Thanks for joining me for this Hot Topic presentation on molecular testing for COVID-19.
Thanks for joining me for this Hot Topic presentation on molecular testing for COVID-19.

I’m Dr. Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice
I’m Dr. Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice

in the Department of Laboratory Medicine and Pathology at Mayo Clinic.
in the Department of Laboratory Medicine and Pathology at Mayo Clinic.

Before we begin, I wanted to mention that I serve as an Advisory Board member for DiaSorin
Before we begin, I wanted to mention that I serve as an Advisory Board member for DiaSorin

molecular.
molecular.

So with that, let’s get started.
So with that, let’s get started.

As the number of cases continues to increase each day of COVID-19, our knowledge of the
As the number of cases continues to increase each day of COVID-19, our knowledge of the

virus itself, as well as how we can and should test for COVID-19, is also rapidly evolving.
virus itself, as well as how we can and should test for COVID-19, is also rapidly evolving.

In this presentation, I want to provide a brief overview of the virus, the current testing
In this presentation, I want to provide a brief overview of the virus, the current testing

options, and finally review what factors may influence the performance of molecular testing
options, and finally review what factors may influence the performance of molecular testing

in patients with possible COVID-19 disease.
in patients with possible COVID-19 disease.

To begin, it’s important to highlight that not all coronaviruses are created equal.
To begin, it’s important to highlight that not all coronaviruses are created equal.

Over the past several decades, we know now that four serotypes of coronaviruses—I’ve
Over the past several decades, we know now that four serotypes of coronaviruses—I’ve

listed those here on this slide—circulate in the population, and are associated with
listed those here on this slide—circulate in the population, and are associated with

annual epidemics of mild-to-moderate upper respiratory tract infection.
annual epidemics of mild-to-moderate upper respiratory tract infection.

The serotypes listed on this slide are most commonly associated with the common cold.
The serotypes listed on this slide are most commonly associated with the common cold.

However, over the past 20 years, there have been outbreaks of novel or variant coronaviruses
However, over the past 20 years, there have been outbreaks of novel or variant coronaviruses

that have been associated with more severe disease.
that have been associated with more severe disease.

In 2002, there was an outbreak of respiratory illness in China that was due to severe acute
In 2002, there was an outbreak of respiratory illness in China that was due to severe acute

respiratory syndrome coronavirus, or SARS-CoV, and SARS-CoV ultimately was confirmed in about
respiratory syndrome coronavirus, or SARS-CoV, and SARS-CoV ultimately was confirmed in about

8,400 cases, and it had a case fatality rate of approximately 10%.
8,400 cases, and it had a case fatality rate of approximately 10%.

A decade later, Middle East Respiratory syndrome coronavirus, or MERS, caused an outbreak effecting
A decade later, Middle East Respiratory syndrome coronavirus, or MERS, caused an outbreak effecting

around 2,500 people, but MERS was associated with a high-case fatality rate of about 35%.
around 2,500 people, but MERS was associated with a high-case fatality rate of about 35%.

MERS cases have occurred primarily in the Arabian Peninsula and have not shown broad
MERS cases have occurred primarily in the Arabian Peninsula and have not shown broad

global spread to this point.
global spread to this point.

And now in 2020, we’re in the midst of a global pandemic caused by severe acute respiratory
And now in 2020, we’re in the midst of a global pandemic caused by severe acute respiratory

syndrome coronavirus 2, or SARS-CoV-2, which has been determined to be a variant of the
syndrome coronavirus 2, or SARS-CoV-2, which has been determined to be a variant of the

2003 SARS virus.
2003 SARS virus.

In other words, the current virus is not believed to be a novel virus, but a relative of the
In other words, the current virus is not believed to be a novel virus, but a relative of the

original SARS virus from 2003.
original SARS virus from 2003.

SARS-CoV-2 has now caused over 3.2 million cases worldwide.
SARS-CoV-2 has now caused over 3.2 million cases worldwide.

It’s killed more than 228,000 people, and that translates to a case fatality rate of
It’s killed more than 228,000 people, and that translates to a case fatality rate of

about 7%.
about 7%.

One common question is “Why do certain coronaviruses cause more severe disease?”
One common question is “Why do certain coronaviruses cause more severe disease?”

It’s known that coronaviruses are zoonotic in origin, which means that they naturally
It’s known that coronaviruses are zoonotic in origin, which means that they naturally

reside in animal reservoirs.
reside in animal reservoirs.

Sequencing and phylogenetic data support the conclusion that many of the coronaviruses
Sequencing and phylogenetic data support the conclusion that many of the coronaviruses

infecting humans have bats as their natural reservoir.
infecting humans have bats as their natural reservoir.

In fact, we have solid data indicating that SARS from 2003 was transmitted from bats to
In fact, we have solid data indicating that SARS from 2003 was transmitted from bats to

palm civets, which served as the intermediate host for spillover of SARS into humans.
palm civets, which served as the intermediate host for spillover of SARS into humans.

Similarly, MERS virus is believed to have been harbored in bats, but the initial transmission
Similarly, MERS virus is believed to have been harbored in bats, but the initial transmission

of MERS virus into humans occurred through contact with dromedary camels in the Arabian
of MERS virus into humans occurred through contact with dromedary camels in the Arabian

Peninsula.
Peninsula.

And finally, while much less is known regarding SARS-CoV-2, it is believed that the virus
And finally, while much less is known regarding SARS-CoV-2, it is believed that the virus

was transmitted from bats to animals that were sold at wet markets in China.
was transmitted from bats to animals that were sold at wet markets in China.

Although the intermediate host for SARS-CoV-2 is still undefined, we have sequencing data
Although the intermediate host for SARS-CoV-2 is still undefined, we have sequencing data

that suggests that the pangolin or turtles may have facilitated the transmission of SARS-CoV-2
that suggests that the pangolin or turtles may have facilitated the transmission of SARS-CoV-2

to humans.
to humans.

Now that we have reviewed some background information on SARS-CoV-2, let’s turn our
Now that we have reviewed some background information on SARS-CoV-2, let’s turn our

attention to laboratory testing.
attention to laboratory testing.

To date, the vast majority of testing for COVID-19 has been performed using a technology
To date, the vast majority of testing for COVID-19 has been performed using a technology

called real-time PCR.
called real-time PCR.

PCR-based assays detect the viral RNA in clinical samples, and are designed to diagnose active
PCR-based assays detect the viral RNA in clinical samples, and are designed to diagnose active

infection with SARS-CoV-2.
infection with SARS-CoV-2.

Recently, there has been a significant amount of attention on serology, or antibody-based
Recently, there has been a significant amount of attention on serology, or antibody-based

assays, for COVID-19.
assays, for COVID-19.

These tests detect antibodies, like IgG, which have been developed by the immune system in
These tests detect antibodies, like IgG, which have been developed by the immune system in

response to SARS-CoV-2 infection.
response to SARS-CoV-2 infection.

Serologic assays are unlikely to play a key role in diagnosis of COVID-19 due to the fact
Serologic assays are unlikely to play a key role in diagnosis of COVID-19 due to the fact

that is requires 10-14 days for antibodies to develop following infection, but serology
that is requires 10-14 days for antibodies to develop following infection, but serology

will be important as a means of determining whether an individual has been exposed to
will be important as a means of determining whether an individual has been exposed to

the virus and has developed an antibody response.
the virus and has developed an antibody response.

With regards to molecular testing for SARS-CoV-2, the majority of the real-time PCR assays that
With regards to molecular testing for SARS-CoV-2, the majority of the real-time PCR assays that

have been developed target a combination of several genes in the SARS-CoV-2 genome.
have been developed target a combination of several genes in the SARS-CoV-2 genome.

Examples of viral gene targets targeted by these PCR assays include the nucleocapsid,
Examples of viral gene targets targeted by these PCR assays include the nucleocapsid,

the open reading frame, and the envelope and RNA-dependent RNA polymerase genes.
the open reading frame, and the envelope and RNA-dependent RNA polymerase genes.

As the primary clinical manifestation of COVID-19 is a respiratory illness, PCR tests are generally
As the primary clinical manifestation of COVID-19 is a respiratory illness, PCR tests are generally

used to test samples obtained from the upper or lower respiratory tract.
used to test samples obtained from the upper or lower respiratory tract.

Appropriate upper respiratory sample types include a nasopharyngeal swab or a throat
Appropriate upper respiratory sample types include a nasopharyngeal swab or a throat

swab.
swab.

If a patient has evidence of a lower respiratory tract infection, or if the patient is in a
If a patient has evidence of a lower respiratory tract infection, or if the patient is in a

later stage of the disease, then a clinical specimen such as sputum, bronchoalveolar lavage
later stage of the disease, then a clinical specimen such as sputum, bronchoalveolar lavage

fluid (BAL fluid), or tracheal secretions may be appropriate for testing.
fluid (BAL fluid), or tracheal secretions may be appropriate for testing.

There are several questions that are common with regards to molecular testing that I’d
There are several questions that are common with regards to molecular testing that I’d

like to address.
like to address.

The first is “When is SARS-CoV-2 shed at the highest amount?”
The first is “When is SARS-CoV-2 shed at the highest amount?”

There is a growing amount of data in the published literature suggesting that the virus is present
There is a growing amount of data in the published literature suggesting that the virus is present

at peak amounts very early in the disease course.
at peak amounts very early in the disease course.

One study that was recently published in Nature Medicine actually showed that peak viral shedding
One study that was recently published in Nature Medicine actually showed that peak viral shedding

occurs about 24 hours prior to a patient’s onset of symptoms.
occurs about 24 hours prior to a patient’s onset of symptoms.

This is very important, in that it likely explains why we have seen such effective community
This is very important, in that it likely explains why we have seen such effective community

spread of this virus, since many people are shedding the virus at high amounts before
spread of this virus, since many people are shedding the virus at high amounts before

they even know they are infected.
they even know they are infected.

Importantly, the viral load tends to decrease in the upper airway after 3-5 days of symptoms.
Importantly, the viral load tends to decrease in the upper airway after 3-5 days of symptoms.

Another common question is “What is the sensitivity of PCR tests for COVID-19?
Another common question is “What is the sensitivity of PCR tests for COVID-19?

Is it really only 60%?”
Is it really only 60%?”

At this point, it is important to underscore that most clinical laboratories only know
At this point, it is important to underscore that most clinical laboratories only know

the analytical sensitivity of the molecular PCR tests.
the analytical sensitivity of the molecular PCR tests.

In other words, laboratories have determined the limit of detection of these assays using
In other words, laboratories have determined the limit of detection of these assays using

control material and known positive samples.
control material and known positive samples.

The clinical sensitivity, or the number of times the test will be positive in patients
The clinical sensitivity, or the number of times the test will be positive in patients

with COVID-19, still needs to be defined.
with COVID-19, still needs to be defined.

The true clinical sensitivity of COVID-19 PCR tests likely depends on several factors,
The true clinical sensitivity of COVID-19 PCR tests likely depends on several factors,

including when the sample is collected in relation to the patient’s disease course,
including when the sample is collected in relation to the patient’s disease course,

the sample type, the quality of the sample that is collected, and the performance characteristics
the sample type, the quality of the sample that is collected, and the performance characteristics

of the test itself.
of the test itself.

With regards of sensitivity of PCR, there was a recent study published in the Journal
With regards of sensitivity of PCR, there was a recent study published in the Journal

of the American Medical Association that assessed the detection rate of PCR using different
of the American Medical Association that assessed the detection rate of PCR using different

sample types in hospitalized patients.
sample types in hospitalized patients.

The detection rate in these various sample types range significantly, and as you can
The detection rate in these various sample types range significantly, and as you can

see on this slide, the highest rate of positive results was achieved when testing BAL fluid
see on this slide, the highest rate of positive results was achieved when testing BAL fluid

and sputum: 93% of the BAL were positive, and 72% of the sputum samples were positive
and sputum: 93% of the BAL were positive, and 72% of the sputum samples were positive

in these patients.
in these patients.

However, the number of positive results decreased in sample types such as nasal swabs and throat
However, the number of positive results decreased in sample types such as nasal swabs and throat

swabs at 63% and 32% respectively.
swabs at 63% and 32% respectively.

I think these data are attributing to some of the information of a 60% sensitivity for
I think these data are attributing to some of the information of a 60% sensitivity for

PCR.
PCR.

But it’s really important to underscore when these patients were tested as these were
But it’s really important to underscore when these patients were tested as these were

mainly hospital patients.
mainly hospital patients.

They were likely further along in their disease course, and we know now from the viral transmission
They were likely further along in their disease course, and we know now from the viral transmission

data, again, that the virus tends to decrease in viral load in the upper respiratory tract
data, again, that the virus tends to decrease in viral load in the upper respiratory tract

further along in the disease.
further along in the disease.

So this data actually makes a lot of sense—being that these are hospitalized patients—the
So this data actually makes a lot of sense—being that these are hospitalized patients—the

virus is likely present at higher amounts in the lower respiratory tract, which we can
virus is likely present at higher amounts in the lower respiratory tract, which we can

see by the positivity rates and sample types such as BAL and sputum.
see by the positivity rates and sample types such as BAL and sputum.

In summary, COVID-19 is continuing to cause a worldwide pandemic involving millions of
In summary, COVID-19 is continuing to cause a worldwide pandemic involving millions of

cases.
cases.

It’s caused by a variant virus called SARS-CoV-2.
It’s caused by a variant virus called SARS-CoV-2.

We now have several laboratory tests for SARS-CoV-2.
We now have several laboratory tests for SARS-CoV-2.

Real-time PCR is used for acute diagnosis of patients with symptoms, and serology can
Real-time PCR is used for acute diagnosis of patients with symptoms, and serology can

be used to determine whether an individual has been exposed to the virus and mounted
be used to determine whether an individual has been exposed to the virus and mounted

an antibody response.
an antibody response.

Importantly, the sensitivity of PCR testing depends on a number of factors, including
Importantly, the sensitivity of PCR testing depends on a number of factors, including

the timing of disease, when the test is performed, the type of sample that is collected, the
the timing of disease, when the test is performed, the type of sample that is collected, the

quality of the sample, and finally, the performance characteristics of the test itself.
quality of the sample, and finally, the performance characteristics of the test itself.

Thanks very much for joining me for this Hot Topic on COVID-19 molecular testing, and if
Thanks very much for joining me for this Hot Topic on COVID-19 molecular testing, and if

you have any questions, you can submit them to Mayo Clinic Laboratories.
you have any questions, you can submit them to Mayo Clinic Laboratories.

Thank you.
Thank you.