Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 31
Filter
1.
Viruses ; 12(6)2020 05 26.
Article in English | MEDLINE | ID: covidwho-1726015

ABSTRACT

The recent outbreak of the Coronavirus disease 2019 (COVID-19) has quickly spread worldwide since its discovery in Wuhan city, China in December 2019. A comprehensive strategy, including surveillance, diagnostics, research, clinical treatment, and development of vaccines, is urgently needed to win the battle against COVID-19. The past three unprecedented outbreaks of emerging human coronavirus infections at the beginning of the 21st century have highlighted the importance of readily available, accurate, and rapid diagnostic technologies to contain emerging and re-emerging pandemics. Real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) based assays performed on respiratory specimens remain the gold standard for COVID-19 diagnostics. However, point-of-care technologies and serologic immunoassays are rapidly emerging with high sensitivity and specificity as well. Even though excellent techniques are available for the diagnosis of symptomatic patients with COVID-19 in well-equipped laboratories; critical gaps still remain in screening asymptomatic people who are in the incubation phase of the virus, as well as in the accurate determination of live viral shedding during convalescence to inform decisions for ending isolation. This review article aims to discuss the currently available laboratory methods and surveillance technologies available for the detection of COVID-19, their performance characteristics and highlight the gaps in current diagnostic capacity, and finally, propose potential solutions. We also summarize the specifications of the majority of the available commercial kits (PCR, EIA, and POC) for laboratory diagnosis of COVID-19.


Subject(s)
Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Asymptomatic Infections , Betacoronavirus , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Humans , Immunoenzyme Techniques , Neutralization Tests , Nucleic Acid Amplification Techniques , Pandemics , Point-of-Care Testing , Reagent Kits, Diagnostic/standards , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Sensitivity and Specificity , Serologic Tests , Tomography, X-Ray Computed , Virus Shedding
2.
Microbiol Spectr ; 10(1): e0078621, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1605388

ABSTRACT

Seroepidemiological studies to monitor antibody kinetics are important for assessing the extent and spread of SARS-CoV-2 in a population. Noninvasive sampling methods are advantageous for reducing the need for venipuncture, which may be a barrier to investigations, particularly in pediatric populations. Oral fluids are obtained by gingiva-crevicular sampling from children and adults and are very well accepted. Enzyme immunoassays (EIAs) based on these samples have acceptable sensitivity and specificity compared to conventional serum-based antibody EIAs and are suitable for population-based surveillance. We describe the development and evaluation of SARS-CoV-2 IgG EIAs using SARS-CoV-2 viral nucleoprotein (NP) and spike (S) proteins in IgG isotype capture format and an indirect receptor-binding-domain (RBD) IgG EIA, intended for use in children as a primary endpoint. All three assays were assessed using a panel of 1,999 paired serum and oral fluids from children and adults participating in school SARS-CoV-2 surveillance studies during and after the first and second pandemic wave in the United Kingdom. The anti-NP IgG capture assay was the best candidate, with an overall sensitivity of 75% (95% confidence interval [CI]: 71 to 79%) and specificity of 99% (95% CI: 78 to 99%) compared with paired serum antibodies. Sensitivity observed in children (80%, 95% CI: 71 to 88%) was higher than that in adults (67%, CI: 60% to 74%). Oral fluid assays (OF) using spike protein and RBD antigens were also 99% specific and achieved reasonable but lower sensitivity in the target population (78%, 95% CI [68% to 86%] and 53%, 95% CI [43% to 64%], respectively). IMPORTANCE We report on the first large-scale assessment of the suitability of oral fluids for detection of SARS-CoV-2 antibody obtained from healthy children attending school. The sample type (gingiva-crevicular fluid, which is a transudate of blood but is not saliva) can be self collected. Although detection of antibodies in oral fluids is less sensitive than that in blood, our study suggests an optimal format for operational use. The laboratory methods we have developed can reliably measure antibodies in children, who are able to take their own samples. Our findings are of immediate practical relevance for use in large-scale seroprevalence studies designed to measure exposure to infection, as they typically require venipuncture. Overall, our data indicate that OF assays based on the detection of SARS-CoV-2 antibodies are a tool suitable for population-based seroepidemiology studies in children and highly acceptable in children and adults, as venipuncture is no longer necessary.


Subject(s)
Antibodies, Viral/analysis , COVID-19/diagnosis , Gingival Crevicular Fluid/immunology , Immunoglobulin G/analysis , SARS-CoV-2/immunology , Adolescent , Child , Child, Preschool , Humans , Immunoenzyme Techniques , Infant , Sensitivity and Specificity , Seroepidemiologic Studies
4.
Ann Med ; 53(1): 34-42, 2021 12.
Article in English | MEDLINE | ID: covidwho-1574894

ABSTRACT

BACKGROUND: Studies have demonstrated the diagnostic efficiency of antibody testing in COVID-19 infection. There is limited data on the IgM/IgG changes in asymptomatic and discharged patients with reoccurring positive nucleic acid test (RPNAT). This study aims to investigate these IgM/IgG changes. METHODS: There were 111 patients with positive nucleic acid test (NAT) and 40 suspected patients enrolled in the study. The serum SARS-CoV-2 specific IgM/IgG antibody levels were retrospectively analysed with the disease progress in asymptomatic and RPNAT patients. RESULTS: The best overall performance was found by combining the IgM, IgG, and CT; 95.1% sensitivity and 75% specificity. This was tested in 111 RT-PCR positive cases. The median IgM and IgG levels were lower in the asymptomatic group compared to the symptomatic group (p < .01). Among 15 RPNAT cases, the IgM levels of the RPNAT group at the time of discharge (IgM2.79, IQR: 0.95-5.37) and retest (IgM 2.35, IQR: 0.88-8.65) were significantly higher than those of the non-reoccurring positive nucleic acid test group (Non-RPNAT) (IgM on discharge: 0.59, IQR: 0.33-1.22, IgG on retest: 0.92, IQR: 0.51-1.58). CONCLUSION: Serum SARS-CoV-2 specific IgM/IgG antibody levels remained at a low level during hospitalisation for asymptomatic patients. Elevated IgM levels may have implications in the identification of RPNAT patients before discharge. Key messages This study determined the IgM/IgG changes in asymptomatic and RPNAT patients. The rate of serum SARS-CoV-2 specific IgM/IgG antibody levels increase in the asymptomatic group was lower than in the symptomatic group during hospitalisation. The IgM level did not decrease significantly at discharge in the RPNAT patients, and was higher than that of the Non-RPNAT group on discharge. These results highlight the importance of timely monitoring of IgM levels to identify RPNAT patients before discharge.


Subject(s)
Antibodies, Viral/blood , Betacoronavirus/isolation & purification , Coronavirus Infections/immunology , Immunoglobulin G/blood , Immunoglobulin M/blood , Pneumonia, Viral/immunology , COVID-19 , COVID-19 Testing , Case-Control Studies , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Female , Humans , Immunoenzyme Techniques/methods , Male , Pandemics , Retrospective Studies , SARS-CoV-2
5.
J Med Virol ; 93(12): 6778-6781, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1544295

ABSTRACT

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Immunoenzyme Techniques/methods , Immunologic Tests/methods , SARS-CoV-2/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , COVID-19/virology , Humans , Mass Screening/methods , Nasopharynx/immunology , Nasopharynx/virology , RNA, Viral/genetics , RNA, Viral/immunology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load/genetics , Viral Load/immunology
6.
J Clin Virol ; 146: 105048, 2022 01.
Article in English | MEDLINE | ID: covidwho-1540751

ABSTRACT

Direct detection of SARS-CoV-2 viral antigens could replace RT-PCR, provided that its clinical performance is validated in different epidemiological settings. Here, we evaluated the performance of the VITROS Antigen test, an enzyme immunoassay detecting a SARS-CoV-2 antigen, in NPSs from 3 cohorts of patients. METHODS: Three cohorts including SARS-CoV-2 RNA-positive samples collected during the first and second wave of the French epidemic between March 2020 and February 2021 (including variant B.1.1.7/α and variant B.1.351/ß). RESULTS: Among the 1763 prospectively tested subjects, 8.2% (145/1763) were SARS-CoV-2 RNA-positive by RT-PCR. Using Ct ≤ 30 and Ct ≤ 35 as thresholds, the sensitivities of the antigen assay were 98.8% (93.6-100%) and 93.5% (87.0-97.3%), respectively. The overall specificity of the assay was 100% (1614/1614; 99.8-100%). In a retrospective cohort of subjects infected with variants of concern, 90.4% (47/52) of NPSs containing B. B.1.1.7/α (Ct ≤ 35) and 100% (7/7) of those containing B.1.351/ß were positive with the VITROS EIA SARS-CoV-2 Antigen test. CONCLUSION: The excellent performance of the EIA Antigen test reported here, including in patients infected with viral "variants of concern", support the use of high-throughput, EIA-based SARS-CoV-2 antigen assays as an alternative or complement to nucleic acid testing in order to scale-up laboratory screening and diagnostic capacities.


Subject(s)
COVID-19 , SARS-CoV-2 , Antigens, Viral , Humans , Immunoassay , Immunoenzyme Techniques , RNA, Viral , Retrospective Studies , Sensitivity and Specificity
7.
J Infect Chemother ; 28(2): 273-278, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1536654

ABSTRACT

BACKGROUND: Levels of 50% neutralizing titer (NT50) reflect the a vaccine-induced humoral immunity after the vaccination against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Measurements of NT50 are difficult to implement in large quantities. A high-throughput laboratory test is expected for determining the level of herd immunity against SARS-CoV-2. METHODS: We analyzed samples from 168 Japanese healthcare workers who had completed two doses of the BNT162b2 vaccine. We analyzed immunoglobulin G (IgG) index values against spike protein (SP) using automated chemiluminescent enzyme immunoassay system AIA-CL and analyzed the background factors affecting antibody titer. SP IgG index was compared with 50% neutralization titers. RESULTS: The median SP IgG index values of the subjects (mean age = 43 years; 75% female) were 0.1, 1.35, 60.80, and 97.35 before and at 2, 4, and 6 weeks after the first dose, respectively. At 4 and 6 weeks after the first dose, SP IgG titers were found to have positive correlation with NT50 titer (r = 0.7535 in 4 weeks; r = 0.4376 in 6 weeks). Proportions of the SP IgG index values against the Alpha, Beta, Gamma, and Delta variants compared with the original strain were 2.029, 0.544, 1.017, and 0.6096 respectively. Older age was associated with lower SP IgG titer index 6 weeks after the first dose. CONCLUSIONS: SP IgG index values were rised at 3 weeks after two doses of BNT162b2 vaccination and have positive correlation with NT50. SP IgG index values were lower in the older individuals and against Beta and Delta strain.


Subject(s)
COVID-19 , Adult , Aged , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 Vaccines , Female , Humans , Immunoenzyme Techniques , Male , SARS-CoV-2 , Vaccination
8.
Sci Rep ; 11(1): 20323, 2021 10 13.
Article in English | MEDLINE | ID: covidwho-1467136

ABSTRACT

This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4-98.5%) and specificity of 100% (95% CI 95.1-100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3-98.1%) and a specificity of 100% (95% CI 95.1-100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P < 0.0001). The in-house and the pre-commercial SARS-CoV-2 nucleocapsid antigen assay demonstrated technical and clinical sensitivity comparable to real-time RT-PCR methods for identifying SARS-CoV-2 infected patients and thus can be used clinically as well as serve as a reference method for antigen Point of Care Testing.


Subject(s)
COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Antigens, Viral/immunology , COVID-19 Serological Testing/methods , Coronavirus Nucleocapsid Proteins/analysis , Denmark , Diagnostic Tests, Routine , Humans , Immunoenzyme Techniques , Nasopharynx/virology , Nucleocapsid/analysis , Nucleocapsid/immunology , Phosphoproteins/analysis , Phosphoproteins/immunology , SARS-CoV-2/pathogenicity , Sensitivity and Specificity , Single Molecule Imaging/methods , Virion/chemistry
9.
Mikrobiyol Bul ; 55(3): 461-464, 2021 Jul.
Article in Turkish | MEDLINE | ID: covidwho-1325957

ABSTRACT

Although the reverse transcriptase polymerase chain reaction (RT-PCR) method has been accepted as the reference method in the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA, it requires special laboratory conditions, complicated and expensive laboratory instruments, competent laboratory staff and long testing duration. Antigen testing methods such as enzyme immunoassay, fluorescent antibody and visually-read immunochromatographic rapid antigen detection (RAD) tests eliminated the above mentioned disadvantages of the RT-PCR. The aim of this study was to determine the performance of a RAD test kit (V-Chek, SGA Ltd, Ankara, Turkey). Two paired nasopharyngeal swabs were collected from each patient, and one of them was used for the RAD test, while the other one (different swab) was used to perform the RT-PCR test. SARS CoV-2 Double Gene RT-PCR kit (Bioeksen, Turkey) was used for RNA amplification on the Light Cycler 480 plate-based RT-PCR instrument (Roche, Switzerland). The SARS-CoV-2 double gene RT-PCR kit targeting the SARS-CoV-2 specific N (nucleocapsid) and Orf1ab gene regions was used for RNA amplification. The human RNaseP gene was used for nucleic acid extraction and inhibition control. The shape of the growth curves was examined and the non-sigmoidal curves were recorded as "negative". Sigmoidal curves with cycle threshold (Ct) <38 were evaluated as "positive". The Ct values of all positive results were recorded. V-Chek RAD test kit uses a colloidal gold enhanced double antibody sandwich type antigen test kit. A SARS-CoV-2 positive specimen produces a distinct color band in the test region, formed by the specific antibody-antigen colored conjugate complex. A positive or negative result is indicated by a colored line appearance on the test region. A colored line appears in the control region, independent of the SARS-CoV-2 presence. The result is visually read 10 minutes after the last drop of the sample liquid is dispensed into the sample well. Specificity and sensitivity values were calculated accepting the RT-PCR results as a standard. Agreement between two different techniques was assessed using Cohen's kappa score. 110 patients were enrolled in this study; 34 (30.9 %) of these patients had positive RT-PCR samples, with the mean of Ct values of 25.8 (95% CI= 24.1-27.5), median of Ct values of 26. In our study population, the overall sensitivity was 61.8% (95% CI= 45.4-78.1), and specificity was 100%. Taking RT-PCR as reference, Cohen's kappa score for the antigen test was 0.691. Fisher's exact test was p<0.001. In conclusion, the RAD kit used in the study determined to be useful for the rapid identification of COVID-19 patients. However, a negative result does not eliminate the possibility of COVID-19 infection and should be confirmed by RT-PCR and clinical findings.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Immunoenzyme Techniques , RNA , Time Factors
10.
J Med Virol ; 93(12): 6778-6781, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1281221

ABSTRACT

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Immunoenzyme Techniques/methods , Immunologic Tests/methods , SARS-CoV-2/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , COVID-19/virology , Humans , Mass Screening/methods , Nasopharynx/immunology , Nasopharynx/virology , RNA, Viral/genetics , RNA, Viral/immunology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity , Viral Load/genetics , Viral Load/immunology
12.
mSphere ; 6(3)2021 05 12.
Article in English | MEDLINE | ID: covidwho-1230164

ABSTRACT

Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43.IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.


Subject(s)
Coronavirus OC43, Human/physiology , Cytopathogenic Effect, Viral , Receptors, Virus/metabolism , Serine Endopeptidases/metabolism , Viral Load/methods , Animals , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus OC43, Human/isolation & purification , Humans , Immunoenzyme Techniques , Receptors, Virus/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/genetics , Vero Cells/virology , Virus Cultivation , Virus Internalization , Virus Replication
13.
Blood ; 138(4): 299-303, 2021 07 29.
Article in English | MEDLINE | ID: covidwho-1228983

ABSTRACT

Vaccination using the adenoviral vector COVID-19 vaccine ChAdOx1 nCoV-19 (AstraZeneca) has been associated with rare vaccine-induced immune thrombotic thrombocytopenia (VITT). Affected patients test strongly positive in platelet factor 4 (PF4)/polyanion enzyme immunoassays (EIAs), and serum-induced platelet activation is maximal in the presence of PF4. We determined the frequency of anti-PF4/polyanion antibodies in healthy vaccinees and assessed whether PF4/polyanion EIA+ sera exhibit platelet-activating properties after vaccination with ChAdOx1 nCoV-19 (n = 138) or BNT162b2 (BioNTech/Pfizer; n = 143). In total, 19 of 281 participants tested positive for anti-PF4/polyanion antibodies postvaccination (All: 6.8% [95% confidence interval (CI), 4.4-10.3]; BNT162b2: 5.6% [95% CI, 2.9-10.7]; ChAdOx1 nCoV-19: 8.0% [95% CI, 4.5% to 13.7%]). Optical densities were mostly low (between 0.5 and 1.0 units; reference range, <0.50), and none of the PF4/polyanion EIA+ samples induced platelet activation in the presence of PF4. We conclude that positive PF4/polyanion EIAs can occur after severe acute respiratory syndrome coronavirus 2 vaccination with both messenger RNA- and adenoviral vector-based vaccines, but many of these antibodies likely have minor (if any) clinical relevance. Accordingly, low-titer positive PF4/polyanion EIA results should be interpreted with caution when screening asymptomatic individuals after vaccination against COVID-19. Pathogenic platelet-activating antibodies that cause VITT do not occur commonly following vaccination.


Subject(s)
Autoantibodies/immunology , COVID-19 Vaccines/adverse effects , COVID-19/prevention & control , Platelet Factor 4/immunology , Polyelectrolytes , Purpura, Thrombotic Thrombocytopenic/etiology , Vaccination/adverse effects , Adult , Asymptomatic Diseases , Autoantibodies/blood , Female , Health Personnel , Humans , Immunoenzyme Techniques , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Platelet Activation , Purpura, Thrombotic Thrombocytopenic/immunology , Seroconversion , Thrombophilia/etiology
14.
Blood ; 137(26): 3656-3659, 2021 07 01.
Article in English | MEDLINE | ID: covidwho-1215090

ABSTRACT

Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.


Subject(s)
Autoantibodies/blood , COVID-19 Vaccines/adverse effects , COVID-19/prevention & control , Flow Cytometry/methods , Immunoglobulin G/blood , Platelet Activation/immunology , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/diagnosis , Receptors, IgG/immunology , SARS-CoV-2 , Vaccination/adverse effects , Antibody Specificity , Autoantibodies/biosynthesis , Autoantibodies/immunology , COVID-19 Vaccines/immunology , Heparin/adverse effects , Heparin/immunology , Humans , Immunoenzyme Techniques , Immunogenicity, Vaccine , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , P-Selectin/analysis , Purpura, Thrombocytopenic, Idiopathic/etiology , Purpura, Thrombocytopenic, Idiopathic/immunology
15.
J Infect Dis ; 224(2): 218-228, 2021 07 15.
Article in English | MEDLINE | ID: covidwho-1203709

ABSTRACT

BACKGROUND: Primary diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is based on detection of virus RNA in nasopharyngeal swab samples. In addition, analysis of humoral immunity against SARS-CoV-2 has an important role in viral diagnostics and seroprevalence estimates. METHODS: We developed and optimized an enzyme immunoassays (EIA) using SARS-CoV-2 nucleoprotein (N), S1 and receptor binding domain (RBD) of the viral spike protein, and N proteins from SARS, Middle East respiratory syndrome (MERS), and 4 low-pathogenic human CoVs. Neutralizing antibody activity was compared with SARS-CoV-2 IgG, IgA, and IgM EIA results. RESULTS: The sensitivity of EIA for detecting immune response in COVID-19 patients (n = 101) was 77% in the acute phase and 100% in the convalescent phase of SARS-CoV-2 infection when N and RBD were used as antigens in IgG and IgA specific EIAs. SARS-CoV-2 infection significantly increased humoral immune responses against the 229E and NL63 N proteins. S1 and RBD-based EIA results had a strong correlation with microneutralization test results. CONCLUSIONS: The data indicate a combination of SARS-CoV-2 S1 or RBD and N proteins and analysis of IgG and IgA immunoglobulin classes in sera provide an excellent basis for specific and sensitive serological diagnostics of COVID-19.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Immunoglobulin A/blood , Immunoglobulin G/blood , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Humans , Immunoenzyme Techniques , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Neutralization Tests , Phosphoproteins/immunology , SARS-CoV-2/immunology , Sensitivity and Specificity
16.
Diagn Microbiol Infect Dis ; 101(3): 115412, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1201522

ABSTRACT

The COVID-19 pandemic has led to the influx of immunoassays for the detection of antibodies towards severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the global market. The Canadian Public Health Laboratory Network Serology Task Force undertook a nationwide evaluation of twelve laboratory and 6 point-of-care based commercial serological assays for the detection of SARS-CoV-2 antibodies. We determined that there was considerable variability in the performance of individual tests and that an orthogonal testing algorithm should be prioritized to maximize the accuracy and comparability of results across the country. The manual enzyme immunoassays and point-of-care tests evaluated had lower specificity and increased coefficients of variation compared to automated enzyme immunoassays platforms putting into question their utility for large-scale sero-surveillance. Overall, the data presented here provide a comprehensive approach for applying accurate serological assays for longitudinal sero-surveillance and vaccine trials while informing Canadian public health policy.


Subject(s)
Antibodies, Viral/blood , COVID-19/epidemiology , Laboratories/standards , Public Health , SARS-CoV-2/immunology , Serologic Tests/standards , COVID-19/blood , Canada/epidemiology , High-Throughput Screening Assays , Humans , Immunoenzyme Techniques , SARS-CoV-2/isolation & purification , Serologic Tests/methods
17.
J Infect Chemother ; 27(7): 1039-1042, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1164052

ABSTRACT

INTRODUCTION: The pandemic of a novel coronavirus disease 2019 (COVID-19) caused by a severe acute respiratory coronavirus 2 (SARS-CoV-2) infection has been problematic worldwide. A new SARS-CoV-2 antigen test (LUMIPULSEⓇ) was licensed and widely used in Japan since May 2020. We conducted this study intending to whether the automated quantitative CLEIA antigen test using a saliva sample is effective and valid for the diagnosis of COVID-19. PATIENTS AND METHODS: We analyzed and compared the diagnostic accuracy of both the automated quantitative CLEIA antigen test and real-time RT-PCR (rRT-PCR) using a saliva sample from individuals suspected as having COVID-19. RESULTS: A total of 305 samples were collected and tested in Aichi Medical University Hospital and affiliated facilities from December 2020 until January 2021 at our institute. Using reverse-transcription PCR as a reference, the AUROC of the automated quantitative CLEIA antigen test was 0.903 (95% confidential interval 0.845-0.962, p < 0.001). The appropriate cut-off antigen level was 4.0 pg/mL and had a sensitivity of 77.8%, a specificity of 99.6%, a positive predictive value of 98%, and a negative predictive value of 94.5%. On the other hand, the diagnostic accuracy of the antigen test decreased among patients among patients with COVID-19 with threshold cycle (Ct-value)≥27, which shows the AUROC was 0.795 (95%CI 0.687-0.907, p < 0.001). CONCLUSION: While the automated quantitative CLEIA antigen test from saliva specimen could be one of the most useful diagnostic tests for the diagnosis of COVID-19 in general practice, clinicians should know the limitations of the antigen test.


Subject(s)
COVID-19 , Saliva , Humans , Immunoenzyme Techniques , Japan , SARS-CoV-2 , Sensitivity and Specificity
18.
J Virol Methods ; 292: 114129, 2021 06.
Article in English | MEDLINE | ID: covidwho-1144852

ABSTRACT

BACKGROUND: At present, the only reliable test for COVID-19 diagnosis is RT-qPCR. Serological assays have been widely used to increase the detection sensitivity of infected population. Hereby, we report the performance of a new pan-IgG multiplex Enzyme Immunoassay (immunodot) method for exploration of discrepant SARS-COV-2 serological results. METHODS: A retrospective study on 38 residual serum samples from recovered COVID-19 subjects with discordant serological results on Roche and Snibe platforms, were reanalyzed on a new semi-automated pan-IgG immunodot Enzyme Immunoassay, namely COVIDOT-TEST, in order to find the source of discrepancies and to evaluate the latter method. All samples were analyzed on the BlueDiver® Instrument and all strips were read by the BlueScan® Scanner using Dr DOT® Software. RESULTS: Based on our data, subject samples showed specific IgG reactions on ≥ 2 different antigens on immunodot strips. Of these 38 samples, 97.4 % of samples showed specific IgG reaction against S1 + S2 antigens, 89.5 % showed against RBD antigen, 86.8 % against S2 antigen reaction on the COVIDOT-TEST kit. Specific IgG-S1 antigen and IgG-N antigen reactions were detected in 73.7 % and 65.8 % of the samples, respectively. CONCLUSION: The new semi-automated pan-IgG immunodot Enzyme Immunoassay method appeared to be a reliable assay to confirm suspicious COVID-19 serological screening results.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoenzyme Techniques/methods , SARS-CoV-2/immunology , Adult , Antibodies, Viral/blood , Female , Humans , Immunoglobulin G/blood , Male , Retrospective Studies
20.
Sci Rep ; 11(1): 5198, 2021 03 04.
Article in English | MEDLINE | ID: covidwho-1117665

ABSTRACT

The quantitative range and reproducibility of current serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are not optimized. Herein, we developed a diagnostic test that detects SARS-CoV-2 IgG and IgM with high quantitativeness and reproducibility and low interference. The system was based on the high-sensitivity chemiluminescence enzyme immunoassay (HISCL) platform and detects IgG and IgM specific to SARS-CoV-2 spike and nucleocapsid proteins. Quantification accuracy and reproducibility were evaluated using serially diluted samples from 60 SARS-CoV-2-infected patients. Assay performance was evaluated using serum samples from the SARS-CoV-2-infected patients and 500 SARS-CoV-2-negative serum samples collected before the emergence of SARS-CoV-2. The system showed high quantification accuracy (range, 102), high reproducibility (within 5%), and no cross-reaction between SARS1- and MERS-S proteins. Detection accuracy was 98.3% and 93.3% for IgG and IgM against spike proteins and 100% and 71.7% for IgG and IgM against nucleocapsid proteins, respectively. Mean antibody levels were > 10 times that in negative samples upon admission and > 100 times that at convalescent periods. Clinical severity upon admission was not correlated with IgG or IgM levels. This highly quantitative, reproducible assay system with high clinical performance may help analyze temporal serological/immunological profiles of SARS-CoV-2 infection and SARS-CoV-2 vaccine effectiveness.


Subject(s)
COVID-19 Testing/methods , Immunoglobulin G/analysis , Immunoglobulin M/analysis , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Female , Humans , Immunoenzyme Techniques , Luminescent Measurements , Male , Middle Aged , ROC Curve , Young Adult
SELECTION OF CITATIONS
SEARCH DETAIL