Dynamics of SARS-CoV-2 seroassay sensitivity: a systematic review and modelling study.

BackgroundSerological surveys have been the gold standard to estimate numbers of SARS-CoV-2 infections, the dynamics of the epidemic, and disease severity. Serological assays have decaying sensitivity with time that can bias their results, but there is a lack of guidelines to account for this phenomenon for SARS-CoV-2.AimOur goal was to assess the sensitivity decay of seroassays for detecting SARS-CoV-2 infections, the dependence of this decay on assay characteristics, and to provide a simple method to correct for this phenomenon.MethodsWe performed a systematic review and meta-analysis of SARS-CoV-2 serology studies. We included studies testing previously diagnosed, unvaccinated individuals, and excluded studies of cohorts highly unrepresentative of the general population (e.g. hospitalised patients).ResultsOf the 488 screened studies, 76 studies reporting on 50 different seroassays were included in the analysis. Sensitivity decay depended strongly on the antigen and the analytic technique used by the assay, with average sensitivities ranging between 26% and 98% at 6 months after infection, depending on assay characteristics. We found that a third of the included assays departed considerably from manufacturer specifications after 6 months.ConclusionsSeroassay sensitivity decay depends on assay characteristics, and for some types of assays, it can make manufacturer specifications highly unreliable. We provide a tool to correct for this phenomenon and to assess the risk of decay for a given assay. Our analysis can guide the design and interpretation of serosurveys for SARS-CoV-2 and other pathogens and quantify systematic biases in the existing serology literature.

Meta-analysis of diagnostic performance of serological tests for SARS-CoV-2 antibodies up to 25 April 2020 and public health implications.

We reviewed the diagnostic accuracy of SARS-CoV-2 serological tests. Random-effects models yielded a summary sensitivity of 82% for IgM, and 85% for IgG and total antibodies. For specificity, the pooled estimate were 98% for IgM and 99% for IgG and total antibodies. In populations with ≤ 5% of seroconverted individuals, unless the assays have perfect (i.e. 100%) specificity, the positive predictive value would be ≤ 88%. Serological tests should be used for prevalence surveys only in hard-hit areas.

Laboratory-Based SARS-CoV-2 Receptor Binding Domain Serologic Assays Perform with Equivalent Sensitivity and Specificity to Commercial FDA-EUA Approved Tests.

During early phases of the SARS-CoV-2 epidemic, many research laboratories repurposed their efforts towards developing diagnostic testing that could aid public health surveillance while commercial and public diagnostic laboratories developed capacity and validated large scale testing methods. Simultaneously, the rush to produce point-of-care and diagnostic facility testing resulted in FDA Emergency Use Authorization with scarce and poorly validated clinical samples. Here, we review serologic test results from 186 serum samples collected in early phases of the pandemic (May 2020) from skilled nursing facilities tested with six laboratory-based and two commercially available assays. Serum neutralization titers were used to set cut-off values using positive to negative ratio (P/N) analysis to account for batch effects. We found that laboratory-based receptor binding domain (RBD) binding assays had equivalent or superior sensitivity and specificity compared to commercially available tests. We also determined seroconversion rate and compared with qPCR outcomes. Our work suggests that research laboratory assays can contribute reliable surveillance information and should be considered important adjuncts to commercial laboratory testing facilities during early phases of disease outbreaks.

Current methods for diagnosis of human coronaviruses: pros and cons.

The current global fight against coronavirus disease (COVID-19) to flatten the transmission curve is put forth by the World Health Organization (WHO) as there is no immediate diagnosis or cure for COVID-19 so far. In order to stop the spread, researchers worldwide are working around the clock aiming to develop reliable tools for early diagnosis of severe acute respiratory syndrome (SARS-CoV-2) understanding the infection path and mechanisms. Currently, nucleic acid-based molecular diagnosis (real-time reverse transcription polymerase chain reaction (RT-PCR) test) is considered the gold standard for early diagnosis of SARS-CoV-2. Antibody-based serology detection is ineffective for the purpose of early diagnosis, but a potential tool for serosurveys, providing people with immune certificates for clearance from COVID-19 infection. Meanwhile, there are various blooming methods developed these days. In this review, we summarise different types of coronavirus discovered which can be transmitted between human beings. Methods used for diagnosis of the discovered human coronavirus (SARS, MERS, COVID-19) including nucleic acid detection, gene sequencing, antibody detection, antigen detection, and clinical diagnosis are presented. Their merits, demerits and prospects are discussed which can help the researchers to develop new generation of advanced diagnostic tools for accurate and effective control of human coronavirus transmission in the communities and hospitals.

Highly Sensitive and Specific SARS-CoV-2 Serological Assay Using a Magnetic Modulation Biosensing System.

Sensitive serological assays are needed to provide valuable information about acute and past viral infections. For example, detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies could serve as the basis for an "immunity passport" that would enable individuals to travel internationally. Here, utilizing a novel Magnetic Modulation Biosensing (MMB) system and the receptor-binding domain of the SARS-CoV-2 spike protein, we demonstrate a highly sensitive and specific anti-SARS-CoV-2 IgG serological assay. Using anti-SARS-CoV-2 IgG antibodies, RT-qPCR SARS-CoV-2-positive and healthy patients' samples, and vaccinees' samples, we compare the MMB-based SARS-CoV-2 IgG assay's analytical and clinical sensitivities to those of the enzyme-linked immunosorbent assay (ELISA). Compared with ELISA, the MMB-based assay has an ~6-fold lower limit of detection (129 ng/L vs. 817 ng/L), and it detects an increase in the IgG concentration much earlier after vaccination. Using 85 RT-qPCR SARS-CoV-2-positive samples and 79 -negative samples, the MMB-based assay demonstrated similar clinical specificity (98% vs. 99%) and sensitivity (93% vs. 92%) to the ELISA test, but with a much faster turnaround time (45 min vs. 245 min). The high analytical and clinical sensitivity, short turnaround time, and simplicity of the MMB-based assay makes it a preferred method for antibody detection.

Performance evaluation of newly developed surrogate virus neutralization tests for detecting neutralizing antibodies against SARS-CoV-2.

We evaluated newly developed surrogate virus neutralization tests (sVNT) for detecting neutralizing antibodies (NAbs) against the receptor binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). VERI-Q SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit (MiCo BioMed, Gyeonggi-do, Republic of Korea, hereafter, "eCoV-CN") is an enzyme-linked immunosorbent assay-based sVNT, and VERI-Q SARS-CoV-2 Neutralizing Antibody Rapid Test Kit (MiCo BioMed, hereafter, "rCoV-RN") is a point-of-care lateral-flow immunochromatography test with auto-scanner. A total of 411 serum samples were evaluated. Both evaluations used a 50% plaque reduction neutralization test (PRNT50) as the gold standard. Compared with PRNT50, the eCoV-CN showed 98.7% positive percent agreement (PPA), 96.8% negative percent agreement (NPA), 97.4% total percent agreement (TPA), with kappa values of 0.942. The rCoV-RN showed 98.7% PPA, 97.4% NPA, 97.8% TPA, and kappa values of 0.951, comparing to PRNT50. Neither assay indicated cross-reactivity for other pathogens, and the signal indexes were statistically significantly correlated to the PRNT50 titer. The two evaluated sVNTs show comparable performances to the PRNT50 with the advantages of technical simplicity, speed, and do not require cell culture facilities.

Evaluation of ten (10) SARS-CoV-2 rapid serological tests in comparison with WANTAI SARS-CoV-2 ab ELISA in Burkina Faso, West Africa.

BACKGROUND: The aim of this study was to evaluate the performance of ten (10) SARS-CoV-2 serological rapid diagnostic tests in comparison with the WANTAI SARS-CoV-2 Ab ELISA test in a laboratory setting. MATERIALS AND METHODS: Ten (10) SARS-CoV-2 serological rapid diagnostic tests (RDTs) for SARS-CoV-2 IgG/IgM were evaluated with two (2) groups of plasma tested positive for one and negative for the other with the WANTAI SARS-CoV-2 Ab ELISA. The diagnostic performance of the SARS-CoV-2 serological RDTs and their agreement with the reference test were calculated with their 95% confidence intervals. RESULTS: The sensitivity of serological RDTs ranged from 27.39 to 61.67% and the specificity from 93.33 to 100% compared to WANTAI SARS-CoV-2 Ab ELISA test. Of all the tests, two tests (STANDARD Q COVID-19 IgM/IgG Combo SD BIOSENSOR and COVID-19 IgG/IgM Rapid Test (Zhejiang Orient Gene Biotech Co., Ltd)) had a sensitivity greater than 50%. In addition, all ten tests had specificity greater than or equal to 93.33% each. The concordance between RDTs and WANTAI SARS-CoV-2 Ab ELISA test ranged from 0.25 to 0.61. CONCLUSION: The SARS-CoV-2 serological RDTs evaluated show low and variable sensitivities compared to the WANTAI SARS-CoV-2 Ab ELISA test, with however a good specificity. These finding may have implications for the interpretation and comparison of COVID-19 seroprevalence studies depending on the type of test used.

Silent Antibodies Start Talking: Enhanced Lateral Flow Serodiagnosis with Two-Stage Incorporation of Labels into Immune Complexes.

The presence of pathogen-specific antibodies in the blood is widely controlled by a serodiagnostic technique based on the lateral flow immunoassay (LFIA). However, its common one-stage format with an antigen immobilized in the binding zone of a test strip and a nanodispersed label conjugated with immunoglobulin-binding proteins is associated with risks of very low analytical signals. In this study, the first stage of the immunochromatographic serodiagnosis was carried out in its traditional format using a conjugate of gold nanoparticles with staphylococcal immunoglobulin-binding protein A and an antigen immobilized on a working membrane. At the second stage, a labeled immunoglobulin-binding protein was added, which enhanced the coloration of the bound immune complexes. The use of two separated steps, binding of specific antibodies, and further coloration of the formed complexes, allowed for a significant reduction of the influence of non-specific immunoglobulins on the assay results. The proposed approach was applied for the serodiagnosis using a recombinant RBD protein of SARS-CoV-2. As a result, an increase in the intensity of test zone coloration by more than two orders of magnitude was demonstrated, which enabled the significant reduction of false-negative results. The diagnostic sensitivity of the LFIA was 62.5% for the common format and 100% for the enhanced format. Moreover, the diagnostic specificity of both variants was 100%.

A review post-vaccination SARS-CoV-2 serological test: Method and antibody titer response.

The development of the Coronavirus disease 2019 (COVID-19) vaccine is one of the most important efforts in controlling the pandemic. Serological tests are used to identify highly reactive human donors for convalescent plasma therapy, measuring vaccine efficacy and durability. This review article presents a review of serology tests and how antibody titers in response to vaccines have been developed. Some of the serological test methods discussed are Plaque Reduction Neutralization Test (PRNT), Enzyme-Linked Immunosorbent Assay (ELISA), Lateral flow immunoassay (LFIA), chemiluminescent immunoassay (CLIA), and Chemiluminescent Micro-particle Immunoassay (CMIA). This review can provide an understanding of the application of the body's immune response to vaccines to get some new strategies for vaccines.

Array-Based Multiplex and High-Throughput Serology Assays.

The detection of antibody responses using serological tests provides means to diagnose infections, follow disease transmission, and monitor vaccination responses. The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, highlighted the need for rapid development of robust and reliable serological tests to follow disease spreading. Moreover, the rise of SARS-CoV-2 variants emphasized the need to monitor their transmission and prevalence in the population. For this reason, multiplex and flexible serological assays are needed to allow for rapid inclusion of antigens representing new variants as soon as they appear. In this chapter, we describe the generation and application of a multiplex serological test, based on bead array technology, to detect anti-SARS-CoV-2 antibodies in a high-throughput manner, using only a few microliters of sample. This method is currently expanding to include a multi-disease antigen panel that will allow parallel detection of antibodies towards several infectious agents.

Real-world performance of SARS-Cov-2 serology tests in the United States, 2020.

BACKGROUND: Real-world performance of COVID-19 diagnostic tests under Emergency Use Authorization (EUA) must be assessed. We describe overall trends in the performance of serology tests in the context of real-world implementation. METHODS: Six health systems estimated the odds of seropositivity and positive percent agreement (PPA) of serology test among people with confirmed SARS-CoV-2 infection by molecular test. In each dataset, we present the odds ratio and PPA, overall and by key clinical, demographic, and practice parameters. RESULTS: A total of 15,615 people were observed to have at least one serology test 14-90 days after a positive molecular test for SARS-CoV-2. We observed higher PPA in Hispanic (PPA range: 79-96%) compared to non-Hispanic (60-89%) patients; in those presenting with at least one COVID-19 related symptom (69-93%) as compared to no such symptoms (63-91%); and in inpatient (70-97%) and emergency department (93-99%) compared to outpatient (63-92%) settings across datasets. PPA was highest in those with diabetes (75-94%) and kidney disease (83-95%); and lowest in those with auto-immune conditions or who are immunocompromised (56-93%). The odds ratios (OR) for seropositivity were higher in Hispanics compared to non-Hispanics (OR range: 2.59-3.86), patients with diabetes (1.49-1.56), and obesity (1.63-2.23); and lower in those with immunocompromised or autoimmune conditions (0.25-0.70), as compared to those without those comorbidities. In a subset of three datasets with robust information on serology test name, seven tests were used, two of which were used in multiple settings and met the EUA requirement of PPA ≥87%. Tests performed similarly across datasets. CONCLUSION: Although the EUA requirement was not consistently met, more investigation is needed to understand how serology and molecular tests are used, including indication and protocol fidelity. Improved data interoperability of test and clinical/demographic data are needed to enable rapid assessment of the real-world performance of in vitro diagnostic tests.

Accuracy of serological tests for COVID-19: A systematic review and meta-analysis.

Objective: To determine the diagnostic accuracy of serological tests for coronavirus disease-2019 (COVID-19). Methods: PubMed, Embase and the Cochrane Library were searched from January 1 2020 to September 2 2022. We included studies that measured the sensitivity, specificity or both qualities of a COVID-19 serological test and a reference standard of a viral culture or reverse transcriptase polymerase chain reaction (RT-PCR). The risk of bias was assessed by using quality assessment of diagnostic accuracy studies 2 (QUADAS-2). The primary outcomes included overall sensitivity and specificity, as stratified by the methods of serological testing [enzyme-linked immunosorbent assays (ELISAs), lateral flow immunoassays (LFIAs) or chemiluminescent immunoassays (CLIAs)] and immunoglobulin classes (IgG, IgM, or both). Secondary outcomes were stratum-specific sensitivity and specificity within the subgroups, as defined by study or participant characteristics, which included the time from the onset of symptoms, testing via commercial kits or an in-house assay, antigen target, clinical setting, serological kit as the index test and the type of specimen for the RT-PCR reference test. Results: Eight thousand seven hundred and eighty-five references were identified and 169 studies included. Overall, we judged the risk of bias to be high in 47.9 % (81/169) of the studies, and a low risk of applicability concerns was found in 100% (169/169) of the studies. For each method of testing, the pooled sensitivity of the ELISAs ranged from 81 to 82%, with sensitivities ranging from 69 to 70% for the LFIAs and 77% to 79% for the CLIAs. Among the evaluated tests, IgG (80-81%)-based tests exhibited better sensitivities than IgM-based tests (66-68%). IgG/IgM-based CLIA had the highest sensitivity [87% (86-88%)]. All of the tests displayed high specificity (97-98%). Heterogeneity was observed in all of the analyses. The detection of nucleocapsid protein (77-80%) as the antigen target was found to offer higher sensitivity results than surface protein detection (66-68%). Sensitivity was higher in the in-house assays (78-79%) than in the commercial kits (47-48%). Conclusion: Among the evaluated tests, ELISA and CLIA tests performed better in terms of sensitivity than did the LFIA. IgG-based tests had higher sensitivity than IgM-based tests, and combined IgG/IgM test-based CLIA tests had the best overall diagnostic test accuracy. The type of sample, serological kit and timing of use of the specific tests were associated with the diagnostic accuracy. Due to the limitations of the serological tests, other techniques should be quickly approved to provide guidance for the correct diagnosis of COVID-19.

Borrelia multiplex: a bead-based multiplex assay for the simultaneous detection of Borrelia specific IgG/IgM class antibodies.

BACKGROUND: Lyme borreliosis (LB) is the most common tick-borne infectious disease in the northern hemisphere. The diagnosis of LB is usually made by clinical symptoms and subsequently supported by serology. In Europe, a two-step testing consisting of an enzyme-linked immunosorbent assay (ELISA) and an immunoblot is recommended. However, due to the low sensitivity of the currently available tests, antibody detection is sometimes inaccurate, especially in the early phase of infection, leading to underdiagnoses. METHODS: To improve upon Borrelia diagnostics, we developed a multiplex Borrelia immunoassay (Borrelia multiplex), which utilizes the new INTELLIFLEX platform, enabling the simultaneous dual detection of IgG and IgM antibodies, saving further time and reducing the biosample material requirement. In order to enable correct classification, the Borrelia multiplex contains eight antigens from the five human pathogenic Borrelia species known in Europe. Six antigens are known to mainly induce an IgG response and two antigens are predominant for an IgM response. RESULTS: To validate the assay, we compared the Borrelia multiplex to a commercial bead-based immunoassay resulting in an overall assay sensitivity of 93.7% (95% CI 84.8-97.5%) and a specificity of 96.5% (95%CI 93.5-98.1%). To confirm the calculated sensitivity and specificity, a comparison with a conventional 2-step diagnostics was performed. With this comparison, we obtained a sensitivity of 95.2% (95% CI 84.2-99.2%) and a specificity of 93.0% (95% CI 90.6-94.7%). CONCLUSION: Borrelia multiplex is a highly reproducible cost- and time-effective assay that enables the profiling of antibodies against several individual antigens simultaneously.

Low infectious risk of re-positive COVID-19 patients: a single-center study.

OBJECTIVE: The aim of this study was to investigate the infectiousness of re-positive coronavirus disease 2019 (COVID-19) patients. METHODS: All nucleic acid testing (NAT) was performed using throat swabs, nasopharyngeal swabs, and anal swabs, which were tested by Fluorescent quantitative realtime PCR. Re-positive cases were defined as a discharged patient who re-tested positive by NAT. Micro-neutralization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was performed based on the methods for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) viruses. IgM and IgG against the N protein of SARS-CoV-2 were determined by ELISA. RESULTS: A total 255 (16.04%) of 1590 COVID-19 patients were re-positive. The re-positive cases were more likely to occur in patients in the 20-39 years age group and in patients with disease of moderate severity. Quantitative PCR showed that cycle threshold (Ct) values and viral loads were both far lower than in the hospitalized COVID-19 patients. The viral load in re-positive cases was very low. Viral culture of the samples from re-positive patients showed no cytopathic effect, and NAT of the culture medium of viral cultures all exhibited negative results. CONCLUSION: The viral load in re-positive cases was very low; patients were not infectious and the risk of human-to-human transmission was extremely low. Discharged COVID-19 patients should undergo home health management for 3 weeks.

Prevalence of molecular and serological tests of the new coronavirus (SARS-CoV-2) in Carlos Chagas laboratory - Sabin group in Cuiabá.

BACKGROUND: Coronavirus disease 2019, which is caused by the new severe acute respiratory syndrome coronavirus 2, became a pandemic in 2020 with a mortality rate of 2% and high transmissibility, thus making studies with an epidemiological profile essential. OBJECTIVES: The aim of this study was to characterize the population that performed the severe acute respiratory syndrome coronavirus 2 molecular and serological tests in Carlos Chagas Laboratory - Sabin Group in Cuiabá. METHODS: A retrospective cross-sectional study was carried out with all the samples collected from nasal swab tested by RT-PCR and serological for severe acute respiratory syndrome coronavirus 2 IgM/IgG from the population served between April and December 2020. FINDINGS: In the analysis period, 23,631 PCR-coronavirus disease 2019 examinations were registered. Of this total number of cases, 7,649 (32.37%) tested positive, while 15,982 (66.31%) did not detect viral RNA and 374 of the results as undetermined. The peak of positive RT-PCR performed in July (n=5,878), with 35.65% (n=2,096). A total of 8,884 tests were performed on serological test SOROVID-19, with a peak of 1,169 (57.16%) of the positive tests for severe acute respiratory syndrome coronavirus 2 in July. MAIN CONCLUSIONS: Molecular positivity and serological tests, both peaked in July 2020, were mostly present in women aged 20-59 years, characterizing Cuiabá as the epicenter of the Midwest region in this period due to the high rate of transmissibility of severe acute respiratory syndrome coronavirus 2.

Review of current in vitro COVID-19 diagnostics methods.

The diagnosis of COVID-19 is considered a significant step in the management of the disease that is causing a major worldwide public health challenge from the time of its emergence in December 2019. Since it has been established that SARS-CoV-2 spreads rapidly, timely detection of the positive cases and isolation of such individuals and their contacts helps in containing viral transmission. In this paper, we review the in vitro technology platforms for testing and diagnosing COVID-19 patients: molecular tests, rapid antigen tests, and serology tests. As part of our review of each category of tests, we discuss the commercialized testing platforms, their analyzing systems, specimen collection protocols, and testing methodologies. Moreover, the efficacy and limitations of each technique are also discussed. The key structural components of the virus are presented to provide an understanding of the scientific principles behind the testing tools.

Diagnostic performance of molecular and serological tests of SARS-CoV-2 on well-characterised specimens from COVID-19 individuals: The EDCTP "PERFECT-study" protocol (RIA2020EF-3000).

BACKGROUND: The SARS-CoV-2 pandemic is a global threat affecting 210 countries, with 2,177,469 confirmed cases and 6.67% case fatality rate as of April 16, 2020. In Africa, 17,243 cases have been confirmed, but many remain undiagnosed due to limited laboratory-capacity, suboptimal performance of used molecular-assays (~30% false negative, Yu et al. and Zhao et al., 2020) and limited WHO-recommended rapid-tests. OBJECTIVES: We aim to implement measures to minimize risks for COVID-19 in Cameroon, putting together multidisciplinary highly-experienced virologists, immunologists, bioinformaticians and clinicians, to achieve the following objectives: (a) to integrate/improve available-infrastructure, methodologies, and expertise on COVID-19. For this purpose, we will create a platform enabling researchers/clinicians to better integrate and translate evidence into the COVID-19 clinical-practice; (b) to enhance capacities in Cameroon for screening/detecting individuals with high-risks of COVID-19, by setting-up effective core-facilities on-site; (c) to validate point-of-care SARS-CoV-2 molecular assays allowing same-day result delivery, thus permitting timely diagnosis, treatment, and retention in care of COVID-19 patients; (d) to implement SARS-CoV-2 diagnosis with innovative/highly sensitive ddPCR-based assays and viral genetic characterization; (e) to validate serological assays to identify COVID-19-exposed persons and follow-up of convalescents. METHODS: This is a prospective, observational study conducted among COVID-19 suspects/contacts during 24 months in Cameroon. Following consecutive sampling of 1,536 individuals, oro/nasopharyngeal swabs and sera will be collected. Well characterised biorepositories will be established locally; molecular testing will be performed on conventional real-time qPCR, point-of-care GeneXpert, antigen-tests and digital droplet PCR (ddPCR); SARS-CoV2 amplicons will be sequenced; serological testing will be performed using ELISA, and antibody-based kits. Sensitivity, specificity, positive- and negative-predictive values will be evaluated. EXPECTED OUTCOMES: These efforts will contribute in creating the technical and clinical environment to facilitate earlier detection of Sars-CoV-2 in Africa in general and in Cameroon in particular. Specifically, the goals will be: (a) to implement technology transfer for capacity-building on conventional and point-of-care molecular assays, achieving a desirable performance for clinical diagnosis of SARS-CoV2; (b) to integrate/improve the available infrastructure, methodologies, and expertise on Sars-CoV2 detection; (c) to improve the turn-around-time for diagnosing COVID-19 infection with obvious advantage for patients/clinical management thanks to low-cost assays, thus permitting timely treatment and retention in care; (d) to assess the epidemiology of COVID-19 and circulating-variants in Cameroon as compared to strains found in other countries.