Prions

Please note that other past theories about TSEs agents include :
• Nemavirus (Narang, 1990) : an enzyme-coding ssDNA surrounded by 2 protein layers (the inner one made up of PrP^c)
• Lentivirus
• virino (Dickinson and Outram, 1979) : a host protein (sinc) protects vDNA from the immune system
• To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids. The study also suggests that the mechanism of resistance against these diseases does not operate at the level of gut absorption. The 50 sheep studied displayed different levels of resistance to scrapie, but all absorbed the prions equally readily. Although the study suggests that prion proteins can be absorbed by the gut, this seems to happen only rarely, at least in sheep. If the same is true of humans, then scientists may need to rethink exactly how the consumption of tainted meat leads to disease. Jeffrey's team predigested a mixture of abnormal PrP and typical sheep stomach contents. But when they then injected this into the gut, almost no rogue protein was absorbed, and subsequent testing found that the mixture contained almost no abnormal PrP. Sheep in the field are probably exposed to very little prion protein in their gut, because their stomachs would dispose of even the minuscule amounts ingested. Another possibility is that absorption occurs higher in the gut, before proteins are digested, perhaps in the mouth^{ref1, ref2}
Pathogenesis : according to such a theory, the CTD is essential in inducing the conformational switch (a-helix => b-fold) of the normal prion glycoprotein (PrP^c) into a disease-causing conformer (PrP^res / PrP^Sc) which aggregates into protease-resistant b-sheets. Host RNA molecules are cofactors for prion replication^ref. Induction of b-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine : antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize PrP^Sc but not the PrP^c isoform (antibody binding to the pathological can be created in vitro by partial denaturation of PrP^c). PrP contains 2 conserved N-glycosylation sites, which are partially occupied by a highly heterogeneous array of complex-type oligosaccharides carrying sialylated and fucosylated antennae. It has been proposed that the occupancy of the N-glycosylation sites can be correlated with differente strains of prions, but this hypothesis remains controversial. For example, prions isolated from the brains of patients with "classical" forms of CJD were found to have a very different sugar pattern from those isolated from patients with a variant form of CJD that struck some patients much earlier in life. But the sugar pattern of variant CJD prions was identical to those that cause BSE - a finding key to establishing that variant CJD was in fact the human form mad cow disease. Deletions of one or both glycosylation sites in transgenic mice result in unusual patterns of PrP^Sc deposition, suggesting that might play a role in the diseases process, but the functions of the sugars remain enigmatic. MALDI-MS molecular weight profiling of N-oligosaccharides released from hamster PrP and PrP^Sc revealed decreased amounts of oligosaccharides with bisecting GlcNAc residues, and increased amounts of tri- and tetraantennary sugars, in the PrP^Sc oligosaccharides compared with those from normal PrP. ES methodology, including ultrahigh-sensitivity MS/MS experiments on a Q-TOF instrument was used to provide detailed information on the occupancy of the second glycosylation site (Asn¹⁹⁶) of murine PrP, thus complementing a previous MS investigation of glycosylation at Asn¹⁸¹ in the hamster. The 2 sites are substituted with more than 60 bi-, tri-, and tetraantennary core-fucosylated structures, many of which are bisected. The oligosaccharides carry a limited number of antennae types. The diversity and difference in site occupancy arise largely from the arrangements of the antennae types in individual oligosaccharides. The NTD binds Cu⁺ preventing oxidative stress. It's the conversion process, not the accumulation of protein, that is key to the disease : depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. The switch may produce a toxic by-product, an intermediate form, or may deplete a factor that is crucial to brain-cell survival^ref. Prnp^-/- animals don't have the disease and the gene loss itself don't create any syndrome : so it may be presumed that PrP^c has no importance in cell metabolism or its functions are substituted by PRND / prion protein 2 / dublet, which codes for doppel (as the latter has no NTD, Prnp^-/- animals suffer more from oxidative stress).
Inherited or new onset point mutations in the human PRNP gene, coding for p27-30^{CD230 / prion protein / PrPc}, may involve different codons, causing clinically different inherited diseases which can transmitted under appropriate circumstances. As the normal protein is usually expressed in Mammalia neurons (GPI-anchored), prions usually cause transmissible spongiform encephalopathies (TSE), without inflammation nor immune reaction
Some examples of species barrier in TSEs are the forms affecting :
• Antilocapra americana
• Tragelaphus strepsiceros
• Tragelaphus angasii
• chronic wasting disease (CWD)
• Cervus dama
• Cervus elaphus
• Odocoileus hemionus
• Odocoileus virginianus
• Mustela vison : transmissible mink encephalopathy (TME)
• Felis catus : feline spongiform encephalopathy (FSE)
• Ovis aries : scrapie
• Bos taurus : bovine spongiform encephalopathy (BSE) / mad-cow disease
• scientists currently have no idea whether Takifugu rubripes, Gallus gallus, turtles (Testudines) and frogs (Xenopus laevis), which also harbour prions, suffer diseases as a result. This is partly because they don't know what symptoms to look for.
Human diseases : 4 common PrP^Sc types have been found in humans to date: types 1-3 in sporadic and iatrogenic CJD, and type 4 associated exclusively with vCJD
• kuru

Epidemiology : the Fore tribe of Papua New Guinea between 1920s and 1950s. There are 4 major regions in Guinea - the coast is called Basse (low) or Maritime Guinea, the next section of the interior is called Moyenne (lesser) Guinea, the more mountainous area to the east is called Haute (high) Guinea, and the tail that drops down to the southeast is called Guinea Forestiere or Forest Guinea. These major division also provide broad cultural division as well - Basse Guinea is primarily Susu people and culture, Moyenne Guinea is more of the Pular people, who originate from nomadic tribes, Haute Guinea is primarily Malinke and Forest Guinea is home to the Toma, Kissi and similar groups who carry on strong traditional practices for medicine and religion. Its incidence has steadily fallen after the abrupt cessation of its route of transmission (endocannibalism) in Papua New Guinea in the 1950s. 11 patients with kuru were identified from July, 1996, to June, 2004, all living in the South Fore. All patients were born before the cessation of cannibalism in the late 1950s. The minimum estimated incubation periods ranged from 34 to 41 years. However, likely incubation periods in men ranged from 39 to 56 years and could have been up to 7 years longer. PRNP analysis showed that most patients with kuru were heterozygous (M/V) at polymorphic codon 129, a genotype associated with extended incubation periods and resistance to prion disease^ref.
Aetiology : cannibalistic funerary rituals (women and babies ate brains of died relatives) (Zigas, 1956; Gajdusek, 1957; Hadlow, 1959). The first in the population to develop kuru shared the genetic feature common to most vCJD victims, i.e. codon 129 (=>Met) homozygosis (dubbed M/M). In the UK, 66% of people are either heterozygous (M/V) or valine homozygous (V/V) and so, some believe, are less susceptible to prion disease. But some of those with M/V and V/V genotypes also developed kuru, often after incubation periods of 20 years or more : hence further cases of vCJD may show longer incubation periods and occur in older individuals with the M/V and V/V genotypes. There is extensive anthropological evidence that cannibalism is not just some rarity that happened in New Guinea : other evidence of prehistoric cannibalism includes cuts and burn marks on Neanderthal bones and biochemical analysis of fossilized human feces. A protective allele exists for both kuru and nvCJD : selection for this SNP has been very widespread or happened very early in the evolution of modern humans, before human beings spread all over the planet : we can't say which of those it is; but the obvious implication is that prion disease has provided the selection pressure.
Symptoms & signs : up to 30 years incubation => spinocerebellar ataxia, dysarthria, head, trunk and limbs tremors disappearing during sleeping => flaccid paralysis, dysphagia
Prognosis : death within 3-9 months.
• familial fatal insomnia (FFI) : an inherited prion disease, transmitted as an autosomal dominant trait, from mutations in codon 129 (=>Met) or 178 (Asp =>Asn)
• sporadic FFI
• familial FFI
Symptoms & signs : average age of insorgence is 49; atrophy of ventral and dorsomedial nuclei of the thalamus, various stages of cerebral ataxia and cerebellar ataxia, progressive insomnia, hallucinations, stupor, and coma; autonomic and motor disturbances are also present.
Prognosis : death within 6 months to 3 years of onset (average 13 months)
• Neumann familial progressive subcortical gliosis : proteinase K (PK)-resistant prion protein with no mutation in the coding region of the PRNP gene. Furthermore, the linkage to chromosome 17 suggests that genes other than the structural gene for prion protein on chromosome 20 are involved in prion metabolism.
• Huntington disease-like 1 (HDL1) : autosomal dominant in individuals heterozygous for a 192-nucleotide insertion within the coding region of the PRNP gene, resulting in an expanded prion protein with 8 extra octapeptide repeats.
• Gerstmann-Sträussler-Scheinker (GSS) syndrome : an inherited prion disease from mutations in codon 102 (Pro=>Leu), A117V, 178 (Asp=>Asn), 200 (Glu=>Lys), D202N, Q212P, Q217R, Q219. Variable amounts of smaller, distinct PrP^res isoforms of ca. 7-15 kDa are seen in all GSS variants

Epidemiology :
• UK : onset at age 50-60; deaths of definite and probable GSS cases :
• 1990 : 0 deaths
• 1991 : 0 deaths
• 1992 : 1 death
• 1993 : 2 deaths
• 1994 : 3 deaths
• 1995 : 3 deaths
• 1996 : 4 deaths
• 1997 : 1 death
• 1998 : 1 death
• 1999 : 0 deaths
• 2000 : 1 death
• 2001 : 2 deaths
• 2002 : 1 death
• 2003 : 2 deaths
• 2004 : 0 deaths
• 2005 : 4 deaths
• USA : no confirmed cases since 1997
Symptoms & signs : common characteristics of cognitive and motor disturbances (cerebello-pyramidal syndrome) and the presence of multicentric amyloid plaques (AP) in the brain. 6 types exist :
• spongiform changes in GSS are related to the presence of high levels of these distinct 21-30 kDa isoforms (i.e. a subset of GSS P102L patients).
• ataxic form, there are progressive cerebellar ataxia and dementia
• telencephalic form, there are dysarthria, dementia, rigidity, tremor, and hyperreflexia
• GSS with neurofibrillary tangles (NFT), there are progressive short-term memory loss and clumsiness.
Prognosis : death occurs in 1 to 5 years.
• prion disease with protracted course : missense mutation in the PRNP gene (176640.0022)
• Creutzfeld-Jakob disease (CJD) : pulvinar atrophy. 0.25-2 cases per million inhabitants / year, expecially between age 55 and 75. 993 people have died of all forms of CJD in Britain between 1990 and 2004. Some 740 of those deaths were attributed to sporadic CJD, the most common form of the disease
• classical CJD (first described in 1920; average age of diagnosis : 68 years (can be manifested in infancy^ref); average duration of disease after diagnosis : 6.5 months)
• sporadic CJD (sCJD) (85-90%) : 117 (Ala=>Val), 178 (Asp=>Asn), 198 (Phe=>Ser), 200 or 210.
• Heidenhain's syndrome or variant : a rapidly progressive degenerative disease

Aetiology : methionine homozygosity at codon 129 and "type 1" protease-resistant prion protein, spongiform encephalopathy
Symptoms & signs : beginning and causing the most pronounced damage in the right occipital lobe, manifested by cortical blindness (=> atrophy of the visual cortex => bilateral inferior hemianopsia), presenile dementia, dysarthria, ataxia, athetoid movements, and generalized rigidity, but less damage in the cingulate gyrus and basal ganglia compared with patients with CJD who have cerebral ataxia as the leading clinical sign. The CSF assay for the 14-3-3 protein may be normal. Disease course : 5.7 months
• myoclonic variant : methionine homozygosity at codon 129 and to "type 1" protease-resistant prion protein
• variant with dementia of long duration : different genotypes at codon 129 and "type 2" protease-resistant prion protein.
• ataxic variant : different genotypes at codon 129 and "type 2" protease-resistant prion protein.
• variant with kuru plaques : different genotypes at codon 129 and "type 2" protease-resistant prion protein
Epidemiology : prevalence : 1 every 1,000,000
• UK : deaths of definite and probable sporadic CJD cases
• 1990 : 28 deaths
• 1991 : 32 deaths
• 1992 : 45 deaths
• 1993 : 37 deaths
• 1994 : 53 deaths
• 1995 : 35 deaths
• 1996 : 40 deaths
• 1997 : 60 deaths
• 1998 : 63 deaths
• 1999 : 62 deaths
• 2000 : 49 deaths
• 2001 : 56 deaths
• 2002 : 73 deaths
• 2003 : 58 deaths
• 2004 : 1 death
• 2005 : 41 cases, 63 deaths
• 2006 : 4 deaths
• USA : 2005 : 46 cases; 876 confirmed cases since 1997
The decrease in referrals is however potentially concerning from the point of view of complete case ascertainment. The reason for this drop is unknown and the numbers will need careful monitoring over 2005. It is particularly notable that the number of recorded sporadic CJD deaths in 2004 is lower than in the 3 previous years; however, the death data for 2004 may be incomplete. Detailed clinical and epidemiological information has been obtained for the great majority of patients. The case-control study for risk factors of CJD has continued and initial analysis has been undertaken. The post mortem rate for patients with suspected CJD is high, although there is ongoing evidence that this rate continues to decline, in line with general autopsy rates in the UK. This is reflected in the reduced number of brain specimens examined in the neuropathology laboratory in 2004. The reduction in sporadic CJD numbers (32 in 2004, 52 in 2003) is another potential concern along with the fall in referral numbers and sporadic CJD deaths noted above. In 1990-2003 mortality rates from sporadic CJD in England, Scotland, Wales and Northern Ireland were, respectively, 0.86, 0.84, 1.08 and 0.57/million/year. The difference between the rates in each country is not statistically significant (p>0.2). These rates are comparable to those observed in other countries in Europe and elsewhere in the world, including countries which are free of BSE. There was some variation in the observed mortality rates between the different regions within the UK but this variation is not statistically significant (p>0.2). The highest and lowest mortality rates from sporadic CJD were observed in the South West (SMR=129) and Northern Ireland (SMR=79).
Only about 3-4 cases appear in New Zealand and 300 cases in the USA each year (30 under age 55 and 270 above age 55), but several studies have suggested that the disorder might be more common than thought and that as many as tens of thousands of cases might be going unrecognized. First and foremost, CJD is not a nationally reportable disease. Even with nationally reportable diseases, one sees significant under-reporting, probably related to the health care delivery system in the USA, which is heavily weighted towards private-sector providers. Unfortunately the private sector is less consistent with disease reporting to state and federal levels. In the absence of a national mandate for reporting of a disease, the incentive becomes negligible, so the traditional "tip of the iceberg" seen in disease reporting shrinks even further. Clusters of sporadic CJD have been reported in various areas of the United States -- Pennsylvania in 1993, Florida in 1994, Oregon in 1996, New York in 1999-2000, and Texas in 1996. In addition, several people in New Jersey developed CJD in recent years, including a 56-year-old woman who died on 31 May 2003. Although in some instances, a BSE link was suspected, all of the cases ultimately were classified as sporadic CJD. Researchers at Yale University, who conducted a 1989 study that found 13% of Alzheimer's disease patients actually had CJD^ref. Although autopsies once were performed on approximately 50% of all corpses, the frequency has dropped to < 15% in the United States. The National Center for Health Statistics (NCHS) -- a branch of the CDC -- stopped collecting autopsy data in 1995.While autopsies have been declining, the number of deaths attributed to Alzheimer's has increased more than 50-fold since 1979, going from 857 deaths then to nearly 50 000 in 2000, partly due to the aging of the population. Switzerland discovered in 2002 that its CJD rate was twice that of any other country in the world. Switzerland had been seeing 8 to 11 cases per year from 1997 to 2000. Then the incidence more than doubled, to 19 cases in 2001 and 18 cases in 2002. In the 18-year period between 1979 and 1996 USA saw a jump from 1 case of sporadic CJD in people under the age of 30 (a warning sign for vCJD) to 5 cases in 5 years between 1997 and 2001. There have been increases in sporadic CJD in France, Germany, and Italy, all of which have detected mad cow disease in their cattle. PrP^Sc is deposited in the neuroepithelium of the olfactory mucosa in patients with sporadic CJD, and olfactory biopsy may provide diagnostic information in living patients. The olfactory pathway may represent a route of infection and a means of spreading prions.
Human remains in cattle feed could have caused the first case of mad cow disease. The hypothesis seeks to answer lingering questions about the fatal infection, which has affected 180,000 cows in Britain alone since the mid-1980s, and has gone on to cause > 100 deaths in humans. During the 1960s and 1970s Britain imported hundreds of thousands of tonnes of whole and crushed bones and animal carcasses. These were used for fertilizer and to feed livestock. Nearly 50% of these imports came from Bangladesh, where peasants gathering animal materials may have also picked up human remains. Other experts in the field view the idea with scepticism, saying that proof remains circumstantial. The argument isn't very compelling because there's no smoking gun evidence. Yet until 1986 no such illness had been spotted in cattle. Once recognized, BSE became widely known as 'mad cow disease'. The incidence of the disease then rocketed, peaking in late 1992. Tests indicated misshapen prion proteins in the brains of the cows as the source of the problem. And when authorities banned the practice of recycling animal remains into cattle feed the number of sick livestock began to drop. But experts continue to puzzle over how BSE arose in the first place. One of the most widely believed theories is that prions responsible for sheep scrapie got incorporated into cattle feed. Scientists argue that ingested scrapie prions radically altered the normal, analogous proteins in one cow, which then developed the first case of BSE.  But the Colchesters point out that cows have been exposed to scrapie for 70 years, so it is hard to explain why BSE emerged only recently. A more likely source is recent exposure to human remains carrying sporadic CJD. Religious customs in Bangladesh and surrounding areas mean that many corpses are disposed of in rivers. Nearly 50 per cent came from Bangladesh, India, and Pakistan, where gathering large bones and carcasses from the countryside and from rivers is an established local trade. Hindu funerary practices require that human remains are disposed of in a river, preferably the Ganges. Although the body should ideally be burned, many people cannot afford enough wood for a full cremation, the report's authors claim. Simply smoking the pelvis in women and the torso in men is sometimes enough. And many complete corpses are thrown into the Ganges. Human remains have been described in material delivered to processing mills. And during the 1960s, human material was confirmed in consignments of bones shipped into French docks from Asia.People may have collected remnants from such bodies when foraging for animal carcasses^ref. Any prions in these corpses might then have caused mad cow disease. Experts agree that the theory needs to be checked. More information needs to be collected about the number of deaths from sCJD in the Indian subcontinent (about 120 Hindu people each year : but the poor, who might account for many corpses in the Ganges, also have a relatively short life expectancy, while human prion diseases, meanwhile, often present themselves in old age), what happened to the bodies, and whether prions could have been transmitted in the way proposed. When human sporadic CJD prions were injected into mice in previous studies, the mice did not become ill^ref. The paper includes an overview of the various theories on the origin of BSE, namely:
• extrinsic origin (acquired from another species):
• human TSE
• sheep scrapie
• other species (such as farm, zoo, game, or wild animal)
• iatrogenic: contaminated medication transmitting TSE from another animal (including human).
• intrinsic origin (spontaneous event within one or more individual animals)
• predisposing factors (factors that could predispose to an extrinsic or intrinsic mechanism)
• host factors
• organophosphate pesticide exposure; high manganese or low copper levels in soil, Acinetobacter bacteria in cattle feed.
For the opinion of the Eu Scientific Steering Committee, Nov 2001, "Hypotheses on The Origin and Transmission of BSE". Readers might be reminded that the inclusion of human tissue in recycled animal feed was first discussed in April 1996, when a Swiss television program, investigating the links between contaminated animal feed and BSE, discovered that thousands of human placentas from hospitals in Switzerland had been turned into animal feed since the 1960s. Subsequently, the City council of Zurich launched an inquiry into how such a practice, which breached hospital guidelines on waste disposal, could remain undetected for so long. The department of health and environment in Zurich indicated that they had evidence that 820 kilograms of human placentas from 2 hospitals in Zurich were mixed in with dead pets and turned into animal feed since the start of 1995. They added the same disposal route was certainly used by hospitals in Winterthur and probably by hospitals in other Swiss cities^ref. It was further reported that the Zurich canton veterinarian pledged to stop the practice immediately and to consider prosecutions and that the feed producer had to destroy 200 tons of MBMs in stock. Was Switzerland the only country where such or similar practice prevailed?
Prognosis : average survival < 6 months
• familial CJD (5-10%) : 129 (=>Val)

Epidemiology :
• UK : deaths of definite and probable familial CJD cases
• 1990 : 0 deaths
• 1991 : 3 deaths
• 1992 : 5 deaths
• 1993 : 3 deaths
• 1994 : 4 deaths
• 1995 : 2 deaths
• 1996 : 2 deaths
• 1997 : 4 deaths
• 1998 : 4 deaths
• 1999 : 2 deaths
• 2000 : 2 deaths
• 2001 : 3 deaths
• 2002 : 4 deaths
• 2003 (7 July) : 1 deaths
• 2004 : 0 deaths
• 2005 : 6 deaths
• USA : 2005 : 22 cases; 159 confirmed cases since 1997
• iatrogenic CJD (< 1%) due to use of contaminated surgical equipments. The risk of contracting CJD from surgery is very low :
• neurosurgery (the greatest risk) : CNS
• cornea allotransplants
• dura mater allotransplants
• Tutoplast^® [Pfrimmer-Viggo GmbH & Co., Erlangen, Germany]
• Lyodura^® [Braun Melsungen AG, Melsungen, Germany] :
• Japan : on 1997, a nongovernment surveillance group for CJD in Japan reported 43 cases of CJD associated with receipt of cadaveric dura mater grafts. In all but one case, the most probable vehicle of transmission was a single brand of dural graft produced before May 1987. The precise number of dura mater grafts used in Japan is unknown, but an estimated 20,000 grafts per year might have been used during 1983 to 1987, when the manufacturer revised collection and processing procedures for the implicated product to reduce the risk for CJD transmission. As of March 2003, ongoing surveillance in Japan had identified an additional 54 dura mater graft-associated cases. All 97 patients received dura mater grafts during the period 1978 to 1991. 3 patients received more than one dural graft during this period, including one patient reported previously : in all 3 cases, the first graft was considered to be the source of infection. Medical conditions leading to the use of dural grafts in these patients included tumor (n = 46), brain hemorrhage (n = 14), Jannetta procedure for facial palsy (n = 13) and for trigeminal neuralgia (n = 6), intracranial aneurysm (n = 8), unspecified anomalies (n = 5), intracranial hematoma (n = 3), injury (n = 1), and ossification of the spinal posterior longitudinal ligament (n = 1). Latency periods ranged from 14 months (receipt in 1987 and onset in 1989) to 275 months (receipt in 1978 and onset in 2001). The median and mean latency periods were 122 and 125 months, respectively. During 1983 to 1987, the estimated minimum risk for CJD within 17 years of receipt of the implicated product in Japan was approximately 1 case per 1250 grafts (0.04%). No cases have been reported among patients who received their first dural graft after 1991; however, because of the long latency period between graft placement and symptom onset, additional cases of graft-associated CJD are likely to be reported. That all Lyodura^®-associated CJD cases to date occurred among patients who received grafts before 1992 suggests that all implicated grafts likely were processed before 1987; the implicated product's expiration date is 5 years after processing. In 1997, the report of 43 cases of dura mater graft-associated CJD in Japan represented the largest cluster of such cases in any one country. In one of the CJD cases reported in Japan, the implicated graft was used in a spinal (not an intracranial) procedure : this case suggests that transmission from contaminated dura might occur in areas of the neuraxis outside of the cranial vault. Subsequently, numerous other dura mater graft-associated cases were identified worldwide; nearly all patients had received the implicated product, including one additional U.S. patient
• USA : Lyodura^® never was produced by the manufacturer for distribution in the USA, and relatively few Lyodura^® grafts were used in this country. In May 1987, after identification of the first dural graft-associated CJD case in a 28-year-old U.S. woman from Connecticut who had received the implicated product 2 years before, the manufacturer revised its procedures for collecting and processing dura mater grafts to reduce the risk for CJD transmission (e.g., by discontinuing the commingling of dura and disinfecting them with sodium hydroxide). B.Braun Melsungen, its Japanese distributor, and the Japanese health ministry were later ordered by the courts to pay out £330,000 to the families of CJD victims in Japan, which had the largest Lyodura-associated outbreak. In 1997, an estimated 4500 dural grafts were distributed for use in the USA. After the TSEAC recommendations were issued, the number of dural grafts distributed for use in the United States declined to an estimated 900 grafts in 2002.
• Australia : Lyodura was introduced in 1972 and withdrawn in 1987, when the product was found to be linked to non-variant CJD. It was also used in Australia during the 1970s and 1980s : Australians who received the product had a 0.23% chance of contracting the disease.
• UK : a 34-year-old man (Simon Stratford) died of CJD > 15 years after an operation in which he was treated with infected Lyodura graft. The father-of-4 from St Neots, Cambridgeshire, needed a repair to his dura mater, a membrane lining between the skull and the brain, when he had a tumour removed at the Neurosurgery Department of Addenbrooke's Western General Hospital, Cambridge, in September 1987. Between October 2002 and April 2003, Mr Stratford had problems sleeping, then lost feeling in his face and arm, and became withdrawn and angry as his condition deteriorated, losing his ability to swallow, walk and speak. Up to 50 former hospital patients in Edinburgh are feared to be at risk of having contracted the human form of mad cow disease during brain surgery. The graft was withdrawn from the market in 1996 ago after it was linked with CJD. But NHS Lothian health bosses have warned that tracing the affected patients will not be possible as case notes were not stored. In Edinburgh, 4-5 patients per year may have had this material used. Many of the notes of these patients have been destroyed so it will make it impossible to know whether this was used. An intelligent guess might be made from the nature of the operation and the research. 3 of the surgeons from Edinburgh who are believed to have used the material retired in 1987, which could add further difficulties to assessing the extent of the problem. Experts will be forced to make only an "intelligent guess" over whether or not people were exposed to any danger. The risk of contamination from Lyodura grafts in the city is thought to stretch between 1982 and 1992 when the product was widely used. Lyodura, was withdrawn from the market 9 years ago following links with CJD.
Recipients of contaminated dura mater grafts might remain at risk for CJD for >22 years after receiving grafts. Patients with a rapidly progressive dementia consistent with CJD and a history of dural graft implantation should be reported through local or state health departments to CDC.
• administration of human-derived GH
• tonsils (see below)
• spleen (33%)
• appendix
• muscles (25%)^ref, may also harbor prions and contaminate surgical instruments
• liver transplantation or the associated albumin transfusions^ref
Surgical incidents occur when instruments that are considered potentially contaminated with the CJD agent during use on an index patient have been subsequently re-used on other patients. 183 surgical incidents were reported to the Panel during its first 4 years :
• sporadic CJD (possible, probable or definite) / 86 (47%)
• variant CJD (possible, probable or definite) / 61 (28%)
• other types of CJD, or 'at risk' of CJD, or CJD type unclear / 34 (19%)
• not CJD / 12 (7%)
Surgical incidents often involve a number of procedures from different specialties :
• gastroenterology / 68 (18%)
• obstetrics and gynaecology / 27 (7%)
• neurology/neurosurgery / 27 (7%)
• orthopaedics / 40 (11%)
• ophthalmology / 28 (8%)
• ear nose and throat / 26 (7%)
• dentistry /  28 (8%) : the UK Health Protection Agency (HPA) will use mice to see if vCJD can be passed on from contaminated dental instruments. They will also see if the mice show signs of the disease in the tissue in their mouths. The risk is thought to be small, but comes after concerns vCJD could be spread via donated blood and tissue. The government has taken measures to protect the blood supply by banning donations from certain groups, including those who have had blood transfusions since 1980, after it emerged 2 people may have been infected after having a blood transfusion. There have also been concerns about whether the prions responsible for vCJD can be spread by surgical instruments, especially when used during brain surgery. The aim of the 3-year experiment is to give the Department of Health more guidance on the risks posed by vCJD in dentistry and to determine whether stricter advice on the best cleaning methods needs to be issued. The study was outlined at the HPA's 2005 conference at the University of Warwick, which heard an estimated 75 million dental procedures are conducted in the UK each year and 2 million of these are invasive root canal treatments. Experts will also look at the different methods of decontaminating surgical instruments, such as manual scrubbing, the use of sound waves and washer disinfection
• other / 122 (34%)
• total /  364 (100%)
Investigation of past surgical incidents occasionally resulted in advice to remove from surgical use on patients other than the index patient (to quarantine or destroy, or donate for research) instruments considered to be potentially contaminated with the CJD agent. Such advice was generally only given for instruments that had not undergone > 10 cycles of use and decontamination since their use on certain index patients. During the last reporting year (year 4, 2003-2004) the Panel advised that instruments be removed from use on other patients following use on an index patient with, or at-risk of, CJD in 2 incidents. For reports to 31 Aug 2004, advice has been given to contact and inform 7 patients who were involved in 10 surgical incidents of their potential exposure to CJD via surgical instruments. Such advice was generally only given for patients who were definitely exposed to potentially contaminated instruments immediately after the instruments had been used on certain index patients. It was advised that these patients should be considered to be potentially at risk of CJD for public health purposes and asked to take certain precautions (i.e. not to donate blood or other tissues, and to inform their medical and dental carers prior to any invasive procedures) in order to reduce the potential risk of onward transmission of the CJD agent by possible
iatrogenic routes.
Patients with extraneural PrP^Sc had a significantly longer duration of disease and were more likely to have uncommon molecular variants of sporadic CJD than were patients without extraneural PrP^Sc. Among the patients registered by the end of 2000 the frequency of methionine-methionine (MM) genotype was much lower in the UK (4%, 1 of 27 cases) than in France (62%, 48 of 77, p=0.0001) : different genotypic distributions of hGH-transmitted CJD in French and UK patients may be due to infection with different contaminating strains of the CJD agent in France and the UK. hGH-transmitted CJD homozygous cases in France (of which 74% (48 of 65) were MM) arose significantly earlier than in heterozygous cases
Prevention : surgical instruments used on people who are known to have CJD are quarantineed after use, and then destroyed
• an enzyme from bacteria found in volcanic pools digests the prions when exposed to a solution with a temperature of 60°C, reducing their infectivity almost a million-fold
• decontamination of surgical instruments by using radio-frequency gas-plasma treatment^ref
Epidemiology :
• UK : deaths of definite and probable iatrogenic CJD cases
• 1990 : 5 deaths
• 1991 : 1 death
• 1992 : 2 deaths
• 1993 : 4 deaths
• 1994 : 1 death
• 1995 : 2 deaths
• 1996 : 4 deaths
• 1997 : 6 deaths
• 1998 : 3 deaths
• 1999 : 6 deaths
• 2000 : 1 death
• 2001 : 3 deaths
• 2002 : 0 deaths
• 2003 : 4 deaths
• 2004 : 0 deaths
• 2005 : 3 deaths
• USA : 2005 : 0 cases; 15 confirmed cases since 1997
• new variant CJD (vCJD / nvCJD) was first recognized in a British 19-year-old boy who died in 1995

It has infectious aetiology via...
• oral route from :
• specified risk material (SRM) from cattle (Bos taurus) affected with BSE : in 1996 the human PrP^Sc was found to resemble the PrP^Sc found in BSE. The Committee for Proprietary Medical Products (CPMP) of the European Agency for the Evaluation of Medicinal Products (EMEA) established, in 2001, 4 BSE risk categories on the basis of examinations of cows and other animals that became infected with similar diseases (for example, sheep which contracted scrapie). These categories have been summarized in the "Note for guidance on minimizing the risk of transmitting animal spongiform encephalopathy agents via medicinal products," as follows :
• category I (very high infectivity):
• brain
• spinal cord
• eyes
• category II (high infectivity):
• cerebrospinal fluid
• hypophysis (pituitary gland)
• ilium (lower part of the small intestine)
• lymph nodes
• tonsils
• spleen
• suprarenal gland (e.g. from the peripheral nerve tissue and the suprarenal gland of the BSE positive 94 month old cow that had died on 9 Mar 2004^ref.)
• upper part of the colon (proximal)
• dura mater ("hard" meninges)
• placenta
• pineal gland
• category III (moderate infectivity):
• bone marrow
• liver
• lung
• mucous membrane of the nose
• pancreas
• peripheral nerves could carry infectious prions from the brain to
• tongue : anyway mice injected with nerve cells from infected cow tongue remain healthy
• there are a number of papers out regarding the issue of prions being found in muscle. Indeed, since prions are believed to be associated with neural tissue, then it stands to reason prions can travel along any nerve path. One needs to understand the fundamentals of US Slaughter facilities to comprehend the situation. Cattle are humanely stunned with a captive bolt stunner that penetrates or piths the brain rendering the animal unable to feel pain. However, the animal is not dead. Depending upon the speed of the slaughter plant the animal remains alive, but unable to comprehend or feel pain, for an average of 2 to 7 minutes before the throat is cut, exsanguinating the animal. During that 2 to 7 minutes the neurological tissue that captive bolt compressed into the brain and into the blood stream can circulate throughout the body, as long as the heart beats. The prion is smaller than a red blood cell. Therefore, it would appear that the prion agent can be in muscle tissue.
• thymus
• lower part of the colon (distal).
• category IV (no detectable infectivity):
• mammary glands
• milk
• testicles
• ovaries
• fetal tissue
• uterus
• heart
• kidney
• urine
• seminal vesicle
• thyroid gland
• blood
• serum
• coagulae
• skeleton muscles
• saliva
• salivary glands
• feces
• gall bladder
• connective tissue
• hair
• skin
• bones
• cartilage tissue
The said list is not similar to the SRM list that covers organs prohibited from entering the food and feed chain. The latter have been selected while considering their relative risk factor as food for man and animals, which is related to quantitative aspects of their BSE-prion contents.
2 ewes (Ovis aries) fed 5 mg of BSE-infected material had lambs that died of BSE after showing signs of infection in their tonsils 546 days after birth. Their mothers had shown no outward signs of the disease at lambing, one showing them 73 days after lambing, and the other 198 days after. But it is still not certain that the lambs were infected while in the uterus, or shortly before or after lambing. The disease may have spread through the birthing fluids or in some other way. The evidence so far suggests this is far more likely than the lambs catching the disease from other apparently unaffected sheep. It is already known that BSE-like diseases can be transmitted via blood in humans as well as animals, but there has been no evidence that it has been handed from dam to calf in cattle, or mother to baby in people. The sheep involved were of a genetic type that in lab tests previously appeared most susceptible to BSE. But it is unclear how many such sheep are in flocks on farms. There are 15 different genotypes, and unlike in BSE in cattle, genetic type seems important. Unfortunately at present there would be no way of identifying resistant sheep in time for them to go into food, while banning others. The fear about sheep has existed for years because, until the late 1980s, they were fed the same sort of feed as cattle. However if it was ever in sheep, there is no suggestion that it ever existed on a large scale. There is some good news. The lambs that seem to have inherited BSE showed a brain signature similar to BSE in cattle. Officials have been worried that some BSE in sheep, if it existed, might have been masked by a similar disease called scrapie, not known to be dangerous to humans^ref
It will be also interesting to note whether results of controlled infection trials in bovines within the UK-based BSE research project, "Experimental production of bovine tissue for the validation of BSE diagnostic tests (SE1736)", have been published or might become available
One assumption lies at the root of efforts to keep the meat we eat safe from mad cow disease: If an animal with BSE falls ill with another infection, its immune response can carry large numbers of prions to organs throughout its body. In 2003, small amounts of prions in the muscle tissue of people who had died from vCJD were detected, suggesting that animal meat might harbour trace amounts of these disease proteins too. When prions were administered to mice with 5 inflammatory diseases of kidney, pancreas or liver, in all cases, chronic lymphocytic inflammation enabled prion accumulation in otherwise prion-free organs. Inflammatory foci consistently correlated with lymphotoxin upregulation and ectopic induction of PrP^C-expressing FDC-M1⁺ cells, whereas inflamed organs of mice lacking lymphotoxin- or its receptor did not accumulate PrP^Sc nor infectivity upon prion inoculation. The amount of prions in these organs was just as high as is generally found in diseased spleens : cells involved in the inflammatory response somehow help the prions to replicate, and to spread to the parts of the body being targeted by the immune reaction. By expanding the tissue distribution of prions, chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission^ref.
The age at which specified risk materials (SRM) (tissue such as brain and spinal cord) are removed from cattle slaughtered for food was set at 12 months in all European Union (EU) member states (except for the United Kingdom) in 2000. This measure was designed to protect consumers of beef from the risk of bovine spongiform encephalopathy (BSE)^{ref1, ref2}. New information supplied by the World Organization for Animal Health (OIE), surveillance data and scientific research was recently considered by members of the European Food Safety Authority Biological Hazards Panel. Based on this evidence and a review of previous discussions, the panel recognized that the bovine central nervous system is unlikely to become infectious until an infected animal is considerably older than 12 months^ref. The average age of cattle with BSE in the EU has increased between 2001 and 2004, from 86 months to 108, most likely due to effective control measures. It is further observed that the number of BSE cases reported at an age less than 35 months in past years in the EU has been only 4 out of a total number of 6520 BSE cases on a total of close to 41 million animals tested. The minimum age of BSE cases in the EU has been 28 and 29 months (2 animals) in 2001, 32 and 34 months in 2002, 36 months in 2003 and 42 months in 2004. The 3 youngest animals were emergency slaughter, whereas the remainder of BSE cases in young animals (i.e. younger then 48 months, table 1) included all target groups. Only 4 cases in cattle under 35 months have been reported since 2001. The lowest age of animals with BSE rose from 28 to 42 months between 2001-4. From pathogenesis studies of infectivity distribution throughout the incubation period, and the earliest age of clinical manifestation, it was concluded that a bovine central nervous system would not become infectious until at least 26 months of age, as it appears that only during the last quarter of the incubation period does the disease become detectable. The panel concluded that setting the age for SRM removal at 21 months would allow a reasonable safety margin. Based on this opinion, the European Commission will consider whether changes to the existing legislation should be proposed. There is currently no scientific basis for raising the age limit for bovine tonsil and intestine. The most significant factor which deserves to be taken into account when considering the age limit for the human consumption of SRM is the pace and rate of the feed ban implementation; in other words -- the time span elapsed since the actual, final withdrawal of ruminant Meat and bone meal (MBM) from cattle feed, and indeed -- in view of potential cross contamination -- also from the feed of other food animals. It seems that effective, full implementation had not taken place in the EU, as a whole, prior to the end of 2002. As stated in the report, breaches have been encountered even later. This delay deserves to be seen as a significant factor in the preference of EFSA'a Scientific Panel on Biological Hazards to choose and recommend a SRM age limit of 21 rather than 30 months. Other "GBR III" countries, such as Canada, have opted to apply a 30-months-age SRM limiti^ref. The EU has completed, since 2000, the assessment of the geographical BSE-risk (GBR) of 66 countries. 3 assessments have been carried out during 2005, relating to Costa Rica, El Salvador and Nicaragua, all 3 obtaining the status of GBR II ("Presence of one or more cattle clinically or pre-clinically infected with the BSE agent is unlikely but not excluded"). 7 other GBR assessments have been issued by EFSA in September 2004, relating to Canada, USA, Mexico, South Africa, Australia, Sweden and Norway^ref
• deers with CWD : efforts to find the infectious prion in the muscle of infected animals by seeing whether antibodies to the prion could find any and bind to them have previously failed. But Telling's lab has now shown, using transgenic mice, that disease prions can reside in the muscle of deer infected with CWD. The team replaced the gene for the normal mouse version of the prion protein with the normal gene from deer, so the mice made the normal, healthy deer protein. They then injected the mouse brains with tissue from infected deer. 12 to 18 months later, the mice developed encephalopathy. Tissues from both the infected deer's brains and thigh muscles caused disease. Muscle took slightly longer to cause disease than brain tissue, showing it had slightly less prion. We don't know that it is transmitted in the wild by animals eating muscle from infected animals. We now have to see where else the prion might be, including saliva and even excrement, using more transgenic mice. Because we tested deer that were already ill, we don't know what the distribution of prion is in animals that are still incubating the disease. Hunters have been warned by wildlife agencies not to kill and eat obviously ill animals, but an animal not yet showing signs of the disease might still carry the abnormal prion, albeit less of it. It is also unknown whether people can catch encephalopathy by eating  CWD-infected meat, as they can from eating BSE-tainted meat^ref
• studies to test the transmissibility of the BSE agent to pigs (Sus scrofa) began in 1989. Parenteral inoculation of the agent by three routes simultaneously (intracranially, intravenously and intraperitoneally) produced disease with an incubation period range of 69-150 weeks. Pre-clinical pathological changes were detected in 2 pigs killed electively at 105 and 106 weeks post-inoculation. Infectivity was detected by bioassay in inbred mice in the CNS of those pigs that developed spongiform encephalopathy. Infectivity was also found in the stomach, jejunum, distal ileum and pancreas of terminally affected pigs. These findings show that pigs are susceptible to BSE. In contrast, disease failed to occur in pigs retained for 7 years after exposure by feeding BSE-affected brain on three separate days, at 1-2 week intervals. The amounts fed each day were equivalent to the maximum daily intake of meat and bone meal in rations for pigs aged 8 weeks. No infectivity was found in tissues assayed from the pigs exposed orally. This included tissues of the alimentary tract. It is suggested that these pigs did not become infected. The relatively high oral exposure used in these experiments compared with feed-borne exposure in the field may explain the absence of an epidemic of spongiform encephalopathy in domestic pigs concurrent with the BSE epidemic in the UK^ref. Pigs and poultry in the United Kingdom have undeniably been exposed to the BSE agent. They consumed the same ruminant protein that gave rise to the BSE epidemic in cattle, but there has been no evidence of an epidemic in these species. Experimental investigations have shown pigs to be susceptible to infection by multiple parenteral challenge, but resistant to oral exposure with BSE-infected cattle brain. Current but incomplete evidence suggests that they are also resistant to oral challenge with sheep scrapie. Studies in domestic chickens indicate that they are resistant to both parenteral and oral challenge. Unfortunately, no published data exists on the susceptibility of fish to infection. Incidental findings in the brains of unexposed pigs are described that could otherwise give rise to concerns aboutthe presence of a transmissible spongiform encephalopathy in pig populations around the world^ref. In an experimental study of the transmissibility of BSE to the pig, seven of 10 pigs, infected at 1-2 weeks of age by multiple-route parenteral inoculation with a homogenate of bovine brain from natural BSE cases developed lesions typical of spongiform encephalopathy. The lesions consisted principally of severe neuropil vacuolation affecting most areas of the brain, but mainly the forebrain. In addition, some vacuolar change was identified in the rostral colliculi and hypothalamic areas of normal control pigs. PrP accumulations were detected immunocytochemically in the brains of BSE-infected animals. PrP accumulation was sparse in many areas and its density was not obviously related to the degree of vacuolation. The patterns of PrP immunolabelling in control pigs differed strikingly from those in the infected animals^ref. A new study details transplanting swine organs to people and the possibility of TSE disease being transmitted. Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies, National Animal Disease Center Virus and Prion Diseases of Livestock, Evaluation of a diet free of animal protein in germfree swine. 2 experiments were conducted in which germfree pigs or pigs monoassociated with Lactobacillus paracasei subspecies paracasei were fed either a traditional milk-based diet (Esbilac) or an experimental diet free of animal protein (DFAP). Throughout the 16-day study, animals' clinical condition, total weight gain, feed conversion, and bacterial contamination were monitored. At the conclusion of the study the animals were killed, necropsied and tissues sampled for L. paracasei isolation. General pig disposition remained consistent between treatment groups and trials, except for two animals that developed mild diarrhoea during trial 1. All pigs remained viable during the study irrespective the diet fed or probiotic inoculation. Germfree pigs fed the Esbilac diet gained on average a total of 1034 +/- 63.0 g, and had a feed conversion ratio of 0.17 +/- 0.01 g of gain per 1 ml of diet. Germfree pigs fed the experimental diet gained on average a total of 599 +/- 151 g, and had a feed conversion ratio of 0.10 +/- 0.02 g of gain per 1 ml of diet. Monoassociated pigs fed the Esbilac diet gained on average a total of 862 +/- 70.3 g, and had a feed conversion ratio 0.14 +/- 0.01 g of gain per 1 ml of diet. Monoassociated pigs fed the experimental diet gained on average a total of 563 +/- 96.8 g, and had a feed conversion ratio of 0.09 +/- 0.02 g of gain per 1 ml of diet. Lactobacillus paracasei established extensively in pigs fed either the Esbilac or experimental diets. Lactobacillus paracasei had no effect (P >0.05) on piglet growth and did not display any interactions based on the diet fed. Measured growth parameters were statistically different (P <0.05) based on the diet fed and variance seen between trials. In conclusion, a DFAP has been developed and has been shown to be capable of sustaining life in piglets up to 16 days of age^ref.
• risk from injection of chicken breast or tumbling of chicken meat with water and other beef proteins to enhance the weight to increase profit margins has still to be assessed
In humans, even if current opinion holds that prions infect people through the stomach, prions might enter via small wounds on the tongue : the incubation period following TME agent inoculation into the lingual muscles was the shortest (around 80 days) among the 5 nonneuronal routes of inoculation, including another intramuscular route and oral route (190 days). Deposition of the abnormal isoform of the prion protein, PrP^Sc, was first detected in the tongue and submandibular lymph node at 1 to 2 weeks following inoculation of the tongue with TME. PrP^Sc deposits in the tongue were associated with individual axons, and the initial appearance of TME in the brain stem was found in the hypoglossal nucleus at 2 weeks postinfection. At later time points, PrP^Sc was localized to brain cell groups that directly project to the hypoglossal nucleus, indicating the transneuronal spread of TME. TME PrP^Sc entry into the brain stem preceded PrP^Sc detection in the rostral cervical spinal cord. These results demonstrate that TME can replicate in both the tongue and regional lymph nodes but indicate that the faster route of brain invasion is via retrograde axonal transport within the hypoglossal nerve to the hypoglossal nucleus. Topical application of TME to a superficial wound on the surface of the tongue resulted in a higher incidence of disease and a shorter incubation period than with oral TME ingestion. Therefore, abrasions of the tongue in livestock and humans may predispose a host to oral prion infection of the tongue-associated cranial nerves^ref. HY strain of the TME PrP^Sc accumulates within skeletal muscle cells, in addition to axons, in the tongue of hamsters following intralingual and intracerebral inoculation. Localization of PrP^Sc to the neuromuscular junction suggests that this synapse is a site for prion agent spread between motor axon terminals and muscle cells. Following intracerebral inoculation, the majority of PrP^Sc in the tongue was found in the lamina propria, where it was associated with sensory nerve fibers in the core of the lingual papillae. PrP^Sc staining was also identified in the stratified squamous epithelium of the lingual mucosa. These findings indicate that prion infection of skeletal muscle cells and the epithelial layer in the tongue can be established following the spread of the prion agent from nerve terminals and/or axons that innervate the tongue. Ingestion of meat products containing prion-infected tongue could result in human exposure to the prion agent, while sloughing of prion-infected epithelial cells at the mucosal surface of the tongue could be a mechanism for prion agent shedding and subsequent prion transmission in animals^ref. These findings suggest that food products containing ruminant or cervid tongue may be a potential source of prion infection for humans. Sensitive western blotting revealed consistent PrPSc accumulation in skeletal muscles from forelimb and hindlimb, head, back and shoulder, and in tongue of hamsters orally infected with scrapie^ref. When the process of eating and digesting infected meat is mimicked by mashing up brain tissue that contained prions from patients who had a form of CJD and exposing it to a range of harsh proteases from the mouth, stomach and intestine, prions escape this attack almost unscathed, as does ferritin, which is abundant in meat. The 2 proteins seem to stick together. By attaching fluorescent tags to the 2 proteins, they showed that they are transported through a growing sheet of human Caco-2 intestinal epithelial cells hand-in-hand^ref. Prions can also pass through the gut unaided, through M cells^ref. The ferritin protein is almost identical in a wide range of species, which might allow it to latch on to and ferry foreign prions from cows : ferritin might transport prions to humans from other animals with prion disease, such as deer and elk suffering from chronic wasting disease. Hobbling the transport of ferritin might block prions from entering the body, but this would be difficult to use therapeutically because people would have to be treated at the time when they were being exposed, which is many years before symptoms of the disease emerge.
Of 2 macaques given a 5 g oral dose of brain homogenate from a BSE-infected cow, 1 macaque developed vCJD-like neurological disease 60 months after exposure, whereas the other remained free of disease at 76 months^ref.
• corneal allograft from infected donor
• maternal transmission : the Chief Medical Officer for England asked SEAC to consider current evidence and comment on the potential transmission of variant Creutzfeldt-Jakob disease (vCJD) from mother to child via human breast milk. In utero transmission was also considered. The committee also commented on the scientific basis of a risk reduction measure for possible transmission of vCJD via banked breast milk. No diagnostic test is currently available for the detection of abnormal PrP in milk. Research is under way to develop tests to screen for the possible presence of abnormal prion protein (PrP) in milk samples from cattle experimentally infected with BSE [A joint FSA/SEAC milk working group is monitoring and providing advice on this research carried out at the Veterinary Laboratories Agency.] These modified tests may also be applicable to human milk. However, it is not yet clear when/if a reliable test will be available. A small number of breast milk banks in the UK supply highly vulnerable premature babies for whom no milk may be available from the mother. A model developed by the Department of Health to assess the effect of pooling breast milk from multiple donors on the possible risks of transmission of vCJD via breast milk banks was considered. There is some, albeit limited, published epidemiological and experimental research on maternal transmission of prion diseases. There are also unpublished surveillance data of children born to vCJD cases from the National CJD Surveillance Unit and UK surveillance of neurological illness in children which might inform on potential risks of maternal transmission. There is no evidence that vCJD infectivity has ever been transmitted through breast milk. However, a theoretical risk exists. Modelling studies clearly show that the practice of pooling breast milk increases the number of donors to which a recipient is exposed and thereby increases the potential risk of an infant receiving milk contaminated with vCJD infectivity. The theoretical risk of infection can be minimised by not pooling the milk, by the use of individual hand operated breast milk pumps for single donors, and by the use of single-use sterilised bottles for collection. In addition, available evidence suggests that infection/inflammation of the breast results in increased lymphocytes in milk and therefore increased risk of infectivity. This risk would be minimised if milk from donors showing signs of infection were not used. The committee suggested that, if practicable, milk could be stored for an appropriate period of time to allow the health status of donors to be monitored, before it is released. However, information was not available to the committee on whether long-term storage of human milk is detrimental to its nutritional quality. There is evidence from animal studies for low-level maternal transmission of prions in cattle and sheep. This transmission may occur in utero, via milk and/or perinatally. However, the possibility that this putative maternal transmission might have been due to another mode of transmission, for example through a contaminated environment or feed, cannot be ruled out. In contrast, in humans there is no evidence for maternal transmission in cases of familial prion disease, other than the transfer of a mutant form of the PrP gene, and there is no evidence of maternal transmission of kuru. However, compared with other human prion diseases vCJD may pose a greater risk because of the greater involvement of the lymphoreticular system in vCJD pathogenesis. Although, breast tissue (and placenta) from a single vCJD case tested negative for PrPvCJD, transfer of infectivity to breast milk may depend on the physiological status of the mammary gland. Similar tests or infectivity bioassays have not been conducted on breast tissue from lactating patients with vCJD. A published study suggesting transmission of sCJD in colostrum^ref was considered unreliable because tissues not normally associated with high levels of infectivity (blood and placenta) showed equivalent infectivity to that of the brain in this study. Analysis of prospective surveillance data of UK children born to mothers with, or that had subsequently developed clinical vCJD, provide no evidence for maternal transmission of vCJD. However, the number of cases is very small and the incubation period of vCJD, if transmitted from mother to child, is unknown and so the children may yet be too young to have developed symptoms. The phenotype of BSE infection in humans expressing PrP genotypes other than M/M at codon 129 is not known. Given recently published studies in mice expressing the human PrP gene^ref, which suggest that the human PrP genotype may affect disease phenotype, the committee considered it very important that undiagnosed neurological diseases be carefully monitored. In this respect, amongst others, it is recommended that the careful monitoring of neurological illnesses through the PIND surveillance of children^ref continue. In summary, there is currently no pidemiological evidence for maternal transmission of vCJD, including transmission via breast milk. However, there is a hypothetical risk. Although available evidence is limited and mostly indirect rather than direct, this risk, if any, appears to be low. As a risk cannot be excluded, a watching brief should be maintained.
• cosmetics tainted with BSE : the FDA is urging cosmetic firms to not use certain cattle-derived substances in their products, because consumers run a slight risk if they apply  to open cuts
• plasma components : epidemiologic evidence of the absence in humans of disease transmission can probably be explained by the absence of significant plasma infectivity until the onset of symptomatic disease, and comparatively low levels of infectivity during the symptomatic stage of disease; the reduction of infectivity during plasma processing; and the need for at least 5-7 times more infectious agent to transmit disease by the intravenous than intracerebral route. These and other factors probably also account for the absence of transmission after the administration of whole blood or blood components. Anyway 2 cases have been reported in UK :
• a 62-yrs old man who received 5 units of erythrocytes during an operation in 1996 developed nvCJD. One of these units was traced to a 24-year-old donor in whom vCJD developed >3 years after the blood was donated, dying shortly afterwards. In 2002 (6.5 years after the transfusion), vCJD developed in the recipient, who 13 months after illness onset. An autopsy confirmed the diagnosis of vCJD. 1 other person is thought to have received blood from the same donor^ref.
• a PRNP codon 129 heterozygous methionine/valine (MV) man received a unit of erythrocytes in 1999 from a donor who developed vCJD 18 months after blood was donated. The recipient died of a ruptured aortic aneurysm 5 years after the transfusion. Tests of the patient's spleen and cervical lymph node detected protease-resistant prion protein with a Western blot mobility pattern and glycoform ratio similar to those seen in other vCJD patients. These results and the absence of neurologic symptoms and brain pathologic findings indicated that this patient had a subclinical vCJD infection. Prion protein gene sequencing showed heterozygosity for methionine and valine at codon 129, which indicated that persons not homozygous for methionine (>50% of the population) can be infected by the vCJD agent^ref. Whether non-homozygotes can also develop vCJD, albeit with a longer incubation period, is as yet unknown : this suggests that wider groups of people could be at risk than was thought. According to a Finnish study, 42% (37% ?) of the UK population is MM homozygous at PRNP codon 129, with 47% heterozygous methionine/valine (MV) and the remaining 11% homozygous for valine (VV)^ref. vCJD was successfully transmitted to mice with all three human genotypes, but behaved differently in each. Transmission occurred least easily in the 16 VV mice compared to the 16 with the MV gene pattern and the 17 with MM. However, most of the MV animals did not develop clinical signs of vCJD during their short lifetime compared to those with MM, most of whom did. Only one mouse with VV showed signs it had contracted the disease, though it had no clinical symptoms.
• an elderly woman who received a blood transfusion in 1999 from a donor who later died from vCJD died from an unrelated medical condition : she harbored prions in her spleen and cervical lymph nodes
The chances that 2 confirmed infections among people who received blood was caused instead by eating mad-cow tainted beef were remote -- 1 in 80,000 to 1 in 1 billion. There is a concern that individuals who are incubating the disease, who themselves don't show any evidence of the disease, could still pose a risk to others, if they were blood donors, or if they had an operation which involved instruments contacting infected tissues : in March 2004 Britain banned people who had had transfusions over the past 24 years from donating blood to reduce the risk of spreading the disease. 9 donors have been identified as having developed vCJD after giving blood. Collectively, they made 23 plasma donations to 14 people. The donated plasma was used to manufacture factor VIII, factor IX, antithrombin, intravenous immunoglobulin G, albumin, intramuscular human normal immunoglobulin, and anti-D. In total, 6000 letters are to be sent out informing patients of the risk assessment -- although only 2/3 are being told they may be at direct risk^ref. The CJD Incidents Panel (CJDIP) advises certain special public health precautions need to be taken for recipients of UK sourced plasma products who have been exposed to a > 1% potential additional risk of vCJD infection, as these patients could pose a risk to others in defined circumstances. These at-risk patients are asked:
• not to donate blood, organs or tissues
• to inform their clinician if they need medical, surgical or dental treatment, so that infection control precautions can be taken to reduce any possible risk of spreading vCJD, and to consider informing their family, in case they (the patients) need emergency surgery in the future^ref
The CJDIP has categorised each batch of implicated plasma products according to the likelihood that special public health precautions need to be taken as follows:
• high: the amount of potential infectivity in product batches was high enough to warrant special public health precautions following the administration of a very small dose. These batches should be traced, and the recipients advised of their exposure and asked to take special public health precautions
• medium: substantial quantities of the material in question would need to have been administered to warrant special public health precautions. Efforts should be made to trace these batches and assess the additional risk to individual recipients to determine whether pecial precautions should be taken
• low: the potential additional risk to recipients is considered negligible. These batches do not need to be traced and the individual recipients do not need to be informed.
Britain has exported blood products that could be contaminated with the human form of mad cow disease to at least 11 countries, including India, in the late 1990s raising fears of further transmission of the deadly condition. Besides India, where 953 vials of albumin were sent, the other 10 recipient countries are Ireland (polio vaccine, 83 500 doses), Brazil (44 864 vials albumin, 80 vials immunoglobulin), Dubai (UAE) (2400 vials albumin), Turkey (840 vials immunoglobulin), Brunei (400 vials albumin), Egypt (144 vials albumin), Morocco (100 vials albumin), Oman (100 vials immunoglobulin), Russia (23 vials factor VIII) and Singapore (3 vials immunoglobulin).
Since 1998, plasma derivatives, such as clotting factors, have been prepared from plasma imported from the USA. Since October 1999, white blood cells (which may carry the greatest risk of transmitting vCJD) have been removed from all blood used for transfusion (leucoreduction) in the U.K. because of concerns about potential infectivity of vCJD in blood, or in blood components. In August 2002 we announced that fresh frozen plasma for treating babies and young children born after 1st January 1996 would be obtained from the USA. In December 2002, the Department of Health completed its purchase of the largest remaining independent US plasma collector, Life Resources Incorporated. This secures long-term supplies of non-UK blood plasma for the benefit of NHS patients.
British blood banks have already taken action to reduce prion content in blood products by, for example, removing white blood cells. They may also consider prion filtering, although this would be very expensive, and they could exclude donors of certain ages, although this would dramatically reduce blood supplies. One study presented to the committee suggested that Britain could be hit by a second wave of vCJD : there could be thousands of Britons infected with the deadly brain disease who do not show any symptoms. These carriers mean the infection could spread widely, through surgery or blood transfusions, even if tainted beef is removed from the food chain. There is a huge discrepancy between the number of people who have died and the number expected to die given the seeming prevalence of prions.
On March 2005 Japan reviewed its ban on blood donations from people who stayed in Britain or France -- 2 countries hit by mad-cow disease in the 1980s and 1990s -- for one day or longer between 1980 and 1996 after a deceased Japanese man was confirmed on February 2005 to have been infected with vCJD was likely to have contracted the human form of mad cow disease during a 24-day stay in Britain in 1990. The man also stayed in France for 3 days at around the same time that France was hit by an outbreak of mad cow disease. However, the Japanese Red Cross Society warned that the expanded ban would cut potential blood donors by several hundred thousand people per year and may have a serious impact on blood supply. The previous ban covered those people who had stayed in Britain for at least one month and in France for at least 6 months between 1980 and now.
An 18-year-old boy died from vCJD in March 1997 -- one of 4 victims in a cluster of cases in the Yorkshire village of Armthorpe. What the public never learned was that the Edinburgh-based National vCJD Unit knew before the 18-year-old's death that he was a registered blood donor. His father told doctors as his son lay in coma but was ordered to keep quiet so that Government inspectors could investigate the cluster without scaring the public. A year later, the father learned the authorities had traced at least 7 people who had been given his son's blood -- but that none of them would be told. For 7 years those known to have received the boy's tainted blood were kept in the dark until scientists decided in September 2004 that they should be informed. They are among 14 people in Britain known to have received vCJD-tainted blood. They have not been identified, but all have been offered counselling. The decision to contact them came only after the 1st case emerged of a patient possibly dying from vCJD contracted through a blood transfusion. The cover-up is confirmed in the Minutes of a meeting of the Doncaster Health Authority on 9 Nov 2001. They reveal how the National vCJD Unit kept a secret log of people given blood from the boy and other donors infected with vCJD. The Minutes, obtained by Jim Oldfield, editor of the Armthorpe Community Newsletter, say the recipients would not be informed and explain that if they later donated blood, it was to be thrown away. At this meeting was Doctor Ken Allen, the consultant in Communicable Disease Control in Doncaster, who led the Armthorpe CJD investigation
100 blood donors who gave blood to 3 people in 1993/4 who have since died from vCJD were warned of vCJD risk and asked to notify their doctors so that extra precautions can be taken if they have surgery or other invasive care. They are also being asked not to donate any more blood, tissue or organs. It is not known whether the source of vCJD in the 3 patients who died was related to the blood that
they received or to BSE-infected meat that they ate. Back in September, the government identified 17 people who received blood transfusions from people who went on to develop vCJD and sent out 6000 letters to others informing them of the potential risk. Since 1997, all cases of vCJD that are reported to the National CJD Surveillance Unit and diagnosed as having probable" vCJD are passed on to the National Blood Service which searches its blood donor records. If the patient has given blood, subsequently any stocks of that blood are immediately destroyed. White blood cells, which it is thought may carry the greatest risk of transmitting the disease, have been removed from all blood used for transfusion since 1999. And blood products have been prepared from plasma imported from the US
since 1998. There is no test for the brain wasting disease, so those at risk have no way of knowing whether they have vCJD^{ref1, ref2}. vCJD infection has been observed in 2 recipients of blood transfusions from donors who later developed vCJD^{ref1, ref2}. One of these recipients did not develop vCJD and died of causes unrelated to the disease. Although other exposures, including, most notably, dietary exposure to BSE, cannot be excluded as the source of these patients' infections, it is considered highly probable that these 2 patients were infected by blood transfusion. These reports added to previous evidence of vCJD infectivity in blood obtained from experiments in animals and led to the conclusion that transfusion should be considered a possible route of vCJD transmission in humans. Over 2 million blood donations are collected each year in the UK by the blood services, and over half a million patients receive transfusions annually. Of the 150 people who have died from vCJD in the UK to 1 July 2005^ref, only 4 have been confirmed as having received blood transfusions that may be associated with their subsequent development of vCJD. (For 2 other cases, symptoms developed before or very shortly after transfusion, and therefore transfusion is not considered a possible source of their infections.) For one of these cases, the probable source of infection has already been identified, as one of the donors went on to develop vCJD. For the remaining 3 cases, transfusion remains a possible source of the recipients' infection. A risk assessment by the Department of Health looked at the probability of donors to vCJD cases being the source of a recipient's infection and, therefore, the probability that the donors themselves are infected^ref. The United Kingdom CJD Incidents Panel^ref considered this risk assessment and recommended that such donors should be considered as potentially at-risk of vCJD for public health purposes' unless the probability of being infected with vCJD (as implied by donation to a vCJD case and estimated with precautionary or worst case assumptions) falls clearly below 1%. Unless there is evidence to the contrary, the assumptions for this calculation are: that an infected donation would certainly infect the recipient (the worst case assumption); that there is no way of distinguishing between the contributing donors (and recipient) in terms of risk of primary vCJD infection (e.g., that all had been resident in the UK during the BSE outbreak), and that there is no other significant alternative infection route for the recipient, such as having been operated on with instruments previously used for healthcare interventions on a patient with vCJD. These individuals are being informed that they are "potentially at-risk of vCJD for public health purposes" so that special public health precautions can be taken to reduce the risk of person-to-person transmission of vCJD during their healthcare. Specifically, these are: not to donate blood, organs or other tissues, and to inform their healthcare providers of their "at-risk" status in order that infection control guidance^ref can be implemented for the instruments used in certain invasive healthcare procedures (and so that certain invasive healthcare procedures that have already been carried out on them can be considered by the CJD Incidents Panel). Since its establishment in 2000, the CJD Incidents Panel has issued advice relating to several groups of patients identified as at increased risk of CJD. Other groups of patients who are considered to be potentially at-risk of vCJD for public health purposes include: certain patients who have been operated on with instruments previously used for healthcare interventions on a patient with vCJD; recipients of blood from donors who later developed vCJD, and patients who have been treated with plasma products that may have been contaminated with vCJD infection. This new notification of donors to vCJD cases is a further precautionary measure to reduce the possible risk of secondary transmission of vCJD in the UK^ref.
• there are no documented cases of anyone getting prion disease through surgery, but there remains a theoretical risk (see above). However, several commercial products are in the pipeline to keep surgical steel free of prions and at least one is expected to be on the market by the end of 2005.
Reported frequent consumption of beef and beef products thought likely to contain mechanically recovered or head meat, or both, including burgers and meat pies, was associated with increased risk for vCJD in UK, as was reported frequent chicken consumption. Surgical operations were generally similarly reported for cases and control subjects, with the exception of a small group of minor operations, possibly attributable to underreporting in control subjects. Cases and control subjects had similar reported occupational histories and exposure to animals^ref.
Epidemiology : 191 cases; average age of diagnosis : 28 years; average incubation time : 12 years.
Estimated range of incubation periods for vCJD cases with presumed route and year of exposure :

presumed route of exposure (estimated range of incubation periods are based on a single vCJD case except for Leicestershire, England, where the Leicestershire Health Authority reported the estimated incubation period for a cluster of 5 vCJD cases)	country of residence	period of potential BSE/vCJD exposure	estimated range of incubation period (y)
foodborne	Canada	1987–1990	11–14
	Ireland	1989–1995	5–10
	Japan (patient was reported to have stayed in the United Kingdom 24 days)	1989	12
	United Kingdom (Leicestershire, England)	1980–1985	10–16
	United States	1980–1992	9–21
bloodborne	United Kingdom	1996	6.5
	United Kingdom (atient with presumed bloodborne transmission died of a nonneurologic disease. The presence of the vCJD agent was detected in the spleen and cervical lymph node; the patient was heterozygous for methionine and valine at the polymorphic codon 129 of the prion protein genep)	1999	>5

At 30 Jun 2005 there have been :

• UK : 161 definite or probable cases and 156 deaths (110 confirmed, 45 without neuropathological confirmation)^ref :
• 1994 : 8 cases, 0 deaths
• 1995 : 10 cases, 3 deaths
• 1996 : 11 cases, 10 deaths
• 1997 : 14 cases, 10 deaths
• 1998 : 17 cases, 18 deaths
• 1999 : 29 cases, 15 deaths
• 2000 : 24 cases, 28 deaths
• 2001 : 16 cases, 20 deaths
• 2002 : 5 cases, 17 deaths
• 2003 : 6 cases, 18 deaths
• 2004 : 8 cases, 4 deaths
• 2005 : 3 cases, 5 deaths
• Feb 6 2006 : 1 cases, 1 death
The geographic distribution of vCJD cases across the United Kingdom (UK) may reflect the path by which they became infected. This may be similar over wide geographic areas, or be associated with localised factors. In 2000, a cluster of 5 cases of vCJD was investigated in Leicestershire. Statistically, these cases were unlikely to have arisen by chance, and extensive enquiries revealed factors that may have augmented the risk of acquiring vCJD in the local population: the consumption of beef purchased from butchers where there was a risk of cross-contamination of carcass meat with BSE-infected bovine brain. Following this investigation a standard public health approach to the investigation of any geographically associated cases (GAC) of vCJD has been adopted. This requires the sharing of information gathered with consent from the families of the cases involved in a multi-disciplinary investigation lead by the local department of public health, with support from the National vCJ!D-GAC Investigations Steering Group. Unlike the Leicestershire investigation, statistical evidence of clustering (i.e., that a geographical association is unlikely to have arisen by chance) is not a pre-condition for investigation. The aim of these vCJD-GAC investigations is to help to identify risk-factors for transmission and possible ongoing risk. Investigations focus primarily on the identification of dietary and medical links between cases and of exposures to the BSE agent, or the possibility of iatrogenic transmission between cases that could plausibly explain their local occurrence. Other explanations are also considered. By the end of March 2005, 154 cases of probable or definite vCJD had been reported in the UK. 5 of the 154 cases were part of the Leicestershire cluster. A further 35 cases had been involved in 12 subsequent vCJD-GAC investigations across the UK. The north of the UK continues to have a higher incidence, although the reason for this is unclear.
The future vCJD death toll for Britain alone could lie between 10 and 7,000. According to some estimates by researchers at Imperial College London at worst only another 540 cases would be reported in UK by 2080. A combination of factors, including the use of older dairy cattle for beef, traditional slaughtering methods in small abattoirs and the sale of meat through small butchers, might explain a cluster of 5 vCJD deaths between 1998 and 2000 in and around Queniborough, Leicestershire, UK, following BSE exposure in the early to mid-1980s, years in which the disease was rampant in herds but unrecognised as a threat to humans^ref. The incidence of deaths since 1994 is consistent with an underlying trend that has reached a peak and is now in decline. Previously, a model with a rise to a plateau gave a similar fit to the data as the model with a peak^ref, however, with only the one case in the 1st quarter of 2004, this model no longer provides a good fit. As was first noted during 2003^{ref1, ref2} the data are not consistent with an exponentially increasing underlying trend. The best fitting statistical model (a quadratic) gives an estimate that a peak in deaths occurred in about September 2000 and that the current incidence is 2.7 deaths per quarter. Extrapolating this model gives a prediction of 8 deaths for the 12 months to March 2005 (95% prediction interval of 3 to 15). The interpretation of these results requires caution, as there may be a long tail or more than one peak to the epidemic. There is also the possibility of human-to-human spread through procedures such as blood transfusions^ref. There remains the possibility of future peaks, which may occur in patients in different genetic groups.
Researchers at Plymouth's Derriford Hospital and the Creutzfeldt-Jakob Disease Surveillance Unit tested 12,674 appendix and tonsil samples removed in the late 1990s from people in the highest-risk group, mainly aged in their 20s : 3 showed PrP^Sc and extrapolating their findings to the whole population, they estimated that 3800 Britons may harbour the disease^ref. Adequate DNA was available for analysis in 2 of the 3 specimens, both of which were homozygous for valine at codon 129 in the PRNP^ref. The estimate should be treated with caution, particularly as 2 of the 3 positive samples showed a pattern of the prion accumulation different from those taken from confirmed vCJD victims. Looking at appendix samples will underestimate the true picture : the government should look again at whether surgeons should use disposable instruments in operations where there may be a risk of transmission, such as the removal of tonsils. The Health Protection Agency is in the process of collecting 100 000 tonsil samples which will be tested for signs of vCJD : the larger scale of the study should provide better estimates of the number of people who may be affected. One explanation is that many people do have the disease, but only 10% show symptoms.
The National Anonymous Tonsil Archive (NATA) is a national unlinked anonymous survey of tonsil tissue that will be used in studies on the prevalence of abnormal prion protein, the agent believed to be responsible for infection with vCJD^ref. A distinctive feature of vCJD is the distribution of abnormal prion protein in the peripheral lymphatic system, particularly tonsil tissue, which may be detected during the pre-clinical phase of the disease during which infection may be transmitted. Through testing tonsil specimens, the study offers an opportunity to measure the population prevalence of asymptomatic infection and to refine projections of vCJD incidence to help plan interventions to limit the impact of infection, and to plan care-provision for those who may develop the disease. NATA began at the end of 2003 and aims to collect 100 000 pairs of tonsils from over 100 NHS hospital trusts during the next 3 years. The study is managed by the Health Protection Agency and sponsored by the Department of Health and the Medical Research Council. Tonsils are being collected from people of all ages during routine tonsillectomies. Only tissue not required for patient care that would normally be discarded is included, and patients are given an opportunity to object to their tissues being used in the archive. The number of tonsil pairs received per month between January 2004 and April 2005 has reached a total of 9970 by the end of April 2005. The number of tonsil pairs received each week should continue to increase as more hospitals are recruited into the study. The number of collection forms that have been completed but against which no tonsil tissue was collected is 431 (369 due to patient objection and 172 due to clinical pathology being requested on the specimen as part of the patient's care). By April 2005, 59 of the 100 NHS hospital trusts that perform over 200 tonsillectomies each year in England were regularly sending tonsil pairs to NATA. A further 41 hospitals trusts received a recruitment visit and are expected to begin collecting tonsils shortly. Recruitment of hospitals continues, with the expectation that the target recruitment rate of 500 tonsil pairs per week will be met. Currently approximately 50,000 tonsillectomies are performed annually in England.
An Imperial College team in 2005 calculated there will be around 70 future deaths : the worst-case scenario could see another 600 deaths, but that this is unlikely. Thousands of people could carry vCJD, but show no symptoms. The higher forecast is based on the possibility that people from different genetic subgroups could be affected by vCJD. Deaths have been declining from their peak of 28 in 2000 to 9 in 2004^ref.
A man's wife, from Walham Green, Sandhurst, Gloucestershire, died last month after battling the sporadic form of the brain disease for 9 months. The man's sister died 2 years ago after contracting the same form of the disease. Both women were 67 when they died. They were both treated in the same ward in the Gloucestershire Royal Hospital. There is 1 in 1 million cases of this form of the disease : the likelihood of such close non-blood relatives getting CJD would be very unusual
• 31 outside UK : 6 of these 31 cases were in people who lived in the UK for > 6 months between 1980 and 1996^ref
• 17 in France (8 died and 3 still alive): 4 men and 2 women aged 20 to 37. 7^th case under investigation^ref. An 8th case gave blood several times between 1993 and 2003 and 10 people received transfusions of red blood cells from him. Plasma from the donations was also used by a French company, LFB, to make drugs based on blood derivatives. LFB has withdrawn all unused stocks of medications made with this plasma and has told pharmacies and clinics to take any remaining items made from suspect batches off their shelves. A 9th case is an individual who had donated blood several times between 1984 and 2002. The 10th and 11th cases were not known to be blood donors.
• The NIH's Laboratory for Central Nervous System Studies (CNSS) received brains from CJD patients from all over the world and has samples dating back to 1963. NIH officials, however, maintain the remaining samples in the collection -- stored in some 30 freezers by the National Institute for Neurological Disorders and Stroke (NINDS) in Bethesda, Md. -- are of little value and may be disposed of if researchers or institutions do not come forward to claim them. Families of patients who died of CJD have reacted with outrage, concerned that the effort mounted to collect the brains in the 1st place has been all for naught. Several have contacted their respective members of Congress and urged them to step in. The brain of a French woman who died in 1971 when 40-50 years old showed in 2000 evidence consistent with vCJD at Western  blotting, a finding that if confirmed would indicate the deadly disease began infecting people more than 20 years earlier than previously thought (the 1st recognized case was detected in the United Kingdom in 1995). A sample of the woman's brain had been injected into a chimpanzee sometime around 1977, and when Bruce Johnson examined the chimpanzee's brain, it, too, showed a pattern consistent with vCJD. Johnson said he never published his finding because he wanted to confirm it, but he never had an opportunity to do so before he retired in 2003. The CNSS lab was officially closed in April 2004. He said he hopes to conduct further examinations of the woman's brain when he starts a new position with the Food and Drug Administration. If we've still got her brain, we can look and see if it's BSE. One possible way is to inject some of the woman's brain into mice. Johnson subscribes to the hypothesis put out by his NIH colleague Joe Gibbs, who thought it was possible that all mammals, including cows, spontaneously develop a mad cow-like disease at the rate of one per million. If that is true, Johnson said, the French woman may have developed her condition from being unfortunate enough to have eaten infected meat from that one in a million animal.
• 4 in Ireland :
• St. Vincent's Hospital in Dublin reported that a patient, who had undergone a gastroscopy earlier in 1999, was diagnosed with vCJD on 31 May 1999. The scope used on the vCJD-infected patient was subsequently used on 49 other patients. Although the patient was not identified by the hospital, she was a mother in her 30s who had been resident in England in the 1980s, the time when the population was exposed to BSE in meat products. Although CJD has been shown to be transmitted from one infected patient to another via certain neurosurgical procedures, there was no subsequent report of transmission of vCJD to other patients in this case^ref
• a man in his early 20s, infected 10-15 years ago, was confirmed in Dublin after suggestive brain MRI and tonsillar biopsy in Oct 2004. The patient never received a blood transfusion nor made a blood donation, and never undergone an operation. The current patient has not lived abroad, and this is therefore Ireland's 1st indigenous case^{ref1, ref2}. Ireland has the 2nd highest rate of BSE in cattle in the world. In 2003, an expert team at the Irish National CJD Surveillance Unit modelled the possible risk to the Irish population based on relative exposure to BSE-contaminated meat and infectivity of bovine tissue^ref. Their analysis indicated that one case of vCJD would be expected in the future. Measures to reduce the risk of exposure to the vCJD causative agent via consumption of infected bovine tissue were applied in farming and meat processing in 1996 and via blood donation since 1999^ref.
• 2 in The Netherlands :
• a 26 year old woman was detected through national surveillance of CJD, coordinated by the Erasmus Medical Centre in Rotterdam. She was treated at Mesos hospital in Utrecht but died on Tue 3 May 2005. The hospital had made a diagnosis of probable vCJD on 15 Apr 2005. Specialists at the Erasmus Medical Centre in Rotterdam confirmed the diagnosis on 18 Apr 2005. The probable diagnosis is based on MRI and clinical signs and was reported on Thu 21 Apr 2005. She has not visited the UK in the past years. The patient has never donated or received blood or tissue, and the likelihood that any other people have been exposed to this infection is reported to be very small^{ref1, ref2}. So far, 77 cases of BSE in bovines have been diagnosed in the Netherlands since 1997, peaking in 2002. Since 1 Sep 2001, all blood-derived products are leucocyte-depleted. Since 1 Nov 2001, blood donors who had stayed in the UK longer than 6 months between 1980-1996 are excluded. Since 1 Feb 2005, blood donors who have received blood transfusion(s) after 1 Jan 1980 are excluded. The feeding of ruminants with ruminant-derived meat & bone meal (MBM) has been banned in 1989. Import of MBMs from the UK was banned in 1990. Since 1997, high-risk organs, derived from bovines, are excluded from the food chain. This includes, among others, the brain, spinal cord, eyes and tonsils. Since 1 Jan 2001, all bovines older than 30 months are BSE-tested in slaughterhouses prior to their release for consumption. The 1st case of BSE in an indigenous cow was reported in the Netherlands in 1998. However, according to the updated opinion of EU's Scientific Steering Committee on the Geographical Risk of Bovine Spongiform Encephalopathy (GBR), adopted on 11 Jan 2002, exports of life bovine or meat & bone meal (MBM) from the Netherlands could have represented an external challenge to the importing country since 1985 (possible) or 1987 (likely)^ref. The Dutch daily NRC Handelsblad stated on Tue 3 May 2005 that Dutch experts were surprised to have their 1st vCJD case that early. This, in view of the fact that in the UK there was one human case per 1300 cows with BSE, while in Holland there have been, so far, only 77 BSE cows. They raise the possibility that the victim consumed (imported) British beef at an earlier date
• on Thursday 22 Jun 2006 the Health Ministry gave no details of a new case, other than to say that the usually fatal disease was probably contracted by eating infected beef some time in the past. The patient has no mutations associated with inherited CJD and is homozygous for methionine at codon 129 of the prion protein gene^{ref1, ref2, ref3, ref4}
• 1 in Italy (a 27-year-old girl from Menfi, Palermo, died on 6 Aug 2003 in Milan's Besta hospital). A suspected case is a 30-year-old woman in the D'Anna clinic in Palermo at the beginning of June 2004
• 1 in Portugal : a 12-year-old boy was diagnosed on June 2005. The patient does not have a history of travel to the UK. It is not surprising, since the risk of human disease is presumed to relate to the amount of bovine meat consumed or extent of exposure to bovine products
• 1 in Spain : a 26-years old woman infected between 1994 and 1995 died on 10 Jul 2005 in the Foundation Alcorcon, Hospital of Madrid. She had no specific risk factors for vCJD (no prior blood transfusions or visits to the United Kingdom). The patient's symptoms began in November 2004, with rapid progression to dementia, normal MRI and EEG, and 14-3-3 protein found in cerebral spinal fluid. Other neurological manifestations including ataxia, dysarthria, apraxia, and myoclonus appeared in early 2005. The 1st abnormal MRI was observed in April 2005. The patient was methionine homozygous at codon 129 of the prion protein gene (PRPN). There were no identified PRPN mutations or family history of CJD. The case was notified to the Spanish CJD state registry in May 2005 and initially fitted the criteria for both probable sporadic CJD (sCJD) and possible vCJD; it was registered as probable sCJD. Brain neuropathology results of tests conducted at Fundacion Hospital de Alcorcon at Madrid subsequently confirmed vCJD. Materials have been sent to the European reference center at the United Kingdom's National CJD Surveillance Unit in Edinburgh for further study, and results are expected later in August 2005. Spain has reported 567 cases of BSE from 2000. The occurrence of the 1st suspected human case of vCJD in Spain is not unexpected, since the risk of contracting vCJD appears to be linked to the amount of bovine meat consumed or extent of exposure to bovine products. According to the latest figure compiled by the European Union and the OIE, Spain ranks 5th in the world in terms of the number of BSE-affected cattle (532), after France with 946, Portugal with 949, Ireland with 1470 and the UK with 184 138. However, by contrast, in the current year [2005], Spain, with 42 cases, ranks 2nd after the UK with 126 cases, followed by Ireland with 29, Portugal with 17 and France with 2 cases of BSE^{ref1, ref2}
• outside Europe : anyway all resided for one or more period in the UK, or parts of Europe with outbreaks of BSE in cattle
• 1 in Canada
• 1 in Hong Kong
• 2 in USA :
• in 2002, the likely occurrence of vCJD was reported in a 22-year-old woman living in Florida and represented the first detection of the disease in North America^ref. In early November 2001, the patient in Florida was evaluated for depression and memory loss that adversely affected her work performance and may have contributed to a traffic ticket she received for failure to yield the right of way at a traffic sign. In December 2001, the patient developed involuntary movements, gait disturbances, difficulty dressing, and incontinence. The following month, she was taken to a local emergency department; a computed tomographic scan of her brain showed no abnormalities, a diagnosis of panic attack was made, and antianxiety medications were prescribed. In late January 2002, the patient was transported to the United Kingdom, where her mother resided. During the ensuing 3 months, the patient's motor and cognitive deficits worsened, which caused falls resulting in minor injuries; she had difficulty taking care of herself, remembering her home telephone number, and making accurate mathematical calculations. She also experienced confusion, hallucination, dysarthria, bradykinesia, and spasticity. An electroencephalogram evaluation showed no abnormalities, but a brain magnetic resonance imaging (MRI) study showed the characteristic signal abnormalities in the pulvinar and metathalamic regions suggestive of vCJD. Western blot and immunohistochemical analyses of tonsillar biopsy tissue demonstrated the presence of protease-resistant prion protein, which supported the diagnosis of vCJD. By September 2002, the patient was bedridden. An experimental treatment with quinacrine was given to the patient for 3 months, but she showed little improvement. She remained in a state of akinetic mutism and died in June 2004, ?32 months after illness onset. The patient was born in Great Britain in 1979 and immigrated to the United States with her family in 1992. She had no history of surgery or receipt of blood or blood products, and she was never a blood donor. Consistent with findings for vCJD patients in the UK associated with potential foodborne exposure, this patient was homozygous for methionine at polymorphic codon 129 of the prion protein gene. A full autopsy was performed, and neuropathologic examination of brain tissue showed the presence of florid plaques and severe cortical atrophy. Immunohistochemical staining for the prion protein showed numerous plaquelike formations along with a synaptic staining pattern similar to that previously described in other vCJD patients. This patient is the only US resident with a confirmed diagnosis of vCJD. She was likely exposed to BSE while growing up in the United Kingdom from 1980 to 1992, which suggests an incubation period of 9–21 years (Table). The illness duration in this patient (32 months) was much longer than the median illness duration for patients in the United Kingdom with vCJD (14 months, range 6–40 months)^ref
• Patrick Hicks, 49, of Riverside, Calif., died late in 2004. U.S. authorities in Jan 2005 ruled out vCJD, but brain samples were sent in May 2005 to Dr. Jean Jacques Hauw at the Laboratoire De Neuropathologie at the Groupe Hospitalier Pitie-Salpetriere in Paris to be re-analyzed. Hicks had symptoms consistent with vCJD, and his relatively young age also made him a possible candidate for the disease. If Hicks did in fact suffer from vCJD, he could be the first case of the disease due to consumption of domestic beef since his family said he was a heavy eater of beef, never traveled outside of the country and had not undergone any medical procedures that would put him at risk of contracting the disease. Relatives thought the National Prion Disease Pathology Surveillance Center in Cleveland, which conducted the initial examination of Hicks' brain, ruled it sporadic CJD without conclusive proof. The NPDPSC arranged for a company called 1-800-AUTOPSY to collect Patrick's brain upon his death. However, the company failed to follow the proper protocol required by NPDPSC and did not freeze any samples from the brain. Instead, the autopsy company fixed the entire organ in formalin, making it difficult to conduct definitive tests that would help distinguish whether Hicks had vCJD or sporadic CJD. The company previously told UPI they did not have the equipment necessary to freeze the tissue and would have made NPDPSC aware of this when they were first contacted
• 2005 : 0 cases
• 1 in Japan : a 51-year-old Japanese man who had spent around 24 days in the United Kingdom in 1990, 3 days in France, and 2 weeks in other European countries where vCJD has not been reported, first showed symptoms of the disease in December 2001, died and was confirmed on Fri 4 Feb 2005. He had no history of surgery or blood transfusion, or a family history of prion disease. Electroencephalogram (EEG) showed diffuse slowing, but no periodic synchronous discharges (PSD). In Dec 2001 (30 months after onset) brain MRI showed hyperintensity in the caudate, putamen, thalamus, and cerebral cortex, with higher intensity in the caudate and putamen than the thalamus. Conversion of a positive to negative pulvinar sign on MRI has been described in a few vCJD patients. EEG suggested the presence of PSD, which have not been reported in other vCJD cases^ref. The diagnosis of probable sporadic CJD was supported by EEG and MRI findings. He died of pneumonia in Dec 2004. Autopsy showed findings characteristic of vCJD, including florid plaques and the Parchi type 2B or Collinge type 4 pattern of protease-resistant PrP. He is known to have eaten mechanically recovered meat during his visit, and although exposure in other European countries he visited, including France and Japan, cannot be excluded, it is thought that he may have been exposed to the BSE agent during his UK visit. If exposure in the UK was the source of his infection, then the incubation period to illness onset was 11.5 years. It is also noted that the patient's illness duration was unusually long, at 42 months (median 14 months)^{ref1, ref2, ref3, ref4}. This case suggests revision of the WHO vCJD case definition 3 to prevent missing cases of vCJD
• 1 in Saudi Arabia : the US National Prion Disease Pathology Surveillance Center confirmed a vCJD diagnosis by analyzing a brain biopsy sample from a 33-year-old Saudi man admitted to a hospital in Saudi Arabia. Although detailed information on
this patient was not available, he may have visited the UK, if at all, only for several days. Thus, the patient may have contracted the disease in Saudi Arabia after eating BSE-contaminated cattle products imported from the UK^ref
• suspected, but unconfirmed, cases in the Czech Republic, Spain, Saudi Arabia (a 65-year-old man from Madinah was admitted to King Faisal Specialist Hospital in Jeddah on 19 Jan 2004), Guadeloupe, New Zealand, Russia, and South Africa, but none has been confirmed so far
• discounted suspected cases in New Zealand, Russia, and South Africa (2000 : a woman in a Johannesburg hospital whose brain biopsy material was consistent with a diagnosis of classical (sporadic) CJD. Pretoria's Military Hospital 1 is investigating the possibility that a female patient died on Sat 8 May 2004 was affected by vCJD : it seems unlikely, in the present case, that the initial diagnosis of vCJD will be sustained by laboratory examination. The age of the victim is not stated in the report, and the basis for the diagnosis of vCJD is unclear. Prolonged residence in the UK, or another European country where BSE has been observed in cattle, might be the reason for considering a diagnosis of vCJD)
• federal and state disease investigators have rejected a theory that a cluster of 17 suspected vCJD cases were linked to eating meat contaminated with the agent of BSE at racetrack restaurants at Garden State Racetrack in Cherry Hill, N.J., USA between 1988 and 1992. Analysis of brain tissue specimens pointed to classic CJD and not to vCJD in 5 of the cases. In 6 other cases, brain tissue specimens were unavailable, but clinical and diagnostic evidence indicated probable CJD. Of the 6 remaining cases, 3 were found to be unrelated to either CJD or vCJD, and 3 remain under investigation. The 14 deaths that were definitely or possibly linked to CJD occurred over about 9.25 years, the report states. Attendance at the racetrack from 1998 through 1992 totaled about 4.1 million, and the overall incidence of CJD in people aged 50 and older is 3.4 cases per million per year. Given these facts, 14 CJD deaths among racetrack attendees in 9.25 years would not be considered unusual. In addition, investigators identified the CJD subtypes in 4 cases and found they were all different, suggesting that the cases did not have a common cause^ref
Pathogenesis :
• all of the 146 British vCJD cases tested thus far have been Met¹²⁹/Met¹²⁹ (MM) homozygotes for PRNP (found in 40% of UK population) and type 4 PrP^Sc. This led some researchers to hope that people with different genotypes (VV (10%) or MV (50%)), who make up 60% of the population, may be protected from the disease. The development of vCJD in transgenic mice expressing the human PrP gene only occurred in those mice that were methionine homozygotes at codon 129 of that gene, according to a recently published study^ref. Mice expressing valine at PrP 129 developed a different type of disease with different neuropathological and molecular phenotypes and a different rate of secondary (within-species) transmission. 4 distinct forms of the disease-related prion protein (PrP^Sc) have been found in the brain tissue of patients with CJD: patients with classical CJD have PrP^Sc types 1-3, and patients with vCJD have type 4 PrP^{Scref1, ref2, ref3}. Codon 129 of the human PrP gene encodes either methionine or valine. This polymorphism appears to critically affect the susceptibility of humans to prion diseases. All vCJD patients (that is, patients with type 4 PrPSc) tested to date have been homozygous for methionine at codon 129. In this study, mouse strains with the human PrP gene instead of the mouse PrP gene (transgenic mice) were given intracerebral inoculation with brain tissue from vCJD patients and cows with bovine spongiform encephalopathy (BSE). All the mice that were homozygous for methionine at human PrP codon 129 developed clinical disease and neuropathological signs typical of vCJD, as well as having type 4 PrP^Sc. Mice that were homozygous for valine at human PrP codon 129 responded quite differently: only 50% became infected, and they developed type 5 PrP^Sc. Type 5 was 1st described by the same research group in 1997^ref, also in mouse studies. Type 5 PrP^Sc has type 4-like glycoform ratios but gives type 2-like digestion products after treatment with proteinase K, and is associated with very weak diffuse PrP deposition in the brain in contrast to the florid PrP plaques associated with type 4. The study went on to investigate the within-species transmission of these PrP types -- that is, transmission without a species barrier -- and as such, a model for human-to-human transmission of prion disease. Within-species transmission of prion disease typically has a high (100%) attack rate. Surprisingly, the brain inocula derived from 4 clinically-affected mice that were homozygous for valine at human PrP codon 129 failed to transmit clinical disease or asymptomatic prion infection to other valine homozygous mice of the same breed. Furthermore, when methionine homozygous mice were inoculated with brain tissue containing type 5 PrP^Sc from the valine homozygous mice, some (10 of 13) developed sub-clinical infections with type 4 PrP^Sc, and 3 of 13 developed clinical prion disease with type 2 PrP^Sc and a neuropathology that resembled that of human sporadic CJD.These findings indicate that codon 129 polymorphism determines the ability of human PrP to form the various types of PrP^Sc, and also the disease phenotype resulting from infection with BSE and vCJD prion. Specifically, human PrP 129 valine appears not to be a compatible substrate for the type of prion (type 4) seen in vCJD. The authors recommend determination of PrP^Sc types among all human patients with prion disease. This may further contribute to our understanding of patterns of human prion disease in relation to exposure to BSE or iatrogenic sources of vCJD, and also guard against missing BSE-related infections that present as disease that resembles sporadic CJD more closely than vCJD. Based on these animal models, and therefore with caution, the authors conclude that human infection with BSE-derived prions may not be restricted to a single disease phenotype, but may result in sporadic CJD-like -- or novel -- phenotypes in addition to vCJD, with the type of disease experienced depending on the genotype of the host source of the infection and the genotype of the recipient.
• transgenic mice expressing human PrP Met¹²⁹, inoculated with either BSE or vCJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. However, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, also resulted in a distinct molecular phenotype that was indistinguishable from that of sporadic CJD with PrP^Sc type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure^ref.
• ... but ... :
• Met¹²⁹/Val¹²⁹ (MV) heterozygotes : a single case of probable iatrogenic vCJD infection has been identified in one patient
• The distinctive neuropathological and molecular phenotype of vCJD can be faithfully recapitulated in Prnp-null transgenic mice homozygous for human PrP M129 but not V129, where a distinct prion strain is propagated. Susceptibility of 129MV heterozygotes, the most common PRNP genotype, was modelled in transgenic mice and show that, remarkably,  propagation of type 4 PrPSc was not associated with characteristicvCJD neuropathology. Depending on the source of the inoculum, these mice can develop 4 distinct disease phenotypes after challenge withbovine spongiform encephalopathy (BSE) prions or vCJD (human-passaged BSE) prions. vCJD-challenged mice had higher attack rates of prion infection than BSE-challenged recipients. These data argue that human PRNP 129heterozygotes will be more susceptible to infection with vCJD prions than to cattle BSE prions and may present with a neuropathological phenotype distinct from vCJD^ref
• homozygous for Val¹²⁹ : a tonsil sample tested positive (see above). People infected with vCJD with a valine homozygous codon 129 PRNP genotype may have a prolonged incubation period, during which horizontal spread of the infection could occur either from blood donations or from contaminated surgical instruments used on these individuals during the asymptomatic phase of the illness^ref.
• transgenic mice expressing human PrP with Val¹²⁹ inoculated intracerebrally with vCJD prions develop a distinct phenotype of vCJD and, remarkably, persistence of a barrier to transmission of BSE-derived prions on subpassage : the brains of animals lacking the MM genes also became spongy and showed clumps of altered protein characteristic of prion disease, although the distribution of these effects was different from that seen in MM mice with vCJD^ref. Type-4 prions, the type associated with vCJD, can only propagate themselves in people that make the M form of the protein. It seems the V form of the protein just cannot adopt the particular molecular shape that characterises type 4. The studies in mice also suggest that if these prions were to pass from person to person (for example by blood transfusion) then, depending on the genetic type of the person becoming infected, at least 3 different patterns of disease might result: type 2 (which is seen in sporadic CJD); type 4 (which causes vCJD) or type 5 (which may cause a new pattern of disease). Thus, primary and secondary human infection with BSE-derived prions may result in sporadic CJD-like or novel phenotypes in addition to vCJD, depending on the genotype of the prion source and the recipient : evidence of vCJD-like infection was found in the autopsy of a non-MM person from the Netherlands. But that could also mean that people without MM may simply carry the prions without developing symptoms, or may develop similar symptoms after a longer incubation period.
• spleen's follicular dendritic cells (FDCs) are the main immune reservoir for prions and the distance between the FDCs and the enervated areas of the spleen determines the velocity and efficiency of neuroinvasion : anything that reduces the distance between FDCs and sympathetic nerves—inflammatory conditions or infections, for instance—could enhance prion disease susceptibility (genetically engineered CXCR5^-/- mice in which prion-rich cells are abnormally close to nerve endings in the spleen develop disease symptoms around 30 days earlier than normal mice). There is a long lag period during which the infectious agent replicates in the FDCs before moving on to brain : switching off the FDCs is a pretty mild treatment as it does not lead to severe immunosuppression^ref.
Symptoms & signs :  the most striking early neurologic manifestation in some vCJD patients was painful sensory symptoms (dysesthesia or paresthesia). Other neurologic signs, such as chorea, dystonia, and myoclonus, commonly develop late in the course of vCJD
Prognosis : average duration of disease after diagnosis : 14 months; 90% dies within 1 year due to cachexia or intercurring infections
Laboratory examinations :
• Microsens has developed the 1st blood test that can detect abnormal prions in the blood of patients with vCJD
• EEG shows periodic sharp wave complexes (PSWC) (specificity = 74% in sporadic CJD ^ref)
• [endogenous proteins]_CSF :
• 14-3-3 (sensitivity = 53 in sporadic CJD^ref-82^ref-86^ref-89.8^ref-90.6 in patients with sporadic CJD (false negatives in patients with familial CJD associated with the 178 codon mutation)^ref-100%^ref; specificity = 84^ref-86^ref-87^ref-92^ref-100%^ref). A positive 14-3-3 result was significantly correlated with a shorter time between disease onset and the lumbar puncture for the 14-3-3 test^ref. The concentrations of 14-3-3 and tau in the CSF were reduced in CJD patients with a long duration of disease (> 1 year; p < 0.05). The concentrations of 14-3-3 or tau were lowest at the onset or at the end stage of the disease, while the beta amyloid concentration remained low throughout the course of the disease^ref. As a marker of neuronal damage, it is also found in the CSF of patients with a variety of neurological disorders, including purulent bacterial meningitis : all patients who recovered cleared 14-3-3 protein from the CSF before discharge from the hospital (this was the first CSF marker to clear), whereas those who died never cleared the protein^ref
• NSE > 25 ng/mL (sensitivity = 79.7%; specificity = 91.5%)^ref
• S-100 > 2.5 ng/mL (sensitivity = 94.2%; specificity = 85.4%)
• tau > 1300 pg/ml (sensitivity = 87%; specificity = 97%)ù
• increased phospho-tau (P-tau)/total tau ratio^ref
• significantly higher positive frequency of pro-MMP-9 and of the active form of MMP-2, along with significantly higher levels of TIMP-1 and TIMP-2, classical inhibitors of MMP-9 and MMP-2, respectively. We also found a positive correlation between 14-3-3 protein concentration and that of TIMP-1 and TIMP-2 levels (correlation coefficients of 0.793 and 0.798, respectively)^ref
• diffusion-weighted MRI : in sCJD patients hyperintense signal in T2-weighted sequences was found to be present in the neostriatum (the caudate nucleus and putamen) with specificity of 93%, while in vCJD cases signal hyperintensity was found in pulvinar thalami. The "pulvinar sign" was evidenced to be highly sensitive (79%) and specific (100%) for the diagnosis of the latter form of CJD. An MRI result with symmetrically increased signal intensity in the pulvinar region relative to the signal intensity in other deep and cortical gray mater areas has been reported in >75% of vCJD patients. The presence of the pulvinar sign may suggest a vCJD diagnosis in the appropriate clinical context.
The chances of all 3 diagnostic testing modalities to be negative in a single case of sporadic CJD are extremely remote^ref. Autopsy with neuropathologic confirmation remains the only definitive way to make a diagnosis of CJD. Patients with vCJD can be distinguished from patients with the more common sporadic CJD by their younger median age at death (28 years and 68 years, respectively), predominantly psychiatric manifestations (mood disorders, personality changes and psychotic symptoms) at illness onset^ref, delayed appearance of frank neurologic signs, absence of a diagnostic electroencephalographic pattern, presence of the pulvinar sign on MRI, and a longer median illness duration (<6 and 14 months, respectively)^{ref1, ref2}. Almost all vCJD patients have died before 55 years of age, compared with only 10% of sporadic CJD patients. A prominent, early involvement of lymphoid tissues has enabled a reasonably accurate premortem diagnosis of vCJD, using tonsillar biopsy
Web resources :
• Monthly CJD statistics by UK Department of Health
• CJD News
• Investigations undertaken in possible CJD cases
• The CJD Foundation
• The UK Creutzfeldt-Jakob Disease Surveillance Unit
• Human BSE Foundation (HBSEF)
• European and Allied Countries Collaborative Study Group of CJD (EUROCJD)
Necroptic findings : amyloid plaques (AP)
• in kuru, plaques are typical "kuru" plaques--stellate structures deposited mostly in the granular- and Purkinje-cell layer of the cerebellum. Many smaller or larger clusters ae visible but, in contrast to GSS, they never merge together to form multicentric plaques.
• in GSS, plaques are localised in the granular- and Purkinje-cell layer and the molecular cell layer.
• unicentric "kuru" plaque consist of stellate arrangements (stars or cores) of amyloid bundles emanating from a densely interwoven centre. Amyloid stars are surrounded by astrocytic processes and invaded by microglial cells but dystrophic neurites are only rarely seen
• clusters of unicentric plaques, which by merging eventually form
• multicentric plaques are often surrounded by dystrophic neurites and are the hallmark of GSS
• neuritic plaques (the rarest type)
Laboratory examination :
• antemortem : there are ethical issues about screening for a disease that has no known cure
• EEG : sensitivity, specificity, and positive and negative predictive values of PSWCs are 64%, 91%, 95%, and 49%, respectively. In the falsely positive cases, Alzheimer's disease and vascular dementia are the underlying diseases. However, only in 20% of cases both clinical and EEG data would have led to the false-positive result to diagnose probable CJD^ref.
• CSF examination is at best suggestive (high 14-3-3, NSE, tau protein (> 1300 pg/mL) and S100B levels in CSF and serum)
• prion conversion assays : adding bases long and single-stranded mammalian RNA increases the test's sensitivity as the nucleic acid functions as a scaffold
• tonsil test has high rates of false positives and false negatives (25-30%)
• a brain biopsy cannot be justified while alive, given we can offer no specific treatment, so it can only be performed post-mortem
• detection of PrP^Sc in blood :
• some scientists have tried extracting blood from live subjects and injecting this into another animal's brain. They then wait, typically for months, to see whether the animal receiving the shot develops the clinical traits of the disease. But this method only succeeds in picking up on an infection 31% of the time it is present. Moreover, it is not clear whether blood taken from humans can cross the species barrier to reproduce the illness in test animals such as hamsters
• amplifying the negligible levels of the misshapen proteins in blood to a detectable level. His team overcame the first barrier to this goal in 2001 years ago, when it managed to replicate deadly prions from hamster brains. The technique involves mixing normal proteins with tiny amounts of the infectious version in a test tube, causing the abnormal molecules to multiply and clump together over a period of about 30'. A pulse of sound then breaks up the clumps, freeing the misshapen proteins to repeat the process. Protein misfolding cyclic amplification (PMCA) technology can be automated and optimized for high-efficiency amplification of PrPSc. 140 PMCA cycles in about 70 hours leads to a 6600-fold increase in sensitivity over standard detection methods. 2 successive rounds of PMCA cycles resulted in a 10-million-fold increase in sensitivity and a capability to detect as little as 8000 equivalent molecules of PrPSc. Tests on 18 diseased and 12 healthy hamsters revealed that this method could detect prions 50% of the time they were present after 2 140-cycle runs. After 6 runs this was boosted to 89% sensitivity and 100% specificity. Notably, serial PMCA enables detection of PrPSc in blood samples of scrapie-afflicted hamsters with 89%. These findings represent the first time that PrPSc has been detected biochemically in blood, offering promise for developing a non-invasive method for early diagnosis of prion diseases^ref. Adapting the technique to run tests on human blood should not take longer than 6 months. The initial injection of infectious prions into the hamsters' brains created a stab wound that may have helped prions spread into the bloodstream. This unusual circumstance may have artificially boosted the test's sensitivity. In any case, the test may help to answer the question of how many silent carriers there are living in Britain. Solving this would help to predict future incidence of disease, and might give pharmaceutical companies reason to invest in a treatment
• PrP^Sc can be detected biochemically in the blood of hamsters infected with scrapie during most of the presymptomatic phase of the disease. At early stages of the incubation period, PrP^Sc detected in blood was likely to be from the peripheral replication of prions, whereas at the symptomatic phase, PrP^Sc in blood was more likely to have leaked from the brain. The ability to detect prions biochemically in the blood of infected but not clinically sick animals offers a great promise for the noninvasive early diagnosis of TSEs^ref.
• cerebral biopsy :
• PrP^Sc profiling quantifies the amount of PrP^Sc in 9 defined brain regions (vs. the usual 2) relative to internal standards, and thereby allows for direct comparison of individual profiles. It was applied as part of the detailed molecular characterization to autopsy samples from 50 patients with sCJD. sCJD types are determined by a patient's PRNP genotype at the polymorphic position 129 of PRNP and by the relative resistance of PrP^Sc to proteolytic degradation. It is thought that most patients only have 1 prion type, but previous reports have described coexistence of 2 different types in some samples^ref. Analysis of this wealth of data revealed correlations between distinct PrP^Sc distribution patterns and sCJD subtypes. Before doing cerebral biopsies, the sCJD subtype should be determined so that the correct brain area is examined. The researchers also found coexistence of 2 different prion types in 20% of their overall samples, and in > 50% of the samples from patients who were heterozygous for the 129 polymorphism in the PRNP gene. These data lend further support to a link between molecular signature and clinical heterogeneity of the disease^ref.
• postmortem :
• the main strategy of current tests is to use proteinase K. A tissue sample from the brain stem of a suspect cow is mixed with enzymes. Normal prions are broken down, but abnormal prions are sturdy and resistant and some are left over after the sample is treated. The key is finding ways to detect residual prions
• western blot with the addition of phosphotungstic acid precipitation step (Bio-Rad Deslys et al.)^{ref1, ref2}
• xenodiagnosis :
• in Rattus spp. symptoms appears 2 years after inoculation of cerebral samples from infected bovines, but incubation period shortens with sequential passages within a species.
• Clethrionomys glareolus has an incubation period of only 6 months
• conformation-dependent immunoassay (CDI) was originally developed to detect various forms of prions in cows, sheep, deer and other animals. CDI not only identifies prions in human brain tissue but is faster and far more precise than the standard immunohistochemistry (IHC) detection methods. Before development of the CDI, all immunoassays for the disease-causing PrP isoform (PrP^Sc) used limited proteolysis to digest the precursor cellular PrP (PrP(C)). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP^Sc, researchers used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP^Sc in all 10-24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, IHC, and the CDI. 3 of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, approximately 90% of the total PrP^Sc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP^c. The CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive^ref
Trial chemotherapeuticals :
• amantadine and derivatives
• acyclovir
• vidarabine
• amphotericin B
• i.c. anionic chelators
• GAG-SO₄^2-
• pentosan polysulphate (PPS) (Cartrophen^®, Elmiron^®, Hemoclar^®, Pentosan^®) : a pump is placed under the skin in the abdomen and the drugs are administered through tubes from the pump going the lateral ventricle of brain. Every few weeks the pump is filled up with the drug. A Belfast patient is the only person known to have survived 3 years while suffering from the disease : as his health rapidly declined his family went to court for him to be allowed to take PPS. The patient has since undergone 2 years of having PPS injected i.c.. Because the drug, which is used in the USA to treat cystitis, has anticoagulant blood thinner and anti-inflammatory properties, it was thought to carry the risk of side effects. About 22 people have been treated so far (10 in Japan) : 1 died despite treatment, some have continued without improvement but the condition of about 6 has stabilised^ref.
• heparan sulfate mimetics (HMs) : HM2602^ref
• quinacrine^ref
• Congo red
• IFNs
• quinacrine and chlorpromazine : in 2001, one British vCJD patient was reported to have made an astonishing recovery during a trial, but the woman has since died
• antisense RNA/DNA
• i.c. humanized mAbs with high affinity for both murine PrP^c and PrP^Sc markedly reduce peripheral PrP^Sc levels and prion infectivity, even when the antibodies are first administered at the point of near maximal accumulation of PrP^Sc in the spleen. Antibodies that grab hold of normal prions and prevent them from transforming into the harmful configuration helped mice survive for over 300 days after equivalent untreated animals had succumbed to the disease after being injected with infectious prions^ref. But antibodies did not enter the brain in that trial, so researchers would not see side-effects on nerve cells : actually such mAbs can kill nerves themselves when injected into the brains of healthy mice as they  mimic one way in which abnormal prions destroy the brain during BSE and vCJD — they bind to normal prions in the cell membrane, somehow triggering signals that knock the cell dead^ref. Researchers are experimenting with different types of antibodies in therapy trials, he points out, some of which might not kill off nerve cells.
Prevention : PrP^Sc is ...
• resistant to :
• ionizing radiations
• water boiling
• formaldehyde
• b-propiolactone
• nucleases
• sensitive to :
• 60' in NaOH 5% (1 N, pH>10) or NaClO 20 000 ppm reduce infectivity by >5-6 log₁₀ lethal doses
• autoclaving at 134°C for 1 hour
• with immersion in water reduces infectivity by >5-6 log₁₀ lethal doses
• without immersion is somewhat less effective (4-4.5 log reduction)
• proteinase K and trypsin
• alkaline cleaner
• phenolic disinfectant
• combination of an enzymatic cleaner and vaporised hydrogen peroxide (VHP)
• VHP alone, which can be compatible with electronic components, achieved an approximately 4·5 log reduction in infectivity (equivalent to autoclaving without water immersion)^ref
• Et₂PC
• SDS
• urea
• K⁺SCN^-
• ether
• the National Institute for Clinical Excellence (NICE) has issued interim guidance stating that surgeons who use diathermy in tonsillectomies should consider changing the way they operate so that they minimize their use of diathermy during tonsillectomies^ref. The risk of complications may also be higher with currently available disposable diathermy equipment for tonsillectomy and, again, surgeons should consider discontinuing their use. Diathermy involves using a high-frequency electrical current that passes through an area of the body to produce heat to remove tonsils and/or to seal up the area where tonsils are removed. The results of the audit suggest that there are higher risks of secondary hemorrhage requiring re-admission and return to theatre after tonsillectomy using diathermy techniques or coblation as compared with techniques that use no diathermy either for dissection or for sealing the blood vessels (the "cold steel" technique). In January 2001, the Department of Health (DH) had recommended that single-use instruments should be used for adenotonsillectomy to minimize the risk of transmission of vCJD. This followed DH advice describing the present state of knowledge of vCJD transmission risk from one patient to another. During the course of 2001, however, various difficulties were highlighted with single-use instruments, and reports of both primary and secondary hemorrhage after surgery increased. As a consequence, the DH reintroduced standard reusable instruments for tonsil and adenoid surgery.
• Gradiflow^® (source : Gradipore) membrane technology can detect and remove PrP^Sc from g-globulins and albumin : the unit pulls the prions through a wafer-like membrane, either by electrical charge or by varying the size of the holes in the membrane.
• leucoreduction alone is not sufficient to remove all infectivity of TSE from donated blood^ref
• Leukotrap Affinity Prion Reduction Filter (source : Pall Corporation), expected to be launched commercially in Europe in spring 2005, can remove WBCs and 99% of all types of prions -- both cell and non cell-associated 99% -- from RBCs, the most widely transfused blood component, below the limit of detection of the western blot assay. An animal infectivity study compared filtered and unfiltered blood infected with scrapie prion. After the 300-day study, 3 of the 18 control hamsters that received the unfiltered blood developed scrapie. Only 2 of these animals had displayed clinical signs of the disease. None of the hamsters receiving filtered blood developed scrapie.
Web resources :
• Molecular evolution of prions
• MRC Prion Unit
• National Prion Disease Pathology Surveillance Center
• Prion diseases
• Vertical and lateral transmission of transmissible spongiform encefalopathies (TSE)
• Host Genetics and Prion Disease program (HGPD) at National Microbiology Laboratory of Canada
• Prionics AG - Switzerland
• NIH factsheet on prion diseases

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