Real Time PCR and Culture-Based Virus Isolation Test in Clinically Recovered Patients: Is the Subject Still Infectious for SARS-CoV2?

Abstract

Background: The highly variable manifestation of the COVID-19 disease, from completely asymptomatic to fatal, is both a clinical and a public health issue. The criteria for discharge of hospitalized patients have been based so far on the negative result of Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) tests, but the persistence of viral fragments may exceed that of the integral virus by weeks. The aim of our study was to verify the clearance of the virus at viral culture in patients hospitalized for COVID-19 that have clinically recovered but are still positive on nasopharyngeal swab.

Methods: The study was conducted in hospitalized patients with positive RT-PCR on nasopharyngeal swab. Patients included were from asymptomatic to severe cases and performed nasopharyngeal control swabbing on day 14 for asymptomatic patient or at least three days after remission of symptoms. RT-PCR positive specimens were sent to a biosafety level 3 laboratory for viral culture.

Results: We performed a combined analysis of RT-PCR and a highly sensitive in vitro culture from 84 samples of hospitalized patients. The average age was 46 ± 20.29, and 40.5% of the subjects had radiologically confirmed pneumonia, with average PaO₂ of 72.35 ± 12.12and P/F ratio of 315 ± 83.15. Ct values for the N gene were lower in the first swab than in the control one (p < 0.001). The samples from 83 patients were negative at viral culture, and RT-PCR on the respective supernatants always confirmed the absence of viral growth.

Conclusions: Our preliminary results demonstrate that patients clinically recovered for at least three days show the viral clearance at viral culture, and presumably they continued to not be contagious.

Keywords: COVID-19; RT-PCR; SARS-CoV-2; cell culture; viral transmission.

Figure 1

Comparison of cycle threshold (Ct) values at T0 (positive nasopharyngeal swab at hospital admission) and at T1 (nasopharyngeal control swab at clinical recovery performed after 19.95 ± 5.71 days from T0) for the N gene in a population of 84 patients described by Violin plot).

Figure 2

Viral cultures examined by a microscope at 5× magnification: (a) Absence of viral growth, as shown by complete confluence of Vero E6 cells; (b) Cytopathic effects consisting of rounding and detachment of cells in Vero E6 cell cultures at 96 h post-seeding.