Next generation sequencing of multiple SARS-CoV-2 infections in the Omicron Era

Abstract

Since the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the need for an effective vaccine has appeared crucial for stimulating immune system responses to produce humoral/cellular immunity and activate immunological memory. It has been demonstrated that SARS-CoV-2 variants escape neutralizing immunity elicited by previous infection and/or vaccination, leading to new infection waves and cases of reinfection. The study aims to gain into cases of reinfections, particularly infections and/or vaccination-induced protection. We conducted a retrospective descriptive study using data collected during the SARS-CoV-2 pandemic. This analysis involved Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) and Next Generation Sequencing (NGS). RT-qPCR was performed on 416,466 naso-oropharyngeal swabs, with 10,380 samples further analyzed using NGS technology. RT-qPCR identified 350 cases of reinfection, of which 228 were subjected to detailed analysis via NGS. Our findings revealed two interesting cases involving pediatric patients who were not vaccinated. Positive results were observed in these cases within a short interval (< 60 days) and the "nature" of the infection, whether attributable to Reinfection or Viral Persistence, was investigated. Specifically, we discuss a case involving an unvaccinated 18-month-old child, which may represent one of the earliest instances of BA.5/BA.5 reinfection identified worldwide.

Keywords: NGS; Omicron; Reactivation; Reinfection; SARS-CoV-2; Vaccine.

Fig. 1

Trend of identified SARS-CoV-2 variants at DIBS from November 2020 to January 2024.

Fig. 2

Double instances of infection. (A) Dynamics of SARS-CoV-2 reinfections (blue columns, right axis) compared to the non-omicron and omicron sequenced cases (orange and green lines, left axis). (B) Double infection cases assessed by RT-qPCR: 344 cases of double infections, two of which characterized by two events of infection with a time interval of less than 60 days and highlighted with Symbol *. The X-axis reports the year of the first infection; the Y-axis shows the number of cases, and each column depicts the number of second infection cases with different colors).

Fig. 3

Reinfection cases evaluated by NGS: 224 cases of double infections sequenced. Symbol * indicates the pediatric cases presenting two infection events with a time interval of less than 60 days (46 days each). The X axis reports the first infection, the Y axis reports the number of second infection cases, each column depicts by different colors the subtypes of SARS-CoV-2 found at the second infection.

Fig. 4

Genome comparison among the sequences of the girl family VS the most similar ones identified in GISAID using SARS-CoV-2 (BA.2) as reference genome (reference: Wuhan-Hu-1 with BA.2 SNPs; dataset name: nextstrain/sars-cov-2/BA.2). (A) Genome comparison among EPI_ISL_14149323 (child in May), EPI_ISL_14149324 (father), EPI_ISL_14149325 (mother), EPI_ISL_13826896 (child in July). The orange arrows indicate the five SNPs present in the July sequence of the child [in ORF1a (C1912T; A3859G; T6979G), in ORF1b (G16181A) and in ORF3a (C25626T)] and the green one indicates the two SNPs present in the May sequence (G28361T and G28371T in Orf9b). (B) Genome comparison between EPI_ISL_14149323 (child in May), EPI_ISL_13204456 (sequence from Belgium). The blue arrows indicate the two different SNPs, present in the Belgium sequence (T22917G in the S gene; A27038G in the M gene). (C) Genome comparison between EPI_ISL_13826896 (child in July), EPI_ISL_14823347 (sequence from INMI L. Spallanzani I.R.C.C.S). The blue arrows indicate the three SNPs present in the INMI L. Spallanzani I.R.C.C.S sequence (C44T in Orf1a; T22917G in the S gene; A27038G in the M gene) and the green one indicates the SNP present in the child (C1912T in ORF1a).