NEURODEGENERATION, Vol. 4, pp 357–368 (1995) Diagnosis and Incidence of Prion (Creutzfeldt-Jakob) Disease: A Retrospective Archival Survey with Implications for Future Research C.J. Bruton,1 R.K. Bruton,2 S.M. Gentleman3 and G.W. Roberts4 1 Corsellis Collection Brain Bank, Dept. of Neuropathy, Runwell Hospital, Wickford, Essex; 2 Department of Pharmacology, Birmingham University, Birmingham; 3Departments of Psychiatry and Anatomy, Charing Cross and Westminster Medical School, London; 4 Department of Molecular Neuropathology, SmithKline Beecham, Harlow, Essex Reliable identification of Creutzfeldt-Jakob disease (CJD) in the UX has become essential following the suggestion that prion disease in cattle (BSE) might transmit, accidentally, to humans who eat contaminated beef. Recent data suggest that some cases of CJD may be clinically unrecognized; in order to examine this proposal we reviewed all cases of dementia (n 5 10001) collected in the Runwell Hospital Brain Archive between 1964 and 1990. We identified 19 cases of spongiform encephalopathy of which only 11 were diagnosed before death. These 11 individuals had a characteristic clinical history of CJD (relentless mental deterioration, prominent motor signs and death within a year). Their brains showed little or no external abnormality. In contrast, only two of the eight clinically unrecognized cases had characteristic symptoms. The remaining six presented atypically; their illness lasted 3 years or more, motor signs were much less evident, and simple dementia was the most prominent feature. The brains showed moderate or severe cerebral atrophy. Our data indicate that only about 60% of prion disease cases with pathologically typical spongiform encephalopathy were identified clinically during life. This suggests that human prion disease may be more common than previously supposed and that a further review of the epidemiology of the disease is required © 1995 Academic Press Limited Key words: human spongiform encephalopathy, Creutzfeldt-Jakob Disease, diagnosis, neuropathology, prion protein THE TERM PRION DISEASE includes the human disorders Creutzfeldt-Jakob disease (CJD), GerstmannSträussler-Scheinker disease (GSS), Kuru, fatal familial insomnia and familial atypical dementia along with similar disorders in sheep (scrapie), cattle (BSE), goats, cats and other species (Prusiner et al., 1992). These conditions are subacute, progressive, transmissible and uniformly fatal degenerative diseases of the central nervous system. They have also been known as ‘subacute spongiform encephalopathies’, ‘slow virus infections’, ‘transmissible dementias’ and ‘transmissible amyloidoses’, all names which signify successive attempts to identify their cardinal features (Nevin et al., 1960; Corsellis, 1976; Prusiner, 1987; Gajdusek, 1991). The central link between all prion diseases is thought to be the presence of an abnormalprotease resistant-isoform (PrPsc) of the normal prion protein (PrPc) expressed by most neurons in the central nervous system (Prusiner et al., 1992). Human prion disease is rare; Kuru is nowadays almost extinct while the worldwide incidence of CJD is thought to be approximately one case per million people, per annum (Masters & Richardson, 1978; Brown et al., 1987; Harries-Jones et al., 1988; Will, 1993). GSS, regarded by many as a familial variant of CJD, affects very few families throughout the world. The importance of the human encephalopathies, however, far exceeds their rarity, not only because they have been transmitted experimentally to various animal species (Gibbs et al., 1968; Beck et al., 1969; Gibbs et al., 1980), but also because accidental person to person Correspondence to: Dr. Clive, J. Bruton, Curator, Corsellis Collection Brain Bank, Department of Neuropathology, Runwell Hospital, Wickford, Essex SS11 7QE Received 5 May 1995; accepted for publication 21 July 1995 © 1995 Academic Press Limited 1055-8330/95/040357 1 12 $12.00/0 357 358 transmission has occurred (Brown, 1988; Cochius et al., 1990; Buchanan et al., 1991; Brown et al., 1992). This raises the further theoretical possibility that one of the naturally occurring animal spongiform encephalopathies could also be transmitted, accidentally, to man. Considerable scientific and public interest has been focused on this last possibility during the recent outbreak of spongiform encephalopathy in cattle (Wells et al., 1987) engendering the fear that BSE might be transmitted to man from ‘infected’ meat (Southwood Committee, 1989). The outbreak of BSE is vast and not yet under control (Harrison & Roberts, 1992); BSE has been transmitted orally to mice (Barlow & Middleton, 1990) and the potential risk to human public health has been the subject of intense debate. Prudently, the British Government has decided to monitor new cases of human spongiform encephalopathy, particularly CJD, as they arise in the community (Southwood Committee, 1989; Will, 1993). This means, in effect, that new cases of CJD must be recognized during life and verified postmortem with a considerable degree of accuracy. The diagnostic criteria for CJD are widely recognized. Clinical diagnosis is provisional (Bell & Ironside, 1993) and neuropathological diagnosis has, until recently, been based on a combination of neuronal loss, glial proliferation and a typical spongiform degeneration in the brain (Prusiner et al., 1992; Bell & Ironside, 1993). Recent work has identified a further, perhaps, definitive molecular marker, the presence of an abnormal isoform of the prion protein (Roberts et al., 1986, 1988). This marker is consistently yet not universally associated with spongiform degeneration (Bell & Ironside, 1993) and is the reason why so many apparently disparate clinical syndromes have now been included in the definition of ‘prion disease’ and why the clinico-pathological spectrum of prion disease is now considered to be broader than previously supposed (Harrison & Roberts, 1991; Prusiner et al., 1992). A recent study undertaken to examine the problem examined 46 cases of ‘non-spongiform’ dementia and reported no evidence of occult prion disease (Brown et al., 1993). The authors concluded that “prion disease and spongiform encephalopathy are one and the same”. Nevertheless, their work has been criticized for its small sample size and inappropriate choice of clinical material (Collinge et al., 1993). Our investigation has been designed to examine two propositions. The first is that prion (CJD) disease may be more common than the current epidemiology suggests (Anon, 1990; Harrison & Roberts, 1991). The second is that patients with prion (CJD) disease who C.J. Bruton et al. have an older age of onset, few motor signs and prominent symptoms of dementia, are more likely to be clinically misdiagnosed (Harrison & Roberts, 1991). We report the results of a large retrospective survey from a neuropathological perspective. We have reexamined the clinical notes and the brains of all the histologically confirmed cases of prion disease in a large neuropathological archive (The Corsellis Collection) and determined whether prion disease was suspected during life. Our results are recorded below. Materials and Methods Runwell Hospital is a large institution for the mentally ill; its neuropathological archive (The Corsellis Collection) contains brain specimens collected not only from the hospital itself, but also from a wide variety of psychiatric and general hospitals in south-eastern England (the combined population for Greater London, South-East England and East Anglia in 1989 was 19.4 million). During the 25 year survey period, a total of 6559 brains were examined in the department. More than 1000 of these were from patients who had been considered, during life, to suffer from dementia. Nineteen individuals (13 females, 6 males; average age at death 62 years) form the basis of this study. They represent all cases of spongiform encephalopathy diagnosed, histologically, in the department of neuropathology at Runwell Hospital during the 25 years beginning 1 January 1964. Their clinical details, abstracted from original hospital notes, their post-mortem records and their referral specialities are summarized in Tables 1 and 2. Neuropathology As part of routine departmental procedure, all brains were fixed in 10% formalin for one month and examined nakedeye. Many were photographed before and after fixation. Blocks of tissue were taken routinely from the frontal, parietal, occipital and temporal lobes and embedded in paraffin wax. Cut sections from each tissue block were stained with Nissl’s method using Cresyl Violet, Haematoxylin and Eosin, Van Gieson, Congo Red, PAS, Heidenhain Woëlcke’s method for myelin and Holzer’s method for fibrous glia. Frozen cryostat sections were stained with von Braunmühl’s method for plaques and tangles. Immunocytochemistry Formalin-fixed, paraffin-embedded sections from the cerebellum of 12 cases in the present series ( Case Nos. 3, 4, 5, 6, 8, 10, 11, 13, 14, 15, 18 and 19) were examined and have been reported as part of an immunocytochemical study by Roberts et al., 1988 (Table 3). These authors used rabbit polyclonal affinity-purified antiserum raised against the hamster scrapie PrP 27–30 kDa. Paraffin-embedded sections from the remaining seven Archival survey of prion disease Table 1. Clinical features and macroscopical appearances of brains Sex Age at death (yrs) Length of illness (yrs) Brain weight (gm) Gyral atrophy 1 2 3 4 5 6 7 8 9 10 11 12 13 M M F F F F F F F M F M F 58 66 67 57 68 64 72 58 48 60 56 55 73 3/12 5/12 1 5/12 6/12 6/12 4/12 1 6/12 3/12 5/12 1 3/12 1040 1125 1140 1030 953 1367 1180 1338 1552 1335 1257 1353 1125 1 1 1 0 1 0 1 0 0 0 0 0 1 1 1 0 0 1 1 1 0 1 1 0 0 1 14 15 16 17 18 19 F F F M* M F** 72 75 48 40 72 79 6 41 3 4 31 ? 910 955 942 1140 900 850 111 111 11 1 11 11 11 111 11 1 11 11 Case no. ExtraDiagnosis Ventricular Abnormal Cerebellar Pyramidal pyramidal suspected dilatation behaviour Dementia Myoclonus ataxia signs signs during life 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 NO YES YES YES NO YES YES YES YES YES YES YES YES NO NO NO NO NO NO *Strong family history; **Schizophrenic. Leucotomy, 34 years before death; 0 5 normal; 1 5 slight; 11 5 moderate; 111 5 severe; Blank 5 absent; 1 5 present. 359 360 Table 2. Referral speciality, investigations and clinical diagnosis Case Year no. of death Referral specialty Length of Cerebral illness atrophy (yrs) (post-mortem) Investigations E.E.G. 1 1965 P 3/12 1 2 1966 GM 5/12 1 Abnormal: ‘Nevin Jones Syndrome’ 3 4 5 1970 1971 1976 N N GM 1 5/12 6/12 1 0 1 Abnormal: ? CJD Abnormal: ‘Diffuse non specific’ Abnormal: ‘Diffuse non specific’ 6 7 8 9 10 11 12 13 1979 1979 1979 1980 1981 1981 1982 1983 GM N N GM N N N N 6/12 4/12 1 6/12 3/12 5/12 1 3/12 0 1 0 0 0 0 0 1 Abnormal: ? CJD Abnormal: ? CJD Abnormal: ? CJD Abnormal: ? CJD Abnormal: ? Not CJD Abnormal: ? CJD Abnormal: ? ‘Diffuse non specific’ 14 1964 P 6 111 Abnormal: ‘Non specific’ 15 16 1977 1978 P P 41 3 111 11 Normal 17 1980 N 4 1 18 19 1983 1984 P P 31 ? 11 11 Other AEG: ‘Atrophy’ Diagnosis before death (clinical notes) ? Cerebral tumour. ? Presenile Dementia Cerebral biopsy: ‘normal’ CT: ‘Mild atrophy’ CT: ‘Normal’ CT: ‘Normal’ CT: ‘Mild atrophy’ CT: ‘Atrophy’ AEG: ‘Atrophy’ CT: ‘Atrophy’ Normal Nevin-Jones subacute vascular encephalopathy CJD CJD ? viral encephalitis ? cerebral degeneration CJD CJD CJD CJD CJD CJD CJD CJD Depressed. Hypochondrical. ? ‘Cerebral arteriosclerosis Senile Dementia Alzheimer’s Disease Familial Dementia. ? Huntington’s Chorea Senile Dementia Schizophrenia 1 5 slight; 11 5 moderate; 111 5 severe; GM 5 General Medicine; N 5 Neurology; P 5 Psychiatry; Blank 5 none. C.J. Bruton et al. Roberts et al. (1988) Remaining cases PrPsc staining Spongiform degeneration in cortex Case No. Roberts case Frontal Temporal Occipital Cerebellar No. PrPsc plaques Cerebellum 1 11 11 1 0 2 111 11 111 1 3 4 5 6 7 0 1 1 11 111 1 1 11 11 11 11 0 11 11 11 11 0 1 11 1 7 3 8 6 8 9 1 1 1 1 1 1 1 1 4 10 11 12 13 11 11 0 1 1 1 1 1 1 111 1 1 11 1 0 1 5 2 Yes Yes 11 Yes 14 15 16 17 1 11 11 1 11 1 11 1 11 0 1 1 11 1 0 11 9 12 Yes Yes 18 19 111 11 1 111 1 1 1 0 10 13 Cerebellum Frontal cortex Equivocal staining of diffuse deposits Intensely stained plaques molecular layer Widespread cellular staining Intense perivacuolar staining Sparse diffuse deposits in molecular layer Intense perivacuolar staining Punctate staining around Purkinje cells Glial and neuronal staining Sparse diffuse deposits Sparse cellular staining Other pathology Archival survey of prion disease Table 3. Histology and immunohistochemistry Yes Yes Yes Yes Yes Senile plaques1 None Rare plaque cores in granule cell layer Yes Yes Extensive cellular staining Diffuse deposits and cellular staining Leucotomy (34 yrs before death) 0 5 None; 1 5 Slight; 11 5 Moderate; 111 5 Severe. 361 362 C.J. Bruton et al. cases (1, 2, 7, 9, 12, 16, 17) were processed for immunocytochemistry to determine the presence of the abnormal isoform of prion protein using the following protocol: 12 µm sections of cerebral and cerebellar cortex were dewaxed and pretreated with an 80% solution of formic acid for 8 minutes. Sections were then incubated overnight at 4°C with a rabbit polyclonal antibody to the abnormal prion protein (IA8, kindly provided by Dr J. Hope as part of the MRC workshop on PrP immunostaining) at a dilution of 1:1800. Sections were further processed using an ABC Elite (Vector Labs, UK) protocol before counterstaining with haematoxylin. Results Microscopical examination of the brain in all 19 cases showed the characteristic appearances of spongiform degeneration of the cerebral cortex associated with neuronal loss, neuronal degeneration and a marked astrocytic gliosis (Fig. 1a–d ) that occurs in almost all cases of CJD (Masters & Richardson, 1978; Richardson & Masters, 1995). In CJD, the distribution and sever- ity of spongiform change is “not consistently present throughout the central nervous system and varies considerably from case to case” (Bell & Ironside, 1993). The severity and distribution of spongiform degeneration in our material was equally variable (Table 3). None of our 19 cases showed the extensive white matter involvement or end-stage gliosis that has been reported in some cases of long clinical duration (Richardson & Masters, 1995). Immunocytochemically, all 19 cases showed an accumulation of the abnormal isoform of the human prion protein (PrP). The 12 cases reported positive by Roberts et al. (1988) are identified in Table 3. The remaining seven cases (1, 2, 7, 9, 12, 16, 17) that were pretreated with formic acid and immunostained with the 1A8 antibody to prion protein revealed varying degrees of immunoreactivity (Table 3). The variety of structures stained in the different cases was similar to that recently described by Hayward et al. (1994). This ranged from plaque-like deposits in the molecular layer of the cerebellum (Fig. 2a), to dense perivacuo- Figure 1. Cerebral cortex from four cases showing the classical appearances of spongiform degeneration, neuronal loss and reactive gliosis. (a) Case No. 11 ‘Typical’ symptoms; survival 5 months. (b) Case No. 14 ‘Atypical’ symptoms; survival 6 years. (c) Case No. 15 ‘Atypical’ symptoms; survival 4 years. (d) Case No. 16 ‘Atypical’ symptoms; survival 3 years. Haematoxylin & Eosin 370, 3160, 3100 and 3160 respectively. Archival survey of prion disease Figure 2. (a) Plaque-like deposits of PrPSC in the molecular layer of the cerebellum in case no. 2; (b) Perivacuolar deposits of the protein in the cerebral cortex of case no. 7. Sections counterstained with haematoxylin. 363 364 C.J. Bruton et al. lar staining in areas of spongiform change in the cerebral cortex (Fig. 2b). In some of the cases there was focal staining of neuronal perikarya while in others occasional diffuse and/or cored plaque-like deposits were seen in the cerebral cortex. Thus far, our 19 cases resembled many others described in the literature. However, careful consideration of their clinical features and the naked-eye appearances of their brains suggested that they could be sub-divided into two main groups in that the brains of case numbers 1–13 showed little or no macroscopic abnormality whereas the remaining six were either moderately or severely atrophic (Figs 3, 4). An examination of the clinical histories of the 13 patients with externally normal brains (Table 1) showed the textbook features of CJD with a relentlessly progressive mental deterioration, prominent neurological motor abnormalities and death usually within a year (Bell & Ironside, 1993). The diagnosis of CJD was suspected during life in 11 of these 13 patients. In contrast, the histories of the other six patients (Case Nos. 14–19) who had notable cerebral atrophy, were very different; their illness lasted 3 years or more, motor signs were much less evident and in no patient was a diagnosis of spongiform encephalopathy suspected during life. The clinical investigations, the presumptive clinical diagnosis taken from the case records, the year of death and the referral pattern of all 19 cases are summarized in Table 2. Of the 13 patients whose brains showed little or no external abnormality, eight had been referred from a department of neurology, four from general physicians and only one had been admitted to a psychiatric hospital. Eleven of the 13 had an electroencephalogram, all 11 electroencephalograms were abnormal and in seven cases the electroencephalographic abnormality suggested a diagnosis of CJD. In contrast, of the six individuals who had cerebral atrophy, five died in a psychiatric hospital, three had been investigated by an electroencephalogram and two of the three electroencephalograms were reported as normal. Discussion Current estimates of the incidence of prion (CJD) disease derive from epidemiological studies where patients have been identified primarily on the basis of clinical criteria (Will, 1993; Bell & Ironside, 1993). These studies are well known and suggest that prion disease (CJD) has an incidence of some 0.5–1 per million of the population (typically 30–40 cases a year in the UK) a rate which, until recently, had remained stable for decades (Will, 1993). Our retrospective survey (n 5 10001 patients with dementia) indicates that clinical diagnosis alone may considerably underestimate the number of cases of prion protein with spongiform encephalopathy (CJD type). Most cases of prion (CJD) disease are thought to present with a consistent and characteristic clinical history. However, atypical cases are known to occur (Corsellis, 1976; Cruz-Sanchez et al., 1987; Kruger et al., 1990, Richardson & Masters, 1995). Some atypical cases are of ‘long duration’ and it has been suggested that the length of illness may possibly affect the degree of atrophy found in the brain (Corsellis, 1976). Nevertheless, cases of long duration have been considered to account for only 5% of patients with CJD (Brown et al., 1986). Brown and his colleagues found that many long duration cases were characterized by a very gradually progressive mental deterioration that, only terminally, gave rise to more rapid mental and physical decay. This description is in accord with the clinical history found in the present series (Table 1) where simple dementia without abnormal motor signs was the most common symptom. Such findings, in the presence of a moderate or severe degree of cerebral atrophy, would suggest a diagnosis of Alzheimer’s disease. It is hardly surprising, therefore, that the true diagnosis of CJD remained unsuspected in all six long duration cases in the present series before results of the neuropathological examination were known. Such atypical, ‘Alzheimer-like’ cases formed no less than 30% of all CJD brains diagnosed histologically in the Runwell Hospital Archive during the last 25 years. The implications of the data are self-evident. They suggest that the strict application of current clinical diagnostic criteria alone for the provisional diagnosis of CJD could lead inevitably to the omission of some cases that are characterized by an older age of onset, slower clinical course and prominent symptoms of cognitive decline (Anon, 1990; Harrison & Roberts, 1991). The consequence of this will be to undermine the validity of the currently accepted figures for the incidence of prion (CJD) disease and the public health measures that are dependent upon them (Anon, 1990). Our conclusions depend, of course, on the validity of the methods for diagnosing prion (CJD) disease and the representative nature of the Runwell Archive. The cases were diagnosed using well recognized histological criteria (Corsellis, 1976; Masters & Richardson, 1978; Bell & Ironside, 1993) in combination with immunocytochemical methods to determine the pres- Archival survey of prion disease 365 Figure 3. (a) Lateral view of left cerebral hemisphere, showing no external abnormality. Case no. 6 ‘Typical’ symptoms; survival 6 months. 30.7 (b) Lateral view of left cerebral hemisphere showing extreme cerebral atrophy. Case no. 14 ‘Atypical’ symptoms; survival 6 years. 30.7. 366 C.J. Bruton et al. Figure 4. (a) Coronal slice of brain at interventricular foramen level showing no external abnormality. Case no. 4. ‘Typical’ symptoms; survival 5 months. 30.7 (b) Coronal slice of brain at interventricular foramen showing severe cerebral atrophy. Case no. 16. ‘Atypical’ symptoms; survival 3 years. 30.7. 367 Archival survey of prion disease ence of PrPsc (Roberts et al., 1986, 1988; Prusiner et al., 1992; Bell & Ironside, 1993). Numerous studies have confirmed that PrPsc is a sensitive and specific molecular marker of prion disease (Prusiner et al., 1992; Nicholl et al., 1995). PrPsc immunocytochemistry is nowadays, commonly used to track the extent of pathological change in both animal and human brains (Prusiner et al., 1992; Lantos et al., 1992; Bell & Ironside, 1993). In addition, the absence of identifiable PrPsc in cerebral tissue has been used to identify the limits of the spectrum of prion pathology (Clinton et al., 1993; Gomi et al., 1994). In this way we believe that we have established as rigorously as possible that the patients in the present survey had indeed suffered from prion disease (CJD). The representative nature of the Runwell archive is, however, more difficult to defend. Runwell is a large mental institution and the 6559 brains examined during the 25 year survey included more than 1000 who were clinically demented. From this sample, 19 patients with spongiform encephalopathy are considerably more than would be expected from an ‘unselected’ series of 1000 clinically demented patients (Jellinger et al., 1990; Tomlinson, 1992) and the suggestion may be made that the archive ‘over-represents’ atypical cases of CJD that mimic Alzheimer’s disease. It is equally possible, however, that atypical ‘Alzheimer-like’ cases have been excluded inadvertently from other published studies (Brown et al., 1987; Harries-Jones et al., 1988) where individuals were collected from neurology clinics rather than from patients in the long stay wards of a psychiatric hospital. In this respect it is pertinent to consider that of the 13 patients in our series who had typical symptoms of CJD, no less than 12 had been referred from general medical hospitals and only one from a psychiatric hospital. Of the six patients with atypical, long duration, ‘Alzheimer-type’ symptoms five had died in a mental hospital. Nevertheless, we do not contend that our data conform to the most strict criteria of epidemiological vigour although the source of all brain material in the Corsellis Collection has been from unselected hospital post-mortems or referrals from other hospitals without special bias towards the diagnosis of CJD. Furthermore, a post-mortem study of over 1000 demented patients from a total of 6559 brains is a sizeable neuropathological sample by any standards and the Corsellis Collection is not only the largest, but also the most broadly based brain archive in the UK. Following the occurrence of BSE in cattle, the iatrogenic transmission of human prion disease in children treated with growth hormone, in women given gonadotrophic hormone and in some neurosurgical patients following the use of dural grafts (Brown et al., 1992), Government health policy designed to prevent the transmission of prion disease has become the subject of considerable debate. Government policy has been framed by our understanding of the epidemiology of the disease in humans. We have shown here that our understanding may be less robust than originally thought. One of the most worrying features of the present series, however, is that long duration ‘Alzheimer-like’ cases formed no less than 30% of all the prion (CJD) disease cases. These cases may slip through the diagnostic net and could represent an unidentified source of future iatrogenic infection. Furthermore, if changing environmental factors (e.g. BSE infected tissues) lead to an alteration in the clinical presentation of prion (CJD), the situation may become more difficult to interpret especially in view of the massive national decline in hospital postmortem examinations that are needed to confirm the diagnosis (The Registrar General’s Statistical Review, 1972; 1982–1987). 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