Prolonged SARS-CoV-2 RNA detection by RT-PCR: variability in cycle threshold values over time

Millions of reverse transcription-polymerase chain reaction (RT-PCR) tests have been performed worldwide during the SARS-CoV-2 pandemic, using various protocols. This study evaluates the duration of SARS-CoV-2 RNA detectability by RT-PCR at body temperature and analyzes changes in cycle threshold (Ct) values over time. Positive nasopharyngeal swabs for SARS-CoV-2 RT-PCR (n = 120) with different Ct values were collected from Hospital Universitario 12 de Octubre (Madrid, Spain, 2020). Samples were processed using an open-access protocol for the Hologic Panther Fusion system, targeting the envelope gene. They were stratified into six Ct value groups (<20, 20.0-25.0, 25.1-30.0, 30.1-35.0, 35.1-40.0, and >40.0) and incubated at 37°C for 15, 30, 45, and 60 days, with RT-PCR repeated at each interval. Samples with Ct <20.0 remained positive throughout the study, whereas those with Ct >40.0 were negative by day 15. A significant proportion of samples with Ct ≤35.0 remained positive at day 60. Positivity during incubation decreased with higher Ct values (P < 0.001). Ct values increased over time but inconsistently by day 60. In conclusion, SARS-CoV-2 RNA can persist for months, leading to prolonged RT-PCR positivity. However, repeated RT-PCR testing introduces variability in qualitative results and Ct values, complicating clinical decisions. Patient monitoring should emphasize clinical assessment and viral viability assays rather than repeated RT-PCR testing.IMPORTANCEThis study investigates the prolonged persistence of SARS-CoV-2 detected by reverse transcription-PCR (RT-PCR) at human body temperature and the evolution of cycle threshold (Ct) values in positive respiratory samples. Our findings indicate that SARS-CoV-2 RNA can persist for months, and that repeated RT-PCR testing over time introduces variability, particularly in samples with high Ct values. This work highlights the necessity of complementing RT-PCR testing with methods assessing viral viability to avoid over-reliance on repeated testing in clinical decision-making. This study addresses a critical issue in COVID-19 diagnostics, with important implications for clinical management and infection control.