key: cord-0828519-zyxsxfyt
authors: Haage, Verena; de Oliveira-Filho, Edmilson Ferreira; Moreira-Soto, Andres; Kühne, Arne; Fischer, Carlo; Sacks, Jilian A.; Corman, Victor Max; Müller, Marcel A.; Drosten, Christian; Drexler, Jan Felix
title: Impaired performance of SARS-CoV-2 antigen-detecting rapid tests at elevated and low temperatures
date: 2021-03-16
journal: J Clin Virol
DOI: 10.1016/j.jcv.2021.104796
sha: 23f52d34a54b4e80ca59e50a39881b9ff7a7a8e1
doc_id: 828519
cord_uid: zyxsxfyt

Rapid antigen-detecting tests (Ag-RDTs) can complement molecular diagnostics for COVID-19. The recommended temperature for storage of SARS-CoV-2 Ag-RDTs ranges between 2-30 °C. In the global South, mean temperatures can exceed 30 °C. In the global North, Ag-RDTs are often used in external testing facilities at low ambient temperatures. We assessed analytical sensitivity and specificity of eleven commercially-available SARS-CoV-2 Ag-RDTs using different storage and operational temperatures, including short- or long-term storage and operation at recommended temperatures or at either 2-4 °C or at 37 °C. The limits of detection of SARS-CoV-2 Ag-RDTs under recommended conditions ranged from 1.0 × 10(6)-5.5 × 10(7) genome copies/ml of infectious SARS-CoV-2 cell culture supernatant. Despite long-term storage at recommended conditions, 10 minutes pre-incubation of Ag-RDTs and testing at 37 °C resulted in about ten-fold reduced sensitivity for five out of 11 SARS-CoV-2 Ag-RDTs, including both Ag-RDTs currently listed for emergency use by the World Health Organization. After 3 weeks of storage at 37 °C, eight of the 11 SARS-CoV-2 Ag-RDTs exhibited about ten-fold reduced sensitivity. Specificity of SARS-CoV-2 Ag-RDTs using cell culture supernatant from common respiratory viruses was not affected by storage and testing at 37 °C, whereas false-positive results occurred at outside temperatures of 2-4 °C for two out of six tested Ag-RDTs. In summary, elevated temperatures impair sensitivity, whereas low temperatures impair specificity of SARS-CoV-2 Ag-RDTs. Consequences may include false-negative test results at clinically relevant virus concentrations compatible with inter-individual transmission and false-positive results entailing unwarranted quarantine assignments. Storage and operation of SARS-CoV-2 Ag-RDTs at recommended conditions is essential for successful usage during the pandemic.

Advantages of SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDTs) include fast results and their applicability on site without dependence on laboratory settings.

With a constantly growing number of commercially available Ag-RDTs on the global market, the number of studies validating Ag-RDTs from different manufacturers is increasing rapidly (1) (2) (3) (4) (5) (6) (7) . However, none have interrogated the performance of Ag-RDTs under conditions that differ from supplier-recommended storage and operation conditions (5-30°C), such as those observed in tropical settings where ambient temperatures routinely exceed 30°C ( Figure 1A) . This is challenging because tropical regions are strongly affected by the SARS-CoV-2 pandemic as evident from total cases reported from India, Brazil, Argentina, and Colombia, four out of the ten most affected countries worldwide by November 2020 (Figure 1B) . On the other hand, the global North is currently affected by the second wave of the COVID-19 pandemic (10, 11). To manage testing demand, different actors have opened external testing stations such as `diagnostic streets´ or drive-through facilities in urban settings (12) . These facilities are often of provisional nature, for example in the form of unheated tents. In the winter months, temperatures in Europe or the U.S. can range from -10°C to 10°C (13, 14) , well below the recommended operating temperatures of most Ag-RDTs.

Most manufacturers of SARS-CoV-2 Ag-RDTs specify storage conditions between 2-30°C, but stipulate that tests be equilibrated to room temperature (15-30°C) at the time of use to guarantee performance. With temperatures around freezing point during the winter months, unheated testing facilities cannot always comply with these conditions. J o u r n a l P r e -p r o o f Temperature tolerance of SARS-CoV-2 diagnostic tools or environmental stability requirements have been previously discussed as hurdles to be addressed according to the World Health Organization (WHO) (15, 16) .

To validate the performance of SARS-CoV-2 Ag-RDTs in both, tropical and cold settings, we compared analytical sensitivity and specificity using recommended conditions and either elevated or low temperatures.

SARS-CoV-2 (BetaCoV/Munich/ChVir984/2020) was grown on Vero E6 cells (C1008;

African green monkey kidney cells), maintained in DMEM (10% FCS) at 37°C with 5% CO2. For quantification, viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) and quantified using photometrically quantified in vitrotranscribed RNA standards (17, 18) . For determining the limit of detection (LOD), SARS-CoV-2 stock (2.2x10 9 copies/ml) was serially diluted in plain DMEM and 5µl per dilution was added to the extraction buffer of the respective kit for validation experiments. For Table 1 ). Consequently, due to a limited number of available tests, experiments were performed in duplicates. LOD was defined as the lowest dilution at which both replicates were positive. A dilution factor correction was applied based on the volume of extraction buffer (range: 100-500µl) provided by each SARS-CoV-2 Ag-RDT kit.

Specificity for tropical conditions was assessed using cell culture supernatant of the ubiquitous human coronaviruses HCoV-229E (2.9x10 7 copies/ml) HCoV-OC43 (1.0x10 6 copies/ml). 5µl of stocks were directly used for validation experiments except for Coris COVID-19 Ag Respi-Strip as described above.

Specificity for cold settings was tested using common cell culture-derived respiratory viruses including HCoV-229E, HCoV-OC43, influenza virus A H1N1 (7.8x10 6 copies/ml) and rhinovirus A (2.2x10 6 copies/ml). 20µl of viral cell culture supernatant were added to proprietary lysis buffer or as an internal control 20µl of lysis buffer were directly applied to test cassettes for validation experiments. Viral concentrations were selected according to the guidelines on analytical specificity testing for SARS-CoV-2 Ag-RDTs published by the German Federal institute for vaccines and biomedicines (19) .

Additionally , ten healthy laboratory members who previously volunteered for a SARS-CoV-2 Ag-RDT validation study were tested (1). Healthy volunteers were without symptoms of respiratory tract infection and tested negative for SARS-CoV-2 by RT-qPCR (20) . All subjects received instructions on self-sampling, recently shown to be a reliable alternative to professional nasopharyngeal swabs for Ag-RDTs (21) . Swabs were dissolved immediately in 1ml PBS and 20µl of PBS were added to proprietary buffer for testing.

For tests with visual readout, results in the form of a band were scored by two researchers independently and in case of discrepancy a third person was consulted to reach a final decision (reader-based tests: Bioeasy 2019-nCoV Ag and ichroma -COVID-19 Ag). Results were defined as borderline when a weak, discontinuous band or smear was observed that could not be clearly defined as a positive or negative result.

Data of maximum temperatures of the hottest month (°C) on country level at the spatial resolution of 2.5 min were obtained from WorldClim 2 (8). Exactextractr package in R version 4.0.2 was used to calculate national means. Data on COVID-19 cases were obtained from Worldmeter (9) and visualized using the GraphPad Prism software.

At present, there are at least 139 SARS-CoV-2 Ag-RDTs commercially available (22), from which 11 were selected for temperature stability validation at elevated temperatures based on the availability of clinical performance data (1) and manufacturing by leading suppliers implying availability on the global market ( Table 1) . First, we determined analytical sensitivity at recommended conditions by determining the limit of detection (LOD) of SARS-CoV-2 Ag-RDTs at room temperature. The dilutionfactor corrected limit of detection (LOD) for validated SARS-CoV-2 Ag-RDTs ranged from 1.0x10 6 copies/ml to 5.5x10 7 copies/ml of SARS-CoV-2 cell culture supernatant ( Table 2) .

Those LODs were consistent with previously published virus concentrations for validation of Ag-RDTs using clinical samples (1), suggesting robustness of our data. Our data also highlight profound differences in analytical sensitivity of up to 50-fold for SARS-CoV-2 Ag-RDTs from different manufacturers. 

1.4x10 6 1.7x10 6 5.9x10 6 1.3x10 6 5.5x10 7 3.2x10 7 1.0x10 6 1.7x10 6 8.4x10 6 1.2x10 6 1. We then assessed analytical sensitivity of SARS-CoV-2 Ag-RDTs following short-and longterm exposure to 37°C (settings (ii), (iii), (v), (vi); Figure 2) . The analytical sensitivity of about half of the evaluated SARS-CoV-2 Ag-RDTs (five out of eleven; 45%) was already compromised by about ten-fold when tests were stored under recommended conditions but exposed to 37°C for only ten minutes prior to testing at 37°C (ii) (Figure 3 ; for LOD refer to Supplementary Table 2 ). This effect was even more pronounced when tests were stored under recommended conditions but exposed to 37°C for ten minutes prior to testing at room temperature (iii), as all eight tested kits showed an about 10-fold reduced sensitivity under this experimental setting. After 19-21 days storage at 37°C and testing at 37°C (v) or testing at room temperature (vi), eight out of the total eleven SARS-CoV-2

Ag-RDTs (73%) showed an about ten-fold reduction in analytical sensitivity when compared to recommended temperatures. In sum, those data indicate that even shortterm exposure of SARS-CoV-2 Ag-RDTs to elevated temperatures affects their sensitivity and that multiple temperature shifts might more seriously affect test sensitivity. Figure 2 ) was examined by testing for cross-reactivity with the ubiquitous human coronaviruses (HCoV) HCoV-229E (2.9x10 7 copies/ml) and HCoV-OC43 (1.0x10 6 copies/ml) (23, 24) . SARS-CoV-2 Ag-RDTs showed no cross-reactivity with HCoV-229E or HCoV-OC43 upon storage and testing at elevated temperatures ( Table 3) . Tests were performed in duplicates.

As the national COVID-19 reference laboratory in Germany, we have been contacted by multiple outside testing facilities across Germany reporting an unusual high number of positive SARS-CoV-2 Ag-RDTs. In order to validate SARS-CoV-2 specificity performance when operated at low outside temperatures (2-4°C) (test conditions vii and viii; Figure   2 ), we selected a subset of six Ag-RDTs for reasons of scarcity of tests and urgency to conduct the testing under the current weather conditions that prevailed at the time of physicians' reports from external testing stations ( Table 1) . Two of the six SARS-CoV-2

Ag-RDTs showed impaired specificity ( Figure 4A ) when stored at room temperature, but when exposed to 2-4°C for 30 minutes prior to testing at 2-4°C (vii) as cross-reactivity with common respiratory viruses, and false-positive results occurred in healthy volunteers in the form of weak, but clearly visible bands (Figure 4B) . In one test (test I),

unspecific reactivity was only observed upon short-term incubation at 2-4°C (vii) followed by test operation at 2-4°C, but not after long-term storage at 2-4°C (viii). In J o u r n a l P r e -p r o o f Additionally, our data on an overall impaired performance of Ag-RDTs at elevated temperatures are consistent across tests and analytical sensitivity for several tests was identical upon usage of either duplicates or higher numbers of replicates.

In sum, it was previously shown that clinically relevant virus concentrations of about 10 6 genome copies per ml suffice for virus isolation and culture and therefore serve as a correlate for infectivity (39, 40) . Our study strongly suggests that short-and long-term exposure to elevated temperatures may compromise sensitivity of SARS-CoV-2 Ag-RDTs to an extent that may lead to false-negative test results at clinically relevant virus concentrations, potentially enhancing SARS-CoV-2 spread in tropical settings. At the same time, false-positive test results owed to test operation at low temperatures might not only lead to unwarranted individual quarantine assignments, but also to potential regional lockdown measures if those results were reported to public health authorities without confirmation by a gold standard test such as RT-PCR (20) . Foundation. The study was further supported in part by the Foundation for Innovative New Diagnostics (FIND), including procurement of some test kits.

The authors declare no conflict of interest. 

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