1. Prions: Proteins Gone Bad
Karen Moody, PT, MS
Creutzfeldt-Jakob Disease Surveillance Coordinator
Texas Department of State Health Services
Diseases in Nature – Ft Worth, TX
June 2, 2009
I am the CJD surveillance coordinator for Texas Department of State Health Services. Prion Diseases otherwise known as Transmissible Spongiform Encephalopathies encompass a variety of illnesses in both humans and animals. Creutzfeldt-Jakob disease is just one type of prion disease in humans and there are four different subtypes of CJD. CJD is not the only human prion disease. And there are a variety of animals also affected by prion disease – cows being just one. So it is easy to get confused. Just keep in mind that Prion Diseases are the same as Transmissible Spongiform Encephalopathies.

2. Outline The Medical Mystery Prions Human Prion Disease
Animal Prion Disease
BSE – Mad Cow Disease
Variant CJD
Texas CJD Cases
Reporting and Investigating
Infection Control
Resources
We will begin by discussing the history of prion disease and the mysteries behind them
Then talk about prions – what are they? How we think they operate? And how do you inactivate them?
I will touch upon human prion diseases
Animal prion diseases
What is BSE or Mad Cow Disease – history
Variant CJD – I think one of the biggest misconceptions surrounding these diseases is that there is an assumption particularly in cattle states that if a person has CJD then they got it from cows and they have mad cow disease. Variant CJD is just one form of CJD. We will talk about that.
World wide number of variant CJD
Texas CJD cases
Reporting and Investigating prion disease in Texas
Resources I use when investigating cases.

3. Prion Disease - A Medical Mystery
Scrapie – sheep
Fatal Familial Insomnia (FFI)
Gerstmann-Straussler-Scheinker
1920s
Hans Gerhard Creutzfeldt and
Alfons Maria Jakob
Prion disease both in animals and humans was and in some ways still is a medical mystery. Scientists and families have traced these diseases back to the 1700s. At that time no one knew what was causing the disease nor did they know what to do about it. Scrapie is the name given to prion disease in sheep. It is a neurological disease in sheep that causes them to scrape themselves incessantly. Scrapie almost wiped out the European sheep industry. A human genetic prion disease called fatal familial insomnia is a prion disease in humans rendering the patient incurable insomnia. The patient literally dies of the inability to sleep. One family in Italy has traced this disease in their family back to the 1700s. This book is about that family and other prion diseases. Gerstmann-Straussler-Scheinker is another genetic prion disease among humans. Although very rare it is characterized by cerebellar ataxia and spastic paraparesis, with dementia developing later in the course of the disease.
No one knew what caused these diseases…..they just knew that the symptoms were similar and the outcome was the same no matter what they did treatment wise.
In the 1920s two German physicians first described the fatal neurodegenerative disease we know as CJD. The criteria they described in pts. Is not the same criteria used today but the name has held.

4. Prion Disease – A Medical Mystery
Kuru – Fore Tribe - New Guinea
1980s
Bovine Spongiform Encephalopathy (BSE)
The Discovery of Prions – Stanley Prusiner
1990s
Prions are transmissible from cattle to humans (variant CJD)
The disease that took its toll on the Fore Tribe in Papua New Guinea was a medical mystery for years. Scientists orginally traveled to New Guinea to study the tribe and while there discovered that people were dying at an alarming rate and mainly women and children. Many of the tribe assumed it was evil spirits. Scientists and epidemiologists struggled to know what was happening to this tribe. While kuru presented similarly to CJD – it was different in many ways. It affected different areas of the brain (lower brain such as cerebellum); happened in much younger age groups (children and adolescents). It was a mystery. After years of research it was determined that this tribe participated in cannibalism as a way of honoring their dead. The women and children would eat the brains and spinal cord while the men would feast on muscle. Since the prions are concentrated in the brain and spinal cord the women and children were dying at a much faster rate than the men. Once they stopped the cannibalism the disease stopped.
Again up until the 1980s no one knew what the exact agent of this disease was. It was initially thought to be a slow virus because of the unusually long incubation periods between exposure and onset of symptoms. However in 1966, they discovered the molecular weight of the agent was too small to be any type of virus or known infectious agent.
Since the 1980s the protein only theory has been led by Stanley Prusiner. He discovered prions following years of research on scrapie infected sheep brains. In 1997 he was awarded the Nobel prize for Physiology and Medicine for his discovery. While there are still scientists who dispute this theory the protein only theory remains the most viable theory behind prion disease.
And finally – following an epidemic of mad cow disease we discover this variant strain of prions not only in cows but humans as well.

5. Prions – Proteins Gone Bad
Not a bacterium or virus
Misfolded protein
Two stable conformations
(PrPc vs. PrPsc)
No nucleic acid
Genetic mutations
Survive routine disinfection &
sterilization procedures
The book I am reading right now called Deadly Feasts says, Prions are the strangest thing in all biology. PRions are not a bacterium or a virus. PRions are literally proteins gone bad…misfolded proteins. PRions exhibit two stable conformations. Prions are found in all mammals and are located in the brain and spinal cord. The function of normal prions is unknown. In the diagram PrPc is the normal cellular prion. PrPsc is the abnormal misfolded protein. This form is infectious rich in beta sheet conformation, insoluablea nd mostly protease resistant. It appears the misfolding is what causes the neurons to die off. But how does the misfolding affect all the prions. Prions have no nucleic acid so they cannot replicate themselves. One way of propogating is by a genetic mutation where the gene actually codes for a misfolded protein. Prions survive routine disinfection & sterilization procedures so special precautions must be taken with surgical instruments and certain medical procedures to assure the prion is inactive.

6. Human Prion Diseases Sporadic Acquired (Infectious) Genetic
Sporadic CJD (sCJD)
Sporadic fatal insomnia
Acquired (Infectious)
Variant CJD (vCJD)
Iatrogenic CJD (iCJD)
Genetic
Fatal Familial Insomnia
Familial CJD (fCJD)
Gertsmann-Straussler-Scheinker
Prion diseases are a closely related group of fatal neurodegenerative disorders that affect humans and other mammals. All prion diseases affect the brain differently (even with different types of CJD) so symptoms differ from type to type. A unique feature of this disease is that they can have three different origins: sporadic, acquired or infectious and genetic. In addition, clinical , epidemiological and neuropathological features can be very different. They are classified together because the key molecular event appears to be the same – the misfolding of the prion protein. Take a look at the diagrams and see that where the brain is affected is different for each disease therefore clinical presentation will be different as well. Notice that there are four kinds of CJD. At least one type falls under all three categories. Sporadic CJD is the most common type of CJD and human prion disease in the US with a rate of one per million. Variant CJD and Iatrogenic CJD are acquired or infectious. Iatrogenic CJD can be acquired through medical procedures such as corneal transplant, dura mater graft or growth hormone derived from cadavers. Familial CJD is genetic.

7. Animal Prion Diseases Chronic Wasting Disease (CWD)
Mule deer, White tailed deer, Moose and Elk
Bovine Spongiform Encephalopathy (BSE)
Cattle
Scrapie
Sheep
A few animal prion diseases are listed here. Chronic wasting disease first identified as wasting syndrome in captive mule deer in 1960s in Colorado; in the wild in 1981 and recognized as a spongiform encephalopathy in NEXT SLIDE. To date no strong evidence of transmission to humans has ever been reported. Endemic in Wyoming and Colorado CWD has been reported in 11 US states and 2 Canadian provinces. TURN TO NEXT SLIDE
BSE or mad cow disease in cattle. Scrapie in Sheep. It is also been recognized in mink and felines as well.

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As far as we know there have been no reports of CWD in Texas. We have it right on our border but I know TP&W monitors it and there have been no reports of CWD in Texas.
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9. Bovine Spongiform Encephalopathy (BSE or Mad Cow Disease)
1985 – recognized progressive neurological disorder in two cattle
1986 – examination of cow brain indicate spongiform changes
1987 – meat-and-bone-meal feed
1988 – Feed ban enacted in UK
1993 – BSE epidemic in UK peaked
with 1,000 cattle affected per week
2007 – 184,500 cattle confirmed in
35,000 herds
Seen by many as a ‘horror story’ BSE or mad cow disease was truly that. Farmers began seeing their cattle fall in the 1980s with the first call to a vet in 1985….the vet tried treating for ovarian cysts and low magnesium but eventually the cow went down was slaughtered and went to the Knackers yard (place where they process carcasses into meat and bone meal). The disease spread affecting all breeds of cattle all over England seemingly simultaneously. In 1986 scientists examined cow brain tissue under electron microscope and found spongiform changes. The first thought was scrapie but most of the farmers in the UK had no sheep. They knew it had something to do with food contamination but what? And….dairy cattle were more affected than dairy cattle. What did dairy cattle have in common that beef cattle did not. Dairy cattle were often given supplemental protein in the form of meant-and-bone-meal. That is, the ground, cooked and dried remains of dead animals including downer cattle and sheep dead of an undiagnosed disease. NEXT SLIDE. A high protein diet was required to maintain high milk productions. Later on, epidemiologists concluded that the combination of processing with lower temperatures and the abandonment of solvent extraction contributed to the infectivity of BSE. By 1988 feed containing ruminant-derived protein was ban in the UK. However, this was not properly policed for years. By 1993 the mad cow epidemic peaked with 1000 cattle affected per week. By the end of 2007 over 184,000 cattle had been confirmed which affected more than 35,000 herds…..devastating.

12. Cases of BSE in USA 2003 – Washington 2005 – Texas 2006 - Alabama
Holstein imported from Canada
2005 – Texas
First endemic case of BSE in US
Alabama
Euthanized and buried on farm (herd of origin not identified)
April 25, 2009 – FDA issued regulation barring high risk material use in animal feed in USA.
Just to let you know we have had several cases of BSE in the USA. In 2003 Washington state reported a case of BSE in an adult Holstein imported from Canada. This cow was slaughtered although high risk tissue was removed prior to it going to food chain.
In 2005 Texas had the first endemic case of BSE in the US in a cow 12 years old. In 2006 Alabama reported a case of BSE although the herd of origin was never identified so we are unsure if this was also and endemic case. After much debate, in April of 2008 the decision was made to bar use of high risk material in any kind of animal feed in the US. To give the farmers and ranchers time to comply to this regulation it was not officially enacted until April of this year.

13. vCJD 1990 – CJD surveillance unit in UK
1995 – Three CJD cases (ages 16, 19, 29)
10 suspected CJD cases <50 years old
1996 – Identified first human case
Distinctive clinical syndrome associated with plaque formation
vCJD prion strain unique to humans
Prion strain similar to BSE strain
Clinical and pathological characteristics different from sCJD
Because of concern of BSE transmission to humans a CJD surveillance unit was established in May of 1990 to monitor cases of CJD in the UK. Then from May to October of 1995 the surveillance unit was notified of three cases of CJD in patients 16, 19, and 29 years of age. When the brain tissue was examined in these cases they saw amyloid plaques, which was unexpected since their occurrences was only seen in 5 -10% of sporadic cases. The youth of these patients was also alarming. By December 1995, the Surveillance Unit had been informed of 10 suspected cases of CJD in persons <50 years of age. Two of these patients were found neuropathologically to have CJD and like the previous 3 patients had extensive plaque deposition. By January of 1996 two additional cases of CJD in young patients were neuropathologically confirmed and a distinctive clinical syndrome with associated plaque formation begin to emerge.

14. Clinical and Pathological Characteristics of vCJD
Amyloid plaques with spongiform degeneration
Clinical characteristics
Psychological / behavioral symptoms
Dysesthesias
Average age of patient – 28 years
Duration of illness – 12 to 14 months
No unique EEG findings
Positive pulvinar sign on MRI
Both pathology and clinical characteristics differ in vCJD from other types of CJD. In vCJD amyloid plaques are seen with spongiform degeneration. These plaques are also seen with kuru and other types of acquired prion disease but not with sporadic origins. Clinical characteristics differ from other types also….why?.....because vCJD affects different areas of the brain than sporadic or familial. Initial symptoms include psychological/behavioral symptoms as well as dyesthesias so it can mimic psychiatric illness. The patient is usually much younger and has a longer duration of illness than sCJD. There are no unique findings on EEG and there is a positive pulvinar sign on MRI.

15. Sporadic CJD vs. Variant CJD
Characteristics
sCJD
vCJD
Age
68 Years
28 Years
Duration
4-5 Months
13-14 Months
Clinical Presentation
Dementia
Psychiatric / Behavioral Symptoms
MRI ‘Pulvinar Sign’
Not Reported
Present (>75%)
Periodic Spikes on EEG
Often Present
Often Absent

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Age groups at death for vCJD and sCJD…..two very similar diseases affecting very different age groups.
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17. vCJD Worldwide As of 2008 Do genetics play a role in susceptibility?
208 cases (4 related to blood transfusions)
Three cases identified in USA – likely exposure in UK
Do genetics play a role in susceptibility?
Polymorphic codon 129 PRNP gene
100% of cases have been Methionine-Methionine
How many more cases might we see?
As of 2008 there have been 208 cases of vCJD worldwide – four of those related to blood tranfusions. Three of those cases were identified in the USA including one in Texas. All three of those have been determined to be exposed in the UK. In the UK – the number of cases peaked in 2000 with 28 cases and by 2008 we have only one documented case and zero for 2009 so far.
Although the amount of infectious tissue ingested must be a critical determinant for the transmission of BSE to humans in the form of vCJD, the human genotype at polymorphic codon 129 of the PRNP gene appears to play an important role in susceptibility to infections. The encoding alternatives are Valine and Methionine. All vCJD cases tested have been homozygous for Methionine. It is possible that the infecting strain may not be able to replicate in any other human genotype. However, it is possible that heterozygotes are resistant to the disease and become ill after longer incubation periods that those of homozygotes. If this is the case then we may see another surge of illness.

18. Reporting and Investigating
CJD – reportable condition in Texas
CSF – elevated protein and positive Tau protein = suspect case of CJD
Medical records – signs and symptoms
Arrange for autopsy (especially those suspected of having vCJD)
Under 55 years – suspect for vCJD
CJD is a notifiable condition in Texas within one week of suspecting a case. However, it is reported differently to DSHS than other reportable conditions. Most of the reporting of CJD comes from the National Prion Disease Pathology Surveillance Center by way of CSF protein and tau protein test results. A physician orders the test and ships it to the prion center. The prion center does the test and sends the results back to the ordering physician and my office. The test can be elevated, not elevated or ambiguous. All elevated test results prompt an investigation. To conduct the investigation I obtain medical records to determine if signs and symptoms meet case criteria. The WHO and CDC have specific guidelines that constitute case status. In other words, to be classified as a confirmed case – there must be examination of tissue either from biopsy or autopsy. If there is no autopsy or biopsy, then my job is to read through the medical record and determine case status based on signs and symptoms and other testing. This sometimes requires that I speak with a family member or physician. If the patient is still living, I will contact the physician and see if CJD is still in the differential dx. If so, I will talk to the physician about autopsy. Autopsy is not mandatory, but necessary to confirm the case. If the family is really interested in what their loved one has they must go for the autopsy. In addition they can look at the DNA for possible mutations. The case status will be listed as confirmed, probable, possible or not a case.
vCJD is suspected until proven not on all cases <55 years of age. That is not to say we have never had a sCJD case under 55 years but that is the cut off for possible variant and we are always diligent about those cases.

19. CJD in Texas Type 2004 2005 2006 2007 2008 Sporadic 8 9 5 10 12 3 1
-Confirmed 8 9 5 10 12
-Probable 3
-Possible 1
Variant
Familial
Iatrogenic
Sporadic Fatal Insomnia
Total 14 18 11 16
Texas has 24,000,000 people. The rate of CJD in the US is 1 per million. We average about 15 cases per year so we are either a lot of cases are not being reported or we have a much lower rate of CJD than the rest of the country.

20. CJD Cases by Age and Gender
2005
2006
2007
2008
Total
Gender
Male 7 5 8 27
Female 2 9
Age
<55 4 3 13
>55 11 6 41
*Based on data as of March 30, 2009
† Includes confirmed, probable, possible sporadic and familial CJD
Looking at the CJD cases by age and gender. Ocasionally there are a few more males than females but for the most part just as many males as females get this disease. By age – the cut off age for possible variant cases is 55. In other words, we investigate under 55 as a potential vCJD case but for the most part the majority of cases are over 55.

21. iCJD and Infection Control
iCJD risk – contaminated surgical instruments & certain medical procedures
Incineration eliminates risk of infectivity
Special procedures - heat resistant surgical devices
Prions not transmissible by touching, kissing or bathing
Possible transmission – ingest or transplant contaminated neural tissue
When talking about prions it is always a good idea to talk about infections control. As I mentioned earlier, prions are very difficult to inactivate in fact that is one of the ways they discovered prions is that they could not be destroyed by the normal disinfectants and sterilization procedures that knock out organisms with nucleic acid. Special procedures should take place when prion infectivity is suspected. Iatrogenic CJD is acquired through contaminated surgical instruments and certain medical procedures (corneal transplant, dura mater transplant and growth hormone from cadaver derived pituitary hormone). Instruments used in these procedures should be destroyed by incineration or disinfected with special procedures for instruments which can’t be destroyed. Prions are not transmitted by casual contact and transmission is possible with neural procedures or ingestion of contaminated neural tissue.

22. Resources
National Prion Disease Pathology Surveillance Center (NPDPSC)
Free CSF testing and autopsy arrangement
CJD Foundation
Family conference
Centers for Disease Control and Prevention (CDC)
Quarterly conference calls
World Health Organization (WHO)