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TitleAdvancing Prion Science - Guidance for the Natl Prion Research Pgm - R. Erdtmann, et al (NAP, 2004) WW
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SCIENCE: Guidance for

the National Prion
Research Program

Rick Erdtmann
Laura B. Sivitz,



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Committee on Transmissible Spongiform Encephalopathies:
Assessment of Relevant Science

Rick Erdtmann and Laura B. Sivitz, Editors

Medical Follow-up Agency

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effect, thereby shrinking the available donor pool. The lack to date of an
approved test to detect prions in human blood has a great deal to do with
the technical challenges of developing a test with sufficient sensitivity to
detect a single infectious unit (IU). The titers of prions circulating in the
blood of patients with sCJD or vCJD are not known at present, nor is the
quanity of prions sufficient to constitute an infectious unit in human blood.
Also unknown is whether the titer of the sCJD or vCJD agent in blood
might change and even revert to zero during the incubation period. This
information is particularly relevant to the agent of vCJD because it is ac-
quired from outside the body and because it travels a circuitous route
through peripheral systems on its way to the CNS. The dynamic nature of
the vCJD agent increases the complexity of designing antemortem diagnos-
tics for the disease.

In addition, the size and number of prion aggregates in a sample affect
the detection and removal of PrPSc from blood, blood products, and blood
derivatives. For example, if a blood or plasma sample contained an IU that
was a single prion aggregate containing 105 PrPSc molecules, the IU would
be relatively easy to filter out but difficult to detect, due to the low prob-
ability that a random sample would contain the aggregate. By contrast, if a
blood or plasma sample contained 1,000 PrPSc aggregates, each comprised
of 100 molecules, the aggregates would be much harder to filter out but
theoretically easier to detect as a result of the higher probability that a
random sample would contain an aggregate—assuming the detection tool
were sensitive enough to detect a 100-molecule aggregate.

Recently, information gained from compartmentalized infectivity stud-
ies in a mouse model and a complex series of mathematical calculations
helped an investigator determine that 100 IU of infectivity (in buffy coat)
was equivalent to 10 picograms/mL of PrPSc (Brown, 2001). Brown used
this figure as an estimate target level that a future successful diagnostic test
would need to achieve, although he gave caveats that might alter this esti-
mate. Other models and methods need to be applied to reach more precise

Recommendation 5.1: Fund research (1) to determine the amount
of sporadic Creutzfeldt-Jakob disease (sCJD) prions and variant
Creutzfeldt-Jakob disease (vCJD) prions in human blood and (2) to
estimate the amount of PrPSc corresponding to one infectious unit
of sCJD and vCJD prions in human blood. [Priority 1]2

Until one IU is determined for sCJD and vCJD in human blood admin-

2The committee denotes each recommendation as priority level 1, 2, or 3 based on the
criteria and process described in the Introduction.

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istered to other humans, animal assays will need to be at least sensitive
enough to detect one mouse IU, within a specified transgenic strain, given a
specified volume and dilution of human blood, administered by the intrac-
erebral route. This would improve the consistency and reliability of an as-
say test.

Once the technical problem of developing a sufficiently sensitive test
has been solved, the other technical challenge is to develop a test so specific
that it can correctly identify a negative subject with a negative test result.
Failure to achieve this high level of specificity will result in false-positive
tests. This problem is especially acute in the case of CJD, which is uniformly
fatal, is associated with a prolonged asymptomatic incubation period and
has no effective prophylaxis or treatment. The psychological and social dam-
age to persons told mistakenly that they have CJD would be staggering.

This concern regarding false-positive test results is based on the statisti-
cal fact that the predictive value (correctness) of a positive test decreases as
the prevalence of the disease decreases in the population. For a rare disease
such as CJD, which occurs in 1 in 1 million persons, this is a thorny di-
lemma (see Table 5-3). If one had an excellent screening test for CJD whose
sensitivity and specificity were both an exceptional 99.9 percent and used
that test to screen 1 million persons, the percent correctness of a positive
test would vary with disease prevalence. If the disease being screened oc-
curred in 1 of every 100 persons, a positive test would be correct 91 percent
of the time. If the disease were rare, on the other hand, affecting 1 of every
1 million persons, the positive test would be correct less than 1 percent of
the time. In this case, with 1 million persons being screened, the true posi-
tive case would be correctly identified, but 1,000 persons would be incor-
rectly identified as positive. Thus only 1 of 1001 (0.1 percent) would be
correctly identified as positive, and virtually all the positive test results
would be false-positives.

The practical solution would be to perform a second- or third-level
confirmatory test that would be highly specific. That is how a similar di-
lemma with HIV screening is being approached. The HIV screening test,
despite having a specificity of 99.8 percent, has a predictive value of only 8
percent for a correct positive test (Dodd and Stramer, 2000). Follow-on
confirmatory tests are then used to verify to the initial screening test. Unfor-
tunately, such confirmatory tests for CJD or other TSEs are not available at

Reporting Results and Counseling Donors Who Test Positive for TSE

There are additional concerns related to proper counseling and report-
ing of TSE screening tests. Most of these concerns focus on management of
consent for use of the test and notification of the test result. It is standard

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protein (PrPC) and its misfolded isoform (PrPSc) as well as the identification
of one or more epitopes on these proteins. Blocking or activating the
epitope(s) could potentially disrupt an essential step in TSE pathogenesis,
such as the conversion of PrPC to PrPSc.

scrapie: A TSE of sheep and goats. The TSE was first described by Scottish
veterinarians in the 1700s, centuries before prions were first recognized.
The modes of transmission are thought to be contact with infected sheep or
goats or their placentas, contact with a scrapie-contaminated environment,
or oral intake of scrapie agent-contaminated material. The ÒScÓ in the term
PrPSc refers to scrapie. PrPSc is used to refer to the abnormal isoform of
PrPC associated with TSEs.

species barrier: The genetic, metabolic, physiological, and physical differ-
ences among species that result in variable susceptibility to an infectious

sporadic Creutzfeldt-Jakob disease (sCJD): The most common variety of
CJD. The cause is unknown. sCJD appears to occur worldwide at a rate of
approximately one case per 1 million population. Most cases involve older

transgene: A gene from one organism that has been transferred and inte-
grated into the DNA of another organism of the same or different species
such that the transferred gene is expressed in the host organism. Investiga-
tors conducting prion transmission studies use transgenes to convey un-
natural molecular characteristics to experimental animals so as to circum-
vent the species barrier. For example, it is easier to transmit BSE to a
transgenic mouse with a bovine transgene than to a normal mouse. These
experiments are further enhanced when the mouseÕs own PrP gene expres-
sion is knocked out (PrP0/0).

transmissible spongiform encephalopathy (TSE): A general term that refers
to all diseases associated with the presence of prions in vacuolated central
nervous system tissue. Prions from TSE-affected brain tissue are believed to
transmit the neurodegenerative disease state from the affected animal to
another host. A synonym for prion disease.

TSE: See transmissible spongiform encephalopathy.

variant Creutzfeldt-Jakob disease (vCJD): A clinical type of CJD first iden-
tified in 1996 and believed to result from the ingestion of beef products

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containing the infectious agent of bovine spongiform encephalopathy (BSE).
The majority of vCJD cases occur in young adults.

Western blot: A technique used in molecular biology to detect and identify
proteins in a test sample; also known as an immunoblot. A mixture of
proteins is embedded in a slab of polyacrylamide gel and subjected to elec-
trophoresis, during which an applied voltage causes the proteins to travel
linearly toward the opposite end of the acrylamide slab at rates dependent
on each proteinÕs mass (measured in kilodaltons [kDa]) and charge. The
pattern on the gel is transferred (blotted) to nitrocellulose paper or a nylon
membrane. This paper is then probed with detector antibodies. This pro-
tein-antibody complex is then bound with a labeled antiglobulin that visu-
alizes a pattern of dark bands that vary in intensity depending upon the
amount of protein in the sample. The test is usually run simultaneously on
the same gel and paper with appropriate control specimens.

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