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British Medical Bulletin 66:267-279 (2003)
© 2003 The British Council

Diagnosis of prion diseases

Eric Kübler, Bruno Oesch and Alex J Raeber

Prionics AG, Schlieren, Switzerland


   Abstract
Top
 Abstract
Overview of TSE diagnosis
 Classical TSE diagnosis
 Molecular TSE diagnoses
 Summary and perspectives
 References
 
Prion diseases are usually diagnosed clinically and confirmed by post-mortem histopathological examination of brain tissue. The only reliable molecular marker for prion diseases is PrPSc, the pathological conformer of the prion protein that accumulates in the central nervous system and, to a lesser extent, in lymphoreticular tissues. For BSE, several commercial diagnostic kits based on the post-mortem immunochemical detection of PrPSc in brain tissue are now available. These rapid screening tests have been used in active surveillance of BSE and have greatly improved the detection of infected cattle before their entry into the human food chain. At present, no diagnostic test exists for the detection of prion diseases in live animals or humans. New diagnostic techniques aimed at increasing sensitivity and specificity of PrPSc detection in body fluids and at identifying novel surrogate markers are under development. In this report, we review the classical diagnostic methods as well as present and future tools for the diagnosis of prion diseases.


   Overview of TSE diagnosis
Top
 Abstract
 Overview of TSE diagnosis
Classical TSE diagnosis
 Molecular TSE diagnoses
 Summary and perspectives
 References
 
Transmissible spongiform encephalopathies (TSEs) encompass a group of fatal neurodegenerative diseases in animals and man, which can be transmitted experimentally to laboratory rodents and primates. The aetiology of naturally occurring TSEs seems to comprise horizontal and vertical transmission as well as genetic predisposition, yet for the majority of cases it is unclear. The onset of clinical illness is preceded by a long incubation period of months to decades. Clinical symptoms of TSEs include dementia and loss of movement co-ordination. Neuropathological examination typically reveals a profound astrocytic gliosis and spongiform changes, sometimes accompanied by the formation of amyloid deposits. In the 1980s, it was established that a common hallmark of TSEs was the accumulation of an abnormal isoform of the host-encoded prion protein (PrP) in the brains of affected animals and humans.

Unlike other infectious diseases caused by viruses or bacteria, prion diseases are difficult to diagnose using conventional methods such as PCR, serology or cell culture assays. This is because the infectious agent or prion lacks a nucleic acid component and consists solely of an abnormally folded conformer of the normal host protein PrP that the infected organism does not recognise as foreign so that neither inflammatory nor immunological responses are observed. Laboratory diagnosis of TSEs is further complicated by the uneven distribution of TSE agents in body tissues, with highest concentrations consistently found in nervous system tissues and very low concentrations in easily accessible body fluids such as blood or urine.


   Classical TSE diagnosis
Top
 Abstract
 Overview of TSE diagnosis
 Classical TSE diagnosis
Molecular TSE diagnoses
 Summary and perspectives
 References
 
Clinical diagnostics

Human prion diseases have been traditionally classified into Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI) and kuru. About 75% of all human prion diseases are sporadic forms of CJD. Sporadic CJD is a rapidly progressive, multifocal dementia, usually with myoclonus (involuntary muscle twitching without unconsciousness)1. Dominant clinical features include fatigue, insomnia, depression, weight loss, headaches, general malaise, and ill-defined pain sensations. In addition, neurological features including extrapyramidal signs, cerebellar ataxia, pyramidal signs, cortical blindness and psychiatric features are frequent; about 12% of sporadic CJD patients are reported to epidemiological surveillance studies by psychiatrists2.

Currently, definite diagnosis of prion diseases is still considered to be possible only after histopathological examination of biopsied or autopsied brain material. However, in recent years, advances in the clinical characterisation of different CJD phenotypes have been made and many diagnostic techniques are under development, helping to achieve a precise diagnosis of this disorder during lifetime. The clinical diagnosis of sporadic CJD is supported by a variety of clinical tests, including detection of 14-3-3 and other proteins in cerebrospinal fluid, the patterns of electroencephalogram (EEG) and neuro-imaging technologies2 such as computer tomography and magnetic resonance imaging. Table 1 shows the sensitivities and specificities for the diagnostic techniques in sporadic CJD.


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  		Table 1 Sensitivity and specificity of diagnostic techniques in sporadic CJD

 
More recently, the spectrum of human prion diseases has been widened by a new variant form of Creutzfeldt-Jakob disease (vCJD) that has been attributed to the consumption of BSE-contaminated meat products3. Some general clinical criteria for the differential diagnosis of sporadic and variant CJD have been established4 and an analysis of the first 100 cases of vCJD identified in the UK revealed that a significant proportion of the patients exhibit a combination of psychiatric and neurological features within 4 months of clinical onset5.

Histopathology and immunohistochemistry

Historically, TSEs have been diagnosed by their most prominent hallmarks including the histological features of spongiform changes, astrocytic gliosis and, albeit not consistently seen in all TSEs6, amyloid plaques. Post-mortem neuropathological examination of brain tissue from an animal or human has remained the ‘gold standard’ of TSE diagnosis. This analysis includes detection of vacuolation within specific brain regions by light microscopy and can be applied to the analysis of different prion strains. Most remarkably, prion strain typing in mice by analysis of the neuronal lesion profiles in brain tissue has provided strong evidence that vCJD in humans is caused by the same prion strain as BSE in cattle7. Similar studies to assess the presence of a BSE strain in sheep have not yielded conclusive results, due to the extensive strain variation in natural scrapie8.

The extent of astrocytic gliosis in brain tissue can be assessed by immunohistochemical staining using antibodies to the astrocytic marker protein glial fibrillary acidic protein (GFAP). Immunohistochemistry can be performed on formalin-fixed or frozen tissue sections to detect the disease-specific marker protein PrPSc in situ9. This sensitive diagnostic tool relies not only on detecting the presence of PrPSc but also on its distribution in the brain and in lymphoid tissues. The profound lymphotropism of some prion strains, such as vCJD, natural scrapie and chronic wasting disease (CWD) in deer and elk has led to suggestions of using immunohistochemistry for PrPSc on lymphoid tissues in the preclinical diagnosis of TSEs. In a recent study, it was shown that pathological PrP can be detected in lymphoid tissues as early as 42 days following oral exposure of deer to CWD prions10. Similarly, vCJD can be diagnosed by detection of characteristic PrP immunostaining and PrPSc on tonsil biopsy11.

PrP-immunopositive amyloid plaques in brain are a highly specific diagnostic feature of some prion diseases. They are found in all GSS cases but only in some patients with CJD13. The identification of ‘florid’ plaques surrounded by spongiform vacuoles in vCJD but not in other forms of human prion diseases indicated that vCJD has a distinctive pathogenesis3. The findings of similar ‘florid’ plaques in macaques infected with BSE further supported the hypothesis that vCJD and BSE are caused by the same prion strain14.

Prion bioassay

Animal bioassays have been used extensively in TSE research and diagnostic testing. However, bioassays are severely limited in their widespread use by the length of time it takes to obtain results and the species barrier effect. Yet animal bioassays remain the only method to measure directly the infectious agent and are, therefore, the most sensitive assay available for the detection of prions.

Although ruminants were used early on to assay for infectivity in sheep and goats15, bioassays for research purposes improved dramatically with the introduction of rodent-adapted scrapie strains and their titration by intracranial inoculation of mice16 and hamsters17.

A further breakthrough in the detection of prions by bioassay was achieved by the genetic engineering of PrP-deficient (knockout), mutant and transgenic mice18. In particular, transgenic mice expressing high levels of heterologous PrPC (e.g. human or bovine) on an otherwise PrP null background have shown great potential as a diagnostic tool for the detection of human or bovine prions. Transgenic mice expressing high levels of bovine PrP are about 10 times more sensitive than cattle and more than 1000 times as sensitive as RIII wild-type mice to infection with BSE prions19.


   Molecular TSE diagnoses
Top
 Abstract
 Overview of TSE diagnosis
 Classical TSE diagnosis
 Molecular TSE diagnoses
Summary and perspectives
 References
 
Properties of PrPSc and PrP27–30

The discovery of PrPSc transformed research on TSEs. It provided a molecular marker that was shown to be specific for all prion diseases as well as the major, and likely the only, constituent of the infectious prion.

PrP27–30 was discovered by enriching proteinase K-treated fractions from Syrian hamster brain for scrapie infectivity9 and recovering a major protein component with an apparent molecular mass of 27–30 kDa. It was later found20 that PrP27–30 was the protease-resistant core of PrPSc. N-terminal sequencing of PrP27–30 led to the cloning of the host-encoded PrP gene and the identification of the protease-sensitive isoform of the prion protein (PrPC) in uninfected animals21. The key pathogenic event in TSEs is the conformational change of the host-encoded prion protein (PrPC) into the pathological isoform named PrPSc (after its first identification in experimentally scrapie-infected rodents). This conformational change involves refolding of {alpha}-helical structures of PrPC into the ß-sheet structure of PrPSc. PrPSc has the same amino acid sequence as well as the same post-translational modifications (e.g. the N-linked glycosylation and the GPI-anchor) as PrPC and, therefore, differs from it only in its tertiary structure22. PrPSc aggregates into amyloid fibrils and accumulates in nervous tissue and, to a lesser extent, in lymphoreticular tissues.

In an infected host, levels of PrPSc are directly proportional to prion titres. After experimental inoculation of rodents with TSE agents, PrPSc is usually detectable in the central nervous system weeks before the appearance of disease, and its level increases to a maximum that is reached up to months before the animal dies23. During the asymptomatic phase, both infectivity and PrPSc are readily detected in lymphoreticular tissues. Recently, PrPSc has also been found to accumulate in muscle tissue of hamsters orally infected with scrapie24 and in experimentally infected transgenic mice25.

Factors determining sensitivity and specificity

The hallmark of any diagnostic test is its diagnostic sensitivity (ability to identify positive samples as positive) and specificity (ability to identify negative samples as negative). This determines the usefulness of a test in the field (i.e. how many false and true positives it will detect). A further, more technical, parameter is the analytical sensitivity, which reflects the level of antigen that can be detected by a particular assay. The analytical sensitivity may or may not correlate with diagnostic sensitivity depending on the appearance and concentration of the analyte during the course of the disease. In the case of protease-resistant PrP as an analyte of TSE diagnosis, the two parameters differ because PrPSc accumulates exponentially and not linearly in the brain of infected animals during the incubation phase of the disease26.

The sensitivity and specificity of TSE tests were evaluated in the European Union as well as individual countries by using a certain number of positive and negative samples from animals previously diagnosed with a reference method such as histology or immunohistochemistry for PrP (<http://europa.eu.int/comm/food/fs/bse/bse12_en.pdf>, <http://europa.eu.int/comm/food/fs/bse/bse42_en.pdf>)27,28. Some of the tests were further evaluated in a field trial in order to determine their performance under normal routine conditions (<http://europa.eu.int/comm/food/fs/sc/ssc/out316_en.pdf>)29.

For all these validation studies, positive material was taken from animals at late stages in the disease. It was, therefore, not possible to determine the time point after infection at which the animals could be diagnosed. There is, however, one study in which cattle were orally infected with 100 g BSE brain and then analysed at different time points30. As a result it became clear that histology is positive only shortly before the appearance of clinical symptoms, whereas immunohistochemical techniques detecting accumulations of PrPSc gave a positive result about 6 months earlier. This is in accordance with the notion that the accumulation of PrPSc is an exponential process going rapidly from below detection level to maximal amounts of PrPSc. For this reason, slight variations in analytical sensitivity do not result in a higher diagnostic sensitivity because the time span between ‘not detectable’ to ‘full levels’ of PrPSc is relatively small. The probability of finding an animal in that stage is, therefore, quite small. This is also illustrated by the field-testing in France (Table 2), which illustrates equal diagnostic sensitivity for two of the BSE tests most commonly used in Europe, despite some claims to the contrary.


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  		Table 2 Numbers of positively and negatively tested cattle from two different populations (normal slaughter and cattle from the risk category) in France using two different assay systems (Prionics and BioRad)

 
Currently used test principles (approved by national authorities)

Presently, the most widely used diagnostic tests exploit the relative protease resistance of PrPSc in brain samples to discriminate between PrPC and PrPSc, in combination with immunological (anti-PrP-antibody-mediated) detection of the proteinase K-resistant part of PrPSc (PrP27–30). Sample preparation is one crucial part of such diagnostic tests and influences the diagnostic sensitivity and specificity as well as the through-put. Because of the membrane attachment of PrPC and PrPSc, tissue solubilisation has to include detergent extraction. In addition, conditions for proteinase K digestion have to be set such that the N-terminus of PrPSc and the whole PrPC peptide can efficiently be digested while PrP27–30 remains resistant. Various other proteases can be used for discriminating between the protease-sensitive PrPC molecules and the protease-resistant PrPSc structure. However, while proteinase K digests the whole N-terminus of PrPSc, other proteases (e.g. trypsin or pronase) do not result in such a well-defined removal of the N-terminus but rather leave the whole PrPSc molecule intact or digest away a few amino acids only31. For non-size-discriminating immuno-assays, this issue is of no direct importance since size shifts are not a part of the result output. In Western blot assays, a size shift is directly visible and constitutes a specificity criterion for a positive assay28. This has the additional advantage of providing a direct in-process control for the performance of the protease. For ELISA-based or similar assays, it is of paramount importance that the digestion conditions are optimal as even slightly incomplete digestion of PrPC can lead to false-positive results. Currently, 5 test kits use the detection of protease-resistant PrP as an assay principle and have been positively evaluated by the European Union in 1999 and 2003.

Prionics-Check Western (Western blot, Prionics AG)
 Simple automated one-step sample preparation followed by protease treatment. Detection occurs after the separation of the treated sample by denaturing polyacrylamide gel electrophoresis and transfer to a membrane using a PrP-specific antibody and an alkaline phosphatase-coupled secondary antibody detection system generating chemiluminescence. The presence of a PrP-immunoreactive signal with the additional two criteria of a reduced molecular weight (due to digestion of the N-terminus of PrPSc) and a typical 3-band pattern (due to different glycosylation forms of PrP) results in a TSE-positive diagnosis.

Platelia test (ELISA, BioRad)
 Sample preparation involving protease treatment followed by precipitation and a centrifugation step for enrichment of the analyte. Detection by sandwich ELISA using a colour-converting enzyme-coupled detection antibody. Cut-off setting with a grey zone that requires repetition of the assay.

Enfer test (ELISA, Enfer)
 One-step sample preparation involving sample transfer into and out of a plastic bag followed by protease treatment. The analyte is coated individually onto ELISA plates followed by detection via polyclonal antibodies and an enzyme-coupled secondary antibody. Detection is by chemiluminescence.

Prionics-Check LIA (ELISA, Prionics AG)
 Simple one-step sample preparation procedure followed by protease treatment. Detection occurs via sandwich ELISA using monoclonal antibodies, one of which is enzyme-labelled. Detection is with chemiluminescence using a plate-specific cut-off setting.

CDI (Inpro)
 In addition to a mild protease-treatment, the CDI uses the differential binding of antibodies to native or denatured PrPSc. The detection antibody recognises a conformation-dependent epitope that, while always exposed in the non-infectious form (PrPC), only becomes exposed in the infectious form of PrP (PrPSc) upon denaturation. Quantification of the binding events leads to a difference between the signals given by denatured PrPSc and native PrPSc, which is used as a diagnostic criterion.

Currently, the most widely used BSE tests in Europe are the Prionics-Check Western test and the Platelia test. In total, about 8 million BSE tests are performed per year.

Future test principles and platforms (not yet approved by national authorities)

One approach invented by the National Disease Center of the US Department of Agriculture uses a competition assay to detect PrP27–3032. A peptide encompassing part of the PrP amino acid sequence and labelled with a fluorescent marker is mixed with an anti-peptide antibody (which can also bind to PrP27–30) at such a concentration that 50% of the peptide is bound to the antibody. The unbound peptide is separated from the antibody-bound peptide by capillary electrophoresis and both the unbound and the bound peptide fractions are detected by laser-induced fluorescence intensity from which the intensity ratio (bound/free peptide) is calculated. Upon addition of a protease-treated tissue sample, any PrP27–30 present would compete with the labelled peptide for binding to the antibody and thus some of the bound peptide would be released from the antibody, the ratio of bound peptide to free peptide would decrease and indicate the presence of PrP27–30. Because fluorescent probes can be detected at very low concentrations, this assay was claimed to be more sensitive than the Western blot and ELISA test. The inventors published data showing that with this method PrP27–30 can be detected in blood of scrapie infected sheep and elk33. However, this assay has not yet been approved by any authorities for large scale screening for TSEs.

Saborio and colleagues34,35 introduced a novel in vitro approach for the detection of PrPSc. They developed a method to convert PrPC molecules into protease-resistant PrPSc-like molecules that depend on the presence of exogenously added PrPSc in the sample. The inventors claim that otherwise undetectable levels of PrPSc are amplified to detectable levels and that they are, therefore, able to detect PrPSc molecules in blood samples from infected animals.

A new platform to detect PrP27–30 is currently being developed by Prionics AG. The test involves a sandwich immunoassay in which the capture anti-PrP antibody is sprayed as a line perpendicular to the long axis of a nitrocellulose strip. A detection antibody coupled to a coloured bead is then brought into contact with a protease-digested sample within a well of a microplate. When the strip is put into the well, the bead-coupled antibody/PrP27–30 complex migrates up the nitrocellulose strip driven by capillary forces and is immobilised at the capture antibody line. The resulting immunosandwich appears as a coloured line and can easily be detected by eye or by a lumi-imager. This detection system reduces assay time significantly. After the protease digestion, results can be obtained in less than 20 min. In addition, assay controls can be added without the use of additional labour or consumables.

Additional detection techniques are being developed that are very technical and not yet applicable for easy handling or high through-put screening. Among these are spectroscopy-based methods such as fluorescence correlation spectroscopy (FCS)36, multispectral ultraviolet fluorescence spectroscopy (MUFS)37, and Fourier transform infrared spectroscopy38.

Surrogate markers

PrPSc is well-suited as a marker for the post-mortem diagnosis of TSEs because high concentrations are found in the CNS of infected animals and humans. However, from the perspective of preclinical diagnosis, the use of PrPSc as a marker is limited because its concentrations outside the CNS and, in particular, in easily accessible body fluids (e.g. blood and urine) are extremely low (if it is present at all). The detection of a protease-resistant PrP-derived molecule in urine of prion-infected animals and humans has been reported39, however; a solid routine test has not yet been established.

Detecting vCJD before patients show clinical symptoms is an urgent priority, as it could dramatically reduce the risk of contaminating blood supplies and hospital equipment. Therefore, disease-indicating markers other than PrPSc from easily available body fluids are needed.

Using differential RNA display technologies, it was found that a novel transcript coding for erythroid differentiation-related factor (EDRF) showed progressively decreased expression during the course of the disease in scrapie-infected mice40. In a similar approach, cellular nucleic acids were identified in the serum of BSE-infected cattle and appeared to correlate with the development of the disease41. If such nucleic acids were indeed present in blood, this might form the basis of a simple PCR-based diagnosis of CJD and related disorders from live organisms.

Another simple, non-invasive test to diagnose TSEs uses a technique known as high-resolution electrocardiography (ECG) to measure a unique heart rate variability signature observed at the early stages of infection42. The test was used on 150 cows experimentally infected with the BSE agent and detected the disease based on increased levels of respiratory sinus arrhythmia (RSA) in 2 animals that died 8 months later.

Strain typing

Additional diagnostic precision has been made possible by the introduction of PrPSc isotypes on the basis of the mobility of the protease-resistant proteinase K fragments in polyacrylamide gel electrophoresis43–45. Based on this method, two major types of PrPSc of all forms of CJD and fatal insomnia have been determined45. PrPSc type 1 migrates to 21 kDa on gels after treatment with proteinase K and deglycosylation; type 2 PrPSc migrates to 19 kDa under these conditions. The different sizes are attributed to different 3-dimensional structures, which lead to exposure of distinct cleavage sites for proteinase K. The two PrPSc types co-distribute with distinct disease phenotypes and are conserved upon transmission to susceptible animals. Additional subtypes of PrPSc have been distinguished on the basis of the ratios of the three PrPSc glycoforms45 and of the profile generated by two-dimensional gel electrophoresis46.

Strain typing may have an additional implication when screening sheep for scrapie. Sheep are susceptible to both sheep scrapie prions and bovine BSE prions. BSE prions in sheep may have similar consequences for human health as if they originate in bovines and thus detection is important. As mentioned above, one difference (in addition to glycosylation differences) between sheep scrapie and bovine BSE PrPSc molecules lies in the different proteinase K cleavage sites and thus in the size of PrP27–30 (with scrapie PrPSc having a slightly larger fragment). Detection of the difference has been established by developing an antibody against the sequence lying between the two proteinase K cleavage sites47. A signal using this antibody on a Western blot suggests the presence of sheep scrapie PrPSc, the absence of a signal suggests the presence of BSE PrPSc. Such an assay could routinely be performed on samples that have previously been screened positive for general protease-resistant PrPSc.


   Summary and perspectives
Top
 Abstract
 Overview of TSE diagnosis
 Classical TSE diagnosis
 Molecular TSE diagnoses
 Summary and perspectives
References
 
Current diagnostic methods are based mainly on the physicochemical differences between PrPC and PrPSc which, to date, are the only reliable markers for TSEs. The tests differ mainly at the level of the detection methods. Due to the slow kinetics of accumulation of PrPSc in the preclinical stage of the disease, the current diagnostic capabilities are markedly limited with respect to the detection of the disease early in the incubation period. Thus, the search for another marker for the detection of TSEs is of utmost importance. Several approaches have already been undertaken to detect additional surrogate markers. It may, however, not be possible to find a single surrogate marker that is absolutely specific. Approaches aimed at detecting differences in the expression pattern of proteins and/or nucleic acids between healthy and diseased organisms might eventually allow detection of TSEs long before clinical signs appear. Such an achievement would be necessary for timely medical treatment of individuals once a therapy is available.


   Footnotes
 
Correspondence to: Dr Alex J. Raeber, Prionics AG, Wagistr. 27a, CH-8952 Schlieren, Switzerland


   References
Top
 Abstract
 Overview of TSE diagnosis
 Classical TSE diagnosis
 Molecular TSE diagnoses
 Summary and perspectives
 References
 

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J. Novakofski, M. S. Brewer, N. Mateus-Pinilla, J. Killefer, and R. H. McCusker
Prion biology relevant to bovine spongiform encephalopathy
J Anim Sci, June 1, 2005; 83(6): 1455 - 1476.
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