key: cord-0831250-4087yiz5
authors: Trombetta, B. A.; Kandigian, S. E.; Kitchen, R. R.; Grauwet, K.; Webb, P. K.; Miller, G. A.; Jennings, C. G.; Jain, S.; Miller, S. M.; Kuo, Y.; Sweeney, T.; Gilboa, T.; Norman, M.; Simmons, D. P.; Ramirez, C. E.; Bedard, M.; Fink, C.; Ko, J.; Peralta, E. J. D. L.; Watts, G.; Gomez-Rivas, E.; Davis, V.; Barilla, R.; Wang, J.; Cunin, P.; Bates, S.; Morrison-Smith, C.; Nicholson, B.; Wong, E.; El-Mufti, L.; Kann, M.; Bolling, A.; Fortin, B.; Ventresca, H.; Zhou, W.; Pardo, S.; Kwock, M.; Hazra, A.; Cheng, L.; Ahmad, R.; Toombs, J. A.; Larson, R.; Pleskow, H.; Luo, N. M.; Samaha, C.; Pandya, U. M.
title: Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2
date: 2021-01-04
journal: nan
DOI: 10.1101/2021.01.02.20248998
sha: cedc568b28dad7c93e366ee997945f44bbac985d
doc_id: 831250
cord_uid: 4087yiz5

Background: COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. Methods: We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. Results: Combined IgG+IgM sensitivities ranged from 33.9% to 94.6%, while combined specificities ranged from 92.6% to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG+IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG+IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 g/mL), followed by a similar LOD of 1.5 g/mL for CareHealth, Cellex, KHB, and Vivachek. Conclusion: We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Coronavirus disease 2019 , caused by infection with the severe 30 acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a global 31 pandemic on March 11 th , 2020 1 . A second wave of the pandemic is well underway 1, 2, 32 3, 4 . However, accurate estimates of transmission rely on accurate and widely available 33 immunosurveillance tools to measure SARS-CoV-2 infection in diverse community 34 settings. Among SARS-CoV-2-infected individuals, 40-45% are estimated to remain 35 asymptomatic 5 , suggesting that prevalence is likely underestimated 6 . Therefore, 36

detecting prior exposure to SARS-CoV-2 as opposed to other viruses, including other 37 coronaviruses is crucial 7 . 38

There are different types of clinical SARS-CoV-2 tests 8 . Diagnostic testing 39 relies on reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and 40

antigen-based immunodiagnostics to detect active infection 9, 10, 11 . Conversely, 41 serological tests can monitor population prevalence and prior exposure by measuring 42 antibodies against SARS-CoV-2 12, 13, 14 . These include enzyme-linked immunosorbent 43 assays (ELISAs), chemiluminescence assays, and lateral flow assays (LFAs) 11, 15, 16 . 44 LFAs are attractive for home testing and population surveillance, since they are 45 affordable, scalable, rely on easily accessible specimens such as fingerstick whole 46 blood and give a result readout within minutes 13 . Since multiple vaccines received 47 emergency use authorization 17 , LFAs could be used to determine whether vaccines 48 elicit a detectable and durable immune response 18, 19, 20, 21, 22 . Furthermore, early 49 seroconversion was shown to predict better clinical outcomes 23, 24 . Hence, easy-to-50 use LFAs will have important applications in the upcoming phases of the pandemic. 51

Since the onset of the COVID-19 epidemic, multiple studies evaluated the accuracy of 52 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 4, 2021. ; https://doi. org/10.1101 org/10. /2021 serological tests 15, 25, 26, 27, 28, 29 . Many of these tests received Emergency Use 53 Authorization (EUA) through the Food and Drug Administration (FDA) 30 . 54

Despite the utility of SARS-CoV2 antibody tests, misinterpretation of results is 55 very likely 31 . A negative serological test result does not preclude prior infection since 56 seroconversion occurs 9-11 days after symptoms onset for IgM and 18-20 days for 57

IgG antibodies, respectively 16, 32, 33, 34 . Conversely, positive results do not indicate 58 active infection 31 . Furthermore, the prevalence of SARS-CoV-2 is highly variable 1, 6 , 59 and known to directly impact the predictive value of a test result. A higher prevalence 60 increases the likelihood that positive test results indicate a real infection (i.e. higher 61 positive predictive value) 35 , but will also decrease the negative predictive value, 62 resulting in more false negative results 35 . Therefore, accessible tools to assist the 63 public with interpreting results based on test accuracy and different prevalence 64 scenarios are critical 31, 36 . 65

In April 2020, the Mass General Brigham Center (MGB) for COVID Innovation's 66 direct-to-consumer working group scanned available serological assays and selected 67 20 lateral flow assays, based on reported assay characteristics and supply chain 68 availability 37 . The LFAs were evaluated by blinded operators using the same samples 69 to standardize the evaluation of their accuracy. Additionally, we developed a user-70 friendly web-tool to provide context for the end user to interpret their results. Here, we 71 report the evaluation data to serve as a public resource to guide implementation of 72

LFAs, and the tool to aid the interpretation of home testing results. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 4, 2021. ; https://doi. org/10.1101 org/10. /2021 Methods 75

We procured 56 SARS-CoV-2 positive, 46 pre-pandemic negative, and 10 HIV+ EDTA Developed Test) 10-40 days prior to sample collection. The participants' charts were 85 reviewed by study staff to identify samples collected ≥10 days after onset of symptoms 86 and to exclude immunosuppressed participants, after which samples were 87 anonymized and stripped of protected health information. Pre-pandemic negative 88 control samples were randomly selected from healthy participants with a Charlson 89

Age-Comorbidity Index 38 score ≤2, with EDTA plasma banked in the MGB Biobank 90 between Jan 1-Dec 1, 2019 from inpatients. HIV-positive control samples were 91 obtained from EDTA plasma samples banked prior to January 2020 in a study on 92 neuropathic pain in HIV. All HIV-positive participants were on antiretroviral therapy. 93

For 8 out of the 10 HIV-positive samples, viral load quantification was available and 94

showed 256 copies/ml or less, and 5 showed either undetectable loads or under 20 95 copies/ml. The study was approved under the Massachusetts General Brigham (MGB) 96

Institutional Review Board (protocol no. 2020P001204). 97 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

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Twenty commercial IgG/IgM lateral flow assays (LFAs) from 18 manufacturers were 99 evaluated (Supplementary Table 1 ). LFAs were analyzed by blinded operators 100 according to manufacturer instructions for use (IFU), with the exception of using 101 micropipettes instead of manufacturer-provided droppers to minimize technical 102 variability. Samples were thawed on ice, randomized, and brought to room 103 temperature. Kit components were also brought to room temperature. The IFU-104 specified volumes of sample and buffer were added to the cassette. Specified sample 105 volumes varied for different LFAs but were typically in the 5-20µL range. The cassettes 106

were run at room temperature on a flat surface and results read immediately after the 107 time interval defined in the IFU (typically ranging from 10-15 minutes). Each cassette 108 was independently scored by two blinded raters as either "positive," "negative," or 109 

For inter-operator reproducibility analysis, separate pools of EDTA plasma were 115 obtained from >30 pre-pandemic healthy individuals (negative pool) and >30 116 convalescent participants collected after symptom resolution at the Massachusetts 117

General Hospital (MGH) respiratory illness clinic (positive pool). Convalescent 118 samples for the positive pool were confirmed to be positive using the COBAS SARS-119

CoV-2 PCR test (Roche Diagnostics, Indianapolis, IN) at MGH. A total of 20 replicates 120 per pool were run by two independent pairs of blinded operators, alternating between 121 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

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Four independent operators working in teams of two on separate days applied each 169 pool 10 times to each LFA, with processing as dictated by the instructions for use 170 (IFU). API version1 LFAs were not assessed for reproducibility due to limited cassette 171 availability and high sample volumes required. Of the remaining 19 LFAs, three 172 (BTNX, Camtech, and Carehealth) had 100% consistent correct outcomes across both 173 isotypes, with an additional three (BioHit, Zhuhai Livzon, and Phamatech) having no 174 incorrect or inconsistent outcomes with one or two invalid tests (Figure 1) Table 3 ). While these kits may not yet be intended for the 187 general public, it will be important to clarify the instructions moving forward and include 188 clearly marked droppers to minimize potential for sample volume errors. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 4, 2021. (Table 3 ). Under the assumption that the likelihood of two randomly occurring false 201 positives for any one individual is low, an IgG/IgM composite score (averaged operator 202 scores, see Methods) was produced to maximize test specificity. Using this composite 203 score, all LFAs except BioHit, Cellex, Edinburgh, InTec and Vivachek achieved a 204 specificity of 100%. This result underscores the potential of considering the outcome 205 in both isotypes to minimize false positives, although it is more likely that a single 206 isotype will be used in clinical testing. 207

We created heatmaps to visualize individual sample outcomes across all LFAs 209 to assess whether we systematically detected the same miscalls across multiple LFAs 210 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 4, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 4, 2021. all LFAs (except Genobio, Oranoxis, OZO, Ray Biotech, and U2U) have an LOD within 240 the linear range of the SIMOA assay (1 -10,000 µg/mL). Lumiquick has the lowest 241 LOD at 0.1 µg/mL, which was extrapolated by dilution to be within the linear range of for these assays spans a large range (from ~30% to 100%). As the population 259 prevalence increases, the burden on specificity is decreased, and at 50% prevalence 260 the PPV of all LFAs is above 87.5%. Posterior PPV can be improved for most LFAs 261 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 4, 2021. In this study, we report a standardized cross-evaluation of LFAs on the same 276 pre-pandemic SARS-CoV-2-negative and PCR-confirmed SARS-CoV-2-positive 277 samples, and rate their reproducibility, usability, and performance characteristics. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 4, 2021. immunity 44 . LFAs also obviate the need for complex laboratories to process the 295 samples 45 . As the pandemic expands to previously unexposed communities, it is 296 critical to use simple tools to monitor exposure dynamics and seroconversion in SARS-297

CoV-2-exposed individuals, as well as vaccine-induced immunity 22 . We tested a 298 mixture of LFAs targeting SARS-CoV2 nucleocapsid, spike proteins, or both. 299

Moderna's mRNA-1273 and Pfizer-BioNTech COVID-19 vaccines encode SARS-300

CoV2 spike proteins to induce anti-spike antibodies 20, 46 . Therefore, LFAs targeting the 301 spike and nucleocapsid proteins of SARS-CoV2 could be used to differentiate vaccine-302 and infection-induced antibody responses, respectively. 303

Rigorous evaluation of these LFAs by manufacturer-independent parties is 304 important. The US FDA independently reviews medical products before 305 commercialization. The FDA used Emergency Use Authorization (EUA) authority to 306 accelerate the implementation of diagnostic products during the pandemic. 307

Commercial manufacturers were required to submit a completed EUA request 30 . 308 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 4, 2021. ; https://doi.org/10. 1101 /2021 Unfortunately, the rush to market introduced many tests that did not meet typical US 309 or international standards 47 . Therefore, the FDA and international regulatory agencies 310 continue to update guidelines for authorization of new serological tests. Our evaluation 311 plan mirrored the FDA guidelines for evaluating serological tests 30 . We included 10 312

HIV-positive samples to test whether they have higher false positive results in SARS-313

CoV2-negative samples 48 , and did not detect higher false positive results. 314

The mere detection of IgG or IgM responses does not guarantee that 315 neutralizing antibodies are present at protective titers 13, 49 . The study demographics 316 suggest a slight over-representation of African Americans among cases, as reported 50 . 317

However, the sample size was underpowered to formally determine the effect of race 318 on test performance. In our analysis, IgM detectability was less sensitive and 319 reproducible than IgGs across multiple LFAs, possibly due to both lower IgM titres, 320 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 4, 2021. ; https://doi.org/10.1101/2021.01.02.20248998 doi: medRxiv preprint rapid immunosurveillance to monitor extent of population transmission, particularly in 332 asymptomatic but SARS-CoV2-exposed individuals 6, 43 . 333

One major concern about the deployment of these tests is the misinterpretation 334 of positive results 13, 49 . As more tests move towards FDA clearance for home use, serological test result may prematurely instill confidence that one has immunity against 345 SARS-CoV-2 infection, thus resulting in behavioral changes that increase risk of 346 transmission 61 . Hence, the probability that a person without antibodies will test 347 negative on a serological test is more important than test sensitivity 59, 60, 61 . 348

Our study presents a few limitations. Although we successfully benchmarked the 349 performance of the LFAs to a quantitative assay 39 , we did not determine the 350 neutralizing potential of these antibodies. Secondly, samples were acquired when 351 PCR testing was restricted to severely ill patients. For epidemiological studies and 352 population surveillance, it will be important to evaluate assay performance on 353 asymptomatic individuals. 354 . CC-BY-NC 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted January 4, 2021. ICU (no intubation) -Recovered 1 (11.1%) 0 (0%) 2 (12.5%) 0 (0%) 3 (5.4%) Outpatient 1 (11.1%) 1 (5.6%) 4 (25%) 1 (7.7%) 7 (12.5%) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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A score was computed for each sample/assay/antibody combination, using the following algorithm:

-Each of N operators reading the LFA were assigned a weight of 1/N to final score -If an operator observed a band, the score would increase by 1/N. If the operator saw no band, the score would decrease by 1/N This process was performed separately for the IgG and IgM channels, with an overall score produced for each antibody. This per-sample/per-antibody score thus has the following values: -1 (all operators agree: no band), 0 (operators disagree), and +1 (all operators agree: band observed). Given that COVID-19 remains, even in late 2020, a low-prevalence disease, we apply a conservative case definition (score ≥0), where discordant operator readings (score=0) are classed as negative for presence of the antibody, in order to favor specificity. A combined IgG and IgM score was computed as the average of the two individual scores: (IgG+IgM)/2.

Reproducibility analysis used the same scoring system above. Tests of the COVID+ pool that received a score >0 were called "correct". COVID-tests were ones that received a score <0. A score of 1 or -1 was called "consistent", with all other scores indicating disagreement between operators called as "inconsistent." These two classifications were concatenated to produce the outcomes "correct consistent", where both operators agreed on the correct outcome, "incorrect consistent," where both operators agreed on the incorrect outcome, and "inconsistent," where operators disagreed on the outcome. The outcome of each individual test was plotted as a proportion of total tests of each pool on each of the two days of testing.

We developed an interactive web-application (https://covid.omics.kitchen) to help visualize false positive and false negative LFA readings based on test accuracy and disease prevalence. We incorporate data from the 20 LFAs reported here as well as the 6 LFAs evaluated by Whitman, et al 16 . County, State, and US-wide disease rates (cumulative numbers of individuals confirmed to have been infected since the start of the outbreak) are pulled dynamically from the New York Times github repository (https://github.com/nytimes/covid-19-data). The webapp is implemented in d3.js JavaScript and hosted on Amazon Web Services AWS/Amplify.

Supplementary 

Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide 472 to molecular and serological in-vitro diagnostic assays

Comparative assessment of multiple COVID-19 serological technologies 476 supports continued evaluation of point-of-care lateral flow assays in hospital and community 477 healthcare settings

Evaluation of SARS-CoV-2 serology assays reveals a range of test 480 performance

The Advisory Committee on Immunization Practices' Interim

Supplementary Includes explanation of intended use 2Storage conditions of provided kit components are listed 3Sample requirements and collection methods (e.g. whole blood, serum, plasma, equilibration temperature, etc.) 4Storage information for specimens 5Precautions/provides warnings and conditions to avoid (e.g. do not use expired cassettes, etc.) 6Test procedure: temperature specified for running sample and cassette 7Test procedure: clearly states when cassette should be read 8Test procedure: clearly states valid time window (e.g. test results invalid after a certain interval of time) 9Test procedure: provides visual diagrams for protocol steps 10Provides instruction on how to proceed if results are invalid (e.g. repeat test, or increase incubation time) 11Includes a rubric for interpretation of results

Provides visual diagrams or cassette images to assist with interpretation of results (has to include at least an example of a positive and negative readout)

Clearly describes correct volume to pipette (e.g. marked pipette, markings explained in the IFU, or dropwise measurement)

Complete instructions provided for lancets or other accessories included for use (e.g. instructions present for all kit components)