What's bugging you? The Intersection of Genomics and Infectious Disease - Jonathan Zenilman

What's bugging you? The Intersection of Genomics and Infectious Disease – Jonathan Zenilman

good morning thanks for inviting me and I just want to check my am 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 technology 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 those 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 have never seen a Gram stain so the loads of the world has changed substantially since the good old days this is this is gonococcal drew this is the kind of chocolate this has got neisseria gonorrhoeae 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 the kulta there can be major transport problems for example if 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 court 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 or in many cases don't exist anymore so in principles the advent of the genomic era has resulted in many opportunities for infectious disease diagnosis and management basically you want to detect the ideas of detect species specific DNA amplify usually by an amplification technology use 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 bacterial DNA have unique 16s ribosomal to pluck bacterial DNA out of a mixture of human animal or other types of DNA this should be match it's the DNA is that matched to libraries which have these specific sequences and right now there's over two thousand sequins bacteria who've been fully sequence in this number is equal 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 pcrs polymerase chain reaction is a typical amplification system this is your target DNA the strands are separated there's a packed up tech polymerase which actually catalyzes the attachment of another Rand and then this goes on usually be a thorough thermocycler which is a heating switch where the system is heated to 95 degree 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 10 to the 8 amplification with 30 cycles what's happened since the 1990s nucleic acid Diagnostics worser first commercialized in the 1990s with the with the early examples with wide commercialization being actually in STDs in my area and HIV VAR lo an HIV viral load is actually a DNA count of HIV Rock virus in the blood it's not an active 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 paste pathogens have been identified for example Whipple'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 solid 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 in practice way two thousand these sea organisms were fully sequence at the present time with with large numbers increasing every year the current trends in this area as 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 that microbial microbiome projects which are looking at the ecology of the mocoso 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 bench top systems basically turnkey systems where 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 patient's there was an aircraft that were other passengers who went to Toronto and to hospitals in Toronto were closed down and there were large numbers of deaths due to due to a novel coronavirus well this virus this is the SARS virus and its relationship to the two AV and other two 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 where in a large-scale outbreak situation very much like a movie who saw who very like much like the movie contagion where this was actually solved within a month but by the event through completely through DNA technology if we go to wear 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 and it's 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 em or doing a control program or if you're in Philadelphia and New Orleans going to have large school based screening programs basically these are done by urine or vag delphinus or vaginal swabs where there's no transport problems the material is stable and you can be transported to the lab and you can get diagnostic accuracy in fact that this resulted the clanky 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 thirty to forty percent higher than culture because you don't have death during transport or you don't have fastidious organism 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 can be turned around in 24 hours in commercial labs are in fact a 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 can identify resistant bugs unless you know specifically what you're looking for so you can 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 gonococcal and chlamydia testing into a field setting this was a household survey in Baltimore which we did in the late 1990s which was a field's population-based field survey it was 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 fifteen percent the women black women in six point four percent of black men or chlamydia positive nine and three percent were GC positive so the Air Force I was combined 650 between between fifteen percent of women and six percent of men were either positive for chlamydia or GC and similarly one point I for non blacked what 2.8 one-point-three percent so basically this that this is prevalent gonococcal and chlamydial infection in baltimore in the late 1990s interesting in this study was repeated three years ago and found substantially lower prevalences and this has study been done there's the same studies been done a number of different areas has been done in job for applicants and military recruits it's part of the end is part of the in haines and because you're not dealing with transport problems it's easy to do see the fee seal Clostridium difficile which is a major pathogen hospital-acquired infections represent a major major advances in diagnostics though the clashes among you will remember that the old way was taking a seat 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 filtrate than 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 Eliza assays but the sensitivity is eighty percent but right now the current standard is P see our 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 that better quality and he probably faced this probably she ate this problem which we faced he's sorry Bob sleepy mas and Chinese has quality under his portfolio we actually this is our CT afraid of Bayview through the 2000s and in 2008 wing student aggressive antibiotic management program which reduced our seed if rates by about fifty percent 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 forty percent increased sensitivity so when you Institute these tests you're going to increase your rate your reporting rate your actual rate stays the same but this has to be explained to people out to you know you have to understand this because otherwise your quality people or 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 there a chlamydia positivity rate and then what they did at this point in time they introduced nucleic acid testing and the 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 what this is saying actually during this period of time they were under estimating the amount of tests we take pcs polymerase chain reaction it's a nucleic it's one of the nucleic acid amplification technologies so we're only a DNA amplification here second what about detecting undetectable or hard to detect organisms I mentioned the team with the causative agent of Whipple's disease there's PCRs therefore he'll go back there in GI ulcers or low actually those were defined by culture and histology using but are highly fastidious bartonella and other facilities bacteria are identified often through pcr mek 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 protests for for a servant you know for in women this is completely DNA based in the STD area treponema pallidum and lymph oh grannie loma venereum 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 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 the 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 we're not even known these are newly discovered DNA's bacterial DNA sequences one subject had 16 strains so to give you a sense from from hard-to-reach areas in small samples this could be very helpful in diagnostic one of the problems implementing this type of stuff clinically besides the fact that it's technically at least at this level is still technically very challenging specificity false positives are a major problem give you a very good example about ten years ago and I was still running my lab for STD pathogens at the main Hopkins campus one of the texts had actually sonicated gonococcal organisms on one side of a lab and our grp crs for the next three months were positive which means that it kind of makes you wonder what was I breathing for during that there in that period of time either all right what about antimicrobial resistance genomics can rapidly detect enemy 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 mrs a 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 using practice has not been worked out yet but I think that we are not we are going to be seeing a lot of interests in this area because of the rapidity in desktop settings of doing this and it has a very high utility in tracking outbreaks or for example if we have a multidrug-resistant Pseudomonas in the hospital if we get the genetic profile and we have half a dozen cases in an ICU if the genetic profile all in those cases match then that means we have an outbreak on the other hand if their divergent that means they're independent cases and we have to get people you know those are the hand-washing intervention is different for both in terms of how we approach that now this is an interesting story it is actually really again from our own experience dealing with gonococcal disease Michael Don here is a collaborator of mine and tell Eve who had a collection of about a hundred gonococcal 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 in the early 2000s late 1900s late 1990s early 2000s of trafficked women who are commercial sex workers 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 online security is pretty tight you say no I got that one covered i can get it i can bring i can get it onto 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 dis 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 adsorbed them onto filter paper and ship them to us by a DHL and we had no problem except for one problem at Kennedy Airport we got a call from the agriculture inspector asking us when we needed an affidavit that was not a pathogen of domestic animals otherwise they'd have no problem honest 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 and actually running mi sees the minimum inhibitory concentrations and what we did is we had probes for quinolone resistance which actually have some very well characterized probes we of the gyre age I race and Part C which are sequences and here you see these are the amino acid changes which occur at which caused quinolone resistance so this is called the QR dr quinolone resistance determining regions in the orion ism and what we weren't able to do is take the organisms which he had he had this part of the he had the left part and we determined the right part and match and you can see that their specific sequences which are associated with different levels in the mi seas and this way we're able to characterize these organisms we also recently did the simile with a study of a former graduate student of mine who had studied who has a large gonococcal collection consumo 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 pallidum cannot be cultured but genomics has facilitated the understanding of the epidemiology resistance so the or quotient 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 uh there's been a lot of interest in traditional therapy of syphilis is better than penicillin there's been a lot of interest in using as if they're mice and macrolides because it's especially it's much easier to do especially in settings where you're not dealing with the formal healthcare system the problem is is that there's a 23s RNA gene mutation which actually causes resistance to as if through mice and it's been associated with the resistance to azithromycin so studies so what our Kela luikart who publishes pay four in New England Journal in 2004 is I did prevalence studies to look how common is this and this is you can see there were changes in San Francisco where there's 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 and 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 as if their minds 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 as if they're meissen for syphilis control and found that it's very effective however what clearly has to happen is that the origin 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 make a swap em Legion 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 the diagnosis of tuberculosis and the diagnosis of resistance on inh and rifampin resistance specifically to determine therapy at initiation at initial diagnosis the sensitivity and specificity for both detection of TB and detection or resistance is ninety-eight 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 pert so this is the first basically its entry 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 rifampin and this is critically important in places like South Africa with our major problems with TV resistance domestically we for example we do see some TV 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 it's actually major presence aus East Asia there's major pounds 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 Maryland 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 inception relax own and cefixime resistance this is actually the current issue of NM micro agents of chemotherapy with a high level treatment failure cetera action resistant GC which was diagnosed in Japan this is actually surveillance from a phenotypic standpoint in terms of mi seas over the past 10 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 bed bug is this one over is the one at the bottom for this 150 for two 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 mi c of one and it changes in eight and what's called the pen a locus and then you have an mi c2 and nico for 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 curios so this isn't just a damage to show you how this is actually done that in a situation wherein quinolones you have a single step where there's one change which causes a resistance which causes a substantial increase here you have Q accumulation of different mutations and if you have your data line that brought you can actually see that I was in France in 2010 and you know and I don't speak French I speak Spanish I suppose don't speak French but it's clean 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 two in the fall 2010 in Howard County the reason I show this is that the French just have a much nicer way of saying bad bugs and we do which is there's an elegance apart there's an elegance about it which we don't have okay has understanding genomics facilitate epidemiology well let me show you a couple of examples which again from the STD area but which I think really demonstrate this nicely the old model was that ninety percent of persons with genital herpes shed virus a symptomatically one to ten percent of the time and if you take studies where people were cultured this is from 2006 from a study by Anna wold and sorry in nineteen sorry 1995-96 these are serial cultures you have people culture themselves every day for about them for months at a time and the evolved old cultures here perianal level here perianal d sir this is and the only time that the pink is when she was symptomatic so you see here it's kind of episodic and it's about one percent of the time usually associated with symptoms now what happens when you do genomics and see what's going on I happen in terms of how often they're setting DNA and what you see is this is the percentage of days for detection of HSV on gentle skin mucosa so here you see basically about twenty percent of people have twenty-five percent of people have our here you can 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 detective like 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 is 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 as the avian and here we said here and basically what happened here is that different people at different organisms but this represented a recombinant event between three different zoonosis of influenza which then combined was diagnosed by genomic sequencing HIV how does genomics guide epidemiological investigation understanding transmission interventions and therapy well this law is actually from my sprout for my colleague Tom Quinn who's also investigate across the street the NIH the important thing is this is the transmission rate in study in as 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 as you see as the viral load goes up the transmission rate goes up so essentially below 400 is almost no transmission between 400 and 4,000 there's transmission approximately five percent per year and once you get up to over 50,000 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 transition rate goes down to zero as well so circumcision in viral load are synergistic in HIV transmission next point what about acute HIV viral of seroconversion syndrome HIV ver is you know acute HIV sir conversion 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 twenty percent before it settles down so here we have viral load is describing to us the natural history of the disease as well as also giving us some insights into transmission and also how to intervene so the intervention is based on the knowledge afforded us by the genomic testing at HIV help us acute it resulted interventions to detect acute HIV cases circumcision and transmission to prevent infection I think the circumcision trials have been well described this is the most one of the most exciting things that happened over the past year and my cone 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 of 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 is 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 quickies cannot be cultured in vitro where most of the work has been done to know quickly genomics die guide detection for example these are you spiral load measures are used to determine whether somebody needs therapy or not they define therapy and therapy outcomes the viral load is a therapeutic outcome resistance is genomically to find similar to HIV HIV for example you when we have an initial patients showing further out for treatment an 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 on the TV 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 we're in hepatitis C the host susceptibility can be genomically defined based on genes in the and the individual patient so just for an example for example code on what changes in the codon of the hcv 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 these slides are providing me by Dave Thomas who's the head of infections at Hopkins in 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 permutations and what you can do is based on you can actually define you can get the genome the genomic outcome of the individual virus and the person developed this grid and see what drugs they're 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 you have a good example of somebody who's been somebody where there is distant virus the virus was diagnosed and here we have instituting the peg interferon ribavirin barrett regimen which it was sensitive to and this is viral load here and what happens after initiation of appropriate therapy now this is ashlyn balaga Paul's work from our group again at the main Hopkins campus shelling that there are these specific snips which are sequences in the human genome which define cept ability for interferon dream for interferon and these determine whether persons are going to be susceptible to therapy if they're infected with type 1 HSS HGV virus and this is again taking that to a larger scale from a surveillance standpoint and looking for the prevalence of this mutation in worldwide you can see that they are predominantly seen in Africa very little in Asia and this is clinically important because this explains why type 1 HCV does not respond to a 2 anti-hcv therapy without the new protease inhibitors and type 1 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 then leave leave it open for questions I think I mentioned that the UN microbiome is a direction where the field is is actually it's not going it's here but this does 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 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 our present live there we have the gut we have the oral mucosa via 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 staffs trapped propionibacterium and perhaps karani but it turns out to be much more complicating 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 are inflammatory bowel disease some increases some people are thinking some auto other autoimmune diseases are associated 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 intensity on a third from a therapeutic standpoint and how to genotype environmental exposures and physiological status effect microbe microbiome composition so the reason I show this the pyrosequencing is because the prior 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 Torrent 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 Spadina 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 of higher 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 actually in one of the things that's been a cardinal rule in this is that sequencing gets twenty percent cheaper every year so as this happens what costs $15,000 five years ago is that costs about five thousand dollars i'm going to show you two examples where we you know from with clinton where we've had clinical interests in this area and then sum up so bacterial 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 Gram stain this is the good stuff this is a swab from a healthy vagina nice lactobacilli healthy-looking epithelial cells this is a clue cell which is one of the diagnostic criteria this is kusal from a woman with a with bacterial vaginosis and there's a ground glass appearance and what you have here are anti ropes which are attaching to the mitt to the outer surface and this is a Gram stain of of the same purpose of 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 the normal floor and the vagina is being replaced by gardnerella anaerobes and michael plasmas and this is what we knew phenotypically you know up to about ten years ago Rebecca brought men who is a former graduate student of mine it was now a professor and professor at the University of Maryland Institute for genomic scientist has been studying the microbiome of the vagina and what you have here the way to look at this is the pH over here and the Newton 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 be V 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 plasma mycoplasma fine goldia which is an ANA rope most of these are anaerobes there's a tight opium is at anaerobe which was first discovered studying women with bacterial vaginosis prep Attila is an organ organ is an anaerobic 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 no formally non cultivable i'm going to show you the same story with chronic wounds we've had a major interest in our group and studying chronic wounds chronic lower extremity wounds these are the bane of an internist existence one pundit said the safest place to hide a twenty-dollar bill on a medical ward is under a large dressing wounds have direct medical costs impact their 25 billion dollars 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 antibiotics 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 unyu and we don't do this anymore and this is I say welcome to my world this is typical person in my clinic chronic venous stasis 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 micro wheel burden by different modalities and I'll show you the the bacterial culture and the DNA and compare the microbial populations in two different sites which I want to show you but we demonstrated that the wounds are pretty homogeneous across the whole world so if either traditional culture you find the usual players mrs a staph aureus Pseudomonas which actually turns out to be present only in people have been treated with antibiotics group strep b and a whole host of other things Marissa's present in forty-five percent Pseudomonas in twenty-eight percent group Street Group B Strep in twenty eight percent I would argue part of this is because we're a reference center we're going to get people who have already failed therapy elsewhere so ninety-seven percent of our wounds had at least one aureus and Pseudomonas as I mentioned here nine very high bacterial load in those with Mercia and so forth now when we do the quantitative microbiology i'm going to show you two different methods i'm going to show you that there's a theme here this is an older way of looking at the quantity 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 without cal genies and bacteria bacteria or ities anaerobes here we have a pseudomonas this is a gram-negative this is Group B Strep this is mobile Uncas now what's interesting if you you probably don't remember but mobile Uncas and atomium are the BV bugs the bacterial 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 culpable 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 seeing 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 Marissa 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 are there ways of correcting the flora if they need to be so the conclusion from it from metagenomics from the wounds are that microbial diversity was significantly lower in patients treated with antimicrobial there's a high proportion of anaerobes and non call to the bowls genomics data is 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 scar ability to discover new pathogens our ability to detect pathogens understanding the epidemiology of these pathogens guarding therapy interventions understanding resistance and understanding how susceptibility with the example of hepatitis C they're rapidly replacing traditional microbiology and I call the cell phone paradigm in the appropriate settings because for example they can be we're seeing bench top and expert systems that are being downsized to cell phones for example the military has biothreat agent detection systems which are the size of a sick you know a cell a cell phone which they carry with them 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 avoid having to set up the whole culture systems this is very similar to what 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 a technological concept that is leading to new understanding infectious diseases based on microbial community concepts so thank you shouting from the rooftops that reduction in viral load actually leads to substantial health benefits but what we have is a this is / HIV perforation uh-huh what we have is a policy from the policy problem that governmental systems are not generally 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 do you have ideas in terms of in sort of the policy sphere how we consider of how we encourage both yeah okay yeah I think it's a good question i think until it's an important questions until the question was 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 in has obviously benefits should be cannot be generalized and should we be moving in a policy direction I'm hearing you right until the Datak 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 antiretroviral 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 a no-brainer 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 there they've run short of funds that the aids drug assistance programs have long waiting lists and so and 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 only change because part of the problem is that health care 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 maybe more but I think these data form the especial 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 you know I think the spy comments you know I'm being taped so I can't let my political feelings be known oh ok well actually the current and the pressure is what the current no sitting and why some bugs remain on call to vote I think I would not say remain I think some lot of these are 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 their biochemical legion the trishul leaves or there or there or some oxygen you know you know for example oxygen may be toxic to them there's been a tremendous amount of work on this and 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 it's more of a technical problem because we're using it actually one of my colleagues says we're using technology now that was developed by pests or 130 years ago yeah okay well the question is you know you're actually you're asking the millionaire I'll 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 Whipple's which is a beautiful paper what they do is they take a series of people with disease and then basically because you can't fulfill Koch's 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 of it in each case with it with the disease that it's paying it 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 to sterile space we're not supposed to have stuff in our brain and you're finding 16 bugs here and somebody with an absence that's easy that's a slam dunk yeah for back year blood is actually we've actually we actually looked at blood of wat we were looking at blood not in febrile neutropenic button 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 turned 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 cultures to problems the concentration of bacteria and blood culture ranges between 1 to 10 CF use per mil so it's you know now there are people with high level staff back to ream you may have a thousand you know but it's not like what we see in viruses so you have to give 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 immunofluorescent amplification technologies which some folks at the University of Maryland Baltimore County were you doing in 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 with this a draw to blue blood 10 in the lab and know whether you're back to remake you know it's not there yet yeah yeah yeah yeah I think the question was the suggestion that there's different bowel floor and obese patients there are you I'm not an expert in this because my ears infection but I think they're absolutely differences in obese you know and 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 back I can't answer that because that's not it's not my area of expertise yeah you clean the skin giving alcohol what are you doing at you can put any puncture what you're producing the bacterial load well skin flora that you're worried about is skin flora in healthy people is staph and strep and sick people may be also gram negatives that are acquired from the hospital setting you're basically removed both mechanically removing and killing 10 to the at least about 10 to the 3 of a bacterial of the load so you're reducing the risk of infection you're actually it's both mechanical and biochemical yeah 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 survey that about six months ago what proportion of patients not only dialysis catheters what proportion of patients who are on a general medicine surface have stuff in them that they warren born with and it's including guy out including catheters i see you know i see these pacemakers prosthetic hips things it's about sixty percent and that does not include bullets so which we have something which we have so i think so this is a big file actually what what scares the living daylights out of me is prosthetic joints actually prosthetic joint infection is one of the areas also where they've been using pcr there's been a lot of research looking piece of you are to identify pathogens but if this is a big problem now this is our world and i don't have a good answer but its 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 treatments day appliance there's a couple of directions one is pathogen directed therapy so probably probably the most important and the TV example is a very good one we know exactly what the bug is the characteristics so right now the approach to infections disease in most hospice it eases you know is broad spectrum which actually is the worst thing we can do for our floor so pathogen directly second focusing on drilling down on that in pathogen directly with antimicrobial susceptibility directed third 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 beguiling with flora and for example is there's been a lot of interest for years on correcting the bacterial flora and back to that correcting the floor and bacterial vaginosis with lactobacilli with yogurt which actually has lactobacilli with lactobacillus course pottis which is actually the specific one and all the studies have fallen flat on their face they haven't worked on the other hand probably the most successful one of reef flora in a mucosal surface has been in the case of seed if we're as my boss Tom Bartlett said the the therapy has is incredibly effective but has aesthetic complications and that is in people with severe recurrence e 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 were doing this now and you can imagine there's obvious regulatory problems with you know how do you do this with the FD you know with the FDA and safe and things like that but it works and you know it has you know and it's actually the way it's done is actually its tools taken from a relative and it's either instilled from below by somebody there up there's actually a drought their job opportunity so that people used to do barium enemas because you want to get it all the way up or through a long canter to and there's another I heard that there's a formulation being developed that we're actually can be in a gel cap it's sort of like it's going to absorb you know what you take but the capsule does not dissolve until it's down into the gut so so that's that that's a very good 0 i think but clearly you're raising for example we see this in your area track you know if we were interested in the wound you know we populate the flora with what what we want to see that's a lot of interest in that last question okay it's hard on individual basis you can't differentiate clinic on clinical yep needs on the settings so for example CDF will be clinically obvious for example anybody in a long-term care facility or who's been on antibiotics recently the recently as a nun you know has a nuance of diarrhea they've looted 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 and 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 accountant and they're usually self-limited so the utility here the diagnostic testing is understand the epidemiology as opposed to guide therapy okay thank you you

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