Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection After Recovery from Mild Coronavirus Disease 2019

Abstract

Background: Positive results from real-time reverse-transcription polymerase chain reaction (rRT-PCR) in recovered patients raise concern that patients who recover from coronavirus disease 2019 (COVID-19) may be at risk of reinfection. Currently, however, evidence that supports reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reported.

Methods: We conducted whole-genome sequencing of the viral RNA from clinical specimens at the initial infection and at the positive retest from 6 patients who recovered from COVID-19 and retested positive for SARS-CoV-2 via rRT-PCR after recovery. A total of 13 viral RNAs from the patients' respiratory specimens were consecutively obtained, which enabled us to characterize the difference in viral genomes between initial infection and positive retest.

Results: At the time of the positive retest, we were able to acquire a complete genome sequence from patient 1, a 21-year-old previously healthy woman. In this patient, through the phylogenetic analysis, we confirmed that the viral RNA of positive retest was clustered into a subgroup distinct from that of the initial infection, suggesting that there was a reinfection of SARS-CoV-2 with a subtype that was different from that of the primary strain. The spike protein D614G substitution that defines the clade "G" emerged in reinfection, while mutations that characterize the clade "V" (ie, nsp6 L37F and ORF3a G251V) were present at initial infection.

Conclusions: Reinfection with a genetically distinct SARS-CoV-2 strain may occur in an immunocompetent patient shortly after recovery from mild COVID-19. SARS-CoV-2 infection may not confer immunity against a different SARS-CoV-2 strain.

Keywords: COVID-19; SARS-CoV-2; reinfection; whole-genome sequencing.

Figure 1.

Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 strains. The maximum likelihood method was used with branch lengths measured in the number of base substitutions per site. Each color indicates each sampling location or sampling time point.

Figure 2.

Temporal profile of the viral load in patient 1 with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection. WGS of SARS-CoV-2 from respiratory specimens was performed at 4 time points (at first admission with initial diagnosis, during follow-up, at second admission with reinfection, at third admission with retest positive again). A lower Ct value corresponds to a higher viral load. The values under the dashed line were interpreted as negative for SARS-CoV-2. Viral culture was conducted by inoculating upper (triangle) or lower (star) respiratory tract specimens onto Vero cells. Blue indicates that SARS-CoV-2 was culturable and gray indicates that SARS-CoV-2 was not culturable. *No intact viral genome was observed at third admission. Abbreviations: Ct, cycle threshold; rRT-PCR, real-time reverse-transcription polymerase chain reaction; WGS, whole-genome sequencing.

Figure 3.

Temporal profile of antibodies in patient 1 with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection. Each antibody was measured at 7 time points. A, IgG antibodies against SARS-CoV-2 S1 (blue) and receptor binding domain (green) protein. The cutoff values for IgG antibody assay were S/CO = 1. B, PRNT titers of neutralizing antibody. A PRNT₅₀ titer was defined as the highest serum dilution that results in a reduction of >50% of the control plaque count. A PRNT₅₀ titer of ≥10 was considered protective. Abbreviations: IgG, immunoglobulin G; PRNT, plaque-reduction neutralization test; rRT-PCR, real-time reverse-transcription polymerase chain reaction; S/CO, signal to cutoff. *No intact viral genome was observed at third admission.