Advancing Prion Science Guidance for the National Prion Research Program: Interim Report (2003) / Chapter Skim
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3. Diagnostics For Transmissible Spongiform Encephalopathies
3. Diagnostics For Transmissible Spongiform Encephalopathies
Pages 41-62
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From page 41... ...
In the case of animals, the ability to diagnose or detect an infection drives food safety interventions, which can prevent the introduction of tainted food into the food chain and offset economic damage to the food production industry. In the case of people, the detection of infection can avert the introduction of potentially infectious blood into the blood supply system and can be used to direct appropriate treatment.
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From page 42... ...
challenges the diagnostician to develop a sufficiently sensitive test. In addition, diagnostic tests must be of sufficient specificity to differentiate between normal and abnormal prion proteins and, for some purposes, to discriminate between one or more strains of PrPSc—a challenge resulting from basic deficiencies in understanding of prion strain diversity and the nature of strain variation.
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From page 43... ...
Differentiation of prion disease from other neurodegenerative diseases and differentiation among different prion strains on clinical grounds are problematic because affected individuals exhibit similar symptoms. Clinical diagnostic criteria have nevertheless been established for sporadic Creutzfeldt-Jakob disease (sCJD)
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From page 44... ...
. per~oc 1C complexes MRI Increased signal Hyper-intense signal in basal ganglia, in pulvinar region of caudate nucleus, the thalamus and putamen CSF 14-3-3 protein 14-3-3 protein levels levels usually not usually elevated elevated Histopathology of brain No amyloid 100% florid plaques tissue plaques PrP immunohistochemical Punctate pattern Widespread plaque staining pattern of brain staining pattern tissue Immunohistochemical Negative staining of tonsil or appendix tissue PrPSC isotvne bv Westen1 blot Type 1A PrP present in tissue~ especially toward late-stage disease Type 2B
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From page 45... ...
When patients are evaluated by T2 MRI, proton-density-weighted MRI, or fluid-attenuated-inversion-recovery MRI, there is an increased signal in the basal ganglia about 80 percent of the time (WHO, 19984. MRI also can be used to help differentiate vCJD from sCJD because the posterior pulvinar region of the thalamus shows a hyperintense signal in patients with vCJD.
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From page 46... ...
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From page 47... ...
Nevertheless, studies of sheep naturally infected with the scrapie agent and of mice experimentally infected with it have demonstrated that PrPSc is detectable in lymphoid tissues long before clinical signs of neurological disease appear. Brain Biopsy In atypical cases of CJD, brain biopsy with histological examination for spongiform changes, immunocytochemical staining, and Western blotting for PrPSc, as well as analysis of the PrP gene, is diagnostic in virtually all cases.
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From page 48... ...
In addition to the PrPSc type, the phenotype of human prion diseases is also influenced by the genotype at codon 129 of the PRNP, the site of a common M/V polymorphism. A classification of sporadic prion diseases has been generated on the basis of the combination of the genotype at codon 129 and the PrPSc type (Parch)
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From page 49... ...
The rapid test most widely used to screen for bovine spongiform encephalopathy (B SK) in Europe is a rapid Western blotting test produced by Prionics AG in Switzerland called Prionics Check Western.
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From page 50... ...
Postmortem, the level of accumulation of PrPSc has reached its peak and PrPSc is most concentrated in brain tissue. Testing for CWD by immunohistochemistry and ELISA of both CNS and peripheral lymphoid tissue samples can provide positive results fairly
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From page 51... ...
Later, the successful use of Syrian hamsters reduced the incubation period to illness even further to 70 days (Marsh and Kimberlin, 19751. Further enhancements to the mouse model produced inbred strains that helped elucidate the role of the mouse Prnp gene in susceptibility, incubation times, and prion transmissibility.
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From page 52... ...
Both sheep and human prions have been propagated in this cell system after the agent was first passaged through mice (Kingsbury et al., 1984; Race et al., 1987~. Other cells reported to have been used in cell culture systems include the GT-1 cell line, which is derived from mouse hypothalamic neurons and which has been used successfully to study the scrapie agent (Schatzl et al., 1997~.
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From page 53... ...
Protein Misfolding Cyclic Amplification A novel in vitro approach introduced by Saborio, Soto and colleagues involves the cyclic amplification of PrPSc by sonication (Saborio et al., 2001; Soto et al., 2002~. PrPSc in a test sample is incubated with an excess of normal prion protein such that prpc converts to PrPSc and aggregates into complexes.
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From page 55... ...
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From page 56... ...
The technique involves ultracentrifugation and PK digestion of a sample containing PrP followed by resuspension in a sodium dodecyl sulfate-buffered gel. The migration times were calculated by using Ferguson plots, which in turn were used to estimate the molecular weights of the proteins in the test material.
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From page 57... ...
They concluded that MUFS has great promise as a rapid, sensitive, and specific tool for the direct detection of PrPSc as well as for the differentiation of disparate prion strains (Rubenstein et al., 1998~. Fourier Transform Infrared Spectroscopy A recently reported spectroscopic approach to the identification of prior-infected hosts used Fourier transform infrared spectroscopy, in combination with a highly sophisticated automated computer-assisted pattern recognition program referred to as artificial neural networks, to detect disease-associated differences in patterns of small molecules in serum (Schmitt et al., 20021.
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From page 58... ...
However, in countries where a false-positive test result represents a sentinel BSE case, the economic, political, and societal consequences of that incorrect result would be monumental. The impact of a falsenegative test result might allow a contaminated beef product to enter the food chain.
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From page 59... ...
2001. Novel differences between two human prion strains revealed by twodimensional gel electrophoresis.
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From page 60... ...
2002. Identification of scrapie infection from blood serum by Fourier transform infrared spectroscopy.
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From page 61... ...
1994. Sodium dodecyl sulfate polyacrylamide gel- and replaceable polymerfilled capillary electrophoresis for molecular mass determination of proteins of pharmaceutical interest.
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