated with the presence of neutralizing antibodies in administrative staff, teachers and students at the University of Alicante by means of a rapid serological test and to learn about their experience with vaccination against COVID-19., Methods: A cross-sectional epidemiological study was designed, based on the prevalence of antibodies against the S protein (spike) of SARS-CoV-2. A total of 888 people participated. The study was carried out with a single test (July 6 to July 22, 2021). Using logistic regression, adjusted Odds Ratios were calculated according to sex, age, type of vaccine, number of vaccine doses received, complete vaccination schedule, and having had COVID-19., Results: The vaccines received mostly were Vaxzevria® and Comirnaty®, with 73.3% between both, although 67.2% presented a complete regimen. The results of the OJABIO rapid neutralizing antibody test gave a positive result in 61.4% of the sample. There was a high association between the variables COVID-19 infection, two doses of vaccine, Spikevax® or Comirnaty® vaccine, and eighteen/twenty-nine years old group with a positive result on the OJABIO test. A total of 712 subjects answered the parallel survey (80%) on adverse effects and preferences between the different vaccines against COVID-19., Conclusions: The vaccination status against COVID-19 in the university community after six months of the start of national immunization strategies reflects low coverage despite the excellent willingness to get vaccinated. Neutralizing antibodies (NAb) rapid tests can be useful to guide immunization strategies and decide when to administer new booster doses.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects many mammals, and SARS-CoV-2 circulation in nonhuman animals may increase the risk of novel variant emergence. Cats are highly susceptible to SARS-CoV-2 infection, and there were cases of virus transmission between cats and humans. The objective of this study was to assess the prevalence of SARS-CoV-2 variant infection of cats in an urban setting. We investigated the prevalence of SARS-CoV-2 variant infections in domestic and community cats in the city of Pittsburgh ( n = 272). While no cats tested positive for SARS-CoV-2 viral RNA, 35 cats (12.86%) tested SARS-CoV-2-antibody-positive. Further, we compared a cat-specific experimental lateral flow assay (eLFA) and species-agnostic surrogate virus neutralization assay (sVNT) for SARS-CoV-2 antibody detection in cats ( n = 71). The eLFA demonstrated 100% specificity compared to sVNT. The eLFA also showed 100% sensitivity for sera with >90% inhibition and 63.63% sensitivity for sera with 40-89% inhibition in sVNT. Using a variant-specific pseudovirus neutralization assay (pVNT) and antigen cartography, we found the presence of antibodies to pre-Omicron and Omicron SARS-CoV-2 variants. Hence, this approach proves valuable in identifying cat exposure to different SARS-CoV-2 variants. Our results highlight the continued exposure of cats to SARS-CoV-2 and warrant coordinated surveillance efforts.

Smartphone-based point-of-care testing (POCT) is rapidly emerging as an alternative to traditional screening and laboratory testing, particularly in resource-limited settings. In this proof-of-concept study, we present a smartphone- and cloud-based artificial intelligence quantitative analysis system (SCAISY) for relative quantification of SARS-CoV-2-specific IgG antibody lateral flow assays that enables rapid evaluation (<60 s) of test strips. By capturing an image with a smartphone camera, SCAISY quantitatively analyzes antibody levels and provides results to the user. We analyzed changes in antibody levels over time in more than 248 individuals, including vaccine type, number of doses, and infection status, with a standard deviation of less than 10%. We also tracked antibody levels in six participants before and after SARS-CoV-2 infection. Finally, we examined the effects of lighting conditions, camera angle, and smartphone type to ensure consistency and reproducibility. We found that images acquired between 45° and 90° provided accurate results with a small standard deviation and that all illumination conditions provided essentially identical results within the standard deviation. A statistically significant correlation was observed (Spearman correlation coefficient: 0.59, p = 0.008; Pearson correlation coefficient: 0.56, p  = 0.012) between the OD450 values of the enzyme-linked immunosorbent assay and the antibody levels obtained by SCAISY. This study suggests that SCAISY is a simple and powerful tool for real-time public health surveillance, enabling the acceleration of quantifying SARS-CoV-2-specific antibodies generated by either vaccination or infection and tracking of personal immunity levels.

The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience is maintained using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 min. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/μL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/μL, 1000 copies/μL, 2000 copies/μL, 2000 copies/μL, and 20,000 copies/μL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious diseases. As an alternative to viral protein detection, we further introduce an HCR lateral flow assay for viral RNA detection.

Monoclonal antibody therapy has been approved for prophylaxis and treatment of severe COVID-19 infection. Greatest benefit appears limited to those yet to mount an effective immune response from natural infection or vaccination, but concern exists around ability to make timely assessment of immune status of community-based patients where laboratory-based serodiagnostics predominate. Participants were invited to undergo paired laboratory-based (Abbott Architect SARS-CoV-2 IgG Quant II chemiluminescent microparticle immunoassay) and lateral flow assays (LFA; a split SARS-CoV-2 IgM/IgG and total antibody test) able to detect SARS-CoV-2 anti-spike antibodies. LFA band strength was compared with CMIA titer by log-linear regression. Two hundred individuals (median age 43.5 years, IQR 30-59; 60.5% female) underwent testing, with a further 100 control sera tested. Both LFA band strengths correlated strongly with CMIA antibody titers (P < 0.001). LFAs have the potential to assist in early identification of seronegative patients who may demonstrate the greatest benefit from monoclonal antibody treatment., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Neutralizing antibodies (NAbs) against SARS-CoV-2 have been shown to correlate with protection against infection. Simple tools such as lateral flow assays (LFA) that can accurately measure NAbs may be useful for monitoring anti-SARS-CoV-2 immunity in the future., Objectives: We assessed the performance of the ichroma™ COVID-19 nAb test, a rapid semiquantitative LFA, for the prediction of serum neutralizing activity against SARS-CoV-2 variants., Study Design: Serum samples were collected from COVID-19 recovered patients and vaccinated individuals. The result of the ichroma assay was provided as inhibition rate, and was compared to anti-SARS-CoV-2 IgG levels, and NAbs against Alpha, Delta and Omicron variants., Results: A total of 90 sera from recovered unvaccinated patients and 209 sera from the vaccine cohort were included in this study. In post-infection samples, the ichroma inhbition rate was found to be correlated with IgG levels (ρ = 0.83), and with anti-Alpha NAbs levels (ρ = 0.78). In the vaccine cohort, a good correlation was also observed between the ichroma inhibition rate and IgG levels (ρ = 0.84), as well as NAbs against Alpha (ρ = 0.62), Delta (ρ = 0.88) and Omicron (ρ = 0.74). An ichroma inhbition rate of 77.2%, 90.8% and 99.6% accurately predicted neutralization against Alpha, Delta and Omicron variants respectively., Conclusions: The ichroma™ COVID-19 nAb assay, with appropriate variant cut-offs, can be useful for the monitoring of anti-SARS-CoV-2 immunization and may provide a rapid prediction of protection, especially in individuals with significant levels of NAbs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mutations in the nucleocapsid of SARS-CoV-2 may interfere with antigen detection by diagnostic tests. We used several methods to evaluate the effect of various SARS-CoV-2 nucleocapsid mutations on the performance of the Panbio™ and BinaxNOW™ lateral flow rapid antigen tests and a prototype high-throughput immunoassay that utilizes Panbio antibodies. Variant detection was also evaluated by immunoblot and BIAcore™ assay. A panel of 23 recombinant nucleocapsid antigens (rAgs) were produced that included mutations found in circulating SARS-CoV-2 variants, including variants of concern. All mutant rAgs were detected by all assays, at a sensitivity equivalent to wild-type control (Wuhan strain). Thus, using a rAg approach, we found that the SARS-CoV-2 nucleocapsid mutations examined do not directly impact antigen detection or antigen assay performance.

Rapid detection of nucleic acids is essential for clinical diagnosis of a wide range of infectious and non-infectious diseases. CRISPR-based diagnostic platforms are well-established for rapid and specific detection of nucleic acids but suffer from a low detection sensitivity without a target pre-amplification step. Our recently developed detection system, called CRISPR-ENHANCE, employs engineered crRNAs and optimized conditions to achieve a significantly higher sensitivity and enable femtomolar levels of nucleic acid detection even without target pre-amplification. Using the CRISPR-ENHANCE platform and following the methodology detailed in this paper, nucleic acid detection for low copy numbers can be achieved in less than an hour through either a fluorescence-based detection or a lateral flow assay. The step-by-step instructions provided, in addition to describing how to perform both assays, incorporate details on a LAMP/RT-LAMP-based target amplification step to enable detection of RNA, ssDNA and dsDNA. Furthermore, a protocol for in-house expression and purification of LbCas12a using CL7/lm7-based affinity chromatography, which has been used to achieve a high yield and purity of the enzyme in a single-step, is provided., (Published by Elsevier Inc.)

Background: A vast majority of the commercially available lateral flow immunoassay (LFIA) is used to detect SARS-CoV-2 antibodies qualitatively. Recently, a novel fluorescence-based lateral flow immunoassay (LFIA) test was developed for quantitative measurement of the total binding antibody units (BAUs) (BAU/mL) against SARS-CoV-2 spike protein receptor-binding domain (S-RBD)., Aim: This study aimed to evaluate the performance of the fluorescence LFIA Finecare TM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113)., Methods: Plasma from 150 reverse trancriptase-PCR (RT-PCR)-confirmed positive individuals and 100 prepandemic samples were tested by Fincare TM to access sensitivity and specificity. For qualitative and quantitative validation of the FinCare TM measurements, BAU/mL results of FinCare TM were compared with results of 2 reference assays: the surrogate virus-neutralizing test (sVNT, GenScript Biotech, USA) and the VIDAS®3 automated assay (BioMérieux, France)., Results: Finecare TM showed 92% sensitivity and 100% specificity compared with PCR. Cohen's Kappa statistic denoted moderate and excellent agreement with sVNT and VIDAS®3, with values being 0.557 (95% CI: 0.32-0.78) and 0.731 (95% CI: 0.51-0.95), respectively. A strong correlation was observed between Finecare TM /sVNT (r = 0.7, p < 0.0001) and Finecare TM /VIDAS®3 (r = 0.8, p < 0.0001)., Conclusion: Finecare TM is a reliable assay and can be used as a surrogate to assess binding and neutralizing antibody response after infection or vaccination, particularly in none or small laboratory settings., Competing Interests: Conflict of interest GKN would like to declare that all test kits used in this study were provided as in-kind support for his laboratory to test seroprevalence of anti-SARS-CoV-2 and antibody response among vaccinated and infected individuals in Qatar., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: The development of easy-to-perform diagnostic methods is highly important for detecting current coronavirus disease (COVID-19). This pilot study aimed at developing a lateral flow assay (LFA)-based test prototype to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in saliva samples., Methods: Mice were immunized using the recombinant receptor-binding domain (rRBD) of SARS-CoV-2 virus spike protein. The combinations of the obtained mouse anti-receptor-binding domain (RBD) polyclonal antibodies (PAbs) and several commercial antibodies directed against the SARS-CoV-2 spike protein were used for enzyme-linked immunosorbent assay (ELISA) to select antibody pairs for LFA. The antibody pairs were tested in a LFA format using saliva samples from individuals with early SARS-CoV-2 infection ( n = 9). The diagnostic performance of the developed LFA was evaluated using saliva samples from hospitalized COVID-19 patients ( n = 111); the median time from the onset of symptoms to sample collection was 10 days (0-24 days, interquartile range (IQR): 7-13). The reverse transcription-polymerase chain reaction (rRT-PCR) was used as a reference method., Results: Based on ELISA and preliminary LFA results, a combination of mouse anti-RBD PAbs (capture antibody) and rabbit anti-spike PAbs (detection antibody) was chosen for clinical analysis of sample. When compared with rRT-PCR results, LFA exhibited 26.5% sensitivity, 58.1% specificity, 50.0% positive prediction value (PPV), 33.3% negative prediction value (NPV), and 38.7% diagnostic accuracy. However, there was a reasonable improvement in assay specificity (85.7%) and PPV (91.7%) when samples were stratified based on the sampling time., Conclusion: The developed LFA assay demonstrated a potential of SARS-CoV-2 detection in saliva samples. Further technical assay improvements should be made to enhance diagnostic performance followed by a validation study in a larger cohort of both asymptomatic and symptomatic patients in the early stage of infection., Competing Interests: The authors report no conflicts of interest., (© 2022 The Author(s). Published by Upsala Medical Society.)

Currently, the most accurate way to diagnose an active SARS-CoV-2 (COVID-19) infection is through detection of viral RNA using reverse transcription polymerase chain reaction (RT-PCR) test. While RT-PCR tests are the most sensitive for identifying infection, there are significant limitations, such as global access to sufficient test kits, turnaround times (TAT) from specimen collection to test result is often greater than 24 h and the need for skilled operators in accredited laboratories requiring specialized equipment. A rapid test performed at the point of care (POC) could provide a result within an approximate time of 30 min post specimen collection, be performed by a health care worker and comprise a simple workflow, improving both turnaround time and potentially decreasing costs (e.g., transport, cold-chain, skilled laboratory staff, complex equipment). Determining the performance of SARS-CoV-2 RT-PCR tests is, however, easier to assess than antigen-based POCT, as residual clinical specimens (swabs in universal transport media [UTM]) are readily available in laboratory environments, and do not require patient informed consent. Evaluating the performance of POCT requires informed-consent driven studies, with patients required to provide a standard of care specimen as well as study evaluation specimens, which is often not acceptable as nasopharyngeal swabbing can be invasive, clinical field trials are costly and time consuming. Many institutions and regulatory bodies also require preliminary data prior to use in field settings. Therefore, we have developed a method to determine the performance of antigen based POCT that can be used by implementers in national healthcare programs, regulators and rapid test developers. The method investigates both quantitative and qualitative parameters, with the latter providing insights into the capability for implementation and national program uptake., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing., Methods: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques., Findings: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test., Interpretation: Taken together, the COVID-19 NAb-test TM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care., Funding: Support was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation., Competing Interests: Declaration of Competing Interest A provisional patent covering the COVID-19 NAb-test(TM) test and underlying technology has been submitted through The University of Melbourne. Dr. Fulford reports a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Mr. Van reports grants from Government of the State of Victoria, during the conduct of the study; In addition, Mr. Van has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Dr. Gherardin reports a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Ms. Zheng reports grants from Government of the State of Victoria, during the conduct of the study; In addition, Ms. Zheng has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Mr. Ciula has nothing to disclose. Prof. Drummer has nothing to disclose. Mr. Redmond has nothing to disclose. Dr. Tan has nothing to disclose. Ms. Boo has nothing to disclose. Dr. Center has nothing to disclose. Dr. Li has nothing to disclose. Dr. Grimley has nothing to disclose. Dr. Wines reports grants from Australian Govt, Medical Research Future Fund, grants from NHMRC, National Health and Medical Research Council GNT1145303, Australia, during the conduct of the study; In addition, Dr. Wines has a patent drafted for use of ACE2-Fc pending. Dr. Nguyen has nothing to disclose. Ms. Mordant has nothing to disclose. Dr. Ellenberg has nothing to disclose. Dr. Rowntree has nothing to disclose. Lukasz Kedzierski has nothing to disclose. Prof. Cheng reports that he is a member of government advisory committees advising on COVID policy. Prof. Doolan has nothing to disclose. Prof. Matthews reports grants from Curran Foundation, during the conduct of the study; grants from Gilead sciences, grants from Abbvie Inc outside the submitted work. Dr. Bond has nothing to disclose. Prof. Hogarth reports grants from NHMRC, grants from MRFF, during the conduct of the study; In addition, Prof. Hogarth has a patent Antiviral pending. A/Prof. McQuilten reports grants from Medical Research Future Fund, during the conduct of the study. Prof. Subbarao has nothing to disclose. Prof. Kedzierska has nothing to disclose. Dr. Juno has nothing to disclose. Dr. Wheatley has nothing to disclose. Prof. Kent has nothing to disclose. Prof. Williamson has nothing to disclose. Prof. Purcell reports grants from Victorian Government DHHS, grants from Medical Research Future Fund, other from Jack Ma Foundation, during the conduct of the study; In addition, Prof. Purcell has a patent PCT/AU2021/050839 pending. Prof. Anderson reports grants from Government of the State of Victoria, during the conduct of the study; other from Nanjing BioPoint Diagnostic Technology, outside the submitted work; In addition, Prof. Anderson has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending. Prof. Godfrey reports grants from Victorian Government DHHS, and from Australian Research Council, during the conduct of the study; grants from Medical Research Future Fund, other from Jack Ma Foundation, grants from National Health and Medical Research Council, outside the submitted work; In addition, Prof. Godfrey has a patent Australian Provisional Patent Application 2021901011 (filed 7 April 2021) “Point of care lateral flow test for COVID19 detection” pending., (Copyright © 2021. Published by Elsevier B.V.)

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: After receiving a COVID-19 vaccine, most recipients want to know if they are protected from infection and for how long. Since neutralizing antibodies are a correlate of protection, we developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies from a drop of blood. The LFA is based on the principle that neutralizing antibodies block binding of the receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2)., Methods: The ability of the LFA was assessed to correctly measure neutralization of sera, plasma or whole blood from patients with COVID-19 using SARS-CoV-2 microneutralization assays. We also determined if the LFA distinguished patients with seasonal respiratory viruses from patients with COVID-19. To demonstrate the usefulness of the LFA, we tested previously infected and non-infected COVID-19 vaccine recipients at baseline and after first and second vaccine doses., Results: The LFA compared favorably with SARS-CoV-2 microneutralization assays with an area under the ROC curve of 98%. Sera obtained from patients with seasonal coronaviruses did not show neutralizing activity in the LFA. After a single mRNA vaccine dose, 87% of previously infected individuals demonstrated high levels of neutralizing antibodies. However, if individuals were not previously infected, only 24% demonstrated high levels of neutralizing antibodies after one vaccine dose. A second dose boosted neutralizing antibody levels just 8% higher in previously infected individuals, but over 63% higher in non-infected individuals., Conclusions: A rapid, semi-quantitative, highly portable and inexpensive neutralizing antibody test might be useful for monitoring rise and fall in vaccine-induced neutralizing antibodies to COVID-19., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Detection methods for monitoring infectious pathogens has never been more important given the need to contain the spread of the COVID-19 pandemic. Herein we propose a highly sensitive magnetic-focus-enhanced lateral flow assay (mLFA) for the detection of SARS-CoV-2. The proposed mLFA is simple and requires only lateral flow strips and a reusable magnet to detect very low concentrations of the virus particles. The magnetic focus enhancement is achieved by focusing the SARS-CoV-2 conjugated magnetic probes in the sample placed in the lateral flow (LF) strips for improved capture efficiency, while horseradish peroxidase (HRP) was used to catalyze the colorimetric reaction for the amplification of the colorimetric signal. With the magnetic focus enhancement and HRP-based amplification, the mLFA could yield a highly sensitive technology for the recognition of SARS-CoV-2. The developed methods could detect as low as 400 PFU/mL of SARS-CoV-2 in PBS buffer based on the visible blue dots on the LF strips. The mLFA could recognize 1200 PFU/mL of SARS-CoV-2 in saliva samples. With clinical nasal swab samples, the proposed mLFA could achieve 66.7% sensitivity and 100% specificity.

The global pandemic of COVID-19 continues to be an important threat, especially with the fast transmission rate observed after the discovery of novel mutations. In this perspective, prompt diagnosis requires massive economical and human resources to mitigate the disease. The current study proposes a rational design of a colorimetric lateral flow immunoassay (LFA) based on the repurposing of human samples to produce COVID-19-specific antigens and antibodies in combination with a novel dye-loaded polymersome for naked-eye detection. A group of 121 human samples (61 serums and 60 nasal swabs) were obtained and analyzed by RT-PCR and ELISA. Pooled samples were used to purify antibodies using affinity chromatography, while antigens were purified via magnetic nanoparticles-based affinity. The purified proteins were confirmed for their specificity to COVID-19 via commercial LFA, ELISA, and electrochemical tests in addition to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polymersomes were prepared using methoxy polyethylene glycol- b -polycaprolactone (mPEG- b -PCL) diblock copolymers and loaded with a Coomassie Blue dye. The polymersomes were then functionalized with the purified antibodies and applied for the preparation of two types of LFA (antigen test and antibody test). Overall, the proposed diagnostic tests demonstrated 93 and 92.2% sensitivity for antigen and antibody tests, respectively. The repeatability (92-94%) and reproducibility (96-98%) of the tests highlight the potential of the proposed LFA. The LFA test was also analyzed for stability, and after 4 weeks, 91-97% correct diagnosis was observed. The current LFA platform is a valuable assay that has great economical and analytical potential for widespread applications.

The development of reliable and robust diagnostic tests is one of the most efficient methods to limit the spread of coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, most laboratory diagnostics for COVID-19, such as enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR), are expensive, time-consuming, and require highly trained professional operators. On the other hand, the lateral flow immunoassay (LFIA) is a simpler, cheaper device that can be operated by unskilled personnel easily. Unfortunately, the current technique has some limitations, mainly inaccuracy in detection. This review article aims to highlight recent advances in novel lateral flow technologies for detecting SARS-CoV-2 as well as innovative approaches to achieve highly sensitive and specific point-of-care testing. Lastly, we discuss future perspectives on how smartphones and Artificial Intelligence (AI) can be integrated to revolutionize disease detection as well as disease control and surveillance.

Background: The novel coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has quickly spread worldwide since its outbreak in December 2019. One of the primary measures for controlling the spread of SARS-CoV-2 infection is an accurate assay for its diagnosis. SARS-CoV-2 real-time PCR kits suffer from some limitations, including false-negative results in the clinic. Therefore, there is an urgent need for the development of a rapid antibody test kit for COVID-19 diagnosis., Methods: The nuclear capsid protein (N) and spike protein 1 (S1) fragments of SARS-CoV-2 were expressed in Escherichia coli, and rapid antibody-based tests for the diagnosis of SARS-CoV-2 infection were developed. To evaluate their clinical applications, the serum from COVID-19 patients, suspected COVID-19 patients, recovering COVID-19 patients, patients with general fever or pulmonary infection, doctors and nurses who worked at the fever clinic, and health professionals was analyzed by the rapid antibody test kits. The serum from patients infected with Mycoplasma pneumoniae and patients with respiratory tract infection was further analyzed to test its cross-reactivity with other respiratory pathogens., Results: A 47 kDa N protein and 67 kDa S1 fragment of SARS-CoV-2 were successfully expressed, purified, and renatured. The rapid antibody test with recombinant N protein showed higher positive rate than the rapid IgM antibody test with recombinant S1 protein. Clinical evaluation showed that the rapid antibody test kit with recombinant N protein had 88.56 % analytical sensitivity and 97.42 % specificity for COVID-19 patients, 53.48 % positive rate for suspected COVID-19 patients, 57.14 % positive rate for recovering COVID-19 patients, and 0.5-0.8 % cross-reactivity with other respiratory pathogens. The analytical sensitivity of the kit did not significantly differ in COVID-19 patients with different disease courses (p < 0.01)., Conclusions: The rapid antibody test kit with recombinant N protein has high specificity and analytical sensitivity, and can be used for the diagnosis of SARS-CoV-2 infection combined with RT-PCR., (© 2021. The Author(s).)

For the control of immunity in COVID-19 survivors and vaccinated subjects, there is an urgent need for reliable and rapid serological assays. Based on samples from 63 COVID-19 survivors up to 7 months after symptom onset, and on 50 serum samples taken before the beginning of the pandemic, we compared the performances of three commercial immunoassays for the detection of SARS-CoV-2 IgA and IgG antibodies (Euroimmun SARS-COV-2 IgA/IgG, Mikrogen recom Well SARS-CoV-2 IgA/IgG, and Serion ELISA agile SARS-CoV-2 IgA/IgG) and three rapid lateral flow (immunochromatographic) tests (Abbott PanBio COVID-19 IgG/IgM, Nadal COVID-19 IgG/IgM, and Cleartest Corona 2019-nCOV IgG/IgM) with a 50% plaque-reduction neutralization test (PRNT50) representing the gold standard. Fifty-seven out of 63 PCR-confirmed COVID-19 patients (90%) showed neutralizing antibodies. The sensitivity of the seven assays ranged from 7.0% to 98.3%, and the specificity ranged from 86.0% to 100.0%. Only one commercial immunoassay showed a sensitivity and specificity of greater than 98%.

Background: Serological testing in the COVID-19 pandemic is mainly implemented to gain sero-epidemiological data, but can also retrospectively inform about suspected SARS-CoV-2 infection., Method: We verified and applied a two-tiered testing strategy combining a SARS-CoV-2 receptor-binding domain (RBD)-specific lateral flow assay (LFA) with a nucleocapsid protein (NCP) IgG ELISA to assess seroconversion in n  = 7241 individuals. The majority had experienced symptoms consistent with COVID-19, but had no access to RT-PCR testing. Longitudinal follow-up in n  = 97 LFA + individuals was performed up to 20 weeks after initial infection using NCP and spike protein S1 domain (S1) IgG ELISAs and a surrogate virus neutralization test (sVNT)., Results: Individuals reporting symptoms from January 2020 onwards showed seroconversion, as did a considerable proportion of asymptomatic individuals. Seroconversion for symptomatic and asymptomatic individuals was higher in an area with a known infection cluster compared to a low incidence area. Overall, 94% of individuals with a positive IgG result by LFA were confirmed by NCP ELISA. The proportion of ELISA-confirmed LFA results declined over time, in line with contracting NCP IgG titres during longitudinal follow-up. Neutralizing antibody activity was considerably more stable than S1 and NCP IgG titres, and both reach a plateau after approximately 100 d. The sVNT proved to be not only highly specific, but also more sensitive than the specificity-focussed two-tiered serology approach., Conclusions: Our results demonstrate the high specificity of two-tiered serology testing and highlight the sVNT used as a valuable tool to support modelling of SARS-CoV-2 transmission dynamics, complement molecular testing and provide relevant information to individuals.

Despite collaborative efforts from all countries, coronavirus disease 2019 (COVID-19) pandemic has been continuing to spread globally, forcing the world into social distancing period, making a special challenge for public healthcare system. Before vaccine widely available, the best approach to manage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to achieve highest diagnostic accuracy by improving biosensor efficacy. For SARS-CoV-2 diagnostics, intensive attempts have been made by many scientists to ameliorate the drawback of current biosensors of SARS-CoV-2 in clinical diagnosis to offer benefits related to platform proposal, systematic analytical methods, system combination, and miniaturization. This review assesses ongoing research efforts aimed at developing integrated diagnostic tools to detect RNA viruses and their biomarkers for clinical diagnostics of SARS-CoV-2 infection and further highlights promising technology for SARS-CoV-2 specific diagnosis. The comparisons of SARS-CoV-2 biomarkers as well as their applicable biosensors in the field of clinical diagnosis were summarized to give scientists an advantage to develop superior diagnostic platforms. Furthermore, this review describes the prospects for this rapidly growing field of diagnostic research, raising further interest in analytical technology and strategic plan for future pandemics.

To combat the enduring and dangerous spread of COVID-19, many innovations to rapid diagnostics have been developed based on proteinprotein interactions of the SARS-CoV-2 spike and nucleocapsid proteins to increase testing accessibility. These antigen tests have most prominently been developed using the lateral flow assay (LFA) test platform which has the benefit of administration at point-of-care, delivering quick results, lower cost, and does not require skilled personnel. However, they have gained criticism for an inferior sensitivity. In the last year, much attention has been given to creating a rapid LFA test for detection of COVID-19 antigens that can address its high limit of detection while retaining the advantages of rapid antibodyantigen interaction. In this review, a summary of these proteinprotein interactions as well as the challenges, benefits, and recent improvements to protein based LFA for detection of COVID-19 are discussed., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As new tests and technologies advance our understanding and diagnostic capabilities of the severe acute respiratory syndrome coronavirus 2 and the coronavirus disease 2019, they must be appropriately validated to make sure test performance is following manufacturer claims. In this study, we evaluated the Vazyme 2019-nCoV IgG/IgM Detection Kit, which is a lateral flow assay (LFA), by the plaque reduction neutralization test (PRNT) using 100 patient plasma/serum samples. As compared to the PRNT results, the Vazyme LFA had 95.9% sensitivity and 96.1% specificity. Along with the increased need for rapid, effective, and affordable point of care tests to help provide meaningful epidemiological data, we demonstrated that the Vazyme LFA performed well on IgG detection but cannot be judged on the performance of IgM detection using PRNT alone. However, our observation of the low IgM-positive rate supported the poor performance of IgM detection of this LFA which led to the disapproval of its Emergency Use Authorization recently., Competing Interests: Conflict of interest All authors have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

There is an ongoing need for reliable antigen assays for timely and easy detection of individuals with acute SARS-CoV-2 infection. Using 75 swabs from patients previously tested positive by SARS-CoV-2 PCR and 75 swabs from patients previously tested negative by SARS-CoV-2 PCR, we investigated the sensitivity and specificity of the SARS-CoV-2 Rapid Antigen Test (Roche). We determined a specificity of 96 %. The assay's sensitivity with samples with a cycle threshold of < 25, 25 - <30, 30 - <35, and> = 35 was 100 %, 95 %, 44.8 % and 22.2 %, respectively. We conclude that sensitivity and specificity of the antigen assay is inferior to the PCR assay. However, the antigen assay may be a quick and easy to perform alternative for differentiation of individuals contagious for SARS-CoV-2 from non-contagious individuals., (Copyright © 2020 Elsevier B.V. All rights reserved.)

BackgroundBreakthrough cases of SARS-CoV-2 infection correlate with decreased antibody immunity following mRNA vaccination. Measuring kinetics of vaccine efficacy using traditional laboratory approaches is more expensive and can be impractical. In this study, we evaluated the diagnostic performance of a validated COVID-19 point-of-care lateral flow assay (LFA) kit in detecting post-vaccination antibody response.MethodsWe conducted a prospective cohort study of whole blood and plasma samples to evaluate the performance of a LFA in detecting SARS-CoV-2-specific antibodies following mRNA vaccination compared to enzyme-linked immunosorbent assays (ELISAs). Health care workers at 2 tertiary centers who completed an initial BNT162b2 (n = 103) or mRNA-1273 (n = 35) vaccine series were enrolled between June and August of 2021. We performed an exploratory analysis to correlate band strength and antibody concentration of LFAs and ELISAs respectively.ResultsWhen compared to the ELISA, LFA results showed similar test positivity for plasma samples (P = 0.55), but not for whole blood samples (P

A lateral flow assay (LFA) strip based on dual 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)–encoded satellite Fe3O4@Au (Mag@Au) SERS tags with nanogap is reported for ultrasensitive and simultaneous diagnosis of two SARS-CoV-2 functional proteins. Composed of Fe3O4 core, satellite gold shell with nanogaps, and double-layer DTNB, the Mag@Au nanoparticles with an average size of 238 nm were designed as multifunctional tags to efficiently enrich the target SARS-CoV-2 protein from complex samples, significantly enhancing the SERS signal of the LFA strip and provide quantitative SERS detection of analyte on test lines. The developed dual DTNB-encoded satellite Mag@Au-based LFA allowed simultaneous quantification of spike (S) protein and nucleocapsid (NP) protein with detection limits of 23 pg mL−1 and 2 pg mL−1, respectively, lower than commercial ELISA kits and reported SERS-LFA detection system–based Au NPs and Fe3O4@3 nm Au MNPs. This magnetic SERS-LFA also showed high performance of multi-variant strain detection and further distinguished clinical samples of Omicron variant infection, demonstrating the potential of in situ detection of respiratory virus diseases. [ABSTRACT FROM AUTHOR]

Point‐of‐care diagnosis is crucial to control the spreading of viral infections. Here, universal‐modifiable probe‐gated silica nanoparticles (SNPs) based lateral flow assay (LFA) is developed in the interest of the rapid and early detection of viral infections. The most superior advantage of the rapid assay is its utility in detecting various sides of the virus directly from the human swab samples and its adaptability to detect various types of viruses. For this purpose, a high concentration of fluorescein and rhodamine B as a reporting material was loaded into SNPs with excellent loading capacity and measured using standard curve, 4.19 μmol ⋅ g−1 and 1.23 μmol ⋅ g−1, respectively. As a model organism, severe acute respiratory syndrome coronavirus‐2 (CoV‐2) infections were selected by targeting its nonstructural (NSP9, NSP12) and envelope (E) genes as target sites of the virus. We showed that NSP12‐gated SNPs‐based LFA significantly outperformed detection of viral infection in 15 minutes from 0.73 pg ⋅ mL−1 synthetic viral solution and with a dilution of 1 : 103 of unprocessed human samples with an increasing test line intensity compared to steady state (n=12). Compared to the RT‐qPCR method, the sensitivity, specificity, and accuracy of NSP12‐gated SNPs were calculated as 100 %, 83 %, and 92 %, respectively. Finally, this modifiable nanoparticle system is a high‐performance sensing technique that could take advantage of upcoming point‐of‐care testing markets for viral infection detections. [ABSTRACT FROM AUTHOR]

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To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and control the spread of coronavirus disease (COVID‐19), there is an urgent need for highly sensitive on‐site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein (Cas)‐based molecular diagnostic method was developed for this purpose. Here, a CRISPR system‐mediated lateral flow assay (LFA) for SARS‐CoV‐2 was established based on multienzyme isothermal rapid amplification, CRISPR‐Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/μl in both fluorescence‐ and immunochromatography‐based assays. To enhance the quality control of the CRISPR‐based LFA method, glyceraldehyde‐3‐phosphate dehydrogenase was detected as a reference using a triple‐line strip design in a lateral flow strip. In total, 52 COVID‐19‐positive and 101 COVID‐19‐negative clinical samples examined by reverse transcription polymerase chain reaction (RT‐PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT‐PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR‐mediated LFA and provides a crucial tool for on‐site detection of SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

Serological assays for SARS-CoV-2 are being utilized at an exponential rate for surveillance programs. This enterprise was designed to develop and validate a qualitative immunochromatographic test, via the Lateral Flow Assay (LFA), for detection of immunoglobulins M and G (IgM and IgG) against both nucleocapsid (N) and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Both targeted proteins were cloned and expressed in baculovirus expression system utilizing insect cells Sf9. The recombinant RBD and N proteins were purified and conjugated with gold nanoparticles (AuNPs) to set up the coating antigens pad. Both anti-human IgG and IgM were dispensed on nitrocellulose membrane to capture human antibodies in serum samples. A home-made dispensing system was developed to draw identical test and control lines. The validity of the developed LFA was verified by testing serum samples from 103 convalescent COVID-19 patients who were PCR positive for SARS-CoV-2 along with 28 control serum samples. The developed strips showed distinctive bands for IgM and IgG of both proteins (RBD and N) in positive samples. The sensitivity of RBD-based LFA was 70.9% and 39.8% for IgG and IgM, respectively, with a specificity of 100% for both. The N-based LFA exhibited a sensitivity of 73.8% and 35.9% for IgG and IgM, respectively, while its specificity was 75% and 100% for IgG and IgM, respectively. Our developed LFA could afford a tool for surveillance programs in low-resource countries. Moreover, it might be functional for rapid and inexpensive monitoring of the anti-SARS-CoV-2 antibodies in the sera of vaccinated individuals. [ABSTRACT FROM AUTHOR]

Background: A vast majority of the commercially available lateral flow immunoassay (LFIA) is used to detect SARS-CoV-2 antibodies qualitatively. Recently, a novel fluorescence-based lateral flow immunoassay (LFIA) test was developed for quantitative measurement of the total binding antibody units (BAUs) (BAU/mL) against SARS-CoV-2 spike protein receptor-binding domain (S-RBD). Aim: This study aimed to evaluate the performance of the fluorescence LFIA FinecareTM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113). Methods: Plasma from 150 reverse trancriptase–PCR (RT-PCR)-confirmed positive individuals and 100 prepandemic samples were tested by FincareTM to access sensitivity and specificity. For qualitative and quantitative validation of the FinCareTM measurements, BAU/mL results of FinCareTM were compared with results of 2 reference assays: the surrogate virus-neutralizing test (sVNT, GenScript Biotech, USA) and the VIDAS®3 automated assay (BioMérieux, France). Results: FinecareTM showed 92% sensitivity and 100% specificity compared with PCR. Cohen's Kappa statistic denoted moderate and excellent agreement with sVNT and VIDAS®3, with values being 0.557 (95% CI: 0.32–0.78) and 0.731 (95% CI: 0.51–0.95), respectively. A strong correlation was observed between FinecareTM/sVNT (r = 0.7, p < 0.0001) and FinecareTM/VIDAS®3 (r = 0.8, p < 0.0001). Conclusion: FinecareTM is a reliable assay and can be used as a surrogate to assess binding and neutralizing antibody response after infection or vaccination, particularly in none or small laboratory settings.

COVID-19 mRNA vaccines protect against severe disease and hospitalization. Neutralizing antibodies (NAbs) are a first-line defense mechanism, but protective NAb responses are variable. Currently, NAb testing is not widely available. This study employed a lateral flow assay for monitoring NAb levels postvaccination and natural infection, using a finger-stick drop of blood. We report longitudinal NAb data from BNT162b2 (Pfizer) and mRNA-1273 (Moderna) recipients after second and third doses. Results demonstrate a third dose of mRNA vaccine elicits higher and more durable NAb titers than the second dose, independent of manufacturer, sex, and age. Our analyses also revealed that vaccinated individuals could be categorized as strong, moderate, and poorly neutralizing responders. After the second dose, 34% of subjects were classified as strong responders, compared to 79% after the third dose. The final months of this study coincided with the emergence of the SARS-CoV-2 Omicron variant and symptomatic breakthrough infections within our study population. Lastly, we show that NAb levels sufficient for protection from symptomatic infection with early SARS-CoV-2 variants were not protective against Omicron infection and disease. This work highlights the need for accessible vaccine response monitoring for use in healthcare, such that individuals, particularly those in vulnerable populations, can make informed vaccination decisions. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects many mammals, and SARS-CoV-2 circulation in nonhuman animals may increase the risk of novel variant emergence. Cats are highly susceptible to SARS-CoV-2 infection, and there were cases of virus transmission between cats and humans. The objective of this study was to assess the prevalence of SARS-CoV-2 variant infection of cats in an urban setting. We investigated the prevalence of SARS-CoV-2 variant infections in domestic and community cats in the city of Pittsburgh (n = 272). While no cats tested positive for SARS-CoV-2 viral RNA, 35 cats (12.86%) tested SARS-CoV-2-antibody-positive. Further, we compared a cat-specific experimental lateral flow assay (eLFA) and species-agnostic surrogate virus neutralization assay (sVNT) for SARS-CoV-2 antibody detection in cats (n = 71). The eLFA demonstrated 100% specificity compared to sVNT. The eLFA also showed 100% sensitivity for sera with >90% inhibition and 63.63% sensitivity for sera with 40–89% inhibition in sVNT. Using a variant-specific pseudovirus neutralization assay (pVNT) and antigen cartography, we found the presence of antibodies to pre-Omicron and Omicron SARS-CoV-2 variants. Hence, this approach proves valuable in identifying cat exposure to different SARS-CoV-2 variants. Our results highlight the continued exposure of cats to SARS-CoV-2 and warrant coordinated surveillance efforts.

Smartphone-based point-of-care testing (POCT) is rapidly emerging as an alternative to traditional screening and laboratory testing, particularly in resource-limited settings. In this proof-of-concept study, we present a smartphone- and cloud-based artificial intelligence quantitative analysis system (SCAISY) for relative quantification of SARS-CoV-2-specific IgG antibody lateral flow assays that enables rapid evaluation (p = 0.008; Pearson correlation coefficient: 0.56, p = 0.012) between the OD450 values of the enzyme-linked immunosorbent assay and the antibody levels obtained by SCAISY. This study suggests that SCAISY is a simple and powerful tool for real-time public health surveillance, enabling the acceleration of quantifying SARS-CoV-2-specific antibodies generated by either vaccination or infection and tracking of personal immunity levels.

Background: Limited commercial LFA assays are available to provide a reliable quantitative measurement of the total binding antibody units (BAU/mL) against the receptor-binding domain of the SARS-CoV-2 spike protein (S-RBD). Aim: This study aimed to evaluate the performance of the fluorescence LFA FinecareTM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113) against the following reference methods: (i) the FDA-approved GenScript surrogate virus-neutralizing assay (sVNT); and (ii) three highly performing automated immunoassays: BioMérieux VIDAS®3, Ortho VITROS®, and Mindray CL-900i®. Methods: Plasma from 488 vaccinees was tested by all aforementioned assays. Fingerstick whole-blood samples from 156 vaccinees were also tested by FinecareTM. Results and conclusions: FinecareTM showed 100% specificity, as none of the pre-pandemic samples tested positive. Equivalent FinecareTM results were observed among the samples taken from fingerstick or plasma (Pearson correlation r = 0.9, p < 0.0001), suggesting that fingerstick samples are sufficient to quantitate the S-RBD BAU/mL. A moderate correlation was observed between FinecareTM and sVNT (r = 0.5, p < 0.0001), indicating that FinecareTM can be used for rapid prediction of the neutralizing antibody (nAb) post-vaccination. FinecareTM BAU results showed strong correlation with VIDAS®3 (r = 0.6, p < 0.0001) and moderate correlation with VITROS® (r = 0.5, p < 0.0001) and CL-900i® (r = 0.4, p < 0.0001), suggesting that FinecareTM can be used as a surrogate for the advanced automated assays to measure S-RBD BAU/mL. [ABSTRACT FROM AUTHOR]

In the presented study, we developed a nanogold lateral glow immunoassay-based technique (LFI-COVID-19 antigen test) for the detection of SARS-CoV-2 nucleocapsid proteins; the developed LFI-COVID-19 Ag test has been tested for limit of detection (LOD), cross-reactivity and interfering substances, and performance. It was found that the performance of the developed LFI-COVID-19 antigen test when it was evaluated by RT-qPCR indicated 95, 98, and 97% for sensitivity, specificity and accuracy, respectively. This complies with the WHO guidelines. It was concluded that the developed LFI-COVID-19 antigen test is a point of care and an alternative approach to current laboratory methods, especially RT-qPCR. It provides an easy, rapid (within 20 min), and on-site diagnostic tool for COVID-19 infection, and it is a cheap test if it is manufactured on a large scale for commercial use. [ABSTRACT FROM AUTHOR]

Respiratory illness caused by influenza virus is a serious public health problem worldwide. As the symptoms of influenza virus infection are similar to those of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it is essential to distinguish these two viruses. Therefore, to properly respond to a pathogen, a detection method that is capable of rapid and accurate diagnosis in a hospital or at home is required. To satisfy this need, we applied loop-mediated isothermal amplification (LAMP) and an isothermal nucleic acid amplification technique, along with a system to analyze the results without specialized equipment, a lateral flow assay (LFA). Using the platform developed in this study, all processes, from sample preparation to detection, can be performed without special equipment. Unlike existing PCR methods, the nucleic acid amplification can be performed in the field because hot packs do not require electricity. Thus, the designed platform can provide rapid results without the need to transport the samples to a laboratory or hospital. These advantages are not limited to operations in developing countries with poor access to medical systems. In conclusion, the developed technology is a promising tool for infectious disease management that allows for rapid identification of infectious diseases and appropriate treatment of patients. [ABSTRACT FROM AUTHOR]

Background: The accuracy and reliability of rapid diagnostic tests are critical for monitoring and diagnosing SARS-CoV-2 infection in the general population. This study aimed to evaluate the analytical performance of the BIOSYNEX COVID-19 Ag BSS (Biosynex Swiss SA, Fribourg, Switzerland) antigen rapid diagnostic test (BIOSYNEX Ag-RDT), which targets the SARS-CoV-2 N-nucleocapsid protein for the diagnosis of COVID-19. The Ag-RDT was compared with a real-time RT-PCR (rtRT-PCR) as gold standard for performance measurement. Methods: Two nasopharyngeal flocked swabs were prospectively collected simultaneously in March and April 2021 from 967 individuals aged ≥ 18 years tested for SARS-CoV-2 in two private laboratories, Paris, France. Results: Overall, the Ag-RDT demonstrated high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 81.8%, 99.6%, 96.6%, and 97.5%, respectively. The agreement (97.0%), reliability assessed using Cohen's κ-coefficient (0.87), and accuracy evaluated using Youden index (J) (81.6%) in detecting SARS-CoV-2 were high. The analytical performance of the Ag-RDT remained high when there was significant viral shedding (i.e., N gene Ct values ≤ 33 on reference RT-PCR). The sensitivity was only 55.2% in case of low or very low viral excretion (Ct > 33). Conclusions: The BIOSYNEX Ag-RDT is a promising, potentially simple diagnostic tool, especially in symptomatic COVID-19 patients with substantial viral excretion in the nasopharynx. [ABSTRACT FROM AUTHOR]

Coronaviruses, that are now well-known to the public, include a family of viruses that can cause severe acute respiratory syndrome (SARS) and other respiratory diseases, such as Middle East respiratory syndrome (MERS). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the seventh member of this coronavirus family, was detected in 2019 and can cause a number of respiratory symptoms, from dry cough and fever to fatal viral pneumonia. Various diagnostic assays ranging from real-time polymerase chain reaction (RT-PCR) to point-of-care medical diagnostic systems have been developed for detection of viral components or antibodies targeting the virus. Point-of-care assays allow rapid diagnostic assessment of infectious patients. Such assays are ideally simple, low-cost, portable tests with the possibility for on-site field detection that do not require skilled staff, sophisticated equipment, or sample pretreatment, as compared to RT-PCR. Since early 2021 when new SARS-CoV-2 variants of concern increased, rapid tests became more crucial in the disease management cycle. Among rapid tests, gold nanoparticle (GNP)-based lateral flow assays (LFAs) have high capacity for performing at the bedside, paving the way to easy access to diagnosis results. In this review, GNP-based LFAs used for either COVID-19 proteins or human response antibodies are summarized and recommendations for their improvement have been suggested. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant outbreaks have highlighted the need of antigen-detecting rapid diagnostic tests (Ag-RDTs) that can be used at the point-of-care (POC). Although many Ag-RDTs have been approved for SARS-CoV-2 detection, studies demonstrating the clinical performance of Ag-RDTs against variants of concern, especially the new Omicron variant, are limited. The aim of this study was to evaluate the diagnostic sensitivity and specificity of the AMAZING COVID-19 Antigen Sealing Tube Test Strip (Colloidal Gold) in 584 early symptomatic and asymptomatic participants (age range 0–90 years). The performance of this Ag-RDT was assessed by comparing its results with reverse transcription RT-PCR (rRT-PCR). One hundred twenty positive samples were also analyzed with rRT-PCR to discriminate Omicron and Delta/Kappa variants (72.50% Omicron; 27.50% Delta/Kappa). Overall, the Ag-RDT showed high positive and negative percent values of 92.52% (95% CI, 86.61–95.95%) and 98.05% (95% CI, 96.41–98.95%), respectively, as well as an overall diagnostic accuracy of 96.92% (95% CI, 95.17–98.16%). Taken together, these data indicate that this inexpensive and simple-to-use Ag-RDT presents excellent analytical performance and can reliably detect Omicron and Delta/Kappa variants. [ABSTRACT FROM AUTHOR]

The presence of neutralizing antibodies (NAbs) against SARS-CoV-2 represent a surrogate marker of immunologic protection in populations at high risk of infection such as healthcare workers caring for hospitalized patients with COVID-19. As recommended by CDC and the European CDC, the use of rapid diagnostic tests during population-based evaluations offers an opportunity to identify individuals with serologic evidence of natural infection or who have undergone vaccination. We carried out a cross-sectional study to assess the presence of neutralizing antibodies against SARS-CoV-2 among medical providers at an intensive care unit of a large referral hospital in Alicante, Spain. In addition, we tested for the presence of neutralizing antibodies compared to serum of uninfected individuals from a Biobank. We were also interested in evaluating the use of a rapid lateral flow immunochromatography (LFIC) test against a surrogate ELISA viral neutralization test (sVNT). This rapid test demonstrated a specificity of 1.000 95% CI (0.91–1.00) and the sensitivity of 0.987 95% CI (0.93–1.00). The negative predictive value was 95%. After six months, this rapid test demonstrated that those immunized with two doses of BioNTech/Pfizer vaccine, maintained optimal levels of neutralizing antibodies. We concluded that all Health Care Workers develop NAbs and the use of this rapid immunochromatographic test represents a potential tool to be used in population-based studies to detect serological antibody responses to vaccination. Vaccination policies could benefit from this tool to assess additional doses of vaccine or boosters among high-risk populations. [ABSTRACT FROM AUTHOR]