The Luciferase Immunoprecipitation System (LIPS) Targeting the Spike Protein of SARS-CoV-2 Is More Accurate than Nucleoprotein-Based LIPS and ELISAs for Mink Serology

Since anthropo-zoonotic outbreaks of SARS-CoV-2 have been reported in mink farms, it is important to monitor the seroprevalence within this population. To investigate the accuracy of nucleo (N) or spike (S) protein-based assays to detect anti-SARS-CoV-2 antibodies in animal serum, we compared four assays, two commercial N-based enzyme-linked immunosorbent assays (ELISA) validated for animal sera and two luciferase immunoprecipitation systems (LIPS-N and LIPS-S), to the reference standard plaque reduction neutralisation test (PRNT). Samples included in this study were derived from a naturally infected mink population. For the first time in this study, serum samples of mink were collected over a 307-day period, at different time points, thus providing an overview of performances of four different rapid serological tests over time. The assays were compared by performing a correlation analysis using R2, Spearman's rank-order correlation coefficient, and Fleiss' and Cohen's kappa for analysis of agreement to PRNT, and an UpSet chart was created to visualize the number of shared positive samples between assays. Cohen's kappa test on categorical data showed an excellent agreement between PRNT and LIPS-S, while agreements between PRNT and N-based methods decreased from fair for LIPS-N to poor agreements for the ELISA kits. In addition, LIPS-S revealed the highest number of true-positive SARS-CoV-2 samples compared to N-based methods. Despite an excellent agreement between LIPS-S and PRNT, a weak correlation was detectable between PRNT titres and relative light units. This study shows that the LIPS-S assay can be used for serological surveillance within a naturally exposed mink population, while N-based serological assays are less accurate providing a higher number of false-negative results, especially at a later stage of infection, thus indicating that N antibodies are less persistent in naturally exposed mink. Our findings provide crucial information for veterinarians and competent authorities involved in surveillance and outbreak investigation in wild and farmed minks. [ FROM AUTHOR] Copyright of Transboundary & Emerging Diseases is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Increase in newly diagnosed type 1 diabetes and serological evidence of recent SARS-CoV-2 infection: Is there a connection?

Several studies have investigated the correlation between the COVID-19 pandemic and the onset of type 1 diabetes (T1D) in children, reporting an increased incidence of T1D and severe diabetic ketoacidosis (DKA). This study aimed to investigate the infection by SARS-CoV-2 in children with newly-diagnosed T1D to explore a possible link between SARS-CoV-2 infection, T1D and DKA. Thirty-nine children with a T1D new onset between October 15, 2020, and April 15, 2021, were enrolled. SARS-CoV-2 infection was investigated through a polymerase chain reaction on the nasal swab, dosage of specific antibodies, and an anamnestic question form. Nine (23%) of them had antibodies directed toward SARS-CoV-2, and five (12%) had a history of recent SARS-CoV-2 infection in themselves or in their family. No molecular swabs were positive. Compared to the general pediatric population, the overall incidence of COVID-19 was 5.6 times higher in the T1D patients' group (p < 0.00001). Referring only to the cases in the metropolitan area, we find a net increase in the incidence of T1D compared to the 5 years preceding our study, by 50% compared to the same months in 2016/2017 and 2017/2018, by 69% compared to 2018/2019 and by 77% compared to 2019/2020. The same trend was observed regarding DKA cases. The attributable risk of the pandemic cohort compared to the previous year is 44%. The abnormal disproportion of SARS-CoV-2 infection between children with T1D and the pediatric reference population, with a ratio of 5.6, appears to support the causative role of SARS-CoV-2 in triggering the immune response underlying diabetes, as often described for other viral infections. The difficulty accessing care services during the pandemic, with a consequent diagnosis delay, does not justify the increase in observed T1D cases, which could to be directly linked to the pandemic. The acceleration of the immune process provoked by SARS-CoV-2 may play a suggestive role in the development of T1D with DKA. Multicenter studies are needed to deepen and fully understand the pathophysiological link between SARS-CoV-2 and the onset of T1D in children.

Pulmonary fibrosis in a dog as a sequela of infection with Severe Acute Respiratory Syndrome Coronavirus 2? A case report.

BACKGROUND: Interstitial lung disease is a heterogeneous group of conditions characterized by severe radiographic changes and clinicopathological findings. However, in the vast majority of cases, the cause remains unknown. CASE DESCRIPTION: In the present study, we reported the clinical case of a 3 years old female Bull Terrier presented in October 2020 to the Advanced Diagnostic Imaging Department of the Turin Veterinary Teaching Hospital with a progressive pulmonary illness characterized by dyspnea, exercise intolerance, and a diffuse and severe pulmonary interstitial pattern at imaging investigations. Considering the clinical findings, the dog was included in a serological survey for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in companion animals, showing positive results. Due to the further clinical worsening, the owners opted for euthanasia. At necroscopy, dog showed severe and chronic bronchopneumonia compatible with a Canine Idiopathic Pulmonary Fibrosis and with serological features linked to a SARS-CoV-2 infection. CONCLUSIONS: The comparison of these lesions with those reported in humans affected by Coronavirus Disease 2019 (COVID-19) supports the hypothesis that these findings may be attributable to the post-acute sequelae of SARS-CoV-2 infection in a dog with breed predisposition to Canine Idiopathic Pulmonary Fibrosis (CIPF), although direct evidence of SARS-CoV-2 by molecular or antigenic approaches remained unsolved.

Bacterial ligands as flexible and sensitive detectors in rapid tests for antibodies to SARS-CoV-2.

Lateral flow immunoassay (LFIA) is widely employed as point-of-care tests (POCT) for the diagnosis of infectious diseases. The accuracy of LFIA largely depends on the quality of the immunoreagents used. Typical LFIAs to reveal the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) employ anti-human immunoglobulin (hIG) antibodies and recombinant viral antigens, which usually are unstable and poorly soluble. Broad selective bacterial proteins, such as Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) can be considered alternatives to anti-hIG to increase versatility and sensitivity of serological LFIAs because of their high binding capacity, interspecies reactivity, and robustness. We developed two colorimetric LFA devices including SpA and SpG linked to gold nanoparticles (GNP) as detectors and explored the use of a specific, stable, and soluble immunodominant fraction of the nucleocapsid protein from SARS-CoV-2 as the capturing agent. The optimal amount of SpA-GNP and SpG-GNP conjugates and the protein-to-GNP ratios were defined through a full factorial experimental design to maximize the diagnostic sensitivity of the LFIAs. The new LFA devices were applied to analyze 105 human serum samples (69 positive and 36 negatives according to reference molecular diagnostic methods). The results showed higher sensitivity (89.9%, 95% CI 82.7-97.0) and selectivity (91.7%, 82.6-100) for the SpA-based compared to the SpG-based LFA. In addition, 18 serum samples from cats and dogs living with COVID-19 patients were analyzed and 14 showed detectable levels of anti-SARS-CoV-2 antibodies, thus illustrating the flexibility of the SpA- and SpG-based LFAs.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

Long-term persistence of neutralizing SARS-CoV-2 antibodies in pets.

We monitored the severe acute respiratory syndrome coronavirus 2 antibody response in seven dogs and two cats by using two multispecies ELISA tests, plaque reduction neutralisation test and virus neutralization. SARS-CoV-2 neutralizing antibodies in pets persisted up to 10 months since the first positive testing, thus replicating observations in COVID-19 human patients.

Possible Human-to-Dog Transmission of SARS-CoV-2, Italy, 2020.

We detected severe acute respiratory syndrome coronavirus 2 in an otherwise healthy poodle living with 4 family members who had coronavirus disease. We observed antibodies in serum samples taken from the dog, indicating seroconversion. Full-length genome sequencing showed that the canine and human viruses were identical, suggesting human-to-animal transmission.

Cross-Sectional Serosurvey of Companion Animals Housed with SARS-CoV-2-Infected Owners, Italy.

We conducted a serologic survey among dogs and cats in Italy to detect antibodies against severe acute respiratory syndrome virus 2 (SARS-CoV-2). We found that SARS-CoV-2 seroprevalence was higher among cats (16.2%) than dogs (2.3%). In addition, seroprevalence was higher among animals living in close contact with SARS-CoV-2-positive owners.

A multi-target lateral flow immunoassay enabling the specific and sensitive detection of total antibodies to SARS COV-2.

A rapid test for detecting total immunoglobulins directed towards the nucleocapsid protein (N) of severe acute syndrome coronavirus 2 (SARS CoV-2) was developed, based on a multi-target lateral flow immunoassay comprising two test lines. Both test lines bound to several classes of immunoglobulins (G, M, and A). Specific anti-SARS immunoglobulins were revealed by a colorimetric probe formed by N and gold nanoparticles. Targeting the total antibodies response to infection enabled achieving 100% diagnostic specificity (95.75-100, C.I. 95%, n = 85 healthy and with other infections individuals) and 94.6% sensitivity (84.9-98.9, C.I. 95%, n = 62 SARS CoV-2 infected subjects) as early as 7 days post confirmation of positivity. Agreeing results with a reference serological ELISA were achieved, except for the earlier detection capability of the rapid test. Follow up of the three seroconverting patients endorsed the hypothesis of the random rise of the different immunoglobulins and strengthened the 'total antibodies' approach for the trustworthy detection of serological response to SARS CoV-2 infection.

Dual lateral flow optical/chemiluminescence immunosensors for the rapid detection of salivary and serum IgA in patients with COVID-19 disease.

To accurately diagnose COVID-19 infection and its time-dependent progression, the rapid, sensitive, and noninvasive determination of immunoglobulins A specific to SARS-CoV-2 (IgA) in saliva and serum is needed to complement tests that detect immunoglobulins G and M. We have developed a dual optical/chemiluminescence format of a lateral flow immunoassay (LFIA) immunosensor for IgA in serum and saliva. A recombinant nucleocapsid antigen specifically captures SARS-CoV-2 antibodies in patient specimens. A labelled anti-human IgA reveals the bound IgA fraction. A dual colorimetric and chemiluminescence detection enables the affordable and ultrasensitive determination of IgA to SARS-CoV-2. Specifically, a simple smartphone-camera-based device measures the colour signal provided by nanogold-labelled anti-human IgA. For the ultrasensitive chemiluminescence transduction, we used a contact imaging portable device based on cooled CCD, and measured the light signal resulting from the reaction of the HRP-labelled anti-human IgA with a H2O2/luminol/enhancers substrate. A total of 25 serum and 9 saliva samples from infected and/or recovered individuals were analysed by the colorimetric LFIA, which was sensitive and reproducible enough for the semi-quantification of IgA in subjects with a strong serological response and in the early stage of COVID-19 infection. Switching to CL detection, the same immunosensor exhibited higher detection capability, revealing the presence of salivary IgA in infected individuals. For the patients included in the study (n = 4), the level of salivary IgA correlated with the time elapsed from diagnosis and with the severity of the disease. This IgA-LFIA immunosensor could be useful for noninvasively monitoring early immune responses to COVID-19 and for investigating the diagnostic/prognostic utility of salivary IgA in the context of large-scale screening to assess the efficacy of SARS-CoV-2 vaccines.

Sequelae of COVID-19 in Hospitalized Children: A 4-Months Follow-Up.

Little is known about the sequelae of SARS-CoV-2 infection in children. In a COVID-19 dedicated clinic, we followed-up for 4 months 25 children previously hospitalized for COVID-19, performing clinical, laboratory, and lung ultrasound evaluation. Mid-term sequelae were rarely observed in our COVID-19 children's cohort.

SARS-CoV-2-Induced Kawasaki-Like Hyperinflammatory Syndrome: A Novel COVID Phenotype in Children.

We describe 2 children with persistent fever and profuse diarrhea who developed signs of mucocutaneous involvement (conjunctivitis, fissured lips, skin rash, erythema, and edema of the hands and feet). Blood tests revealed elevated markers of inflammation, lymphopenia, thrombocytopenia, and complement consumption. Afterward, diffuse edema with hypoalbuminemia appeared in the context of a capillary leak syndrome. In both patients, repeated nasal swabs were negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but each patient had high titers of immunoglobulin G and immunoglobulin M against the SARS-CoV-2 virus. The negative PCR results in the presence of immunoglobulin M and immunoglobulin G suggested that the inflammatory response developed in the late phase of viral infection, when SARS-CoV-2 was not detectable in the upper airway. In this report, we describe patients with what we propose to name as SARS-CoV-2-induced Kawasaki-like hyperinflammatory syndrome. SARS-CoV-2-induced Kawasaki-like hyperinflammatory syndrome seems to be caused by a delayed response to SARS-CoV-2. It resembles Kawasaki disease complicated by macrophage activation syndrome, although it has peculiar features, such as prodromal diarrhea, capillary leak syndrome, and myocardial dysfunction. Intravenous corticosteroid treatment appears to be helpful.