Genomic analysis
Two positive SARS-CoV-2 RT-PCR from E1 (Specimens 2 and 3) and one positive specimen (Specimen-4) from E2 were available for whole genome sequencing (WGS).
A B.1.258 lineage strain (Strain 1, Figure) was identified in E1 from the Specimen 2. When analysing the data from the Specimen-3, many heterozygous calls were obtained (Figure), suggesting the presence of two SARS-CoV-2 strains. Cross-contamination with a positive specimen from another patient was ruled out by confirming identical human genetic content (short tandem repeat human DNA profile analysis, Supplementary Figure) in specimens 2 and 3. Heterozygous calls indicated superinfection with a different strain (Strain 2; (lineage B.1)) differing in 13 single nucleotide polymorphisms (SNPs) from Strain 1. Frequency determination of alternative alleles in the heterozygous calls allowed us to determine that superinfecting Strain 2 was overrepresented (59-79% frequency, Supplementary Table). Strain 1 and 2 sequences were analysed along with 2,249 SARS-CoV-2 sequences from specimens collected from COVID-19 cases among the Madrid population throughout the pandemic. A tree with the results was created and Strains 1 and 2 are presented on the branch corresponding to the strains circulating in the first COVID-19 wave (before July 2020, Figure).
A new strain (Strain 3; lineage B.1.177) was identified in E2 from Specimen-4, with 16 SNPs not shared with Strains 1 or 2, and without the 14 SNPs identified in Strains 1 and 2 (Figure). This indicated that COVID-19 E2 was a reinfection. Strain 3 sequence was positioned in the phylogenetic tree among the sequences from strains circulating after July 2020, ruling out its circulation during our case´s first episode. Short tandem repeat human DNA analysis confirmed that specimens from the two sequential COVID-19 episodes were from the same individual (Supplementary Figure).
E1 SARS-CoV-2 superinfection occurred during the first COVID-19 wave, when prevalence of SARS-COV-2 among our population was very high (1,182 cases/100,000 inhabitants) and most hospitalized cases were COVID-19 patients. Moreover, our patient was highly dependent due to neurological damage and remained hospitalized in two different buildings. Thus, the patient was probably exposed to different nosocomial circulating strains that might have caused E1 superinfection. To the best of our knowledge, only another likely SARS-CoV-2 superinfection has been reported (Tarhini, 2021), in an immunosuppressed patient, for whom two SARS-CoV-2 strains were identified on Day 56. There is higher risk of prolonged viral shedding in immunosuppressed SARS-CoV-2 positive cases (Choi et al., 2020), which may explain the superinfection. Our patient was not immunosuppressed and superinfection was detected only one week after the identification of a single-strain infection in a previous specimen. Two coexisting SARS-CoV-2 strains have been also reported in a COVID-19 case who was reinfected 26 days after the first infection (Lee JS, 2020).
E1 was severe and identification of two coinfecting strains coincided with patient´s clinical deterioration, suggesting some clinical impact of superinfection on the severity of E1. On the other hand, E2, associated to a third different strain, was milder, similar to other reinfection reports (Van Elslande et al., 2020), with no consolidation on X-ray and mild hypoxemia. E2 infection resulted from a nosocomial exposure, as Strain 3 was responsible for a hospital outbreak involving at least 11 cases (0 SNPs among them) from three wards. An epidemiological survey confirmed that the health care worker who attended the patient who had shared a room with our case was also infected with the same strain (0 SNPs), suggesting a potential role in the reinfection.
Summarizing, we describe a COVID-19 case with unprecedented viral complexity in SARS-CoV-2 infection. Initially, the patient was superinfected by two different strains within a short period, followed months later by a COVID-19 reinfection by a third distinct strain.