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BACKGROUND: Prions are renowned for their distinct resistance to chemical or physical inactivation, including steam sterilization. Impaired efficacy of inactivation poses a risk to patients for iatrogenic transmission of Creutzfeldt-Jakob disease (CJD) via contaminated surgical instruments. AIMS: Most established prion inactivation methods were validated against scrapie agents, although those were found to be generally less thermostable than human prions. Thus, knowledge gaps regarding steam-sterilization kinetics of CJD prions should be filled and current guidelines reviewed accordingly. METHODS: Prion inactivation through widely recommended steam sterilization at 134°C was assessed for several holding times by analysing the residual prion seeding activity using protein misfolding cyclic amplification (PMCA). FINDINGS: Scrapie 263K was found to be the least thermoresistant prion strain showing no seeding activity after 1.5 min at 134°C, while variant CJD was the most stable one demonstrating some seeding activity even after 18 min of steam sterilization. Sporadic CJD subtype VV2 exhibited residual seeding activity after 3 min, but no detectable activity after 5 min at 134°C. CONCLUSION: Validated steam sterilization for 5 min at 134°C as previously recommended for the routine reprocessing of surgical instruments in contact with high-risk tissues is able to substantially reduce the seeding activity of CJD agents, provided that no fixating chemical disinfection has been performed prior to sterilization and that thorough cleaning has reduced the protein load on the surface to less than 100 µg per instrument.

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BACKGROUND: Cerebral deposition of abnormally misfolded and aggregated alpha-synuclein (αSyn) is a neuropathological hallmark of Parkinson's disease (PD). Pathologically aggregated αSyn species of PD (αSynPD) can act, in a 'prion-like' manner, as proteinaceous nuclei ('seeds') which are capable of self-templated propagation. This has raised concerns that αSynPD seeds transmitted iatrogenically between humans may stimulate αSyn pathologies or clinically harmful effects in the recipients. Effective decontamination when reprocessing medical devices could significantly counteract such risks. Steam sterilization at 134°C is recommended as an essential pathogen inactivation step in many reprocessing guidelines for medical devices, and also shows effectiveness against prions, the self-propagating biological agents long thought to exhibit the highest resistance to steam sterilization. METHODS: This study examined the reduction in αSynPD seeding activity in brain tissue homogenates from patients with PD after steam sterilization at 134°C using a specifically adapted real-time quaking induced conversion assay. FINDINGS: Titres of approximately 1010 50% seeding doses per gram were detected in non-steam-sterilized caudate nucleus tissue of patients with PD by endpoint titration. Five minutes of steam sterilization reduced this titre by only 2.25 ± 0.15 decadic-logarithmic units, with an extension of the sterilization time to 90 min not causing additional inactivation. These findings reveal that αSynPD species are disease-associated biological agents with seeding activity that has higher resistance to steam sterilization than prions. CONCLUSION: The remarkable heat resistance of αSynPD seeds calls for thoroughly validated cleaning and disinfection methods that reliably remove or inactivate possible contaminations of seeding-active αSyn aggregates when reprocessing medical devices.

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In the wake of the bovine spongiform encephalopathy (BSE) epidemic, variant Creutzfeldt-Jakob disease (vCJD) has emerged as a previously unknown prion disease of humans. The initial occurrence of vCJD was observed in 1995/1996, and, so far, a total of 219 vCJD cases have been reported worldwide from seven European and four non-European countries. Of these, 172 cases were observed in the United Kingdom. The exact prevalence of sub- or pre-clinical vCJD infections is unclear. Despite effective measures that have been implemented against both BSE in ruminants and its transmission to humans, there is now a theoretical risk of secondary vCJD transmissions from human to human, for example via blood and blood products, organs and tissues, or contaminated surgical instruments and medical devices. Four cases of probable vCJD transmissions via blood have been described, as well as one case of secondary infection via a plasma product. This article provides an overview of the surveillance and epidemiology of vCJD and outlines public health strategies for the risk assessment and risk management of this novel BSE-associated prion disease in humans.

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Fourier -transform infrared microscopic spectra of scrapie-infected nervous tissue measured at high spatial resolution (approximately 6 microm) were compared with those obtained from the purified, partly proteinase K digested scrapie isoform of the prion protein isolated from nervous tissue of hamsters infected with the same scrapie strain (263K) to elucidate similarities/dissimilarities between prion structure investigated in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP(90-232) after in vitro conformational transition from the predominantly alpha-helical isoform to beta-sheet-rich structures. It is shown that prion protein structure can be investigated within tissue and that detectability of regions with elevated beta-sheet content as observed in microspectra of prion-infected tissue strongly depends on spatial resolution of the experiment.

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Although the ultimate target of infection is the central nervous system (CNS), there is evidence that the enteric nervous system (ENS) and the peripheral nervous system (PNS) are involved in the pathogenesis of orally communicated transmissible spongiform encephalopathies. In several peripherally challenged rodent models of scrapie, spread of infectious agent to the brain and spinal cord shows a pattern consistent with propagation along nerves supplying the viscera. We used immunocytochemistry (ICC) and paraffin-embedded tissue (PET) blotting to identify the location and temporal sequence of pathological accumulation of a host protein, PrP, in the CNS, PNS, and ENS of hamsters orally infected with the 263K scrapie strain. Enteric ganglia and components of splanchnic and vagus nerve circuitry were examined along with the brain and spinal cord. Bioassays were carried out with selected PNS constituents. Deposition of pathological PrP detected by ICC was consistent with immunostaining of a partially protease-resistant form of PrP (PrP(Sc)) in PET blots. PrP(Sc) could be observed from approximately one-third of the way through the incubation period in enteric ganglia and autonomic ganglia of splanchnic or vagus circuitry prior to sensory ganglia. PrP(Sc) accumulated, in a defined temporal sequence, in sites that accurately reflected known autonomic and sensory relays. Scrapie agent infectivity was present in the PNS at low or moderate levels. The data suggest that, in this scrapie model, the infectious agent primarily uses synaptically linked autonomic ganglia and efferent fibers of the vagus and splanchnic nerves to invade initial target sites in the brain and spinal cord.

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In this report a new approach for the identification of pathological changes in scrapie-infected Syrian hamster brains using Fourier transform infrared microspectroscopy is discussed. Using computer-based pattern recognition techniques and imaging, infrared maps with high structural contrast were obtained. This strategy permitted comparison of spectroscopic data from identical anatomical structures in scrapie-infected and control brains. Consistent alterations in membrane state-of-order, protein composition, carbohydrate and nucleic acid constituents were detected in scrapie-infected tissues. Cluster analysis performed on spectra of homogenized medulla oblongata and pons samples also reliably separated uninfected from infected specimens. This method provides a useful tool not only for the exploration of the disease process but also for the development of rapid diagnostic and screening techniques of transmissible spongiform encephalopathies.

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Infection of the central nervous system (CNS) is a defining feature of scrapie. Several findings suggest that scrapie agent invades the CNS via the splanchnic and vagus nerve after ingestion of infectivity. Here we address the involvement of the enteric nervous system (ENS) and gut-associated lymphoid tissue (GALT) in this pathogenetic process. Immunocytochemistry was used for the detection of pathological PrP in the duodenum and ileum of hamsters fed with 263K scrapie and sacrificed at different stages of incubation. The experiments revealed early infection of various GALT components and of submucosal and myenteric ENS ganglia. These results provide evidence for an important role of the ENS in scrapie neuroinvasion and for centripetal vagal spread of infection from the gut to the brain after oral uptake of agent.

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There is some evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The TSE-specific abnormal prion protein (PrP(sc)) is considered as surrogate marker for infectivity. We traced the deposition of PrP(sc) by immunocytochemistry in sheep and hamsters inoculated intraperitoneally with scrapie. The trigeminal, dorsal root, celiac, thoracic, and nodose ganglia contained ganglion cells and fewer satellite cells with prominent granular PrP(sc) deposition. As a novel deposition pattern, punctate deposits in adaxonal location were seen along nerve fibers of peripheral nerve adjacent to ganglia. Such prominent involvement of the PNS in two different experimental scrapie models emphasizes the need to consider the PNS in natural scrapie and other TSEs including bovine spongiform encephalopathy as potential source of infectivity.

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Following recent reports of elevated serum S100 beta protein (S100 beta) levels in patients with genetic and sporadic Creutzfeldt-Jakob disease and in rodents parenterally infected with scrapie, the suitability of serum S100 beta as a preclinical marker for transmissible spongiform encephalopathies was assessed in time-course studies. Syrian hamsters were orally and intraperitoneally challenged with scrapie and assayed for serum S100 beta levels at various times after infection. Although elevated serum S100 beta levels were consistently observed in terminally ill animals for both routes of infection, the experiments failed to detect significantly increased S100 beta serum concentrations prior to the manifestation of clinical symptoms. Thus, in this animal model, serum S100 beta does not appear to be an appropriate marker for the preclinical detection of scrapie, but it may provide a convenient laboratory aid for the diagnosis of transmissible spongiform encephalopathy in naturally or accidentally infected animals and humans.

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Although the ultimate target of infection is the CNS, there is evidence that the peripheral nervous system (PNS) is involved in the pathogenesis of Transmissible Spongiform Encephalopathies (TSEs). We used immunocytochemistry to identify the presence of pathological accumulations of a host protein, PrP, in the CNS and PNS (sensory and autonomic ganglia) of hamsters orally infected with 263K scrapie. All hamsters showed pathological deposition of PrP in most brain areas, along the length of the spinal cord, in nodose (NG) and dorsal root (DRG) ganglia and in the coeliac mesenteric ganglion complex (CMGC). In one case, scant deposition was observed along a few axons of the vagus nerve. This finding suggests that, after oral challenge, TSE infectious agent uses neural pathways and ganglia of the peripheral nervous system to reach target sites in the CNS.

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The pathogenesis of scrapie and other transmissible spongiform encephalopathies (TSEs) following oral uptake of agent is still poorly understood and can best be studied in mice and hamsters. The experiments described here further extend the understanding of the pathways along which infection spreads from the periphery to the brain after an oral challenge with scrapie. Using TSE-specific amyloid protein (TSE-AP, also called PrP) as a marker for infectivity, immunohistochemical evidence suggested that the first target area in the brain of hamsters orally infected with scrapie is the dorsal motor nucleus of the vagus nerve (DMNV), rapidly followed by the commissural solitary tract nucleus (SN). The cervical spinal cord was affected only after TSE-AP had been deposited in the DMNV, SN and other medullary target areas. For the first time, these results demonstrate conclusively that, in our animal model, initial infection of the brain after oral ingestion of scrapie agent occurs via the vagus nerve, rather than by spread along the spinal cord.

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The development of a transmissible spongiform encephalopathy upon uptake of the infectious agent in feed was studied in the model system scrapie in hamsters. Compared to single dosing, repeated dosing caused disease at a considerable higher incidence. The risk of infection was higher when the time interval between repetitive dosing was short. There was a statistically significant trend of clearance of infectivity with time.

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Scrapie is a disease which occurs naturally in sheep and goats and belongs to a group of neurodegenerative disorders known as transmissible spongiform encephalopathies, or TSEs. There is currently no cure for TSEs, and the causative agent has not yet been identified. Numerous experiments, however, have addressed the pathogenetic process following a TSE infection. In this paper we present a study of the spread of the scrapie agent after intraperitoneal infection of hamsters. The accumulation of TSE-specific amyloid protein, TSE-AP (also known as PrP), was used as a marker for infectivity. The data suggested three points of agent entry into the spinal cord: the most important one between thoracic vertebrae T7-9, and two minor ones in the lower cervical spinal cord and between vertebrae T13-L2. Further, strong evidence was found for the existence of a direct route of access to the brain which bypasses the spinal cord and most likely terminates in the medulla oblongata. The indication of an alternative pathway to the brain was confirmed by the data from orally infected hamsters. The spleen appeared to play a potential, but non-essential role in pathogenesis after intraperitoneal infection in our animal model.

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An efficient purification protocol for infectivity causing a transmissible spongiform encephalopathy (TSE) is described. From fractions purified by this protocol about 3 x 10(8) LD50 but only 3 ng of nucleic acids per gram of brain material can be isolated from all TSE-affected brains (hamster, human, sheep, cattle). By PAGE such fractions from brains of infected and control hamsters contained only one distinct nucleic acid band of 1.5 kg together with some broader smear of nucleic acid material. Although distilled water was used for such purifications, quite often a similar nucleic acid band was isolated from blanks containing no brain material. In all instances this material proved to be DNA. The result challenges the potentially important claim that purified infectious preparations of TSE-specific amyloid are free of nucleic acids of viral size. Nucleic acids isolated by other groups from diseased brain were not detected in preparations isolated by the new protocol. The application of this purification protocol in future studies will be helpful to decide whether TSEs are caused by agents containing nucleic acid or by protein only.

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Both infectivity and TSE-specific amyloid protein (also referred to as protease resistant- or prion protein, PrP) are pathognomonic markers for transmissible spongiform encephalopathies (TSE). This paper presents a new densitometric method for the quantification of TSE-specific amyloid protein and its application to studying the pathogenesis of scrapie in Syrian hamsters after infection with scrapie strain 263K. A first study established a close correlation between infectivity and TSE-specific amyloid protein with a doubling time of 2-2.6 days in the brain and cervical spinal cord for both markers. The ratio of infectivity and TSE-specific amyloid protein was relatively constant at a mean value of about 10(6) protein molecules per infectious unit during the course of infection. A subsequent study addressed the temporal-spatial spread of infection in the central nervous system by tracing the accumulation of the pathological protein. The pathogenetic process was first detected in the spinal cord between vertebrae T4 and T9, and then showed an anterograde and retrograde spread with a rate of 0.8-1.0 mm/day. There were also some indications for a possible alternative route of spread of infection from the periphery to the brain, other than via the spinal cord. Involvement of the spleen did not appear essential for the early pathogenesis in hamsters orally infected with the 263K strain of scrapie.

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SAF-protein, an amyloid, is the main constituent of scrapie-associated fibrils (SAF) and a specific marker for transmissible spongiform encephalopathies (TSE). Using an improved extraction method and Western blot detection, the disease-specific amyloid was found in various parts of the central nervous system of hamsters orally infected with scrapie, of squirrel monkeys orally infected with kuru, sporadic Creutzfeldt-Jakob disease (CJD) and scrapie, of human patients with sporadic CJD, of a sheep with natural scrapie and of a cow with bovine spongiform encephalopathy (BSE). In human CJD samples, the concentration of TSE-specific amyloid was estimated to be 1000- to 10 000-fold lower than in the central nervous system of hamsters with scrapie. The extraction method has a yield of 70% and allows Western blot detection of the TSE-specific amyloid in samples representing 1-10 micrograms of brain tissue from intracerebrally infected hamsters, as well as in individual spleens from hamsters with terminal scrapie infected by the intracerebral, oral or intraperitoneal route. A 20-100 mg sample of material is sufficient for the extraction of the pathological protein from different rodent, monkey, ovine, bovine and human tissues. The results reported here demonstrate the potential suitability of the method for the routine diagnosis of TSE as well as for the detailed analysis of distribution patterns of the TSE-specific amyloid in experimental approaches to the investigation of these diseases.

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