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

Abstract

Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa. 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, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA developed in the US, using two-antigen SARS-CoV-2 spike protein and receptor-binding domain. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative). Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43.4% (135/311) for spike protein, 22.8% (71/312) for RBD, and 33.9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73.9% (51.6-89.8), Specificity: 99.4% (97.7-99.9)]. In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures.

Figure 1:

SARS-CoV-2 antigen reactivity by ELISA in COVID-19 naïve samples. A) US adult samples (n=20) B) Malian samples: Spike protein (n=311), RBD (n=312), NCP (n=233). OD: optical density, RBD: receptor binding domain, NCP: nucleocapsid protein Dotted lines represent provisional thresholds of mean plus three standard deviations of US pre-pandemic samples in panel A. US adult samples: Coefficient of variation: spike protein 40.9%, RBD 63.5%, NCP 79.7%. Malian samples: Coefficient of variation: spike protein 86.2%, RBD 148.2%, NCP 109.2%.

Figure 2:

SARS-CoV-2 antigen reactivity by site (age=/>18 years) in COVID-19 naïve Malian samples. A) Spike protein B) RBD C) NCP. SARS-CoV-2 antigen reactivity by age group (Kalifabougou site) D) Spike protein E) RBD F) NCP. Med: median OD value, OD: optical density, RBD: receptor binding domain, NCP: nucleocapsid protein, O’bougou: Ouelessebougou site, K’bougou: Kalifabougou site **** represents p<0.0001, * represents p=0.01-0.05 using Kruskal-Wallis test.

Figure 3:

Correlation matrix of SARS-CoV-2 antigens and four other betacoronavirus spike protein reactivity in COVID-19 naïve Malian samples. SARS2: SARS-CoV-2, RBD: receptor binding domain, NCP: nucleocapsid protein, SARS: SARS-CoV-1.

Figure 4:

Functional activity of antibodies in COVID-19 naïve Malian samples (n=89) and positive control samples (n=11; recombinant nAb n=1, US convalescent serum n=10) A) Pseudovirus neutralization test of samples at lowest dilution (1:20) B) SARS-CoV-2 spike protein and RBD reactivity by ELISA in COVID-19 naïve Malian samples tested by pseudovirus neutralization test C) Pseudovirus neutralization test IC50 versus spike protein and RBD OD in positive control US clinical samples. IC50: half maximal inhibitory concentration, LLOD: lower limit of detection, OD: optical density, RBD: receptor binding domain, recombinant nAb: recombinant neutralizing antibody H4 a-RBD hIgG1.

Figure 5:

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 [14]. 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