Variant Creutzfeldt-Jakob Disease Research Articles

Human prion disease

AbstractHuman prion diseases (HPDs) are progressive and fatal neurodegenerative diseases caused by abnormal prion protein (PrPSc). They can be sporadic, genetic or acquired. Sporadic HPDs include sporadic Creutzfeldt–Jakob disease (CJD) and sporadic fatal insomnia. Genetic HPDs include genetic CJD, Gerstman–Sträussler–Scheinker disease and fatal familial insomnia. Acquired HPDs include Kuru, variant CJD and iatrogenic CJD. The World Health Organization clinical diagnostic criteria for HPDs include clinical findings, cerebrospinal fluid protein markers and electroencephalography, and the UK and European clinical diagnostic criteria include a combination of clinical findings, 14‐3‐3 protein in the cerebrospinal fluid, magnetic resonance imaging diffusion‐weighted imaging and electroencephalography. Recently, the development and clinical application of real‐time quaking‐induced conversion has allowed pre‐mortem diagnosis of sporadic CJD. The real‐time quaking‐induced conversion assay allows detection of >1 fg of PrPSc in diluted CJD brain homogenate, as well as a variety of biological tissues and cerebrospinal fluid. However, the real‐time quaking‐induced conversion assay does not provide prognostic data and has lower diagnostic accuracy for some rarer, atypical prion diseases. Furthermore, recent advancements in laboratory testing and magnetic resonance imaging diffusion‐weighted imaging have shown improved diagnostic accuracy for prion diseases. Clinical trials of prion disease treatments have recently begun in many countries. The therapeutic window consists mainly of the early stage of disease, and few clinical studies have examined the role of biomarkers at this stage. Therefore, biomarkers, imaging and clinical symptoms might be extremely important for the diagnosis of prion diseases.Clinical trials of prion disease treatments have recently begun in many countries. The therapeutic window mainly consists of the early stage of disease, and few clinical studies have examined the role of biomarkers at this stage. Therefore, biomarkers, imaging and clinical symptoms might be extremely important for the diagnosis of prion diseases.

The Challenge of Preventing a Rare but Potentially Devastating Iatrogenic Exposure to Variant Creutzfeldt-Jakob Disease: An Ambi-directional Observation with Policy Intervention

Although rare, patients with variant Creutzfeldt-Jakob Disease (vCJD) in their differential diagnosis of progressive dementia and movement disorder could continue to present to hospitals for care. However, U.S.-based infection control guidelines do not fully address the possibility of vCJD. After near-misses involving increasing numbers of patients with clinical findings and epidemiologic risks compatible with vCJD, or exposures to chronic wasting disease, we sought to improve recognition and prevention of iatrogenic spread of these prion-related diseases.

Variant CJD: Reflections a Quarter of a Century on.

Twenty-five years has now passed since variant Creutzfeldt-Jakob disease (vCJD) was first described in the United Kingdom (UK). Early epidemiological, neuropathological and biochemical investigations suggested that vCJD represented a new zoonotic form of human prion disease resulting from dietary exposure to the bovine spongiform encephalopathy (BSE) agent. This hypothesis has since been confirmed though a large body of experimental evidence, predominantly using animal models of the disease. Today, the clinical, pathological and biochemical phenotype of vCJD is well characterized and demonstrates a unique and remarkably consistent pattern between individual cases when compared to other human prion diseases. While the numbers of vCJD cases remain reassuringly low, with 178 primary vCJD cases reported in the UK and a further 54 reported worldwide, concerns remain over the possible appearance of new vCJD cases in other genetic cohorts and the numbers of asymptomatic individuals in the population harboring vCJD infectivity. This review will provide a historical perspective on vCJD, examining the origins of this acquired prion disease and its association with BSE. We will investigate the epidemiology of the disease along with the unique clinicopathological and biochemical phenotype associated with vCJD cases. Additionally, this review will examine the impact vCJD has had on public health in the UK and the ongoing concerns raised by this rare group of disorders.

Evaluation of biological proxies for surgical instruments and their accuracy for acoustic decontamination techniques

Surgical instruments go through rigorous protocols involving decontamination and sterilization. Of particular concern is Variant Creutzfeldt-Jakob Disease, because (unlike viruses and bacteria) the prions responsible for this are not destroyed by the high temperatures, cleaning chemistries, and radiation used. This paper is part of a study on the effectiveness of ultrasonically-activated streams to remove dangerous prions from surgical instruments to prevent infections from spreading between patients. Cleaning techniques are often evaluated against standard surgical instrument substitutes in a test such as animal bioassays and amplification assays. These test uses thin wires (0.016 mm diameter), which are pre-contaminated, put through the cleaning process, and then sent for analysis to determine how much contamination remains. Despite being an industry standard with proven effectiveness as a proxy for cleaning surgical instruments using heat/chemicals/radiation, thin wires are shown to be a poor proxy when ultrasonically cleaned. The acoustics of thin wires and surgical instruments differ significantly and therefore the performance of acoustic cleaning methods can fail this test while being ultimately effective on surgical instruments. We present measurements on and in the vicinity of thin wires and steel plates to illustrate the differences between these two targets and discuss the reasons for this disparity.

Prion diseases, always a threat?

Prion disease is caused by prion which was identified as a causative factor of transmissible spongiform encephalopathy such as Creutzfeldt-Jakob disease (CJD) and means proteineaceous infectious particle. Prion diseases are devastating neurodegenerative disorders of human and many animals including sheep, cows, deer, cats and camels. CJD presents usually as a rapidly progressive dementia with other symptoms inevitably resulting in death often in months with no available therapy. Like other neurodegenerative diseases, prion diseases are classified into sporadic and genetic forms. In addition, there is the third category of environmentally acquired form. This type includes kuru and iatrogenic CJD due to human dura mater grafts or human pituitary derived hormones as well as variant CJD transmitted through food contaminated with prion of bovine spongiform encephalopathy (BSE). BSE and variant CJD has been almost controlled but chronic wasting disease, prion disease of deer, is spreading widely in North America and South Korea and recently in northern Europe. Recently Aß, α-synuclein and other proteins related to Alzheimer's, Parkinson's and other diseases were reported to have prion features such as transmission to animals. Aß transmission to human was suggested in iatrogenic CJD cases as well as in cerebral amyloid angiopathy cases with cerebral bleeding long after childhood neurosurgery with or without cadaveric dura. These findings indicate prion diseases due to various prions, namely various transmissible proteins appear to be a threat particularly in this longevity society. To overcome prion diseases is one of the top priority in our society.

Histotype-Dependent Oligodendroglial PrP Pathology in Sporadic CJD: A Frequent Feature of the M2C "Strain".

In sporadic Creutzfeldt-Jakob disease, molecular subtypes are neuropathologically well identified by the lesioning profile and the immunohistochemical PrPd deposition pattern in the grey matter (histotypes). While astrocytic PrP pathology has been reported in variant CJD and some less frequent histotypes (e.g., MV2K), oligodendroglial pathology has been rarely addressed. We assessed a series of sCJD cases with the aim to identify particular histotypes that could be more prone to harbor oligodendroglial PrPd. Particularly, the MM2C phenotype, in both its more “pure” and its mixed MM1+2C or MV2K+2C forms, showed more frequent oligodendroglial PrP pathology in the underlying white matter than the more common MM1/MV1 and VV2 histotypes, and was more abundant in patients with a longer disease duration. We concluded that the MM2C strain was particularly prone to accumulate PrPd in white matter oligodendrocytes.

Propagation of CJD Prions in Primary Murine Glia Cells Expressing Human PrPc.

There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt–Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrPSc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments.

Creutzfeldt-Jakob disease surveillance in Australia: update to 31 December 2020.

Nationwide surveillance of Creutzfeldt-Jakob disease and other human prion diseases is performed by the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR). National surveillance encompasses the period since 1 January 1970, with prospective surveillance occurring from 1 October 1993. Over this prospective surveillance period, considerable developments have occurred in pre-mortem diagnostics; in the delineation of new disease subtypes; and in a heightened awareness of prion diseases in healthcare settings. Surveillance practices of the ANCJDR have evolved and adapted accordingly. This report summarises the activities of the ANCJDR during 2020. Since the ANCJDR began offering diagnostic cerebrospinal fluid (CSF) 14-3-3 protein testing in Australia in September 1997, the annual number of referrals has steadily increased. In 2020, 510 domestic CSF specimens were referred for 14-3-3 protein testing and 85 persons with suspected human prion disease were formally added to the national register. As of 31 December 2020, just over half (44 cases) of the 85 suspect case notifications remain classified as 'incomplete'; 27 cases were excluded through either detailed clinical follow-up (9 cases) or neuropathological examination (18 cases); 18 cases were classified as 'definite' and eleven as 'probable' prion disease. For 2020, sixty percent of all suspected human-prion-disease-related deaths in Australia underwent neuropathological examination. No cases of variant or iatrogenic CJD were identified. The SARS-CoV-2 pandemic did not affect prion disease surveillance outcomes in Australia.

Les maladies à prions ou encéphalopathies spongiformes transmissibles

Prion diseases or transmissible spongiform encephalopathies (TSEs) are human and animal diseases naturally or experimentally transmissible with a long incubation period and a fatal course without remission. The nature of the transmissible agent remains debated but the absence of a structure evoking a conventional microorganism led Stanley B. Prusiner to hypothesize that it could be an infectious protein (proteinaceous infectious particle or prion). The prion would be the abnormal form of a normal protein, cellular PrP (PrPc) which will change its spatial conformation and be converted into scrapie prion protein (PrPsc) with properties of partial resistance to proteases, aggregation and insolubility in detergents. No inflammatory or immune response are detected in TSEs which are characterized by brain damage combining spongiosis, neuronal loss, astrocytic gliosis, and deposits of PrPsc that may appear as amyloid plaques. Although the link between the accumulation of PrPsc and the appearance of lesions remains debated, the presence of PrPsc is constant during TSE and necessary for a definitive diagnosis. Even if they remain rare diseases (2 cases per million), the identification of kuru, at the end of the 1950s, of iatrogenic cases in the course of the 1970s and of the variant of Creutzfeldt-Jakob disease (CJD) in the mid-1990s explain the interest in these diseases but also the fears they can raise for public health. They remain an exciting research model because they belong both to the group of neurodegenerative diseases with protein accumulation (sporadic CJD), to the group of communicable diseases (iatrogenic CJD, variant of CJD) but also to the group of genetic diseases with a transmission Mendelian dominant (genetic CJD, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia).

The importance of ongoing international surveillance for Creutzfeldt-Jakob disease.

Creutzfeldt–Jakob disease (CJD) is a rapidly progressive, fatal and transmissible neurodegenerative disease associated with the accumulation of misfolded prion protein in the CNS. International CJD surveillance programmes have been active since the emergence, in the mid-1990s, of variant CJD (vCJD), a disease linked to bovine spongiform encephalopathy. Control measures have now successfully contained bovine spongiform encephalopathy and the incidence of vCJD has declined, leading to questions about the requirement for ongoing surveillance. However, several lines of evidence have raised concerns that further cases of vCJD could emerge as a result of prolonged incubation and/or secondary transmission. Emerging evidence from peripheral tissue distribution studies employing high-sensitivity assays suggests that all forms of human prion disease carry a theoretical risk of iatrogenic transmission. Finally, emerging diseases, such as chronic wasting disease and camel prion disease, pose further risks to public health. In this Review, we provide an up-to-date overview of the transmission of prion diseases in human populations and argue that CJD surveillance remains vital both from a public health perspective and to support essential research into disease pathophysiology, enhanced diagnostic tests and much-needed treatments.

Genetic Risk Factors of Creutzfeldt-Jakob Disease in the Population of Newborns in Slovakia.

The most frequent human prion disease is Creutzfeldt–Jakob disease (CJD). It occurs as sporadic (sCJD), genetic (gCJD), iatrogenic (iCJD) form and as variant CJD. The genetic form represents about 10–15% of confirmed cases worldwide, in Slovakia as much as 65–75%. Focal accumulation of gCJD was confirmed in Orava region. The most common point mutation of the prion protein gene (PRNP) is E200K. CJD has a long asymptomatic phase and it is not known when the carriers of the mutation E200K become infectious. Precautions to prevent iCJD are focused especially on clinical CJD cases, but asymptomatic CJD-specific mutation carriers cannot be excluded, and represent a potential genetic CJD-risk group. The aim of this study was to determine the occurrence, frequency and geographic distribution of the E200K mutation among the newborns, comparing the areas of focal accumulation of gCJD with extra-focal ones, as well as distribution of the polymorphism M129V of the PRNP gene. A total of 2915 samples of dry blood spots from anonymous newborns were analyzed. We used RealTime PCR method to determine the presence of the E200K mutation and the M129V polymorphism. Genetic testing revealed 13 carriers of the E200K mutation. Investigation of the M129V polymorphism affirmed higher representation of methionine homozygotes (48% MM, 44% MV, 8% VV). Achieved results fully confirmed our previous observations concerning both the specific and nonspecific genetic CJD risk among the Slovak general population. The 48% of methionine homozygotes and 4 carriers of the E200K mutation among 1000 live-born children in Slovakia underline the benefits of genetic testing.

Prionopathies and Prionlike Protein Aberrations in Neurodegenerative Diseases

Protein misfolding has been an area of intense research and is implicated in a number of neurodegenerative diseases. Key proteins in the brain lose their native ability to fold and instead assume abnormal conformations. Misfolded proteins cluster to form pathologic aggregates, which cause cellular dysfunction, neuronal death, and neurodegeneration. The prionopathies are best known among the neurodegenerative diseases for their ability to misfold, self-propagate, and infect other organisms. There is increasing evidence of a rationale for a prionlike mechanism of spread of other neurodegenerative diseases through a similar seeding mechanism. In this review, we detail the role of a key protein aberration known to the various prion diseases, including sporadic, variant, and iatrogenic Creutzfeldt-Jakob disease; variably protease-sensitive prionopathy; Gerstmann-Straussler-Scheinker disease; fatal familial insomnia; and kuru. We also discuss the clinical presentation, the available, and emerging imaging options for these diseases. In the second part of this review, we delineate how a prionlike seeding process may be driving the progression of other neurodegenerative diseases, including Parkinson disease, Alzheimer disease, and Huntington disease. A discussion of clinical presentation and imaging features of these example diseases follows to make a case for a common approach to developing imaging biomarkers and therapies of these diseases.Learning Objective: Upon completion of this article, one should be able to describe the various types of prion diseases, recognize and identify the common the neuro-imaging findings in prion diseases, describe seeding mechanism of prion disease, list the common amyloid PET tracers used for Alzheimer’s disease, and list common imaging biomarkers in neurodegenerative diseases.

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<106 WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrPSc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.

Improved surveillance of surgical instruments reprocessing following the variant Creutzfeldt–Jakob disease crisis in England: findings from a three-year survey

Sensitive, direct protein-detection methods are now recommended for the inspection of reprocessed reusable surgical instruments in England to reduce the risk of prion transmission. To implement an established, highly sensitive method to quantify proteinaceous residues on reprocessed instruments in a Sterile Services Department (SSD) and evaluate its potential impact on service provision. We introduced highly sensitive epifluorescence (EDIC/EF) microscopy in a large SSD. Over three years, we periodically tested two models of washer disinfector using stainless-steel tokens spiked with mouse brain homogenate or Browne test soil for comparison. We also obtained data and feedback from staff who had been using EDIC/EF to examine almost 3000 reprocessed instruments. All reprocessed test surfaces harboured residual contamination (up to 258.4 ng from 1-μg spikes). Proximity between surfaces affected decontamination efficacy and allowed cross-contamination. Up to 50 ng de novo proteinaceous contamination was deposited on control surfaces after a single automated washer disinfector (AWD) cycle. The test soil behaved differently than real tissue contamination. SSD staff observed proteinaceous residues on most reprocessed instruments using EDIC/EF, which can detect far smaller amounts than the currently accepted national threshold of 5 μg per side. Implementing recent national guidelines to address the prions concern proved an eye-opener. Microscopic levels of proteins remain on many reprocessed instruments. The impact most of these residues, potentially including prions, may have on subsequent patients after sterilization remains debatable. Improving surveillance capability in SSDs can support decision making and raise the standards of surgical instruments reprocessing.

Heidenhain Variant of Creutzfeldt-Jakob Disease with Concurrent Findings of Posterior Reversible Encephalopathy Syndrome: A Case Report and Literature Review

Although Creutzfeldt-Jakob disease is not yet proved to be related to the development of posterior reversible encephalopathy syndrome, prion invasion to the endothelium has been shown to change blood-brain barrier permeability. We present a case of Heidenhain variant of Creutzfeldt-Jakob disease that had associated imaging findings of posterior reversible encephalopathy syndrome, with a review of the literature, raising the question of whether Creutzfeldt-Jakob disease instead represents another cause for the development of posterior reversible encephalopathy syndrome.

Human prion disease surveillance in Spain, 1993-2018: an overview

ABSTRACT In Spain, human transmissible spongiform encephalopathies (TSEs) have been undergoing continuous surveillance for over 25 years. In 1995, the system was launched as an EU Concerted Action, with EU surveillance network procedures being incorporated from 2002 onwards. The aim of this report was to describe performance and outcomes of this surveillance system across the period 1993–2018. Neurology and public health specialists from every region reported cases to a central hub at the Carlos III Health Institute, Madrid. In all, eight accidentally transmitted cases and five definite variant Creutzfeldt-Jakob disease (vCJD) patients were reported. All vCJD cases were diagnosed between 2005 and 2008. Two of these were family/dietary-related and spatially linked to a third. Yearly incidence of sporadic CJD per million was 1.25 across the period 1998–2018, and displayed a north-south gradient with the highest incidence in La Rioja, Navarre and the Basque Country. Genetic TSEs were observed to be clustered in the Basque Country, with a 4-fold incidence over the national rate. A total of 120 (5.6%) non-TSE sporadic, conformational, rapidly progressing neurodegenerative and vascular brain disorders were reported as suspect CJD. We conclude that TSEs in Spain displayed geographically uneven, stable medium incidences for the sporadic and genetic forms, a temporal and spatial family cluster for vCJD, and decreasing numbers for dura-mater-associated forms. The vCJD surveillance, framed within the EU network, might require continuing to cover all prion disorders. There is need for further strategic surveillance research focusing on case definition of rapid-course, conformational encephalopathies and surgical risk.

Creutzfeldt-Jakob Disease: An Overview and a Preliminary Study on Its Impacts on the Beef and Cattle Production Industry in the United Kingdom and the United States

Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal neurodegenerative disease, that affects 1 in 1,000,000 people, with there being 350 new cases of the pathology every year. In specific, it is a prion disease that is caused by a misfolded prion protein, termed PrPSc, which is the infectious form of the prion protein PrPC. Rather than being recycled by the body, the PrPSc aggregates in the brain as plaques, leading to neurodegeneration of surrounding cells and the spongiform characteristics of the pathology. This paper gives an overview of the current understanding of CJD, by looking at the pathophysiology genetic features of CJD, the different forms of CJD, before exploring the societal impact of CJD. In specific, this societal impact is inspected by investing the correlation between the beef and cattle production industry and the number of CJD cases in the United Kingdom and the United States, through the lens of the 1996 outbreak of variant Creutzfeldt-Jakob disease (vCJD), a form of CJD, in the United Kingdom (UK). This outbreak was caused by an infection of bovine spongiform encephalopathy in the UK’s cattle, which was then transmitted via cattle products. In its wake and seeing that it is always fatal, the outbreak forced many societal changes in an effort to prevent further infection and spread, with an emphasis being placed on the beef and cattle production industries. Hence, it is important to understand the correlation between the beef and cattle production industry and CJD cases. To do this, I looked at the correlation between the United States and United Kingdom’s cattle and beef production industries and CJD cases (vCJD cases for the UK), over the course of the past 25 years. This was done by carrying out a Pearson Correlation test between the number of CJD cases in each country and the rate of cattle production during this period of time and running a linear regression on the dataset. In doing so, it was discovered that the linear regression model fit the dataset with very low accuracy (R2 ≈ 0.5664). Moreover, it was revealed that there was only a moderative negative correlation between the number of CJD cases in the United States and the rate of production of the United States’ beef and cattle production industry (r = -0.7418), however, it was also found that there was a strong upward correlation between the number of vCJD cases in the UK and the rate of production of the UK’s beef and cattle production industry (r = 0.7632), indicating that vCJD does have a strong impact on the UK’s beef and cattle production industry.

Exposure Risk of Chronic Wasting Disease in Humans.

The majority of human prion diseases are sporadic, but acquired disease can occur, as seen with variant Creutzfeldt–Jakob disease (vCJD) following consumption of bovine spongiform encephalopathy (BSE). With increasing rates of cervid chronic wasting disease (CWD), there is concern that a new form of human prion disease may arise. Currently, there is no evidence of transmission of CWD to humans, suggesting the presence of a strong species barrier; however, in vitro and in vivo studies on the zoonotic potential of CWD have yielded mixed results. The emergence of different CWD strains is also concerning, as different strains can have different abilities to cross species barriers. Given that venison consumption is common in areas where CWD rates are on the rise, increased rates of human exposure are inevitable. If CWD was to infect humans, it is unclear how it would present clinically; in vCJD, it was strain-typing of vCJD prions that proved the causal link to BSE. Therefore, the best way to screen for CWD in humans is to have thorough strain-typing of harvested cervids and human CJD cases so that we will be in a position to detect atypical strains or strain shifts within the human CJD population.

Glossary

Glossary

Improving the Predictive Value of Prion Inactivation Validation Methods to Minimize the Risks of Iatrogenic Transmission With Medical Instruments.

Prions are pathogenic infectious agents responsible for fatal, incurable neurodegenerative diseases in animals and humans. Prions are composed exclusively of an aggregated and misfolded form (PrPSc) of the cellular prion protein (PrPC). During the propagation of the disease, PrPSc recruits and misfolds PrPC into further PrPSc. In human, iatrogenic prion transmission has occurred with incompletely sterilized medical material because of the unusual resistance of prions to inactivation. Most commercial prion disinfectants validated against the historical, well-characterized laboratory strain of 263K hamster prions were recently shown to be ineffective against variant Creutzfeldt-Jakob disease human prions. These observations and previous reports support the view that any inactivation method must be validated against the prions for which they are intended to be used. Strain-specific variations in PrPSc physico-chemical properties and conformation are likely to explain the strain-specific efficacy of inactivation methods. Animal bioassays have long been used as gold standards to validate prion inactivation methods, by measuring reduction of prion infectivity. Cell-free assays such as the real-time quaking-induced conversion (RT-QuIC) assay and the protein misfolding cyclic amplification (PMCA) assay have emerged as attractive alternatives. They exploit the seeding capacities of PrPSc to exponentially amplify minute amounts of prions in biospecimens. European and certain national medicine agencies recently implemented their guidelines for prion inactivation of non-disposable medical material; they encourage or request the use of human prions and cell-free assays to improve the predictive value of the validation methods. In this review, we discuss the methodological and technical issues regarding the choice of (i) the cell-free assay, (ii) the human prion strain type, (iii) the prion-containing biological material. We also introduce a new optimized substrate for high-throughput PMCA amplification of human prions bound on steel wires, as translational model for prion-contaminated instruments.