Impaired performance of SARS-CoV-2 antigen-detecting rapid diagnostic tests at elevated and low temperatures

Abstract

Antigen-detecting rapid diagnostic 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 1.0×10⁶- 5.5×10⁷ genome copies/mL of infectious SARS-CoV-2 cell culture supernatant. Despite long-term storage at recommended conditions, 10 min 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, again including an Ag-RDT recommended by the WHO. 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 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.

Keywords: Rapid antigen test; SARS-CoV-2; Sensitivity; Specificity; Temperature stability; Tropics; Winter.

Fig. 1

COVID-19 case numbers and maximum temperatures globally. A. World map representing global temperature distribution based on maximum temperature of the warmest month (°C) freely available from WorldClim 2 [8]. B. Graph represents total COVID-19 cases in the ten most affected countries globally by 25^th of November 2020 [9].

Fig. 2

Experimental setup. A. Validation of SARS-CoV-2 Ag-RDTs at elevated temperatures. (i): storage at recommended conditions (room temperature (rt); 15-30 °C) for 1-6 months and test operation at recommended conditions (rt; 15-30 °C). (ii): storage at recommended conditions (rt; 15-30 °C) for 1-6 months, 10 min pre-incubation of tests at 37 °C prior to operation at 37 °C so as to mimic recommended storage of kits prior to test usage under non air-conditioned conditions in tropical settings. (iii): storage at recommended conditions (rt; 15-30 °C) for 1-6 months, 10 min pre-incubation of tests at 37 °C prior to operation at room temperature. (iv): storage at recommended conditions (rt; 15-30 °C) for 1-6 months and test operation at 37 °C. Settings (v) and (vi) covered storage under tropical conditions (37 °C) for 3 weeks followed by either test operation at 37 °C to mimic non air-conditioned storage and test operation in tropical settings (v) or test operation at room temperature to mimic non air-conditioned storage and test application at room temperature (vi). B. Validation of SARS-CoV-2 Ag-RDTs at low temperatures. (i): storage at recommended conditions (room temperature (rt); 15-30 °C) for 1-6 months and test operation at recommended conditions (rt; 15-30 °C). (vii): storage of tests at recommended conditions, pre-incubation of tests for 30 min at cold temperatures (2-4 °C) and operation at cold temperatures (2-4 °C). (viii): storage of tests at 2-4 °C for 3 days followed by testing at 2-4 °C. rt = room temperature.

Fig. 3

Sensitivity of SARS-CoV-2 Ag-RDTs decreases at elevated temperatures.Analytical sensitivity of SARS-CoV-2 rapid antigen tests upon different storage and operation conditions; i: storage and test operation at recommended conditions (rt; 15-30 °C); ii: storage at recommended conditions (rt; 15-30 °C), 10 min pre-incubation at 37 °C prior to operation at 37 °C; iii: storage at recommended conditions (rt; 15-30 °C), 10 min pre-incubation at 37 °C prior to operation at recommended conditions (rt; 15-30 °C); iv: storage at recommended conditions (rt; 15-30 °C) and test operation at 37 °C; v: storage and testing at 37 °C; vi: storage at 37 °C and testing at recommended conditions (rt; 15-30 °C); ++ positive; + weak positive; borderline: unclear result; - negative;. rt: room temperature. n/a: data not available.

Fig. 4

Specificity of SARS-CoV-2 Ag-RDTs decreases at low temperatures. A.i: storage and test operation at recommended conditions (rt; 15-30 °C); vii: storage at recommended conditions (rt; 15-30 °C), 30 min pre-incubation of tests at 2-4 °C prior to test operation at 2-4 °C; viii: storage at 2-4 °C for 3 days and testing at 2-4 °C; red: positive; white: negative; salmon: weak band, result unclear. rt: room temperature. Tests were performed in duplicates. B. Example for observed cross-reactivity of the ActivXpress test with Influenza virus A H1N1 and SARS-CoV-2 nucleoprotein as positive control (SARS-CoV-2-N; 5 μg/mL) when tested under condition vii: storage at recommended conditions (rt; 15-30 °C), 30 min pre-incubation of tests at 2-4 °C prior to test operation at 2-4 °C.