Articles, Blog

PACCARB 13th Public Mtg, Day 1 Pt 2: PANEL 1: Emerging Resistant Fungi – Candida auris

August 13, 2019

>>Martin Blaser: So, good morning. We are now going to go back into session,
and we’re going start with our panel on emerging resistant fungi, a focus on candida auris. I’m going to turn over the microphone to our
moderator, Dr. Angie Caliendo. Dr. Caliendo.>>Angela Caliendo: Thank you. Hello, everybody. As you know, candida, particularly candida
auris, has been in the news quite a bit in the United States. Our panel today will give the council and
all of you a comprehensive perspective on what the U.S. government is doing about it,
a global perspective on managing an outbreak, and then its etiology in agriculture. For all the panelists we have a timer here
that will indicate when your time is up to allow for everyone’s turn and a great discussion
to follow. So, thank you for abiding by the time limitations. I will introduce our first two speakers who
are kind of tag-teaming it. Dr. Tom Chiller, who is the chief of the mycotic
division’s branch at the CDC, and Dr. Dennis Dixon, the chief of bacteriology and mycology
branch at the NIAID at the NIH. So, gentlemen take it away.>>Tom Chiller: Great, thank you. Thanks, Dr. King and Dr. Blaser, Dr. Musmar,
and the Council for inviting us to talk about fungi. We’re excited to be here. I’m going to lead off and give, hopefully,
a little bit of background on candida auris and its emerging situation, both in the U.S.
and globally and then the others will add in and give other aspects. So, next slide. As mentioned, you might have seen or heard
about this organism recently in the news. This was a New York Times story, front page
of the Sunday New York Times, which, for those of us who work in the fungal world, that was
pretty remarkable. We don’t often see this kind of press for
a fungus. Next slide. This is, I think, normally what we think about
when we think about fungi. [laughter] Certainly fungi, athlete’s foot, and toenail
fungus, are probably some of the most common infections on earth. We know that there are 5 to 6 million different
species of fungi but only a couple hundred cause disease. So, there’s a lot of potential for emergence. Switching to candida, candida is a very common
bloodstream infection. In fact, in some of the work we’ve done over
the past years it is the most common bloodstream infections in many of our ICUs and our hospitals. So, candida is a common organism. We deal with it in the ICU, and we’re treating
it. Next slide. This particular type of candida, candida auris,
really has emerged in the past decade. It was first reported in Japan, although there
were earlier isolates now identified both in Japan and Korea in the late ’90s. It then spread pretty rapidly or emerged pretty
rapidly in multiple locations, and in 2016 we actually put out an alert in the United
States to see whether there were issues and problems with this infection in the U.S. I
will talk a little bit more about that in a moment. Next slide. I think one of the things I wanted to start
out with is why are we concerned about candida auris, and one of the reasons is because it’s
highly drug resistant, more resistant than any fungi we have ever seen in human medicine. It likes to be resistant. It’s very happy bathing in antifungals. It thrives in environments that are rich in
both antifungals and antibacterials, and it’s hard to kill both in and on humans and in
the environment as a disinfectant. We’ve had a hard time getting rid of it. Patients become colonized with this and are
colonized for long periods of time, and that colonization, we now know, can lead to invasive
infections. So, colonization is a problem. And, finally, what’s different about this
organism is that it spreads readily in healthcare settings, and we’ll talk about that in a moment. Next slide. So, I like to say that sort of a paradigm
shift for those of us in medicine for candida infections, because this is really a yeast
that is acting like a bacteria. So, I want you all to think about this in
CARB, as combatting antimicrobial bacterial, that there are fungi that are acting just
like those bacteria that we’re very concerned about. In this particular case, this fungus is where
resistance is the norm. It thrives on skin. It contaminates patient rooms, and it’s transmitted
in healthcare settings, which is not typical for candida at all. Next Slide. So far, now, we’re up to, I think, 35 countries
where this has been reported in, and this is just a map to show you that some of these
countries it’s quite endemic, and in other countries there are just a case or two that
have been reported. Next slide. The other thing that’s fascinating about this
organism, as we’ve been studying it now over the past five years, is that if you do hold
genome sequencing, so if you sequence the entire genome of these organisms, you see
an interesting phylogeographic structure emerge. There are four, of course now actually five,
clades. We just recently reported a new clade. But four clades, for the most part, that make
up the majority of the strains across the world. These four different clades have really emerged
simultaneously over the past decade. We can’t exactly describe a common origin
for them, and so they’ve really come out in these three continents at a very similar time. Next slide. Just to give you some examples of some of
the things that worry us about candida auris as we began to learn more about it early on. This was a study out of India in 27 ICUs. You can see that 19 of them were already heavily
colonized and had issues with candida auris infections, many of them with a 5 percent
prevalence of all their candidemia was candida auris but some of them as high as 50 percent. Half of all candidemias were caused by candida
auris. Next slide. Here’s an example of some recent data from
colleagues in South Africa. You can see the blue are public hospitals. The orange are private hospitals. These are, again, cases of candidemia, so
bloodstream infection. I’m sorry, the circle’s a little low, but
it’s circling candida auris, and it’s showing you that it’s the second most common cause
of candidemia in private hospitals in South Africa and the fifth in public but moving
up. Next slide. Here’s an outbreak that was recently described
in Spain, and you can see here that before 2016 in this particular hospital, the candidemia,
so the bloodstream infections caused by candida, were made up of what we think of as typical
candida species. There was this shift, beginning with this
outbreak, to the point where, after this, in the next 150 candidemia, or bloodstream
infections, almost half of them were candida auris. So, we know that this organism, when it sets
up shop, when it gets into a facility, it can really take off. Next slide. We put out our alert, as I mentioned, in 2016
here in the U.S. We identified in an isolate collection, actually
one isolate, that had gone back to 2013. It was misidentified in that particular case
series, and we were able to go back and identify it. And subsequently, next slide, where we are
today, or at least as of March, you see we’re at about 650 clinical cases, another 1,200
or so screening cases, or patients that are colonized, and in about 12 states. But the majority are really in three distinct
locations, New York, New Jersey, and the Chicago area of Illinois. Next slide. And you can see here, this illustrated on
a map of the U.S. where the current clinical cases are occurring. Next slide. Again, this strong phylogeographic structure
relates perfectly to what we’re seeing in the United States. You can see that every single strain in the
U.S. relates to one of those four clades that I talked about. And you can see here that we have actually
all four clades in the U.S., again, some of which are circulating now more than others. Next slide. I think it’s important to think about the
patient that this affects. This really affects the sickest of the sick,
people who have had tracheostomies and are on ventilators colonized with other multidrug-resistant
organisms, so these bugs tend to travel in packs. It makes some sense because most patients
have recently received antibacterials and/or antifungals, and so they’re at risk for these
highly resistant organisms. The mortality in these patients is around
50 percent at 90 days, but these are very sick people. So, I don’t want to put attributable mortality
that high to candida auris at all. They’re sick people. They are not doing well, but candida auris
is often there, you know, when they are this sick. I’m not worried about candida auris, as of
yet, as a threat to the general public or in healthy individuals. Again, this is really something where patients
who are very sick or are very medically experience tend to get. Hearing that story from Dr. Patterson just
a couple of hours ago is very telling, and I saw that he had two candida species, you
know, in his fluid along with that Acinetobacter. Thankfully those were highly susceptible. Phage therapy, unfortunately, doesn’t work
for candida yet, and, you know, I’m glad that he wasn’t dealing with a resistant candida
like candida auris. But that would be a patient that I would be
worried about. Next slide. Again, the majority of cases don’t have direct
contact with travel or with healthcare abroad, but we certainly have traced a bunch our cases
to that. So, we have traced them back to a country
where they received care, a country that’s highly endemic for candida auris, and then
they’ve come to the United States. But we know that all of the original cases
originated from abroad because of this strong phylogeographic structure that I outlined
before. Next slide. I think it’s really important to note that
one of the things that we found, not just in candida auris but prior dealing with other
MDROs, is that healthcare in the United States, as we all know, has shifted over the past
several decades to a lot of long-term acute care. In those long-term acute care facilities,
we’re finding very high levels of candida auris, and you can see here ventilated skilled
nursing facilities, so these are facilities where patients are ventilated and being taken
care of for longer periods of time, have a much higher colonization rate of candida auris
than do non-ventilated skilled nursing facilities. This was a study that we did looking at many
facilities in New York City. Next slide. And here’s an example of what I’m talking
about. This is a floor of a ventilated skilled nursing
facility, or VSNF as we call them, that we looked at patients for colonization of candida
auris, and you can see here there was one patient that was positive. We screened quite a few of them, as you can
see by the purple hollow circles, and then the one solid circle is the candida-auris-positive
patient. Next slide. About nine months later we went back, and
this is the kind of phenomena that we see in these facilities, where you had one positive
patient and now almost half of the patients in this facility are colonized with this organism. Next slide. And you also see, as I mentioned, they often
travel with other MDROs, and you can see here examples of other MDRO bacteria that are in
these patients as well. So, we are very concerned about these types
of facilities being reservoirs for many of these organism, as we know that these patients
tend to go in and out of facilities, including in and out of tertiary care hospitals from
these facilities because, although they are not sick in tertiary care facilities, these
are ventilated patients. They are very medically exposed and experienced
and often need more acute care. Next slide. As I mentioned, patients are colonized with
this organism, which is sort of atypical for candida. We think of candida more as a commensal in
our gut, not as much of a commensal on our skin, although it can be found. Interestingly, candida auris can persist for
months. We have patients that have been colonized
for over a year now, and we don’t have good decolonization strategies. That’s something that we really need to work
on. As I already mentioned, colonization now,
we know, can lead to invasive disease, and we also know colonization is what causes transmission
to others, probably through contaminated surfaces, which I’ll show you on the next slide please. And you can see we’ve found candida auris
everywhere in patient rooms, especially on beds and bedrails. There’s a direct correlation with the amount
of candida on a patient’s skin and the amount of candida on the bedrail. I think bedrails are things we, as physicians,
don’t often realize we’re touching a lot, but we lean on beds. We touch bedrails. So, another potential real interesting source
for how some of these organisms can be transmitted. The other thing is that the normal disinfectants,
quaternary ammoniums, do not kill this organism. They tend to just spread it around, and so
you really have to use higher level. Right now we are recommending C. diff sporicidal
agents and/or 10 percent bleach. You obviously can’t bleach equipment in hospitals,
so we’re struggling a bit with this infection strategies. Next slide. Many of you know about the Antimicrobial Resistance
Laboratory Network, seven regional labs. Candida auris can now be identified and susceptibility
can be done in all of these labs. That was a great recent addition to the ARLN
network. Next slide. Just to remind us, there are three classes
of antifungals that really treat invasive disease. That’s it, azoles, polyenes, and echinocandins. That’s all we have. Next slide. If you look at candida auris, almost all of
it is resistant to azole, a third is resistant to amphotericin b, and, thankfully, still
only a small percentage is resistant to echinocandins. We’ve isolated two pan-resistant isolates
in 2019. Next slide. And you can see that pan resistance, in this
case, is all three classes. They were identified in New York. The cases were unrelated. They developed resistance to the echinocandin
while on treatment. So, they were already resistant to two drugs,
and they developed echinocandin resistance. No transmission of resistance was seen, and
pan resistance has been reported in a number of other countries. Next slide. So, it’s a new bug with old tricks. Some of the same things that we see with bacteria,
as I mentioned. Next slide. It comes back to the same principles of infection
control, hand hygiene, personal protective equipment, and environmental cleaning. Next slide. So, what keeps me up at night is that I’m
worried that this organism, this candida species is going to leap ahead of the other candida
species and cause major infections. So, thanks for your time.>>Angela Caliendo: Great. Thank you, Dr. Chiller. Dr. Dixon.>>Dennis Dixon: Thank you. So I’m pleased to summarize the NIH, NIAID
activities on candida auris, and I think everybody would imagine the NIAID is the lead institute
for microbes, including candida, at the NIH. I’m going to be telling you about primarily
the extramural community research, that is the grants, contracts, and other resources
we make available to people external to the NIH around the world, and I have a little
bit of the time I’ve given to the intramural efforts, which would be that from the NIH
scientists on campus who are studying this fungus too. So, next, please. We take a general approach at the NIAID of
conceptualizing the research we do into the categories of basic, translational, and clinical,
and, in all instances, we keep our eye toward the goal of diagnosis, prevention, and treatment. We’ve really been pushing our research from
the foundational basic through to product development, over the last 20 years in particular. So, on the left-hand side of the slide, that
thing you see there is the first human vaccine for candida, and it came out of a basic research
activity long before candida auris was known to exist. That candida vaccine just happens to cross
protect in animal models against Staphylococcus aureus and against candida albicans. So, it shows a good example of fundamental
research benefitting entities that aren’t identified until much later in time. It’s not always linear in the development
course. So, I’m going to say quite a bit at the end
about this vaccine. It was developed by Dr. John E. Edwards, Jack
Edwards, and is nibbling at the edges of a larger phase clinical trial, but I’ll get
back to that. Next slide, please. So, just going through the portfolio of research
in NIAID, and most of it is in my branch. We have a team of people working on the fungi. Candida represents about a third of the fungal
portfolio along with Cryptococcus and aspergillus. So, those are like the big three, and they’re
roughly about the same. I think you could also buy the argument that
most of the candida albicans research is going to be informing candida auris research because
of the similarity of the species within the genus at the broad level. Yet, we have seen a relatively rapid uptake
in our portfolio of candida auris creeping into other grants. So, a number of them now have one or more
aims on candida auris. It’s caught people’s attention. They’re wanting to look at it. That’s a good thing, so they are building
it into their programs. And there are some dedicated candida auris
only grants as well. So, I’ve been really impressed as our team
was pulling this together on how much has happened so quickly in response to this public
health need. And you can see here the types of basic research,
or the standard for most microbes in the division, genetic tools, studying the evolution of drug
resistance, looking at pathways that might be exploited for interventions, and looking
at host-pathogen interactions. So, next, please. Translational research, in terms of therapeutics,
and those are all housed by convenience in one portfolio, and in my group, just as a
matter of interest, we actually separate out the programs by basic research, translational
research, and then into clinical. Because we really have that focus on moving
things forward to public health gain. And, so the one portfolio analysis shows that
60 percent of the antifungals in that are dedicated to candida species. And with candida auris, specifically, the
types of projects are library screening, lead optimization, pre-IND studies, and host-based
immune therapy. Diagnostics are also part of what we do, and
there are two open-program announcements, which means we have special interests and
are willing to give special consideration to special public health needs. Fungal diagnostics are listed explicitly. These two numbered ones you see here are open
until 2022 through the regular review cycles, and the first batch is under review. And we will hopefully have some things addressing
the relevant fungi there. Next, please. I want to say a little bit about what our
preclinical service is. It’s something that I don’t think is 100 percent
understood in the community but should be for anybody who is moving toward product development. About — nearly 20 years ago, our division
recognized that you can’t splice everything together by different grant mechanisms and
small-business grants. You sometimes need rapid revelation of data
to move something forward. So, for product development, our division
is providing a suite of preclinical services. Just think of the analogy of a big drug company
that has made multiple different drugs, and they have these different components in the
company, all the way from in vitro screening through to animal models, through to medicinal
chemistry, and structure function modifications, toxicity testing, and scale-up manufacturing,
formulation, and so forth. We provide all of those through a suite of
contracts that are funded and ready to go for an entity that comes forward with a bona
fide product development need. So, it could be a small company, or it could
be an academic researcher who’s seriously moving toward the steps of product development,
often in partnership. So, these are advertised on our website. They are advertised here today for contacting
program staff with a phone call or an email and then being given the forms to fill out. Then there is a selection process where we
look at available services and the competitive need and give access, for free, to have the
services completed. We’re impressed at the number of times our
fingerprints are on products that are in the clinical pipeline throughout the antimicrobial
arena. I have been very pleased at the traction we
have gotten with the fungal community and companies, and, so, pushed a number of things
through these services and on toward later development. So, most of the clinical stage antifungals
that you see here targeting candida species, have utilized NIAID preclinical services. So, if you think of a project in Phase I,
II, or III clinical trial, chances are it got some of our services or some NIH funding
to get to that point. And you can see that in 2017, we provided
a specific C. auris mouse model. Four different products have been through
that mouse model, and fifteen different products have been evaluated in vitro for susceptibility
to — in candida auris. Next, please. For clinical services, we have two different
types of networks. One is Phase I clinical trial units. The other is vaccine and treatment evaluation
units. Both of which can do Phase I clinical trials,
and there are now two Phase I clinical trials about to go live in So, you can be watching that and look to see
what the companies are that are doing first-in-human Phase I trials with products that have activity
against candida auris. Next. Intramural support, so if you look in the
lower right-hand corner, the largest structures there are the NIH Clinical Center, and enormous
building. Seeing the aerial photograph here helps me
to feel better about getting lost so many times, and I’m trying to find the new part
of that mammoth structure. Our researchers there were part of the report
of the first seven cases in the United States, and the NIH received a patient colonized with
C. auris that went on to become infected and was listed in that report published by MMW
and R [sic], I believe. Tom? And then there’s also laboratory research
taking place with several of our researchers on the campus, and they’re interested in looking
at the skin biology and the immunity. Why is it that candida is so effective in
persisting on the skin? What does that have to do with host-pathogen
interaction? What does it have to do with the microbiome? So, there’s looking at the bacterial-fungal
microbiome and looking at strategies for decolonization in mice. Next. Now, spend a little time on this complex model
to show the multiple different ways that a basic R01 grant, that’s the bread-and-butter,
competitive, peer-reviewed grant, made its way all the way through to Phase I, Phase
II clinical trials as a vaccine candidate. So, this is back to Jack Edward’s candida
project, and I can recall talking with him back in the early ’90s at our very first mycology
workshop series. We did five workshops to try and enrich the
field, bring people in, bring model systems people in to bring the fungi up to the contemporary
standards. And we’re talking about the potential for
vaccines for this group of microbes. So, Jack continued to pursue antigens that
were functional in protection in candida albicans, and, after a decade of research, got to zoom
in on attachment antigens, identified the first candidates that worked effectively in
mice to prevent against candida albicans and also Staphylococcus aureus challenge. And we worked with him — so the green is
all NIH funding, and so you can see that the basic research and then some of the preclinical
services in scale-up manufacturing, GMP manufacturing, and putting in vials, the thing you saw in
the plastic block, commemorating the first one that made it to the clinic, supported
by us, along with stability testing, tox testing, assay development. A lot of different things to help a researcher
get from the bench into clinical utility in which Phase I and Phase IIb trials were done
with other’s money, and so you can see that the company did some of these on their own. DOD is interested in the Staphylococcus portion
and is funding some early work with Staphylococcus, and the company in the far-right circle funded
a Phase IIb clinical trial on recurrent vulvovaginal candidiasis. Why? That’s an important disease, and for people
who have it once and get it again, they’re likely to get it again, and again, and again. So, that’s a very great disadvantage to the
patient and advantage to trying to target people because you know who’s coming back
at some point. And you can vaccinate to look for prevention
of future recurrences. When he did that in a sample size of somewhere
between 1 and 200 patients, he found an 80 percent efficacy in the early phases following
vaccination for women under 40 for preventing recurrence. So, that’s where most of the infections occur. I think that some of the comments in the paper
were, “Well, it wasn’t that great for people over 40,” but most of the people who have
this problem are under 40. So, it’s very encouraging. I think he could move that forward there. It’s a matter of company finances, and he
would like to try that in candida auris patients. And we are talking with him about helping
to manufacture some more for that major endeavor of, “How are you going to find the next group
of people who gets this? How many do you have to vaccinate? Do you have enough material to vaccinate all
of them in order to see a signal?” But it’s something that’s under discussion,
and it’s just an example of preparedness and the reason we do this kind of work. Okay, do we have anything else? I think I may be at the end. Yes. So, I’ll leave you on that high note and give
back my three minutes to the rest of the panel.>>Angela Caliendo: [laughs] Thank you, Dr.
Dixon, and just to remind everybody, the panel will have questions after everybody has had
a chance to speak. So, our next speaker is Dr. Derrick Crook. He’s a professor of microbiology at the University
of Oxford’s Nuffield Department of Medicine, and he’s also an infectious disease physician
at Oxford University Hospital. So, welcome, Dr. Crook.>>Derrick Crook: Thank you very much, Angela. I’m really grateful to be here and be speaking
to you. It’s particularly apposite because this is
up Route 29 from UVA where I spent three years doing a general internal medicine residency. And they used to say, “You’ll be back now,”
so I suppose that gives meaning to that statement. It made no sense to me at the time. I’m going to walk you through two observations;
one is in a hospital, which is the hospital that I work in, and the other is the national
English observations around candida auris. I have to say that in this context it is not
as alarming as we might have thought it would have been when Cliff McDonald from the CDC
phoned me up and said, “You’ve got a big international problem with candida auris,” and it seemed
to be a huge defecting situation, and I hope I can convince you in a particular setting
that that might not necessarily be the case. There are two settings; one is the hospital
and one is nationally. Now if I could have the next slide please. So I work in both settings although I’ve just
stood down from Public Health England. But at the time candida auris emerged, I was
the director of the National Infection Service with a mandate to reorganize Public Health
England, which is sort of quite a difficult job to say the least. And I happened to maintain a research interest
at the University of Oxford and the Oxford teaching hospitals. Now the Oxford healthcare system is, as many
of you know, is run by the NHS and delivers health for the whole health economy. Be it secondary or primary care. And it provides acute care for somewhere between
500,000 and 600,000 people and it offers specialist services for somewhere around about a 10th
of England. Public Health England provides public health
services for 55 million people of the 65 million in the United Kingdom and it is dedicated
to controlling communicable disease and protecting the public from infectious disease. Just a very simple mandate. Next slide please. The context of the outbreak in Oxford happened
in a small neuro-surgical intensive care unit which had three isolation bays and 13 beds
on an open unit. So that’s quite helpful to keep in mind. Furthermore, this unit treats about 650 admissions
a year, so not huge, but enough. And what’s interesting about this, Cliff McDonald
called me a month before we had our first case because I said to him, “I’ve never heard
of this before.” And then a month later we had a case in this
unit. Next slide please. This figure is not projecting perfectly, but
sufficiently well, is in the histogram that two epochs; one retrospective and one prospective. Retrospective is in blue and prospective is
in an ochre yellow color, and what’s crucial here is the period in blue, we didn’t have
a means of identifying candida auris. And come 2016, the ability to do so using
mass spectroscopy became available and we identified a case. And then we looked back at all the invasive
isolates we had and we recognized that there’d been the presence of candida auris in the
unit for almost a year-and-a-half, which in its own right was interesting and we had those
isolates preserved. And then working prospectively, we were able
to design a sampling frame that focused on the assets that we had available to us at
the University of Oxford at that time. We had pretty substantial epidemiological
skills in the group that I work with. We had granular electronic data for the entire
patient population using an electronic using an electronic patient record system which
the Cerner system that comes out of the United States. And we had pretty sophisticated whole-genome
sequencing capability that meant that we could generate a reference sequence, which is important
for determining the biology of an organism within a week. So we were then basically kitted out to undertake
a very detailed study, which I checked with Cliff McDonald, who is a good friend of mine,
to make sure that we had designed it as well as we could in the circumstances. We in total identified 73 cases, 70 cases
existed on the neuro-intensive care unit, and these were all of the one clade that emanated
from South Africa. It’s funny because I’ve got a South African
accent if you could recognize, but I had nothing to do with this. [laughter] There were three that had infected CNS devices;
one with an orthopedic pin-site infection and five candidemias. And curiously, given the set of interventions
that I’m going to come to in a moment and is represented on the figure, which is temperature
probes removed, we had no further cases from October 2017, and we haven’t had any cases
since. Next slide please. Okay, this is a simple representation of the
kind of infection control measures that we took. I’ve divided it up into four categories; sort
of operational management, detection of and isolation of cases to minimize contact, which
was necessary on the kind of unit that we had, general control of environment, and sort
of other measures. There’s nothing clever here. I think I must emphasize, this is bog standard
infection control techniques. I’m not going to read through them all, but
they’re very, very straightforward. Just to follow on from a point that was made
earlier by Dr. Keller is that the drug that we used on the unit from identifying candida
auris’ presence was Micafungin, which we knew organisms were susceptible to, whereas to
azoles that were uniformly resistant. Next slide please. We were able to do a fairly careful case control
investigation using a multi-variable logistic regression model to a whole range of variables,
but out of the multi-variate analysis, only two factors were strongly associated with
colonization by candida auris. And the most prominent was the use of axillary
temperature monitoring. That was simple, straightforward, epidemiological-type
investigation. There was a statistically significant effective
use of azoles; Fluconazole particularly, but only a small number of cases received it. So this was not particularly strong statistical
evidence, but is intuitive and plausible. Next slide please. What was clear, of the items that were reused
on the unit, all were associated with candida auris being isolated. So hoists for wearing patients were, bear-huggers
were, and air oximeters were. But in those instances we only found one of
many devices positive. These temperature probes we found a large
number positive. So our view was that reused devices were a
major reservoir and source for ongoing transmission. Next slide please. We were able to use whole genome sequencing
to contextualize the outbreak and we had a number of sampling frames here. One is the individual isolates; then we — for
individual cases, we were able to isolate multiple organisms, and therefore, for a given
individual, you might have five or six isolates to sequence. We had a bioinformatics methodological way
to segregate out different lineages from the same sequencing run, which harks back to work
that we did on clostridium difficile, so that you could de-convolute if there was more than
one strain colonizing somebody. And we were able to do the same for the temperature
probes. And I’m not going to go through the detail
of the slide, but the bottom line is there were mixed populations everywhere you looked,
in the patients and in the temperature probes, which would then intuitively make you wonder
where the temperature probes were picking up multiple strains from multiple patients
and inoculating them onto the next patient. We don’t have proof of that, but it’s the
most parsimonious conclusion you could draw from the data, and was consistent with the
elimination of acquisition with the withdrawal of the temperature probes. And it bears on the statements made earlier
that environmental sources are incredibly important in the ongoing acquisition of candida
auris. And it may reflect greatly on these community
ventilator units that one has where I suspect there will be a whole set of environmental
exposures that are going on in the background of those patients in that particular setting. Next slide please. So the duration of colonization we could study
fairly deliberately. And this is an A-panel on the left-hand side
of the slide and depending on the criterion you used, whether you define clearance as
two negative screens or three negative screens, the median period of colonization was 60 days
or 82 days respectively, so somewhere around about two to three months of median carriage
for candida auris. It means patients probably lost it, which
was helpful. Then in panel B what is very apparent, there
is a large number of patients that were indistinguishable from patients that acquired candida auris,
who did not acquire it. So it’s not inevitable that everybody in a
unit such as this neuro-intensive care unit would acquire candida auris. It was somewhere between a quarter and a fifth. So that was a little bit reassuring, not entirely,
but a little bit reassuring. And then in panel C what is evident there,
there is two blue-colored sections of the histogram bars. Those were individuals that were in the associated
neurology ward and neurosurgery ward, that had acquired candida auris without going on
to the intensive care unit. Intuitively you would believe they acquired
from patients who had been discharged from the neuro-intensive care unit, but crucially
the ward never developed autonomous acquisition of candida auris, and there were no special
interventions in that ward. So it was quantitatively rarely different
from the intensive care unit and I think this has been mentioned already. It is very ill patents, debilitated patients
who are likely susceptible to acquisition. Next slide please. Mortality was no different between carried
and non-carriers. This would have been a shocking result if
it was. Quite frankly, it is not a very discerning
measure of mortality; but nonetheless for what it’s worth, there was no difference. Next slide please. So nationally in England what is really interesting
is that we’ve gone through a process of typing up observation, surveillance, and issuing
guidance in the two boxes that you can see where two different categories of advice and
guidance. The first on the left was general advice,
and the one on the right was to community care homes, which would bear on what’s been
going on in the United States. And the major peak in incidents was the Oxford
outbreak alongside two other outbreaks. And since then we’ve been observing cases
at a very low rate, and furthermore, the majority of those are imported into the country. So the evidence for ongoing, autonomous outbreak
in the U.K. is diminishing. Last slide. I’ve got a lot of people to thank for all
the huge contribution they made to this work. Thank you very much.>>Angela Caliendo: Thank you, thank you very
much Dr. Crook. Okay, so our next speaker is going to be Dr.
Wayne Jurick. He is the lead scientist and research plant
pathologist at the USDA in their quality laboratory — in their food quality laboratory.>>Wayne Jurick: Thank you for the introduction. It’s a sheer pleasure to be here today and
to represent the U.S. Department of Agriculture, Agriculture Research Service. Next slide please. So a little bit about myself, I got my PhD
at the University of Florida, 2006, Department of Plant Pathology. I found myself at the USDA-ARS in Beltsville,
Maryland working for ARS which is, if people don’t know, the in-house research arm of the
USDA. So I’m currently the lead researcher on multiple
projects dealing with anti-microbial resistance, all dealing with fungal plant pathogens of
fruit. Okay, so I work on mycotoxigenic molds primarily
penicillium botrytis so those are my favorite group of fungi. All right, next slide please. So, a little bit about azoles because that’s
what you guys came to learn about. First thing is azoles are also known as the
sterol biosynthesis inhibitors because that’s how they work. This is the only fungicide class that is used
in agriculture and medicine to my knowledge, which is an interesting factoid. Third point here is if are people are familiar
with the FRAC grouping? FRAC stands for Fungicide Resistance Action
Committee. And these azole fungicides are of medium risk
for developing resistance in these fungi, okay. And if you read the literature, the greatest
usage of these azole fungicides that control plant pathogens is in the E.U. and the United
States on a per acre basis. Next slide please. So what are azoles used for? They’re used in crop protection. So if you haven’t thought about it, people
like to eat high quality fruits and vegetables, and that comes at the point of using azoles
control fungal plant pathogens not only in the field, but on seeds and in stored fruit,
which is my area of expertise. The azole resistance in agriculture, so when
you have a failure of an azole fungicide to control said disease, it manifests in crop
loss, reduced food quality, lower grower profits, and increased chemical applications. So to give you some ideas of what azoles are
used specific examples include, tetraconazole that’s used to control Cercospora leaf spot
in sugar beet. Ipconazole controls a disease called smut,
believe it or not, of barley. And lastly one example difenoconazole in a
mixture with fludioxonil for blue mold, which is caused by my favorite fungus, penicillium
species for apple and pear. And notice that those are all the active ingredients
for specific chemistries that are marketed under different trade names by different agricultural
chemical manufacturers. Next slide please. So to give you an example of what some of
these diseases look like, at top left, I like to go root through bins in storage facilities
and that’s a blue mold of apple on the left caused by penicillium species. You can see the very characteristic circular
lesion with the bluish-green colored spores that are persisting on the surface of that
lesion. If you get up real close to them, it actually
has a nice musty smell, which is from the geosmin volatile that the fungus produces. Top right is Cercospora leaf spot of sugar
beet. That’s caused by the fungus Cercospora beticola,
a very, very destructive fungus for sugar beet production in the North Dakota, Minnesota
area. In the bottom center is that smut fungus Ustilago
hordei on barley. Okay so it makes this really ugly looking
brownish-colored distortion of the seed head there on your small grain crop. Next slide please. So now that we’ve gone through what azoles
are; what they’re used for, and how are how are these things applied? So I’m pretty sure you probably have some
ideas on this, but the pre-harvest meaning in the field, is accomplished by air blast
sprayers, backpack, and airplanes. So they still crop dust these days. Number two, post-harvest in storage to protect
your stored fruits and vegetables, that’s going to be as drenches, line sprays, or fogs. Next slide please. So a collage here of application technologies
for not only azoles but other mode of action fungicides. We’ll start with the top left, that’s a crop
dusting plane, aerial application of fungicides. Bottom left is more my venue, that is a line
spray of apples that are being sorted and graded just before packaging. Middle is a typical boom sprayer on what appears
to be a John Deere tractor spraying in large mass quantities of the fungicides on these
booms. My top right is backpack sprayers. So this is typically used in small-plot applications
for, you know, soybeans, corn, any crops that some person applicator can walk through. Bottom right is out in the Pacific Northwest
and you’ll notice the background. That is a typical truck-drencher. So they put the fungicides in with the water
and then they actually pull the truck up to the drencher with the apples, or pears, or
peaches, whatever they are drenching and they let it rip. And you can see it’s about ready to dump a
bunch of fungicides onto the fruit, hence called a drench. Next slide please. So trying to make sort of a full-circle here,
how did these azole fungicides work? So my biochemistry brain it makes sense that
these are sterol biosynthesis inhibiters. So what does that mean specifically? So they target ergosterol biosynthesis in
fungi via CYP51 inhibition and ergosterol is kind of a kin to your cholesterol in human
body, but ergosterol is essential for fungal cell membrane permeability and fluidity. So without ergosterol the fungus is, as they
say, dead. And the CYP51 is the target of this fungicide. And the CYP51 is a highly conserved enzyme
in biochemical pathways. You have them in your liver, fungi have them,
mammals have them, but this is the target of our compound. Next slide. So just to summarize here, I don’t want to
bog you down too much, but if you start with a biochemical pathway here from A. fumigatus
here on the left, starts with one of the squalene which is a polyketide compound; going through
several biochemical steps leading to the ergosterol, the steroid nucleus here on the bottom right. And if you remember your structures that that
pretty much looks like a cholesterol molecule with a few different modifications. Back to the top part of the pathway here,
ERG11, which is encoded by CYP51, has multiple isoforms in fungi. And aspergillus fumigatus there’s two forms;
CYP51-A1 and CYP51-B. This is your target. So basically the azole fungicide binds to
the CYP51 enzyme and inhibits the ergosterol biosynthetic pathway here, it’s pretty straightforward. In penicillium, we are learning that expansum,
we’re learning that there are three isoforms here and that the A isoform is one that’s
most responsive to difenoconazole treatment. And I just want to mention, so nobody’s going
to nail me for stealing this, but this is from a figure one from Alcazar-Fuoli, Frontiers
in Micro, pretty nice paper. Next slide please. So azole resistance mechanisms again harken
back to mutations in that CYP51 gene target. Overexpression of the CYP51 target; efflux,
basically fungi, and I think bacteria do too, have an incredible capacity to pump out fungicides
and toxins that they don’t like. And this is an energy dependent process that
requires a pump or pumps. And then finally detoxification of the target
which I don’t see a whole lot of evidence in for fungi, but this is becoming more and
more in light of newer mechanisms. But the traditional mechanisms again are the
top three bullets here. Next slide please. Okay, so a diagrammatic representation of
what I just told you. Sort of bring it home here, I lifted from
the Crop Protection magazine. It’s noted on the bottom left. And what you have here are these red triangles
and this is your fungicide. This is a fungal cell, pretty creative, and
as you see the fungicide is able to permeate the cell membrane and get in. And you have the fungicide hanging out here
and first mechanism is a mutated target site. So this Pac-Man looking thing here actually
cannot bind any longer to — cannot bind to this azole fungicide and therefore you have
resistance. Step two can be overexpressed of the target
site. So you just have a lot more of the enzyme
firing off making the end product. And you don’t have complete inhibition of
this process. Mechanism number three, again I talked about
the pumps here. You have the triangle being pushed out by
the two blue blobs here and it basically just moving this compound out, which in our case
is azole, azole fungicide. And topic number four would be detoxification
which would be either conjugation or simple mechanistic detoxification of the target so
that it’s no longer toxic. So basically this is a nice little overview
of the azole resistant mechanisms and filamentous fungi. Next slide please. Okay, so with that I’d like to end my talk. I’d like to thank Jomana Musmar for the invitation
to speak, Dr. Tim Widmer, who is my national program leader. And then also my lab which is a very small,
but effective unit. Dr. Franz Lichtner, a post-doc at my lab right
now, my right-hand person, Verneta Gaskins who is my support scientist, and my student
researcher from University of Maryland who is a math major, Otilia Macarisin. And with that I’d like to end my presentation
and yield the four minutes to my esteemed colleague, Dr. Cox.>>Angela Caliendo: Okay, thank you very much. So our last speaker in this group is Dr. Kerik
Cox. He’s a tree, fruit, and berry research, extension,
and teaching at Cornell University’s New York State agricultural experiment station. So welcome Dr. Cox.>>Kerik Cox: Yes, thank you for the nice
introduction. I’m going to tell a couple of stories about
the triazole and multi-fungicide resistance that we’re working with the agricultural pathosystems. Go ahead and give me the next slide. This is where I work at Cornell AgriTech. It’s a stakeholder driven specialty crop research
facility and we focus on both field laboratories, digital laboratories, and molecular laboratories
to attempt to achieve the transition from basic research to practice for growers. And in particular today I’ll talk a lot about
my field lab-type research. And it focuses on two major components; antibiotic
resistance and bacterial pathogens such as on the left and the one that we do the most
research on and I’m happy to talk about today is fungicide resistance research. And go ahead and give me the next slide. The perennial fruit crops make a nice model
system for fungicide resistance research. The hosts are long-lived, sometimes as much
as 30 years always for more than five unless something kills them. And the management periods often last nine
to 12 months except when the plants go dormant, which allows a lot of time for repeat treatments
and repeat instances of disease. Many of the pathogens that we study have numerous
infection cycles, which means the hosts, or my patients, are constantly exposed to the
pathogens in a continual system, where they, you know, they can’t leave the environment. And it allows for a lot of repeat treatments
and the look at how treatment influences selection for fungicide resistance. The other thing that’s kind of nice is that
although these pathogens that we’ll show below, can move around to a limited extent. They’re often stuck within their localized
populations of a field, or a block, or even a couple of neighboring blocks. And it’s — and since the host doesn’t move
as well, it’s little influx possibility of new members, allowing you to really see what
happens to a population over treatments. And some of the bigger systems that we work
with include things like cherry leaf spot which will defoliate cherry trees, brown rot
of stone fruit which is a very aggressive, I guess, R-type pathogen that will rot the
fruit off the trees long before you see them. And even in the tropical arena, the black
sigatoka banana is sort of one of the landmark pathosystems for multi and fungicide resistance. Next slide. And then the one I principally focus on is
apple scab. And you can see that on the right it really
marks up the fruit making them unfit for fresh market consumption and it will also defoliate
the trees themselves. If you look at the bottom right corner the
leaves will get so many lesions that they’ll fall off. The fruit become malformed and inedible. The problem with it is being a sort of perennial
problem because we’re always planting susceptible cultivars, which is what is favored by both
the consumer and the producer and the breeder. Resistance hasn’t been very popular for eating
unfortunately. Which allows the system to end up accumulating
a lot of fungicide apple applications. More than 10 a year and we have resistance
reported in most fungicide classes. And we have about seven to 10 that we can
rotate between that are fairly different. In the old days, we used to wander around
orchards throughout the state and, you know, grab a couple of islets here and there and
say, oh wow, we found resistance, the whole thing is over. But recently we begin to shift to sort of
a more population-based approach. And we found that if you look at the whole
population within an orchard, the phenotype distributions range from highly susceptible
to highly resistant. And what we’ve started to look at is how the
populations are shifting in terms of the fungicide resistance phenotype to the point where we
get a management practice failure under appropriate use practices. Next slide. And before we go into talking a little bit
more about this practical fungicide resistance, I’ll sort of highlight some of the two phases
that we focus on in the agricultural world. Emergence which is the presence or existence
of a member with a mutation that confers resistance and establishment. And it’s important to note that the fungicides
themselves aren’t inherently mutagenic. So they’re not causing the mutations, they’re
occurring, you know, infrequently. Random mutation, maybe UV light, at a natural
level and the fungicides are selecting. And one of the things that we’ve found in
agriculture is that oftentimes the population size is one of the most important factors. So, the least clean orchards, the dirtiest
orchards, the ones with the bigger numbers of populations, will have a higher percentage
chance of having isolates, or members with these advantageous mutations. And they often go to resistance much more
quickly than a nicely sanitized orchard. And it kind of goes back to some of the earlier
comments that we saw from the CDC highlighting the need for sanitation. And we find that the cleanest places less
likely to have resistance. And go to the next slide, where we begin to
actually look at — looking at practical resistance. And what we end up doing is we’re looking
at distributions of phenotype responses, and we compare them to reference distributions. And like in some of the other — this looks
like the graph is a little distorted, but that’s fine, you get the point — and what
we often do is we use statistical tests to compare one distribution to our sensitive
standard and our resistant standard — where we’ve actually confirmed proper application
practice and level of disease. And so, all of the different populations we
look at are classified by how statistically equivalent they are to one of the two. An example of this, we’ll see on the right
side of slide, is for apple orchards in New York, looking at azoles. One of the more popular ones was myclobutanil. And what I’ve done is I’ve taken 121 of these
population distribution comparisons and sort of crammed it into these little histograms
that you see up top, where it’s the percentage of orchards on the Y axis, and the phenotype
response on average across the bottom. The key thing to note is that that red line
typically will institute a part where an orchard will have a failure if someone were to use
that product. The green line is — represents wild populations
that have never seen an azole fungicide or any others. But you can see for the azole myclobutanil
that the majority of those 121 populations would expect a failure. Interestingly enough, we find that chemistry
is a big player and that the intrinsic binding ability of the different azoles can mask what
one might actually see as a resistance reaction in a population of apples. And so if you look at the difenoconazole,
these are the same isolates in the same orchards, tested at the same type of dose, just that
the material’s much more intrinsically active. And in this particular instance, if growers
were using that, they would never know that there was an insensitivity problem occurring
in most of the orchards. The nice thing about this is they can use
this other azole and completely be successful. Next slide. We’ve also begun to learn that what we believe
as a resistance response in the actual orchard environment is a combination of environment
host and the chemistry. And so these are some of our disease trials
using azoles. One in McIntosh on the left in red, and one
in Cortland — for both hot, dry years, which is not favorable to the fungus; and cool,
wet, which is. And, what you can see is the incidence on
fruit. And it can go up almost up to 100 per cent
fairly quickly for these five azole fungicides. You can see that difenoconazole, I mentioned
earlier on McIntosh, in a hot, dry, year — it can almost be effective. The far right, the captan, mancozeb, is multisite,
and we don’t expect to see resistance to those. That’s why it’s our positive control, and
then the untreated is the negative. But on the McIntosh, which is highly susceptible,
they can all end up failing, giving a resistance reaction in both types of years. But in a cool, wet year, what we perceive
as resistance is even more apparent. On the Cortland, however, you may go on with
the same isolates. These trees are planted next to each other
and share the same population; however, in a hot, dry year, using the right azole chemistry,
you can completely mask the ability. And in that case, it actually can perform
better than a multisite resistance standard. So we’re finding that what we believe is resistant
is not just complete genetics, but it’s an interaction between the host, the environment,
and the chemistry we use. Next slide. And, from that I’ll sort of transition a bit
into cross- and multiple-fungicide resistance, because that’s a problem we’re dealing with. Cross-resistance is resistance to multiple
fungicides that share a same target site — like all the azoles, for example. Multiple is resistance that develops to two
or more unrelated classes, resulting from sequential selection, or some sort of multi
drug resistant mechanism. And some of the ones that we’ve already mentioned
today, like candida, black sigatoka apple scab, and another one I’ll mention briefly
called gray mold of strawberry, particularly suffer from this type of problem. Next slide. What we’ve had here is one of my colleagues
in the eastern United States — two of whom got together and they worked with growers
to do a fungicide resistance management program, and they sent sample test kits out. They received samples from growers over four
years, and made fungicide resistance management recommendations. But unfortunately, what they ended up finding
in the end was that all of the isolates that were coming back after four years — there
was an increased chance, increased probability of finding multiple fungicide resistance. And they became very confused, because, “We
followed all the resistance management practices, and we recommended them. Well, how did this end up happening?” So, what I ended up doing is doing a multiple
logistic aggression analysis of 2,000 of their isolates. And what we ended up finding is if you look
at that odd-colored chart at the top of the slide, is that anytime one of the isolates
from red — fungicide from that red was used, more than 50 per cent of the isolates had
resistance to it. And if, anytime one of those red ones was
used, it was always associated with a high level of resistance, and oftentimes matched. So, for the yellow ones, it was often 20 to
50 percent. So, for example, if you found an isolate was
cyprodinil resistant in that middle column that runs down the slide, almost 50 percent
of the other isolates in that column also were resistant to another class. And so, by telling growers — if we look on
the bottom parts, you know the slide right there, and you’re one — okay, rotate all
the chemistries once, all the others will clean the other ones up. But it turns out that if you are using a chemistry
with which most of the population, more than 80 per cent, has resistance, the chance of
you grabbing and dragging along resistance to another fungicide was incredibly high. So every time they used one of those red or
yellows, it seemed that over the course of four years, they were constantly pulling along
other isolates that had multiple fungicide resistance. Just for merit, that — almost every isolate
had resistance to two of the red classes. So what we’re now telling growers, based on
this, is you need to focus on the blue and the green chemistries. And any time you use a red one, you’re pulling
— risking the chance of getting multiple fungicide resistance, which is kind of scary. Let’s go to the next slide. Some of the other things that we’ve found
is that managed isolates often seem like these super isolates with multiple resistance. And this is a study that we were looking at,
actually about five chemistries in a different class of fungicides. And the — on the graph over to the side,
you have sort of phenotype on the Y axis, and the isolate number that we were looking
at on the X. And for the most part, the first isolates
are from baseline, which means they are wild. They’ve never seen fungicides ever in their
life. The second actually come from commercial orchards,
and while they’ve seen fungicides, they’ve never seen this class. But the scary thing that we were finding was
a high level of insensitivity in these isolates that have just been exposed to different classes
of fungicides over time. You can see from the picture on the right
side, those wild isolates just get completely wiped out, while isolates that have just been
exposed to fungicides in general have a high level of insensitivity, which was kind of
scary. Now, if you click to the next slide, we can
see that this insensitivity — I’ve just shortened the number of isolates we’re looking at — but
this resistance, or insensitivity, extended to other classes, as well. We don’t ever see mutations in the targets,
or anything like that. But what we’re finding is the wild isolates
are still sensitive, while these super, or exposed, isolates will be insensitive to azoles,
another chemistry class, or even respiration inhibitors. So, from this, we begin to start looking into,
how can we get to the molecular aspect of multi-drug resistance mechanisms? And if you go to the final slide, we’re beginning
to sort of peel into the genomics of this particular pathosystem. It’s not a model organism, so it hasn’t been
heavily sequenced. But one of the key features that we’ve found
is when you go for these wild isolates, you can look at the genome size in green for that
baseline. It’s 39 megabases. And it has 48 — interestingly enough — I
highlight the bottom colony — transposable elements that are stuck in other coding genes. As you see that we increase to these isolates
that have multiple resistance, you’ll see that the genome sizes are going up, as well
as the number of transcriptional — transposable elements that are getting stuck in coding
regions is increasing, making me think that some of these exposed isolates are very plastic,
and the fungus is using transposable elements to shift the genome around to give it success
against any fungicides that come up, which is kind of a scary thought. And then, we’ll go ahead and take to the last
slide, and we’ll sort of move to my takeaways. What we’ve found so far; tree fruit and other
fruit are long-lived. They get a lot of treatments, and they get
exposed to the pathogen in constant, compromising environments, making them a nice system to
look at. I also wanted to point out, we do a lot of
epiphyte and endophyte studies, in stone fruit and apples and small fruit. And we never find any candida. There are epiphytes, but they’re — they usually
predominate one to two species. And none of them are often human pathogens. So, it’s hard to say what that means for azole
use in agriculture bleeding into medical problems. The other thing that we’ve found is that fungicide
resistance is often, is — population size affects the risk. Sanitation is incredibly important. It will increase — or reduce the time to
selection by the fungicides in leading to failures. And the multi-fungicide resistance that we’ve
often found is the biggest problem is using a fungicide to which most of the populations
already have resistance. That seems to be the best chance at dragging
isolates that happen to have one or two resistance to other chemistries. And with that, we’ll go ahead and switch. And I’ll thank all of my graduate students,
my technicians and postdocs, and all of the actual farmers and grower organizations that
fund the research that I’ve shared with you today. And thank you.>>Angela Caliendo: Great, thank you. Thank you, gentlemen, all, for a very interesting
and informative presentations.>>Female Speaker: Produced by the U.S. Department
of Health and Human Services at taxpayer expense.

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