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

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.

Reinfection in COVID-19: A scoping review

“…It is also ideal that a negative swab is documented between the episodes. 2 A “reinfection” was defined as one in which paired specimens underwent WGS and were compared and found to be different. We also collected data for “possible reinfections” in which a reinfection was postulated, but not confirmed.…”

“…Genomic variations observed through whole genome sequencing are then correlated with clinical presentation and can be confirmed a reinfection of SARS-CoV-2. 2 …”

“…The disadvantages of the Ct value are as follows: (1) there is no absolute Ct cut-off value; and (2) Ct cut-off values are different for each diagnostic reagent even for the same gene. 2 Ct value cannot be a tool to confirm reinfection, but can be useful in cases of persistent positivity as an indication of a possible reinfection that would need confirmation by whole genome sequencing.…”

“…A healthcare worker at our institution was reinfected with SARS-CoV-2 and we collaboratively published a series of four such reinfections. 2 …”

“… 23 33/male/no comorbidities Mild infection Asymptomatic second infection 142 days 2-Tillett R, Sevinsky J, et al. 24 25/male/no comorbidities Mild infection Increased clinical severity during the second episode 42 days 3-Jayanthi Shastri, et al 2 , 25 Series of 4 patients: Patient 1 —27/male/no comorbidities Patient 2 —31/male/no comorbidities Patient 3 —27/male/no comorbidities Patient 4 —24/female/no comorbidities All 4 patients had mild infection All 4 patients had increased clinical severity during the second episode Between 19 and 65 days 4-Van Elslande, et al. 26 51/female/asthmatic Mild infection Similar symptoms but milder in the second episode 60 days 5-Prado-Vivar, et al.…”