key: cord-0983561-f1w4adyw
authors: Ochola, Lucy; Ogongo, Paul; Mungai, Samuel; Gitaka, Jesse; Suliman, Sara
title: Performance evaluation of lateral flow assays for COVID-19 serology
date: 2021-11-03
journal: Clin Lab Med
DOI: 10.1016/j.cll.2021.10.005
sha: 99eebe6e45ec5f367ad2cf6741772621b6a51609
doc_id: 983561
cord_uid: f1w4adyw

The Coronavirus disease of 2019 (COVID-19) pandemic, caused by infection with the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has undoubtedly resulted in significant morbidities, mortalities, and economic disruptions across the globe. Affordable and scalable tools to monitor the transmission dynamics of the SARS-CoV-2 virus and the longevity of induced antibodies will be paramount to monitor and control the pandemic as multiple waves continue to rage in many countries. Serological assays detect humoral responses to the virus, to determine seroprevalence in target populations, or induction of antibodies at the individual level following either natural infection or vaccination. With multiple vaccines rolling out globally, serological assays to detect anti-SARS-CoV-2 antibodies will be important tools to monitor the development of herd immunity. To address this need, serological lateral flow assays (LFAs), which can be easily implemented for both population surveillance and home use, will be vital to monitor the evolution of the pandemic and inform containment measures. Such assays are particularly important for monitoring transmission dynamics and durability of immunity generated by natural infections and vaccination, particularly in resource-limited settings. In this review, we discuss considerations for evaluating the accuracy of these LFAs, their suitability for different use cases, and implementation opportunities.

(IO) authorizations or Conformité Européenne (CE) marks, with evaluations that were often based on samples from a small number of patients, which were not always representative of the entire susceptible population (e.g. symptomatic patients only) 25 . Therefore, these evaluations limited the reliability and generalizability of tests to estimate the true extent of SARS-CoV-2 transmission in diverse community settings. Hence, standardized protocols for rigorous evaluations of these tests by manufacturer-independent third parties became crucial to determine their accuracy and usability in an unbiased way 26 . Importantly, the increased reliance on antibody tests as "immunity passports" demands their careful evaluation, as well as community education on the interpretation of the test results, to prevent premature assumptions of immunity against SARS-CoV-2 [27] [28] [29] .

Since mid-2020, the WHO has advocated for countrywide serosurveys to determine the extent of SARS-CoV-2 spread globally 30 . To guide this process, the WHO developed an interim guidance policy document stating that serological assays would be crucial to support serosurveillance efforts aimed at estimating transmission to inform public health responses 31 .

However, in this document 30 , the WHO cautioned against using serological assays to determine antibody titers as surrogates for protective immunity, or as tools for contact tracing or diagnosis of active infections 30, 31 .

In order to support country wide serosurveillance efforts, the WHO partnered with the Centers for Disease Control (CDC), the Foundation for Innovative New Diagnostics (FINDdx), African Society for Laboratory Medicine (ASLM), and others to evaluate and roll out COVID-19 diagnostics 32 . As a result, FIND created a centralized repository of available SARS-CoV-2 serological assays 32 , which measures both performance accuracy and feasibility for scale-up in low and middle-income countries. This effort resulted in standardized protocols to evaluate the J o u r n a l P r e -p r o o f accuracy and suitability of serological assays to achieve the following: triaging suspected COVID- 19 patients, assessing recovery of convalescent COVID-19 patients, and implementation of these assays in broader seroprevalence initiatives to inform public health actions, such as prioritizing regions of high transmission, for COVID-19 vaccination. Easy-to-use serological assays, such as lateral flow assays, which are also affordable and scalable, will be key to decentralizing access to these tests 33 .

Several reports conducted in different populations with varied demographics showed a wide range of seroprevalence estimates of antibodies against SARS-CoV-2, as highlighted herein 5 . In Wuhan, China, a study on samples from 18,712 asymptomatic participants collected between January to February 2020 found a seroprevalence of 3%-8% for IgG titers 34 , while another study in the same area from March-April 2020 described rates of 0.3% in 9,442 community resident males 35 . In the US one study had 4,675 outpatients 36 , another 177,919 community samples 37 , and in the UK, 365,000 community samples yielded rates that varied from 0-20% 38 . In a Spanish teaching hospital in Madrid, seroprevalence estimates ranged from 25% to 33% among 2,919 health care workers 39 . In a slum in India, the seroprevalence was as high as 57 .9% in 470 individuals 13 . In Pakistan, the estimates in Karachi ranged from 8.7% to 15.1% for 3,005 community samples 40 .

In Sub-Saharan Africa (SSA), most economies adopted systematic lockdowns, social distancing and donning of masks to reduce transmission 41, 42 . As a result, SSA countries saw overall lower rates of severe disease in the early stages of the pandemic [43] [44] [45] . However, following the economic pressure to re-open and relaxation of social distancing measures, infection rates have risen, with seroprevalence estimates in Kenya between 5% for 3,174 blood donor samples 46 and 50% for 196 antenatal clinical samples 47 52 . However, in most cases these estimates are based on studies of target groups, such as healthcare workers, truck drivers and small populations of less than 3,000 5, 53 . Therefore, the number of participants in SARS-CoV-2 serosurveys in low-and middle-income countries has been generally lower than those of wealthier counterparts. The true extent of COVID-19 spread, particularly in rural settings with little active case finding and surveillance remains undetermined, especially where social distancing measures are more difficult to enforce 54 . These seroprevalence studies collectively demonstrate that SARS-CoV-2 spread, estimated by molecular test positivity rates, severely underestimate true transmission rates 5, 55 . Therefore, there is a need for more systematic sampling to determine the evolving seroprevalence of COVID-19 across various communities.

Serological tests that detect antibodies against SARS-CoV-2 include enzyme-linked immunosorbent assays (ELISAs), chemiluminescence assays, and LFAs [56] [57] [58] [59] . ELISAs are platebased assays to detect an analyte, such as an antibody against a SARS-CoV-2 antigen. Several commercial and non-commercial tests have been developed to measure antibodies to SARS-CoV-2, which include both ELISA 60 and chemiluminescence immunoassays 61 . These assays target antibodies against the receptor binding domain (RBD), spike (S) or nucleocapsid (N) proteins 60 67 . In another study, 30 SARS-CoV-2-positive inpatients were subdivided into severe and mild, based on whether they needed intensive care or not, respectively, and a total of 151 samples were collected 68 . In these samples, evaluation of IgG titers of RBD, S and N proteins showed that antibodies against RBD and N proteins more accurately reflected disease status, and were higher in samples from inpatients with severe than mild COVID-19 68 . For chemiluminescent assays, the sensitivity was 96% in 1,338 clinical samples collected at a median of 47 days 69 . Although ELISAs and chemiluminescent assays can quantify antibodies, they remain primarily a research tool, particularly in resource-limited areas, since they require expensive equipment, trained personnel, and central laboratories that preclude their use in decentralized community testing programs.

Serological LFAs are best suited as point-of-care tests for assessing prior exposure to SARS-CoV-2 4, 70 . LFAs were thus developed as tools to detect SARS-CoV-2-specific antibodies in patient sera, plasma, or whole blood. Earlier in the pandemic, serological LFAs were proposed as alternatives to the expensive and time-intensive RT-qPCR, to complement COVID-19 diagnosis 71, 72 . However, molecular, and rapid antigen tests remain the gold standard for FINDdx repository continues to be updated with new SARS-CoV-2 serological tests and their performance characteristics, as evaluated by multiple partner institutions 32 , (Figure 1 and Table1). The performance of these assays relies on the ability of SARS-CoV-2-infected individuals to mount antibodies against the virus as described below.

Innate and adaptive immunity play an important role in controlling SARS-CoV-2 infection 75 .

Adaptive immunity creates durable memory responses to reinfection with SARS-CoV-2, through T cell-mediated cellular immunity [75] [76] [77] , and B cell-mediated humoral immunity 78, 79 . B cells differentiate into plasma cells, which produce antibodies that target viral antigens. Binding of antibodies to the virus can neutralize it and block its replication in host cells, which forms the basis for proposed antibody therapeutics against SARS-CoV-2 80, 81 . Antibodies against SARS-CoV-2 include multiple isotypes 82 : immunoglobulin-M (IgM), IgG, IgA, which start to appear in COVID-19 patients 7-14 days post-infection and persist for weeks after virus clearance 83 . The most detected antibodies recognize either the internal N protein, or the highly immunogenic external S protein 84 .

The RBD is the component of the spike protein, which binds to the human Angiotensin Converting Enzyme-2 (ACE2) receptor to enter and replicate in the host cell 85, 86 . Therefore, neutralizing antibodies against the RBD of SARS-CoV-2 are particularly important to block entry and replication in host cells 87 .

Given the integral role of the S protein and RBD in facilitating viral entry, these antigens form the basis of many immunoassays described to date 87, 88 and inform rational COVID-19 vaccine design 89 . Recent data from immunoassays based on the SARS-CoV-2 nucleocapsid protein show high sensitivity 33, 58, 90, 91 . However, the higher sequence homology of the SARS-CoV-J o u r n a l P r e -p r o o f 2 nucleocapsid to other coronaviruses, compared to the S protein, could increase the possibility of cross-reactivity against N proteins from related coronaviruses [92] [93] [94] .

IgM antibodies are usually the first humoral response upon SARS-CoV-2 infection 95 .

Travel requirements in China have required a negative IgM test to permit travel (http://www.chinaembassy.org/eng/notices/t1841416.htm). However, using IgM as an indicator of early infection is still likely to miss individuals within 5 days of exposure 62 . Serum levels of SARS-CoV-2-specific IgM antibodies decrease precipitously over time, compared to IgG response, as shown in longitudinal serosurveys of households in Wuhan, China 95 , and longitudinal studies of convalescent patients after discharge 77 . In contrast, SARS-CoV-2 RBD-specific IgG antibodies were durable in convalescent COVID-19 patients and showed minimal cross-reactivity against other widely circulating coronaviruses (HKU1, 229E, OC43, NL63) 96 .

LFAs are effective point-of-care tools to detect immune responses to widely transmitted infections like SARS-CoV-2 97, 98 . Serological LFAs measure pathogen-specific antibodies in accessible biological specimens using simple platforms, where gold or other material-based nanoparticles are often used to label secondary antibodies 98, 99 . LFAs are ideal for mass population surveillance for antibody responses, induced by either natural infection or vaccination, because they are cost-effective, portable, rapid, can be designed to measure more than one antibody isotype in the same sample, and do not require sophisticated equipment to produce results 4, 97 . Furthermore, LFAs are easy-to-use and do not require specialized training for implementation 100 . Additional developments to improve their sensitivity include the use of smartphone apps to detect positive LFA results, which could enable aggregation of data in centralized databases to report disease exposure and inform public health intervention 99,101 . J o u r n a l P r e -p r o o f Therefore, LFAs are useful candidates for population serosurveillance and to monitor longevity of vaccine and SARS-CoV-2-induced antibodies to understand the real extent of herd immunity in a population 6, 102 . However, before implementation of LFAs, the performance of these assays must be systematically evaluated (Table 2) , including the impact of factors such as temperature and humidity 25, 32, 103 .

Since LFAs are designed to be qualitative tests, an important question is whether the band strength (that is, the color intensity of the bands) should be evaluated. The interpretation of band strength can be subjective, but perhaps can be improved by incorporating smartphone apps, as done recently for a rapid antigen test 104 . Variation in band strength across multiple samples raises the question of whether band strength correlates with titers of antibody titers 105 . Antibody levels, determined by Optical Density (OD) ratios, were initially low following symptom onset, then increased over time where IgM, IgG and IgA levels correlated with clinical disease severity 106 . A rapid decay of anti-SARS-CoV-2 antibodies, particularly for patients with mild symptoms implies that the band intensity could serve as a biomarker for disease severity 107 . Since suboptimal antibody titers may promote pathology through antibody dependent-enhancement 108 , correlating LFA band strength with symptom severity could provide a use case for LFAs to inform clinical management. Because LFAs are best suited for population surveillance, the importance of the analytical sensitivity, also known as the limit of detection (LoD) of LFA, i.e., the lowest antibody titers in each sample to give a positive LFA result, cannot be understated. High analytical sensitivity is important in cases that present late with milder symptoms and in patients suspected of COVID-19 despite a negative SARS-CoV-2 RT-qPCR test result 90 .

J o u r n a l P r e -p r o o f LFAs have the potential of deployment outside of clinical care settings due to their affordability and ease of use 4 . The number of people infected with SARS-CoV-2 is known to be underestimated, especially in low-and middle-income countries 47 , due to the high rate of unreported and asymptomatic cases which can spread the infection within the community 109 . The availability of molecular testing and public health restrictions that follow especially for the informal labor sector and rural communities, pose real barriers to testing 9 . Thus, LFAs provide a cheap and scalable alternative to estimate the spread in diverse communities. The presence of anti-SARS-CoV-2 antibodies can identify presumably immune individuals and could thus serve as a tool to release individuals from isolation or lockdown 103 . However, it is important to note that LFAs do not quantify antibody titers or their neutralizing potential. Hence, LFAs are not ideal surrogates for herd immunity 6,102 , but are better suited for estimating SARS-CoV-2 transmission in diverse communities.

On the individual level, LFAs can complement efforts for retrospective diagnosis of presumably exposed individuals 94 . Positive LFA results can confirm exposure to a SARS-CoV-2infected individual, and so LFAs can complement contact-tracing tools, but cannot replace molecular or antigen tests 94 . LFAs are also ideal as direct-to-consumer at-home serological tests that empower individuals to test for anti-SARS-CoV-2 antibodies 110 . The Food and Drug Administration (FDA) has already approved several LFAs, such as Cellex qSARS-CoV-2 IgG/IgM Rapid Test and others for home use 111 . Interestingly, Cellex partnered with Gauss to launch a parallel rapid SARS-CoV-2 antigen test, which was the first to be approved by the FDA for home use 112 . It is very likely that serological LFAs will follow suit. Although home use of serological tests can be a vital instrument in empowering users, the risk of result misinterpretation is very high 33 , and may result in premature behavioral changes that could increase risk of SARS-CoV-2 transmission. More dangerously, ineffective immunity has the chance of exerting selection pressure to increase spontaneous mutations of SARS-CoV-2, and transmission of SARS-CoV-2 variants of conern 7, 113 . A positive result is prone to be false when the prevalence of the disease J o u r n a l P r e -p r o o f is low, or if the specificity of the assay is suboptimal for reasons such as cross-reactivity with related coronaviruses 114 . Therefore, deployment of LFAs for home-use requires inclusion of educational materials that facilitate interpretation as explained below.

The identity of target SARS-CoV-2 antigens in the LFAs is critical 22, 33, 57, 58 . Some LFA kits target the N protein 115 , others the RBD 116 Table 2 ). Vaccines that are based on complete inactivated virus, such as BBV152/COVAXIN or N antigen only will not allow this use case 121 . The variety of antigenic targets for the LFAs, as well as more complex serological assays, allow for this application 33, 122 . LFAs targeting the S protein only include COVID-19 IgM/IgG tests from: Camtech, Oranoxis and Ozo, and N-specific LFAs include CareHealth, KHB, Phamatech and Ray Biotech, while several LFAs target both and would not be suitable for this use case 33, 58 . The tests overall show high sensitivity and specificity for IgG antibodies in samples collected 10 days or more following a positive SARS-CoV-2 RT-qPCR result 33 . The sensitivity was generally higher for IgG than IgM, which motivates for using IgG LFA readouts for serosurveys or home use 33 . Overall, FINDdx reports that most LFAs target the N antigen (Table 1) Table 2 .

It is important that cohorts used for LFA evaluations reflect the characteristics of the intended populations for implementation. For example, if the intended application is testing longevity of vaccine-induced response, the study design should include control pre-vaccination samples, proximal post-vaccination samples to assess seroconversion (e.g., 1-and 2-weeks postvaccination) and remote samples (e.g., 6 months or 1-year post-vaccination). In this situation, quantitative serological assays such as ELISAs should be used as a reference to benchmark the LFA performance 33 Symptomatic SARS-CoV-2 infection increases the pre-test probability that someone was which may be due to cross-reactivity to other related coronaviruses, or real transmission of SARS-CoV-2 before molecular testing was widely implemented. Therefore, controls should be collected from earlier samples, even before October or November of 2019, to rule out unreported SARS-CoV-2 infection. One possible way to avoid including SARS-CoV-2 exposed individuals as negative controls is to use pre-pandemic bio-banked samples. Alternatively, individuals who are routinely tested for SARS-CoV-2, who have never had a positive test result would be the suitable "matched" uninfected group. This prospective evaluation of LFA effectiveness is especially critical as new variants circulate and may compromise the performance accuracy of the LFAs under evaluation [134] [135] [136] . However, in situations where controls are enrolled from the same SARS-CoV-2 exposed communities, repeat testing with highly sensitive molecular tests as well as complementary serological tests that are more sensitive 137 , would be important to rule out prior J o u r n a l P r e -p r o o f exposure to the virus. This is particularly critical in prospective studies where LFAs are evaluated using freshly collected samples, such as whole blood from finger pricks or saliva 104 .

In addition to the choice of population of interest for LFA evaluation, the samples can either be collected cross-sectionally or longitudinally or using a hybrid of the two designs 83, 95 . As above, the sampling scheme should address the intended use case. Evaluating LFAs to measure durability of vaccine-induced antibodies will require longitudinal sampling 138 , whereas crosssectional samples from confirmed SARS-CoV-2 exposed and unexposed individuals would suffice for evaluation of LFAs for implementation in seroprevalence studies. For positive cases, samples should be collected at least 10 days 33 to 3 or more weeks 58 after symptom onset, for those with clear COVID-19 symptoms, to allow sufficient time for seroconversion 74 . Asymptomatic study participants should be diagnosed by a positive RT-qPCR results using a sensitive molecular test. In general, very low sensitivity and higher variability in accuracy was reported for LFAs measuring IgM and IgG from samples collected within a week post symptoms-onset 103 . This is consistent with the often-delayed seroconversion in COVID-19 patients which occurs around day 11-19 post symptoms onset 139 . Consequently, additional effort is required to improve the sensitivity of these assays for early detection of antibodies following symptoms onset.

One of the biggest limitations with the initial FDA EUA process for evaluation of COVID-19 diagnostics was the small number of clinical samples needed from confirmed SARS-CoV-2infected individuals 23 . Initial evaluations included fewer than 100 SARS-CoV-2 positive cases, which would only detect extreme differences in the accuracy of diagnostic platforms 23,32 . This is particularly critical where the prevalence of SARS-CoV-2 infections in various communities is still relatively low, since lower prevalence reduces the positive predictive value of these tests 114 .

Sample sizes to ensure adequate power are inversely correlated with the effect size differences to be detected at a pre-specified significance level 140 . Consequently, a larger sample size will be required to compare the performance of two LFAs with close sensitivity levels (i.e., small effect size), than comparing two LFAs with poor and excellent sensitivities (i.e., large effect size).

Considering the initial limited sample sizes for LFA evaluations, it is critical to expand sample sizes to validate the performance of LFAs to increase the confidence of assay performance before rollout.

Unlike nasopharyngeal swab samples that are hard to collect and have variable quality 141 , serology assays rely on serum and/or plasma samples collected from whole blood that is drawn by widely standardized procedures. Therefore, it is conceivable that some samples tested by RT-qPCR turn negative or indeterminate because of the quality of the sample tested or RNA degradation, leading to false negative classifications. Serology is less likely to be impacted by sample quality. Furthermore, saliva samples have been evaluated for serology, particularly for induction of IgA responses, but they are not the norm for LFA evaluations 141 . Since the success of a diagnostic test depends on the quality of the biological specimen tested, serological assays are appealing alternative tests because of the reliability of samples needed.

To evaluate the accuracy of LFAs, several performance metrics need to be assessed based on the intended use cases. These characteristics include sensitivity, specificity, positive predictive value (PPV) or precision, and negative predictive values (NPV), inter and intra-operator reproducibility and finally, analytical sensitivity, also known as limit of detection.

Sensitivity refers to the proportion of positive cases, defined by a gold standard test like a SARS-CoV-2 RT-qPCR, that are detected accurately by the test. A highly sensitive test will detect 

Analytical sensitivity or limit of detection refers to the minimum SARS-CoV-2-specific antibody titers that are detectable by the LFA. Quantitative platforms such as ELISAs, can be used to establish the limit of detection for LFAs, by adding titrated amounts of serological standards with known antibody titers, and running them concurrently on the ELISA, or other quantitative platforms, and the LFAs under evaluation. Since LFAs are intended to be qualitative, it is worth considering whether a positive band needs to be detected by the naked eye, or whether additional smartphone apps or instruments can detect faint bands that correspond to low antibody concentrations in the sample 99 . The limit of detection of LFAs is higher than known sensitive quantitative methods such as the ultrasensitive Single Molecule Array (SIMOA) platforms 33, 137, 145 .

However, a high limit of detection reduces the chance of misusing LFAs to ascribe immunity passports to individuals with low antibody titers to conservatively prevent over-estimation of seroprevalence, and herd immunity 27, 28 . Finally, testing reproducibility of test results run by the same operator multiple times (intra-operator reproducibility), or between different operators (interoperator reproducibility), as well as reproducibility across different reagent lots would instill confidence in the reliability of the manufacturing quality of the LFAs.

The emergence of SARS-CoV-2 variants is an important consideration in the evaluation of SARS-CoV-2 diagnostic tests, since SARS-CoV-2 antigenic drift may reduce the sensitivity of these tests 146 Variants of SARS-CoV-2 with the D614G mutation in the spike (S) protein that increases receptor-binding avidity have also been reported globally 151 (Table 3A) The mature SARS-CoV-2 Spike trimer is composed of the exterior S1 and transmembrane S2 subunits 153 . The S1 subunit uses the RBD to interact with the ACE2 receptor, while the S2 subunit governs the fusion between the viral and cellular membranes. Spike is considered the major target of the cellular and humoral responses against SARS-CoV-2 upon natural infection 84, 96 . Of all SARS-CoV-2 variants, the D614G mutant accounts for 75.7% of all circulating strains and is associated with severe clinical presentation 153 . SARS-CoV-2 Spike D614 had a more severe impact on antibody binding than G614 compared to the wildtype strain 154 . Studies using monoclonal antibodies (mAbs) have shown that V483A in the receptor-binding domain has a mutation frequency of over 0.1% 151 . It showed decreased reactivity to the two mAbs (P2B-2F6 and X593) and the A475V is significantly resistant to several neutralizing antibodies 151 Other LFA target S protein coded by S gene which recent data shows that it has majority of mutations, including spike mutation E484K that affect antibody response, and hence could affect the LFA performance. Collectively, whether these mutations reduce the sensitivity of LFAs needs to be systematically evaluated (Table 3B) .

In order to evaluate the detection of SARS-CoV-2 variant-specific antibodies, the mutated antigen from the variants should be included in the kit, especially when amino acid changes in the SARS-CoV-2 antigens are sufficient to alter antibody binding 154 . Hence, recombinant antigens reflecting the pseudotypes of the emerging variants should be incorporated in the next generations of LFAs. Subsequent evaluation efforts for LFAs should perhaps analyze both the conserved and mutated antigens to distinguish whether an infection has occurred, and whether antibodies were generated in response to a mutant strain. It is important to note that the difference in antigenicity may be too subtle to influence detection of antibody responses. However, since new SARS-CoV-2 variants are still emerging, it is imperative to iteratively develop and improve LFA assays to detect variant-specific serological responses.

Following the evaluation of the accuracy of LFAs, they need to be assessed for implementation effectiveness and fitness for use 72, 155 . Effectiveness reflects to whether the LFA is fit for implementation in the intended population and settings by evaluating relevant factors, J o u r n a l P r e -p r o o f including required storage conditions and affordability, particularly in resource-limited countries communities 4 . For instance, if LFAs require refrigeration in hot regions with little access to stable electricity or testing in temperature-controlled settings as reported for rapid antigen LFAs 156 , they may not be fit for implementation in those contexts. It is also important to evaluate whether the kit manufacturers or governments have assumed the financial responsibility to ramp up the supply chain to avail the LFAs to communities. If communities assume the financial burden of evaluation and cost for large scale implementation, it is unlikely that results would meaningfully improve the public health outcomes of these communities.

The WHO's standard for point-of-care tests, including LFAs, need to be ASSURED-"Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users" 157 . Gaps in any of these criteria compromise successful implementation of the evaluated LFAs, as previously reported for diagnostics of sexually-transmitted infections 158 .

Hence, if sustainable scale up of LFAs is intended, then pilots for LFA design and implementation should consider "beginning with the end in mind" framework that enhances its potential for future large-scale impact 159 LFA evaluation studies should consider appropriate theoretical frameworks towards achieving adoption and sustainability. These should ideally guide evidence generation, contextualize implementation and facilitate iteration, adoption, and sustainability.

In conclusion, serological LFAs can be useful tools for estimating the true extent of SARS-CoV-2 globally, which is estimated considering inaccuracies in reporting, limited availability of molecular Issues and questions to address in the evaluation Target population -Will the study include both symptomatic and asymptomatic individuals? -Inclusion of vulnerable and high-risk populations (e.g., immunocompromised individuals and those with comorbidities)? -Diverse ethnic and socio-economic participants -Different age groups (children and the elderly) -Implementation in occupational settings: e.g., for testing health care workers and education staff -Inclusion of travelers (e.g., for border crossing restrictions) Sampling scheme -Cross sectional schemes for direct evaluation of LFA performance characteristics (e.g., sensitivity and specificity) -Longitudinal schemes particularly of highly exposed individuals to allow analysis of seroconversion, durability of vaccine and infection-induced antibody responses.

Type of sample -Is this an easy to collect sample? (e.g., finger prick whole blood, urine, saliva)? Invasiveness? -Does the sample collection require trained personnel? -Access to storage and transport conditions to preserve the sample quality -Infection control: Does the sample expose the 'collector' to SARS-CoV-2 or other pathogens? -Can the end user collect the samples themselves?

Confirmed SARS-CoV-2 exposure and time between confirmed RT-qPCR test and sample collection for serology. -What is the demand landscape for the LFA? -Does the LFA inform social distancing guidelines? -Can the evaluation protocol determine fitness for implementation? -Is the LFA high on the priority list for tools in the fight against the COVID-19 pandemic? -What are the cold chain requirements for storage and distribution? -Can the LFA adapt to different temperatures/climates? Feasibility and adoption -Is there a political will to adopt LFAs? -What is the available infrastructure for rolling out LFAs? -Are they fit for the proposed use cases? -What is the balance between feasibility, practicality and actual fit that ensure utility of adoption? -Will the evaluation assess adoption-uptake (decision to use the LFA and trialability (ability to attract utilization and ease of use-for direct-to-consumer testing) 160, 161 ? J o u r n a l P r e -p r o o f 

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Measuring the Serologic Response to Severe Acute Respiratory Syndrome Coronavirus 2: Methods and Meaning

COVID19 antibody detection using lateral flow assay tests in a cohort of convalescent plasma donors

Practical Considerations for Implementation of SARS-CoV-2 Serological Testing in the Clinical Laboratory: Experience at an

Serology characteristics of SARS-CoV-2 infection after exposure and post-symptom onset

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

Adaptive immunity to SARS-CoV-2 and COVID-19

T Cell Memory: Understanding COVID-19

Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals

Comorbid illnesses are associated with altered adaptive immune responses to SARS-CoV-2

T cell and antibody functional correlates of severe COVID-19. medRxiv

Recent advances in therapeutic applications of neutralizing antibodies for virus infections: an overview

Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody

Antibody profiling and prevalence in US patients during the SARS-CoV2 pandemic

Antibody response to SARS-CoV-2 infection in humans: A systematic review

The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19)

The antigenic anatomy of SARS-CoV-2 receptor binding domain

A serological assay to detect SARS-CoV-2 seroconversion in humans

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness

Interpreting Diagnostic Tests for SARS-CoV-2

Performance Characteristics of the Abbott Architect SARS-CoV-2 IgG Assay and Seroprevalence in

Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection

Serology assays to manage COVID-19

Evaluating ELISA, Immunofluorescence, and Lateral Flow Assay for SARS-CoV-2 Serologic Assays

Seroprevalence and humoral immune durability of anti-SARS-CoV-2 antibodies in Wuhan, China: a longitudinal, population-level, cross-sectional study

Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients

Technical considerations to development of serological tests for SARS-CoV-2

A multi-target lateral flow immunoassay enabling the specific and sensitive detection of total antibodies to SARS COV-2

Dual lateral flow optical/chemiluminescence immunosensors for the rapid detection of salivary and serum IgA in patients with COVID-19 disease

Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis

Smartphone-based enzymatic biosensor for oral fluid L-lactate detection in one minute using confined multilayer paper reflectometry

The bumpy road to achieve herd immunity in COVID-19

Performance of Commercially Available Rapid Serological Assays for the Detection of SARS-CoV-2 Antibodies

Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer

Clinical Evaluation of a COVID-19 Antibody Lateral Flow Assay using Point of Care Samples. medRxiv

Comparison of six commercially available SARS-CoV-2 antibody assays-Choice of assay depends on intended use

Rapid Decay of Anti-SARS-CoV-2 Antibodies in Persons with Mild Covid-19

The potential danger of suboptimal antibody responses in COVID-19

Deciphering the power of isolation in controlling COVID-19 outbreaks

Diagnostic technology for COVID-19: comparative evaluation of antigen and serology-based SARS-CoV-2 immunoassays, and contact tracing solutions for potential use as at-home products

Diagnostics for SARS-CoV-2 detection: A comprehensive review of the FDA-EUA COVID-19 testing landscape

Gauss and Cellex Partner for First At-Home SARS-CoV-2 Antigen Test

SARS-CoV-2 Sequence Characteristics of COVID-19 Persistence and Reinfection. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

StatPearls. Treasure Island (FL)

Two-tiered SARS-CoV-2 seroconversion screening in the Netherlands and stability of nucleocapsid, spike protein domain 1 and neutralizing antibodies

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

An mRNA Vaccine against SARS-CoV-2 -Preliminary Report

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

What do we know about India's Covaxin vaccine?

SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses. medRxiv

Guidelines on newly identified limitations of diagnostic tools for COVID-19 and consequences

A systematic review of the use of the Consolidated Framework for Implementation Research

Persistence and Evolution of SARS-CoV-2 in an Immunocompromised Host

Cross-reactive neutralization of SARS-CoV-2 by serum antibodies from recovered SARS patients and immunized animals

The genetic sequence, origin, and diagnosis of SARS-CoV-2

Prevalence of Asymptomatic SARS-CoV-2 Infection : A Narrative Review

Serological follow-up of SARS-CoV-2 asymptomatic subjects

Racial Disparity of Coronavirus Disease 2019 in African American Communities

Racial and Ethnic Disparities in Viral Acute Respiratory Infections in the United States: Protocol of a Systematic Review

Seroprevalence of SARS-CoV-2 in Hong Kong and in residents evacuated from Hubei province, China: a multicohort study

Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland

Genetic variants and source of introduction of SARS-CoV-2 in South America

SARS-CoV-2 variants show resistance to neutralization by many monoclonal and serum-derived polyclonal antibodies

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

Ultrasensitive high-resolution profiling of early seroconversion in patients with COVID-19

Durability of Responses after SARS-CoV-2 mRNA-1273 Vaccination

Evaluation of a COVID-19 IgM and IgG rapid test; an efficient tool for assessment of past exposure to SARS-CoV-2

Power and sample size

Evaluation of specimen types and saliva stabilization solutions for SARS-CoV-2 testing

Estimation of the diagnostic accuracy of real-time reverse transcription quantitative polymerase chain reaction for SARS-CoV-2 using re-analysis of published data

Detection of SARS-COV N2 Gene: Very low amounts of viral RNA or false positive?

Are Seroprevalence Estimates for Severe Acute Respiratory Syndrome Coronavirus 2 Biased?

Evaluation of two commercial and two noncommercial immunoassays for the detection of prior infection to SARS-CoV-2. medRxiv

Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants

Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom

Introduction of SARS-CoV-2 variant of concern 20h/501Y.V2 (B.1.351) from Malawi to Italy

Imported SARS-CoV-2 Variant P.1 in Traveler Returning from Brazil to Italy

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

Emergence of SARS-CoV-2 B.1.1.7 Lineage -United States

S gene dropout patterns in SARS-CoV-2 tests suggest spread of the H69del/V70del mutation in the US

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity

SARS-CoV2 envelop proteins reshape the serological responses of COVID-19 patients

COVID-19 new diagnostics development: novel detection methods for SARS-CoV-2 infection and considerations for their translation to routine use

Impaired performance of SARS-CoV-2 antigen-detecting rapid diagnostic tests at elevated and low temperatures

A guide to aid the selection of diagnostic tests

Mapping the landscape of diagnostics for sexually transmitted infections: Key findings and recommendations. World Health Organization

Beginning with the end in mind: plannig pilot projects and other programmatic research for successful scaling up

Outcomes for implementation research: conceptual distinctions, measurement challenges, and research agenda

Outcomes for implementation science: an enhanced systematic review of instruments using evidencebased rating criteria

Detection of the new SARS-CoV-2 variants of concern B.1.1.7 and B.1.351 in five SARS-CoV-2 rapid antigen tests (RATs)

T-cell quality in memory and protection: implications for vaccine design

J o u r n a l P r e -p r o o f % of kits authorized Ochola, et al.