New Paper (Diagnostic): Orthogonal immunoassays for IgG antibodies to SARS-CoV-2 antigens reveal that immune response lasts beyond 4 mo post illness onset

Questions to answer about each paper:

Please provide 1-2 sentences introducing the study and its main findings

This study analyzed IgG levels after SARS-CoV2 infection in 52 patients and found that the immune reaction could last up to four months after the initial exposure and that IgG levels correlate with clinical features such severity of the disease.

Study question(s) being investigated:

The study investigated the sustained presence of four Immunoglobulins (RBD, S1, virus neutralizing (VN), and NP antibody) in the convalescent plasma of 52 individuals after SARS-CoV-2 infection.

What type of testing scenario is being considered?

This test was performed on individuals divided into four groups: no illness, mild, moderate, severe, with a follow up of 4 months.

Study population:

What is the model system (e.g., human study, animal model, cell line study)?

This is a human study. The presence of four IgG was tested on blood samples (convalescent plasma) taken from the patients.

What is the sample size?

52 members, 31 were female, 39 were Caucasian, 9 were Asian, 3 were Hispanic, and 1 was African American.

What is the "pre-test" probability of disease in the study population (i.e., what is the anticipated prevalence of the disease?)

Since this study looked at the post diagnosis biomarkers, the diagnosis has been established by the time of the analysis.

For human studies, the following are related to the pre-test probability:

What countries/regions are considered?

Boston suburbs, MA, USA

What is the age range, gender, other relevant characteristics?

The average age of the cohort was 43.9 y. Of the 52 members, 31 were female, 39 were Caucasian, 9 were Asian, 3 were Hispanic, and 1 was African American.

What is the setting of the study (e.g., random sample of school children, retirement communities, etc.)?

This is a retrospective analysis of blood samples donated by randomly selected healthy individuals and diagnosed patients. Since this is a longitudinal study, the analyses were done on convalescent plasma, because the acute phase of the disease has been already well studied.

What other specific inclusion-exclusion criteria are considered?

Minors, pregnant women, vulnerable population, patients in the acute phase of COVID-19 illness, and medically ill individuals were excluded.

Reference test:

What reference test is considered as a "gold standard" comparator for the test under investigation?

Reference antibody for S1 and RBD ELISA is a control monoclonal antibody that binds S1 (in the RBD) and prevents binding of the ACE2 receptor to the RBD developed in-house. Similarly, reference antibody for the nucleocapsid ELISA was SARS-CoV NP Antibody (Sino Biologicals Catalog #40143)

Test assignment:

How are the new and reference tests assigned?

All the samples were controlled using the reference antibodies.

Are there any other relevant details about the study design?

The study design would only perform longitudinal IgG analysis If initial serologic testing was negative. Then there were no more samples collected; if positive, then one or two more longitudinal samples, each 3 wk to 4 wk apart, were collected.

Test conduct:

How were tests performed?

The test looked at the longitudinal levels of IgGs against four proteins (Spike protein, RBD, NP, VN). The gold standard protocol was modified to improve the specificity of detection of anti−SARS-CoV-2 S1 spike, RBD and NP IgG antibodies. Briefly, flat-well Nunc Maxisorp high protein binding plates (ThermoFisher, 44-2404-21) were coated with either 50 ng of recombinant S1 spike protein (Sino Biologicals, 40591-V08H), 25 ng of spike protein RBD antigen (ACRO Biosystems, SPD-C52H3), or 200 ng of NP (ACRO Biosystems Catalog #NUN-C5227) in coat buffer (50 mM sodium bicarbonate buffer, pH 9.6).

Test Assessment

Describe how individuals are classified as positive or negative, e.g. if a threshold is used.

This study is relevant to assess the length of the immune response post diagnosis, therefore, the status of the individuals was known beforehand.

Is there evidence that the test is precise/reproducible when repeated more than once?

Since this is a longitudinal study, the measurements were cross-validated in each individual. Also, the robustness and reproducibility were optimized before testing on patients. To minimize the background effects from the serum, all four assays were initially optimized using at least 10 negative control serum samples. Furthermore, the specificity of the assays was established using 20 cross-reactive serum samples that were positive for endemic coronaviruses and other respiratory viruses.

Are measurements complete?

Yes, all the individuals in the study were included in the follow-up. However, some of them had two blood samples, while other had a third sample. Initial sample collection for all 52 samples occurred between 32 d and 175 d (mean 83 d) PSO. Twenty-eight members of the cohort who exhibited antibody titer on our assays had additional blood samples collected a second time between 64 and 140 d PSO (mean 102 d). Twenty-two of the 28 had a third sample collected between 92 and 142 d PSO (mean 122 d).

Results summary:

What are the estimated sensitivity, specificity, positive predictive value (PPV), and negative predicted value (NPV)?

The sensitivity and specificity of IgGs were those assessed by the manufacturer. However, the whole point of the paper was that using 4 orthogonal IgGs, we can reduce the discrepancies observed in the follow up studies after SARS-CoV-2 infection.

What are the confidence bounds around these intervals?

Interpretation of results for study population:

How good is the test at ruling in or ruling out a disease based on the post-test probabilities?

This is not relevant for this study because the diagnosis has been already establisedh.

Are there identified side affects of the test?

None mentioned.

Is patient adherence to the test likely to be an issue?

The test was not self-administered so patient adherence was not a factor mentioned in the study. However, some patients had three blood samples while others had two or one, possibly because of issues of adherence.

Extrapolation of conclusions to other groups of individuals

How well is the test likely to work in populations with different pretest odds?

The study discussion mentioned the need for a larger cohort to assess the generality of the results.

How costly is the test?

Not relevant because this is not a publicly available test. This is a test for the investigation of the transience of the immune reaction to SARS-CoV-2.

How difficult is it to perform the test in different settings?

The test has to be made in clinical lab setting.

Could the test be combined with other existing tests?

This is a combination of four IgG tests, which is the main novelty of this work.

Summary of reliability

The study addresses the lack of consistency in reporting results in IgG levels in SARS-CoV-2 patients. To address this question, the authors conducted a longitudinal study of the levels of IgGs against four proteins. The approach was deemed "orthogonal" because the four proteins had various roles and their kinetics were different. Therefore, analyzing the four IgG could resolve inconsistencies in the follow-up of patients. Finally, IgG levels were correlated with clinical outcomes such as severity of the disease. Finally, this study looked at 52 individuals from the Boston area, which limits the interpretation of the results to cities and countries with different social and economic conditions.

Progress

Check off the components as they are completed. If the component is not applicable, check the box as well.

[x] 1-2 sentences introducing the study and its main findings
[x] Describe testing scenario
[x] Describe model system
[x] Sample size
[x] Describe prevalnce of disease
[x] Describe countries/regions are considered
[x] Describe age range, gender, other relevant characteristics
[x] Describe setting of the study
[x] Describe other specific inclusion-exclusion criteria
[x] Describe "gold standard"
[x] Describe how the new and reference tests assigned
[x] Describe other relevant details about the study design
[x] Describe how the tests were performed
[x] Describe how individuals are classified as positive or negative
[x] Describe if test is precise/reproducible
[x] Describe whether measurements are complete
[x] What are the estimated sensitivity, specificity, positive predictive value (PPV), and negative predicted value (NPV)?
[x] What are the confidence bounds around these intervals?
[x] Describe post-test probabilities
[x] Describe side affects of the test
[x] Describe patient adherence
[x] Describe how it will extrapolate
[x] How costly is the test?
[x] How difficult is it to perform the test in different settings?
[x] Could the test be combined with other existing tests?
[x] Summary of reliability

greenelab / covid19-review