College of American Pathologists
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Stepping up to the plate in prion surveillance

December 2003
Karen Titus

For researchers enmeshed in prion disease, the world is a dark and complex place. For pathologists, however, dealing with prion disease just gets easier and easier.

An exaggeration, yes. But it’s not completely off the mark, either. Every recent advance in prion research has been accompanied by a bundle of new questions, and prion disease still stirs up fears in many. But for pathologists, matters aren’t nearly as difficult. A good thing, too, since they’re arguably the principal players in prion surveillance efforts.

"Pathology is the way to confirm cases, and to diagnose prion disease definitively," says Lawrence Schonberger, MD, MPH, assistant director for public health, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention. "It’s the gold standard."

Prion disease testing can include a number of options. In cerebrospinal fluid, it means searching for the presence of the 14-3-3 protein, which is a marker for some (though not all) prion diseases, including Creutzfeldt-Jakob disease, when other conditions are excluded. It means looking for a mutation in the prion protein gene in DNA extracted from blood, brain, or other tissue and determining the polymorphism at codon 129, among other sites. It means hunting for the presence of protease-resistant or scrapie protein (PrPSc) and establishing its type. And on fixed brain tissue, it means excluding or confirming/characterizing the prion disease by microscopic examination, after histological/immunohistochemical procedures.

Information about this array of options comes courtesy of the National Prion Disease Pathology Surveillance Center, whose staff know a thing or two about prion disease. The center, founded in 1997 and located at the Division of Neuropathology of Case Western Reserve University, Cleveland, is supported by the CDC and sponsored by the American Association of Neuropathologists. Its goal is clear, if not simple: to have an autopsy performed in all suspected cases of prion disease.

That doesn’t mean you have to perform it.

The center itself is one of the few institutions capable of performing the full array of prion diagnostics, a sort of for all things prion. They link pathologists to institutions that are willing to perform autopsies, and help pay for the cost of the autopsy and body transportation, if necessary. And they’ll provide equipment to those who request it.

"They’re great," says Barbara Crain, MD, PhD, associate professor of pathology and director of the autopsy service, Johns Hopkins, and chair of the CAP Neuropathology Committee. She’s a member of the center’s advisory committee-"So I can say they’re great-I’m not on staff," she says, disclaiming any conflict of interest. "It’s a very professional effort, and they’re very good about telling you how to do the sending," be it tissue, blood, or CSF. There’s no charge for diagnosis or shipping, and turnaround times are quick, she notes. "They even help you fill out your FedEx paperwork," Dr. Crain says. "It’s so simple even I can do it-with a little help," she says with a laugh.

The ease with which pathologists can help track prion disease doesn’t minimize the impact of their role. Pathologists are on the frontlines, says Dr. Schonberger, and have been since 1996, with the identification of what was then called new variant CJD in the United Kingdom. (Its "new" status aging, it’s now known as variant CJD, or vCJD.)

"There’s an increased consensus that this new form of prion disease results from consumption of contaminated food," says Dr. Schonberger. "This problem is diagnosed and requires for confirmation the work of neuropathologists, of people who are able to examine the brain tissue of deceased patients who are clinically suspected of having this disease."

Tracking vCJD is not the only concern. Because the animal form of one prion disease, BSE, or mad cow disease, can jump the species barrier and affect humans, there’s fear that other prion diseases might do likewise. Chronic wasting disease, or CWD, which affects the deer and elk population in the United States, heads the list of worrisome diseases.

"That disease among our deer and elk is nowhere near as prevalent as was the BSE in England and Europe," which have been hit hardest by vCJD, says Dr. Schonberger. But everyone is eyeing deer and elk warily nonetheless, concerned about the possibility that CWD might occasionally dip into the human population. Because of this, he notes, the public health community is trying to increase surveillance of prion diseases in general. "We want to get a better handle on what is normal, and what may emerge as something new," Dr. Schonberger says. "And quite frankly, the central group of specialists we depend on includes pathologists."

It’s a message repeated by Pierluigi Gambetti, MD, director of the surveillance center, as he pleads for more cases.

Though the number of referrals to the center have increased over the last couple years, they’re still too low. By his estimates, the center examines approximately 50 percent of the cases of prion disease thought to occur in the United States each year. His goal is to reach 70 to 80 percent coverage, which he says is the norm at prion surveillance centers in Europe, Canada, Israel, and Australia. Some countries boast case examination rates of nearly 100 percent, which in turn has led to better surveillance-and fresh concerns.

"One finding that is very intriguing, if not worrisome, has been that Switzerland has reported an increase in the incidence of prion disease," Dr. Gambetti says. In the past, incidence has been estimated at roughly one per million. "But Switzerland all of a sudden, for the last three years, reported an increase to almost three per million, which is sizable," he notes. It may mean that incidence has actually increased-obviously a troubling possibility. Or, says Dr. Gambetti, "It could be that Switzerland is very good at surveillance, like they are in making watches," with the higher numbers reflecting more meticulous surveillance than can be done in larger countries.

Persuading U.S. pathologists to step up their efforts hasn’t been easy. "There is the fear of contamination," Dr. Gambetti admits. "And then there’s the reluctance to do something that is a little bit out of the ordinary, something that requires precautions."

Such fear is not groundless, but neither should it be blown out of proportion. Basic precautions have remained consistent and clear, Dr. Crain notes, pointing to the protocols provided by the CAP Laboratory Accreditation Program. "A lot of people are concerned that there’s not a way to decontaminate after you have a case of CJD in your area," she says. "But actually there is. It just involves fixing the tissue in formalin, then treating with at least 95 but preferably 98 or 99 percent formic for an hour.That’s all it takes. And all the glassware and everything else can be decontaminated with 100 percent bleach.

"Once you get the hang of it, it’s really easy," she says.

That’s not to say laboratories should feel compelled to tackle prion diagnostics on their own. Although there’s nothing exotic about Western blot analyses, setup requires use of known positives and negatives and a biosafety level two or three lab-and an institution needs to handle enough cases to make it financially worthwhile, Dr. Crain notes. The government-funded surveillance center, on the other hand, is not dependent on clinical fees. "Even a big hospital like Hopkins isn’t going to do enough to make it worthwhile setting it up," she says. Moreover, most labs are not technically capable of performing the more sophisticated followup tests.

She suggests additional reasons for the less-than-ideal surveillance numbers. "One, of course, is that not all the cases are documented by pathology. There’s the clinical diagnosis, the patient dies, and no autopsy is done to confirm or refute the hypothesis that it’s a prion disease."

Other cases just slip through the cracks, she says; even those who know better occasionally fail to take frozen tissue. When that happens, "It’s still better to send them [the center] the report and send them recuts, so they can at least keep track and get a feeling for how things are going."

Pathologists can also help clinicians identify cases that may otherwise be overlooked. "Unfortunately, I don’t think prion disease is any better recognized clinically than it was even 10 years ago," says Daron Davis, MD, associate professor of neuropathology, University of Kentucky, Lexington. "The index of suspicion is not where it should be with some of our clinical colleagues."

Dr. Davis says he usually "bumps into CJD cases" in his autopsy practice when subjects are referred to him for dementia neuropathologic examination. Before beginning the autopsy, he routinely questions the referring physician or family member about the length of the patient’s dementing illness. "When the clinical course is less than one year, I am highly suspect of the case being compatible with a prion disease" rather than one of the other dementing degenerative neurologic diseases, he says, and appropriate precautions are taken.

Though Dr. Davis is comfortable making the diagnosis of CJD on routine histologic sections, such an exam does not reveal whether the case is familial or sporadic. "We send samples of all our cases to Dr. Gambetti’s lab for molecular analysis, and we did so even before the surveillance program was in place," Dr. Davis says.

Prick the surface of CJD, and it’s quickly evident that it’s more than one disease. Determining the relative frequency of all the CJD variants, says Dr. Schonberger, will "require the cooperation of many pathologists submitting their cases, so that people like Dr. Gambetti can describe the relative ratios and identify any cases that don’t fit any of the known categories. So at least for a while, it’s useful, even for the perfectly classic cases, to still submit the brain tissue to Dr. Gambetti."

Diagnostics are evolving, to be sure. The future may bring highly sensitive tests capable of detecting minuscule amounts of PrPSc, the so-called bad protein, which may eventually allow pathologists to make a diagnosis, using noninvasive methods, at the early stages of disease. "It would be fantastic to have a reliable blood test," says Dr. Gambetti. "And many people, many laboratories, are working toward that."

Those efforts have been aided by researchers’ newfound abilities to generate antibodies that recognize exclusively the abnormal prion protein. "It’s really spectacular," says Dr. Gambetti, whose center has discovered one such antibody, which appears to recognize multiple types of scrapie prion protein-"human, animals, you name it," he says. "It has a strong affinity for all of them but does not recognize the normal prion protein at all. And it has generally high affinity." Such qualities make it relatively easy to detect even the tiniest amounts of abnormal protein. "This would be important not only for diagnostic purposes for the patient, but also to detect whether there is contaminated blood," he says. "Because what is becoming more and more evident with these better detection methods is that the protein, even in sporadic prion disease, may be present with a more widespread distribution than was believed. There is the potential of transmitting the disease by blood transfusion."

This concern is based, in part, on an experiment involving sheep; specifically, a direct transfusion using a considerable amount of blood from a sheep sick with prion disease. Humans aren’t sheep, everyone is careful to point out.

But no one is resting easy.

CWD in deer and elk has turned out to be more widespread than first thought, affecting the Midwest and the South in addition to Wyoming and Colorado. Can it jump the species barrier to humans?

Much attention has been focused on hunters who have developed a prion disease-Dr. Gambetti reports seeing at least 10 such cases at the surveillance center. But the type of prion disease that affected these men remains unknown. Several years ago researchers noted a variability in the prion protein gene that turns out to have tremendous influence in the way vCJD presents, depending on what combination of amino acids the gene encodes. Another disease modifier is the type of abnormal scrapie prion protein, known as type 1 and type 2, that patients develop in their brains. All in all, "We were able to determine six types of sporadic CJD," Dr. Gambetti says.

In the case of the hunters, he says, "The first question we ask ourselves is, Does it fit comfortably well into the known, well-established sporadic prion disease?" If the glove fits, a diagnosis of CWD is less likely.

Less likely, but not impossible. Simply put, if CWD can make the jump to humans, no one knows for sure what those cases would look like.

"If CWD goes into humans, will it look like vCJD? Probably," says Judd Aiken, PhD, professor of animal health and biomedical sciences, School of Veterinary Medicine, University of Wisconsin-Madison.

He pauses. "But what if it doesn’t?"

Humans who acquire vCJD from eating contaminated beef share uniform characteristics, says Dr. Gambetti, including type 2 proteins and the same prion protein genotype. It would thus be reasonable, he says, to expect that if a human form of CWD occurred, it, too, would share those characteristics. But in the case of the aforementioned hunters, each one had different molecular features.

Researchers are plunging into experiments that may help pinpoint prion identification. At the University of Kentucky, for example, Glenn Telling, PhD, associate professor of microbiology, immunology, and molecular genetics, is making transgenic mice that express cervid (deer or elk) PrP, and also chimeric forms between mouse and cervid. At this point, he and his colleagues are waiting to see when the inoculated mice will develop disease.

They’re also interested in the mechanism of prion propagation, which involves using cell culture in vitro approaches as well as transgenic models. Currently Dr. Telling is studying the post-translational processing of PrPc (the normal form of the prion protein) and PrP scrapie (the abnormal form). "It’s clear they’re very different," he says. "So we’re trying to define these processing events in the infected and uninfected states." He and his colleagues have recently discovered an enzyme group called calpains, which are calcium-activated cysteine proteases and cleave the scrapie form of PrP. "We think this is a major advance," he says. "It could open up new therapeutic avenues for inhibiting prion propagation, because if we can inhibit calpains that cleave PrP scrapie, then we can possibly abrogate prion disease. But," he acknowledges, "we need to do a lot of work in vivo to substantiate that, and that’s what we’re doing at the moment."

Other therapeutic possibilities are more developed, especially immunotherapies. Researchers have developed specific antibodies against PrP and demonstrated, in vitro and in animal models, that certain antibodies can inhibit prion replication. "They’ve shown they could treat animals with specific monoclonal antibodies peripherally, at least, and inhibit the development of clinical features of prion disease," Dr. Telling says.

It’s not just the medical professionals who are concerned about prion disease-they’re getting a boost from the other scientists whose research may offer helpful insight into prion diseases.

The species barrier has been a Gordian knot for these researchers as well. The barrier, unfortunately, has not proved to be solid in the case of bovine spongiform encephalopathy, or mad cow disease, which in humans has caused vCJD. Sheep scrapie, on the other hand, has a fairly strong species barrier. Though it’s been around for hundreds of years, "There’s no evidence that sheep scrapie causes disease in humans," says Dr. Aiken. Incidentally, he adds, this is one of the reasons the scientific community failed to appreciate the risks of mad cow disease for humans-BSE was initially thought to have a similarly solid barrier.

CWD appears to fall closer to sheep scrapie on the barrier spectrum-though given the BSE/CJD disaster, no one is saying "case closed," either. Even those who downplay the dangers of CWD aren’t sitting down to platters of venison from CWD-endemic areas. "My guess is that the jump probably won’t happen-but that is quite different from saying it won’t happen," Dr. Aiken cautions.

"The one thing I’ve learned with prion disease is you never say ’never,’" he adds.

Prion diseases also differ in their distribution of the disease agent, as well as in their transmission patterns. With BSE, infectivity is primarily located in nervous system tissue; not coincidentally, neither BSE nor CJD are contagious diseases. With CWD, however, there appears to be high infectivity in lymph nodes, including the lymph tonsil, in deer; it can be transmitted laterally in deer, possibly through saliva. And with sheep scrapie, placenta may be a means of transmission.

Other researchers are trying to get a line on the environmental fate of prions. Strong circumstantial evidence suggests an environmental reservoir may be involved in animal-to-animal transmission of sheep scrapie, says Joel Pedersen, DrEnv, assistant professor in soil biochemistry and environmental toxicology, University of Wisconsin-Madison. "There have been several cases where animals have become infected by being exposed to fields or pens that formerly contained infected animals," he says. Also important, he says, is that wildlife and livestock consume soil; this, coupled with clear evidence of early GI tract involvement in CWD and scrapie infection, suggests that the infectious agent is taken up orally and might be shed through feces.

The nitty-gritty of Dr. Pedersen’s work is indeed gritty-establishing the extent to which the infectious protein associates with common soil constituents; looking at how various types of soil particles (soil, as it turns out, is as heterogeneous as blood) protect prions from degradation; and investigating the length of infectivity of particle-associated prions, before and after they’re exposed to degrading microorganisms.

His work may also sound a bit removed from the realm of medicine and pathology labs, though Dr. Pedersen could be mistaken for a physician when he talks about matters like the detection limits of Western blots. Indeed, along with Drs. Gambetti, Telling, and others, he’s well versed in the devilish nature of prions.

"Usually when you study how virus or bacteria behaves in the environment, you’re looking at a fairly well-defined entity," he says. "But in this case, you have a protein with an undetermined tertiary structure. You’re unable to purify it completely. You have a range of aggregate sizes. And there’s at least eight different charge isomers. It makes conducting experiments very difficult, and it’s forcing us to do things in a different way than we normally would."

For now, however, prions are not imposing drastic new routines onto pathologists. If anything, the demand for autopsies may be prodding them into a more old-fashioned role of caregiver.

"It is the right thing for the patients and their families to get a diagnosis," says Dr. Crain. "The families very much want to know. It’s a matter of taking care of the family and patient, and you shouldn’t overlook that."

Karen Titus is CAP TODAY contributing editor and co-managing editor.