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The Impact of Freeze-Thaw Cycles on the Integrity of SARS-CoV-2 Viral Culture Fluids and Clinical Remnant Samples in Antigen or Nucleic Acid Testing

Hajirah Noor Hussain, Hali Weeks, Derek Zhou, Divya Joseph, Brooke Lam, Haidong Xu, Chushi Zhang, Keqin Gregg, Wenli Zhou*

Preservation at ultra-low temperatures has been a gold standard for long-term storage of many types of clinical specimens including the SARS-CoV-2 virus. The frozen specimens can be easily transported and tested later. In addition, de-identified frozen remnant samples are resources for many preclinical or clinical studies. It is therefore crucial to understand whether Freeze-Thaw Cycles (FTCs) can adversely affect SARS-CoV-2 test performance when frozen samples are tested. Some early studies suggest that FTCs increased the Cycle threshold (Ct) of reverse transcription real-time PCR (RT-PCR or RT-qPCR), indicating the potential degradation of SARS-CoV-2 nucleic acid after FTCs, while the others did not report any significant changes in SARS-CoV-2 nucleic acids after FTCs. Moreover, the impact of FTCs on the performance of SARS-CoV-2 antigen test is scarcely reported.

In this study, we performed paired nucleic acid and rapid antigen tests on the same samples to investigate and directly compare how FTCs affect the performance of two types of tests. Both inactivated viral culture fluid samples and clinical remnant samples were studied. Our results showed that FTCs had minimal negative effects on the performance of the rapid SARS-CoV-2 antigen test, and the test results remained largely consistent throughout FTCs, whereas the Ct values of RT-PCR increased with the increase of FTC numbers. In addition, our data also demonstrated that SARS-CoV-2 is preserved better in Viral Transport Medium (VTM) than in Phosphate-Buffered Saline (PBS) during FTCs regarding nucleic acid testing.