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.