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1.
BMC Infect Dis ; 21(1): 187, 2021 Feb 18.
Article in English | MEDLINE | ID: covidwho-1090687

ABSTRACT

BACKGROUND: Thresholds for SARS-CoV-2 antibody assays have typically been determined using samples from symptomatic, often hospitalised, patients. In this setting the sensitivity and specificity of the best performing assays can both exceed 98%. However, antibody assay performance following mild infection is less clear. METHODS: We assessed quantitative IgG responses in a cohort of healthcare workers in Oxford, UK, with a high pre-test probability of Covid-19, in particular the 991/11,475(8.6%) who reported loss of smell/taste. We use anosmia/ageusia and other risk factors as probes for Covid-19 infection potentially undiagnosed by immunoassays by investigating their relationship with antibody readings either side of assay thresholds. RESULTS: The proportion of healthcare workers reporting anosmia/ageusia increased at antibody readings below diagnostic thresholds using an in-house ELISA (n = 9324) and the Abbott Architect chemiluminescent microparticle immunoassay (CMIA; n = 11,324): 426/906 (47%) reported anosmia/ageusia with a positive ELISA, 59/449 (13.1%) with high-negative and 326/7969 (4.1%) with low-negative readings. Similarly, by CMIA, 518/1093 (47.4%) with a positive result reported anosmia/ageusia, 106/686 (15.5%) with a high-negative and 358/9563 (3.7%) with a low-negative result. Adjusting for the proportion of staff reporting anosmia/ageusia suggests the sensitivity of both assays in mild infection is lower than previously reported: Oxford ELISA 89.8% (95%CI 86.6-92.8%) and Abbott CMIA 79.3% (75.9-82.7%). CONCLUSION: Following mild SARS-CoV-2 infection 10-30% of individuals may have negative immunoassay results. While lowered diagnostic thresholds may result in unacceptable specificity, our findings have implications for epidemiological analyses and result interpretation in individuals with a high pre-test probability. Samples from mild PCR-confirmed infections should be included in SARS-CoV-2 immunoassay evaluations.


Subject(s)
Antibodies, Viral/analysis , /diagnosis , Immunoglobulin G/analysis , Adult , Ageusia/virology , Asymptomatic Infections , Enzyme-Linked Immunosorbent Assay/standards , Female , Health Personnel , Humans , Immunoassay/standards , Male , Middle Aged , Sensitivity and Specificity , Undiagnosed Diseases , United Kingdom
2.
Epidemiol Infect ; 149: e44, 2021 02 10.
Article in English | MEDLINE | ID: covidwho-1075129

ABSTRACT

Much of our current understanding about novel coronavirus disease 2019 (COVID-19) comes from hospitalised patients. However, the spectrum of mild and subclinical disease has implications for population-level screening and control. Forty-nine participants were recruited from a group of 99 adults repatriated from a cruise ship with a high incidence of COVID-19. Respiratory and rectal swabs were tested by polymerase chain reaction (PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sera were tested for anti-SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA) and microneutralisation assay. Symptoms, viral shedding and antibody response were examined. Forty-five participants (92%) were considered cases based on either positive PCR or positive ELISA for immunoglobulin G. Forty-two percent of cases were asymptomatic. Only 15% of symptomatic cases reported fever. Serial respiratory and rectal swabs were positive for 10% and 5% of participants respectively about 3 weeks after median symptom onset. Cycle threshold values were high (range 31-45). Attempts to isolate live virus were unsuccessful. The presence of symptoms was not associated with demographics, comorbidities or antibody response. In closed settings, incidence of COVID-19 could be almost double that suggested by symptom-based screening. Serology may be useful in diagnosis of mild disease and in aiding public health investigations.


Subject(s)
Antibodies, Viral/blood , /virology , Ships , Symptom Assessment , Virus Shedding , Adult , Aged , Aged, 80 and over , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Female , Humans , Incidence , Male , Middle Aged , Neutralization Tests , Tourism , Uruguay , Victoria/epidemiology
3.
Medicina (Kaunas) ; 57(2)2021 Feb 06.
Article in English | MEDLINE | ID: covidwho-1069846

ABSTRACT

Background and objective: Serologic testing is a useful additional method for the diagnosis of COVID-19. It is also used for population-based seroepidemiological studies. The objective of the study was to determine SARS-CoV-2 seroprevalence in healthcare workers of Kaunas hospitals and to compare two methods for specific SARS-CoV-2 antibody testing. Materials and Methods: A total of 432 healthcare workers in Kaunas hospitals were enrolled in this study. Each participant filled a questionnaire including questions about their demographics, contact with suspected or confirmed COVID-19, acute respiratory symptoms, and whether they contacted their general practitioner, could not come to work, or had to be hospitalized. Capillary blood was used to test for SARS-CoV-2 specific immunoglobulin G (IgG) and immunoglobulin M (IgM) a lateral flow immunoassay. Serum samples were used to test for specific IgG and IgA class immunoglobulins using semiquantitative enzyme-linked immunosorbent assay (ELISA) method. Results: 24.77% of study participants had direct contact with a suspected or confirmed case of COVID-19. A total of 64.81% of studied individuals had at least one symptom representing acute respiratory infection, compatible with COVID-19. Lateral flow immunoassay detected SARS-CoV-2 specific IgG class immunoglobulins in 1.16% of the tested group. Fever, cough, dyspnea, nausea, diarrhea, headache, conjunctivitis, muscle pain, and loss of smell and taste predominated in the anti-SARS-CoV-2 IgG-positive group. Using ELISA, specific IgG were detected in 1.32% of the tested samples. Diarrhea, loss of appetite, and loss of smell and taste sensations were the most predominant symptoms in anti-SARS-CoV-2 IgG-positive group. The positive percent agreement of the two testing methods was 50%, and negative percent agreement was 99.66%. Conclusions: 1.16% of tested healthcare workers of Kaunas hospitals were anti-SARS-CoV-2 IgG-positive. The negative percent agreement of the lateral flow immunoassay and ELISA exceeded 99%.


Subject(s)
/epidemiology , Immunoglobulin G/blood , Personnel, Hospital , /immunology , Adult , Aged , /diagnosis , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoassay/methods , Immunoglobulin M/blood , Lithuania/epidemiology , Male , Middle Aged , Sensitivity and Specificity , Seroepidemiologic Studies
4.
Epidemiol Prev ; 44(5-6 Suppl 2): 184-192, 2020.
Article in English | MEDLINE | ID: covidwho-1068138

ABSTRACT

BACKGROUND: since the beginning of the COVID-19 pandemic, the importance of developing a serological test has emerged and a debate on test accuracy and reliability become an issue widely discussed in the media. The importance of communication during this pandemic has been strongly underlined by public health experts, epidemiologists, media expert, psychologists, sociologists. In the case of serological tests, there are several aspects that have to be considered: why we perform the test, what population is tested, which are the parameters conditioning the results and their interpretation. OBJECTIVES: to show how to quantify the uncertainty related to the validity of the serological test with respect to its predictive value and in particular the positive predictive value. METHODS: the evaluation of a qualitative diagnostic test includes four distinct assessments: accuracy, empirical evidence, practical importance, and prevalence of the pathology. Accuracy is measured by the sensitivity and specificity of the test; empirical evidence is quantified by the likelihood ratio, respectively for a positive and negative test result; the practical importance of the result of a diagnostic test is assessed by the positive or negative predictive value. Prevalence of COVID-19 is substantial uncertainty and it is possible to estimate the apparent prevalence starting from the results obtained with a diagnostic test. RESULTS: at the moment, the knowledge about the accuracy of serological tests is limited and little attention is paid to confidence interval on point estimates. In terms of practical importance of testing at individual level, while negative predictive values are high whatever the level of sensitivity of the test, the interpretation of a positive results is very cumbersome. Positive predictive values above 90% can be reached only by tests with specificity above 99% at the expected prevalence rate of 5%. There is a linear relationship between apparent - testing positive - prevalence and real prevalence. The apparent prevalence in the context of serological test for COVID-19 is always larger than real prevalence. The level of specificity is crucial. CONCLUSIONS: the main applications of the serological test in the epidemic contest are: to study the seroprevalence of the virus antibodies in the general population; to screen the healthcare workers for the early identification of contagious subjects' health care settings and to screen the general population in order to identify new incident cases. In the first two cases, seroprevalence study and screening of a high-risk population, the consequences of the uncertainty associated to the statistics are already accounted for in the first situation, or are overcome by repeating the screening on the healthcare workers, and using the molecular test to verify the presence of the virus in those tested positive. The case of screening of general population is more complex and of major interest for the implication it may have on individual behaviours and on the implementation of public health interventions by the political decision makers. A positive result has, per se, no practical value for individuals since the probability of being really infected by the virus is low. The uncertainty associated with the different estimates (sensitivity, specificity and disease prevalence) play a double role: it is a key factor in defining the informative content of the test result and it might guide the individual actions and the public policy decisions.


Subject(s)
Antibodies, Viral/blood , Communication , Immunoassay , Luminescent Measurements , Medical Overuse , Pandemics , /immunology , /epidemiology , Confidence Intervals , Decision Making , Enzyme-Linked Immunosorbent Assay , False Positive Reactions , Health Policy , Humans , Italy/epidemiology , Likelihood Functions , Mass Screening , Predictive Value of Tests , Prevalence , Sensitivity and Specificity , Seroepidemiologic Studies , Uncertainty
5.
Virol J ; 18(1): 12, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1067244

ABSTRACT

BACKGROUND: Chemokine levels in severe coronavirus disease 2019 (COVID-19) patients have been shown to be markedly elevated. But the role of chemokines in mild COVID-19 has not yet been established. According to the epidemiological statistics, most of the COVID-19 cases in Shiyan City, China, have been mild. The purpose of this study was to evaluate the level of chemokines in mild COVID-19 patients and explore the correlation between chemokines and host immune response. METHODS: In this study, we used an enzyme-linked immunosorbent assay to detect serum levels of chemokines in COVID-19 patients in Shiyan City. Expression of chemokine receptors and of other signaling molecules was measured by real-time polymerase chain reaction. RESULTS: We first demonstrated that COVID-19 patients, both sever and mild cases, are characterized by higher level of chemokines. Specifically, monocyte chemotactic protein 1 (MCP-1) is expressed at higher levels both in severe and mild cases of COVID-19. The receptor of MCP-1, C-C chemokine receptor type 2, was expressed at higher levels in mild COVID-19 patients. Finally, we observed a significant negative correlation between expression levels of interferon (IFN) regulatory factor 3 (IRF3) and serum levels of MCP-1 in mild COVID-19 patients. CONCLUSION: Higher expression of MCP-1 in mild COVID-19 patients might be correlated with inhibition of IFN signaling. The finding adds to our understanding of the immunopathological mechanisms of severe acute respiratory syndrome coronavirus 2 infection and provides potential therapeutic targets and strategies.


Subject(s)
/immunology , Chemokine CCL2/blood , Chemokines/blood , Interferon Type I/metabolism , Adult , /physiopathology , China , Disease Progression , Enzyme-Linked Immunosorbent Assay , Female , Humans , Interferon Regulatory Factor-3/blood , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Receptors, CCR2/blood , Signal Transduction/immunology
6.
Science ; 371(6530): 735-741, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1066809

ABSTRACT

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2-RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD-immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2-RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.


Subject(s)
Antibodies, Viral/immunology , Betacoronavirus/immunology , Nanoparticles , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Coronavirus Infections/immunology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Female , Immune Sera/immunology , Immunization , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , Neutralization Tests , Protein Domains , Receptors, Antigen, B-Cell/immunology , Spike Glycoprotein, Coronavirus/chemistry , /virology
7.
Infect Genet Evol ; 88: 104684, 2021 03.
Article in English | MEDLINE | ID: covidwho-1065474

ABSTRACT

We document two cases of viremic and prolonged active infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) where the viral genome was conserved for two months, but infection was with little or no symptoms. The first infection persisted for 80 days and the second for 62 days. Clearance of infection occurred 40 and 41 days, respectively, after development of detectable antibodies. Both cases were identified incidentally in an investigation of reinfection in a cohort of 133,266 laboratory-confirmed infected persons.


Subject(s)
Antibodies, Viral/blood , Genome, Viral , RNA, Viral/blood , Viremia/immunology , Adult , Asymptomatic Diseases , /virology , Enzyme-Linked Immunosorbent Assay , Female , Humans , Infectious Disease Incubation Period , Male , Reverse Transcriptase Polymerase Chain Reaction , /immunology , Time Factors , Viremia/diagnosis , Viremia/virology
8.
Viruses ; 13(2)2021 02 04.
Article in English | MEDLINE | ID: covidwho-1063429

ABSTRACT

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.


Subject(s)
Antibodies, Viral/blood , Enzyme-Linked Immunosorbent Assay , Neutralization Tests , /immunology , /diagnosis , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , ROC Curve , Sensitivity and Specificity
9.
mSphere ; 6(1)2021 02 03.
Article in English | MEDLINE | ID: covidwho-1063057

ABSTRACT

Reported coronavirus disease 2019 (COVID-19) case counts likely underestimate the true prevalence because mild or asymptomatic cases often go untested. Here, we use a sero-survey to estimate the seroprevalence of IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the St. Louis, MO, metropolitan area in a symptom-independent manner. Five hundred three adult and 555 pediatric serum/plasma samples were collected from patients presenting to Barnes-Jewish Hospital or St. Louis Children's Hospital between 14 April 2020 and 12 May 2020. We developed protocols for in-house enzyme-linked immunosorbent assays (ELISAs) using spike and nucleoprotein and used the assays to estimate a seroprevalence rate based on our samples. Overall IgG seropositivity was estimated to be 1.71% (95% credible interval [CI], 0.04% to 3.38%) in pediatric samples and 3.11% (95% CI, 0.92% to 5.32%) in adult samples. Seropositivity was significantly lower in children under 5 years of age than in adults, but rates between adults and children aged 5 or older were similar. Of the 176 samples tested from children under 4 years of age, none were positive.IMPORTANCE This study determined the percentages of both children and adult samples from the greater St. Louis metropolitan area who had antibodies to SARS-CoV-2 in late April to early May 2020. Approximately 1.7 to 3.1% of the tested individuals had antibodies, indicating that they had previously been infected by SARS-CoV-2. These results demonstrate that the extent of infection was about 10 times greater than the number of confirmed cases at that time. Furthermore, it demonstrated that by 5 years of age, children were infected to an extent similar to that of adults.


Subject(s)
Antibodies, Viral/blood , Antibodies, Viral/immunology , /immunology , /immunology , Adolescent , Adult , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Infant , Male , Middle Aged , Missouri/epidemiology , Seroepidemiologic Studies , Young Adult
10.
Sci Rep ; 11(1): 2776, 2021 02 02.
Article in English | MEDLINE | ID: covidwho-1061095

ABSTRACT

The accurate and prompt diagnosis of SARS-CoV-2 infection is required for the control and treatment of the coronavirus infection disease 2019 (COVID-19). In this study, we aimed to investigate the time courses of the anti-severe acute corona respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM and IgG titers and to evaluate the sensitivity and specificity of such tests according to the specific day after the onset of COVID-19 among a patient population in Japan. We measured the titers of SARS-CoV-2 IgM and IgG in sera from 105 subjects, including 26 symptomatic COVID-19 patients, using chemiluminescent immunoassay (CLIA) methods utilizing magnetic beads coated with SARS-CoV-2 nucleocapsid protein and spike protein. The results of a ROC analysis suggested the possibility that the cutoff values in Japan might be lower than the manufacturer's reported cutoff (10 AU/mL): 1  AU/mL for IgM and 5  AU/mL for IgG. The sensitivity of the test before Day 8 after symptom onset was less than 50%; at Days 9-10, however, we obtained a much higher sensitivity of 81.8% for both IgM and IgG. At 15 days or later after symptom onset, the SARS-CoV-2 IgG test had a sensitivity of 100%. These results suggest that if the number of days since disease onset is taken into consideration, these antibody tests could be very useful for the diagnosis of COVID-19 and similar diseases.


Subject(s)
Antibody Specificity , Immunoglobulin G/immunology , Immunoglobulin M/immunology , /immunology , /virology , Enzyme-Linked Immunosorbent Assay , Humans , Japan
11.
PLoS One ; 16(2): e0246346, 2021.
Article in English | MEDLINE | ID: covidwho-1059627

ABSTRACT

BACKGROUND: In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation. AIM: To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples. METHODS: In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined. RESULTS: The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen's kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases. CONCLUSION: The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.


Subject(s)
Antibodies, Viral/analysis , Enzyme-Linked Immunosorbent Assay/methods , /isolation & purification , /diagnosis , Enzyme-Linked Immunosorbent Assay/instrumentation , Humans , Immunoglobulin G/analysis , Phosphoproteins/analysis , Sensitivity and Specificity
12.
Commun Biol ; 4(1): 129, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-1054066

ABSTRACT

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , /chemistry , Spike Glycoprotein, Coronavirus/chemistry , Adult , /immunology , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , /virology , Convalescence , /metabolism , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Female , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Immune Sera/chemistry , Immunity, Humoral , Lentivirus/genetics , Lentivirus/immunology , Male , Middle Aged , Neutralization Tests , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/metabolism , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/immunology , Receptors, Virus/metabolism , /pathogenicity , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Survival Analysis
13.
Arch Virol ; 166(3): 715-731, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1046770

ABSTRACT

Coronaviruses (CoV) are a family of viral pathogens that infect both birds and mammals, including humans. Seven human coronaviruses (HCoV) have been recognized so far. HCoV-229E, -OC43, -NL63, and -HKU1 account for one-third of common colds with mild symptoms. The other three members are severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. These viruses are responsible for SARS, MERS, and CoV disease 2019 (COVID-19), respectively. A variety of diagnostic techniques, including chest X-rays, computer tomography (CT) scans, analysis of viral nucleic acids, proteins, or whole virions, and host antibody detection using serological assays have been developed for the detection of these viruses. In this review, we discuss conventional serological tests, such as enzyme-linked immunosorbent assay (ELISA), western blot (WB), immunofluorescence assay (IFA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA), as well as biosensor-based assays that have been developed for diagnosing HCoV-associated diseases since 2003, with an in-depth focus on COVID-19.


Subject(s)
Antibodies, Viral/blood , /diagnosis , Middle East Respiratory Syndrome Coronavirus/immunology , Severe Acute Respiratory Syndrome/diagnosis , Antibodies, Viral/immunology , Biosensing Techniques/methods , Blotting, Western/methods , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Humans , Luminescent Measurements/methods , SARS Virus/immunology , Severe Acute Respiratory Syndrome/virology
14.
Cytokine ; 140: 155431, 2021 04.
Article in English | MEDLINE | ID: covidwho-1033094

ABSTRACT

OBJECTIVE: To compare the levels of various cytokines between pregnant women with confirmed coronavirus disease (COVID-19) infection and pregnant women without any defined risk factor. MATERIALS AND METHODS: Pregnant women with confirmed COVID-19 infection (study group)(n = 90) were prospectively compared to a gestational age-matched control group of pregnant women without any defined risk factors (n = 90). Demographic features, clinical characteristics, laboratory parameters, interferon-gamma (IFN γ), interleukin (IL-2), IL-6, IL-10, and IL-17 levels were compared between the groups. Additionally, a correlation analysis was performed in the study group for the assessment of IFN γ, IL-2, IL-6, IL-10, and IL-17 levels with disease severity and CRP levels. RESULTS: Study group had significantly higher pregnancy complication rate, erythrocyte sedimentation rate, C-reactive protein, procalcitonin, ferritin, D-dimer, lactate dehydrogenase, IFN γ, and IL-6 values (p < 0.05). On the other hand, the control group had significantly higher hemoglobin, leukocyte, platelet, lymphocyte, IL-2, IL-10, and IL-17 values (p < 0.05). Statistically significant differences were found between the groups for IFN γ, IL-2, IL-10, and IL-17 values between the trimesters (p < 0.05). Statistically significant positive correlations were found for IFN γ and IL-6 with disease severity (r = 0.41 and p < 0.001 for IFN γ and r = 0.58 and p < 0.001 for IL-6). On the other hand, a moderate negative correlation for IL-2 and a weak negative correlation for IL-10 were present (r = -0.62 and p < 0.001 for IL-2 and r = -0.19 and p = 0.01 for IL-10). A statistically significant positive moderate correlation was found between IL-6 and CRP (r = 0.40 and p < 0.001) CONCLUSION: COVID-19 infection seems to have an impact on the cytokine profile of pregnant women varying according to pregnancy trimesters and cytokine levels seem to be correlated with disease severity.


Subject(s)
/blood , Cytokines/blood , Pregnancy Complications, Infectious/blood , /virology , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Female , Host-Pathogen Interactions , Humans , Interferon-gamma/blood , Interleukin-10/blood , Interleukin-17/blood , Interleukin-2/blood , Pregnancy , Prospective Studies , /physiology
15.
PLoS Pathog ; 17(1): e1009161, 2021 01.
Article in English | MEDLINE | ID: covidwho-1029612

ABSTRACT

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used for clinical trials and therapies across the country. Using this protocol, about 80% of convalescent donor plasmas were potentially suitable for therapies. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs.


Subject(s)
/virology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation , Argentina/epidemiology , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Longitudinal Studies , Male , Middle Aged , Pandemics , Seroepidemiologic Studies
17.
Sensors (Basel) ; 21(2)2021 Jan 08.
Article in English | MEDLINE | ID: covidwho-1016225

ABSTRACT

The outbreak of the coronavirus disease (COVID-19) pandemic caused by the novel coronavirus (SARS-CoV-2) has been declared an international public health crisis. It is essential to develop diagnostic tests that can quickly identify infected individuals to limit the spread of the virus and assign treatment options. Herein, we report a proof-of-concept label-free electrochemical immunoassay for the rapid detection of SARS-CoV-2 virus via the spike surface protein. The assay consists of a graphene working electrode functionalized with anti-spike antibodies. The concept of the immunosensor is to detect the signal perturbation obtained from ferri/ferrocyanide measurements after binding of the antigen during 45 min of incubation with a sample. The absolute change in the [Fe(CN)6]3-/4- current upon increasing antigen concentrations on the immunosensor surface was used to determine the detection range of the spike protein. The sensor was able to detect a specific signal above 260 nM (20 µg/mL) of subunit 1 of recombinant spike protein. Additionally, it was able to detect SARS-CoV-2 at a concentration of 5.5 × 105 PFU/mL, which is within the physiologically relevant concentration range. The novel immunosensor has a significantly faster analysis time than the standard qPCR and is operated by a portable device which can enable on-site diagnosis of infection.


Subject(s)
Biosensing Techniques/instrumentation , /diagnosis , Point-of-Care Testing , Spike Glycoprotein, Coronavirus/analysis , Antigens, Viral/analysis , Biosensing Techniques/methods , Dielectric Spectroscopy , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Enzyme-Linked Immunosorbent Assay/instrumentation , Enzyme-Linked Immunosorbent Assay/methods , Equipment Design , Graphite , Humans , Limit of Detection , Pandemics , Proof of Concept Study , Protein Subunits , Single Molecule Imaging/instrumentation , Single Molecule Imaging/methods , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Time Factors
18.
J Immunol Methods ; 490: 112958, 2021 03.
Article in English | MEDLINE | ID: covidwho-1012442

ABSTRACT

The current Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic is a public health emergency of international concern. Sensitive and precise diagnostic tools are urgently needed. In this study, we developed a SARS-CoV-2 spike (S1) protein enzyme-linked immunosorbent assay (ELISA) to detect SARS-CoV-2-specific antibodies. The SARS-CoV-2 S1 ELISA was found to be specific [97.8% (95% CI, 96.7% - 98.5%)], reproducible and precise (intra-assay coefficient of variability (CV) 5.3%, inter-assay CV 7.9%). A standard curve and the interpolation of arbitrary ELISA units per milliliter served to reduce the variability between different tests and operators. Cross-reactivity to other human coronaviruses was addressed by using sera positive for MERS-CoV- and hCoV HKU1-specific antibodies. Monitoring antibody development in various samples of twenty-three and single samples of twenty-nine coronavirus disease 2019 (COVID-19) patients revealed seroconversion and neutralizing antibodies against authentic SARS-CoV-2 in all cases. The comparison of the SARS-CoV-2 (S1) ELISA with a commercially available assay showed a better sensitivity for the in-house ELISA. The results demonstrate a high reproducibility, specificity and sensitivity of the newly developed ELISA, which is suitable for the detection of SARS-CoV-2 S1 protein-specific antibody responses.


Subject(s)
/methods , Epithelial Cells/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antibodies, Neutralizing/blood , Chlorocebus aethiops , Enzyme Assays , Enzyme-Linked Immunosorbent Assay , Humans , Reproducibility of Results , Sensitivity and Specificity , Seroconversion , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
19.
Cell ; 184(2): 476-488.e11, 2021 01 21.
Article in English | MEDLINE | ID: covidwho-1012326

ABSTRACT

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.


Subject(s)
Antibodies, Neutralizing/immunology , Biomarkers/analysis , /physiopathology , Adult , Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , Antibodies, Viral/blood , Biomarkers/blood , /epidemiology , Comorbidity , Coronavirus/classification , Coronavirus/physiology , Cross Reactions , Cytokines/blood , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin A/analysis , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Male , Massachusetts/epidemiology , Middle Aged , Protein Domains , /physiology , Severity of Illness Index , Spike Glycoprotein, Coronavirus/chemistry , Survival Analysis , Treatment Outcome
20.
Nat Struct Mol Biol ; 28(2): 128-131, 2021 02.
Article in English | MEDLINE | ID: covidwho-1010060

ABSTRACT

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible 'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.


Subject(s)
Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Viral/chemistry , Cold Temperature , Cryoelectron Microscopy , Enzyme-Linked Immunosorbent Assay , Humans , Protein Denaturation , Protein Domains , Protein Stability , Spike Glycoprotein, Coronavirus/ultrastructure , Surface Plasmon Resonance
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