Clinical, Serological, Whole Genome Sequence Analyses to Confirm SARS-CoV-2 Reinfection in Patients From Mumbai, India.

Jayanthi Shastri,Sachee Agrawal,Vivekanand A,Nirjhar Chatterjee,Anurag Agrawal,Akshay Kanakan,Swapneil Parikh,Rajesh Pandey,Lipi Thukral,Janani Srinivasa Vasudevan,Lancelot Pinto,Ranjeet Maurya,Sakshi Chaudhary,Saman Fatihi,Chetan Sharma,Sujatha Sunil,Manish Pathak

doi:10.3389/fmed.2021.631769

Abstract

Background: SARS-CoV-2 infection may not provide long lasting post-infection immunity. While hundreds of reinfections have reported only a few have been confirmed. Whole genome sequencing (WGS) of the viral isolates from the different episodes is mandatory to establish reinfection.Methods: Nasopharyngeal (NP), oropharyngeal (OP) and whole blood (WB) samples were collected from paired samples of four individuals who were suspected of SARS-CoV-2 reinfection based on distinct clinical episodes and RT-PCR tests. Details from their case record files and investigations were documented. RNA was extracted from the NP and OP samples and subjected to WGS, and the nucleotide and amino acid sequences were subjected to genome and protein-based functional annotation analyses. Serial serology was performed for Anti-N IgG, Anti- S1 RBD IgG, and sVNT (surrogate virus neutralizing test).Findings: Three patients were more symptomatic with lower Ct values and longer duration of illness. Seroconversion was detected soon after the second episode in three patients. WGS generated a genome coverage ranging from 80.07 to 99.7%. Phylogenetic analysis revealed sequences belonged to G, GR and “Other” clades. A total of 42mutations were identified in all the samples, consisting of 22 non-synonymous, 17 synonymous, two in upstream, and one in downstream regions of the SARS-CoV-2 genome. Comparative genomic and protein-based annotation analyses revealed differences in the presence and absence of specific mutations in the virus sequences from the two episodes in all four paired samples.Interpretation: Based on the criteria of genome variations identified by whole genome sequencing and supported by clinical presentation, molecular and serological tests, we were able to confirm reinfections in two patients, provide weak evidence of reinfection in the third patient and unable to rule out a prolonged infection in the fourth. This study emphasizes the importance of detailed analyses of clinical and serological information as well as the virus's genomic variations while assessing cases of SARS-CoV-2 reinfection.

Highlights

In December 2019, a novel coronavirus (n-CoV-19) sparked an outbreak in Wuhan, China
We identified four individuals who had tested RT-PCR positive for SARS-CoV-2 between April to June 2020 and who tested RT-PCR positive for SARS-CoV-2 once again between July to September after presenting with symptoms suggestive of COVID-19
A handful of confirmed SARS-CoV-2 reinfection have been published on the basis of genome variation observed in the viruses between the two episodes with varying clinical manifestations between the episodes [2, 3, 23, 24]

Summary

Introduction

In December 2019, a novel coronavirus (n-CoV-19) sparked an outbreak in Wuhan, China. This virus was subsequently named SARS-CoV-2 and the disease COVID-19. While there have been many reports of putative reinfections based on RT-PCR positivity, this has been confounded by prolonged shedding of viral RNA in the absence of replication competent virus [4] which can continue to cause RT-PCR positivity for up to at least 83 days [5]. The samples from the two episodes can be sequenced and genomic analysis may demonstrate genetic variation that can’t be explained by short term in vivo evolution, which when combined with epidemiological and clinical evidence, may confirm reinfection [2, 3]. Whole genome sequencing (WGS) of the viral isolates from the different episodes is mandatory to establish reinfection

Methods

Results

Conclusion