A microfluidic biosensor architecture for the rapid detection of COVID-19

The lack of enough diagnostic capacity to detect severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has been one of the major challenges in the control the 2019 COVID pandemic;this led to significant delay in prompt treatment of COVID-19 patients or accurately estimate disease situation. Current methods for the diagnosis of SARS-COV-2 infection on clinical specimens (e.g. nasal swabs) include polymerase chain reaction (PCR) based methods, such as real-time reverse transcription (rRT) PCR, real-time reverse transcription loop-mediated isothermal amplification (rRT-LAMP), and immunoassay based methods, such as rapid antigen test (RAT). These conventional PCR methods excel in sensitivity and specificity but require a laboratory setting and typically take up to six hours to obtain the results whereas RAT has a low sensitivity (typically at least 3000 TCID50/ml) although with the results with 15 mins. We have developed a robust micro-electro-mechanical system (MEMS) based impedance biosensor fit for rapid and accurate detection of SARS-COV-2 of clinical samples in the field with minimal training. The biosensor consisted of three regions that enabled concentrating, trapping, and sensing the virus present in low quantities with high selectivity and sensitivity in 40 minutes using an electrode coated with a specific SARS-COV-2 antibody cross-linker mixture. Changes in the impedance value due to the binding of the SARS-COV-2 antigen to the antibody will indicate positive or negative result. The testing results showed that the biosensor's limit of detection (LoD) for detection of inactivated SARS-COV-2 antigen in phosphate buffer saline (PBS) was as low as 50 TCID50/ml. The biosensor specificity was confirmed using the influenza virus while the selectivity was confirmed using influenza polyclonal sera. Overall, the results showed that the biosensor is able to detect SARS-COV-2 in clinical samples (swabs) in 40 min with a sensitivity of 26 TCID50/ml.

Persulfate-Mediated Dual-Emitting Conjugated Polymers for Electrochemiluminescence Ratio Analysis of SARS-CoV-2 RdRp Gene

Polymers have attracted attention as luminophores due to their excellent electrochemiluminescence (ECL) properties. However, the current research and application of polymers mainly focus on anode emission, and ECL efficiency is not high enough, thus showing a limited application. This work exploited the persulfate-mediated dual-emission characteristics of poly[2,5-dioctyl-1,4-phenylene] polymer nanoparticles (PDP PNPs). The two ECL emissions were collected synchronously at -2.0V and +1.0V with persulfate (S2O82-) as cathodic coreactant and 3-(dibutylamino) propylamine (TDBA) as anodic coreactant, respectively. Interestingly, S2O82- can simultaneously mediate the double emissions, significantly enhancing both cathode emission and anode emission. The dual-emission mechanism was explored carefully and enhancement mechanism of cathodic coreactant S2O82- to anodic emission was hypothesized to be attributed to SO4∙− radicals, which was produced from S2O82- during cathodic potential scanning and oxidized PDP PNPs to generate more cation radical, thus enhancing anodic emission of PDP PNPs. Moreover, the black hole quencher-2 (BHQ2) was exploited as dual-function moderator to quench dual emissions of PDP PNPs synchronously. PDP PNPs coupled with BHQ2 to build ECL ratiometric system for detecting SARS-CoV-2 RdRp gene and its limit of detection was 25.1 aM. Persulfate-mediated double emissions provided a new way to improve the efficiency of ECL emission from polymers and expand their application. The clever integration of dual-emitting PDP PNPs and dual-regulating BHQ2 created a promising single-luminophore-based ratiometric ECL platform, developed an attractive ECL method for detecting SARS-CoV-2 RdRp gene.

Functional Antibody Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Variants in Children With Coronavirus Disease 2019, Multisystem Inflammatory Syndrome in Children, and After Two Doses of BNT162b2 Vaccination.

BACKGROUND: Although neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) correlate with protection against coronavirus disease 2019 (COVID-19), little is known about the neutralizing and antibody-dependent cell-mediated cytotoxicity (ADCC) responses to COVID-19, multisystem inflammatory syndrome in children (MIS-C), and COVID-19 vaccination in children. METHODS: We enrolled children 0-21 years of age with a history of COVID-19 (n = 13), MIS-C (n = 13), or 2 doses of BNT162b2 vaccination (n = 14) into a phlebotomy protocol. We measured pseudovirus neutralizing and functional ADCC antibodies to SARS-CoV-2 variants, including Omicron (B.1.1.529). RESULTS: The primary BNT162b2 vaccination series elicited higher neutralizing and ADCC responses with greater breadth to SARS-CoV-2 variants than COVID-19 or MIS-C, although these were diminished against Omicron. CONCLUSIONS: Serologic responses were significantly reduced against variants, particularly Omicron.

Performance comparison of two nucleic acid amplification systems for SARS-CoV-2 detection: A multi-center study.

BACKGROUND: Many rapid nucleic acid testing systems have emerged to halt the development and spread of COVID-19. However, so far relatively few studies have compared the diagnostic performance between these testing systems and conventional detection systems. Here, we performed a retrospective analysis to evaluate the clinical detection performance between SARS-CoV-2 rapid and conventional nucleic acid detection system. METHODS: Clinical detection results of 63,352 oropharyngeal swabs by both systems were finally enrolled in this analysis. Sensitivity (SE), specificity (SP), and positive and negative predictive value (PPV, NPV) of both systems were calculated to evaluate their diagnostic accuracy. Concordance between these two systems were assessed by overall, positive, negative percent agreement (OPA, PPA, NPA) and κ value. Sensitivity of SARS-CoV-2 rapid nucleic acid detection system (Daan Gene) was further analyzed with respect to the viral load of clinical specimens. RESULTS: Sensitivity of Daan Gene was slightly lower than that of conventional detection system (0.86 vs. 0.979), but their specificity was equivalent. Daan Gene had ≥98.0% PPV and NPV for SARS-CoV-2. Moreover, Daan Gene demonstrated an excellent test agreement with conventional detection system (κ = 0.893, p = 0.000). Daan Gene was 99.31% sensitivity for specimens with high viral load (Ct < 35) and 50% for low viral load (Ct ≥ 35). CONCLUSIONS: While showing an analytical sensitivity slightly below than that of conventional detection system, rapid nucleic acid detection system may be a diagnostic alternative to rapidly identify SARS-CoV-2-infected individuals with high viral loads and a powerful complement to current detection methods.

An electrochemiluminescence biosensor based on morphology controlled iridium complex nanomaterials for SARS-CoV-2 nucleocapsid protein detection

The morphology of electrochemiluminescence (ECL) emitters is closely related to ECL properties, thus the control of their morphology is greatly essential for ECL applications. Herein, a facile nanoprecipitation method was developed to realize controllable morphology of iridium complex nanomaterials by modulating the volume ratio of poly(styrene-co-maleicanhydride) (PSMA) to tris (2-phenylpyridine) iridium(Ⅲ) (Ir(ppy)3). Furthermore, ECL properties of iridium complex nanomaterials with different morphologies were explored through a series of experiments. Iridium complex nanoparticles (Ir NPs) with excellent ECL performance were selected as admirable ECL emitters to construct biosensors. Ir NPs served as the matrix to stepwise capture primary antibody, antigen of SARS-CoV-2 nucleocapsid protein (ncovNP) and secondary antibody bioconjugate coupled with dual quenchers and detection antibody. Taking advantage of the significant quenching effect of noradrenaline (NA) and gold nanoparticles (Au NPs) in the secondary antibody bioconjugate on ECL emission from Ir NPs, the developed ECL biosensor realized the sensitive detection of ncovNP and the detection limit was as low as 47 fg/mL. The integration of morphology-controlled iridium complex nanomaterials and dual quenchers NA and Au NPs provides a promising ECL platform.

The Neutrophil-to-Lymphocyte Ratio is Associated with the Requirement and the Duration of Invasive Mechanical Ventilation in Acute Respiratory Distress Syndrome Patients: A Retrospective Study.

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality and most ARDS patients require ventilatory support. Applying appropriate ventilation strategies based on patients' individual situations has a direct impact upon patients' outcome. The neutrophil-to-lymphocyte ratio (NLR) has been shown to predict the early requirement of invasive mechanical ventilation (IMV) in patients with coronavirus disease 2019 (COVID-19). Our study aimed to investigate the relationship between baseline NLR and IMV in ARDS. Methods: A retrospective study was performed on patients who were diagnosed with ARDS using the Berlin definition and admitted to the First Affiliated Hospital of Soochow University from 2017 to 2022. Clinical data within 24 h after the ARDS diagnosis were collected from the medical record system. Based on the ventilation strategies during hospitalization, patients were divided into three groups and their clinical characteristics were compared. Furthermore, logistic regression analysis was used to screen the independent risk factors for IMV. STROBE checklist was used for this manuscript. Results: 520 ARDS patients were included and the median NLR value in IMV group was significantly higher than that of other groups (P < 0.001). NLR was significantly associated with the requirement of IMV in ARDS patients (OR, 1.042; 95% CI, 1.025-1.060; P < 0.001), other independent risk factors included PaO2/FiO2, Hb, lactate, and use of vasoactive drugs (all P < 0.05). Moreover, we found that the duration of IMV was longer in patients with high NLR (8[IQR, 3-13], 10[IQR, 6-16], respectively, P=0.025). Conclusions: Our results revealed that high baseline NLR level was significantly correlated with an increased risk of IMV in patients with ARDS. Furthermore, higher NLR was associated with prolonged duration of IMV in patients with ARDS.

Recent advances of functional nucleic acid-based sensors for point-of-care detection of SARS-CoV-2.

This review focuses on critical scientific barriers that the field of point-of-care (POC) testing of SARS-CoV-2 is facing and possible solutions to overcome these barriers using functional nucleic acid (FNA)-based technology. Beyond the summary of recent advances in FNA-based sensors for COVID-19 diagnostics, our goal is to outline how FNA might serve to overcome the scientific barriers that currently available diagnostic approaches are suffering. The first introductory section on the operationalization of the COVID-19 pandemic in historical view and its clinical features contextualizes essential SARS-CoV-2-specific biomarkers. The second part highlights three major scientific barriers for POC COVID-19 diagnosis, that is, the lack of a general method for (1) designing receptors of SARS-CoV-2 variants; (2) improving sensitivity to overcome false negatives; and (3) signal readout in resource-limited settings. The subsequent part provides fundamental insights into FNA and technical tricks to successfully achieve effective COVID-19 diagnosis by using in vitro selection of FNA to overcome receptor design barriers, combining FNA with multiple DNA signal amplification strategies to improve sensitivity, and interfacing FNA with portable analyzers to overcome signal readout barriers. This review concludes with an overview of further opportunities and emerging applications for FNA-based sensors against COVID-19.

Hydrophobic Localized Enrichment of Co-reactants to Enhance Electrochemiluminescence of Conjugated Polymers for Detecting SARS-CoV-2 Nucleocapsid Proteins.

The enrichment of co-reactants is one of the keys to improving the sensitivity of electrochemiluminescence (ECL) detection. This work developed a novel hydrophobic localized enrichment strategy of co-reactants utilizing the inner hydrophobic cavity of ß-cyclodextrin (ß-CD). Pt nanoparticles (Pt NPs) were grown in situ on the coordination sites for metal ions of ß-CD to prepare the ß-CD-Pt nanocomposite, which could not only enrich co-reactant 3-(dibutylamino) propylamine (TDBA) highly efficiently through its hydrophobic cavity but also immobilize TDBA via the Pt-N bond. Meanwhile, the carboxyl-functionalized poly[2,5-dioctyl-1,4-phenylene] (PDP) polymer nanoparticles (PNPs) were developed as excellent ECL luminophores. With SARS-CoV-2 nucleocapsid protein (ncovNP) as a model protein, the TDBA-ß-CD-Pt nanocomposite combined PDP PNPs to construct a biosensor for ncovNP determination. The PDP PNPs were modified onto the surface of a glassy carbon electrode (GCE) to capture the first antibody (Ab1) and further capture antigen and secondary antibody complexes (TDBA-ß-CD-Pt@Ab2). The resultant biosensor with a sandwich structure achieved a highly sensitive detection of ncovNP with a detection limit of 22 fg/mL. TDBA-ß-CD-Pt shared with an inspiration in hydrophobic localized enrichment of co-reactants for improving the sensitivity of ECL detection. The luminophore PDP PNPs integrated TDBA-ß-CD-Pt to provide a promising and sensitive ECL platform, offering a new method for ncovNP detection.

Tetrahedron-Based Constitutional Dynamic Network for COVID-19 or Other Coronaviruses Diagnostics and Its Logic Gate Applications.

Considering the large-scale outbreak of the coronavirus, it is essential to develop a versatile sensing system for different coronaviruses diagnostics, such as COVID-19, severe acute respiratory syndrome-related coronavirus (SARS-CoV), and bat SARS-like coronavirus (Bat-SL-CoVZC45). In this work, a tetrahedron-based constitutional dynamic network was built as the sensing platform for coronavirus detection. Four different DNA probes were used to construct the tetrahedron structure. DNAzyme and the fluorophore modified substrate strand were used to generate different fluorescence signals, which can be used to distinguish different coronaviruses. The coronavirus biosensor shows a high sensitivity for COVID-19, Bat-SL-CoVZC45, and SARS-CoV detection, with detection limits of 2.5, 3.1, and 2.9 fM, respectively. Also, the platform is robust, and the possible interference from clinical samples was negligible. Using different coronaviruses as inputs, we have fabricated several concatenated logic gates, such as "AND-OR", "INHIBIT-AND", "AND-AND-AND", and "AND-INHIBIT". Importantly, our logic system can also be used to identify SARS-CoV-2 Delta and Lambda variants in the logic operations. Due to the unique advantages of high sensitivity and selectivity, multiple logic biocomputing capabilities, and multireadout mode, this flexible sensing system provides a versatile sensing strategy for intelligent diagnostics of different coronaviruses with low false-negative rates.

Dynamic changes of IgM and IgG antibodies in asymptomatic patients as an effective way to detect SARS-CoV-2 infection.

BACKGROUND: COVID-19 has become a global pandemic, and close contacts and asymptomatic patients are worthy of attention. METHODS: A total of 1844 people in close contacts with 76 COVID-19 patients were investigated, and nasopharyngeal swabs and venous blood were collected for centralized medical quarantine observation. Real-time fluorescence was used to detect SARS-CoV-2 nucleic acid in nasopharyngeal swabs of all close contacts, and the colloidal gold method was used to detect serum-specific antibodies. Levels of IgM- and IgG-specific antibodies were detected quantitatively through chemiluminescence from the first nucleic acid turned negative date (0 week) and on weekly intervals of ≤1 week, 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, and 6-7 weeks. RESULTS: The total positive rate of the colloidal gold method (88.5%, 23/26) was significantly higher (χ2  = 59.182, p < 0.001) than that of the healthy control group (2.0%, 1/50). There was significant difference in IgG concentration at different time points (0-7 weeks) after negative nucleic acid conversion (χ2  = 14.034, p = 0.029). Serum IgG levels were significantly higher at weekly time points of 4-5 weeks (Z = -2.399, p = 0.016), 5-6 weeks (Z = -2.049, p = 0.040), and 6-7 weeks (Z = -2.197, p = 0.028) compared with 1-2 weeks after negative nucleic acid conversion. However, there was no significant difference (χ2  = 4.936, p = 0.552) in IgM concentration between time points tested (0-7 weeks) after negative nucleic acid conversion. The positive rates of IgM and IgG in asymptomatic patients (χ2  = 84.660, p < 0.001) were significantly higher than those in the healthy control group (χ2  = 9.201, p = 0.002) within 7 weeks of negative nucleic acid conversion. CONCLUSIONS: The IgG concentration in asymptomatic cases remained at a high level after nucleic acid turned negative. Nucleic acid detection combined with IgM and IgG antibody detection is an effective way to screen asymptomatic infections.

SARS-CoV-2: Origin, Intermediate Host and Allergenicity Features and Hypotheses.

The goal of this study is to investigate the probable intermediate hosts and the allergenicity of the notorious virus SARS-CoV-2 to understand how this virus emerged. The phylogenetic analysis of the virus spike proteins indicates that SARS-CoV-2 falls into various small subclades that include a bat coronavirus RaTG13, suggesting bats as a likely natural origin. Refined alignment of the spike protein in NCBI found several fragments that are specific to SARS-CoV-2 and/or SARS-CoV are specific to Rattus norvegicus and/or Mus musculus, suggesting that rodents are the intermediate reservoir of SARS-CoV-2 and SARS-CoV. To evaluate the allergenicity values, the binding affinities of human leukocyte antigen (HLA) class I or II molecules with the spike proteins were calculated, and the results showed that both SARS-CoV-2 and SARS-CoV are predicted to bind to fourteen HLA class I and II molecules with super-high HLA allele-peptide affinities. The infection rate of individuals who have HLA alleles with very high binding affinities who might become infected and develop into refractory patients if there were no medical or non-medical interventions is about 7.36% and 4.78% of Chinese and Americans, respectively. Extremely high temperature and exceptionally low precipitation, the common climate factors between the outbreak sites of COVID-19 in Wuhan in 2019 and SARS in Guangdong in 2002, might have promoted coronavirus evolution into more virulent forms. Our hypothesis suggests that early immunization with an allergenically-engineered virus, in combination with continued surveillance of meteorological factors and viral mutations, may be one of the most powerful prophylactic modalities to fight this virus.

Indications for and contraindications of immune checkpoint inhibitors in cancer patients with COVID-19 vaccination.

The COVID-19 pandemic has lasted over 1 year and will not disappear in a short time. There is no specific remedy against the virus as yet. Vaccination is thus far one of the most important strategies for preventing COVID-19. Cancer patients with COVID-19 have a higher mortality because of immunosuppression. Immune checkpoint inhibitors (ICIs) are a novel anticancer strategy for blocking inhibitory pathways, which are related to the immune response. There is a question regarding whether COVID-19 vaccination and ICI treatment impact each other in cancer patients. This review explores both sides of the relationship between ICI treatment and COVID-19 vaccination and suggests good efficacy and safety of ICI treatment after COVID-19 vaccination as well as little impact on the virus protection and toxicity associated with COVID-19 vaccination during ICI treatment.

Lay abstract The COVID-19 pandemic has lasted over 1 year. Vaccination is a promising strategy for preventing COVID-19. Cancer patients are prone to infection with COVID-19, and these patients have high mortality. Immune checkpoint inhibitors (ICIs) are a novel anticancer strategy. Whether COVID-19 vaccination and ICI treatment impact each other in cancer patients remains unknown. This review explores both sides of the relationship between ICI treatment and COVID-19 vaccination and suggests good efficacy and safety of ICI treatment after COVID-19 vaccination as well as little impact on the virus protection and toxicity associated with COVID-19 vaccination during ICI treatment.

An angiotensin-converting enzyme-2-derived heptapeptide GK-7 for SARS-CoV-2 spike blockade.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global concern and necessitates efficient drug antagonists. Angiotensin-converting enzyme-2 (ACE2) is the main receptor of SARS-CoV-2 spike 1 (S1), which mediates viral invasion into host cells. Herein, we designed and prepared short peptide inhibitors containing 4-6 critical residues of ACE2 that contribute to the interaction with SARS-CoV-2 S1. Among the candidates, a peptide termed GK-7 (GKGDFRI), which was designed by extracting residues ranging from Gly353 to Ile359 in the ligand-binding domain of ACE2, exhibited the highest binding affinity (25.1 nM) with the SARS-CoV-2 spike receptor-binding domain (RBD). GK-7 bound to the RBD and decreased SARS-CoV-2 S1 attachment to A549 human alveolar epithelial cells. Owing to spike blockade, GK-7 inhibited SARS-CoV-2 spike pseudovirion infection in a dose-dependent manner, with a half-maximal inhibitory concentration of 2.96 µg/mL. Inspiringly, pulmonary delivery of GK-7 by intranasal administration did not result in toxicity in mice. This study revealed an easy-to-produce peptide inhibitor for SARS-CoV-2 spike blockade, thus providing a promising candidate for COVID-19 treatment.

Monocyte-to-lymphocyte ratio is associated with 28-day mortality in patients with acute respiratory distress syndrome: a retrospective study.

BACKGROUND: Systemic inflammation relates to the initiation and progression of acute respiratory distress syndrome (ARDS). Neutrophil-to-lymphocyte ratio (NLR) and red blood cell distribution width (RDW)/albumin ratio have been reported to be predictive prognostic biomarkers in ARDS patients. However, the role of monocyte-to-lymphocyte ratio (MLR) as a prognostic inflammatory biomarker in a variety of diseases is rarely mentioned in ARDS. In this study, we explored the relationship between MLR and disease severity in ARDS patients and compared it with other indicators associated with 28-day mortality in patients with ARDS. METHODS: We retrospectively included 268 patients who fulfilled the Berlin definition of ARDS and were admitted to a single institute from 2016 to 2020. Clinical characteristics and experimental test data were collected from medical records within 24 h after the ARDS diagnosis. MLR, NLR, and RDW/albumin ratio levels were calculated. The primary clinical outcome was 28-day mortality. Logistic regression analysis was used to illustrate the relationship between indicators and 28-day mortality. Receiver operating characteristic (ROC) curve was used to evaluate the area under the curve (AUC), and propensity score matching (PSM) was employed to validate our findings. RESULTS: The median MLR values were higher for non-survivors than for survivors before and after matching (P<0.001, P=0.001, respectively). MLR values were significantly associated with 28-day mortality (OR 2.956; 95% CI 1.873-4.665; P<0.001). MLR and NLR indicators were combined for predictive efficacy analysis, and its AUC reached 0.750. There was a significant increase in 28-day mortality depending on the increasing MLR level: low MLR group 38 (20.4%), high MLR group 47 (57.3%) (P<0.001). CONCLUSIONS: Higher MLR values were associated with 28-day mortality in patients with ARDS. Further investigation is required to verify this relationship with prospectively collected data.

Determination of human COVID-19 total antibodies in serum using a time-resolved fluorescence immunoassay.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading rapidly around the world. Antibody detection plays an important role in the diagnosis of COVID-19. Here, we established a new time-resolved fluorescence immunoassay (TRFIA) to determine COVID-19 total antibodies. A double-antigen sandwich TRFIA was optimized and established: recombinant nucleocapsid phosphoprotein (N protein) and spike protein (S protein) of COVID-19 immobilized on 96-well plates captured human COVID-19 antibodies and then banded together with the N/S proteins labeled with europium(III) (Eu3+ ) chelates, and finally, time-resolved fluorometry was used to measure the fluorescence values. We successfully established a TRFIA method for the detection of human COVID-19 total antibodies, and the cutoff value was 2.02. There was no cross-reactivity with the negative reference of the National Reference Panel for IgM and IgG antibodies to COVID-19. The CV of the precision assay was 3.19%, and the assay could be stored stably for 15 days at 37°C. Compared with that of the colloidal gold method and chemiluminescence method, the sensitivity of the TRFIA method was higher, and the false positive/negative rate was lower. This established TRFIA has high sensitivity, accuracy, and specificity, which indicates that this method provides a new detection method for the high-throughput routine diagnosis of COVID-19.

Translating daily COVID-19 screening into a simple glucose test: a proof of concept study.

Home testing is an attractive emerging strategy to combat the COVID-19 pandemic and prevent overloading of healthcare resources through at-home isolation, screening and monitoring of symptoms. However, current diagnostic technologies of SARS-CoV-2 still suffer from some drawbacks because of the tradeoffs between sensitivity, usability and costs, making the test unaffordable to most users at home. To address these limitations, taking advantage of clustered regularly interspaced short palindromic repeats (CRISPRs) and a portable glucose meter (PGM), we present a proof-of-concept demonstration of a target-responsive CRISPR-PGM system for translating SARS-CoV-2 detection into a glucose test. Using this system, a specific N gene, N protein, and pseudo-viruses of SARS-CoV-2 have been detected quantitatively with a PGM. Given the facile integration of various bioreceptors into the CRISPR-PGM system, the proposed method provides a starting point to provide patients with a single-device solution that can quantitatively monitor multiple COVID-19 biomarkers at home.

An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip.

CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction. Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection. Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.

Correction: Health Care Cybersecurity Challenges and Solutions Under the Climate of COVID-19: Scoping Review.

[This corrects the article DOI: 10.2196/21747.].

Health Care Cybersecurity Challenges and Solutions Under the Climate of COVID-19: Scoping Review.

BACKGROUND: COVID-19 has challenged the resilience of the health care information system, which has affected our ability to achieve the global goal of health and well-being. The pandemic has resulted in a number of recent cyberattacks on hospitals, pharmaceutical companies, the US Department of Health and Human Services, the World Health Organization and its partners, and others. OBJECTIVE: The aim of this review was to identify key cybersecurity challenges, solutions adapted by the health sector, and areas of improvement needed to counteract the recent increases in cyberattacks (eg, phishing campaigns and ransomware attacks), which have been used by attackers to exploit vulnerabilities in technology and people introduced through changes to working practices in response to the COVID-19 pandemic. METHODS: A scoping review was conducted by searching two major scientific databases (PubMed and Scopus) using the search formula "(covid OR healthcare) AND cybersecurity." Reports, news articles, and industry white papers were also included if they were related directly to previously published works, or if they were the only available sources at the time of writing. Only articles in English published in the last decade were included (ie, 2011-2020) in order to focus on current issues, challenges, and solutions. RESULTS: We identified 9 main challenges in cybersecurity, 11 key solutions that health care organizations adapted to address these challenges, and 4 key areas that need to be strengthened in terms of cybersecurity capacity in the health sector. We also found that the most prominent and significant methods of cyberattacks that occurred during the pandemic were related to phishing, ransomware, distributed denial-of-service attacks, and malware. CONCLUSIONS: This scoping review identified the most impactful methods of cyberattacks that targeted the health sector during the COVID-19 pandemic, as well as the challenges in cybersecurity, solutions, and areas in need of improvement. We provided useful insights to the health sector on cybersecurity issues during the COVID-19 pandemic as well as other epidemics or pandemics that may materialize in the future.

The development and kinetics of functional antibody-dependent cell-mediated cytotoxicity (ADCC) to SARS-CoV-2 spike protein.

Since the COVID-19 pandemic, functional non-neutralizing antibody responses to SARS-CoV-2, including antibody-dependent cell-mediated cytotoxicity (ADCC), are poorly understood. We developed an ADCC assay utilizing a stably transfected, dual-reporter target cell line with inducible expression of a SARS-CoV-2 spike protein on the cell surface. Using this assay, we analyzed 61 convalescent serum samples from adults with PCR-confirmed COVID-19 and 15 samples from healthy uninfected controls. We found that 56 of 61 convalescent serum samples induced ADCC killing of SARS-CoV-2 S target cells, whereas none of the 15 healthy controls had detectable ADCC. We then found a modest decline in ADCC titer over a median 3-month follow-up in 21 patients who had serial samples available for analysis. We confirmed that the antibody-dependent target cell lysis was mediated primarily via the NK FcγRIIIa receptor (CD16). This ADCC assay had high sensitivity and specificity for detecting serologic immune responses to SARS-CoV-2.