Severe Acute Respiratory Syndrome Coronavirus 2 Seroassay Performance and Optimization in a Population With High Background Reactivity in Mali

Abstract

Background: False positivity may hinder the utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests in sub-Saharan Africa.

Methods: From 312 Malian samples collected before 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and 4 other betacoronaviruses by enzyme-linked immunosorbent assay (ELISA). In a subset of samples, we assessed antibodies to a panel of Plasmodium falciparum antigens by suspension bead array and functional antiviral activity by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA using SARS-CoV-2 spike protein and receptor-binding domain developed in the United States using Malian positive and negative control samples. To optimize test performance, we compared single- and 2-antigen approaches using existing assay cutoffs and population-specific cutoffs.

Results: Background reactivity to SARS-CoV-2 antigens was common in prepandemic Malian samples. The SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly/weakly with other betacoronavirus and P falciparum antibodies. No prepandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a 2-antigen approach. Test performance was optimal using a 2-antigen assay with population-specific cutoffs (sensitivity, 73.9% [95% confidence interval {CI}, 51.6-89.8]; specificity, 99.4% [95% CI, 97.7-99.9]).

Conclusions: We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a 2-antigen assay with cutoffs defined by performance in the target population.

Keywords: Africa; COVID-19; SARS-CoV-2; malaria; serology.

Figure 1.

Study flow chart. CoV, coronavirus; NCP, nucleocapsid protein; P falciparum, Plasmodium falciparum; RBD, receptor binding domain; ROC, receiver operator characteristic; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Figure 2.

Severe acute respiratory syndrome coronavirus 2 antigen immunoglobulin G reactivity by enzyme-linked immunosorbent assay in coronavirus disease 2019-naive samples Malian samples: spike protein (n=311), receptor binding domain ([RBD] n=312), nucleocapsid protein ([NCP] n=233). OD, optical density.

Figure 3.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen immunoglobulin G reactivity by site in coronavirus disease 2019-naive Malian samples (≥18 years only). (A) Spike protein (B) receptor binding domain (RBD), (C) nucleocapsid protein (NCP). Groups compared using Kruskal-Wallis test. P values corrected adjusted multiple comparisons using the Holm-Sidak method. K’bougou, Kalifabougou site; Med, median optical density (OD) value; O’bougou, Ouelessebougou site.

Figure 4.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen immunoglobulin G reactivity by age group in coronavirus disease 2019-naive Malian samples (<10, 10–17, ≥18 years, Kalifabougou site). (A) Spike protein (B) receptor binding domain (RBD), (C) nucleocapsid protein (NCP). Groups compared using Kruskal-Wallis test. P values corrected for multiple comparisons using the Holm-Sidak method. K’bougou, Kalifabougou site; Med, median optical density (OD) value.

Figure 5.

Correlation matrix of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen immunoglobulin G reactivity in coronavirus disease 2019-naive Malian samples with (A) 4 other betacoronavirus spike proteins (Pearson r). (B) A panel of 11 Plasmodium falciparum antigens (Spearman r). Plasmodium falciparum antigen panel: MSP1.19, merozoite surface, long-lived antibody response; MSP-2.Dd2, merozoite surface; MSP-2.CH150, merozoite surface; GLURP.R2, merozoite, long-lived antibody response; AMA-1, sporozoite/merozoite, long-lived antibody response; Rh2.2030, merozoite, short-lived antibody response; GEXP18, gametocyte, short-lived antibody response; HSP40.Ag1, infected erythrocyte/gametocyte, short-lived antibody response; Hyp2, infected erythrocyte/parasitophorous vacuole membrane; Etramp5.Ag1, infected erythrocyte/parasitophorous vacuole membrane, short-lived antibody response; Etramp4.Ag2, infected erythrocyte/parasitophorous vacuole membrane. MERS, Middle East respiratory syndrome coronavirus; NCP, nucleocapsid protein; RBD, receptor binding domain; SARS, SARS-CoV-1; SARS2, SARS-CoV-2.

Figure 6.

The performance of US cutoffs in Malian positive control (n=23) and negative control (n=311) samples. OD, optical density; RBD, receptor binding domain. Dotted lines represent US assay cutoffs for spike protein: 0.674 and RBD: 0.306 [10]. Quadrant A (spike negative, RBD positive): positive control: 1/23, negative control: 65/311. Quadrant B (spike negative, RBD negative): positive control: 3/23, negative control: 225/311. Quadrant C (spike positive, RBD positive): positive control: 18/23, negative control: 8/311. Quadrant D (spike positive, RBD negative): positive control: 1/23, negative control: 13/311.