Relating Numbers of Foodborne Pathogens to Human Illness: Question and Answer Sessions - Dennis Lang

Food Safety Initiative Technical Workshop:

Relating Numbers of Foodborne Pathogens to Human Illness

Tuesday, August 4, 1998
Atrium, Stamp Student Union
University of Maryland
College Park, MD

Question and Answer Sessions

Open Discussion

DR. LONG: Welcome back for our final discussion this afternoon. I will remind you one more time that you have these yellow cards in your folders and Dr. Bradlaw and myself will be sort of walking the aisles and looking for people who want to pass cards to us if you'd rather put a card forward than stand up and ask a question.

This afternoon's panel discussion will be moderated by Dr. Dennis Lang. Dr. Lang is the Enteric Disease Program officer at the National Institute of Allergy and Infectious Disease of NIH. He's charged with the development, coordination and direction of the extramural grants and contracts program in enteric diseases, which includes the development of vaccines and therapeutic agents against enteric pathogens.

Dennis is a member of the Risk Assessment Consortium and a member of the Dose Response Work Group of the Risk Assessment Consortium and he has helped us out a lot and he's helping us out again this afternoon. Thanks, Dennis.

DR. LANG: I thought I would introduce this session by reviewing for myself, as well as yourselves, how I got involved in this process. I was asked to participate in the Risk Assessment Consortium as an NIH representative by virtue of the fact that I direct the Enteric Diseases program at NIAID. The organisms studied by our investigators and supported by NIAID are the ones that are of most concern in foodborne disease.

When I went to the first meeting I sat around the table with statisticians and epidemiologists and wondered what I was doing there. It quickly became apparent during discussions that there was a real lack of information to estimate the risk of the human population to foodborne disease.

The research that NIH supports is both basic and clinical research on the genetics, microbiology, pathogenesis, and virulence of these organisms and ways to control them. Mike Levine has been one of the pioneers in developing human challenge studies for the purpose of studying microbial pathogenesis and for measuring vaccine efficacy. Dr. Tribble alluded to a lot of the work that Mike pioneered, and to which others have contributed.

In performing such challenge studies we seldom work at low doses. The goal of such studies is to approach 100% attack rates in a small number of volunteers. We do not typically study a small number of organisms where it might take 1,000 people to be exposed in order to see an illness.

There is a real disconnect, in a way, between the real life human experience and available human dosing data. These questions are going to become important in defining the human risk of foodborne diseases which, for the most part, result from low dose exposures.

As part of the recommendations from the RAC committee, the FDA has published a request for applications calling for research to try to make the correlation between human exposure at low doses and infection and disease. Parallel work will also be done in animal models, with the goal of developing correlative dose-response curves for some enteric organisms.

They have received applications and I'm not going to say any more about them because that process is still on going. But suffice it to say, that there is going to be an investment by FDA in trying to make some of these extrapolations and measurements.

Therefore, the question to the panelists and to the people in the audience is what kind of recommendations can we make regarding the design of those studies? Which organisms should we study? What animal models are the most appropriate to use for comparison to the human data that we get? What kind of human data should we be looking for?

If we're not going to be seeing full-blown diarrheal disease, are there other end points such as shedding or immune correlates that would be a more sensitive assay that would be quantifiable and meaningful?

These are questions I'm almost certain we are not going to answer today. Part of the reason for having this meeting was to start a dialogue and get the people who, like Dr. Slauch, study pathogenesis in animals to talk with the people who are doing human challenge studies and come up with recommendations that are compatible, with an approach that makes sense.

I will just toss that out first to the panelists, whoever would like to comment about what they see as of common themes that have emerged after today's presentations about pathogenesis, and where we should be going.

Would anyone like to volunteer? Peg, why don't you start? Peg is our risk assessment person and probably as familiar with the trials and tribulations and limitations of risk assessment models.

MS. COLEMAN: Thank you, Dennis.

I'm actually very excited to see this kind of interest in dose-response modeling and to be hearing from the people who can generate data and help us draw inferences that really are plausible in our modeling.

So I agree, Dennis, even if we haven't generated a list of solutions today, we have started the dialogue. It has been helpful for me to hear from some of the basic researchers about what kinds of interests and their own research work might bear on dose-response modeling.

DR. LANG: I have a question, actually, that was generated in the audience that I'd like some response from the panelists. Is threshold dose determination useful for regulation, for regulatory agencies? Let us assume that threshold in this case is a disease-causing threshold and not a threshold of infection.

DR. WILSON: Let me ask, does this refer to some--is there some specific technique that leads to something called a threshold dose? Does that refer to some--is that a term of art in the microbiological business?

DR. LONG: I don't think so and maybe you should define threshold dose for us, for the audience and for the panel, as you interpret it.

DR. WILSON: Mathematically it's a discontinuity in the dose-response curve at the point at which the response as a function of exposure goes from zero to some value greater than zero. I don't think that defines dose in any way.

Within toxicology we talk about no observed effect level of no observed adverse effect doses, but as Dr. Gaylor would tell me if I didn't say this, all that means is that the response is less than the observational uncertainty.

So those two concepts of threshold and dose don't necessarily go together, as I understand them.

DR. LANG: Perhaps the person who asked the question might like to elaborate.

DR. GAYLOR: Actually I asked the question. I heard a lot of discussion today about what's the minimum number of organisms that'll cause illness or infection or maybe illness, and that hasn't served us very well in chemical safety assessment--how many micrograms or milligrams will cause an adverse effect and below that we're okay.

This is pretty illusive and it's coming up with a noninfective dose or a dose that doesn't cause illness--illness to whom and when and under what conditions?

I think it's worthwhile knowing whether 1,000 organisms or 100 organisms or 10 organisms, sort of an order of magnitude. I think that's worth knowing but whether three, four or five E. coli in hamburger are safe or dangerous, I don't think it's worth our effort trying to answer that question. Even if we figure out that three are safe for everybody, say; what about the person that's already exposed to three coli from eating lettuce? Where does that leave us with hamburger?

So there's a lot of regulatory problems even if we could answer the scientific question.

MS. COLEMAN: Can I add a little bit to that discussion? I agree with you. In fact, your language really implies that there is a distribution of thresholds for the population and there's not just one threshold, that each individual may have a threshold.

But the sense in which we've used threshold in our work was how many bacteria does it really take to cause symptoms? If you have one cell infected in your GI tract, will that cause you to show gastroenteritis? Or is it an effect of an accumulation of damage to cells that is necessary to cause symptoms?

So that's the sense that we've used it. And the example of a threshold of four was just meant to illustrate that even low thresholds can have tremendous effects in dose-response modeling.

So we weren't trying to presume that four cells were safe and three were not but that assuming that one cell can cause illness is quite an assumption. So that's the sense that I was raising that point. Should we really look a little closer at the mechanisms of pathogenicity to address that kind of issue?

DR. LANG: Any other comments on that question from the audience?

DR. TATINI: I would like to comment something on that. If theoretically one cell has the potential to cause infection and it's a question of whether this single cell present in the food can reach the target site within the intestinal tract, for example, maybe all problems in the threshold are related to how many should be there in the food such that at each of these barriers, as it goes through, how many of them survive? And if one goes through, that would be adequate if it has the potential to establish in the gut and cause the illness.

So maybe the threshold is related to what happens to these organisms in food. Can they reach the targets? If one reaches the [inaudible] illness, depending upon what [inaudible] the other conditions that exist.

DR. LANG: Another question. Where are the data describing the susceptibility of the human population with respect to the genetic components and their acquired resistance? There is probably very little data available in terms of the human genetic components involved in any of this, but perhaps with the human genome project, in another 10 years we'll be in a better position to assess that component.

But I'll ask Mike Levine to respond initially to that question. Mike?

DR. LEVINE: I brought a couple of slides--it's old technology apparently but we'll give it a try--that addresses this question. It addresses it in the following way.

Let's consider vibrio cholerae as a paradigm of looking at an organism that's incriminated as causing diarrheal disease in humans and let's just ask the questions of what's involved on the host side and on the bacteria side in leading to clinical response.

Let me give as a bit of background there was a guy named Robert Koch a bit more than a century ago who discovered vibrio cholerae in Egypt in association with a large outbreak of cholera, which he called the vibrio comma. He received great publicity with this observation.

There was a more senior individual in Germany at the same time named Max Pettenkoffer and he looked upon the publication of Koch and he decided that in his view, this organism by itself couldn't explain the epidemiologic and clinical features. There was something missing. This is the famous XYZ theory of Pettenkoffer.

And to make a long story short, Pettenkoffer, who was quite an elderly gentleman at the time, got into this very, very acrimonious debate with the younger Koch and sought to prove that Koch was wrong by drinking a pure culture of vibrio cholerae. He did it and he didn't get sick, which raised a few questions. And I'll show you some data that, looking back a century later, we now know why that was so.

About two decades ago we were asked by the NIH to set up a model of El Tor vibrio cholerae 01 biotype El Tor to demonstrate or investigate whether that biotype was a cause of diarrheal disease.

It had been demonstrated some years earlier that if you took vibrio cholerae 01, the classical biotype, and gave it to volunteers without buffer, just gave it to fasting volunteers, if you gave a million organisms or 10 million organisms, nobody got infected and nobody got sick. You had to go up to 100 billion organisms and really enormous amounts to get a healthy, fasting young adult ill. But it was found that if you buffered the gastric acid, suddenly a 10⁶ infectious dose was a 90 percent clinical attack rate dose.

So we looked at an El Tor strain called El Tor Naba N1691 and for the first time with buffer, carried out a dose response in which we went down, went down to 10³ of the three. And even at doses of 10⁴ and 10³, the attack rate remained high--80 at 10⁴, 67 percent at 10³. But if you look in the far right column, the severity of illness in these small numbers went down as the dose went down.

These data were important because this was the first time that there was a link between volunteer studies and the epidemiologic data, which suggested that in the field in endemic areas like Mafla Bazaar in the Kamilla district of Bangladesh, the infecting dose of vibrio cholerae in nature is probably 10², maybe 10³ organisms. Next slide, please.

Now the next thing we did was to ask, with this dose bicarb that causes a 90 percent clinical infectious disease attack rate, what happens if we give the same inoculum with plain water--in this instance 300 milliliters of water in which the inoculum is suspended, given to a fasting volunteer--or if we give the same inoculum, 6 logs with food. The food is a sort of quasi-Bangladeshi meal of fish, rice, custard, a little skim milk.

And with buffer, 90 percent attack rate. With water, just water, no buffering, nobody got infected; nobody got sick.

Now, this is with distilled water--not distilled--with sterile water in which we put the inoculum. In nature there's a lot of incrimination epidemiologically of water but in those instances, sort of river water, for example, or tank water in Bangladesh, that water has zooplankton. It has entities to which vibrios can attach and it's possible that those zooplankton suspended in the water get the organisms through. But just water by itself will not do the job.

And a meal, food, gives you the same clinical attack rate and the same degree of severity as buffer. Next slide, please.

Now, the next slide, which is probably unreadable, just makes the point--it's a listing of a bunch of different strains. This is one of the most important points that I would make. We have tested, over the past two decades in volunteer model probably 10 different wild-type strains, maybe more. And what is quite interesting is that there is no in vitro assay, there's no animal model that predicts what will happen in humans.

And what you see is that some strains are red hot in volunteers, cause very severe illness. Other strains that do the same thing in rabbit RITARD model will cause moderate illness and others will be virtually nonpathogenic or will cause a mild illness that we would not clinically call cholera. Next slide, please. So that's some food for thought.

Selection of the strain going into volunteers is very important and I would say if you want to look, if the question is is such a genus and species a disease-causing pathogen, if you want to answer that for humans, you can't do that with a single strain. You have to look at multiple strains.

That was the pathogen side. Now we have the host side and here, this is also very important in terms of genetic susceptibilities and other susceptibilities.

We know, for example, that gastric acid very much determines severity of cholera. We also know today that blood group O is the single most important host factor that determines severity.

Nutrition status plays a role in developing areas. The more malnourished, the more the severe illness. And background immunity also plays a role.

So all of these, as asked of the question, all of these play a role. Next slide, please.

I want to show you some data with blood group to give an example. Actually, before getting to blood group, hypochlorhydria or low gastric acid can come from several different sources.

One of the things we found back in the 1970s when our volunteers were University of Maryland students, there were a lot of what we used to call grasshoppers. Dave Nalen, who was involved in these clinical studies, first came upon the possible association. In rounding with these volunteers he came up with the suggestion that it appeared that the heavy grass smokers--gave a history of heavy grass smoking--had more severe illness.

So over ensuing studies in a prospective way, he looked at that and in this Lancet publication clearly showed that severity of diarrhea in that model was related to the proclivity or the history of how much grass you smoked, which was, in turn, related to gastric acid because marijuana diminishes gastric acid secretion, and David showed that quantitatively. Next slide, please.

Let's go to a more biological host factor. This may not portray very well so I'll summarize what it shows.

In 1991 cholera came to the Western Hemisphere, returning after an absence of about a century. We knew by this time, from studies in Asia and in volunteers, that blood group O is a critical risk factor, making an individual much more prone to developing cholera gravis, severe cholera.

The lowest prevalence of blood group O in the world, as you might guess, is in the ancestral home of cholera, in Bangladesh, lowest prevalence in the world. The highest prevalence of blood group O in the world, unfortunately for 1991, is on the west coast of South America, where the native South American Indians have a blood group O prevalence of 85 or 90 percent.

So the cholera appeared to be quite severe, appeared to spread and we took some of those strains early on and fed those to volunteers, North American volunteers, and asked the question of whether there was a relationship in North Americans between blood group O and severity.

And in these studies, which summarize results with a couple of different strains from South America, the attack rate in persons of blood group O was 93 percent; the attack rate in non-O was 44 percent. The mean diarrheal stool volume in the blood group O was 5.23 liters, more than five liters. Five liters is an adult human blood volume. This is cholera gravis.

The mean stool volume in the non-O was less than two liters. The number of individuals--of the 15 individuals who were challenged who are blood group O, one-third of them developed, by our definition, cholera gravis, a five-liter purge, and of the non-O it was zero of nine.

So in the North American volunteer, as well, we see the genetic susceptibility playing a critical role in the clinical response. I just thought I'd show some of these data as a response to whoever asked that question and I'd be happy to expand with respect to any other organism they might be interested in.

DR. WILSON: Let me follow up with that and perhaps if people don't know, perhaps you could speculate.

It looked as though the distribution of susceptibilities, at least as measured by severity, was not log normal, was not a simple distribution. It was at least bimodal in some of the other talks that we heard this afternoon.

In contrast, although the theory for dose response to chemicals is not very well developed, it's beginning to look as though a log normal distribution does characterize the susceptibility of people to chemical toxicity.

This is not going to be a log normal distribution where you have all the O's here and everybody else over here. Is that likely to be the rule or at least a common occurrence?

DR. LEVINE: I think that with many organisms, that will turn out to be true. There are other examples. Helabactapilori, for example, where Lewis blood group B are the individuals to whom the helabactapilori preferentially adhere and cause a chronic infection.

There can be indirect effects, as well. You can have a propensity--for example, helabacterine infection by itself may modulate the susceptibility to other enteric pathogens by modifying the gastric acid secretion.

We know from Shigella studies that when you give Shigella without buffer, there's a rather flat curve. There's not a whole lot of difference between two logs and four logs. When you give Shigella with buffer, you suddenly make it a very vertical--a quite striking attack rate.

So there are ways of kind of leveling the playing field so that dose starts to appear. I think the more that we look at bacterial enteric pathogens, the same may also be true of viral; the same is to a great degree true of protozoal. As we look at organisms, we'll find that there are genetic factors that lead to increased infection and/or severity of illness.

DR. LANG: Does anyone else on the panel or the audience want to comment on that issue?

I should point out that in the human controlled feeding studies we purposefully select very healthy individuals for those studies. In fact, there is a whole list, and Mike can attest to this, of exclusion criteria to eliminate people that would be predictably more sensitive to the effects of a particular organism.

In a way, we are stacking the cards against detecting the utmost susceptibility of the human population to these organisms when we do those controlled feeding trials.

For ethical purposes, we are never going to be in a position to do otherwise. The best we can probably hope for is to model animal systems where the animals can be genetically or experimentally manipulated to more closely mimic susceptible humans. Extrapolations of animal data may approximate what would happen in a comparable human.

Does anybody want to add to that? Any of the animal model people? Jim, you spoke a bit about the Salmonella genetic system and also somewhat on the other, the Listeria system, as well.

DR. SLAUCH: There was a recent paper and the reason I'm reluctant to talk about it too much, because I've only read the abstract--reported looking at humans that had come down with Salmonella or other diseases and looking at genetic markers and suggesting, in the abstract, anyway, that the markers that they're finding do correlate with the mouse markers that also suggest disease.

So I think that's the first hint that genome-wide scanning, that there is going to be a correlation, as there are in many other diseases, what we've learned from animal models, that we can manipulate and apply that maybe directly to human studies.

DR. LANG: At least to identify potential humans that would be at increased risk for particular infections.

I'm wondering about the other question. Perhaps the epidemiologist from Minnesota can answer this.

One of the things that we discussed in the Risk Assessment group is the more intensive study of natural foodborne outbreaks as they occur. We need to get involved in more active data acquisition of those populations, not only the people that come down with the disease but those people that have sampled the same food but were not ill. We need to look for immune responses to those organisms, get more proactive about blood collections or data analysis on both the ill and the non-ill from those occurrences.

I wonder if you could make some comments about what that might tell us about human susceptibilities.

DR. SMITH: I think it would tell us a lot. I think the information is out there, especially in these big multi-state outbreaks where we have hundreds of cases.

I do think the information is there. It's just a matter of getting it, asking the appropriate questions to the appropriate people and getting the stool cultures on the ill and the exposed but non-ill and so on and so forth. I do think there's a well of information out there but coordination among all the participating states and the federal agencies that are helping out, I think is very critical.

I mean, all of this obviously needs to be done in a very timely manner and I think it can be, but I don't know if it will be. That's the thing. You know, whose responsibility is it to coordinate all this to make sure all the agencies are coordinated and getting the most--aggregating all their data?

DR. LANG: Is Morris Potter still in the audience? Did he leave? Sorry about that. Maury would have something to say about that from CDC's perspective, I'm sure.

I think that the CDC is becoming more aware of foodborne outbreaks and taking a more proactive role in terms of the Foodnet surveillance and Pulsenet identification of strains. I think we will be in a position, if we're not already, to become more involved early in those outbreaks as they occur. Hopefully, we will be able to generate some of that data.

MS. COLEMAN: Can I expand on that before you move on? It seems also that we ought to talk a little bit about sporadic illness with Campylobacter and how might the passive systems and the active systems be combined to generate some information.

DR. LANG: Question, Dave? Introduce yourself.

MR. NAYLOR: One of the possible ways to tie all these threads together might be it is true that there's a relatively small number of strains that have been tested in human volunteers, and maybe cholera is the one exception, where maybe a dozen or so strains have been tested but we're talking about just a couple of strains completely useful to test one of these newly emerging outbreak-associated or non-outbreak-associated strains.

If we have a larger battery of strains and then we did find that they were variable in terms of their pathogenicity, then to go back and try to tease out what makes these hotter strains, that probably would be a very useful thing to do. To have a larger number of strains as we start thinking about ways to prophylax, et cetera, we would have a strain, number of stains available.

DR. LANG: Maybe that question or that comment is an introduction. I cannot withhold saying something at this point about a recent NIH effort, at least in terms of 0157:H7 strain collection, that might be relevant to the comment that Dave just made. We are in the process of setting up a reference collection of bacterial strains. This collection will serve as a reference for people to use. The reference collection will contain 0157:H7’s and non-0157 STEC strains.

The criteria for entering strains into that collection will be that they come from very well characterized outbreaks and from clinical cases, ones where the patient, clinical outcomes and characteristics are well known. Some of those strains already exist. Others will be collected from future outbreaks. This collection will be maintained at Penn State University. Dr. Tom Witham is the Principal Investigator for this project. Data will be maintained on a website. Strains will be a characterized by a whole range of biochemical and genetic assays, gene sequences, and will include results from in vitro assays and animal studies.

With 0157:H7, an attempt will be made to correlate measurable strain characteristics and a range of clinical outcomes. Whether a similar approach may be appropriate for some other organisms, is open for discussion.

DR. KOPECKO: There's a piece of information that's missing for me. I'm not sure how much is known. We're talking about testing different strains of a pathogen in humans, setting up some animal models, mixing them with various types of foods. The number of volunteers, the cost of doing these kinds of things would get to be enormous with the large number of pathogens involved and the large number of strains involved.

When one thinks about enteric pathogens in food, I just wrote down a series of things. There's certainly vibrio cholerae, vonificus, maybe perihemaliticus, perhaps a couple of other vibrios, a variety of Salmonella strains, Campylobacter, Listeria, Shigella and hemorrhagic E. coli, pathogenic E. coli and toxigenic E. coli, helacobacterin.

Perhaps to get a better handle to help prioritize, what information do we have currently on the amount of disease burden that each of these pathogens causes from foodborne outbreaks? What does that translate into in terms of lost work days, physician visits, cost, mortality?

And then, knowing that for any group of organisms, set up a priority list and start targeting those things that are the biggest problems, if that's possible.

Anyway, I raise that as an issue because looking at all the pathogens and different strains of each and running a variety of tests seems like an onerous task.

DR. WILSON: Let me say a word for expert judgment. Doing what you suggest systematically is probably beyond the resources, if it's even possible. I think it's probably good enough for present purposes to ask the people who are involved with this which ones stick up the most and which ought to have the highest priority.

DR. SMITH: Well, I think the Foodnet program actually gets at some of that. I mean, you have incidence of the major bacterial pathogens. We know that. They also look at the percent of the cases that are hospitalized. I don't think they get into the amount of detail as to how many days of work are lost and so on and so forth. But we do have some, I think, relatively crude indicators as to what are the important pathogens.

DR. KOPECKO: Could you list the top four or five?

DR. SMITH: Oh, I probably could. Just in the way of incidence, Campylobacter is the most commonly recognized cause of bacterial enteritis in the country. I don't think there's any question about that. Salmonella, I think, would be second.

E. coli 157, of course, is much less common but you'll have higher mortality rates, et cetera, in children and more life years lost and so on and so forth. So you have to balance a lower incidence with the more adverse outcomes and so on and so forth. But those would be the three. I'm trying to think. Shigella is also quite common so it would have to be thrown in the mix, but a lot of that is more person to person probably than foodborne.

MS. COLEMAN: Shigella or Listeria might actually be [inaudible] 0157:H7 perhaps or in the '97 data.

DR. LANG: Any other comments before we move to another question?

DR. SLAUCH: That's for the U.S. This is a federally sponsored meeting, so it's pertinent. In Japan vibrio parahemoliticus jumped right to the top of the list because there's so much raw seafood, more seafood eaten.

So again, as far as the FDA is concerned, that may not be relevant.

DR. LANG: Mike, what is the experience with other vibrios and human exposures? Anything other than cholera?

DR. LEVINE: Glenn Morris did some studies with non-01 Vibrio cholerae in a couple of different strains that were published in the Journal of Clinical Investigation seven or eight years ago, I guess. There's limited data.

In terms of disease burden and surveillance, I would just make a caution. For certain organisms the surveillance is relatively easy because the clinical bacteriologic methods allow a ready, simple selection, like a Shigella, like different types of vibrios.

If I asked you what the burden of disease was for enterotoxigenic E. coli or enteropathogenic E. coli or enteroaggregative E. coli in Minnesota, I don't think you could give me a fair answer because toxigenic E. coli, for example, most of that diarrheal disease is going to be indistinguishable from other agents causing a relatively mild illness and unless special surveys are carried out, there's not a quantitation of the burden.

Now, this was not very important until recently, when there's been a change, a globalization in the food system. Now when you go into a supermarket in the wintertime, for example, in Maryland, most or much of the fresh produce comes from Latin America.

We have a very global food supply and I think it's time once again to carry out systematic surveys like were carried out in the early 1980s before there was this globalization, and the surveys at that time suggested that toxigenic E. coli, for example, was very, very rare as a cause of enteric disease in mainstream U.S. population. I would bet that right now things are different.

DR. SMITH: I think that's apparent. We actually are, through Foodnet, taking steps to try to look at that, culturing every stool collected from a health maintenance organization and so on and so forth and we do probe for the enterotoxins and so on and so forth. We're planning to do that definitely.

But you're right. My boss, Mike Osterholm, likes to say you can get traveler's diarrhea without leaving home. That's true. Most of our produce, especially seasonally in winter, comes from Center and South America.

DR. LANG: Okay, I'll move on to a different question. This is for Dr. Tribble.

What was the reason for undertaking the Campylobacter jejuni trial and what did the human review board--IRB, say about the possibility of Guillain-Barre syndrome?

DR. TRIBBLE: Well, as I said in the discussion, the reason we were doing the trial was--the first objective of the trial was actually to go back in and modify the model that we used, that was used in the CVD study, for the main purpose of actually evaluating a vaccine candidate, so that was the reason for the trial.

As far as the Institutional Review Board, as I said, ethical concerns, volunteer risk are major issues related to making decisions to go forward with the steps of experimental infection studies.

As far as that particular strain that we used, it actually never had a post-infectious sequelae associated with it in the outbreaks and the prior infection studies and it's actually used to kill host cell fast strain for the vaccine.

So it's been given, as well, to those individuals, but that would probably not be an adequate thing to make someone feel necessarily all that safe about that aspect because of the fact that the event, such as GBS-associated Guillain-Barre-associated disease with Campylobacter is probably a fairly rare occurrence as a whole. So that would not be enough to make someone feel that comfortable.

So we actually did do some screening with Tony Moore at the University of Galway in Ireland, who has done a lot of work on the LPS outer structure, the mimicry that is the main hypothesis for what the association with Campylobacter-associated Guillain-Barre is and actually looked at the specific gangliocytes that seem to be associated with that, such as GM-1, GD-1A and some of the other gangliocytes, and we actually did not find any of that mimicry, you know, on that specific strain that we used for the challenge, which really increased the complexity of going forward with that trial.

I think that's just another point showing that some of the issues--I didn't go through all of that in detail for the purposes of this discussion but there's a lot of issues that have to be considered to go forward with these types of studies.

And then we, of course, informed volunteers about that process. So that's what we did for the trial.

DR. LANG: Peg, this is one for you, I think. Please comment on the existence of and access to statistical methods for dose-response modeling.

MS. COLEMAN: Well, probably the first paper that people cite when they start talking about dose-response modeling is Chuck Haas's paper from 1983 and Chuck is a professor at Drexel University and he continues to be working in this area and is active in the Society for Risk Analysis.

I think that probably the discussions that start on Friday about the Risk Assessment Clearinghouse, that that's a vehicle to address that question, to actually put some models available on a web page that the clearinghouse would manage so that people can test them.

I'd also give a plug for the SE risk assessment. The report, I understand, is on the FSIS web page, though I've heard some people have trouble downloading it. It is a 250-page document so it is also available in written form. And the intention, I think, in my agency is to put that whole model up on our web page so people can download it and play with it themselves. But that presupposes that the users have Excel and At Risk. So those are the pieces of software that we use for that particular assessment.

But people in the field use all kinds of different software systems. You can do this programming in SAS and basic programming languages, in Mapcad, in Matlab, Mathematica Analytica. There are a large number of commercially available software packages for actually doing the modeling, which is why I have a Mathematica collaborator.

DR. GAYLOR: With the precision of the data that's available, a piece of graph paper and a French curve will work pretty well, too.

DR. WILSON: And the basic Mathematica models have been available for decades, at least since SAS was invented. The models themselves are not hard to come by. The kind of stuff that's in the egg evaluation has more to do with propagation probabilities in one step of the process to another. That's more difficult to do but that's not dose response.

DR. LANG: Do we have any other questions or comments from the audience? Everybody's falling asleep out there. We're doing all the talking up here. And we're running out of things to say.

There's a brave volunteer.

MR. ZHU: I'm Yiliang Zhu of the University of South Florida. Just by listening to the presentations and looking at the data that are presented in today's workshop, I think several issues surface about dose-response modeling.

One issue seems to be the large variation in data measurements in terms of the estimation of the number of organisms and so on.

The second issue is typically very small sample size in terms of animal study and human volunteer studies.

The third issue seems to be the large extent of individual-specific susceptibility and dose thresholds.

Now, I don't think there are good statistical methods that are powerful enough to give you reliable estimation of dose-response relationships, given the nature of [inaudible].

I was involved in an NIMH-sponsored intervention program in which it was done by a social science researcher. The story is the two physicists went to other social scientists who are neighbors to talk about the research and when they asked the sociologists what are you doing now, the sociologist says, "Well, we are still collecting data" and the physicist says, "Don't you have about 10 years data?" and the sociologist said, "Yes; in six months we're going to finish collecting data and in another six months we're going to complete the study and then we'll have another project in which we'll collect another 15 years of data."

So the physicist says, "Well, that's interesting. It looks like you are the least efficient people in analyzing data. We typically collect one year of data and then spend the next five years studying the data."

Okay, the point is that I think it's absolutely correct and fascinating that we decided to collect more data, get enough information so that there will be a basis for dose-response modeling but, on the other hand, dose-response modeling could only be as good as the worst segment of the whole process.

So if there's not powerful enough and adequate statistical methods, then your dose-response modeling will be not as reliable as we wish.

So in other words, it's my opinion that people here, the risk assessors, toxicologists and microbiologists, pathologists, probably we should look at the available statistical method and statistical literature. For example, there was recently developed a method to talk about how to analyze or develop the dose-response model with relatively small sample size, with large individual to individual variation and so on.

So that's my comment. So I don't believe that we are in the field utilizing the best available methods yet. And I think that that's also an important component of this assessment process.

DR. WILSON: Correct me if I'm wrong but I believe that these methods bring in ancillary information other than that which is extracted from the kinds of studies that have been described here. A very good thing to do, I might say.

MS. COLEMAN: And I'd also say that there are agencies that are doing microbial risk assessment right now, so the whole point is to be able to work from the data available and to describe risk but also to describe uncertainties and that maybe that's the answer and not necessarily whatever risk estimate you choose to propose, but how much uncertainty is really associated with that.

And there is going to be a lot of uncertainty at low doses, even if we do use data from animal models or in vitro studies to give us more information at the low dose end.

DR. LANG: Does anybody on the panel feel like they are in grasp of what has been said today sufficiently to summarize where we are and what the recommendations for going forward should be?

Well obviously, the complexity of this problem is a daunting one and I think you could easily get very discouraged if you look at the breadth of the problem and how much information is lacking.

The challenge that we all face, I think, is not to be discouraged by that, but to look upon it as an opportunity to think about new ways or additional ways, additional bits of data that can be collected, to start to address these questions.

Clearly, there are human studies being done wherein human volunteers are challenged with enteric pathogens. These studies are being performed at the University of Maryland and other NIH supported clinical facilities, and by the Department of Defense at different locations. There are also lots of people working on animal models and with in vitro assays with the same organisms. One of the keys, is to get all of these people collaborating so that the same organisms, prepared in the same way can be used in their respective experiments so that correlative data with different models can be obtained and more directly compared to human challenge studies. I think the challenge we face is to try to make connections between those systems and it is a difficult problem but I think we are taking the first steps in that direction today.

Since the audience is dwindling and eyes are closing, we'll wrap it up at that point unless anybody else would care to add two cents worth.

If not -- oh, there's a hand at the back.

VOICE: I would just add that the conduct of risk assessment in this area is not discretionary. It is going to be done. It's a question of how can it be done better.

DR. LANG: Okay. With that, thank you all for your attention and participation.

[Whereupon, at 4:20 p.m., the workshop was adjourned.]

Relating Numbers of Foodborne Pathogens to Human Illness: Question and Answer Sessions - Dennis Lang