Whole Genome Sequencing Confirmed SARS-CoV-2 Reinfections Among Healthcare Workers in India with Increased Severity in the Second Episode

Abstract

Background: Frontline healthcare workers (HCWs) are exceedingly exposed to SARS-CoV-2 and reinfections are a possibility. A RT-PCR positive test does not confirm reinfection. Whole genome sequencing (WGS) of the viral isolates from the different episodes can confirm a reinfection. Methods: RNA was extracted from nasopharyngeal plus oropharyngeal samples from four HCWs who were SARS-CoV-2 RT-PCR positive in May/June and then again in July. Anti-NC antibody testing was performed after the second infection in three HCWs. The RNA was subjected to whole genome sequencing and comparative genome and protein-based functional annotation analyses were performed on the nucleotide and amino acid sequences. Findings: Whole genome sequencing of the eight SARS-CoV-2 viral samples generated a genome coverage ranging from 82.55 to 98.23%. Phylogenetic analysis revealed that sequences belonged to the L clade and within this major clade; they clustered into India-specific A2a and A4 clades. A total of 39 mutations were identified within the eight genomes, including 22 non-synonymous, 16 synonymous, and 1 stop-coding substitutions. Comparative genomic and protein-based annotation analyses revealed differences in the presence and absence of specific mutations in the virus sequences from the first and second episode in all four paired samples. Three HCWs were negative for anti-NC antibodies after the second infection.Interpretation: Genomic variations observed through whole genome sequencing coupled with clinical presentation confirm reinfections of SARS-CoV-2 in healthcare workers. Funding: Municipal Corporation of Greater Mumbai, ICGEB core funding, MLP-2005 and Fondation Botnar grants.Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The study was approved by the Institutional Review Board of Kasturba Hospital of Infectious Diseases; IRB number 015/2020. The patients provided written informed consent.