Assessment of SARS-CoV-2 RNA Test Results Among Patients Who Recovered From COVID-19 With Prior Negative Results

Some patients who have recovered from coronavirus disease 2019 (COVID-19) with documented negative real-time polymerase chain reaction (RT-PCR) results at the time of recovery have had subsequent positive RT-PCR test results for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)^1,2 in the absence of any symptoms suggestive of new infection.³ It is unknown whether such patients are infectious and whether they should be quarantined. Real-time PCR is not a viral culture and does not allow determination of whether the virus is viable and transmissible. We investigated RT-PCR retested positive nasal/oropharyngeal swab (NOS) samples from recovered patients with COVID-19 with prior negative results for the presence of replicative SARS-CoV-2 RNA.⁴

We studied 176 recovered patients with COVID-19 who were admitted to the postacute outpatient service of our institution (Rome, Italy) from April 21 to June 18, 2020, for COVID-19 follow-up.^5,6 Before that, patients had discontinued isolation according to current criteria,⁵ which require no fever for 3 consecutive days, improvement in other symptoms, and 2 negative RT-PCR results for SARS-CoV-2 RNA 24 hours apart.

Nasal/oropharyngeal swab samples from patients at follow-up were analyzed for total (genomic) and replicative (subgenomic) SARS-CoV-2 RNA using RT-PCR assays (eMethods in the Supplement). For patients with positive results for total RNA, samples previously obtained at the time of COVID-19 diagnosis and kept at −112 °F until testing were also tested for replicative RNA. Serological testing was performed for SARS-CoV-2 IgG/IgA detection (eMethods in the Supplement). The ethics committee of the Fondazione Policlinico Universitario A. Gemelli IRCCS (Rome, Italy) approved the study, and written informed consent was obtained from each patient.

As shown in the Table,⁴ 32 of 176 NOS samples (18.2%) tested positive for total SARS-CoV-2 RNA, with viral loads ranging from 1.6 × 10¹ to 1.3 × 10⁴ SARS-CoV-2 RNA copies per mL. One of the 32 samples (3.1%) had replicative SARS-CoV-2 RNA. Samples from the 32 patients at the time of COVID-19 diagnosis were also tested and, expectedly, had replicative SARS-CoV-2 RNA. All but 1 of 32 patients had a positive serology result against SARS-CoV-2 (Table), as well as 139 of remaining 144 patients (data not shown), at COVID-19 follow-up. The patient who tested serologically negative was not the one with a positive test result for replicative SARS-CoV-2 RNA. The mean (SD) time from COVID-19 diagnosis to follow-up was 48.6 (13.1) days in 32 patients (Table) and 57.7 (16.9) days in 144 patients (data not shown).

Similar to that reported elsewhere,² 18% of patients with COVID-19 in our institution became RT-PCR positive for SARS-CoV-2 RNA after clinical recovery and previous negative results.⁵ As positivity in the patients was suggestive, but not necessarily a reflection, of viral carriage, we used replicative SARS-CoV-2 RNA detection as a proxy for virus replication in culture.⁴

Only 1 of 32 patients retesting positive had replicating virus in the NOS sample, suggesting either recurrent infection or reinfection, which is impossible to separate because no whole-genome sequencing and phylogenetic analyses were performed.³ The patient retested positive 16 days after COVID-19 recovery (ie, 39 days from COVID-19 diagnosis) and was symptomatic. The patient was an older adult with hypertension, diabetes, and cardiovascular disease but no evidence of close contacts with people with SARS-CoV-2 infection or persons who became RT-PCR positive. In the 31 remaining patients (who were asymptomatic), their positive result likely represented either recurrent or resolving infection, but in either case, they were unlikely to be infectious. The limitations of our study are the lack of data from viral cultures or whole-genome sequencing analysis and the small sample size.

This study highlights that many patients who recovered from COVID-19 may be still positive (albeit at lower levels) for SARS-CoV-2 RNA, but only a minority of the patients may carry a replicating SARS-CoV-2 in the respiratory tract. Further studies are needed to verify whether such patients can transmit the virus.

Accepted for Publication: October 25, 2020.

Published Online: November 12, 2020. doi:10.1001/jamainternmed.2020.7570

Corresponding Author: Brunella Posteraro, PhD, Dipartimento di Scienze Mediche e Chirurgiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, Italy (brunella.posteraro@unicatt.it).

Author Contributions: Drs Sanguinetti and Cattani had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drs Liotti and Menchinelli contributed equally to the study. Drs Sanguinetti and Cattani contributed equally as senior authors.

Concept and design: Liotti, Posteraro, Landi, Sanguinetti, Cattani.

Acquisition, analysis, or interpretation of data: Liotti, Menchinelli, Marchetti, Posteraro, Sanguinetti, Cattani.

Drafting of the manuscript: Liotti, Menchinelli, Posteraro, Sanguinetti, Cattani.

Critical revision of the manuscript for important intellectual content: Liotti, Marchetti, Landi, Sanguinetti, Cattani.

Statistical analysis: Menchinelli.

Obtained funding: Sanguinetti.

Supervision: Posteraro, Landi.

Other: Liotti.

Conflict of Interest Disclosures: None reported.

Funding/Support: This work was funded by donations from Reale Group and Fondazione Valentino Garavani & Giancarlo Giammetti to support the COVID-19 research in our institution.

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We thank Franziska Lohmeyer, PhD (Scientific Direction, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy) for English revision of the manuscript. She was not compensated for her contributions.