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

Biomedical personnel can become contaminated with nonhazardous reagents used in the laboratory. We describe molecular studies performed on nasal secretions collected longitudinally from asymptomatic laboratory coworkers to determine if they were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulating in the community or with SARS-CoV-2 DNA from a plasmid vector. Participants enrolled in a prospective study of incident SARS-CoV-2 infection had nasal swabs collected aseptically by study staff at enrollment, followed by weekly self-collection of anterior nasal swabs. SARS-CoV-2 diagnosis was performed by a real-time PCR test targeting the nucleocapsid gene. PCR tests targeting SARS-CoV-2 nonstructural protein 10 (nsp10), nsp14, and envelope and three regions of the plasmid vector were performed to differentiate amplification of SARS-CoV-2 RNA from the plasmid vector's DNA. Nasal swabs from four asymptomatic coworkers with positive real-time PCR results for the SARS-CoV-2 nucleocapsid targets were negative when tested for SARS-CoV-2 nsp10, nsp14, and envelope protein. However, nucleic acids extracted from these nasal swabs amplified DNA regions of the plasmid vector used by the coworkers, including the ampicillin and neomycin/kanamycin resistance genes, the promoter-nucleocapsid junction, and unique codon-optimized regions. Nasal swabs from these individuals tested positive repeatedly, including during isolation. Longitudinal detection of plasmid DNA with SARS-CoV-2 nucleocapsid in nasal swabs suggests persistence in nasal tissues or colonizing bacteria. Nonviral plasmid vectors, while regarded as safe laboratory reagents, can interfere with molecular diagnostic tests. These reagents should be handled using proper personal protective equipment to prevent contamination of samples or laboratory personnel.

Automated summary

This study found that nasal secretions from biomedical personnel contained SARS-CoV-2 DNA, even though these individuals did not show any symptoms. It suggests that they may have been infected with SARS-CoV-2 circulating in the community or with SARS-CoV-2 DNA from a plasmid vector. The presence of nonviral plasmid vectors can interfere with molecular diagnostic tests.