Persistent Nonviral Plasmid Vector in Nasal Tissues Causes False-Positive SARS-CoV-2 Diagnostic Nucleic Acid Tests

“Introduction: Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be asymptomatic or cause mild, moderate, or severe disease. To determine the biomarkers predictive of these varied outcomes, we conducted a prospective study of incident SARS-CoV-2 infection. Biomedical researchers and/or household members were enrolled, and the study participants self-collected anterior nasal swabs and delivered them to the testing site weekly. Testing of the nasal swabs by PCR detected SARS-CoV-2 in 14 cases within the cohort, including four asymptomatic coworkers and one of their household members who had no known exposures to SARS-CoV-2-infected individuals. When this 'outbreak' was noted, epidemiologic investigation revealed that the four coworkers within this research laboratory were using a plasmid vector to express the SARS-CoV-2 nucleocapsid in the month prior to enrollment. In this report, we describe the molecular studies performed to determine if these five case participants were infected with SARS-CoV-2 circulating in the community or with the laboratory plasmid...

Discussion: SARS-CoV-2 was amplified from longitudinally collected nasal specimens from five asymptomatic individuals, four coworkers and one of their household members, which suggested person-to-person transmission of pandemic coronavirus. However, after the study team learned that a plasmid vector had been used in the month prior to enrollment by all four case coworkers in the laboratory outbreak, additional studies were performed to differentiate SARS-CoV-2 RNA from virus circulating in the community from DNA from the laboratory plasmid. Our inability to amplify regions of SARS-CoV-2 RNA outside the nucleocapsid that serve as targets in other diagnostic assays did not support the presence of the pandemic SARS-CoV-2 in these nasal swabs. Multiple molecular assays detected DNA sequences, including a codon-optimized region unique to the plasmid used by this research group to produce SARS-CoV-2 nucleocapsid protein, in each of the case coworker’s nasal specimens (Fig. 1). Taken together, these results indicate that the plasmid DNA encoding the SARS-CoV-2 nucleocapsid gave false-positive results in our SARS-CoV-2 diagnostic assay…”