Torofy Blog

Depression and Heart Disease

Changes in Clinical Diagnostics and Tracking Infectious Diseases

>>Thank you for joining us. I’m Dr. Phoebe Thorpe and it’s
my pleasure to welcome you to CDC, Public Health Grand
Rounds for October 2016, “Changes in clinical diagnostics and tracking infectious
diseases”. We have a very exciting session. So let’s get started. But first, a few
housekeeping slides. Public Health Grand Rounds has
continuing education credits available for many disciplines,
physicians, nurses, pharmacists, veterinarians, health
educators, and others. Please see the Public
Health Grand Rounds website for more details. In addition to our website,
we’re also available on social media and we
are live tweeting today. So please use
#CDCGrandRounds for all your tweeting needs. We also have a featured video
segment on YouTube called “Beyond the Data” which is
posted after the session. And we are partnered with
the CDC Public Health Library and Information Center to feature scientific articles
relevant to this session. The full listing is available
at Here is a preview of our upcoming Public
Health Grand Rounds session. Please plan to join
us live or on the web. In addition to today’s
outstanding speakers, I’d also like to take a moment
to thank the many contributions of the individuals listed here. Thank you. And now for a few words from
CDCs Director Dr. Tom Frieden.>>Technology continues
to advance, change and improve our lives. One example is a new
generation of Culture, Independent Diagnostic
Tests or CIDTs. These CIDTs make it possible
for healthcare providers to determine which pathogen
is causing a patient disease, often while the patient is
still at the doctor’s office. When a specific cause is known
quickly, patients benefit from faster diagnosis
and targeted treatment. CIDTs are available
for many infections, including tuberculosis
and chlamydia. They can confirm infections
that weren’t easy to test or culture before such as
legionella and C. difficile. But as with other
technological advances, there are unintended
consequences. Although, CIDTs provide
rapid diagnosis, there is no organism
available for further testing, including by traditional
culture. If clinical care ends
with the CIDT results, we may not get answers
to important questions. We can’t determine whether
one person’s illness is part of a larger outbreak. Whether a bacteria is
resistant to antibiotics and how likely it is to
cause severe illness. Information that’s
key to understanding where infections come from and how they spread is
especially important in the case of infections spread
through our food. Additional laboratory testing is
still needed for positive CIDTs, without it the food
supply will be less safe. Every year, 48 million
Americans get sick from food borne illness; 128,000
are hospitalized and 3000 die. Twenty years ago, along with
our partners CDC established PulseNet and FoodNet
to track the spread of food borne diseases
and work with partners to trace illness back
to contaminated food. Over that time many
outbreaks have been stopped, but not before infecting too
many people across the US and in other countries. In 2015, there were 35
multistate outbreaks of foodborne disease. And each year, the number
of outbreaks increases. Only by connecting these
individual infections can public health investigators trace
illness back to the source and stop further spread. Without cultures and
detailed genetic information, connecting affected individuals in multiple states is more
difficult, takes longer and leaves contaminated
food on the shelf. As you’ll hear today, our scientists are using
many approaches including collaborating to
develop new technologies to help address these issues. But these advances take time
to implement and until they’re in place, public health will
have to continue to rely on cultures to detect, trace
and stop foodborne outbreaks, and keep our food supply safe.>>And now for our
first speaker, Dr. Chris Braden.>>Good afternoon. Today I will be giving you an
introduction to the advances in Culture, Independent
Diagnostic Tests. Culture-based diagnostic
tests have been used for over 150 years, having
been invented by Louie Pasteur. More recently, culture
independent tests have been developed with antigen-based
tests coming into wide use in the 1980s and 1990s. These tests detect
antigens which are specific to a certain pathogen type. In the 2000’s, molecular
detection test based upon polymerase chain reaction
methods were developed. These tests detected short
genetic sequences specific to a single pathogen type. Since 2010, we have seen a major
expansion in the production of multiplex PCR
panels which detect one or multiple pathogens
simultaneously. These panels are often designed
around disease syndromes such as gastroenteritis,
respiratory diseases, meningitis, or bloodstream
infections. Though we will be focusing
mainly on diagnostics for enteric bacterial
pathogens today, culture independent
tests based upon antigen or nucleic acid detection
have been applied to many different
types of pathogens and are often the only practical
way to detect viral pathogens. For enteric pathogens
the number and type of culture independent
diagnostic tests have increased rapidly. In the last 5 years there
has been an increase from 5 antigen-based
tests for Campylobacter and Shiga-toxin-producing
E. coli or STEC to 8 antigen-based tests, several laboratory
developed PCR test used in large commercial
laboratories, and to date 5 multiplex PCR
panels have been FDA cleared. Still others see CIDTs including
multiplex PCR panels have been FDA cleared for respiratory,
meningitis and bloodstream pathogens. FoodNet, a network
covering about 15% of the US population
monitors CIDT use as part of its surveillance activities. For the bacterial
infections tracked by FoodNet, on average 7% were diagnosed
by CIDT in 2012 through 2014. As depicted by the blue
bars in this graph. This more than doubled
to 16% in 2015. Uptake of CIDTs in clinical
laboratories varied by pathogen and the use of multiplex
PCR panels is growing. The use of CIDTs may
impact surveillance trends. Compared with the average
incidents in 2012 to 2014. In 2015, the incidence
of Cryptosporidium and non-0157 sugar
toxin producing E. coli almost doubled. This increase might,
in part be caused of increased use of CIDTs. As you can see from this
graphic, the workflow and CIDTs including multiplex
PCR panels is relatively simple and fast. The clinical specimen
is prepared for testing in a sample tube containing
reagents, which is inserted into the automated
diagnostic instrument. Detection of specific PCR
products yields a positive result for one or more
pathogens included on the panel. Automated analysis
can produce a result in as few as 1 to 2 hours. Reflex cultures should be
done to provide an isolate of the pathogen for
additional testing such as conventional
identification, antimicrobial susceptibility,
and pathogen characterization. More on why this is
important in a moment. However, conducting
reflex culture of CIDT positive specimens
may not always be clinically necessary and entails
additional costs. If reflex culture is done
for public health reasons, it is not clear whether
the clinical or public health laboratories
should conduct the culture testing and bear these costs. Also, in some instances,
it may not be possible to conduct reflex cultures. For example, if the original
specimens obtained with a swab, the entire specimen may be used
and inactivated when transferred to the sample tube
with reagents. Test procedures should include
the option for reflex culture from the original specimen. The benefits of culture
independent diagnostic tests are impressive, especially
for clinical care. They can provide
much faster results for targeted early treatment of
infections, including whether or not antibiotics are needed. The panel test can detect or
rule out multiple pathogens in a single test and are likely
more sensitive than culture. These tests may provide
faster information for local public health action. However, CIDTs alone do not
provide cultured isolates. Isolates are needed to
characterize the pathogen. In addition to the
clinical utility of antimicrobial susceptibility
results to tailor treatment, hospitals and public health
programs need this information to track resistance trends in their facilities
and jurisdictions. Determination of virulence
factors can inform clinicians and public health officials of
the likely severity of illness. Serotype and genotype
information is used to identify specific strains, whether a strain is
causing an outbreak and possible routes
of transmission. Genotype information has been
used extensively in programs such as PulseNet to connect
cases, identify outbreaks and prevent over 270,000
illnesses each year. PulseNet is in the
midst of transformation to more detailed DNA
fingerprint analysis to facilitate outbreak detection and investigation using
next generation whole genome sequencing technology. But this technology also
requires cultured isolates. Each year, 48 million people get
sick, 128,000 are hospitalized and 3000 die from
foodborne disease. Public health programs
need to enhance efforts to make our foods
safer and we need to conduct DNA fingerprint
surveillance of foodborne pathogens to do so. Other possible drawbacks of culture independent
diagnostic tests are related to the difficulty interpreting
test results in some situations. Because DNA from dead microbes
can produce a positive test result, clinicians may not know
a patient is still contagious and public health officials
won’t know if it is safe for a patient to return
to work or to daycare. Also, a single test may
detect multiple pathogens, some of which may not
be causing illness. A study of one brand of a
multiplex PCR GI panel found that over 30% of positive
tests detected more than 1 enteric pathogen. This may lead to
diagnostic confusion and unnecessary treatment,
and to confusion for public health
surveillance systems. CDC has strategies to meet the
surveillance challenges posed by culture independent
diagnostic testing. First, we need to support
the use of reflex cultures for positive CIDT samples
by encouraging states to update referral and
reporting guidelines and loss. We also need to decide who performs the reflex
culture and who pays for it. We can develop expedited
isolate recovery methods to make culture simpler and
cheaper, and we need to work with the diagnostic
device industry to make sure specimens can be
cultured using our procedures. Second, in the short term, we need to implement a whole
genome sequencing technologies for surveillance. Though this will also
require continued use of isolates from
reflex cultures. Developing large whole genome
sequence databases will allow the type of research
and development required to achieve the third tier of
our strategy for the long term, the development of
metagenomic diagnostic tests. Meta-genomic technology
has the potential to provide both the
essential clinical and public health information
direct from specimens without the use of cultures. Dr. Besser, will be
providing some insight on different methods
for metagenomics, including amplicon sequencing,
and shotgun metagenomics. As technology advances,
we must pursue a path that benefits both patient care
and public health surveillance. The solution includes
building a broad understanding of the issue among many
partners, including experts from public health,
clinical medicine, diagnostic device makers,
Congress, regulators, and insurance officials. In 2012, CDC convened a
multi-stakeholder forum to raise awareness of
the impact of CIDTs on public health programs. In 2013, the infectious
disease Society of America published
specific recommendations to improve diagnostics for
infectious diseases in a way that also benefits public
health surveillance. FoodNet partners are tracking
the growing use of CIDTs in evaluating changes over time. And The Council for State and Territorial Epidemiologists
is discussing how best to count infections
identified by CIDT and public health surveillance in updating surveillance
case definitions. And now I’d like
to turn the program over to Alicia Cronquist. [ Applause ]>>Good afternoon. CIDT is having a major impact
on surveillance in Colorado. Our most recent data show that 15 labs are using
multiplex PCR testing. To put this in perspective 40% of enteric bacterial pathogen
cases reported in Colorado so far during 2016 have
been tested using PCR, 89% of the salmonella Shigella and Shiga-toxin-producing E.
coli are xTAG tested using PCR have also had a reflex
culture tested performed either at the clinical lab, or the
state public health lab. In Colorado laboratories of
various sizes and from urban and rural settings are adopting
multiplex PCR since 2013. And we’ve been busy
responding to this switch. Our activity falls
loosely into 3 categories, ensuring accurate
case reporting, facilitating isolate recovery and adapting our day-to-day
public health practice in light of the new types of
cases being reported. Until recently surveillance for enteric bacterial
pathogens captured only culture confirmed cases. The classification scheme
we use in all 50 states, or the case definitions
have been modified to incorporate CIDT positive
results as probable cases and we need to adjust
accordingly. We monitor the local
uptake of CIDT by regularly serving
clinical laboratories. It’s important to know the
types of tests each lab is using so we can understand the
surveillance information we receive from them and
ensure we’re receiving that information correctly. We started this in late 2009 when a few Colorado laboratories
began using antigen-based tests to detect Campylobacter. Currently, we survey
laboratories within our FoodNet
catchment area which is the 7 county Denver
metropolitan area, twice a year. We do a similar survey once
a year with laboratories in the rest of the state where
resources are not as robust. While this activity
is very important, it is also labor-intensive. Knowing which labs are using see
CIDTs is only the first step. Next, we needed to modify our
disease surveillance database to capture the new tests, and ensure data are properly
exported when queried. For us, this required working
with our IT department. We have discovered there are
many ways in which errors in reporting culture
independent results occur. And we continue to work
with disease reporters to improve the quality of the
CIDT case reports we receive. For example, some labs using
electronic lab reporting needed to change their electronic
settings so that CIDT positives
flowed to us correctly. Other laboratories had trouble
modifying printed reports they sent to us. They would report culture
positive for salmonella, for example, when we knew
they were no longer performing culture for salmonella. And finally the thorniest
issue is one of incorrect test
interpretation and reporting. Some multiplex panels
include pathogens that are not reportable, but sound like conditions
that are reportable. We continue to have cases
incorrectly reported to us. Shigella, Shiga-toxin-producing
E. Coli or STEC, and Plesiomonas shigelloides
sound similar, but only two are
reportable in Colorado, and each is a different
pathogen. We’ve created guidance documents
and held several meetings and webinars with infection
preventionists, laboratories and local public
health partners. We reach out individually when
we identify a particular issue and we continue with
frequent stakeholder outreach. We feel we cannot
over communicate with our partners
about these issues. Of course case reporting is
only one part of surveillance, we need isolates for
subtyping for cluster detection and monitoring trends. The big question for us is
where the culture happens. We’ve taken a hybrid approach
to isolate recovery in Colorado. Historically, we have had
excellent relationships with our clinical labs. We received approximately
95% of the isolates needed without having any regulations
for isolate submission in place. We’re building on these
established relationships to find a path forward. We prefer when isolation
is performed at the clinical laboratory. Culture results are available
faster, it decreases concerns about transit of raw
specimens, and the chance that pathogens will
die in transit. It also means that susceptibility testing
can still be performed at the clinical level where
it could impact patient care. We encourage each
lab that adopts CIDT to perform reflex
cultures at a minimum for salmonella, Shigella
and Vibrio. We review our isolate submission
protocols with them to ensure that any isolates that
are generated are sent to the public health lab. When reflex cultures
are not done, clinical laboratories are asked
to forward clinical material which is generally
stool, that tests positive to the state public
health laboratory, where we can then attempt
to isolate the organism. A lot has gone into
creating a workable plan. First we had to prioritize
pathogens. We chose STEC, salmonella,
Shigella, and Vibrio. These organisms have
the highest likelihood of public health action based on confirmation or
subtyping results. Next, we sought and continue
to seek additional funding to perform culture which adds
considerably to our costs. To ensure we would receive
specimens we ask the Board of Health to modify our
disease reporting regulations to require submission
of isolates or clinical material
for select pathogens. This is the first time specimen
submission has been required rather than voluntary
in Colorado. We’ve also worked to
facilitate rapid delivery of viable specimens to the
state public health lab. We reviewed the courier
service we provide to ensure couriers are regularly
going to all labs that need it, and we provide transport media
and written guidance about how to ship clinical material to us. This is an area that’s evolving
as APHO is performing studies to determine the best
methods for isolate recovery. However, none of
this is perfect. Despite all this effort are
specimen submission rate has dropped to 90% and we continue
to see a range of issues. For example, stereotyping during
an outbreak investigation was substantially delayed when
one rural lab mailed clinical material instead of sending
it to us via the courier. The final area is the one where we still need
to do the most work. With CIDT, the data we’re acting
on has changed and we need to figure out how to use
the information we receive. We see an increase
in case reports with less certainty
about each one. Reporting is faster
with respect to the time between specimen collection
and public health notification. However, we now have
a longer period between the time public
health first learns of a case and we have subtyping
information to pair it with. First off, we need to implement
the new CSC case definitions to classify and transmit
surveillance data correctly. To do this, we routinely
collect more detailed test data. In Colorado, we attempt to
collect negative results when they’re pertinent. For example, if a
PCR is positive and culture was negative,
we try to capture that in our surveillance data. We’ve worked to train internal
and local public health staff to appropriately
assign case status based on the new criteria. This includes creating new cheat
sheets and guidance documents for information that’s
ever more complex. We’re working to establish
and evaluate guidance for which cases should
be investigated based on their case status. While it would be great
to interview all suspected and probable cases, we need
to consider local resources and prioritize across
the spectrum of diseases they are
asked to investigate. For example, a suspected, highly infectious respiratory
case would be a higher priority than a probable PCR
positive Campylobacter case. The timing of case
investigation is also critical. For the case categories
we decide to investigate we launch
an investigation based on PCR results and
don’t wait for culture. Other jurisdictions might make
different choices about that. When should we initiate
worker and childcare exclusion or restriction for
PCR positive results? We have chosen to treat a PCR
positive the same as culture in terms of excluding
or restricting workers. Yet, is that’s the right choice? For follow-up testing we accept
a PCR or a culture result. Although culture is
often the test used, since much of the follow-up
testing is performed at the SPHL at no charge to the patient. And how do we handle patients
who are CIDT positive for 2 or more reportable conditions. From a public health
perspective, we need to decide what type of
exclusion might be necessary and which questionnaire to use. Our guidance is currently to choose the disease control
measures for the pathogen with the greatest
risk of transmission, and to interview the patient
using the pathogen specific questionnaire that is
most comprehensive, or merge the 2, when applicable. For example, when a patient is
supported with a PCR positive for Shigella and Campylobacter,
we instruct local public health to follow Shigella exclusion
policies, and interview with a questionnaire that’s
a blend of the Campylobacter and Shigella questionnaires. But clearly much more
research is needed. We have focused our response to
CIDT on accurate case reporting, isolate recovery and
adapting public-health actions to the new types of
cases being reported. These areas have
several factors in common which are a critical assessment
of resources, prioritizing based on public health
goals of detecting and mitigating disease risk and
frequent ongoing communication with partners using a variety of modalities including
guidance documents, phone calls, and training sessions. Thanks. [ Applause ] And now I’d like to introduce
Brad Spring.>>Thank you Alicia and
good afternoon everybody. I’m going to be representing
industry, although I work for BD. I do represent an organization
called AdvaMedDx which I’ll provide
a little bit more detail later. But I’m going to give you
the industry perspective on how we develop tests,
but also how we can partner with public health and other
stakeholders to solve some of the challenges we’re seeing. So, as described earlier,
we do have advantages and challenges of CIDTs. Multiplex PCR test can be
more sensitive than culture and provide results
in a few hours. They may also save costs and significantly
improve healthcare. But because PCR tests require
the organism to be lysed, or the cells be broken apart
to extract the DNA or RNA, we don’t have viable
organisms available for surveillance purposes. Companies do recognize
the need to partner with public health agencies and
FDA to collaborate on solutions. And AdvaMedDx which is an
association represented by over 50 diagnostic companies, can leverage our
strong relationship with lab communities,
government agencies and public health laboratories
to work towards a common goal of providing rapid diagnostic
solutions while meeting the public health needs. So I just want to give you an
overview of how we develop tests in the industry by covering our
product development process. And we typically use what’s
called a phase gate approach. And as you can see here we
start in the concept phase, go through definition,
development, qualification of the validation stages, and
then we’ll launch the product. But the first step in concept
and then through definition is to conduct what we call voice
the customer activities. And this is where we build
upon what are the requirements for the new test. And this is really where we
come up with the ideas and some of the challenges our customers,
or future customers may have and how to address them. So as we do these voice
the customer activities, we gather requirements. And the requirements
may not be specific. A customer may say I want
a test that’s easy to use. May provide fast results. Or it’s small in size. Now these aren’t measurable. So we translate those
requirements into specifications. So for example, a fast result
may be translated into a result within 2 hours including
specimen processing. And we go back to the
customer to verify that essentially
meets their requirements. But certain needs may only be
met by existing technologies that could create what we
call conflicting requirements. So for example, to
achieve a fast result, PCR may be the technology
of choice. And in the cases we’re
discussing here today, you’re not able to get a viable
organism when you use PCR. But just because we have
a conflicting requirement, meaning using PCR doesn’t allow
us to culture the organism, at least from the PCR specimen, we don’t abandon
these requirements and we try to find workarounds. So while requirements
may conflict, that doesn’t mean we just
give up as an industry. We try and find other
ways to address them. So I think, you know going
forward, and even looking back on previous product
development activities, we need to encourage engaging
public health laboratories, CDC, state labs, and others in these
voice of customer activities. So as another example, we
may go to a clinical lab, gather the data, but
rarely are we getting data that says we need to be
able to culture the organism for submission of public health. And then we go to public health,
we’ll hear, well we’ve got to make sure that the
organism’s cultured. So, I’m going to cover too, a couple of potential
opportunities to solve this problem and I think the other speakers
will be addressing these as well. So one example is our
instructions for use need to remind laboratories that
they have to follow state and federal requirements for
collection and preservation of organisms needed
for epidemiological or the public health purposes. So in those instructions for
use, and a customer needs to follow those instructions
for use, we will hopefully achieve a
little bit better compliance to state requirements. There are opportunities in product development
for new technologies. As you’ll hear more about later. There are some on the horizon that will help reduce
compatibility issues between CIDTs and public health. Metagenomics holds the
most promise, today, as it may be possible to
both detect the organism in a metagenomic specimen
and differentiate strains for epidemiological purposes. However, there is much
work still required to develop this technology and you’ll hear more
about this later. One thing to consider, though, is when an industry actually
may decide not to create a test for manufactured test
for clinical lab, a clinical lab may create
that test on their own. So there are a number of lab
developed tests out there today that are basically put in
place to meet an unmet need. So, if there is an
enteric pathogen that isn’t currently
detected using one of these multiplex PCR
tests, a lab may create that test on their own. So as we look at
culturing organisms, it’s not just the industry
instructions for example, they might have to
change, we have to do more to educate the labs on what
their requirements are, especially for LDTs or
lab developed tests. So, just to kind of cover in
more detail the instructions for use opportunity here. Some manufacturers, not all, do put specific precautions
in their labeling. And under the clinical
laboratory improvement amendments, which
are regulations that govern laboratories,
CLIA does require that labs follow
manufacturer’s instructions. So if our instructions
state, for example, the APHL recommended language around laboratories must
follow state and/or local rules pertaining to reportable
pathogens, the lab must do that. Otherwise they during
inspection may get observation regarding that. So going forward, you know we
as an industry do agree we need to put such labeling and
standardize it, these statements in our labelling going
forward and that will help. It doesn’t do everything,
but it will help with the public health
labs and ensuring that these pathogens
are also cultured. So we believe this type
of information is one step in the right direction. We do believe there are
continuing opportunities on these collaborations. So we will, as an
industry, work with CDC, other public health laboratory
agencies, as well as FDA to discuss efforts to
explore additional measures to aid in the surveillance. So, you know, we’ve
talked about the labeling. There may be other
opportunities that we can, as we come together
collectively, figure out how best to
meet this challenge. We have conducted
and will continue to conduct educational outreach
meeting with key stakeholders. So like I said, the
public health labs, other microbiology groups
such as maybe the IDSA, ASM and through other
industry meetings. And as a manufacturer, you
know we have a salesforce, a pretty large salesforce
out there. And we currently use those
salesforces obviously to, not just sell our products
but to also educate customers in a variety of issues. One example being the get smart about antibiotics week
coming up here in November. We’ll distribute material to our
customers to help educate them on antimicrobial resistance. So this is another opportunity
to at least increase awareness and educate customers. And then finally, one thing
we are looking at is just to provide better informational
resources and we’re talking, collectively talking to FDA
about posting a list of approved or cleared molecular
diagnostics on the FDA website. It will serve essentially as
a one-stop shop to finding out what CIDTs are out there
and what are the procedures used to conduct those CIDTs. So, and thank you very much. And now I’d like to
introduce Dr. John Besser. [ Applause ]>>Thank you Brad
and good afternoon. I’m going to be speaking today
on efforts to develop tests for characterization of
our public health pathogens that can be done
directly from specimens. I’m going to be focusing on the
bacterial enteric pathogens, our PulseNet pathogens. And I will be attempting
to squeeze a graduate level in molecular biology
into 8 minutes. So let’s see how that goes. But first why are
we developing tests. Well, most importantly we want
to ensure continue compatibility between our public health tests,
and the commercial systems even if the specimens are
biologically inactivated. And the second compelling
reason is that these tests have the
potential of being quick, which is of course one
of the same reasons that makes CIDTs desirable
for clinical diagnosis. The process of detecting
outbreaks through pathogen
based surveillance, can be a lengthy process,
as illustrated here. Cases must be identified
and linked to other cases, food vehicles identified,
and depending on how a direct specimen tests
are implemented they can have the potential for significantly
reducing the amount of time to an actionable result,
which in turn means that more outbreaks would
be solved more quickly. More illnesses prevented. Developing direct tests for public health is
not a trivial task. Stool is an immensely
complex environment, containing a wide variety of
different DNA and RNA species. There could be significant
human DNA from white blood
cells, epithelial cells. All the food that
you eat, the plants, the animals they all have DNA. There’s bacteria,
parasites, viruses, fungi. Every one of us has an average
of 1000 species of microbes in our gut at any one time. And every gram of stool can have up to 100,000 organisms
per gram. And of particular
concern to us is that many of our PulseNet pathogens are
virtually identical to some of the commensal flora. Every line in this figure
represents the genome of a bacterium that
might be in our gut. The commercial developers
need to find a marker which is conserved within
the species of interest. And specific to it. Meaning, that that marker
doesn’t appear in any of the commensal flora. For public health tests, we must
also specifically detect the pathogen, but also detect
other markers for strain type, resistance virulence, and
also detect variability within those markers so that
we can detect differences between strains, illustrated
here in the colored bars. And these should not be
confused with similar sort of regions in our pathogens. Of course, when we’re
actually doing these tests, we don’t have any
of that information. In fact, what we’re actually
dealing with is many millions of pieces, little pieces
of DNA or short reads as in some of our assays. And there’s two major
problems that we confront. One is signal-to-noise,
finding our pathogen which may be very
rare when compared to the background flora. And the second is
called phasing. Getting all the pieces together
that we find of the pathogen, specifically connected
to the marker, and not including any
pieces that may be from some of the commensal flora. There’s three general
strategies that we’re pursuing to accomplish this task. The first is called
amplicon sequencing, a method that’s been around, especially for the viral
pathogens for quite a while. We believe that this can be
accomplished using technology that is readily available today. And we’re currently
pursuing two approaches. The first, we’re
looking at for STEC, we’ve identified a
heterogeneous region in Shiga-toxin-converting phage,
which is normally integrated into the circular chromosome. And here is a map
of the phage genome. Within the phage is a region
containing the regulatory genes, and also the Shiga-toxin gene,
which defines the pathotype, which helps us solve
the phasing problem. Our scientists have developed
a multiplex PCR assay using conserved primer binding sites which yield overlapping
amplicons covering an 11 to 17 KB region. These amplicons can
then be assembled into a single continuous strand
of DNA or consensus sequence, also known as the contig. The contig, then can be compared
to sequences from other patients to find genetic differences,
represented here in red, which in turn can be used
to assign strain types. And the strain type
information is what we use to detect clusters
and are used as part of epidemiological
case definitions. For salmonella, we’re taking
a slightly different approach, since it’s less related to
commensal flora of an STEC. We’ve developed a pipeline,
or a series of processes that are listed in the bullets
here, to identify specific and informative targets to approximate a whole genome
multilocus sequence typing or wgMLST. The second major strategy that we’re pursuing is
shotgun metagenomics, as was mentioned earlier. What is metagenomics,
it’s the sequencing of all the nucleic
acids in a sample. This is widely used already to characterize the whole
microbial community, or microbiome. In our case, the
stool environment. Researchers here at
CDC, and elsewhere, have shown that it is possible
to both detect pathogens and differentiate strains for
epidemiological studies directly in stool using shotgun
metagenomics. However, the method is
still fairly insensitive, expensive, slow. It produces a lot of data
that has to be handled. But the potential power of metagenomics really
can’t be overestimated. It likely will make it
possible to find solutions to a whole array of
public health problems that are currently
difficult to address. Our group, and other groups, here at CDC are exploring
multiple approaches at various points in the
specimen to sequence process, to solving these signal-to-noise
and phasing problems, some of which are listed here. These approaches, if successful
should also reduce the amount of sequencing and data for
public health surveillance. We’ve also begun to explore
with external collaborators, droplet based single cell
sorting or barcoding, which should allow us
to obtain the same type of information we’re
currently obtaining through whole genome
sequencing of isolates. Individual cells
are encapsulated in the lipid droplets which
are subject to PCR and run through a high-speed
cell sorter. In this animation, there’s
a laser below the red arrow, which elicits a signal
from PCR positive cells. Positive cells, which are
shown here as solid circles, are then routed down a different
path from the other bacteria, shown as empty circles. At least in theory, this
technology could result in a tube of cells
from a single pathogen, which could then be subject
to whole genome sequencing. In summary, direct from
specimen tests could increase compatibility between CIDTs and
our public health activities. And we believe that the current
technological limitations will likely be overcome with
research that’s occurring with multiple partners. And in the end, we
believe that all of these advancements
will make PulseNet and our food supply safe, more
efficient and more effective. In summary for our
entire session today, CIDT technology has been used
for improving patient care and as Dr. Braden has showed
us, and public health is in the process of adapting to these changes in
a variety of ways. Advances in technology
in the public and private realms will make
our lives safer in general, but we must pursue a path that
benefits both patient care and the public’s health. Now I’d like to turn the podium over to Dr. Braden
again, thank you. [ Applause ]>>Well I’ll stay here at the
panelist desk and as we go into the question and answer
session, so first of all I would like to know if we
have any questions from the external audience.>>We do, thank you. Reports are being
charted as final results at some facilities, and I suspect they should be
considered screening tests. On average do these panels
have acceptable sensitivities and specificities, precision
and accuracy, etcetera?>>I’m going to turn to
Dr. Besser and Brad for comments.>>There are some
studies in the literature that address the sensitivity
and specificity of these tests. Frankly, we don’t
know all the answers. Some kits are more specific
and sensitive than others. And this actually has
created some problems in creating the probable and confirmed case
definitions with CIDTs. But I think as time goes
on we will learn more and it’s incumbent upon
us to encourage studies of the performance
characteristics of these tests so that this question
can be answered.>>Yeah, just a comment
on industry. So our tests do go through
the FDA review process and the FDA looks at
what’s our intended use. Is it for screening,
diagnosis, and the sensitivity and specificities related
to that intended use. So FDA would not
approve or clear a test that didn’t meet a
certain threshold. That said, I think
the FDA and industry, as well as labs will
look at is it better than what we have today. So I think when you
look at that, it’s hard to say
is it good enough, but at least it may be better
than what we have now, so. Can’t quite answer on that
on the screening tests.>>However, to add from a
disease reporting perspective, it it’s a reportable condition that the person asking the
question is asking about, then yes, we would still
want that reported to us, along with what type
of test was performed. We need to have all
of that reported to us so we can understand
what’s going on with disease surveillance
and with these tests.>>I would also add that
as the tests, culture and diagnostic tests
have advanced over time, I think the sensitivity and
specificity has improved. And these new multi-analyte
PCR panels have been shown to be quite sensitive. Another question? How about from the audience? Yes, Steve.>>I’m Stephen Rose
Associate Director for Laboratory Science
and Safety. My question is really directed
toward Alicia and falls on the previous question about
sensitivity and specificity. And I think if I heard
you correctly you said that if the CIDT only,
then it’s a probable case. Do you code the cases
that you alluded to that are CIDT positive,
culture negative any differently from one that is CIDT only?>>We try to collect
that level of information and we strongly encourage
laboratories to report pertinent
negatives to us. So if it’s antigen positive
and culture negative, we surely would like to have that culture negative
piece in our dataset. From a coding standpoint to
meet case classification, those would both be
coded as a probable case.>>Peter Gunersmidt from Enteric Diseases
Laboratory branch here at CDC. This is more a question
for Dr. Spring I guess. The metagenomics that
we’re working on trying to develop some methods. Those methods, they
really belong in the clinical laboratories,
don’t they? So my question to
you is where are the, what are the diagnostic industry
doing with this technology and how far are you before
we can implement metagenomics in the clinical laboratories.>>Yeah, I’m probably not going to give you the answer
you’re expecting, which is I don’t really know. But I will say this, that
there are companies out there, and most of the time
they’re startups, right. They’ll get venture capital
funding to investigate this. And I know there is one
company at least that I’m aware of out there that does
provide testing services using metagenomics. I don’t believe they have a
commercial kit for laboratories. I don’t know how
far away we are. Again I haven’t seen
any come through FDA. I think FDA has to set
up standards for these. You know how do you
validate them analytically and clinically. So I think they’re probably
going to be shown more in a laboratory maybe as
an LDT before they show up as a commercially
available assay.>>I believe that metagenomics
will be used regularly, sooner rather than
later for specimens from normally sterile
sites like CSF and blood. And I think that
in the short term that probably will
occur in laboratories. But I would imagine in the very
long-term it’s probably most appropriate for this to happen
in the clinical setting. And then the state and local
public health agencies retrieve information that they need, federal agencies
retrieve what they need. But it seems closer to the patient the more
efficient the system gets. But I think the technology
is a long way from operating at that level.>>Do we have another
question external?>>We do from Twitter. Are there any national
summaries on CIDT surveillance and impact on patient care?>>So there are some summaries
of CIDT use that are provided by the FoodNet publications. Every April FoodNet, as a surveillance system
provides interim reports of the previous year’s
surveillance data and within that data there is
reports of CIDT use. However, we don’t
gather the information about how it’s affecting
clinical care, which I think is the other
part of that question. Putting on my clinician’s hat, you know if I can get the
information about the pathogen that may be causing an illness
faster, same-day or even within hours, that makes
a lot of difference. And so I just, you know in that
antidotal way it really does improve what we can
do with patient care. Any other comments?>>There’s a very good
publication by IDSA [inaudible] on CIDTs and clinical care
that one can find for free on the web, that’s
worth reading. Another question from
the external audience.>>We get laboratory
reports indicating more than one positive on
the same specimen. Do you know if the labs are then
obligated to perform cultures, serology tests, etcetera,
depending on the microorganism?>>Brad, do you know that? Or Alicia?>>I’m not sure I
understand the question. It seems like the laboratories,
if the condition is reportable, they need to report,
I would guess all of the conditions is
that correct Alicia?>>Yeah. Yep. So from a public health
standpoint, they would need to report anything
that was positive. And depending on the state
where they’re located, they might be required to
perform reflexive culture. Or, they might be
required to send some of the clinical material to
the state public health lab, depending on the state
and the pathogen. But I think the question is
more from a clinical perspective and asking about determining which pathogen is
making the patient ill. And I think that’s a
very difficult question that I believe clinicians are
trying to sort out as well.>>As we are hearing from the
community that the incidents of multiple positive signals
from CIDTs is fairly hard. And I think there are
many aspects of CIDTs that we’re going to need
to resolve over the years in interpretation,
that being one of them, and it could be the
polymicrobial infections are much more common than
we knew, or perhaps some of these findings are spurious and we just don’t know,
and time will tell.>>I think, there’s
not some kind of regulatory responsibility
for the laboratories to do more testing to see which
pathogen may be causing illness, but certainly the
physician may follow up with additional tests
requested in order to clarify.>>Any other questions
from the audience?>>David Bell, Division
of Viral Diseases. Could you say anything
more about the role of quantifying some
of these results and assisting with
the diagnosis? You know in PCR we have CT
values and I don’t know as much about the other, but in some
of the issues you raised, it might be relevant if
the pathogen was present in a higher quantity as
opposed to a lower quantity. You know some of these
dual findings and so on.>>So just one comment,
that I mean as you look at the microbium
and normal flora, you know sometimes you
have to distinguish between what is the causative
agent and what is normal flora. So quantification, or
quantitation is required for some of these assays, because you may be actually
detecting a healthy level of some bacteria. So it depends on the test, and depends on what the
treatment options may be. But I think there is
value in quantitation.>>If you’re referring to
clinical diagnosis, is that? Or the? Is that what
you’re referring to?>>Well, well both. Also surveillance because
it’s not clinically relevant. It may or may not be public
health, but it’s both.>>Sure, I believe most of the,
or all of the CIDTs operate with thresholds built
into the system already. So the positive result you
get is a quantitative result. In the area of metagenomics,
and sequencing, and shotgun metagenomics,
quantification is, quantitation is an approach
that we’re exploring extensively to help with phasing, have been
re-binning clinical quantitation of pathogens to align our
various bits together. So for characterization of
our pathogens, what we call in situ pathogen
characterizations, quantitation is an important
part of our overall strategy.>>So to follow up on David
Bell’s question on quantitation and the issue of exclusion
either from workplace or daycare, and of course, my
experience is with norovirus where it’s quite clear that
the virus is detectable much, much longer than somebody
is probably a real risk for transmission. And so is there any thought
of using quantitation in making those determinations
about when somebody is or is not excluded from the
workplace or from daycare?>>It would be great. I don’t know if it’s at all in
the horizon, but even before that I think that some follow up
studies are desperately needed to track how long people shed. I think that we exclude
people fairly routinely from occupations if they have,
say E. coli 0157 or Shigella, we will exclude them
from their jobs, which has a tremendous impact
on them and their lives. And so form a public
health standpoint, we are very concerned
about doing that based on a PCR positive and are very
eager to participate in studies, perhaps where stool is
collected, and both cultured and then put through
a multiplex PCR so we understand that better. I don’t believe those
studies have been done yet.>>Do we have another question?>>FoodNet depends on public
health lab surveillance to describe changes in
illness incident rates overtime and the incident rates
have targets that come from healthy people 2020. How has CDC incorporated
the impact of CIDT in tracking illness
incidents overtime and marking our progress in meeting foodborne illness
national health objectives.>>I will start and
Alicia may comment also. So there were a couple of times,
and especially Alicia alluded to the fact that CSTE is
revising case definitions. When we revise case
definitions it is going to affect the surveillance
data trends. What is going to have to occur
is to the extent possible to take the data that we
have about the increase in specificity and sensitivity
of some of these tests, and apply that to the
models that we use to then generate our
surveillance trends to be able to translate what
we’ve seen before with what we’re seeing now. That’s not an easy task, but
it is something that a number of groups are trying to do. So that we can then determine if
we are actually still on track with meeting our
goals, or we’re not. Alicia?>>As Dr. Braden alluded,
we’re working on a variety of data collection projects and
thinking about different ways to model the data to assess
the impact of these changes on our trends so that we can
accurately say whether disease is going up or down to meet
these national objectives. One key piece of information
that we didn’t have time to discuss during the formal
presentations, is another way in which we hope to partner
with clinical microbiology labs, which is the collection
of information from them about how much testing
they’re performing and who they’re performing
testing on. And this is absolutely
critical information, so that we can understand the
data that’s flowing to us. One area with CIDT it’s possible that more testing
is being performed, or testing is being performed
on different groups of people than it used to be performed in. And we need to understand that to understand the
surveillance data that’s coming to us. So in addition to the
partnerships that we’ve talked about with clinical microbiology
labs and submitting isolates or clinical material to state
public health laboratories, another key area for
participation in partnership between public health
and clinical micro labs in the near future is going to
be working with us to figure out how to obtain this, what
we’re calling test volume data.>>Another question
from the audience.>>Hi. Lee Katz
Foodborne Diseases Laboratory branch. I was wondering from the panel if there are any products being
used for source attribution. So in Dr. Besser’s
presentation, he noted that some food
might also be in a sample, in a stool sample, if there
are products that might be used to detect them that are being
in research and development. And if those food sources
are detected, I’m wondering if at Colorado and other state
health labs, if it might be used to inform epidemiology.>>That’s a very good question. No there is no such test
out there that I’m aware of, but it’s an excellent
suggestion. If you were able, I’m sure
that if Alicia were able to tell her cases what
they ate and ask them where they got it from,
that would be very helpful.>>I think there are
some technologies that may adapt a
clinic test, right, and maybe a sample preparation
that has to be modified and then allow it
to work in a CIDT. I know there are some
next generation sequencing technology, well the technology
itself of NGS is used in say in some food screening. I know they even use it in like
looking for male and female eggs in selecting, you know which ones go forward
and which ones don’t. I think they’re also using those in detecting potential
pathogens.>>One area where this
technology has been used for a while has been
in food fraud. So for instance, fish,
the fist that you buy at the supermarket may or may
not be what it says it is. And the FDA has been using
technology of the sort to confirm the identification
of seafood.>>Thank you so much for joining
us and I’d like to take a moment to thank our speakers. Thank you very much. [ Applause ] And please join us next month
for Public Health Grand Rounds.

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