>> Welcome to module 2.5,
"Confirming SARS-CoV-2 Reinfection
with Whole Genome Sequencing."
This presentation is a part
of the COVID-19 Genomic Epidemiology Toolkit
from CDC's Office of Advanced Molecular Detection.
My name is Dr. Megan Crumpler, and I am the laboratory director
at the Orange County, California Public Health Laboratory.
In this module, we will present a case study
that demonstrates how whole genome sequencing can be used
as an investigative tool
to identify cases of true reinfection.
Be sure to check out the toolkit's other modules,
which include a combination of case studies
and training materials to help you get started supplementing
epidemiology with genome sequence data.
SARS-CoV-2 reinfection is thought to be a rare event
and may be due to waning immunity to the virus over time.
A study published in December 2020 identified 243
out of 133,266,000 laboratory-confirmed cases
that had one or more positive tests at least 45 days
after the first positive test.
On further investigation of these 243 cases, strong evidence
for reinfection was found for 54 or 22%.
Based on these findings,
the risk of reinfection was estimated at 0.02%
and the reinfection incident rate was 0.36 per
10,000 person-weeks.
CDC's common investigation protocol
for suspected SARS-CoV-2 reinfection prioritizes one,
persons with or without COVID-19-like symptoms
who have a positive test more than 90 days
after their initial infection or illness and two,
persons with COVID-19-like symptoms 45 to 89 days
after initial infection or illness.
Genomic sequencing of paired specimens is recommended
to confirm reinfection.
A link to the CDC protocol is included here
and on the toolkit webpage.
In the case we present here, a 32-year-old man who had resided
at two separate homeless shelters in June
and October 2020 was found to have a COVID-19 reinfection.
This person's first infection was identified
in June 2020 during a facility-wide serial testing
program at Shelter A after a staff member was reported
to have tested positive for COVID-19.
When tested, our case individual had symptoms consistent
with COVID-19 including fever,
sore throat, cough, and headache.
He recovered and did not require hospitalization.
This person's second infection occurred during an outbreak
at another facility, Shelter B, in October 2020.
As before, he was tested along with all shelter staff
and residents after another resident was reported
to have tested positive.
Although he was symptomatic at the time,
he once again recovered without hospitalization.
Case investigators recognized
that this patient met the criteria for investigation
for reinfection because it had been more than 90 days
since the person's initial infection.
As part of routine SARS-CoV-2 surveillance in Orange County,
California, whole genome sequencing is performed
on all positive specimens that are tested
at the public health lab that meet specific,
technical criteria for whole genome sequencing.
It's important to note that some specimens
from an outbreak may not be eligible for sequencing if,
for example, they have a high cycle threshold (CT) value
or other features predictive of poor sequence quality.
On this slide, we have a phylogenetic tree
of various SARS-CoV-2 sequences
in Orange County calculated with Nextstrain.
Focusing your attention on the two highlighted branches
in blue boxes, you can see that the June and October sequences
from our case individual are genetically distinct.
The June 2020 specimen falls into clade 20A
and the October 2020 specimen falls into clade 20G.
Let's take a closer look
at a direct comparison of the sequence data.
As shown on the previous slide, the two specimens fall
into different clades as determined
by Nextclade 20A and 20G.
Their pangolin lineage assignments are also different.
The June specimen is B.1 and the October specimen is B.1.2.
Examining the specific amino acid substitutions
in these two sequences, we saw that both contain the spike D14G
and the nonstructural proteins 12: P323L mutations.
Overall, the October sequence contained many more mutations
relative to the Wuhan WHO1 reference sequence
than the June sequence.
This is to be expected given
that the virus continues to mutate over time.
These observed genetic differences, in conjunction
with epidemiologic data,
suggested that this was indeed a true reinfection.
We had further evidence that these infections
in the case-patient were distinct
because each was part of a larger outbreak.
Shelter A experienced an outbreak from May to July
when 19 residents and seven staff were infected.
Shelter B had an outbreak from mid-October to early November
when 14 residents and one staff were infected.
The public health lab was able to retrieve sequencing data
for specimens from four persons infected in the first outbreak
and nine in the second.
First, we will review the 2020 outbreak
at shelter A. The phylogenetic tree here,
which displays the number of mutations on the x-axis,
shows relatedness among the four outbreak sequences including the
one from our case under investigation
for potential reinfection, marked by the blue arrow.
The first specimen was collected on May 22,
2020 and the last was the June 3rd, 2020 specimen
of our case-patient,
all of which were closely related according
to sequencing data, differing by only a couple of mutations.
Next, we will review the October 2020 outbreak
at shelter B. This phylogenetic tree shows relatedness among the
nine outbreak specimens including the one
from our patients suspected of reinfection.
The date range of collection
for these specimens was October 15th,
2020 through October 23rd, 2020.
Remember that sequences could only be obtained from nine
of the 16 specimens collected from the cases in this outbreak.
As with the other outbreak, these sequence data suggest
that the cases were related.
However, the longer branches within the cluster
which indicate more mutations
between the viral sequences could reflect an undersampling
in the context of ongoing transmission.
In summary, reinfection with SARS-CoV-2 appears to be rare.
However, we still don't have much individual-level
information on how well immunity developed
from a previous SARS-CoV-2 infection might protect
against infection with a different viral variant.
Most cases of reinfection documented so far have been
in highly exposed populations undergoing routine testing
such as healthcare workers.
Our case study shows that whole genome sequencing
and epidemiologic data together can be helpful
in confirming suspected cases of SARS-CoV-2 reinfection.
The sequence data from specimens collected in June 2020
and October 2020 revealed clear differences in both clade
and lineage assignments.
Specific differences in mutation patterns also indicated
that the two infections resulted from distinct viral genotypes.
Alternatively, if the two sequences had been similar
or nearly identical, we would have suspected instead
that our case-patient suffered from persistent infection.
The sequencing results were consistent
with epidemiologic data which indicated
that the patient had been exposed in two outbreaks
of COVID-19 occurring more than three months apart
in separate facilities.
Persons experiencing homelessness may be at high risk
for SARS-CoV-2 reinfection because of repeated exposures
in multiple homeless shelters.
In general, settings that bring people from the wider community
into close proximity create the potential
for re-exposure to SARS CoV-2.
This concludes module 2.5.
Part two of this toolkit contains case studies
that review the use of genomics in response to COVID-19.
Please visit the COVID-19 Genomic Epidemiology Toolkit
page, where you can find further reading on this topic as well
as a short survey to provide feedback about this module.
On the toolkit page, you can also subscribe
to our mailing list and receive announcements as new modules
and materials are released.
Thank you.