 Good morning Thanks for inviting me and I just want to check my I'm I okay with the sounds and stuff like that. Okay, cuz we're being taped What I was asked to do this is actually part of the NIH series and I was asked to Provide some insights on how genomics has changed infectious diseases So what I thought I would do this morning is take you on a little bit of an odyssey Which includes my personal odyssey and some of the work that I've done and how my life has changed how our Diagnostics have changed as well and then show you a couple of case examples where The the advent of genomic sex analogy has changed our ability to diagnose manage treat and also Understand the epidemiology and implications of infections. First of all, I have no conflicts 2003 was an interesting year besides Dr. Collins's editorial in the New England Journal. It was also the year when the human genome was sequenced and published and It was also a year where we had the first major case where infectious diseases were rapidly diagnosed in an outbreak setting exclusively via genomics and I'll show you Now I hope some of you probably remember this And these still exist But you know culture there are many For example, I have infectious disease fellows now who've never seen a gram stain So the world has changed substantially since the good all days. This is this is gonna cockle This is the gonna cockle. This is nice theory gonorrhea and here we have a candle jar Which used to be used for transporting these bugs However, as you know Cultures take 24 to 48 hours to process in an era when we need rapid diagnostic information. This is a problem Cultures are prone to overgrowth in all kinds of problems and as we see centralization of laboratory services It's difficult There can be major transport problems. For example, if you I did this once which may indicate that I'm a bit of a strange guy if you go to the FAA website at four o'clock in the morning and see what planes are in the air It's FedEx and quest labs Because your laboratory Your laboratory work may go to elaborate may go to a quest or a lab port lab in Salt Lake City Depending on what specifically you're ordering So the days when you can go down to your local hospital microbiology lab and speak to the tech and find out What's going on are in many cases don't exist anymore? so In principles the advent of the genomic era Has resulted in in many opportunities for infectious disease diagnosis and management basically You want to detect the ideas to detect species specific DNA Amplify usually by an amplification technology you'd often polymerase chain reaction, but there are a whole host of newer technologies Which have come online over the past few years? One of the key elements is that bacteria DNA have unique 16s ribosome sequences Which can be used to pluck bacterial DNA out of a mixture of? Human animal or other types of DNA this should be matched It's the DNA is then matched to libraries which have these specific sequences and right now there's all over 2000 sequence bacteria who've been fully sequenced and this number is Increasing exponentially it's linked to a detection system and the tech the detection system technology has been Really advancing incredibly rapidly to the point where these are now desktop Machines with small footprints, and I'll show you and just for those of you for review I know most of you have seen this PCR polymerase chain reaction is the typical amplification system This is your target DNA the strands are separated. There's a pack to attack polymerase Which actually catalyzes the attachment of another strand, and then this goes on usually be a thermo a thermocycler, which is a heating set which is a Where the system is heated to 95 degrees centigrade and then rapidly cooled which? anneals the DNA and basically within usually an hour you can get 30 Amplification steps if you remember back from when you're a kid if you take a penny and double it after a month You'll have a million dollars So therefore this is a tenth of the eighth amplification with 30 cycles What's happened since the 1990s nucleic acid? Diagnostics were searched first commercialized in the 1990s with the with the early examples with wide Commercialization being actually an STD is my area and HIV viral load and HIV viral load is actually a DNA count of HIV Virus in the blood. It's not an act and that's one of the first Wide cool commercial uses of DNA technology to manage patients But there's been since the early 1990s many Non-cultivable pace pathogens have been identified for example whippel's disease is actually caused by a non-cultivable GI pathogen After 2001 there are major investments in technology and microbial detection for obvious reasons There's been some simultaneous investment in the human genome project and other sequencing projects such as the NIH's investment in Tiger Which is the sequence repository and as I mentioned approximately 2000 these seek Organisms were fully sequenced at the present time with with large numbers increasing every year The current trends in this area we see are the commercialization of discovery into diagnostic tests There's rapid the development of rapid and and actually we're going to see in the next five years point of care diagnostics Genomics as clinical management tools. I'll show you a couple of examples Bacterial population genomics and its impact understanding the popular bacterial populations and the ecology bacteria Also, there's an interest in host genomics. For example, there are certain host genotypes Which dictate how people will respond to infections or therapy hepatitis C is a very good example and I'll show you an example of that Microbial microbiome projects, which are looking at the ecology of the mucosal bacteria And we're looking at these as communities as opposed to single organisms And then we're seeing through the commercialization process the development of expert and benchtop systems Basically turnkey systems were basically all you do is insert a sample and you get a result Where all this is done behind the scenes through the black box and there's a great example with TB which I'll show you So this is remember SARS back in 2003 This was the Index case in Hong Kong in the Hong Kong hotel There was a Vietnamese physician who took care of him who died the patients There was an aircraft that were there are passengers who went to Toronto and two hospitals in Toronto were closed down And there were large numbers of deaths due to it due to a novel coronavirus. Well, this virus This is the SARS virus in its relationship to the to avian other to avian and other corona coronaviruses This problem was solved within 30 days Using sequence technology which the pathogen was identified a diagnostic test was developed And control measures were instituted This virus was never cultured. This was solely identified from DNA So this is the first example we're in a large-scale outbreak situation very much like the movie who saw who very like much like the movie contagion Where this was actually solved within a month But by the avian through completely through DNA technology If we go to where to my area in terms of sexually transmitted diseases Nucleic acid amplification test which is the acronym we use are the dominant mode of gonococcal and chlamydia testing It's very hard to get a culture anymore This can be used the advantage of this which we fit which we identified very early in this widely used is that this can be used for General and non-general samples, but in field settings such as schools So if you're in Africa sub-Saharan Africa doing an NIH funded study And there's a lot of them or doing a control program Or if you're in Philadelphia, New Orleans where they have large school-based screening programs Basically, these are done by urine or vaginal self-administer vaginal swabs where there's no transport problems. The material is stable And you can it can be transported to the lab and you can get diagnostic accuracy In fact that this resulted nucleic acid amplification Resulted in a conundrum for the FDA because it turns out that the sensitivity of Nucleic acid testing For STDs and most other infections is actually 30 to 40 percent higher than culture Because you don't have death during transport or you don't have fastidious organisms So therefore from a regulatory standpoint, there was a lot of discussions because using traditional reference standards ie culture the specificity was low However, in fact, the problem was is that the reference standard which was used had a low sensitivity So this has caused a rethinking from a regulatory standpoint of how you define the gold standard These also could be turned around in 24 hours in commercial labs So in fact the test takes six hours to run it can be multiplexed, but And this is a big problem now with gonorrhea and with other organisms You cannot identify resistant bugs unless you know specifically what you're looking for So you can't under you won't know if it's resistant to tetracycline or penicillin Because you're not growing the bug now you can identify if you know what the genes are you can identify them But this is a challenge So this is a great example of how we Adapted gonicoclon chlamydia testing into a field setting. This was a household survey in Baltimore, which we did in the late 1990s Which was a fields population-based field survey. This is published in JAMA and What we did is is a household prevalence study where we did a population-based sample knocked on people's doors Took a behavioral survey and as by the way, would you pee in a cup for us? and And what you find is that among This is for chlamydia. This is for chlamydia and gonorrhea We had 15% of women black women and six point four percent of black men were chlamydia positive nine and three percent War GC positive So therefore sorry so this combined six so 50 between between 15 percent of women and six percent of men Were either positive for chlamydia or GC and similarly one point For non-black two point eight and one point three percent. So Basically this that this is prevalent Gonicoclon chlamydia infection in Baltimore in the late 1990s Interesting that this study was repeated three years ago and found substantially lower prevalences and this is study been done There's the same studies been done in a number of different areas has been done in job poor applicants and military recruits It's part of the end as part of the end Haynes and because you're not dealing with transfer problems. It's easy to do See the feast seal costume the feast seal which is a major pathogen hospital acquired infections Represents a major major advances in diagnostics But the clinicians among you will remember that the old way was taking a seed if Culture which we recommend not to do because Culture may be false positive because the bug may be there but not causing Diarrhea the toxin assay which was the most common one required a stool filled Trait then being laid on tissue culture cells in the laboratory This takes about a day or two to do meanwhile the patient has got lots of diarrhea. It's also technically complicated and expensive About eight years ago. There were a lot of assays, but the sensitivity is eighty percent But right now the current standard is PCR of the toxin a toxin B genes which has a which can have a six-hour turnaround time So this has all happened again within ten years Now, but what happens when you start using the new tests? Well, dr. Rothstein is the head that equality and he probably faced this problem. He'll appreciate this problem, which we faced He's the sorry Bob's the VP ma's and chart. He's he has quality under his portfolio We actually this is our C diff rated Bayview through the 2000s and in 2008 we instituted an aggressive antibiotic management program which reduced our C diff rates by about 50% and that's that dip here and then it went up In late 2009, but what happened what happened was we changed the testing algorithm in the lab and went from the EIA To the PCR the PCR has about a 40% increase sensitivity So when you institute these tests you're going to increase your rate your reported rate your actual rate stays the same But this has to be explained to people to you know, you have to understand this because otherwise your Quality people administrators may actually have a heart attack when they see this now similarly This is what happened. This is actually a Series of clinics from Germany. This was published in sexually transmitted infections in 2006 This is their chlamydia Positivity rate and then what they did at this point in time. They introduced nucleic acid testing in their chlamydia positivity rate almost doubled This does not represent a change in sexual behavior. This does not represent a change in clinical In any kind of clinical protocols This is solely a result of testing or what this is saying actually during this period of time. They were underestimating the amount of tests PCS polymerase chain reaction. It's a new clay. It's one of the nucleic acid amplification technologies So we're looking at DNA amplification here second. What about Detecting undetectable or hard to detect organisms. I mentioned the T with the causative agent of Whipple's disease There's PCRs are for helicobacter In GI ulcers, although actually those were identified by culture and histology using that but are highly fastidious Bartonella and other fastidious bacteria are identified often through PCR mech through through nucleic acid amplification TB is increasingly being diagnosed by nucleic acid amplification and I'll show you a very exciting technology for that HPV the famous HPV that causes cervical cancer, and that's in the vaccine. This is a non-cultivable virus So here we have a vaccine which was developed On a non-cultivable virus and is identified if you're doing diagnostic assays for example protest for For servant, you know for in women. This is completely DNA based In the STD area trepanema pallidum and lymphogranuloma venerium are completely DNA based now because this is at least when you're looking for the direct organism Because this is non-cultivable even though people have been trying for a hundred years This is a great example of one of the most interesting things that I've seen in this area This is a study in France in which they look at 20 patients with brain abscess Traditional cultures found 22 strains of organisms However, when they PCR the samples they did nucleic acid amplification and find the bacterial DNA and match it up against the libraries They got 72 different strains in these 20 patients Including 27 species that were not previously seen in brain abscess About five were not even that were not even known These are newly discovered DNA spectral DNA sequences one subject had 16 strains So to give you a sense of from from hard to reach areas In small samples, this could be very helpful and diagnostic now one of the problems Implementing this type of stuff clinically besides the fact that it's technically at least at this level. It's still technically very challenging Specificity false positives are a major problem. We'll give you a very good example About 10 years ago, and I was still running my lab For STD pathogens at the main Hopkins campus One of the techs had actually sonicated gynecoccal organisms on one side of a lab and our PCRs for the next three months were positive Which means that kind of makes you wonder what was I breathing for during that during that period of time either all right What about antimicrobial resistance? Genomics can rapidly detect antibiotic resistance when you know what you're looking for So if you know specifically what you're looking for this can be a great tool It can be used as rapid screens and this is Increasingly being thought of for screening in areas where we know we have resistance problems such as Multiple drug resistant gram negatives in the hospital setting screening for MRSA and so forth in the appropriate setting I would posit to you that the exact Way that this is used these are going to be used in practice has not been worked out yet But I think that we are we are going to be seeing a lot of interest in this area because of the rapidity in desktop settings of doing this and it has a very high Utility in tracking outbreaks So for example if we have a multi drug resistant pseudomonas in the hospital if we get the genetic profile And we have a half a dozen cases in an ICU if the genetic profile of all those cases match Then that means we have an outbreak on the other hand if they're divergent That means they're independent cases and we have to get people you know There's a the hand-washing intervention is different for both in terms of How we approach that? Now this is an interesting story and it's actually Relate again from our own experience dealing with gona cockle disease Michael Don here is a collaborative mind in Tel Aviv who had a collection of about a hundred gona cockle organisms Which were obtained from commercial sex workers who were originally from the former Soviet Union There have been a large problem there around the central bus station the early 2000s late 1900s late 1990s early 2000s of trafficked women Who are commercial sex workers and with high levels of antibiotic resistance and we were interested in looking at the organisms? The problem was he was going to send them to us and then 2009 11 happened and memory and the antibiotic the major infectious disease meetings Were postponed until December and he told me he was he was able and he was going to come to Chicago And he was going to bring the plates with him And I told him you know israeli on security is is pretty tight He said no, I got that one covered I can get it I can bring I can get it on to the plane And I told him if you come from the Middle East in December of 2001 with 50 or 60 bacterial plates in your bag You're going to this you're going to be remanded. We're not going to see you So we came up with an alternate plan in which he actually took the organisms and actually Exorbed them on to filter paper and ship them to us by DHL And we had no problem except for one problem at Kennedy Airport We got a call from the agriculture inspector asking us went to we needed an affidavit This was not a pathogen of domestic animals Otherwise they had no problem This I couldn't make this up But what we have here we have this is these are the bugs that we see So what we did was he had actually run the MICs the minimum inhibitory concentrations and What we did is we had probes for quinolone resistance, which actually have some very well characterized probes We have the gyrate gyrate and part C which are sequences and here you see these are the amino acid changes which occur Which cause quinolone resistance? So this is called the QRDR quinolone resistance determining regions in the organism and what we're actually able to do is take the organisms Which he had he had this part of the he had the left part and we determine the right part and Match and you can see that there are specific sequences which are associated with different levels in the MICs And in this way we're able to characterize these organisms We also recently did the similarly with a study of a former graduate student of mine Who had studied who has a large conical collection in Kisumu, Kenya and we did the same work for her So here you have situation. We have well characterized mutations. You can develop probes to specifically look for them This is an example of syphilis where t-palatum cannot be cultured, but genomics has facilitated the understanding of the epidemiology of resistance So the or what syphilis has been around for obviously a long time if you want to culture it You have to culture it in rabbit testicles a live animal model The rabbits are not happy nor are the people who take care of them but What's happened is is that there's sorry there's There's been a lot of interest in traditional therapy of syphilis is benzothene penicillin There's been a lot of interest in using azithromycin and macrolides because it's especially it's much easier to do Especially in settings where you're not dealing with the formal health care system The problem is is that there's a 23 s RNA gene mutation Which actually causes resistance to azithromycin? This has been associated with the resistance to azithromycin So studies so what Sheila Lucard who published this paper in New England Journal in 2004 is they did prevalent studies to look how common is this and this is you can see there were changes in San Francisco Where there was a lot of syphilis at that time in Dublin in Ireland for some reason There was also a lot of resistance and this tend to segregate more in gay community So here we have a situation where syphilis in gay men was much more resistant than syphilis in straight heterosexuals What's interesting is that since that time this has changed almost completely resistant in San Francisco I was completely resistant in most of the areas where you have Large groups of gay men so azithromycin is not a good therapeutic option This is important because NIH funded a large study Three or four years ago in sub-Saharan Africa, which looked at azithromycin For syphilis control and found that it's very effective However, what clearly has to happen is that the autism needs to be monitored from a surveillance perspective And here you can see these are data from all over the world where if you have access to the biological material And the biological material is just a swab from a lesion This is so basically all you do is take a swab from a lesion dry it out and ship it and you can determine the Point prevalence so this can be very useful as a surveillance tool This is a really exciting area and many of you may know about this This is where the same technology same application is applied to to the diagnosis of tuberculosis and the diagnosis of Resistance an INH and rifampin resistance specifically to determine therapy at initiation at initial diagnosis The sensitivity and specificity for both Detection of TB and detection of resistance is 98 percent And this has gone in two hours from a sputum sample So patients coughing in front of you or hopefully not in front of you to the side of you You take a sputum sample. It's inserted into this machine, which is called the gene expert So this is the first basically it's entered into a cassette Inside the box everything is taken care of and you get a readout of whether this is TB or not And whether it's resistant to INH or rifampin and this is critically important in places like South Africa Where there are major problems with TB resistance? domestically we for example, we do see some TB in Baltimore you see some here a lot, you know But you know we're not that concerned about resistance in our population But in South Africa and other parts where there's actually major problem Southeast Asia where there's major problems with resistance This is important because if you start somebody on the incorrect therapy You're going to not know about it for six weeks for TB and another six weeks for the for the For the susceptibility test up to 12 weeks to get a full panel all this has been compressed into two weeks This is my favorite bug is in gonorrhea again This is actually there's been an increased problem in Susceptibility and now we're seeing you may have seen this in the press major problems in septriaxone and suffixin resistance This is actually the current issue of NMI curly agents chemotherapy where there's a high level treatment failure Sceptriaxone resistant GC which was diagnosed in Japan This is actually surveillance from a phenotypic standpoint in terms of MIC's Over the past ten years and you see that the numbers have actually been going up when I was at CDC This number was actually one log lower, so there's been a gradual creep over the time But this is a hard slide to read But the bad bug is this one over is the one at the bottom for the this one 5042 and This is changes in sequences from the wild type and what you see here in the middle are Intermediate so for example This is a these are the sequences of the bugs which would have an MIC of one and it changes in a in what's called the pen a Locust and then you have an MIC of two an MIC of four the one that's eight is the treatment failure one And you can see that this guy is accumulating different Mutations and eventually these are cumulative So this is a just a damage to show you how this is actually done that in a situation Where in quintalones you have a single step where there's one change which causes a resistance which causes an eight substantial increase Here you have accumulation of different mutations and if you have your data lined up right you can actually see that I Was in France in 2010 And you know and I don't speak French. I speak Spanish. I spoke don't speaks French But it's clear I can understand this and this was actually when they had the NTM metallo beta-lactamase Organism which was the pan-resistant bug and in fact there was one of those which was seen in the fall of 2010 in Howard County The reason I show this is that the French just have a much nicer way of saying bad bugs than we do Which is there's an elegance of part. There's an elegance about it, which we don't have Okay, how does understanding genomics facilitate epidemiology? Well, let me show you a couple of examples, which again from the SED area, but which I think really demonstrate this nicely The old model was that 90% of persons with genital herpes shed virus asymptomatically 1 to 10 percent of the time and If you take studies where people Were cultured this is from 2006 from a study by Anna Wald and sorry in 1995 96 These are Serial cultures you have people culture themselves every day for about them for months at a time and the involved cultures here Peri anal Volatile here peri anal these are this is and the only time that the pink is when they're when she was symptomatic So you see here. It's kind of episodic and it's about 1% of the time usually associated with symptoms Now what happens when you do genomics and see what's going on? In terms of how often they're shedding DNA and what you see is this is the percentage of days for detection of HSV on genital skin mucosa. So here you see basically about 20% of people have 25% of people have Are shedding here you could see so basically what the point being here is that people are shedding virus With herpes about a third to half of the time It may not be detected by culture, but it's subclinical So therefore this changes the way you think about the disease process the disease process is not something which turns on and turns off But something which is actually on all the time, but just modulating itself Swine flu many of you remember that from a couple of years ago This is an interesting one because what it actually represented was a Recombinant event between a human virus a pig virus and a bird virus So the typical flu is usually is the avian and here we said here and basically what happened here is that? Different people had different organisms, but this represented a recombinant event between three different Zoonoses of influenza, which then combined was diagnosed by genomic sequencing HIV has genomics guide epidemiological Investigation understanding transmission interventions and therapy Well, this slide is actually from my friend from my colleague Tom Quinn who's also a investigator cross-street the NIH The important thing is this is the transmission rate in study in a large study when this has been replicated a number of times between dichotomous couples so Sexual transmission between couples one partner has the virus one partner does not and you see as The viral load goes up the transmission rate goes up So essentially below four hundred there's almost no transmission Between four hundred and four thousand there's transmission approximately five percent per year and once you get up to over fifty thousand It's twenty five percent a year. This has profound implications Furthermore what happens when you put circumcision into the mix if you have circumcised people You see the transmission rate goes down to zero as well So circumcision and viral load are synergistic in HIV transmission Next point. What about acute HIV viral of seroconversion syndrome? HIV as you know acute HIV seroconversion syndrome within a week or two after you get the virus You have your viral load may be as high as a million and the transmission rate may at that time may be as high as 20% before it settles down So here we have viral load is describing to us the natural history of the disease as well It's also giving us some insights into transmission and also how to intervene So the interventions based on the knowledge afforded us by the genomic testing in HIV help us acute result in interventions to detect acute HIV cases Circumcision and transmission to prevent infection Think the circumcision trials have been well described This is the most that one of the most exciting things that happened over the past year and Mike Hone led this group from the University of North Carolina and what happened in group one was early HIV Intervention so therefore again, we have dichotomous couples people where one partner had the infection the other partner did not The partners were the infected partners were treated even if they did not meet criteria yet for initiation Treatment and the control group received standard of care. This is HIV transmission in the intervention group HIV transmission in the control group. I think basically this tells the story very clearly HIV treatment reduces viral load below 400 Reduces transmission as probably you're going to see a lot of focus on prevention from that standpoint Similarly with hepatitis C, but with other with other you know with other slants hepatitis C is an infection Which again is another virus which has not been which is cannot be cultured in vitro Where most of the work has been done genomically genomics die guide detection for example These are used viral load measures are used to determine whether somebody needs therapy or not They did they define therapy and therapy outcomes. The viral load is a therapeutic outcome Resistance is genomically defined similar to HIV And HIV for example you when we have an initial patient showing further out for treatment In HIV viral load is obtained and also the genomic profile of the viruses obtained Which will tell us which resistance mutations they have so we very similar to what you saw in the TB situation So we don't treat them with with drugs to which they are resistant Therapy strategies are based on genomic testing and here we have an interesting thing where in hepatitis C The host susceptibility can be genomically defined based on genes in the in the individual patient so Just for an example for example codon Changes in the codon of the HCB protease will render And this is the wild type and this is the protease will render the individual Resistant to a series of protease inhibitor drugs and therefore understanding this can help guide therapy This is actually slides are provided to me by Dave Thomas who's the head of infectious disease at Hopkins and an hepatitis C expert here We have the drug classes For hepatitis C and again, this is not to be expert Just to give you an example and we have the protein mutations and what you can do is based on you can actually define You can get the genome the genomic outcome of an individual virus in the person Develop this grid and see what drugs they are going to be sensitive to and define therapy Based on actual knowledge of the resistance profile of the drugs of the virus Without ever having to grow up the virus is all done based on genetic sequencing And this does not take long to do either. This is also going to be done in a single run This is for example here I have a good example of somebody who's been somebody with a resistant virus the virus was diagnosed and here We have instituting the pegging to fear and rob of iron vet regimen We were to which it was sensitive to and this is viral load here and what happens after initiation of appropriate therapy Now this is asher and Balaga Paul's work from our group Again at the at the main Hopkins campus showing that there are these specific snips which are sequences in the human genome Which defines? susceptibility for interferon dream for interferon and these determine whether persons Are going to be susceptible to therapy if they're infected with type one HSS Hcv virus and this is again taking that to a larger scale from a surveillance standpoint and looking for the prevalence of this mutation Worldwide you can see that They are Predominantly seen in Africa very little in Asia and this is clinically important because it's this explains why type one Hcv does not respond to To eight to anti Hcv therapy without the new protease inhibitors and type one is most commonly seen in African Americans So With that very rapid overview I'll spend the next 10 minutes kind of going over some microbiome projects And some of the things that we've done and leave the leave it open for questions I Think I mentioned that the human microbiome is a direction where the field is is actually it's not going It's here, but this is not look at single organisms. This looks at Organism community so think of bacteria as bacterial communities. This is especially relevant on mucosal surfaces Without understanding the interactions between our human microbial genomes It is impossible to obtain a complete picture of our biology and the important part here is that each part of our body has a different suite of Organisms which are present live there. We have the gut. We have the oral mucosa. We have it. There's the vagina There's the skin and even different parts of the skin have different organisms now We were traditionally taught for example that skin organisms our staff strep Propionobacterium and perhaps Corini, but it turns out to be much more complicated than that C. Diff is a problem in the gut because it is a because the normal flora have been eradicated by antibiotic therapy furthermore, there's some non-infectious diseases which are associated with microbiome changes for example Inflammatory bowel disease some increasingly some people are thinking some auto other autoimmune diseases are associated with microbiome abnormalities as well So the critical questions are how do we acquire maintain our microbial communities? How does it respond to stress? Can we use this information to intentionally on a threat from a therapeutic standpoint? And how do genotype environmental exposures and physiological science affect my crop microbiome composition? so The reason I show this the pyro sequencing is because the pyro sequencing is basically DNA DNA analysis on steroids Here we're not looking for a specific organism, and there's actually other other methods now as well including Something called the Illumina the titanium and there's recently something called ion Tarn. They're all variants on the same theme essentially what these technologies allow you to do is Sequence the entire DNA From a from a specimen not just looking for one organism, but do the complete bacterial sequencing on Spits on a host of DNA and that's going to result in a large number of different organisms So for example, if I take a swab of my mouth and put it into a piece a pyro sequencing machine I'm going to get 50 60 different bacterial sequences, and it'll tell me how many millions of each ones were there So this is actually an enormous proposition And you know it actually and one of the things that's been a cardinal rule And this is that sequencing gets 20% cheaper every year So as this happens that what costs $50,000 five years ago is now costs about $5,000 I'm going to show you two good two examples where we've you know from with clinic where we've had clinical interests in this area and Then sum up so bacteria vaginosis is the most common cause of inflammatory vaginal disorders And it's actually it's actually an ecological disturbance of the vaginal flora It's not an STD in the traditional standpoint and diagnosis is based on clinical criteria or Graham's thing This is the good stuff. This is a swab from a healthy vagina nice lactobacillus a healthy looking epithelial cells This is a clue cell, which is one of the diagnostic criteria. This is Clue cell from a woman with a with bacterial vaginosis and there's a ground glass appearance and what you have here anaerobes, which are attaching to the met to the outer surface and this is a gram stain of Of the same purpose of an individual with the same condition. Now. This one looks a lot different Than this I'd say this looks sicker than the first one And what happens is is that you have lactobacillus, which is a normal flora in the vagina is being replaced by Gardenarella anaerobes and mycoplasmas, and this is what we knew phenotypically You know up to about ten years ago Rebecca Brotman, who is a former graduate student of mine and who's now a professor professor at the University of Maryland Institute for genomic sciences has been studying the The microbiome of the vagina and what you have here the way to look at this is the pH over here and The new gen score both of which are indirect measures of BV Here we have that you see as you go. This is so going from healthy to BV healthy to BV And this is the microbiome of individuals In each category when you see here we have here that there's mostly it's lactobacillus Here we have lots of organisms which many of which were non-cultivable Prior to using this technology lots of things like urea plasm and mycoplasma the fine goldia Which is an anaerobic most of these are anaerobes Atopium is an anaerobic which was first discovered studying women with bacterial vaginosis Prevotella is an organ is an anaerobic which is also found in the mouth So what we see here is a shift from a normal flora towards Anaerobes and a lot of anaerobes with a lot of anaerobic Diversity and a lot of anaerobes which are reformally non-cultivable I'm going to show you the same story with chronic wounds We've had a major interest in our group in studying chronic wounds chronic low extremity wounds These are the bane of an internist existence One pundit said the safest place to hide a $20 bill on a medical ward is under a large dressing Wounds have direct medical cost impact. They're $25 billion a year. My brother will take the dressing off wounds have substantial indirect cost benefit This is there's little research being done in this area and this is a great opportunity because nobody knows what's really going on here From the infection standpoint, there's no good definition of infection in chronic wounds How do you define colonization versus infection people give antibodies because bugs are there But they have no idea really what's going on there people propose quantitative culture as one modality But this is on actually when you look at the data to support this It's really not good and we don't do this anymore and this is I say welcome to my world This is typical person in my clinic chronic menostasis ulcer non-healing And uses compression or slowly healing and does this wound require treatment or not And what is the bacterial community of that wound and how is that different from what we see elsewhere? I think we saw that so our research program objectives were to describe the prevalence of bacterial species in chronic wounds To assess the microbial burden by different modalities Now I'll show you the the bacterial culture and the DNA and Compare the microbial populations of two different sites, which I won't show you But we demonstrated that the wounds are pretty homogeneous across the whole wound So if you do a traditional culture you find the usual players MRSA staff aureus pseudomonas Which actually turns out to be present only in people who've been treated with antibiotics Groups to be and a whole host of other things Merces present in 45 percent pseudomonas in 28 percent group street group B strep in 28 percent I would argue part of this is because we're a reference center. We're gonna get people who've already failed therapy elsewhere So 97 percent of our wounds had at least one aureus and pseudomonas as I mentioned to here night Very high bacterial load in those with MRSA and So forth now when we do the quantitative microbiology, I'm gonna show you two different methods I'm gonna show you that there's a theme here This is an older way of looking at the quantitative at the sorry at the at the microbial DNA here We took the microbial DNA of the wound and typed it out and speciated and see what was there and we find That these are skin bugs now calgenies and bacteria bacteriitis or anaerobes here We have a pseudomonas This is a gram negative. This is group B strep This is mobile lancas Now what's interesting if you probably don't remember but mobile lancas and atobium are the BV bugs the bacteria vaginosis bugs So we're seeing so we have again your chronic mucosal infection characterized by large numbers of anaerobes including some which are non-cultivable This is what's called a heat map in which each Vertical column is one of our specimens and this is the horizontal Which are the different organisms that are seen again. You see there's large diversity of organisms Again, if we went through a lot of these are pseudomonas because we have a group which was treated with antibiotics previously But the important role is that there's a large diversity of anaerobic bugs. So we're not just seeing Mercer in there We're not just seeing strep We're seeing a whole host of other things the question that we're interested in now Is that if you on how does this? Impact on wound healing and can these patterns can we develop patterns are their ecological patterns? Which will predict whether a wound will heal or not and almost And and are there ways of correcting the flora if they need to be So the conclusion from from metagenomics from the wounds are that microbial diversity was significantly lower in patients treated with Anomicrobials, there's a high proportion of anaerobes and non-cultivables Genomic status suggests that anaerobes are critically important and this may represent synergistic infections However, and here we get into the next step in genomic studies. This is DNA only so dead bugs are going to be there RNA Indicates what's active. So for example in the STD area We don't recommend people do traditional tests of cures Because you'll shed bacterial DNA from dead bugs for two weeks So therefore Interpretation of these things has to recognize that DNA is also Present in dead bugs and may be present for a while So in conclusion genomics has impacted the discover ability to discover new pathogens our ability to detect pathogens understanding the epidemiology of these pathogens guiding therapy the interventions understanding resistance and understanding how susceptibility with the example of hepatitis C they're rapidly replacing traditional microbiology and I call it the cell phone paradigm in the appropriate settings because for example They can be we're seeing benchtop and export systems that are being downsized to cell phones For example, the military has bio threat agent detection systems which are the size of a signal a cell phone Which they carry with them in the field But also if you're in a developing country and you're looking for specific pathogens for example if you're fielding TB diagnostics You get the capital funding to basically fund the the box which actually does the whole thing You you avoid having to set up the whole culture systems. This is very similar to what's seen in developing countries really It's much cheaper now to install a cell phone system than to install a landline system because you don't need all that infrastructure So I think that's it. So the microbiome and microbiome is an ecological concept that is leading to new understanding Infectious diseases based on microbial community concepts. So thank you Yeah But what we have is a this is this is for HIV Governmental systems are not generally willing to invest in Prevention as treatment and so and that that's actually applicable to a lot of potential health benefits of genomic medicine So you have ideas in terms of in sort of the policy sphere how we consider How we encourage policy yeah, okay, I think it's a good question. I think until It's an important question is until the Question was run until the data came out. There was a very valid argument to be made and the question is Reducing viral load as prevention should that you know Has obviously benefits should be can that be generalized and should we be moving in a policy direction if I'm hearing you right? Until the data was published last year. There was a valid question to be a valid question Is that first of all there's treating people who are not meeting thresholds for treatment yet? result in unintended consequences in terms of Inter-retroval resistance down the line drug side effects and so forth I think with the data now, this is very compelling and I think any program from my standpoint, this is at no brain You know for example and we but we do see your issue there's this policy disconnect because for example And we see we've seen this over the past year that in states where they where they've run short of funds that The age drug assistance programs have long waiting lists and certain things like that and I think what we now have is a business case to make That is a very strong business case that this results in further in reduction of further infection Now this all may change because part of the problem is that Healthcare has been funded prevention the prevention side of health care is funded from a different pot than the treatment side With the integration of health care that's going to happen over the next five years This is going to partially resolve itself. I think people may be more responsive But I think these data form the special especially convincing for now From a policy standpoint of policymakers don't want to make that decision after being presented with the data Then the only solution is to replace the policymaker that I don't have any You know, we you know, I think despite comments, you know, I'm being taped so I can't let my political feelings be known Oh, okay Well, actually the current time the question is what's the current understanding on why some bugs remain uncultable? I think I would not say remain. I think some of these are on were undiscovered Until the DNA technology became available and they have growth requirements, which are extremely fastidious so for example, they just they you know, and Either they're biochemical leaves and nutritional leaves or their or their or some oxygen, you know You know, for example oxygen may be toxic to them There's been a tremendous amount of work a lot on this in environmental microbiology Because if you go into the environmental side, you see, you know, you take, you know, river water and grow What's in there? There's an enormous number of things that are in there The microbiome is enormous So I think this is it's more of a technical problem because we're using and actually one of my colleagues Says we're using technology now that was developed by pasture 130 years ago. Yeah. Okay. Well the question is you know, you're actually you're asking the million-dollar question And basically what constitutes colonization? What constitutes disease? So I think what happens is is that? Just fine, and this is our this is my whole issue with wounds and a lot of wound care Just finding the organism there doesn't mean it's doing anything and it's just you know I think if you look at the papers where they've identified organisms, for example whipples, which is a beautiful paper What they do is they take a series of people with disease And then basically you cost you can't fulfill Cox postulates in this But what they do is they and they demonstrate that there's a Consistent and that the organism is found consistently and then there's a lot in each case with that with the disease that It's in terms of where it is in the organism. It's there when you treat them. It goes away So I think you have to establish a case That this is actually causing invasive infection the brain abscess is a very you know is a very easy one because This is a brain abscess. It's a sterile space We're not supposed to have stuff in our brain and you're finding 16 bugs here and somebody with an abscess. That's easy. That's a slam Yeah, you forgot. Yeah Blood is actually we've actually we actually looked at blood a while We were looking at blood not in febrile nutrients, but in burn patients blood is very difficult So if you look at the blood literature, you'll see that there's a lot of work done on DNA technology once the blood cultures turn positive So once if it's in the machine and it turns positive there's a lot of work being done to identify what's growing in there with DNA technology at Admission at time, but I'm interested in what happens at time of the initial blood culture The problem is is what is the concentration of bacteria and blood culture is two problems The concentration of bacteria and blood culture ranges between 1 to 10 CFUs per mil So it's you know now there are people with high-level staff factor emu may have a thousand You know, but it's not like what we see in viruses So you have to have higher volumes and second is the background of human DNA is And so is enormous so you have to sort that out So I think there's other people looking at this. I think there's a lot of technical problems I think there are there are some very promising technologies including some micro-minifluorescent Amplification technologies which some folks at the University of Maryland Baltimore County Are you doing and some other types of things, but it's not there yet? I mean I ideally you would love to have somebody in the you know somebody would this say There are two with blood in the lab and know whether you're back to remic, you know, it's not there yet Yeah, yeah Yeah, I think the question was a suggestion that there's different bowel flora and obese patients There are I'm not an expert in this because my ears an infection, but I think they're absolutely differences in obese You know an obese patients. There's differences in animal models for that And I think the question is and how that regulates food intake And I think there's been a lot of interest in can you change the flora? You know, what is it a chicken or the egg problem? Besides that I can't answer that because that's not it's not my area of expertise. Yeah You're reducing the bacterial load won't skin flora that you're worried about is Skin flora in healthy people is staff and strep and sick people It may be also gram negatives that are acquired from the hospital setting. You're basically removed both mechanically removing and killing 10 to this at least about 10 to the 3 of a back to of the load, so you're reducing the risk of infection It's both mechanical and biochemical The question was after after my lecture, how can you live with dialysis catheters? Well You know again, we run into the question what constitutes Infection and what constitutes colonization and what a topic which I did not discuss is the whole issue of biofilm because actually organisms on Surfaces do not live in a planktonic state like they do in the lab. They're actually in film situations Where they may be relatively inactive We actually surveyed in patients at Bayview I had a medical student surveyed about six months ago. What proportion of patients? It's not only dialysis catheters what proportion of patients who are on a general medicine service have stuff in them that they weren't born with and It's including dialy including catheters IC you know ICDs pacemakers prosthetic kips things like it's about 60% and that does not include bullets So which we have something which we have so I think so this is a big problem Actually what what scares the living daylights out of me is prosthetic joints actually prosthetic joint infections is one of the areas Also where they've been using PCR there's been a lot of research looking PCR to identify Pathogens, but if this is a big problem, you know, this is our world and I don't have a good answer But it's an enormous problem. You know what I'm the clinicians. I'm just the translating guy Okay, okay good question the question is how do I see this going from the treatment standpoint I see there's a couple of directions one is Pathogen directed therapy So probably probably the most important and this the TV example is a very good one We know what exactly what the bug is the characteristics So right now the approach to infection disease in most hospital settings is you know is Broad spectrum which actually is the worst thing we can do for a floor. So pathogen directed second focusing on drilling down on that and Pathogen directed with antimicrobial susceptibility directed Thorough it is from an epidemiological standpoint if they're if you're worried about specific outbreak situations being able to identify Specific outbreak bugs and deal with them in a rapid way the quite the other question which you asked which is interesting is the be dealing with flora and For example, there's been a lot of interest for years on correcting the bacterial flora and back to the Correcting the flora and bacterial vaginosis with lactobacilli with yogurt, which actually has lactobacilli with Lactobacillus crispatus, which is actually the specific point and all the studies have fallen flat on their face They haven't worked on the other hand Probably the most successful one of reflora in a mucosal surface has been in the case of C. Diff where As my boss Don Bartwood said the the therapy has is incredibly effective But has aesthetic complications and that is in people with severe recurrence C. Diff And there are these people you probably know some of them who cannot get off of vancomycin or You know or other therapies and they recur all the time the therapy which works is a stool transplant And it fixes it within 24 hours And There are people who are doing this now and you can imagine There's obvious regulatory problems with the you know, how do you do this with the you know with the FDA and safe and things like that But it works And you know it has you know, it's actually the way it's done is actually it's still taken from a relative But it's either instilled from below by somebody You there are there's actually a drop their job opportunities so that people used to do barium enemas Because you want to get it all the way up or through a long canter tube And there's another I heard that there's a formulation being developed of where actually can be in a gel cap But it's sort of like it's gonna And absorb you know, so what you take but the capsule does not dissolve until it's down into the gut So so that's that's very good. You know, I think but clearly you're raising for example We'd see this in urinary tract, you know, we were interested in the wound You know, can we repopulate the flora with what we what we want to say that's a lot of interest in that Last question It's hard to on an individual basis. You can't differentiate clinic clinically after you've done settings. So for example C. Diff Would be clinically obvious for example anybody in a long-term care facility or who's been on antibiotics recently as a recently You know has a new onset diarrhea that generally if they have leukocytosis a white cell count generally C. Diff until proven otherwise So what we would do is we would get the test for C. Diff and then treat empirically But in terms of viral outbreaks one of the problems with viral outbreaks is that they don't the viruses don't grow But so from a public and they're usually self-limited So the utility here of the diagnostic testing is to understand the epidemiology as opposed to guide therapy Okay, thank you