SARS-CoV-2 seroassay performance and optimization in a population with high background reactivity in Mali

Woodford, John; Sagara, Issaka; Dicko, Alassane; Zeguime, Amatigue; Doucoure, M’Bouye; Kwan, Jennifer; Zaidi, Irfan; Doritchamou, Justin; Snow-Smith, Maryonne; Alani, Nada; Renn, Jonathan; Kosik, Ivan; Holly, Jaroslav; Yewdell, Jonathan; Esposito, Dominic; Sadtler, Kaitlyn; Duffy, Patrick

BACKGROUND: False positivity may hinder the utility of SARS-CoV-2 serological tests in sub-Saharan Africa. METHODS: From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA. In a subset of samples, we assessed antibodies to a panel of P. 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 US using Malian positive and negative control samples. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs. RESULTS: Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic Malian samples. SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly-weakly with other betacoronavirus and P. falciparum antibodies. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs [Sensitivity: 73.9% (51.6-89.8), Specificity: 99.4% (97.7-99.9)]. CONCLUSIONS: We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a two-antigen assay with cutoffs defined by performance in the target population.