Persistence of SARS-CoV-2 RNA in lung tissue after mild COVID-19.

On Dec 1, 2020, we reported a successful case of double-lung transplantation from a SARS-CoV-2 seropositive donor 105 days after the onset of mild COVID-19. Although repeated quantitative (q)RT-PCR analyses of donor nasopharyngeal swabs were negative, this technique detected RNA of the SARS-CoV-2 N gene (delta Ct 35) from a biopsy of the right lung taken during organ procurement. Viral culture of this biopsy was negative and donor-to-recipient transmission did not occur. Complementary orthogonal methods were needed to corroborate and interpret the qRT-PCR results.

Therefore, we did ultrasensitive single-molecule fluorescence RNA in-situ hybridisation with RNAscope technology on formalin-fixed paraffin-embedded sections of the same lung biopsy (appendix p 1), and compared the results with those of a lung biopsy from a deceased patient with acute COVID-19 (figure A and B ; appendix p 2). We stained 14 slides of the donor lung biopsy, each containing one 5 μm section, as follows: five slides with a probe for the N gene; five slides with a probe for the S gene; and four slides with probes for N and S. A probe for the basigin gene, which has been proposed to encode an alternative host recipient for SARS-CoV-2, served as a positive control on the ten slides stained for N or S only. We identified characteristic RNAscope puncta in three out of nine slides for the N probe, and in six out of nine slides for the S probe (figure C and D). These puncta appeared to be located in clumps of sloughed-off material, and no cells or cell nuclei could be discerned in this debris-like tissue.

Figure

Ultrasensitive single-molecule fluorescence RNA in-situ hybridisation with RNAscope

Formalin-fixed paraffin-embedded sections of a lung biopsy from a patient with COVID-19 who died 5 days after a positive quantitative RT-PCR result from a nasopharyngeal swab (A, B). Red puncta show RNA for the SARS-CoV-2 N gene (A) or the SARS-CoV-2 S gene (B), and green puncta show RNA for BSG. The nuclear stain DAPI is shown in grey. Scale bar is 20 μm. Formalin-fixed paraffin-embedded sections of the right lung biopsy from the lung donor (C, D). Red puncta show RNA for the SARS-CoV-2 N gene (C) or the SARS-CoV-2 S gene (D), and green puncta show RNA for BSG. The nuclear stain DAPI is shown in grey. The dotted traces denote areas with debris-like tissue that contains puncta for SARS-CoV-2 N or S and for BSG. Scale bar is 10 μm. BSG=basigin.

To our knowledge, this is the first report of long-term (>100 days) persistence of SARS-CoV-2 RNA in lung tissue of an immunocompetent patient after convalescing from COVID-19. The debris-like tissue that contained SARS-CoV-2 RNA might be composed of degenerated endothelial cells that had detached from vessel walls, dysmorphic syncytial elements of pneumocytes, or dead neutrophilic plugs in the interstitium.3, 4 We speculate that this debris-like tissue might shield SARS-CoV-2 RNA from degradation.

Data on sputum, nasopharyngeal swabs, and bronchoalveolar lavage fluid indicate that prolonged detection of SARS-CoV-2 RNA is rare and limited to a few weeks. By contrast, SARS-CoV-2 RNA persisted in the lung parenchyma for 105 days after the onset of a mild course of COVID-19. Nonetheless, at the time of writing, 11 months after transplantation, the recipient is in good health. Our data show that the persistence of SARS-CoV-2 RNA in this donor lung tissue has been inconsequential.

LJC is supported by a Katholieke Universiteit Leuven University Chair funded by Medtronic and a post-doctoral grant from University Hospitals Leuven (KOOR-UZ Leuven). LVG is supported by a post-doctoral grant from University Hospitals Leuven (KOOR-UZ Leuven). AV is supported by a fundamental research grant from the Research Foundation Flanders (FWO). DVR is supported by a grant from the Broere Charitable Foundation. RV and JW are supported as senior clinical research fellows by the FWO Belgium. EW is supported by Stichting tegen Kanker (basic and clinical oncology research). All other authors declare no competing interests.