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1.
Pathogens ; 11(10)2022 Sep 25.
Article in English | MEDLINE | ID: covidwho-2043896

ABSTRACT

Mucosal immunity, including secretory IgA (sIgA), plays an important role in the early defence against SARS-CoV-2 infection. However, a comprehensive evaluation of the local immune response in tears in relation to blood antibody reservoirs has not yet been conducted. A total of 179 symptomatic laboratory-confirmed COVID-19 patients were included in this single-centre study. Conjunctival swabs were analysed by a reverse transcription polymerase chain reaction for the detection of SARS-CoV-2 RNA. In parallel, tear samples collected by Schirmer test strips and plasma samples were analysed by ELISA to detect anti-S1 IgA levels. The concentrations of selected inflammatory cytokines in tears were determined by a magnetic bead assay. Anti-SARS-CoV-2 sIgA was present in the tears of 81 (45.25%) confirmed COVID-19 patients, and the tear IgA levels were correlated with the plasma IgA levels (Rs = +0.29, p = 0.0003). SARS-CoV-2 RNA in the conjunctival sac was identified in 18 COVID-19 patients (10%). Positive correlations between the tear IgA level and the concentrations of several cytokines TNF-α (Rs = +0.23, p = 0.002), IL-1ß (Rs = +0.25, p < 0.001), IL-2 (Rs = +0.20, p = 0.007), IL-4 (Rs = +0.16, p = 0.04), IL-5 (Rs = +0.36, p < 0.001), IL-6 (Rs = +0.32, p < 0.001), IL-8 (Rs = +0.31, p < 0.001), VEGF (Rs = +0.25, p < 0.001) and GM-CSF (Rs = +0.27, p < 0.001) were also found. Quantitative tear film-based sIgA could potentially serve as a rapid and easily accessible biomarker of external mucosal immunity to SARS-CoV-2. The concentration of sIgA is directly related to individual host immune responses to SARS-CoV-2 infection.

2.
Methods Mol Biol ; 2511: 257-271, 2022.
Article in English | MEDLINE | ID: covidwho-1941381

ABSTRACT

Serological assays have been a useful tool for detection of antibodies to SARS-CoV-2 during the COVID-19 pandemic. These assays are used for epidemiology and serosurveillance to monitor the progression of the pandemic, to identify and differentiate individuals who have developed antibodies from natural infection versus vaccine-induced immunity, and to identify potential donors of convalescent plasma for therapeutic purposes. In this chapter, we describe a commercially available bead-based serological assay, the Luminex® xMAP® SARS-CoV-2 Multi-Antigen IgG Assay, that detects and identifies antibodies against three SARS-CoV-2 antigens. In addition to the assay principle and workflow, we describe modifications that may be used to evaluate alternate sample types, antibody isotypes, and potential neutralizing antibody responses.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/diagnosis , COVID-19/therapy , Humans , Immunization, Passive , Immunoglobulin G , Pandemics , Sensitivity and Specificity
3.
Methods Mol Biol ; 2511: 133-147, 2022.
Article in English | MEDLINE | ID: covidwho-1941372

ABSTRACT

SARS-CoV-2 causes generally mild symptoms, with approximately 10-20% of cases progressing to severe disease. The pathophysiologic mechanisms by which SARS-CoV-2 causes severe disease are largely unknown. Data have indicated the involvement of different immunogenetic markers such as HLA, T, and B cells, to be associated with disease outcome. This has led to interest in these genes as potential biomarkers of SARS-CoV-2 susceptibility and for predicting prognosis and response to vaccines and other therapeutic strategies. In this chapter, we discussed outline protocols for characterizing these potential biomarkers and methods for identifying SARS-CoV-2 biomarkers using the Luminex® 100/200 technology and next-generation sequencing.


Subject(s)
COVID-19 , SARS-CoV-2 , Biomarkers , COVID-19/diagnosis , High-Throughput Nucleotide Sequencing , Humans , Immunogenetics , SARS-CoV-2/genetics
4.
Microbiol Spectr ; 10(4): e0124822, 2022 08 31.
Article in English | MEDLINE | ID: covidwho-1909613

ABSTRACT

This study compares three of the most inclusive and widely used panels for respiratory syndromic testing in the United States, namely, Luminex NxTAG Respiratory Pathogen Panel (RPP), BioFire FilmArray Respiratory Panel (RP), and GenMark eSensor Respiratory Viral Panel (RVP). We compared the three assays using nasopharyngeal swab samples (n = 350) collected from symptomatic patients (n = 329) in the pre-coronavirus disease 2019 (COVID-19) era. There was no significant difference in the overall accuracies of BioFire and Luminex assays (P = 0.6171); however, significant differences were found between BioFire and GenMark (P = 0.0003) and between GenMark and Luminex (P = 0.0009). The positive percent agreement of the BioFire RP assay was 94.1%, compared to 97.3% for GenMark RVP and 96.5% for Luminex RPP. Overall negative percent agreement values were high for all three assays, i.e., 99.9% for BioFire and Luminex and 99.5% for GenMark. The three assays were equivalent for adenovirus, human metapneumovirus, influenza A, and respiratory syncytial virus. Increased false-positive results were seen with BioFire for the endemic coronaviruses and with GenMark for influenza B and the parainfluenza viruses. IMPORTANCE Clinical laboratories have multiple choices when it is comes to syndromic respiratory testing. Here, the Luminex NxTAG RPP is compared to the BioFire FilmArray RP and GenMark eSensor RVP for overall and per-target accuracy. As new tests come to market, it is important to ascertain their performance characteristics, compared to other widely used in vitro diagnostic products.


Subject(s)
COVID-19 , Influenza, Human , Respiratory Tract Infections , Viruses , Humans , Molecular Diagnostic Techniques/methods , Respiratory Tract Infections/diagnosis , Viruses/genetics
5.
Diagnostics (Basel) ; 12(5)2022 May 07.
Article in English | MEDLINE | ID: covidwho-1862747

ABSTRACT

Interleukin-6 (IL-6) has been linked to several life-threatening disease processes. Developing a point-of-care testing platform for the immediate and accurate detection of IL-6 concentrations could present a valuable tool for improving clinical management in patients with IL-6-mediated diseases. Drawing on an available biobank of samples from 35 patients hospitalized with COVID-19, a novel quantum-magnetic sensing platform is used to determine plasma IL-6 concentrations. A strong correlation was observed between IL-6 levels measured by QDTI10x and the Luminex assay (r = 0.70, p-value < 0.001) and between QDTI80x and Luminex (r = 0.82, p-value < 0.001). To validate the non-inferiority of QDTI to Luminex in terms of the accuracy of IL-6 measurement, two clinical parameters-the need for intensive care unit admission and the need for mechanical intubation-were chosen. IL-6 concentrations measured by the two assays were compared with respect to these clinical outcomes. Results demonstrated a comparative predictive performance between the two assays with a significant correlation coefficient. Conclusion: In short, the QDTI assay holds promise for implementation as a potential tool for rapid clinical decision in patients with IL-6-mediated diseases. It could also reduce healthcare costs and enable the development of future various biomolecule point-of-care tests for different clinical scenarios.

6.
J Clin Med ; 11(9)2022 May 08.
Article in English | MEDLINE | ID: covidwho-1847361

ABSTRACT

BACKGROUND: This study investigated the presence and duration of ophthalmic symptoms in the early phase of COVID-19 to assess the corresponding local immune response on the ocular surface. METHODS: The study included data from 180 COVID-19 patients and 160 age-matched healthy controls. The main finding was the occurrence of ophthalmological manifestations at the time of admission to the hospital and during the preceding 7 days. Tear film concentrations of TNF-α, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p70, GM-CSF, and IFN-γ were determined by a magnetic bead assay. RESULTS: Among the COVID-19 patients, 12.64% had at least one ocular symptom at the time of admission, and 24.14% had symptoms within the preceding 7 days (p < 0.001 vs. controls). We found that the COVID-19 patients complained more frequently about eye tearing (p = 0.04) and eye pain (p = 0.01) than controls. A multivariate analysis of the patients and controls adjusted for age and sex revealed that COVID-19 was an independent factor associated with higher VEGF and IL-10 tear film concentrations (ß = +0.13, p = 0.047 and ß = +0.34, p < 0.001, respectively) and lower IL-1ß, IL-8, and GM-CSF levels (ß = -0.25, p < 0.001; ß = -0.18, p = 0.004; and ß = -0.82, p = 0.0 respectively). CONCLUSIONS: SARS-CoV-2 does not attract a strong local response of the conjunctival immune system; therefore, ophthalmic symptoms may not constitute a substantial element in the clinical picture of novel COVID-19 infection.

7.
J Clin Lab Anal ; 36(5): e24363, 2022 May.
Article in English | MEDLINE | ID: covidwho-1763247

ABSTRACT

BACKGROUND: Serological tests can be used to detect antibodies in the serum of subject's after SARS-CoV-2 infection and vaccination. Currently, variability in antibody titers and the availability of a multiplicity of serological tests have made it necessary to highlight their appropriateness and limitations in various diagnostic settings. METHODS: This study is part of Covidiagnostix, a multicenter project aimed at the assessment of the health technology used in SARS-CoV-2 serological tests. Based on data gained from the analysis of over 5000 subjects, a selected number of serum samples, representative of different diagnostic settings, were analyzed first by qualitative immunoassays (IgA, M, and G MILLIPLEX® SARS-CoV-2 tests based on Luminex® ) to define the immunoglobulins serum composition and subsequently by four serological diagnostic tests (Elecsys Anti-SARS-CoV-2 and Elecsys Anti-SARS-CoV-2 S by Roche, SARS-CoV-2 IgG by Siemens Healthcare, and CHORUS SARS-CoV-2 "NEUTRALIZING" Ab by DIESSE). The first WHO International Standard for SARS-CoV-2 was also analyzed using the same methods. RESULTS: This study evaluated the antibody content and titer of the WHO Standard and serum of subjects with/without previous infection and before/after vaccination for SARS-CoV-2. CONCLUSION: The definition of antibodies in the WHO standard and the analysis of serum samples allowed for the identification of the appropriateness of serological tests in each diagnostic setting, increasing the effectiveness of the resulting laboratory data. Furthermore, we found that it would be optimal to produce new international standards against the S1 domain and RBD of the SARS-CoV-2 spike protein for a more effective serological monitoring of vaccination.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/diagnosis , Humans , Serologic Tests , Spike Glycoprotein, Coronavirus
8.
J Transl Med ; 20(1): 134, 2022 03 18.
Article in English | MEDLINE | ID: covidwho-1745446

ABSTRACT

BACKGROUND: A thorough understanding of a patient's inflammatory response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is crucial to discerning the associated, underlying immunological processes and to the selection and implementation of treatment strategies. Defining peripheral blood biomarkers relevant to SARS-CoV-2 infection is fundamental to detecting and monitoring this systemic disease. This safety-focused study aims to monitor and characterize the immune response to SARS-CoV-2 infection via analysis of peripheral blood and nasopharyngeal swab samples obtained from patients hospitalized with Coronavirus disease 2019 (COVID-19), in the presence or absence of bamlanivimab treatment. METHODS: 23 patients hospitalized with COVID-19 were randomized to receive a single dose of the neutralizing monoclonal antibody, bamlanivimab (700 mg, 2800 mg or 7000 mg) or placebo, at study initiation (Clinical Trial; NCT04411628). Serum samples and nasopharyngeal swabs were collected at multiple time points over 1 month. A Proximity Extension Array was used to detect inflammatory profiles from protein biomarkers in the serum of hospitalized COVID-19 patients relative to age/sex-matched healthy controls. RNA sequencing was performed on nasopharyngeal swabs. A Luminex serology assay and Elecsys® Anti-SARS-CoV-2 immunoassay were used to detect endogenous antibody formation and to monitor seroconversion in each cohort over time. A mixed model for repeated measures approach was used to analyze changes in serology and serum proteins over time. RESULTS: Levels of IL-6, CXCL10, CXCL11, IFNγ and MCP-3 were > fourfold higher in the serum of patients with COVID-19 versus healthy controls and linked with observations of inflammatory and viral-induced interferon response genes detected in nasopharyngeal swab samples from the same patients. While IgA and IgM titers peaked around 7 days post-dose, IgG titers remained high, even after 28 days. Changes in biomarkers over time were not significantly different between the bamlanivimab and placebo groups. CONCLUSIONS: Similarities observed between nasopharyngeal gene expression patterns and peripheral blood biomarker profiles reveal a connection between the circulation and processes in the nasopharyngeal cavity, reinforcing the potential utility of systemic blood biomarker profiling for therapeutic monitoring of patient response. Serological antibody responses in patients correlated closely with reductions in the COVID-19 inflammatory protein biomarker signature. Bamlanivimab did not affect the biomarker dynamics in this hospitalized patient population.


Subject(s)
COVID-19 , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Neutralizing , Antibodies, Viral , Biomarkers , COVID-19/drug therapy , Gene Expression , Humans , Nasopharynx , SARS-CoV-2
9.
Emerg Infect Dis ; 28(4): 878-880, 2022 04.
Article in English | MEDLINE | ID: covidwho-1708502

ABSTRACT

To determine when severe acute respiratory syndrome coronavirus 2 arrived in Congo, we retrospectively antibody tested 937 blood samples collected during September 2019-February 2020. Seropositivity significantly increased from 1% in December 2019 to 5.3% in February 2020, before the first officially reported case in March 2020, suggesting unexpected early virus circulation.


Subject(s)
COVID-19 , SARS-CoV-2 , Congo/epidemiology , Humans , Retrospective Studies
10.
Vet Sci ; 9(2)2022 Jan 27.
Article in English | MEDLINE | ID: covidwho-1674883

ABSTRACT

We tested 144 pet rabbits sampled in France between November 2020 and June 2021 for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by microsphere immunoassay. We reported the first evidence of a natural SARS-CoV-2 infection in rabbits with a low observed seroprevalence between 0.7% and 1.4%.

11.
Methods Protoc ; 4(4)2021 Oct 09.
Article in English | MEDLINE | ID: covidwho-1463776

ABSTRACT

Serology tests are extremely useful for assessing whether a person has been infected with a pathogen. Since the onset of the COVID-19 pandemic, measurement of anti-SARS-CoV-2-specific antibodies has been considered an essential tool in identifying seropositive individuals and thereby understanding the extent of transmission in communities. The Luminex system is a bead-based technology that has the capacity to assess multiple antigens simultaneously using very low sample volumes and is ideal for high-throughput studies. We have adapted this technology to develop a COVID-19 multi-antigen serological assay. This protocol described here carefully outlines recommended steps to optimize and establish this method for COVID-19-specific antibody measurement in plasma and in saliva. However, the protocol can easily be customized and thus the assay is broadly applicable to measure antibodies to other pathogens.

12.
J Virol Methods ; 297: 114228, 2021 11.
Article in English | MEDLINE | ID: covidwho-1294030

ABSTRACT

High-throughput serological tests that can detect neutralizing antibodies against SARS-CoV-2 are desirable for serosurveillance and vaccine efficacy evaluation. Although the conventional neutralization test (cVNT) remains the gold standard to confirm the presence of neutralizing antibodies in sera, the test is too labour-intensive for massive screening programs and less reproducible as live virus and cell culture is involved. Here, we performed an independent evaluation of a commercially available surrogate virus neutralization test (sVNT, GenScript cPass™) that can be done without biosafety level 3 containment in less than 2 h. When using the cVNT and a Luminex multiplex immunoassay (MIA) as reference, the sVNT obtained a sensitivity of 94 % (CI 90-96 %) on a panel of 317 immune sera that were obtained from hospitalized and mild COVID-19 cases from Belgium and a sensitivity of 88 % (CI 81-93 %) on a panel of 184 healthcare workers from the Democratic Republic of Congo. We also found strong antibody titer correlations (rs>0.8) among the different techniques used. In conclusion, our evaluation suggests that the sVNT could be a powerful tool to monitor/detect neutralising antibodies in cohort and population studies. The technique could be especially useful for vaccine evaluation studies in sub-Saharan Africa where the basic infrastructure to perform cVNTs is lacking.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Humans , Neutralization Tests , Serologic Tests
13.
ACS Infect Dis ; 7(6): 1596-1606, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-1135641

ABSTRACT

The presence of antibodies against endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We developed a serum screening platform using a bead-based Western blot system called DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. Characterization of the immunoassay for detection of SARS-CoV-2 specific antibodies revealed a sensitivity of 90.3% and a diagnostic specificity of 98.1%. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates comparable assay performances (Cohen's κ ranging from 0.8874 to 0.9508). Analogous assays for OC43, 229E, and NL63 were established and combined into one multiplex with the SARS-CoV-2 assay. Seroreactivity for different coronaviruses was detected with high incidence, and the multiplex assay was adapted for serum screening.


Subject(s)
COVID-19 , Coronaviridae , COVID-19 Testing , Humans , Plant Extracts , SARS-CoV-2
14.
J Clin Microbiol ; 59(2)2021 01 21.
Article in English | MEDLINE | ID: covidwho-1041778

ABSTRACT

Reliable serological tests are required to determine the prevalence of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to characterize immunity to the disease in order to address key knowledge gaps in the coronavirus disease 2019 (COVID-19) pandemic. Quantitative suspension array technology (qSAT) assays based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and enzyme-linked immunosorbent assays (ELISAs) with their higher precision, dynamic range, throughput, miniaturization, cost-efficiency, and multiplexing capacity. We developed three qSAT assays for IgM, IgA, and IgG against a panel of eight SARS-CoV-2 antigens, including spike protein (S), nucleocapsid protein (N), and membrane protein (M) constructs. The assays were optimized to minimize the processing time and maximize the signal-to-noise ratio. We evaluated their performances using 128 prepandemic plasma samples (negative controls) and 104 plasma samples from individuals with SARS-CoV-2 diagnosis (positive controls), of whom 5 were asymptomatic, 51 had mild symptoms, and 48 were hospitalized. Preexisting IgG antibodies recognizing N, M, and S proteins were detected in negative controls, which is suggestive of cross-reactivity to common-cold coronaviruses. The best-performing antibody/antigen signatures had specificities of 100% and sensitivities of 95.78% at ≥14 days and 95.65% at ≥21 days since the onset of symptoms, with areas under the curve (AUCs) of 0.977 and 0.999, respectively. Combining multiple markers as assessed by qSAT assays has the highest efficiency, breadth, and versatility to accurately detect low-level antibody responses for obtaining reliable data on the prevalence of exposure to novel pathogens in a population. Our assays will allow gaining insights into antibody correlates of immunity and their kinetics, required for vaccine development to combat the COVID-19 pandemic.


Subject(s)
Antigens, Viral/immunology , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin Isotypes/blood , SARS-CoV-2/immunology , Adult , Antibodies, Viral/blood , COVID-19/blood , Cross Reactions , Female , Humans , Immunoassay , Male , Middle Aged , Reproducibility of Results , Sensitivity and Specificity , Viral Structural Proteins/immunology
15.
J Clin Microbiol ; 58(8)2020 Jul 23.
Article in English | MEDLINE | ID: covidwho-999203

ABSTRACT

In December 2019, the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in the Hubei province of China and later spread all over the world. There was an urgent need of a high-throughput molecular test for screening the COVID-19 patients in the community. The Luminex NxTAG CoV extended panel is a high-throughput FDA emergency use-authorized molecular diagnostic assay for SARS-CoV-2 detection. This system targets three genes (ORF1ab, N, and E genes) of SARS-CoV-2, the ORF1ab region of SARS-CoV, and the ORF5 region of MERS-CoV. In this study, we evaluated the diagnostic performance of this system with nasopharyngeal swab specimens of 214 suspected COVID-19 patients in Hong Kong. The results were compared with our routine COVID-19 reverse transcription-PCR (RT-PCR) protocol with a LightMix SarbecoV E-gene kit and an in-house RdRp/Hel RT-PCR assay. The NxTAG CoV extended panel demonstrated 97.8% sensitivity and 100% specificity to SARS-CoV-2 in nasopharyngeal specimens. On low-viral load specimens, the sensitivity of the NxTAG panel could still maintain at 85.71%. Strong agreement was observed between the NxTAG panel and the routine COVID-19 RT-PCR protocol (kappa value = 0.98). Overall, the E gene target of the NxTAG panel demonstrated the highest sensitivity among the three SARS-CoV-2 targets, while the N gene targets demonstrated the least. In conclusion, the NxTAG CoV extended panel is simple to use, and it has high diagnostic sensitivity and specificity to SARS-CoV-2 in nasopharyngeal specimens. We recommend this diagnostic system for high-throughput COVID-19 screening in the community.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , High-Throughput Nucleotide Sequencing/methods , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/diagnosis , Adult , Aged , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , Coronavirus Infections/virology , Female , Hong Kong , Humans , Male , Middle Aged , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Sensitivity and Specificity
16.
Front Microbiol ; 11: 603931, 2020.
Article in English | MEDLINE | ID: covidwho-979023

ABSTRACT

Despite efforts to develop anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody (Ab) immunoassays, reliable serological methods are still needed. We developed a multiplex addressable laser bead immunoassay (ALBIA) to detect and quantify anti-Spike S1 and nucleocapsid N Abs. Recombinant S1 and N proteins were bound to fluorescent beads (ALBIA-IgG-S1/N). Abs were revealed using class-specific anti-human Ig Abs. The performances of the test were analyzed on 575 serum samples including 192 from SARS-CoV-2 polymerase chain reaction-confirmed patients, 13 from seasonal coronaviruses, 70 from different inflammatory/autoimmune diseases, and 300 from healthy donors. Anti-S1 IgM were detected by monoplex ALBIA-IgM-S1. Comparison with chemiluminescent assays or enzyme-linked immunosorbent assays was performed using commercial tests. Multiplex ALBIA-IgG-S1/N was effective in detecting and quantifying anti-SARS-CoV-2 IgG Abs. Two weeks after first symptoms, sensitivity and specificity were 97.7 and 98.0% (anti-S1), and 100 and 98.7% (anti-N), respectively. Agreement with commercial tests was good to excellent, with a higher sensitivity of ALBIA. ALBIA-IgG-S1/N was positive in 53% of patients up to day 7, and in 75% between days 7 and 13. For ALBIA-IgM-S1, sensitivity and specificity were 74.4 and 98.7%, respectively. Patients in intensive care units had higher IgG Ab levels (Mann-Whitney test, p < 0.05). ALBIA provides a robust method for exploring humoral immunity to SARS-CoV-2. Serology should be performed after 2 weeks following first symptoms, when all COVID-19 (coronavirus disease 2019) patients had at least one anti-S1 or anti-N IgG Ab, illustrating the interest of a multiplex test.

17.
J Virol Methods ; 288: 114025, 2021 02.
Article in English | MEDLINE | ID: covidwho-939116

ABSTRACT

Large-scale serosurveillance of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) will only be possible if serological tests are sufficiently reliable, rapid and affordable. Many assays are either labour-intensive and require specialised facilities (e.g. virus neutralization assays), or are expensive with suboptimal specificity (e.g. commercial ELISAs and RDTs). Bead-based assays offer a cost-effective alternative and allow for multiplexing to test for antibodies against multiple antigens and against other pathogens. Here, we compare the performance of spike (S) and nucleocapsid (NP) antigens for the detection of SARS-CoV-2 specific IgG, IgM and IgA antibodies in a panel of sera that includes recent (up to six weeks after symptom onset, severe n = 44; and mild cases n = 52) and old infections (five months after symptom onset, mild n = 104), using a Luminex-bead based assay and comparison to a virus neutralization test. While we show that neutralizing antibody levels are significantly lower in mild than in severe cases, we demonstrate that a combination of the recombinant nucleocapsid protein (NP) and receptor-binding domain (RBD) results in highly specific (99 %) IgG antibody detection five months after infection in 96 % of cases. Although most severe Covid-19 cases developed a clear IgM and IgA response, titers fell below the detection threshold in more than 20 % of mild cases in our bead-based assay. In conclusion, our data supports the use of RBD and NP for the development of SARS-CoV-2 serological IgG bead-based assays.


Subject(s)
Antibodies, Viral/immunology , COVID-19/diagnosis , Immunoassay , Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Neutralizing , COVID-19/immunology , COVID-19/virology , Humans , Immunoassay/methods , Immunoassay/standards , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Neutralization Tests , ROC Curve
18.
One Health ; 11: 100192, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-907097

ABSTRACT

In a survey of household cats and dogs of laboratory-confirmed COVID-19 patients, we found a high seroprevalence of SARS-CoV-2 antibodies, ranging from 21% to 53%, depending on the positivity criteria chosen. Seropositivity was significantly greater among pets from COVID-19+ households compared to those with owners of unknown status. Our results highlight the potential role of pets in the spread of the epidemic.

19.
Transbound Emerg Dis ; 68(4): 2628-2632, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-901200

ABSTRACT

Many infectious pathogens can be transmitted by highly mobile species, like bats that can act as reservoir hosts for viruses such as henipaviruses, lyssaviruses and coronaviruses. In this study, we investigated the seroepidemiology of protein antigens to Severe acute respiratory syndrome virus (SARS-CoV-1) and Middle eastern respiratory syndrome virus (MERS-CoV) in Grey-headed flying foxes (Pteropus poliocephalus) in Adelaide, Australia sampled between September 2015 and February 2018. A total of 301 serum samples were collected and evaluated using a multiplex Luminex binding assay, and median fluorescence intensity thresholds were determined using finite-mixture modelling. We found evidence of antibodies reactive to SARS-CoV-1 or a related antigen with 42.5% (CI: 34.3%-51.2%) seroprevalence but insufficient evidence of reactivity to MERS-CoV antigen. This study provides evidence that the Grey-headed flying foxes sampled in Adelaide have been exposed to a SARS-like coronavirus.


Subject(s)
Chiroptera , Coronavirus Infections , SARS Virus , Animals , Coronavirus , Coronavirus Infections/epidemiology , Coronavirus Infections/veterinary , Lyssavirus , Middle East Respiratory Syndrome Coronavirus , Seroepidemiologic Studies
20.
J Clin Virol ; 129: 104521, 2020 08.
Article in English | MEDLINE | ID: covidwho-610625

ABSTRACT

BACKGROUND: Knowledge of the COVID-19 epidemic extent and the level of herd immunity is urgently needed to help manage this pandemic. METHODS: We used a panel of 167 samples (77 pre-epidemic and 90 COVID-19 seroconverters) and SARS-CoV1, SARS-CoV2 and MERS-CoV Spike and/or Nucleopcapsid (NC) proteins to develop a high throughput multiplex screening assay to detect IgG antibodies in human plasma. Assay performances were determined by ROC curves analysis. A subset of the COVID-19+ samples (n = 36) were also tested by a commercial NC-based ELISA test and the results compared with those of the novel assay. RESULTS: On samples collected ≥14 days after symptoms onset, the accuracy of the assay is 100 % (95 % CI: 100-100) for the Spike antigen and 99.9 % (95 % CI:99.7-100) for NC. By logistic regression, we estimated that 50 % of the patients have seroconverted at 5.7 ± 1.6; 5.7 ± 1.8 and 7.9 ± 1.0 days after symptoms onset against Spike, NC or both antigens, respectively and all have seroconverted two weeks after symptoms onset. IgG titration in a subset of samples showed that early phase samples present lower IgG titers than those from later phase. IgG to SARS-CoV2 NC cross-reacted at 100 % with SARS-CoV1 NC. Twenty-nine of the 36 (80.5 %) samples tested were positive by the commercial ELISA while 31/36 (86.1 %) were positive by the novel assay. CONCLUSIONS: Our assay is highly sensitive and specific for the detection of IgG antibodies to SARS-CoV2 proteins, suitable for high throughput epidemiological surveys. The novel assay is more sensitive than a commercial ELISA.


Subject(s)
Antibodies, Viral/blood , Betacoronavirus/immunology , Immunoglobulin G/blood , Middle East Respiratory Syndrome Coronavirus/immunology , Serologic Tests/methods , /immunology , Adult , Aged , Aged, 80 and over , Antigens, Viral/immunology , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Coronavirus Infections/virology , Female , Humans , Male , Middle Aged , Nucleocapsid Proteins/immunology , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , ROC Curve , SARS-CoV-2 , Sensitivity and Specificity , Severe Acute Respiratory Syndrome/diagnosis , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/immunology , Time Factors
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