key: cord-0715748-vnybikvc authors: Yadouleton, Anges; Sander, Anna-Lena; Moreira-Soto, Andres; Tchibozo, Carine; Hounkanrin, Gildas; Badou, Yvette; Fischer, Carlo; Krause, Nina; Akogbeto, Petas; de Oliveira Filho, Edmilson F.; Dossou, Anges; Brünink, Sebastian; Aïssi, Melchior A. Joël; Djingarey, Mamoudou Harouna; Hounkpatin, Benjamin; Nagel, Michael; Drexler, Jan Felix title: Limited Specificity of Serologic Tests for SARS-CoV-2 Antibody Detection, Benin date: 2021-01-03 journal: Emerg Infect Dis DOI: 10.3201/eid2701.203281 sha: 63f2abd0346ea7495503563acd64c41d522d6069 doc_id: 715748 cord_uid: vnybikvc We used commercially available ELISAs to test 68 samples from coronavirus disease cases and prepandemic controls from Benin. We noted <25% false-positive results among controls, likely due to unspecific immune responses elicited by acute malaria. Serologic tests must be carefully evaluated to assess coronavirus disease spread and immunity in tropical regions. We used commercially available ELISAs to test 68 samples from coronavirus disease cases and prepandemic controls from Benin. We noted <25% false-positive results among controls, likely due to unspecific immune responses elicited by acute malaria. Serologic tests must be carefully evaluated to assess coronavirus disease spread and immunity in tropical regions. nonstructural protein 1 (NS1) antigen (IgG), the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) (IgG), and the EBV viral capsid (CA) antigen (IgM and IgG), as well as real-time PCR tests (TIB MOLBIOL, https://www. tib-molbiol.com) for all human pathogenic Plasmodium species, EBV, and cytomegalovirus (CMV). Plaque-reduction neutralization tests (PRNTs) were performed by using similar methods for SARS-CoV-2 and ZIKV as described (4, 6) . We used previously described recombinant S-based immunofluorescence assays (7) to test for specific antibodies to common cold betacoronavirus human coronavirus (HCoV) OC43 and HCoV-HKU1. Among the 8 patients with RT-PCR-confirmed SARS-CoV-2 infection, seroconversion ranged from 62.5%-100% (95% CI 30.8%-100.0%), depending on the ELISA used ( Figure 1 , panel A), suggesting differential sensitivity of ELISAs on the basis of immunoglobulin detected and the commercial kit used. Indeed, early after infection, IgA-based tests had a higher sensitivity than most IgG-based SARS-CoV-2 ELISAs; only the InBios IgG-based kit was positive for all RT-PCR-confirmed patients (Figure 1 , panel A). A total of 87.5% (7/8) of ELISA results were confirmed by a highly specific SARS-CoV-2 PRNT (Figure 1, panel B) . When summarizing all antibody classes, antigens, and kits among the 60 prepandemic controls, we observed 25.0% (15/60; 95% CI 15.7%-37.3%) positive or borderline ELISA results (8) . Different from RT-PCRconfirmed cases, ELISA reactivity in those samples contrasted with the complete lack of SARS-CoV-2-specific neutralizing antibodies, suggesting unspecific ELISA reactivity ( Figure 1, panel B) . Unspecific SARS-CoV-2 ELISA reactivity might be consistent with, but not limited to, 3 scenarios. First, antibodies elicited by common infections with endemic human coronaviruses might cross-react with SARS-CoV-2 antigens (1). However, a Fisher exact test showed no statistically significant difference in the frequency of antibody reactivity with common cold coronavirus antigens between SARS-CoV-2 ELISA-positive serum samples compared with SARS-CoV-2 ELISA-negative samples. In detail, reactivity with HCoV-OC43 was Figure 3 ). Finally, polyclonal B cell activation also can be caused by acute malaria, which is widespread in Africa (10). More (71.4%) persons with SARS-CoV-2-positive ELI-SAs than those with negative ELISAs (54.3%) were positive for Plasmodium in a highly sensitive PCR test, but the difference was not statistically significant by Fisher exact test (p = 0.35; Figure 1 , panel C). However, parasite loads were statistically significantly higher among SARS-CoV-2 ELISA-positive than ELISA-negative persons by Student t-test (p = 0.035; Figure 1 , panel C). In malaria, higher parasite loads are detected at early stages of infection and decrease over time, suggesting a higher proportion of acute malaria in SARS-CoV-2 ELI-SA-positive patients compared with likely subacute or chronic malaria in SARS-CoV-2 ELISA-negative patients (11) . Thus, acute malaria is the most plausible explanation for unspecific SARS-CoV-2 ELISA reactivity in prepandemic controls. To assess the breadth of Limited Specificity of Tests for SARS-CoV-2, Benin unspecific reactivity, we tested the serum samples from prepandemic controls by using a ZIKV IgG ELISA, for which unspecific reactivity has been reported in cases of acute malaria (10) . We found that 57.1% of samples that elicited potentially unspecific SARS-CoV-2 ELI-SA results also showed ZIKV ELISA-positive results, whereas only 23.9% of samples that were SARS-CoV-2 ELISA-negative were ZIKV ELISA-positive. This difference was statistically significant by Fisher exact test (p = 0.019) (Figure 1 , panel D; Appendix Figure 4 ). From the prepandemic controls that were SARS-CoV-2 ELISA positive, no ZIKV ELISA-positive serum samples showed ZIKV-specific neutralizing antibodies, suggesting unspecific reactivity of those samples in the ZIKV ELISA, similar to the discrepant results of SARS-CoV-2 ELISA and PRNT observed in those serum samples ( Figure 1 , panel E; Figure 2 ). We assessed SARS-CoV-2 antibody-based serologic diagnostics in Benin and noted unspecific reactivity in up to 25% of febrile patients, possibly due to acute malaria. Limitations of our study include the small sample size and limited patient metadata. Testing of serum samples for CMV and EBV by PCR might not have been sensitive due to lack of cell-associated viral nucleic acid; therefore, we cannot exclude potential herpesvirus reactivation affecting serologic testing. Nevertheless, our analyses point to acute malaria as the likely cause of the unspecific serologic reactivity, although we cannot exclude other coexisting conditions in the tropics, such as dengue virus, which also can affect testing (12). Unspecific reactivity in serologic tests might affect public health interventions in tropical regions, leading to overestimates of SARS-CoV-2 circulation in regions where malaria is endemic and to misidentification of SARS-CoV-2 hotspots. In addition, due to false-positive SARS-CoV-2 results, target populations for vaccine campaigns might be missed when vaccines become available, and coexistent diseases, such as malaria, might be overlooked, leading to higher mortality rates from endemic diseases (13, 14) . The robustness of current and future SARS-CoV-2 serologic tests should be further assessed by multicentric seroepidemiologic studies from different tropical regions (15) . This article was preprinted at https://www.medrxiv.org/ content/10.1101/2020.06.29.20140749v1. Serological assays for emerging coronaviruses: challenges and pitfalls Temporal dynamics in viral shedding and transmissibility of COVID-19 Early detection of SARS-CoV-2 antibodies in COVID-19 patients as a serologic marker of infection Severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease 2019 patients The difficulties in obtaining reliable Zika virus diagnostics High Zika virus seroprevalence in Salvador, northeastern Brazil limits the potential for further outbreaks Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections Comparison of serologic assays for Middle East respiratory syndrome coronavirus Analysis of the virus-specific and nonspecific B cell response to a persistent B-lymphotropic gammaherpesvirus Van den Bossche D. Letter to the editor: Specificity of Zika virus ELISA: interference with malaria Multiplex real-time PCR for the diagnosis of malaria: correlation with microscopy Viral emergence and immune interplay in flavivirus vaccines Effect of the Ebola-virusdisease epidemic on malaria case management in Guinea, 2014: a cross-sectional survey of health facilities Serological cross-reactivities between the retroviruses HIV and HTLV-1 and the malaria parasite Plasmodium falciparum We thank Arne Kühne, Wendy Jo-lei, and Patricia Tscheak from the Institute of Virology, Charité, Berlin, Germany for laboratory assistance and Olfert Landt from TIB MOLBIOL GmbH, Germany for providing diagnostic reagents.