Center for drug evaluation and research



Download 373.06 Kb.
Page5/6
Date conversion21.02.2016
Size373.06 Kb.
1   2   3   4   5   6

Serous pleural effusion, often in great amount, pretty equally in both pleura, usually unaccompanied by any signs of pleural inflammation, and in the lungs the changes are but slight.

So we've unfortunately had an opportunity to rediscover this disease with Sverdlovsk,b ut it was well known to the physicians of the early or late 19th Century.

Slide off, please.

Now, there have been, as you've heard, very few cases in the U.S. There are some 18 reported cases, a few others throughout the world. Up until Sverdlovsk about 100 cases worldwide, and now with Sverdlovsk maybe 200 or so in humans that have been reported.

There have been several animal models that have been used to study this disease, but the non-human primate, particularly the Rhesus macaque, has been the model that has been used in the '50s and again more recently.

We've had an opportunity to study this because after the Gulf War began, we were asked to address a simple question: how to treat someone who had been exposed to an aerosol of anthrax spores?

And as a result of that, we generated some additional data which, together with the previous data in the non-human primate, gave us some more information about the pathology of this disease.

The next slide, please.

I hope you can see this. If not, I'll point out the highlights here. Is that a little out of focus?

PARTICIPANTS: Yeah.

DR. FRIEDLANDER: Good, good. And I'll just go over the highlights here.

Intrathoracic lymph nodes. There are two tables I'll show. These were compiled by Dr. Gary Zauchar, a veterinary pathologist at USAMRID, and they summarize the experience in the literature of the human disease and in the Rhesus monkey. This is all data at USAMRID, 25 animals, which includes the controls from the experiment I will describe, plus some others.

Intrathoracic lymph node involvement. Of the 72 cases he could find in the literature, that is to say the 41, 42 from Sverdlovsk, plus about 30 others, about 90 percent; in Sverdlovsk this was 100 percent.

In the Rhesus, about 80 percent of animals have involvement of the intrathoracic nodes. In the mediastinum, various changes have been noted in about 80 percent; again, in the Sverdlovsk series 100 percent.

Here this is somewhat lower, about 40 percent.

If you look at primary pneumonia, 30 percent, and this does include as pointed out by Dr. Walker nonbacterial pneumonia, that is to say some hemorrhagic pneumonia without bacilli being present.

In the Rhesus it's about 16 percent.

In that regard, I should point out that there is some old data in the Rhesus macaques from the '60s that were infected with mites in the lung, and a characteristic lesion was described, not unsimilar perhaps in some cases to the arc welders. That is to say localization at the site of previous damage in the bronchial where that may be a source of entrance of the organism where it could persist.

In terms of the brain, the total CNS involvement, about 50 percent, and again in the Rhesus about 50 percent.

There are some differences in terms of mesenteric lymph node involvement. Whether these are due to how careful the specimens are looked at, of course, is very difficult to tell.

And as was pointed out, there's one other point to mention here, and that is the survival time in the humans versus the various animal models.

In the Rhesus macaque, this is the average, about 4.8 days, five days post exposure at the time of death. That's about the same post onset of illness to death in the summary of all the human cases reported.

So there's a longer incubation period. There's a longer time to death in the human model than there is probably in the primate.

Now, that has to be couched particularly with Sverdlovsk, in my view, that we really don't know the details of these cases. We really do not know. There's been disinformation that's been given out before, as you're all well aware, and we don't know the specifics of the treatment of all the case as fairly quickly, as I understand it, after the diagnosis was made at least household contacts and others received antibiotics.

So it's unclear whether the longer incubation period is, in fact, modified by individuals once this was known to the community going out either on their own or through the physicians and getting antibiotics.

Next slide, please.

In regard to the pathology, there's one point I'd like to make, and that's this question of the relationship between the duration of the illness and the pathologic findings, and if you just concentrate on the Rhesus monkeys here, these are the total number of animals. They're small numbers, but there's a suggestion.

Here's the mean survival time, from three days out to seven to eight days. As the animal survives longer, the incidence of mediastinal disease increases -- there's only one animal out here -- as you might expect.

That is to say if the disease is first in the node, the longer the animal lives, the more likely it is for it to spread to the mediastinum and for you to see the pathological changes at autopsy.

And, similarly, if you look at these six animals that died on day three, CNS involvement was 17 percent. As you go to day four, five, six, seven, the incidence of CNS involvement again goes up, suggesting the longer the animal survives, the more inflammatory cells you see and the more extensively disease is, approaching more that of the human.

Next, please.

Now, this shows some examples of the characteristic finding to be anticipated in this disease, and that is the widened mediastinum.

Next slide, please.

Relatively clear lungs.

This is another case, again, a widened mediastinum with pleural effusion.

Next.

This is an over penetrated chest X-ray of a Rhesus macaque. Here's a normal animal, and here's the widening of the mediastinum that, again, is really quite evident.



Next, please. Could you -- that's okay. I'm a little disoriented here.

This is the trachea. The head is here. Phrenic nerves. That's the business end of this disease, very analogous to what you saw Dr. Walker present in the human cases, that hemorrhagic enlarged node.

It's difficult to tell about this glistening edema of the mediastinum. Unless it's hemorrhagic, it's not something that one would easily pick up.

Also notice the pink lungs. So this is the disease we're really talking about. It's really mediastinitis.

Next, please.

This is the same brain that must be traveling around the world now very frequently from a case in Sverdlovsk.

(Laughter.)

DR. FRIEDLANDER: Next, please.

This is one of the Rhesus monkeys, entirely comparable lesions.

Next, please.

Now, as soon as the Gulf War, as I said, started -- you can take that slide off -- we were asked to design studies to address this question. There had been prior studies in the literature by a group in England, as well as the U.S., that attempted to address this issue in the Rhesus monkey model, and they used post exposure antibiotic treatment for varying periods of time, for five days and ten days, and what they discovered is that the animals survived while they were on treatment, but once the treatment stopped, the animals died of anthrax.

There was one experiment done with a 20 day course of antibiotics. It was complicated by various other infections that the animals had. Again, about a third of the animals died, but they were very small numbers.

What they did show was that if you gave antibiotics a vaccine, you did protect the animals post exposure.

Now, a rational treatment of inhalational anthrax has to take into account a couple of obvious facts that I think most of you are now well aware of, and that is that the spore, as we know, can survive for decades and probably hundreds of years in the environment, but it also can survive in the host for extended periods of time, and that creates a very difficult therapeutic situation because while it can survive, once you discontinue antibiotics, the spore may then germinate.

And this was really established by Barnes in 1947, and that's what the next slide shows. I should point out that in the first paper by Abraham, Chain and Florey on penicillin in 1941, one of the organisms, in fact, they looked at was Bacillus anthracis, and shortly after the first human cases in '44, I believe, were treated cutaneous with penicillin.

Barnes studied this in the mouse and pointed out that one of the main factors in the therapy of inhalational anthrax is the persistence of spores in the tissues and their germination after the blood penicillin level has fallen, and that remains the dilemma that we have.

Next slide.

And this is another issue that also remains an unknown. Unfortunately we have lots of unknowns, no assurity. That is, the conditions which govern the germination of anthrax spores in vivo remain completely obscure.

I should say they're almost completely obscure in vitro. People are just now beginning to look at the germination genes in Bacillus anthracis, and an operant that's involved in germination, at least one set of genes involved in germination has recently been discovered. But we don't know what causes the spore to germinate, why it sits around, and why it may at day 20 or 30 appear to germinate.

Next, please.

Now, this idea was given further empirical support from the data of Henderson, and what Henderson showed was that in the Rhesus monkey, you could recover spores. These were done in treated and vaccinated animals, but you could recover viable spores for extended periods of time.

And what I've done here is basically plot. This is from Henderson's data. He had data showing about 15 percent of the inoculum surviving at 42 days, about two percent at 50 days, and there were traces even at 100 days.

Excuse me. I just want to get water.

And what I've done is show this graphically here. If you start with ten LD-50s, you're below an LD-50 at about a month or so, but if you're up at 100 or 1,000 LD-50s, even out at two and a half months or so, you're still above an LD-50.

Now, there are a lot of assumptions based on this data, but there is support for it both from Henderson's work and as I'll show you from our work that animals can die after an extended period of treatment when you stop the antibiotics.

So I wouldn't put too much credence in the -- there are no error bars here. There are few animals. Nevertheless, conceptually the idea, I think, is a valid one.

Next, please.

So this basically summarizes the point. The spore may persist in a viable but ungerminated state for extended periods of time, and that antibiotics do not act on the spore. They act only after it begins to germinate.

So we sought to determine, as I said, whether a more prolonged course of therapy could be effective alone or in conjunction with vaccination. Dr. Meyerhoff asked me to describe briefly some of the effort and urgency that went into performing this experiment. Given the nature of the events that occurred during the Gulf War, I can tell you that we were truly on a wartime footing working seven days a week for months to try to get this experiment done as quickly and as well as we could because an answer needed to be given.

Next slide, please.

This shows the chronology of the events. Iraq invaded Kuwait on the 2nd of August. The first challenge in two Rhesus monkeys took place on the 29th of August. During that period of time we had to develop an aerosol model for the monkey. We had not done that at USAMRID in modern times.

We had to get 68 monkeys from around the country, in addition to writing animal protocols and getting them approved. That was a lot easier actually than getting the monkeys.

And during this period of time, we also performed a preliminary pharmacological study because we had very limited data, and that took place about the same time.

So within about two weeks we demonstrated that we could aerosolize spores with a lethal dose in two monkeys and did preliminary pharmacology in six monkeys, two each with two antibiotics, and the experiment began on the 13th of September.

Next slide.

There were more than 60 people that were involved in the design and the implementation of the experiments. There were 68 monkeys used, eight in the preliminary experiments and 60 in the post exposure prophylaxis experiment.

There were 3,780 courses of anesthesia given to these monkeys; 1,550 quantitative blood cultures; parenteral medications, 720; oral gastric medications, 1,920.

One animal died from aspiration pneumonia, and one animal died from unknown causes. It really was an effort for the veterinary support and the animal handlers here, I think, to accomplish this so quickly and without significant side effects really to the animals.

Next, please.

This is the experimental design. It was quite simple. On day zero the animals were challenged with eight LD-50s, lethal dose 50s, by aerosol. Day one, treatment was begun with antibiotic alone, vaccination alone, or the combination in one group. This is actually day 31. They got 30 days of antibiotics, and then it was discontinued.

They were rechallenged about three and a half months later with a higher dose, 50 LD-50s by aerosol.

Next.


There were ten controls. They got saline. They were basically a control for the penicillin group. They got saline intramuscularly every 12 hours beginning one day after exposure until the time of death.

The penicillin group, there were ten animals treated with penicillin G, IM every 12 hours for 30 days. This dose -- I'll mention that in a moment.

Ciprofloxacin, there were ten animals treated at a dose of 125 milligrams every 12 hours, again, for 30 days.

Doxycycline, the same regimen, except 30 milligrams by oral gastric tube every 12 hours for 30 days.

Doxycycline, the same regimen, but in addition, they got a half an mL of the AVA vaccine on days one and 15 following aerosol exposure.

There was another group that just received post exposure vaccine on days one and 15 following aerosol exposure if they survived. As a control they received water by oral gastric tube every 12 hours. The oral gastric medications required anesthesia.

On the basis of the initial pharmacology, which was based -- there was no literature that we could find about tetracycline and penicillin in the Rhesus. There was a little bit of data on ciprofloxacin. We based the dosage on body surface area and modified it slightly based upon two animals per group.

So that we upped the dose of penicillin a little bit, of doxycycline, and we gave loading doses of ciprofloxacin. The first dose was double the dose, and this was based just upon the initial two animals.

Next, please.

I just point out a couple of things here. There were daily blood cultures from the untreated controls in the vaccination groups until death or for 14 days. In the antibiotic treated groups, the blood was cultured every other day until 80 percent of the controls died, and then twice weekly until day 30.

When the antibiotics were discontinued, the were cultured every other day until day 60, and then once a week until rechallenge.

ELISAs were done. All of the animals were observed at least twice daily until death or euthanasia, and a diagnosis was confirmed in all of the animals that died by isolation of Bacillus anthracis from the blood, and in all the deaths in which the cultures were negative, cultures were obtained at autopsy of the blood, spleen, liver, lung, intrathoracic nodes, and brain.

Next, please.

The antibiotic sensitivity test that we performed with this strain showed that in Mueller-Hinton broth the MIC was 0.08 micrograms for penicillin. For ciprofloxacin, this strain, 0.08, and the values for doxycycline are given here.

The NBC was equivalent to the MIC for ciprofloxacin.

The serum levels were determined by bioassay. Peak levels were determined at one hour pos dose for cipro and two hours for penicillin and doxycycline after doses on day five through 30, five, nine, 20 and 30.

The trough levels were determined 12 hours after a dose.

Next, please.

In the central panel, which you can make out in B here, this is a log scale of the geometric mean serum levels. I mentioned that, I think in the read-ahead package. As we discovered, this is presented slightly differently as arithmetic means that you may hear about.

But the MIC was 0.8 about across here, and these are the geometric means of the trough on day three, five, nine, and 20, and as you can see, they're above the MIC. The trough is for all -- throughout the period of the study.

These are the peak levels, the means, and then they are at least tenfold higher than the MIC and MBC throughout the course of the experiment.

Next slide, please.

And that's just reiterated here. The actual values, geometric mean, peak levels were between 0.98 to 1.69, while the trough levels were between 0.12 to 0.19 micrograms per mL, and the MIC and MBC for this strain was 0.08.

Next, please.

This shows some of the findings in these animals. This is the control group. Nine of the ten control animals died, with the mean time to death of 5.6 days. This issue that animals are not ill until time of death is fallacious in the Rhesus macaque. These animals are ill. They're ill for anyplace from one to four days before death.

There's decreased spontaneous activity. They go off their feed. They're weak. They're anorexic, not unlike the situation in humans.

Bacteremia occurs for a mean of 1.8 days before death with low to fairly high levels, ten to the one to ten to the fifth colony forming units per mL.

Terminal bacteremias are usually quite high. There was one animal with a low terminal bacteremia of 200 organisms per mL that had meningitis with two times ten to the seventh CFUs per gram of brain tissue. Five of nine of these animals had gross findings of mediastinitis and intrathoracic, hemorrhagic lymphadenitis, and in five of nine meningitis was present, and it was hemorrhagic in three of the cases.

One animal survived, had persistently negative blood cultures.

Next.


In most of the animals the organisms were all over the place. This is an easy diagnosis to make.

In a few animals, the organisms were more difficult to find, and I just point this out. This is immuno-histochemistry by EM with an antibody to a polysaccharide in the cell wall that clearly outlines degraded organisms in a macrophage.

Next.

This is just a higher magnification showing degraded bacilli that are coated with this antibody, with gold particles.



Next.

Now, these are the results of the experiment. The control, as I mentioned, nine out of ten animals died. With vaccine alone post exposure, eight out of ten died. With penicillin, three out of ten animals died.

Now, this is at three and a half months. this is 30 days of treatment, off drug for three to three and a half months. This is the long term survival.

None of the animals died while on antibiotics from anthrax. They all died subsequent to discontinuing taking the antibiotic.

In the ciprofloxacin group, one out of nine animals died after going off ciprofloxacin. I'll talk about these animals in a little more detail subsequently, but there was one animal that died five days after exposure from an aspiration pneumonia, had no evidence of anthrax on autopsy, and this animal was excluded from analysis.

As I'll mention, there's a second animal that died 73 days after stopping ciprofloxacin. This was due to urethral obstruction, and there was no evidence of anthrax at autopsy, and this animal was included in our analysis as a survivor.

For doxycycline, again, none of the animals died while on treatment. One animal died when the doxycycline was discontinued.

In the group of doxycycline plus vaccine, none of the animals died due to anthrax. There was one animal that died six days after discontinuing the doxycycline, but had no evidence of anthrax on autopsy. The cause of death in that animal is unknown. There was some mild myocardial degeneration, mild, but we don't know why that animal died, but the animal was excluded from statistical analysis because it had only been off antibiotics for six days.

So there was a statistically significant increase in survival in all the groups that had received any antibiotic.

Next slide, please.

So the conclusions from this part of the study were that vaccination alone begun after exposure to anthrax spores did not protect animals; that all of the antibiotics, including ciprofloxacin, provided complete protection when given after the aerosol exposure to spores, as long as the animals remained on treatment.

An extended 30 day treatment period with either penicillin, ciprofloxacin or doxycycline alone provided significant long term protection upon discontinuance of therapy, with from 70 to 90 percent -- that's 89 percent for the ciprofloxacin group -- of the animals surviving.

That post exposure vaccination when combined with doxycycline protected all of the animals. This difference was not statistically significant because most of the animals survived with just antibiotic.

Now, the animals that survived exposure were examined for evidence of an immune response. None of the animals treated with just antibiotic had any evidence that they had seen anthrax by the antibody assay that we used, which was an antibody to protective antigen. That is, they behaved as if the infection had been aborted, and they did not generate an immune response.

The only animals that generated an immune response was the group that received doxycycline plus vaccine, and so it appeared, as I said, that the antibiotics totally suppressed the infection.

The next slide, please.

We looked at the resistance of the survivors at three to three and a half months after discontinuance of the antibiotics, and they basically confirmed what the antibody data had predicted, namely, that the animals that were treated, whether penicillin, ciprofloxacin, or doxycycline alone succumb to rechallenge. They were not immune. Only the doxycycline plus vaccine group survived, and these differences, again, are statistically significant.

Next slide, please.

The overall results are shown graphically. I like to show this slide because it's one slide that has all the data. It's six months of work with 60 people. So for a short lecture, I just show this.

This shows the control group. Here's the time of exposure. The vaccine alone group, the animals die. Antibiotic treatment for 30 days, the animals all survive.

I want to point out this animal in the ciprofloxacin group. This ciprofloxacin group is the open triangles, and we'll focus on that. Just first these are the three penicillin animals, the closed triangles, day nine, 12, and 20. Following discontinuance of the drug three animals died. Again, on rechallenge these animals die.

The doxycycline alone group, one animal dies at 28 days after discontinuing the antibiotics. That's 58 days after the challenge. Again, these animals die on rechallenge.

And now the ciprofloxacin group. This animal was the animal that died from aspiration pneumonia. There was one animal that died on day six, I believe. I just want to make sure I got -- this animal died of inhalational anthrax with hemorrhagic necrotic lymphadenitis, intrathoracic nodes and mediastinitis.

This animal died on day 73, and that is 103 days after exposure. The animal developed urinary obstruction. The urine culture showed non-hemolytic staphylococcus. The blood culture was negative. There were attempts to relieve the obstruction, but the animal was euthanized five days later.

1   2   3   4   5   6


The database is protected by copyright ©essaydocs.org 2016
send message

    Main page