Comparison of one single-antigen assay and three multi-antigen SARS-CoV-2 IgG assays in Nigeria.

Objectives: Determining an accurate estimate of SARS-CoV-2 seroprevalence has been challenging in African countries where malaria and other pathogens are endemic. We compared the performance of one single-antigen assay and three multi-antigen SARS-CoV-2 IgG assays in a Nigerian population endemic for malaria. Methods: De-identified plasma specimens from SARS-CoV-2 RT-PCR positive, dried blood spot (DBS) SARS-CoV-2 RT-PCR positive, and pre-pandemic negatives were used to evaluate the performance of the four SARS-CoV-2 assays (Tetracore, SARS2MBA, RightSign, xMAP). Results: Results showed higher sensitivity with the multi-antigen (81% (Tetracore), 96% (SARS2MBA), 85% (xMAP)) versus the single-antigen (RightSign (64%)) SARS-CoV-2 assay. The overall specificities were 98% (Tetracore), 100% (SARS2MBA and RightSign), and 99% (xMAP). When stratified based on <15 days to ≥15 days post-RT-PCR confirmation, the sensitivities increased from 75% to 88.2% for Tetracore; from 93% to 100% for the SARS2MBA; from 58% to 73% for RightSign; and from 83% to 88% for xMAP. With DBS, there was no positive increase after 15-28 days for the three assays (Tetracore, SARS2MBA, and xMAP). Conclusion: Multi-antigen assays performed well in Nigeria, even with samples with known malaria reactivity, and might provide more accurate measures of COVID-19 seroprevalence and vaccine efficacy.

Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries.

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries.

Performance of SARS-CoV-2 Antigens in a Multiplex Bead Assay for Integrated Serological Surveillance of Neglected Tropical and Other Diseases.

Serosurveillance can provide estimates of population-level exposure to infectious pathogens and has been used extensively during the COVID-19 pandemic. Simultaneous, serological testing for multiple pathogens can be done using bead-based immunoassays to add value to disease-specific serosurveys. We conducted a validation of four SARS-CoV-2 antigens-full-length spike protein, two receptor binding domain proteins, and the nucleocapsid protein-on our existing multiplex bead assay (MBA) for enteric diseases, malaria, and vaccine preventable diseases. After determining the optimal conditions for coupling the antigens to microsphere beads, the sensitivity and specificity of the assay were determined on two instruments (Luminex-200 and MAGPIX) when testing singly (monoplex) versus combined (multiplex). Sensitivity was assessed using plasma from 87 real-time reverse transcription polymerase chain reaction (rRT-PCR) positive persons collected in March-May of 2020 and ranged from 94.3% to 96.6% for the different testing conditions. Specificity was assessed using 98 plasma specimens collected prior to December 2019 and plasma from 19 rRT-PCR negative persons and ranged from 97.4% to 100%. The positive percent agreement was 93.8% to 97.9% using 48 specimens collected > 21 days post-symptom onset, while the negative percent agreement was ≥ 99% for all antigens. Test performance was similar using monoplex or multiplex testing. Integrating SARS-CoV-2 serology with other diseases of public health interest could add significant value to public health programs that have suffered severe programmatic setbacks during the COVID-19 pandemic.

Validation of xMAP SARS-CoV-2 Multi-Antigen IgG assay in Nigeria.

OBJECTIVE: There is a need for reliable serological assays to determine accurate estimates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence. Most single target antigen assays have shown some limitations in Africa. To assess the performance of a multi-antigen assay, we evaluated a commercially available SARS-CoV-2 Multi-Antigen IgG assay for human coronavirus disease 2019 (COVID-19) in Nigeria. METHODS: Validation of the xMAP SARS-CoV-2 Multi-Antigen IgG assay was carried out using well-characterized SARS-CoV-2 reverse transcription polymerase chain reactive positive (97) and pre-COVID-19 pandemic (86) plasma panels. Cross-reactivity was assessed using pre-COVID-19 pandemic plasma specimens (213) from the 2018 Nigeria HIV/AIDS Indicator and Impact Survey (NAIIS). RESULTS: The overall sensitivity of the xMAP SARS-CoV-2 Multi-Antigen IgG assay was 75.3% [95% CI: 65.8%- 82.8%] and specificity was 99.0% [95% CI: 96.8%- 99.7%]. The sensitivity estimate increased to 83.3% [95% CI: 70.4%- 91.3%] for specimens >14 days post-confirmation of diagnosis. However, using the NAIIS pre-pandemic specimens, the false positivity rate was 1.4% (3/213). CONCLUSIONS: Our results showed overall lower sensitivity and a comparable specificity with the manufacturer's validation. There appears to be less cross-reactivity with NAIIS pre-pandemic COVID-19 specimens using the xMAP SARS-CoV-2 Multi-Antigen IgG assay. In-country SARS-CoV-2 serology assay validation can help guide the best choice of assays in Africa.

Cross-Reactivity of Two SARS-CoV-2 Serological Assays in a Setting Where Malaria Is Endemic.

Accurate SARS-CoV-2 serological assays are critical for COVID-19 serosurveillance. However, previous studies have indicated possible cross-reactivity of these assays, including in areas where malaria is endemic. We tested 213 well-characterized prepandemic samples from Nigeria using two SARS-CoV-2 serological assays, Abbott Architect IgG and Euroimmun NCP IgG assay, both targeting SARS-CoV-2 nucleocapsid protein. To assess antibody binding strength, an avidity assay was performed on these samples and on plasma from SARS-CoV-2 PCR-positive persons. Thirteen (6.1%) of 212 samples run on the Abbott assay and 38 (17.8%) of 213 run on the Euroimmun assay were positive. Anti-Plasmodium IgG levels were significantly higher among false positives for both Abbott and Euroimmun; no association was found with active Plasmodium falciparum infection. An avidity assay using various concentrations of urea wash in the Euroimmun assay reduced loosely bound IgG: of 37 positive/borderline prepandemic samples, 46%, 86%, 89%, and 97% became negative using 2 M, 4 M, 5 M, and 8 M urea washes, respectively. The wash slightly reduced avidity of antibodies from SARS-CoV-2 patients within 28 days of PCR confirmation; thereafter, avidity increased for all urea concentrations except 8 M. This validation found moderate to substantial cross-reactivity on two SARS-CoV-2 serological assays using samples from a setting where malaria is endemic. A simple urea wash appeared to alleviate issues of cross-reactivity.

Validation of Commercial SARS-CoV-2 Immunoassays in a Nigerian Population.

Validated assays are essential for reliable serosurveys; however, most SARS-CoV-2 immunoassays have been validated using specimens from China, Europe, or U.S. populations. We evaluated the performance of five commercial SARS-CoV-2 immunoassays to inform their use in serosurveys in Nigeria. Four semiquantitative enzyme-linked immunosorbent assays (ELISAs) (Euroimmun anti-SARS-CoV-2 nucleocapsid protein [NCP] immunoglobulin G [IgG], Euroimmun spike SARS-CoV-2 IgG, Mologic Omega COVID-19 IgG, Bio-Rad Platelia SARS-CoV-2 Total Ab) and one chemiluminescent microparticle immunoassay (Abbott Architect SARS-CoV-2 IgG) were evaluated. We estimated the analytical performance characteristics using plasma from 100 SARS-CoV-2 PCR-positive patients from varied time points post-PCR confirmation and 100 prepandemic samples (50 HIV positive and 50 hepatitis B positive). The Bio-Rad assay failed the manufacturer-specified validation steps. The Euroimmun NCP, Euroimmun spike, and Mologic assays had sensitivities of 73.7%, 74.4%, and 76.9%, respectively, on samples taken 15 to 58 days after PCR confirmation and specificities of 97%, 100%, and 83.8%, respectively. The Abbott assay had 71.3% sensitivity and 100% specificity on the same panel. Parallel or serial algorithms combining two tests did not substantially improve the sensitivity or specificity. Our results showed lower sensitivity and, for one immunoassay, lower specificity compared to the manufacturers' results and other reported validations. Seroprevalence estimates using these assays might need to be interpreted with caution in Nigeria and similar settings. These findings highlight the importance of in-country validations of SARS-CoV-2 serological assays prior to use to ensure that accurate results are available for public health decision-making to control the COVID-19 pandemic in Africa. IMPORTANCE This study used positive and negative sample panels from Nigeria to test the performance of several commercially available SARS-CoV-2 serological assays. Using these prepandemic and SARS-CoV-2-positive samples, we found much lower levels of sensitivity in four commercially available assays than most assay manufacturer reports and independent evaluations. The use of these assays with suboptimal sensitivity and specificity in Nigeria or countries with population exposure to similar endemic pathogens could lead to a biased estimate of the seroprevalence, over- or underestimating the true disease prevalence, and limit efforts to stop the spread of SARS-CoV-2. It is important to conduct in-country validations of serological SARS-CoV-2 assays prior to their widespread use, especially in countries with limited representation in published assay validations.