key: cord-0942012-v6rc4bmu
authors: Valdivia, Arantxa; Torres, Ignacio; Latorre, Víctor; Francés‐Gómez, Clara; Ferrer, Josep; Forqué, Lorena; Costa, Rosa; de la Asunción, Carlos Solano; Huntley, Dixie; Gozalbo‐Rovira, Roberto; Buesa, Javier; Giménez, Estela; Rodríguez‐Díaz, Jesús; Geller, Ron; Navarro, David
title: Suitability of two rapid lateral flow immunochromatographic assays for predicting SARS‐CoV‐2 neutralizing activity of sera
date: 2020-12-17
journal: J Med Virol
DOI: 10.1002/jmv.26697
sha: a9897eb53306f09e6341567ffa6c577086018ff3
doc_id: 942012
cord_uid: v6rc4bmu

Assessment of commercial severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially available lateral flow immunochromatographic assays (LFIC; Wondfo SARS‐CoV‐2 Antibody test and the INNOVITA 2019‐nCoV Ab test) in comparison with a SARS‐CoV‐2 neutralization pseudotyped assay for coronavirus disease 2019 (COVID‐19) diagnosis in hospitalized patients and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Ninety sera were included from 51 patients with moderate to severe COVID‐19. A green fluorescent protein (GFP) reporter‐based pseudotyped neutralization assay (vesicular stomatitis virus coated with SARS‐CoV‐2 spike protein) was used. Test line intensity was scored using a 4‐level scale (0 to 3+). The overall sensitivity of LFIC assays was 91.1% for the Wondfo SARS‐CoV‐2 Antibody test, 72.2% for the INNOVITA 2019‐nCoV IgG, 85.6% for the INNOVITA 2019‐nCoV IgM, and 92.2% for the NtAb assay. Sensitivity increased for all assays in sera collected beyond day 14 after symptoms onset (93.9%, 79.6%, 93.9%, and 93.9%, respectively). Reactivities equal to or more intense than the positive control line (≥2+) in the Wondfo assay had a negative predictive value of 100% and a positive predictive value of 96.4% for high NtAb(50) titers (≥1/160). Our findings support the use of LFIC assays evaluated herein, particularly the Wondfo test, for COVID‐19 diagnosis. We also find evidence that these rapid immunoassays can be used to predict high SARS‐CoV‐2‐S NtAb(50) titers.

Serological testing is increasingly recognized as a useful tool for control of the coronavirus disease 2019 (COVID- 19) pandemic; beyond complementing reverse-transcription polymerase chain reaction (RT-PCR) assays for disease diagnosis in symptomatic patients, detection of specific antibodies allows for estimating SARS-CoV-2 infection incidence and virus spread in a given population, inferring protection against reinfection, evaluating vaccine efficacy, and selecting appropriate plasma specimens from convalescent COVID-19 patients for passive transfer therapies. [1] [2] [3] Numerous SARS-CoV-2 serological tests have been commercialized, 4 among which lateral flow immunochromatographic assays (LFIC) are particularly appealing because of their rapid turnaround times, simplicity of use, and suitability for point of care testing.

SARS-CoV-2 neutralizing antibodies (NtAb) are presumed to play a major protective role against SARS-CoV-2 infection. [5] [6] [7] Unfortunately, virus neutralization assays, whether using wild-type SARS-CoV-2, engineered SARS-CoV-2 pseudotypes, or chimeric viruses, 8 are unsuited for routine testing, thus creating a need to assess commercial SARS-CoV-2 immunoassays for their capacity to provide reliable information on sera neutralizing activity. Several studies have evaluated the performance of LFIC in comparison with NtAb assays in subjects with past or ongoing SARS-CoV-2 infection. [9] [10] [11] [12] Nevertheless, to the best of our knowledge, only one has attempted to quantitatively correlate results yielded by the two assay types by analyzing the strength of test line reactivity in LFIC devices and NtAb 50 titers. 9 Here, we sought to evaluate the performance of two commercially available LFIC, widely used in our country, compared with a SARS-CoV-2 neutralization pseudotype assay for COVID-19 diagnosis in hospitalized patients, and determine whether the intensity of the test band in LFIC was associated with the levels of NtAb, recognizing the SARS-CoV-2 Spike (S) protein.

The current study included 90 sera from 51 patients with moderate to severe laboratory-confirmed (RT-PCR) COVID-19 RT-PCR admitted to Hospital Clínico Universitario of Valencia between March 5 and April 30, 2020. 13 Sera were grouped according to the timing of collection after symptoms onset: 41 were obtained within < 15 days (median, 11 days; range, 5-14 days), and 49 later on (≥15 days, at a median of 23 days; range, 15-41 days). In addition, a total of 20 prepandemic sera from healthy individuals were collected within 2019, of which 10 belonged to patients with prior endemic coronavirus infections (HCoV-229E, n = 8; HCoV NL63, n = 1; HCoVH-KU, n = 1) and were included as controls. Sera had been cryopreserved at -20°C and were thawed for the analyses described below. This study was approved by the Research Ethics Committee of Hospital Clínico Universitario INCLIVA (March, 2020).

A green fluorescent protein (GFP) reporter-based pseudotyped neutralization assay with a nonreplicative vesicular stomatitis virus (VSV) backbone coated with SARS-CoV-2 spike (S) protein was used for neutralization assays on Vero cells, using heat-inactivated sera and a viral input of 1250 focus-forming units, as previously described. 13 Sera that did not reduce viral replication by 50% at 1/20 dilution were considered non-neutralizing and were arbitrarily assigned a value of 1/10. The antibody dilution resulting in 50% virus neutralization (NtAb 50 ) was calculated using the drc package (version 3.0-1) in R via a two-parameter log-logistic regression model (LL.2 model). 

Here, NtAb titers ≥ 1/160 were deemed as high, in line with the minimum NtAb 50 titer of plasma from COVID-19 convalescent individuals recommended by FDA for therapeutic use. 14

Test performances were evaluated by the sensitivity with the associated 95% confidence interval (CI). Cohen's κ statistic was used to evaluate the qualitative agreement between immunoassays. Differences between medians were compared using the Mann-Whitney U-test. The Spearman rank test was used for the assessment of correlations between the intensity of reactivity of sera in LFIC assays and the NtAb titers. A p < .05 was considered statistically significant.

The analyses were performed using SPSS version 20.0 (SPSS).

Categorical results obtained by the LFIC immunoassays and the NtAb assay are shown in Table 1 A handful of studies have compared the performance of LFIC assays versus SARS-CoV-2 neutralization assays, [9] [10] [11] [12] but none of the LFIC evaluated herein was included. These studies differed in many aspects, namely, the timing of sera collection, clinical presentation of COVID-19, neutralization antibody assay employed, NtAb 50 titer cutoff value for positive results, and the reference method for sensitivity calculations (either the NtAb assay itself or RT-PCR results).

Not surprisingly, overall sensitivities reported for these LFIC assays vary widely, ranging from 46% to 100%. 10, 11 Here, we used a SARS-CoV-2-S-pseudotype as the viral input; nevertheless, NtAb levels F I G U R E 2 Median neutralizing antibody titers (NtAb 50 ) in sera from hospitalized COVID-19 patients according to their strength of reactivity in lateral flow immunochromatographic assays (grading scale of test lines from 0 to 3+). p values for selected comparisons are shown measured by this assay have been shown to correlate strongly with those from assays using live SARS-CoV-2. 8 Although the overall sensitivity of both LFIC assays was lower than the neutralization assay, it was comparable when sera collected ≥15 days were analyzed separately.

A novel contribution of the current study is that a strong association was found between the strength of test line reactivity and NtAb 50 titers, notably when employing the Wondfo SARS-CoV-2

Antibody assay. Using a simple grading scale, we could discriminate reasonably well between sera containing high and low NtAb 50 titers (≥1/160 or <1/160, respectively). In sera giving reactivities ≥ 2+

(comparable to or more intense than the control line), we identified high-NtAb level sera with excellent PPV and NPV. Weidner and colleagues were the first to prove the suitability of this approach. By using a 4-level intensity scale on the Wantai SARS-CoV-2 Ab rapid test, they were able to predict NtAb 50 titers (live SARS-CoV-2) > 1/200 with NPV and PPV of 92%. 9 The Wantai assay detects anti- 

The authors have no conflict of interest. Conceptualization, supervision, writing the original draft, review, and editing.

Data available on request from the authors.

David Navarro http://orcid.org/0000-0003-3010-4110

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