On March 3 2021, the woman, whose medical history was unremarkable, was admitted to the OLV Hospital in the Belgian city of Aalst after a spate of falls. She tested positive for COVID-19 on the same day. She lived alone and received nursing care at home, and had not been vaccinated against COVID-19.
Initially, there were no signs of respiratory distress and the patient had good oxygen saturation. However, she developed rapidly worsening respiratory symptoms, and died five days later.
When the patient’s respiratory sample was tested for VOCs with PCR, they discovered that she had been infected by two different strains of the virus – one which originated in the UK, known as B.1.1.7 (Alpha), and another that was first detected in South Africa (B.1.351; Beta).
The presence of both strains was confirmed by PCR on a second respiratory sample, by sequencing of the S-gene and by whole genome sequencing.
“This is one of the first documented cases of co-infection with two SARS-CoV-2 variants of concern”, says lead author and molecular biologist Dr. Anne Vankeerberghen from the OLV Hospital in Aalst, Belgium.
“Both these variants were circulating in Belgium at the time, so it is likely that the lady was co-infected with different viruses from two different people. Unfortunately, we don’t know how she became infected.”
On December 14, 2020, the UK authorities informed WHO that a variant (B.1.1.7; Alpha) had been detected in the south east of England (Kent). Within a few weeks, this variant took over from the viral strains circulating in this region, and has since spread to more than 50 countries, including Belgium.
On December 18, 2020, the South African authorities reported that a variant (B.1.351; Beta) had been detected and was spreading rapidly throughout three provinces of South Africa, and has now been identified in at least 40 countries, including Belgium.
In January 2021, scientists in Brazil reported that two people had been simultaneously infected with two different strains of the coronavirus – the Brazilian variant known as B.1.1.28 (E484K) and a novel variant VUI-NP13L, which had previously been discovered in Rio Grande do Sul.
But the study has yet to be published in a scientific journal [1]. Previous research has reported people infected with different influenza strains [2].
“Whether the co-infection of the two variants of concern played a role in the fast deterioration of the patient is difficult to say”, says Vankeerberghen. “Up to now, there have been no other published cases. However, the global occurrence of this phenomenon is probably underestimated due to limited testing for variants of concern and the lack of a simple way to identify co-infections with whole genome sequencing.”
She continues, “Since co-infections with variants of concern can only be detected by VOC-analysis of positive samples, we would encourage scientists to perform fast, easy and cheap VOC-analysis by PCR on a large proportion of their positive samples, rather than just whole genome sequencing on a small proportion. Independent of the technique used, being alert to co-infections remains crucial.”
Biology of SARS-CoV-2 re-infection
Early experimental study showed that re-infection by coronaviruses is possible. However, little was known about the possibility of re-infection by SARS-CoV-2 since the COVID-19 pandemic is still in its early phase [33]. A study on rhesus macaques showed that re-infection could not occur after challenging the monkeys with the same dose of SARS-CoV-2 strain as the first infection [34]. However, recent findings have shown that re-infection is possible, specifically with a different strain, with confirmed cases of re-infection in Hong Kong [1] and Nevada [3]. To et al. [1] and Edridge et al. [33] suggested that natural infection may be responsible for re-infection in coronaviruses (HCoV-NL63, HCoV-229e, HCoV-OC43 and HCoV-HKU1), and this could be a general occurrence for all coronaviruses, including SARS-CoV-2.
Differences were observed in the genomes of confirmed re-infection cases of SARS-CoV-2. The differences between the initial and subsequent infection of the viral genomes are attributed to the clade/lineage, the number of single nucleotide variants (SNVs), the difference in the amino acids and number of dinucleotide multi-nucleotide variant (MNV) [1, 3]. Although the re-infection case in Nevada, USA belongs to the same clade as the first infection, clade 20C, there are some genomic differences as obtained from the genomic sequence analysis (Table 1).
Table 1
Studies that reported cases of SARS-CoV-2 re-infection.
| Country (Citation) | Age/gender/general health condition | Period between episodes (RT-PCR positive outcomes) | No. of cases | Key Clinical findings | Scheduling of RT-PCR and Ct figures | Sequencing | Mutation | Immunoglobulin testing |
|---|---|---|---|---|---|---|---|---|
| Hong Kong, China (To et al) [1] | 33-year-old Immunocompetent male | 142 days | 1 | First episode: dry cough, fever, headache. Second episode: no symptoms | First episode: positive outcome 3 days after symptom onset with Ct of 30.5 Second episode: positive outcome 1–3 days (Ct 26–28) & 5 days (Ct 32) post symptom onset | 1st and 2nd viral genomes from dissimilar lineages and differentiated by 24 nucleotides. First episode: Next strain 19A/GISAID V/Rambout clade B.2 (Hong kong) Second episode: Next strain 20A/GISAID G/Rambout B.1.79 (Spain) | Amino acid variations in Spike protein (N-terminal domain, upstream helix, subdomain 2), nucleoprotein, non-structural proteins (NSP3, NSP5-6, NSP12), accessory proteins (ORF3a, ORF8, ORF10). | First episode: negative for IgG 10 days after symptom commenced. Second episode: negative for IgG 1–3 days following hospitalization with a reactive outcome on the 5th day. |
| Washoe, Nevada, USA (Tillet et al) [3] | 25-year-old immunocompetent male | 48 days | 1 | First episode: less severe symptoms (dry cough, sore throat, diarrhea, headache). Second episode: more severe symptoms (pyrexia, headache, dry cough, dizziness, hypoxia) with stronger immune response. | First episode: positive outcome on the 24th day following the commencement of symptoms (Ct 35.2). Second episode: positive outcome on the 6th day following the commencement of symptoms (Ct 35.3). | 1st and 2nd viral genomes originated from a common lineage (Next strain 20C) and differentiated by 7 nucleotides. | SNVs (25563G>T, 3037C>T, 1059C>T, 23403A>G, 14408C>T) | First episode: no immunoglobulin test was done. Second episode: reactive for IgG/IgM on day 7 post symptom onset. |
| Belgium (Van Elslande et al.) [38] | 51-year-old immunocompetent female on corticosteroid for asthma management | 93 days | 1 | First episode: pyrexia, migraine, dry cough, dyspnea, chest pain. Second episode: migraine, dry cough, fatigue. | First episode: N1-gene (Ct 25.6). Second episode: N1-gene (Ct 32.6) | 1st and 2nd viral genomes from dissimilar lineages and differentiated by 11 nucleotides. First episode: Rambout clade B.1.1. Second episode: Rambout clade A. | Amino acid variations in Spike protein [G23403A, A23873G, C24726T], nucleoprotein [A28881G, A28882G, C28883G], accessory proteins [ORF1a (C3037T, C8782T, C11654T), ORF1b (T14408C, T17427G)]. | First episode: no immunoglobulin test was done. Second episode: reactive for IgG on day 7 with a value of 134 and for neutralizing antibodies on the 6th week with a value of 1/320 following symptom onset. |
| Ecuador (Prado-Vivar et al.) [52] | 46-year-old immunocompetent male | 63 days | 1 | First episode: less severe symptoms (migraine, drowsiness). Second episode: more severe symptoms (pyrexia, dry cough, dyspnea, sore throat). | First episode: positive outcome on the 11th day following the commencement of symptoms (Ct 36.85, ORF3a gene). Second episode: positive outcome on the 4th day following the commencement of symptoms. | 1st and 2nd viral genomes from dissimilar lineages. First episode: Next strain 20A/GISAID B1.p9 clade. Second episode: Next strain 19B/GISAID A.1.1 clade. | No common mutations between the viral sequences of both first and second episodes. First episode: 8 SNPs (C2113T, C3037T, C7765T, C14408T, C17690T, C18877T, A23403G, G25563T) which results in 4 amino acid changes [NSP12 (P323L), NSP13 (S485L), SP (D614G), ORF3a (Q57H). Second episode: 10 SNPs (C1457T, C8782T, T9445C, C17531C, C17747T, A17858G, C18060T, G18756T, A24694T, T28144C) which result in 5 amino acid changes [NSP2 (R218C), NSP (I432T, P504L, Y541C), ORF8 (L84S) | First episode: negative for IgG 4 days following symptom onset. Second episode: positive for IgG on the 30th day with a value of 34.1 following symptom onset. |
| India (Gupta et al.) [39] | 25-year-old immunocompetent male. | 108 days | 2 | First & second episodes: no symptoms. | First episode: positive outcome (Ct 36). Second episode: positive outcome (Ct 16.6). | 1st and 2nd viral genomes with 9 distinctive variants. | Synonymous mutations: C241T, C6445T, G11383A, T11408C, C18877T, C25207T, C26735T. Nonsynonymous mutations: T1947C, G17584T, A23403G, C23934T, G25563T, C26456T. | First and second episodes: no immunoglobulin test was done. |
| 28-year-old immunocompetent female | 111 days | First & second episodes: no symptoms. | First episode: positive outcome (Ct 28.16). Second episode: positive outcome (Ct 16.92). | 1st and 2nd viral genomes with 10 distinctive variants. | Synonymous mutations: C241T, C18877T, C23929T, C26735T, C29215T. Nonsynonymous mutations: C3267T, C13730T, C14408T, G17584T, G19109T, T22882G, A23403G, G24794A, G25563T. |
According to Jain et al. [35], clade 20C possess genomic variants in the regions C14408T, A23403G, C1059T, and G25563T. However, viral particle B has an additional variant in the region C3037T [3]. The mutation in the viral genome A is missense; however, viral genome B included a synonymous mutation in the C3037T region [27].
While the A23403G occurred in the S region, the other mutation occurred in the ORF1ab region, which is the longest ORF in the SARS-CoV-2 genome [36]. The extra change in the S region could be responsible for the severity in the re-infection case in the Nevada case.
According to the To et al. [1], they highlighted that the viral genome in the first episode of infection in the re-infection case in Hong Kong was of the GISAID Clade V, Next strain clade 19A, Pangolin lineage B.2 and with a probability of 0.99; while the re-infection viral genome was of the GISAID Clade G, Next strain 20A, Pangolin lineage B.1.79 with a probability of 0.70. Specifically, lineage B.1 belongs to variant G614, which is widely distributed globally with substitution in the regions C241T, C3037T, C14408T and G23403A; while lineage B.2 belongs to the variant V251 [37].
Furthermore, these two viral genomes differ from one another by changes in the amino acids in the spike protein, accessory proteins (ORF3a, ORF8 and ORF10), nucleoprotein, membrane protein and non-structural proteins (NSP3, NSP5, NSP6, NSP12) [1]. The genome of the first virus is related to the Clade GR obtained in England between March and April 2020 [1], with varying nucleotide and amino acids mutations (GISAID Database).
Analysis of 10,022 samples to understand the genomic variability of SARS-CoV-2 also showed that G614 variant had been the most common variant since the onset of the pandemic in December 2019 {Koyama, 2020 #462} [36]. Evidence has shown that G614 has a higher titre of viral particles in upper respiratory tract specimens [15, 37], but it is associated with lower RT-PCR cycle thresholds and not necessarily increased diseases severity [15].
The re-infection case in Belgium showed that the first infection belongs to the lineage B.1.1 while the second infection belong to lineage A, with eleven genomic mutations identified in the two strains [38]. According to Gupta et al. [39] there were some genetic variations in the case of re-infection among the two healthcare workers identified in India.
Using WGS, there were six non-synonymous (NS) variations in patient 1 when the two virus strains were compared. Virus strains in patient 2 showed nine NS variations between the two strains in the different genomic regions. Most of these variations were observed between the orf1b and envelope protein region.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810769/
More information: [1] Pervasive transmission of E484K and emergence of VUI-NP13L with evidence of SARS-CoV-2 co-infection events by two different lineages in Rio Grande do Sul, Brazil | medRxiv www.medrxiv.org/content/10.110 … 021.01.21.21249764v1
[2] Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus – ScienceDirect www.sciencedirect.com/science/ … ii/S1386653215007404
ECCMID ABSTRACT 04978: Case report: a 90-year-old lady infected with two CoVID-19 VoCs: 20I/501Y.V1 and 20H/501Y.V2
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