1
Title: Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point of Care
1
Testing
2
Running Title –Validation of SARS-CoV-2 POCT
3
4
Authors: Steven E. Conklin*
a
Kathryn Martin*
a
, Yukari C Manabe
b
,
Haley A Schmidt
a
, Jernelle
5
Miller
a
, Morgan Keruly
c
, Ethan Klock
b
, Charles S Kirby
a
, Owen R Baker
c
, Reinaldo E
6
Fernandez
b
, Yolanda J Eby
a
, Justin Hardick
b
, Kathryn Shaw-Saliba
d
, Richard E Rothman
d
,
7
Patrizio P Caturegli
a
, Andrew R Redd
b,c
, Aaron AR Tobian
a,b
, Evan M Bloch
a
, H Benjamin
8
Larman
a
, Thomas C Quinn
b,c
, William Clarke#
a
and Oliver Laeyendecker#
b,c
9
* These authors contributed equally to this manuscript
10
# These authors contributed equally to this manuscript
11
12
Affiliations:
13
a) Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore,
14
Maryland, USA
15
b) Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore
16
Maryland, USA
17
c) Division of Intramural Research, National Institute of Allergy and Infectious Diseases,
18
National Institutes of Health, Baltimore, Maryland, USA.
19
d) Department of Emergency Medicine, Johns Hopkins University School of Medicine,
20
Baltimore, Maryland, USA
21
22
Keyword: SARS-CoV-2 serology; point of care test; performance; cross-reactivity
23
24
Corresponding author:
25
Oliver Laeyendecker MS, MBA, PhD
26
855 North Wolfe St.
27
Rangos Building, room 538A
28
Baltimore MD, 21205
29
Phone: 410-502-3268
30
Email: olaeyen1@jhmi.edu
31
32
33
34
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted August 4, 2020. ; https://doi.org/10.1101/2020.07.31.20166041doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
2
ABSTRACT
35
Background. Rapid point-of-care tests (POCTs) for SARS-CoV-2-specific antibodies vary in
36
performance. A critical need exists to perform head-to-head comparison of these assays.
37
Methods. Performance of fifteen different lateral flow POCTs for the detection of SARS-CoV-
38
2-specific antibodies was performed on a well characterized set of 100 samples. Of these, 40
39
samples from known SARS-CoV-2-infected, convalescent individuals (average of 45 days post
40
symptom onset) were used to assess sensitivity. Sixty samples from the pre-pandemic era
41
(negative control), that were known to have been infected with other respiratory viruses
42
(rhinoviruses A, B, C and/or coronavirus 229E, HKU1, NL63 OC43) were used to assess
43
specificity. The timing of seroconversion was assessed on five POCTs on a panel of 272
44
longitudinal samples from 47 patients of known time since symptom onset.
45
Results. For the assays that were evaluated, the sensitivity and specificity for any reactive band
46
ranged from 55%-97% and 78%-100%, respectively. When assessing the performance of the
47
IgM and the IgG bands alone, sensitivity and specificity ranged from 0%-88% and 80%-100%
48
for IgM and 25%-95% and 90%-100% for IgG. Longitudinal testing revealed that median time
49
post symptom onset to a positive result was 7 days (IQR 5.4, 9.8) for IgM and 8.2 days (IQR 6.3
50
to 11.3).
51
Conclusion. The testing performance varied widely among POCTs with most variation related to
52
the sensitivity of the assays. The IgM band was most likely to misclassify pre-pandemic
53
samples. The appearance of IgM and IgG bands occurred almost simultaneously.
54
55
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted August 4, 2020. ; https://doi.org/10.1101/2020.07.31.20166041doi: medRxiv preprint
3
Introduction
56
The respiratory illness Coronavirus disease-19 (COVID-19) is caused by severe acute
57
respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection.[1] The COVID-19 pandemic
58
has challenged the diagnostic testing capacity of the global healthcare industry. Though the
59
initial burden of disease was most pronounced in high-income countries, the pandemic has since
60
spread to middle- and low-income countries that lack substantial laboratory infrastructure.
61
Despite major efforts to contain and slow down the viral spread, the limited testing capability of
62
hospitals, public health laboratories, and government agencies remains a major challenge.
63
Accurate serological tests for SARS-CoV-2 infection are used to estimate the numbers of
64
individuals who have been infected and have developed a humoral immune response
65
(seroconvert). Understanding seroprevalence is important to determine the spread of the disease
66
and to identify populations with a high burden of infection.[2] Furthermore, if previous infection
67
provides immunity to the disease, these assays could be used to determine those who would be
68
vulnerable or protected from infection.
69
Broadly, there are two types of assay formats to detect antibodies against SARS-CoV-2
70
infection enzyme-linked immunosorbent assays (ELISAs) and serologic lateral flow assays
71
(LFA). ELISAs, with or without a chemiluminescent signal, offer high throughput testing, but
72
require substantial laboratory infrastructure and trained personnel for operation.[3] LFAs that
73
detect antibodies against SARS-CoV-2 are easy to use, rapid, portable and often qualify as point
74
of care tests (POCT) that can be used outside of a centralized laboratory facility. [4] POCT can
75
be used at home or in a doctor’s office and take minutes to complete. Unfortunately there is a
76
great deal of variation on the performance of these POCT assays for the accurate detection of
77
antibodies to SARS-CoV-2 infection.[5] Serologic LFAs can have wide ranging performance
78
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted August 4, 2020. ; https://doi.org/10.1101/2020.07.31.20166041doi: medRxiv preprint
4
based on the viral antigens used and how they were elaborated, and the construction of the
79
cassette.
80
Lack of standardization makes performance comparison of serological assays
81
challenging. Structurally, SARS-CoV-2 possesses four main structural proteins: spike
82
glycoprotein (S), envelope glycoprotein (E), membrane glycoprotein (M), and nucleocapsid
83
protein (N).[6,7] These proteins are immunogens capable of inducing the generation of the IgA,
84
IgG, and/or IgM antibodies targeted by LFAs. Unstandardized iterations in the antigen/antibody
85
combination used by LFAs is a key contributor for performance variation among platforms. Poor
86
understanding of immunological response kinetics to SARS-CoV-2 infection further complicates
87
evaluation. The impact of these variables was evident upon initiating comparison of different
88
LFAs for SARS-CoV-2 antibody detection. [8–11] Initial reports are mixed: some report LFAs
89
as being unsuitable for use, while others profess their potential for rapid screening of patients for
90
acute infection.[9–11] Many of these studies were constrained by small sample sizes, failure to
91
evaluate for cross-reactivity and failure to assess sensitivity of the assays by stage of infection,
92
all of which could influence the findings.
93
Many LFAs were released into the market quickly due to US Food and Drug
94
Administration (FDA) emergency use authorization (EUA) as a response to the COVID-19
95
pandemic without a comprehensive assessment of performance. Since then, stricter criteria for
96
approval have been in place due to greater US FDA oversight of the antibody testing EUA
97
process. [12] These include evaluation of cross-reactivity (specificity >95% to other
98
coronaviruses), specificity approaching 100%, high positive/negative predictive agreement
99
(
≥
90%). Regardless of EUA approval, assessing the performance characteristics of LFAs is
100
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted August 4, 2020. ; https://doi.org/10.1101/2020.07.31.20166041doi: medRxiv preprint
5
necessary for understanding the longitudinal thresholds for sensitivity and specificity, and
101
potential cross-reactivity with other non-SARS-CoV-2 viruses.
102
For SARS-CoV-2, antibody reactivity or presence is generally measured as time from
103
symptom onset.[13] While consensus on the optimal time to perform the POCT is lacking, the
104
majority of reports suggest that the tests are best undertaken >14 days post-symptom onset.[14–
105
19] Furthermore, studies on samples from convalescent plasma donors, who had a documented
106
positive RT-PCR test, demonstrate that some individuals have undetectable antibody
107
responses.[20] In terms of specificity, false positive results may occur for a variety of reasons,
108
particularly due to cross reactivity to other coronaviruses (229E, HKU1, NL63, and
109
OC43).[9,12-22]
110
Despite increasing reports on the performance of individual POCTs to detect SARS-
111
CoV-2 antibodies, the overall performance of all the commercially available POCTs is still
112
unclear. To further expand POCT evaluation, we compared the performance of multiple POCTs
113
for SARS-CoV-2 antibody detection. To this end we used the same set of samples from known
114
infected and uninfected individuals to perform a head-to-head analysis of 15 POCT assays. We
115
further evaluated seven of these assays to assess the time window between onset of symptoms
116
and detection of antibodies to SARS-CoV-2 infection. Overall, the goal of our work is
117
highlighting the performance characteristics of a series of LFAs to further expand general
118
understanding of LFA utility and serve as an informative reference for potential deployment
119
efforts.
120
121
Materials and Methods
122
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted August 4, 2020. ; https://doi.org/10.1101/2020.07.31.20166041doi: medRxiv preprint
