key: cord-0993161-4356uufe
authors: Moeller, Maria E.; Fock, Jeppe; Pah, Pearlyn; Veras, Antia De La C.; Bade, Melanie; Donolato, Marco; Israelsen, Simone B.; Eugen‐Olsen, Jesper; Benfield, Thomas; Engsig, Frederik N.
title: Evaluation of commercially available immuno‐magnetic agglutination in comparison to enzyme‐linked immunosorbent assays for rapid point‐of‐care diagnostics of COVID‐19
date: 2021-02-17
journal: J Med Virol
DOI: 10.1002/jmv.26854
sha: 26d6fcfb1c266d3a7b120a6c9b50f650f6c19cab
doc_id: 993161
cord_uid: 4356uufe

INTRODUCTION: Coronavirus disease 2019 (COVID‐19) is caused by Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). Fast, accurate, and simple blood‐based assays for quantification of anti‐SARS‐CoV‐2 antibodies are urgently needed to identify infected individuals and keep track of the spread of disease. METHODS: The study included 33 plasma samples from 20 individuals with confirmed COVID‐19 by real‐time reverse‐transcriptase polymerase chain reaction and 40 non‐COVID‐19 plasma samples. Anti‐SARS‐CoV‐2 immunoglobulin M (IgM)/immunoglobulin A (IgA) or immunoglobulin G (IgG) antibodies were detected by a microfluidic quantitative immunomagnetic assay (IMA) (ViroTrack Sero COVID IgM + IgA/IgG Ab, Blusense Diagnostics) and compared to an enzyme‐linked immunosorbent assay (ELISA) (EuroImmun Medizinische Labordiagnostika). RESULTS: Of the 33 plasma samples from the COVID‐19 patients, 28 were positive for IgA/IgM or IgG by IMA and 29 samples were positive by ELISA. Sensitivity for only one sample per patient was 68% for IgA + IgM and 75% IgG by IMA and 80% by ELISA. For samples collected 14 days after symptom onset, the sensitivity of both IMA and ELISA was around 91%. The specificity of the IMA reached 100% compared to 95% for ELISA IgA and 97.5% for ELISA IgG. CONCLUSION: IMA for COVID‐19 is a rapid simple‐to‐use point‐of‐care test with sensitivity and specificity similar to a commercial ELISA.

Coronavirus disease 2019 is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread globally since its discovery in Wuhan, China in December 2019. 1, 2 In spite of advances in antiviral treatment, it remains a disease with considerable morbidity and mortality. 3, 4 Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of SARS-CoV-2 RNA is the recommended test to diagnose active COVID-19, but several serological tests for have been developed. [5] [6] [7] [8] Immunoassays detect different antibodies to SARS-CoV-2, namely antibodies to different parts of the spike or the nucleocapside protein. [9] [10] [11] [12] Although SARS-CoV-2 RNA can be demonstrated at the onset of COVID-19 symptoms, antibodies against SARS-CoV-2 can in most cases be demonstrated after 11 days (interquartile range [IQR] = 7.0-14.0). 13 So serological testing, in general, cannot replace RT-PCR for diagnosing acute COVID-19 but may serve as a valuable supplement in persons with classical symptoms of COVID-19 and repeated negative RT-qPCR for clarification of diagnosis, although its main application is to assess immunity.

Enzyme-linked immunosorbent assay (ELISA) tests may take hours to perform, are usually batched, and require laboratory facilities and skilled personnel. Lateral flow assays for antibody detection are quick single sample tests but have lower sensitivity compared to ELISA, the read-out is operator dependent, and the result is qualitative. [14] [15] [16] An automated, real-time, and quantitative point-of-care (POC) test using capillary blood with high sensitivity would offer the ability of testing for SARS-CoV-2 antibodies both within and outside of a hospital setting.

In this study, we used a novel POC analysis for SARS-CoV based on automated immunomagnetic assay (IMA) technology. The analysis is performed on a portable POC testing device. Readout of results is automated, real-time, and quantitative using capillary blood.

We compared the performance of a well-tested commercial ELISA for COVID-19 with IMA for rapid testing for COVID-19 antibodies. The aim was to establish the sensitivity and specificity of the IMA, for future use in the clinic during the COVID-19 pandemic.

We included individuals with confirmed COVID-19 by RT-qPCR for SARS-CoV-2 RNA on naso-/oropharyngeal swabs or lower respiratory tract specimens, from March 20 to May 1, 2020, with at least one available plasma samples. 17 and reagent resuspension by the design of microfluidic chambers and channels and control over the angular velocity profile of the cartridge rotation. 21 The optomagnetic signal is obtained by measuring the modulated transmitted light through a suspension of magnetic nanoparticles in response to an alternating magnetic field. 22 The magnetic particles are covalently coupled to antigens or antibodies. Upon target induced magnetic particle agglutination, the change in optical and magnetic anisotropy results in a change in the optomagnetic signal which can be used to quantify the target concentration. [23] [24] [25] Incubating the particle in homogeneous magnetic fields speeds up the reaction kinetics. 23 

Patient characteristics were presented as median with IQR or count with percentage. The data analysis included calculation of the following parameters: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy. Confidence intervals for sensitivity and specificity were calculated using the Exact Binomial method. 27 As case samples were taken from individuals identified as having COVID-19 from a positive RT-PCR, these were considered "true positive." The plots including receiver operating characteristics (ROC)curves were constructed in python using the matplotlib, seaborn, and sklearn packages. Differences in titers were calculated by Fisher's exact test using SPSS statistical software, Version 25.0 (Norusis; SPSS Inc.).

We included a total of 20 individuals, who contributed 33 plasma samples. The individuals were mostly male (65%) with a median age of 71 (IQR = 53-76) years, where most had at least one comorbidity (75%) ( Table S1 ). All had chest radiograph infiltration, and of these, five persons (25%) and more than 30 L; 13 persons (65%). Thirty-five percent of the patients had a "do-not-resuscitate" order and 20%

were limited in treatment in terms of intensive care unit (ICU) admission. Forty-five percent of the patients were admitted to the ICU.

Twenty percent of the patients died within 30 days (Table S1) . (Table S1 ). 

The specificity of IMA was 100.0% for the IgM + IgA and the IgG assays. Three of the 40 control samples (two IgA and one IgG) were positive by ELISA resulting in a specificity of 95.0% and 97.5%, respectively (Table 1) . Additionally, three non-COVID-19 samples were borderline positive by ELISA for IgA corresponding to a lower specificity of 87.5% (Table S1 ). All borderline and false-positive ELISA results were from HIV-negative samples. No sample was indeterminant by IMA.

The PPV of ELISA is likely to be 85.3%-96.7% and of the IMA it is 100%. The NPV for ELISA is measured to be 89.7%-90.5% and of the IMA 88.9%-90.5% (Table 1 ).

The distance of data points from the cut-off values and confidence in assigning a positive or negative result differed between the IMA and ELISA assays (Figure 2 ). The distribution of positive and negative data points was distinct for the IMA cartridge, with a cut-off value above all the negative samples, which allowed for unequivocal interpretation of all measurements. In contrast, the ELISA data had less separation, especially for IgA, resulting in a "grey zone" of borderline data points to which a positive or negative result could not be assigned. Both positive and negative samples have borderline data points. ROC curves of the assays all showed an area under the curve above 0.93 (see Figure S1 ). individuals testing negative but presenting clinical symptoms of COVID-19 need to be retested using another serological test or RT-qPCR. 28, 32, 33 Here, we found that the NPV of the IMA is likely to be comparable to that of a commercially available ELISA test (90%-91%).

The limitation of a given sample producing a false-negative result is correlated to different factors, such as time of testing in relation to symptom onset, changes in antibody levels during illness, and severity of the disease. Several studies covering the use of ELISA, Chemiluminescence immunoassays (CLIA), and qualitative assays show that full diagnostic sensitivity for neither IgM nor IgG is reached before approximately 14-22 days from onset of symptoms. 30, 31, [34] [35] [36] It has been reported that IgM detection was more variable than IgG, and detection was the highest when IgM and IgG results were combined for both ELISA and POCs. 28 The addition of IgA may improve sensitivity as it has been found to have higher titers than IgM. 37 Using IMA cartridges, we observed better performance of the IgA + IgM/IgG combination in terms of sensitivity while keeping the specificity at 100%.

In this study, we compared two different serological technologies against two different antigens. Preferably it should have been two different serological technologies against the same antigen but this was not available at the time. Two previous studies have shown that antibodies to the nucleocapside antigen, which is smaller than the spike protein and lacks a glycosylation site, can be measured earlier than antibodies to the spike protein antigen. 13, 38, 39 This could increase the sensitivity of the IMA but in this study, we only found The detailed clinical data including symptom onset and disease severity improved the interpretation of the results as antibody titers were found to be affected by both as previously reported. 33, 44 Comparison of a well-tested commercial ELISA strengthens the evaluation of the novel IMA. 

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Evaluation of commercially available immuno-magnetic agglutination in comparison to enzyme-linked immunosorbent assays for rapid point-of-care diagnostics of COVID-19