key: cord-0709302-o3tx4zno
authors: Emmerich, Petra; Murawski, Carolin; Ehmen, Christa; von Possel, Ronald; Pekarek, Neele; Oestereich, Lisa; Duraffour, Sophie; Pahlmann, Meike; Struck, Nicole; Eibach, Daniel; Krumkamp, Ralf; Amuasi, John; Maiga‐Ascofare, Oumou; Rakotozandrindrainy, Raphael; Asogun, Danny; Ighodalo, Yemisi; Kann, Simone; May, Jürgen; Tannich, Egbert; Deschermeier, Christina
title: Limited specificity of commercially available SARS‐CoV‐2 IgG ELISAs in serum samples of African origin
date: 2021-03-05
journal: Trop Med Int Health
DOI: 10.1111/tmi.13569
sha: 70419df7a991118c452fab935d0bf26c925a2b9d
doc_id: 709302
cord_uid: o3tx4zno

OBJECTIVES: Specific serological tests are mandatory for reliable SARS‐CoV‐2 diagnostics and seroprevalence studies. Here, we assess the specificities of four commercially available SARS‐CoV‐2 IgG ELISAs in serum/plasma panels originating from Africa, South America, and Europe. METHODS: 882 serum/plasma samples collected from symptom‐free donors before the COVID‐19 pandemic in three African countries (Ghana, Madagascar, Nigeria), Colombia, and Germany were analysed with three nucleocapsid‐based ELISAs (Euroimmun Anti‐SARS‐CoV‐2‐NCP IgG, EDI(TM) Novel Coronavirus COVID‐19 IgG, Mikrogen recomWell SARS‐CoV‐2 IgG), one spike/S1‐based ELISA (Euroimmun Anti‐SARS‐CoV‐2 IgG), and in‐house common cold CoV ELISAs. RESULTS: High specificity was confirmed for all SARS‐CoV‐2 IgG ELISAs for Madagascan (93.4%‐99.4%), Colombian (97.8%‐100.0%), and German (95.9%‐100.0%) samples. In contrast, specificity was much lower for the Ghanaian and Nigerian serum panels (Ghana: NCP‐based assays 77.7%‐89.7%, spike/S1‐based assay 94.3%; Nigeria: NCP‐based assays 39.3%‐82.7%, spike/S1‐based assay 90.7%). 15 of 600 African sera were concordantly classified as positive in both the NCP‐based and the spike/S1‐based Euroimmun ELISA, but did not inhibit spike/ACE2 binding in a surrogate virus neutralization test. IgG antibodies elicited by previous infections with common cold CoVs were found in all sample panels, including those from Madagascar, Colombia, and Germany and thus do not inevitably hamper assay specificity. Nevertheless, high levels of IgG antibodies interacting with OC43 NCP were found in all 15 SARS‐CoV‐2 NCP/spike/S1 ELISA positive sera. CONCLUSIONS: Depending on the chosen antigen and assay protocol, SARS‐CoV‐2 IgG ELISA specificity may be significantly reduced in certain populations probably due to interference of immune responses to endemic pathogens like other viruses or parasites.

In February and early March 2020, the first COVID-19 cases were reported from African countries with a high level of international contacts (Egypt, Algeria, Nigeria, Morocco, South Africa) (1) . Following these primary importation events, case numbers rapidly increased due to intra-continental and intra-national transmission as well as further importation mainly from Europe and Asia (1, 2) . As of February 14 th 2021, 2,723,431 laboratory-confirmed SARS-CoV-2 infections and 68,294 deaths caused by COVID-19 have been reported from the WHO Africa region (3) .

Nevertheless, the observed incidence rates are still well below the estimated numbers that were expected based on the socioeconomic challenges many African countries are facing (4) and the numbers observed in Europe and the Americas (3) . Although testing capacities vary widely between African countries, this observation is not solely based on underreporting, but probably reflects both the lower median age of the African population and a different immune status induced by contact with endemic pathogens (4) . Besides modulating COVID-19 morbidity and mortality rates, the latter may also influence performance data of SARS-CoV-2 serological tests; in particular assay specificity may be challenged by previous or current infections with other endemic pathogens (5) (6) (7) . Therefore, analyses of this parameter in different populations are an important prerequisite to ensure reliable diagnostic procedures and seroprevalence studies.

Up to now, a plethora of ELISA tests for detection of anti-SARS-Cov-2 antibodies has been developed and commercialized (8) . Usually, these assays employ one of the major immunogenic coronavirus proteins (9) as antigen, i.e. the nucleocapsid protein (NCP) or defined subdomains of the spike protein (e.g. S1 or the isolated receptor binding domain (RBD)). In this study, we investigate the specificity of three NCP-based SARS-CoV-2 IgG ELISA kits and one spike/S1-based kit in pre-COVID-19 serum/plasma panels from three African countries (Ghana, Madagascar, Nigeria) as well as from South America (Colombia) and Europe (Germany).

This article is protected by copyright. All rights reserved Human serum/plasma samples: The study was performed using stored human serum/plasma samples collected before the COVID-19 pandemic ( Table 2 ), line blotting (Euroline Anti-SARS-CoV-2 Profile IgG line blot (Euroimmun)), and surrogate neutralization assays (sVNT, Genscript, US) were performed and evaluated according to the manufacturers' instructions.

: testing was performed as described previously (10) using SARS-CoV-2 infected Vero cells fixed with acetone/methanol.

IgG antibodies were detected using a patented platform technology (11, 12) .

Briefly, diluted patient serum/plasma samples were co-incubated together with a biotinylated recombinant antigen for 24 h at 4 °C in a microwell plate coated with a recombinant IgG immune complex specific capture molecule.

Following a washing step, the bound IgG/antigen immune complexes were visualized by subsequent application of horseradish peroxidase (HRP)-labeled streptavidin and the colorimetric HRP substrate tetramethylbenzidine (TMB).

After stopping the enzymatic reaction, the assay result was generated by measuring the optical density of the solution in the well at 450/620 nm. 

Statistical analyses (calculation of 95% confidence intervals, Fisher's exact test) were performed using GraphPad Prism.

To assess the specificities of commercially available SARS-CoV-2 IgG ELISA tests in sample panels of different origin, a priori SARS-CoV-2 IgG negative samples (Table 1) Table 2) . While IgG ELISA specificities where good to excellent for pre-COVID-19 samples originating from Colombia, Madagascar, and Germany, increased false positive rates were observed in a priori SARS-CoV-2 IgG negative sera from Ghana and Nigeria ( Figure 1 , Table 3 ).

The index values obtained with the three assays employing the same antigen (recombinant NCP) showed a clear correlation ( Figure 2 ). In contrast, only a small number of 15 out of 600 African samples (Ghana 1: 4/150, Ghana 2:

3/133, Madagascar: 0/167, Nigeria: 8/150) were concordantly classified as positive by both the NCP-based and the spike/S1-based Euroimmun IgG ELISA ( Figure 3A -C), a criterion that is fulfilled by the vast majority of sera from PCRconfirmed COVID-19 patients in the convalescent phase (13) . Positive or borderline serum reactivity with recombinant SARS-CoV-2 NCP (respectively spike/S1) was confirmed for 5/7 (7/7) Ghanaian and 6/8 (7/8) Nigerian samples using a commercially available line blot (Euroline Anti-SARS-CoV-2 Profile IgG line blot (Euroimmun)) ( Figure   3D ). In addition, antibodies binding to SARS-CoV-2 spike S2 domain were found in 7/7 Ghanaian and 4/8 Nigerian samples ( Figure 3A -D). However, all 15 samples tested negative in IgG IIFT using SARS-CoV-2-infected Vero cells (10) and showed no or only very weak activity in a SARS-CoV-2 surrogate virus neutralising test (sVNT, Genscript, US) ( Figure 3D ).

To investigate the influence of previous infections with common cold CoVs on SARS-CoV-2 ELISA specificity, the complete sample panel (n=882) was assayed for IgG antibodies interacting with the C-terminal dimerization domains of the OC43, HKU1, NL63, and 229E NCPs using an in-house ELISA. In concordance with the worldwide occurrence of these viruses, considerable fractions of all serum panels, including the ones from Germany and Colombia, reacted positive in these tests ( Figure 4 ).

In addition, the 15 African sera concordantly classified as positive by both the NCP-based and the spike/S1based Euroimmun ELISA ( Figure 3A -C) were also analysed with the Euroline Anti-SARS-CoV-2 Profile IgG line blot (Euroimmun) ( Figure 3D ). All of them showed a strong signal with OC43 NCP (mean index value 5.6) that by far exceeded the signals detected for the NCPs of HKU1 (mean index value 0.68), NL63 (mean index value 1.2), 229E

(mean index value 1.0), and also SARS-CoV-2 (mean index value 1.3). Strikingly, only 2/7 Ghanaian samples but 7/8

Nigerian samples reacted positive in the in-house OC43 ELISA employing an N-terminally truncated OC43 NCP as

This article is protected by copyright. All rights reserved antigen ( Figure 3D ).

Information about Plasmodium parasitaemia was only accessible for one of the Ghanaian panels (Ghana 1, 55/150 samples from symptom-free children with microscopically detectable parasitaemia) and the Madagascan (4/167

Plasmodium-PCR positive samples) panel. Here, a reduced specificity in Ghanaian parasitaemic vs. non-parasitaemic samples was observed for the Euroimmun Anti-SARS-CoV-2-NCP-IgG ELISA but not for the other SARS-CoV-2 IgG ELISAs (Table 4 ).

In our study, we report a markedly reduced specificity of four SARS-CoV-2 IgG serological assays in serum samples originating from countries of sub-Saharan Africa, i.e. Ghana and Nigeria. This observation is in concordance with recent reports from Benin (7), Malawi (14), Tanzania (6), and Zambia (6) describing performance data of commercially available ELISAs (7, 14) and IIFT using eukaryotic cells overexpressing SARS-CoV-2 proteins (6), respectively. Positive reactivity with both SARS-CoV-2 NCP and spike/S1 detected by ELISA could be confirmed by line blotting for 11/15 and 14/15 African sera, respectively. Thus, the line blot is slightly less sensitive in picking up the apparently false positive signals in the pre-COVID-19 African sera. As has previously been recognized for serological tests aiming at detection of anti-SARS-CoV-1 antibodies (9), our study revealed a higher rate of false positive assay results in the NCP-based IgG ELISAs than in the spike/S1-based IgG ELISA. This finding may reflect the higher degree of sequence conservation between the immunodominant regions of the coronavirus NCPs (9), rendering assays based on this antigen more prone to cross-reactivity than tests employing the less conserved spike protein. As has already been reported for ZIKV (16) and SARS-CoV-2 IgG ELISAs (7), hypergammaglobulinaemia resulting from polyclonal B-cell activation induced by pathogens like Plasmodia can challenge assay specificity. Indeed, we observed a slightly reduced specificity of the Euroimmun Anti-SARS-CoV-2-NCP-IgG ELISA in Ghanaian parasitaemic vs. non-parasitaemic samples (83.6% (95% CI: 71.5%-91.4%) vs. 94.7% (95% CI: 88.0%-98.0%), p = 0.0387)). While

Plasmodium falciparum malaria is holoendemic in many regions in Ghana and Nigeria (17), length and intensity of malaria transmission varies significantly between different areas in Madagascar (17, 18) . In Colombia, 78% of the population lives in malaria free areas, and Plasmodium falciparum accounts only for 58% of malaria cases (42%:

Plasmodium vivax) (19) . None of the 134 Colombian donors included in our study reported a previous malaria Figure 3D ).

Our study has some limitations that have to be acknowledged when interpreting the presented data. First of all, the sample panels (originating from previous studies with different scientific objectives) do not reflect representative cross-sections of the respective countries' populations and also comparability between sample panels is limited.

Although in total 600 African samples have been analysed, the number of samples per country giving rise to false

This article is protected by copyright. All rights reserved positive SARS-CoV-2 IgG assay results is still relatively small, impeding statistical analyses. Furthermore, limited accessible sample volumes prevented us from performing material-intensive assays as the SD Bioline Dengue Duo IgG Rapid Test or Plasmodium-specific RT-PCR for the complete panel.

Our work shows that several commercially available SARS-CoV-2 IgG ELISAs, especially those employing recombinant NCP as antigen, are prone to generate a high number of false positive results when testing serum/plasma samples originating from Sub-Saharan Africa. Beside other factors, high antibody titres resulting from previous infections with other coronaviruses and/or acute or previous malaria episodes may cause this phenomenon.

Based on these findings, the following recommendations should be considered when testing sera from African 

This article is protected by copyright. All rights reserved NCP as antigen. Bold numbers: % of samples for which an iv ≥ 1.3 was obtained, numbers in brackets: 95%

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Correspondence: Christina Deschermeier, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str

Phone +49-40-42818-438

The authors thank M. Panning for providing common cold coronavirus RNAs. J. Hansen is thanked for expert technical assistance. The study was supported by the German Federal Ministry of Education and Research (Grant no.