CJD diagnosis just got easier Test for Creutzfeldt–Jakob disease raises hopes of speedier diagnosis
Published online 30 January 2011 Nature doi:10.1038/news.2011.59
News
CJD diagnosis just got easier Test for Creutzfeldt–Jakob disease raises hopes of speedier diagnosis.
Tiffany O'Callaghan
In prion diseases such as CJD, an isomer of a prion protein takes on an abnormal shape. AP Photo/Professors Stanley Prusiner/Fred Cohen, University of California San Francisco Medical SchoolInvasive biopsy is currently the only sure way to diagnose the degenerative neurological condition Creutzfeldt–Jakob Disease (CJD). But a highly sensitive assay could change that, providing a fast, accurate alternative for early diagnosis of this rare but deadly condition.
In its most common form, known as sporadic CJD, the disease affects roughly one in a million people. Beginning in the 1990s, several cases of a variation of CJD known as vCJD were reported among people who had consumed beef from cows infected with another disease, bovine spongiform encephalopathy (BSE).
The findings, published online in Nature Medicine1, also suggest that the assay — developed by microbiologist Ryuichiro Atarashi of Nagasaki University, Japan, and his team — could pave the way for the screening of broad sectors of the population.
CJD is a prion disease, in which an isomer of a common protein known as the prion protein (PrP) takes on an abnormal shape and becomes an infectious variant called PrPSc. This variant is thought to trigger the subsequent malformation of other PrP proteins. Unlike their normal counterparts, PrPSc prions cannot be broken down, and instead accumulate — often clustering in brain tissue.
The pockets of abnormal tissue that result cause brain tissue to develop a sponge-like appearance, and because prion conditions can be spread by affected humans or animals, the diseases are often referred to as transmissible spongiform encephalopathies (TSEs). Humans can be affected by several such conditions, while in addition to BSE in cows, there are several other such disorders among animals, including a condition called scrapie in sheep and hamsters.
No false positives One problem that has plagued developers of non-biopsy diagnostic techniques is that it is often difficult to avoid false positives among samples taken from patients with neurodegenerative disorders other than CJD.
So Atarashi and his colleagues used a new assay known as a real-time quaking-induced conversion (RT-QUIC) assay. 'Quaking-induced' refers to in vitro shaking, which researchers believe helps to accelerate the reactions, enabling the assay to produce results more quickly.
The team tested cerebrospinal fluid samples from 18 people with CJD and 35 people with other neurodegenerative diseases. This pilot group produced no false positives, and CJD was correctly diagnosed more than 83% of the time.
The researchers compared these results with those obtained using an existing assay that tests for levels of a protein known as 14-3-3, which is a marker for sporadic CJD. When tested on patient samples, the accuracy of 14-3-3 was 72.2%, whereas the specificity was 85.7%.
In a subsequent blind trial on 30 cerebrospinal fluid samples from Australia, RT-QUIC showed 100% specificity, resulting in no false positives among the 14 control samples, and correct diagnoses of 87.5%. 14-3-3 was equally accurate, but the rate of false positives was much higher.
"This technique allows definitive ante-mortem confirmation of CJD," says Atarashi, adding that this is currently difficult because it demands the detection of PrPSc in patients' biopsy specimens.
Supersensitivity The RT-QUIC assay is also extraordinarily sensitive — detecting the presence of harmful prions at very high dilutions — and speedy, yielding results within 48 hours.
Atarashi first began developing RT-QUIC as a researcher at the National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, in Montana. In 2008, he and his team successfully used it to screen cerebrospinal fluid taken from scrapie-infected hamsters2.
In December, Atarashi co-authored a paper reporting the efficacy of the RT-QUIC assay on nasal secretions and cerebrospinal fluid from hamsters with prion disorders3.
Byron Caughey, chief of prion/TSE research at the Rocky Mountain lab and a co-author of the two hamster studies, is encouraged by the application of the assay to human cerebrospinal fluid samples. "Of course it will also be important to detect prion diseases in other species, but human diagnosis is of pre-eminent importance," Caughey says.
And although the next step is to replicate the findings in a much larger sample, the promise shown by RT-QUIC in analysing substances other than spinal fluid in hamsters suggests a potentially fertile area for future research in humans. If RT-QUIC could be used to screen blood samples, or cheek or nasal swabs, for example, it could open up the possibilities of much earlier diagnosis and more widespread screening of donated blood.
"The earlier you're able to detect the presence of an infection in humans or animals, the more chance you have of preventing transmission to others and treating the disease in those who are infected," Caughey says.
References 1.Atarashi, R. et al. Nature Med. doi:10.1038/nm.2294 (2011). 2.Atarashi, R. et al. Nature Methods 5, 211-212 (2008). 3.Wilham, J. M. et al. PLoS Pathog. 6, e1001217 (2010).
http://www.nature.com/news/2011/110130/full/news.2011.59.html#B1
Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays
Jason M. Wilham1, Christina D. Orrú1,2, Richard A. Bessen3, Ryuichiro Atarashi4, Kazunori Sano4, Brent Race1, Kimberly D. Meade-White1, Lara M. Taubner1, Andrew Timmes1, Byron Caughey1*
1 Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, Hamilton, Montana, United States of America, 2 Department of Biomedical Sciences and Technologies, University of Cagliari, Monserrato, Italy, 3 Veterinary Molecular Biology, Montana State University, Bozeman, Montana, United States of America, 4 Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Kyushu, Japan
Abstract
A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrPC to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD50). Brain tissue from 263K scrapie-affected hamsters gave SD50 values of 1011-1012/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD50 values of 103.5–105.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD50 values of 102.0–102.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.
Author Summary
Prion diseases are deadly infectious neurodegenerative disorders of mammals which involve the misfolding of host prion protein. To better manage these diseases, we need to be able to detect and quantify the infectious particles, or prions, in biological samples. However, current tests lack the sensitivity, speed and/or quantitative capabilities required for many important applications in medicine, agriculture, wildlife biology and research. To address this problem, we have developed a new prion assay that is highly sensitive, rapid, and quantitative. This assay takes advantage of the ability of miniscule amounts of infectious prions to seed the misfolding of large excesses of normal prion protein in test tube reactions. Quantitation is achieved by testing a range of sample dilutions and determining loss of seeding activity, i.e. the end-point dilution. Similar analyses have long been used to quantify prions by inoculation into animals; however, such bioassays take months or years to perform and are both animal-intensive and expensive. Our new method provides a more practical means of detecting and quantifying prions. So far, we have applied this assay to prions from sheep, deer, and hamsters, and have found surprisingly high levels of prions in the nasal and cerebral spinal fluids of infected hamsters.
Citation: Wilham JM, Orrú CD, Bessen RA, Atarashi R, Sano K, et al. (2010) Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays. PLoS Pathog 6(12): e1001217. doi:10.1371/journal.ppat.1001217
Editor: David Westaway, University of Alberta, Canada
Received: May 4, 2010; Accepted: November 3, 2010; Published: December 2, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This research was funded by the Intramural Research Program of the NIAID, NIH. J.M.W. was supported in part by the Undergraduate Scholarship Program of the NIH and C.D.O. was partially supported by the Master and Back Program of the Regione Sardegna (Italy). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: BCAUGHEY@niaid.nih.gov
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1001217;jsessionid=0A1AAA0F70627D3CF7689F79025DD839.ambra02
Friday, January 21, 2011
Strain-Specific Barriers against Bovine Prions in Hamsters
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/strain-specific-barriers-against-bovine.html
Tuesday, January 25, 2011
Generation of a new form of human PrPSc in vitro by inter-species transmission from cervids prions
http://chronic-wasting-disease.blogspot.com/2011/01/generation-of-new-form-of-human-prpsc.html
Saturday, January 29, 2011
Atypical L-Type Bovine Spongiform Encephalopathy (L-BSE) Transmission to Cynomolgus Macaques, a Non-Human Primate
Jpn. J. Infect. Dis., 64 (1), 81-84, 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/atypical-l-type-bovine-spongiform.html
Wednesday, January 19, 2011
EFSA BIOHAZ Scientific Opinion on the revision of the quantitative risk assessment (QRA) of the BSE risk posed by processed animal proteins (PAPs)
EFSA Journal 2011;9(1):1947
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/efsa-biohaz-scientific-opinion-on.html
Monday, January 17, 2011
MAD COW Update on Feed Enforcement Activities to Limit the Spread of BSE January 13, 2011
January 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/mad-cow-update-on-feed-enforcement.html
Thursday, November 18, 2010
Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy following passage in sheep
http://bse-atypical.blogspot.com/2010/11/increased-susceptibility-of-human-prp.html
Tuesday, January 18, 2011
Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/agent-strain-variation-in-human-prion.html
Thursday, December 23, 2010
Molecular Typing of Protease-Resistant Prion Protein in Transmissible Spongiform Encephalopathies of Small Ruminants, France, 2002–2009 Volume 17, Number 1–January 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2010/12/molecular-typing-of-protease-resistant.html
Wednesday, January 19, 2011
EFSA and ECDC review scientific evidence on possible links between TSEs in animals and humans Webnachricht 19 Januar 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/efsa-and-ecdc-review-scientific.html
Friday, January 21, 2011
Strain-Specific Barriers against Bovine Prions in Hamsters
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/strain-specific-barriers-against-bovine.html
Saturday, December 18, 2010
OIE Global Conference on Wildlife Animal Health and Biodiversity - Preparing for the Future (TSE AND PRIONS) Paris (France), 23-25 February 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2010/12/oie-global-conference-on-wildlife.html
Monday, November 30, 2009
USDA AND OIE COLLABORATE TO EXCLUDE ATYPICAL SCRAPIE NOR-98 ANIMAL HEALTH CODE
http://nor-98.blogspot.com/2009/11/usda-and-oie-collaborate-to-exclude.html
http://bseusa.blogspot.com/2010/04/usda-and-oie-out-of-touch-with-risk.html
Tuesday, November 02, 2010
BSE - ATYPICAL LESION DISTRIBUTION (RBSE 92-21367) statutory (obex only) diagnostic criteria CVL 1992
http://bse-atypical.blogspot.com/2010/11/bse-atypical-lesion-distribution-rbse.html
USA
5 Includes 16 cases in which the diagnosis is pending, and 18 inconclusive cases;
6 Includes 21 (19 from 2010) cases with type determination pending in which the diagnosis of vCJD has been excluded.
2010
PLEASE NOTE REFERENCE LINES 5. AND 6.
Monday, August 9, 2010
National Prion Disease Pathology Surveillance Center Cases Examined (July 31, 2010) Year Total Referrals2 Prion Disease Sporadic Familial Iatrogenic vCJD
1996 & earlier 51 33 28 5 0 0
1997 114 68 59 9 0 0
1998 88 52 44 7 1 0
1999 120 72 64 8 0 0
2000 146 103 89 14 0 0
2001 209 119 109 10 0 0
2002 248 149 125 22 2 0
2003 274 176 137 39 0 0
2004 325 186 164 21 0 1(3)
2005 344 194 157 36 1 0
2006 383 197 166 29 0 2(4)
2007 377 214 187 27 0 0
2008 394 231 204 25 0 0
2009 425 259 216 43 0 0
2010 204 124 85 20 0 0
TOTAL 3702(5) 2177(6) 1834 315 4 3
1 Listed based on the year of death or, if not available, on year of referral;
2 Cases with suspected prion disease for which brain tissue and/or blood (in familial cases) were submitted;
3 Disease acquired in the United Kingdom;
4 Disease was acquired in the United Kingdom in one case and in Saudi Arabia in the other case;
5 Includes 16 cases in which the diagnosis is pending, and 18 inconclusive cases;
6 Includes 21 (19 from 2010) cases with type determination pending in which the diagnosis of vCJD has been excluded.
http://www.cjdsurveillance.com/pdf/case-table.pdf
Monday, August 9, 2010
National Prion Disease Pathology Surveillance Center Cases Examined (July 31, 2010)
(please watch and listen to the video and the scientist speaking about atypical BSE and sporadic CJD and listen to Professor Aguzzi)
http://prionunitusaupdate2008.blogspot.com/2010/08/national-prion-disease-pathology.html
Tuesday, December 14, 2010
Infection control of CJD, vCJD and other human prion diseases in healthcare and community settings part 4, Annex A1, Annex J, UPDATE DECEMBER 2010
http://creutzfeldt-jakob-disease.blogspot.com/2010/12/infection-control-of-cjd-vcjd-and-other.html
Saturday, June 13, 2009
Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States 2003 revisited 2009
http://cjdusa.blogspot.com/2009/06/monitoring-occurrence-of-emerging-forms.html
Saturday, January 2, 2010
Human Prion Diseases in the United States January 1, 2010 ***FINAL***
http://prionunitusaupdate2008.blogspot.com/2010/01/human-prion-diseases-in-united-states.html
my comments to PLosone here ;
http://www.plosone.org/annotation/listThread.action?inReplyTo=info%3Adoi%2F10.1371%2Fannotation%2F04ce2b24-613d-46e6-9802-4131e2bfa6fd&root=info%3Adoi%2F10.1371%2Fannotation%2F04ce2b24-613d-46e6-9802-4131e2bfa6fd
14th ICID International Scientific Exchange Brochure -
Final Abstract Number: ISE.114
Session: International Scientific Exchange
Transmissible Spongiform encephalopathy (TSE) animal and human TSE in North America
update October 2009
T. Singeltary
Bacliff, TX, USA
Background:
An update on atypical BSE and other TSE in North America. Please remember, the typical U.K. c-BSE, the atypical l-BSE (BASE), and h-BSE have all been documented in North America, along with the typical scrapie's, and atypical Nor-98 Scrapie, and to date, 2 different strains of CWD, and also TME. All these TSE in different species have been rendered and fed to food producing animals for humans and animals in North America (TSE in cats and dogs ?), and that the trading of these TSEs via animals and products via the USA and Canada has been immense over the years, decades.
Methods:
12 years independent research of available data
Results:
I propose that the current diagnostic criteria for human TSEs only enhances and helps the spreading of human TSE from the continued belief of the UKBSEnvCJD only theory in 2009. With all the science to date refuting it, to continue to validate this old myth, will only spread this TSE agent through a multitude of potential routes and sources i.e. consumption, medical i.e., surgical, blood, dental, endoscopy, optical, nutritional supplements, cosmetics etc.
Conclusion:
I would like to submit a review of past CJD surveillance in the USA, and the urgent need to make all human TSE in the USA a reportable disease, in every state, of every age group, and to make this mandatory immediately without further delay. The ramifications of not doing so will only allow this agent to spread further in the medical, dental, surgical arena's. Restricting the reporting of CJD and or any human TSE is NOT scientific. Iatrogenic CJD knows NO age group, TSE knows no boundaries. I propose as with Aguzzi, Asante, Collinge, Caughey, Deslys, Dormont, Gibbs, Gajdusek, Ironside, Manuelidis, Marsh, et al and many more, that the world of TSE Transmissible Spongiform Encephalopathy is far from an exact science, but there is enough proven science to date that this myth should be put to rest once and for all, and that we move forward with a new classification for human and animal TSE that would properly identify the infected species, the source species, and then the route.
http://ww2.isid.org/Downloads/14th_ICID_ISE_Abstracts.pdf
DID EVERYONE FILL OUT THEIR CJD QUESIONNAIRE FROM THE CDC AND OR THE CJD FOUNDATION ???
Friday, November 30, 2007
CJD QUESTIONNAIRE USA CWRU AND CJD FOUNDATION
http://cjdquestionnaire.blogspot.com/
Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA 77518
News
CJD diagnosis just got easier Test for Creutzfeldt–Jakob disease raises hopes of speedier diagnosis.
Tiffany O'Callaghan
In prion diseases such as CJD, an isomer of a prion protein takes on an abnormal shape. AP Photo/Professors Stanley Prusiner/Fred Cohen, University of California San Francisco Medical SchoolInvasive biopsy is currently the only sure way to diagnose the degenerative neurological condition Creutzfeldt–Jakob Disease (CJD). But a highly sensitive assay could change that, providing a fast, accurate alternative for early diagnosis of this rare but deadly condition.
In its most common form, known as sporadic CJD, the disease affects roughly one in a million people. Beginning in the 1990s, several cases of a variation of CJD known as vCJD were reported among people who had consumed beef from cows infected with another disease, bovine spongiform encephalopathy (BSE).
The findings, published online in Nature Medicine1, also suggest that the assay — developed by microbiologist Ryuichiro Atarashi of Nagasaki University, Japan, and his team — could pave the way for the screening of broad sectors of the population.
CJD is a prion disease, in which an isomer of a common protein known as the prion protein (PrP) takes on an abnormal shape and becomes an infectious variant called PrPSc. This variant is thought to trigger the subsequent malformation of other PrP proteins. Unlike their normal counterparts, PrPSc prions cannot be broken down, and instead accumulate — often clustering in brain tissue.
The pockets of abnormal tissue that result cause brain tissue to develop a sponge-like appearance, and because prion conditions can be spread by affected humans or animals, the diseases are often referred to as transmissible spongiform encephalopathies (TSEs). Humans can be affected by several such conditions, while in addition to BSE in cows, there are several other such disorders among animals, including a condition called scrapie in sheep and hamsters.
No false positives One problem that has plagued developers of non-biopsy diagnostic techniques is that it is often difficult to avoid false positives among samples taken from patients with neurodegenerative disorders other than CJD.
So Atarashi and his colleagues used a new assay known as a real-time quaking-induced conversion (RT-QUIC) assay. 'Quaking-induced' refers to in vitro shaking, which researchers believe helps to accelerate the reactions, enabling the assay to produce results more quickly.
The team tested cerebrospinal fluid samples from 18 people with CJD and 35 people with other neurodegenerative diseases. This pilot group produced no false positives, and CJD was correctly diagnosed more than 83% of the time.
The researchers compared these results with those obtained using an existing assay that tests for levels of a protein known as 14-3-3, which is a marker for sporadic CJD. When tested on patient samples, the accuracy of 14-3-3 was 72.2%, whereas the specificity was 85.7%.
In a subsequent blind trial on 30 cerebrospinal fluid samples from Australia, RT-QUIC showed 100% specificity, resulting in no false positives among the 14 control samples, and correct diagnoses of 87.5%. 14-3-3 was equally accurate, but the rate of false positives was much higher.
"This technique allows definitive ante-mortem confirmation of CJD," says Atarashi, adding that this is currently difficult because it demands the detection of PrPSc in patients' biopsy specimens.
Supersensitivity The RT-QUIC assay is also extraordinarily sensitive — detecting the presence of harmful prions at very high dilutions — and speedy, yielding results within 48 hours.
Atarashi first began developing RT-QUIC as a researcher at the National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, in Montana. In 2008, he and his team successfully used it to screen cerebrospinal fluid taken from scrapie-infected hamsters2.
In December, Atarashi co-authored a paper reporting the efficacy of the RT-QUIC assay on nasal secretions and cerebrospinal fluid from hamsters with prion disorders3.
Byron Caughey, chief of prion/TSE research at the Rocky Mountain lab and a co-author of the two hamster studies, is encouraged by the application of the assay to human cerebrospinal fluid samples. "Of course it will also be important to detect prion diseases in other species, but human diagnosis is of pre-eminent importance," Caughey says.
And although the next step is to replicate the findings in a much larger sample, the promise shown by RT-QUIC in analysing substances other than spinal fluid in hamsters suggests a potentially fertile area for future research in humans. If RT-QUIC could be used to screen blood samples, or cheek or nasal swabs, for example, it could open up the possibilities of much earlier diagnosis and more widespread screening of donated blood.
"The earlier you're able to detect the presence of an infection in humans or animals, the more chance you have of preventing transmission to others and treating the disease in those who are infected," Caughey says.
References 1.Atarashi, R. et al. Nature Med. doi:10.1038/nm.2294 (2011). 2.Atarashi, R. et al. Nature Methods 5, 211-212 (2008). 3.Wilham, J. M. et al. PLoS Pathog. 6, e1001217 (2010).
http://www.nature.com/news/2011/110130/full/news.2011.59.html#B1
Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays
Jason M. Wilham1, Christina D. Orrú1,2, Richard A. Bessen3, Ryuichiro Atarashi4, Kazunori Sano4, Brent Race1, Kimberly D. Meade-White1, Lara M. Taubner1, Andrew Timmes1, Byron Caughey1*
1 Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, Hamilton, Montana, United States of America, 2 Department of Biomedical Sciences and Technologies, University of Cagliari, Monserrato, Italy, 3 Veterinary Molecular Biology, Montana State University, Bozeman, Montana, United States of America, 4 Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Kyushu, Japan
Abstract
A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrPC to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD50). Brain tissue from 263K scrapie-affected hamsters gave SD50 values of 1011-1012/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD50 values of 103.5–105.7/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD50 values of 102.0–102.9/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.
Author Summary
Prion diseases are deadly infectious neurodegenerative disorders of mammals which involve the misfolding of host prion protein. To better manage these diseases, we need to be able to detect and quantify the infectious particles, or prions, in biological samples. However, current tests lack the sensitivity, speed and/or quantitative capabilities required for many important applications in medicine, agriculture, wildlife biology and research. To address this problem, we have developed a new prion assay that is highly sensitive, rapid, and quantitative. This assay takes advantage of the ability of miniscule amounts of infectious prions to seed the misfolding of large excesses of normal prion protein in test tube reactions. Quantitation is achieved by testing a range of sample dilutions and determining loss of seeding activity, i.e. the end-point dilution. Similar analyses have long been used to quantify prions by inoculation into animals; however, such bioassays take months or years to perform and are both animal-intensive and expensive. Our new method provides a more practical means of detecting and quantifying prions. So far, we have applied this assay to prions from sheep, deer, and hamsters, and have found surprisingly high levels of prions in the nasal and cerebral spinal fluids of infected hamsters.
Citation: Wilham JM, Orrú CD, Bessen RA, Atarashi R, Sano K, et al. (2010) Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays. PLoS Pathog 6(12): e1001217. doi:10.1371/journal.ppat.1001217
Editor: David Westaway, University of Alberta, Canada
Received: May 4, 2010; Accepted: November 3, 2010; Published: December 2, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This research was funded by the Intramural Research Program of the NIAID, NIH. J.M.W. was supported in part by the Undergraduate Scholarship Program of the NIH and C.D.O. was partially supported by the Master and Back Program of the Regione Sardegna (Italy). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: BCAUGHEY@niaid.nih.gov
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1001217;jsessionid=0A1AAA0F70627D3CF7689F79025DD839.ambra02
Friday, January 21, 2011
Strain-Specific Barriers against Bovine Prions in Hamsters
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/strain-specific-barriers-against-bovine.html
Tuesday, January 25, 2011
Generation of a new form of human PrPSc in vitro by inter-species transmission from cervids prions
http://chronic-wasting-disease.blogspot.com/2011/01/generation-of-new-form-of-human-prpsc.html
Saturday, January 29, 2011
Atypical L-Type Bovine Spongiform Encephalopathy (L-BSE) Transmission to Cynomolgus Macaques, a Non-Human Primate
Jpn. J. Infect. Dis., 64 (1), 81-84, 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/atypical-l-type-bovine-spongiform.html
Wednesday, January 19, 2011
EFSA BIOHAZ Scientific Opinion on the revision of the quantitative risk assessment (QRA) of the BSE risk posed by processed animal proteins (PAPs)
EFSA Journal 2011;9(1):1947
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/efsa-biohaz-scientific-opinion-on.html
Monday, January 17, 2011
MAD COW Update on Feed Enforcement Activities to Limit the Spread of BSE January 13, 2011
January 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/mad-cow-update-on-feed-enforcement.html
Thursday, November 18, 2010
Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy following passage in sheep
http://bse-atypical.blogspot.com/2010/11/increased-susceptibility-of-human-prp.html
Tuesday, January 18, 2011
Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/agent-strain-variation-in-human-prion.html
Thursday, December 23, 2010
Molecular Typing of Protease-Resistant Prion Protein in Transmissible Spongiform Encephalopathies of Small Ruminants, France, 2002–2009 Volume 17, Number 1–January 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2010/12/molecular-typing-of-protease-resistant.html
Wednesday, January 19, 2011
EFSA and ECDC review scientific evidence on possible links between TSEs in animals and humans Webnachricht 19 Januar 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/efsa-and-ecdc-review-scientific.html
Friday, January 21, 2011
Strain-Specific Barriers against Bovine Prions in Hamsters
http://transmissiblespongiformencephalopathy.blogspot.com/2011/01/strain-specific-barriers-against-bovine.html
Saturday, December 18, 2010
OIE Global Conference on Wildlife Animal Health and Biodiversity - Preparing for the Future (TSE AND PRIONS) Paris (France), 23-25 February 2011
http://transmissiblespongiformencephalopathy.blogspot.com/2010/12/oie-global-conference-on-wildlife.html
Monday, November 30, 2009
USDA AND OIE COLLABORATE TO EXCLUDE ATYPICAL SCRAPIE NOR-98 ANIMAL HEALTH CODE
http://nor-98.blogspot.com/2009/11/usda-and-oie-collaborate-to-exclude.html
http://bseusa.blogspot.com/2010/04/usda-and-oie-out-of-touch-with-risk.html
Tuesday, November 02, 2010
BSE - ATYPICAL LESION DISTRIBUTION (RBSE 92-21367) statutory (obex only) diagnostic criteria CVL 1992
http://bse-atypical.blogspot.com/2010/11/bse-atypical-lesion-distribution-rbse.html
USA
5 Includes 16 cases in which the diagnosis is pending, and 18 inconclusive cases;
6 Includes 21 (19 from 2010) cases with type determination pending in which the diagnosis of vCJD has been excluded.
2010
PLEASE NOTE REFERENCE LINES 5. AND 6.
Monday, August 9, 2010
National Prion Disease Pathology Surveillance Center Cases Examined (July 31, 2010) Year Total Referrals2 Prion Disease Sporadic Familial Iatrogenic vCJD
1996 & earlier 51 33 28 5 0 0
1997 114 68 59 9 0 0
1998 88 52 44 7 1 0
1999 120 72 64 8 0 0
2000 146 103 89 14 0 0
2001 209 119 109 10 0 0
2002 248 149 125 22 2 0
2003 274 176 137 39 0 0
2004 325 186 164 21 0 1(3)
2005 344 194 157 36 1 0
2006 383 197 166 29 0 2(4)
2007 377 214 187 27 0 0
2008 394 231 204 25 0 0
2009 425 259 216 43 0 0
2010 204 124 85 20 0 0
TOTAL 3702(5) 2177(6) 1834 315 4 3
1 Listed based on the year of death or, if not available, on year of referral;
2 Cases with suspected prion disease for which brain tissue and/or blood (in familial cases) were submitted;
3 Disease acquired in the United Kingdom;
4 Disease was acquired in the United Kingdom in one case and in Saudi Arabia in the other case;
5 Includes 16 cases in which the diagnosis is pending, and 18 inconclusive cases;
6 Includes 21 (19 from 2010) cases with type determination pending in which the diagnosis of vCJD has been excluded.
http://www.cjdsurveillance.com/pdf/case-table.pdf
Monday, August 9, 2010
National Prion Disease Pathology Surveillance Center Cases Examined (July 31, 2010)
(please watch and listen to the video and the scientist speaking about atypical BSE and sporadic CJD and listen to Professor Aguzzi)
http://prionunitusaupdate2008.blogspot.com/2010/08/national-prion-disease-pathology.html
Tuesday, December 14, 2010
Infection control of CJD, vCJD and other human prion diseases in healthcare and community settings part 4, Annex A1, Annex J, UPDATE DECEMBER 2010
http://creutzfeldt-jakob-disease.blogspot.com/2010/12/infection-control-of-cjd-vcjd-and-other.html
Saturday, June 13, 2009
Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States 2003 revisited 2009
http://cjdusa.blogspot.com/2009/06/monitoring-occurrence-of-emerging-forms.html
Saturday, January 2, 2010
Human Prion Diseases in the United States January 1, 2010 ***FINAL***
http://prionunitusaupdate2008.blogspot.com/2010/01/human-prion-diseases-in-united-states.html
my comments to PLosone here ;
http://www.plosone.org/annotation/listThread.action?inReplyTo=info%3Adoi%2F10.1371%2Fannotation%2F04ce2b24-613d-46e6-9802-4131e2bfa6fd&root=info%3Adoi%2F10.1371%2Fannotation%2F04ce2b24-613d-46e6-9802-4131e2bfa6fd
14th ICID International Scientific Exchange Brochure -
Final Abstract Number: ISE.114
Session: International Scientific Exchange
Transmissible Spongiform encephalopathy (TSE) animal and human TSE in North America
update October 2009
T. Singeltary
Bacliff, TX, USA
Background:
An update on atypical BSE and other TSE in North America. Please remember, the typical U.K. c-BSE, the atypical l-BSE (BASE), and h-BSE have all been documented in North America, along with the typical scrapie's, and atypical Nor-98 Scrapie, and to date, 2 different strains of CWD, and also TME. All these TSE in different species have been rendered and fed to food producing animals for humans and animals in North America (TSE in cats and dogs ?), and that the trading of these TSEs via animals and products via the USA and Canada has been immense over the years, decades.
Methods:
12 years independent research of available data
Results:
I propose that the current diagnostic criteria for human TSEs only enhances and helps the spreading of human TSE from the continued belief of the UKBSEnvCJD only theory in 2009. With all the science to date refuting it, to continue to validate this old myth, will only spread this TSE agent through a multitude of potential routes and sources i.e. consumption, medical i.e., surgical, blood, dental, endoscopy, optical, nutritional supplements, cosmetics etc.
Conclusion:
I would like to submit a review of past CJD surveillance in the USA, and the urgent need to make all human TSE in the USA a reportable disease, in every state, of every age group, and to make this mandatory immediately without further delay. The ramifications of not doing so will only allow this agent to spread further in the medical, dental, surgical arena's. Restricting the reporting of CJD and or any human TSE is NOT scientific. Iatrogenic CJD knows NO age group, TSE knows no boundaries. I propose as with Aguzzi, Asante, Collinge, Caughey, Deslys, Dormont, Gibbs, Gajdusek, Ironside, Manuelidis, Marsh, et al and many more, that the world of TSE Transmissible Spongiform Encephalopathy is far from an exact science, but there is enough proven science to date that this myth should be put to rest once and for all, and that we move forward with a new classification for human and animal TSE that would properly identify the infected species, the source species, and then the route.
http://ww2.isid.org/Downloads/14th_ICID_ISE_Abstracts.pdf
DID EVERYONE FILL OUT THEIR CJD QUESIONNAIRE FROM THE CDC AND OR THE CJD FOUNDATION ???
Friday, November 30, 2007
CJD QUESTIONNAIRE USA CWRU AND CJD FOUNDATION
http://cjdquestionnaire.blogspot.com/
Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA 77518
posted by Terry S. Singeltary Sr. at
1:23 PM
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