Detection of SARS‐CoV‐2 in Semen Months After COVID‐19: Potential Evidence of Low‐Level Replicative Persistence

BACKGROUND: Viral persistence is considered one of the key mechanisms driving long COVID symptoms. The male genital tract (MGT), particularly the testis, is an immune-privileged site that may serve as a viral reservoir for SARS-CoV-2. However, the presence of SARS-CoV-2 in semen and its long-term infectiousness remains controversial. OBJECTIVES: We aimed to investigate the long-term presence and potential replicative activity of SARS-CoV-2 in semen, evaluating whether the virus can persist and maintain low-level culture signals in the MGT well beyond the acute phase of infection. MATERIAL AND METHODS: This observational, prospective, cross-sectional study included 38 men treated at Mater Dei Hospital, Brazil, between October 2020 and October 2021, who had recovered from mild to moderate COVID-19 and consented to participate. Semen samples were collected from mildly symptomatic participants at approximately 1 month (15- 45 days), 3 months, and 6 months after symptom onset. The moderately symptomatic group provided a single sample approximately 6 months post-infection. Semen analysis and SARS-CoV-2 detection were performed using RT-qPCR, followed by viral isolation in Vero CCL-81 cells. Potential viral persistence and replication signals were evaluated through immunofluorescence and transmission electron microscopy (TEM). RESULTS: SARS-CoV-2 was detected by RT-qPCR in 21 of 38 patients (55%), with high cycle threshold (Ct 38-39) values observed for more than 6 months post-infection. Following inoculation into Vero cell cultures, a low-level in vitro expansion of viral copies was identified in a subset of samples from mild and moderate patients. The potential presence of the virus was further supported by the presence of viral proteins via immunofluorescence and the identification of structures morphologically compatible with coronavirus-like particles by TEM. CONCLUSION: This study provides evidence of the long-term persistence of viral RNA and culture-derived signals suggestive of restricted or low-level replicative activity in semen. These findings suggest that the MGT may serve as a viral reservoir, highlighting the importance of longitudinal semen analysis for monitoring viral persistence and reservoir dynamics after clinical recovery.