>>>They found infectivity in the red and white blood cells and plasma of a variant CJD patient and in the plasma of two of four sporadic CJD patients tested. These findings indicate the need to continue assessing the possible risk for CJD transmission via transfusion of blood products.<<<
3. Detection of Infectivity in Blood of Persons with Variant and Sporadic Creutzfeldt-Jakob Disease, Jean Yves Douet, et al. Creutzfeldt-Jakob disease (CJD) is a rare but fatal brain disease of humans. Over the past 60 years, this disease has developed in several hundred patients who had received tissue (mainly growth hormone or nervous tissue grafts) from infected cadaver donors. A variant form of CJD, primarily occurring in Europe, has been causally linked with bovine spongiform encephalopathy (commonly known as mad cow disease). Recent research, which used a relatively new type of highly sensitive laboratory mice, enabled researchers to measure infectivity in blood. They found infectivity in the red and white blood cells and plasma of a variant CJD patient and in the plasma of two of four sporadic CJD patients tested. These findings indicate the need to continue assessing the possible risk for CJD transmission via transfusion of blood products.
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Jean Yves Douet, Saima Zafar, Armand Perret-Liaudet, Caroline Lacroux, Séverine Lugan, Naima Aron, Herve Cassard, Claudia Ponto, Fabien Corbière, Juan Maria Torres, Inga Zerr, and Olivier Andreoletti
Author affiliations: Ecole Nationale Vétérinaire Toulouse, France (J.Y. Douet, C. Lacroux, S. Lugan, N. Aron, H. Cassard, F. Corbière, O. Andréoletti); National Reference Center for Transmissible Spongiform Encephalopathy, Georg August University, Göttingen, Germany (S. Zafar, C. Ponto, I. Zerr); Hospices Civils de Lyon, France (A. Perret-Liaudet); BioRan, Bron, France (A. Perret-Liaudet); Centro de Investigación en Sanidad Animal, Madrid, Spain (J.M. Torres)
We report the presence of infectivity in erythrocytes, leukocytes, and plasma of 1 person with variant Creutzfeldt-Jakob disease and in the plasma of 2 in 4 persons whose tests were positive for sporadic Creutzfeldt-Jakob disease. The measured infectivity levels were comparable to those reported in various animals with transmissible spongiform encephalopathies.
Among humans, Creutzfeldt-Jakob disease (CJD) is a low incidence disease (≈1 case per million per year) that occurs as either a sporadic (sCJD) or a familial/genetic (fCJD) form. Whereas familial disease forms are linked to a mutation in the prion protein gene (Prnp), no clear epidemiologic risk factors have been identified for sporadic disease forms. sCJD is not a uniform disorder in terms of clinical and neuropathological phenotype. sCJD cases are classified as type 1 or 2 according to the polymorphism at codon 129 of the protease-resistant prion protein (PrP) sequence (methionine/valine) and to the electromobility of the proteinase K–resistant core of the abnormal PrP (PrPres) (1). Type 1 and type 2 isoforms in sCJD are believed to correspond to different transmissible spongiform encephalopathy (TSE) agents
Despite their relative rarity, several hundred iatrogenically transmitted CJD cases were identified during the past 60 years (2). Some data supporting the presence of infectivity in the blood of sCJD-affected patients were reported following the intracerebral inoculation of blood fractions from affected patients into rodents. These observations remain ambiguous because other studies did not confirm them (3,4).
In 1996, a new form of CJD, named variant CJD (vCJD), was identified in humans. Variant CJD was demonstrated to be caused by the agent that causes bovine spongiform encephalopathy in cattle (5). In the United Kingdom, 4 vCJD transmissions (3 clinical cases and 1 asymptomatic infection) were probably caused by the transfusion of non–leuco-depleted erythrocyte concentrates prepared from donors who later had positive test results for vCJD (6). More recently, a presumed additional case of vCJD infection was reported in the United Kingdom in a hemophilic patient who had received fractionated plasma products, including some units linked to a donor who later was diagnosed with vCJD (7). Despite the epidemiologic evidence of bloodborne transmission in vCJD, bioassays performed on conventional rodent models failed to demonstrate the presence of infectivity in the blood (8). The lack of TSE transmission in conventional rodent models could be a consequence of a low infectivity level in blood from vCJD- and sCJD-affected patients (as described in sheep and rodent TSE models) (9) or of the existence of the species barrier phenomenon that limits the transmission of human prions to these animal models. The development during the last decade of transgenic mice models expressing PrP from others species that abrogate the species barrier now offers the potential to detect low level of infectivity (10).
In this study, we used 2 transgenic mouse models that displayed a high sensitivity to the vCJD or sCJD TSE agents to estimate the infectious titer in certain blood fractions from vCJD- and sCJD-affected patients. According to legislation of the United Kingdom, Germany, and France, the experimental protocol, including the use of human samples, was approved by UK National CJD Research & Surveillance Unit tissue bank: REC reference number 2000/4/157-German TSE reference center: Ref Nr 11/11/93, PHRC ref 2004-D50-353 for patient from France.
Previous studies reported a high sensitivity in transgenic mice overexpressing bovine PrP (tgBov) for the detection of the bovine spongiform encephalopathy agent. To demonstrate that tgBov also displays a high sensitivity to vCJD infection, we titrated to endpoint a vCJD isolate (10% brain homogenate) by intracerebral inoculation in this model (Tg110) (11). Considering the potential diversity of TSE agents that may cause sCJD, we decided to focus only on type 1 homozygous for methionine at codon 129 of the PRP gene (MM1) sCJD cases. An endpoint titration of a MM1 sCJD 10% brain homogenate was performed in a mouse model that express the methionine 129 variant of the human PrP gene (tgHu:Tg340) (12). This enabled confirmation of the capacity of the tgBov and tgHu models to detect the vCJD and sCJD MM1 agent, respectively, up to a 10−6 dilution of the reference brain homogenates (Table 1; 13). This value was within the range of the brain/blood relative infectivity reported in various TSE animal models (9,14).
Thumbnail of Abnormal prion protein (PrPres) detection by using Western blot (WB) and paraffin-embedded tissue (PET) blot in the brain of transgenic mice expressing the methionine 129 variant of the human prion protein (PrP) (tgHu) or bovine PrP (tgBov). A, B) PET blot PrPres distribution in coronal section (thalamus level) of tgHu mice inoculated with sporadic Creutzfeldt-Jakob disease (sCJD) MM1 isolates (10% brain homogenate): A) reference isolate used for the endpoint titration in Table 1; B Figure. [[caption]]
In the next step of our experiment, blood fractions (erythrocytes, plasma, and leukocytes) from 1 vCJD-confirmed patient were injected intracerebrally in tgBov mice. Similarly, plasma samples from 4 sCJD MM1 patients were inoculated with tgHu (Table 2). The blood fraction preparation was performed by using laboratory scale hematologic protocols (Technical Appendix Adobe PDF file [PDF - 31 KB - 3 pages]), not by following the procedure applied by blood banking services. This method implies that the leucodepletion that is applied to blood labile products in most countries to reduce the vCJD bloodborne transmission risk was not performed. Brain tissue samples from each of the 4 sCJD cases were also inoculated with tgHu. On the basis of the incubation period (Table 2) and PrPres distribution pattern in the brain as assessed by using paraffin-embedded tissue blot, the TSE agents in those isolates were indistinguishable from those in the MM1 sCJD case that was used for endpoint titration (Figure, panel A).
No TSE clinical signs or PrPres accumulation were observed in the tgBov or tgHu mice inoculated with phosphate-buffered saline or brain and plasma from healthy human controls. The 3 blood fractions from the vCJD-affected patient caused a positive result but low attack rate among tgBov mice (Table 2). On the basis of these results, infectivity in erythrocytes and plasma was estimated to be 2.12 infectious dose (ID)/mL of inoculum. In leukocytes, the infectious titer was estimated to be 2.23 ID/mL of whole blood. According to these values and the hematocrit of the sample (Technical Appendix Adobe PDF file [PDF - 31 KB - 3 pages]), the global infectious titer whole blood in the tested patient would be ≈4.45 ID/mL. Such infectious level is approximately equivalent to 1.4 µg of the reference vCJD brain sample that was endpoint-titrated (Table 1).
In tgHu mice, positive transmission was observed among mice inoculated with 2 of 4 plasma samples (Table 2). The infectious titers in both positive plasma samples were estimated to be 2.12 and 3.7 ID/mL of plasma, which is equivalent to 0.3–0.5 µg of the reference sCJD MM1 brain sample that was endpoint titrated (Table 1). However, because of the limited number of mice inoculated (n = 24) and the overall sensitivity of the assay (upper CI limit 6.24 ID/mL), the absence of transmission in mice inoculated with the 2 other plasma samples cannot be interpreted conclusively
In tgBov inoculated with vCJD and tgHu inoculated with sCJD, the PrPres banding patterns observed by Western blot in animals challenged with brain homogenate and blood components were identical (Figure, panels C, D). These results support the contention that the TSE agent propagated in tgBov mice and tgHu were vCJD and sCJD agents, respectively.
The data reported here confirm the presence of infectivity in erythrocytes, leukocytes, and plasma from vCJD-affected patients and demonstrate unambiguously the presence of infectivity in the plasma of some, but not all, sCJD-affected patients. The infectivity levels that we measured in the tested vCJD and sCJD blood components were comparable to those reported in various TSE animal models. The number of cases included in our study was limited; a new experiment that would include a larger number of cases and different blood fractions from sCJD cases will be necessary to refine the data. However, these results represent a substantial input for assessing the risk for interindividual bloodborne transmission of sCJD and vCJD.
Mr Douet is assistant lecturer in ophthalmology at the National Veterinary School of Toulouse and a PhD student in the TSE group in the UMR INRA ENVT 1225 unit. His primary research interests are the pathogenesis of the prion disease with special emphasis on the iatrogenic risk of transmission.
The authors are greatly indebted to the National Creutzfeldt-Jakob Disease Surveillance Unit (UK-Edinburgh) for providing variant CJD brain samples.
This work was supported by a grant from the European Commission: Protecting the food chain from prions: shaping European priorities through basic and applied research (PRIORITY, N°222887; project no. FP7-KBBE-2007-2A) and by grants from the JPND program (DEMTEST: Biomarker based diagnosis of rapid progressive dementias-optimization of diagnostic protocols, 01ED1201A). The study in Germany was funded by the Robert Koch-Institute through funds of the Federal Ministry of Health (grant no. 1369-341).
Article DOI: http://dx.doi.org/10.3201/eid2001.130353
Detection of Infectivity in Blood of Persons with Variant and Sporadic Creutzfeldt-Jakob Disease
Biochemical Typing and PrP ORF Sequencing of Sporadic and Variant Creutzfeldt-Jakob Disease Genes Confirmation of the disease diagnosis, PrPres WB typing and PrnP gene sequencing in the patients were performed by the national CJD reference center of the country of origin of each patient. All patients were Methionine/Methionine at codon 129 and no other mutation was observed. sCJD cases were all originating from Germany. The vCJD case whose blood was tested by bioassay was originating from France. The vCJD case that was used in the endpoint titration experiment was provided by the UK CJD reference center in Edinburgh.
Blood Collection and Fractionation
sCJD blood samples were collected by using S-Monovette® Coagulation Sodium Citrate 1 in 3 mL tubes according to manufacturer instruction (SARSTEDT AG & Co. · www.sarstedt.com) . Tubes were centrifuged for 20 minutes at 2000 rpm, plasma was then collected and cell-free fraction underwent another centrifugation step at 13000 rpm for 10 minutes. Supernatant was collected and stored frozen. The hematocrit values corresponding to the different samples were: sCJD case 1: 37.6%, sCJD case 2: 39.7%, sCJD case 3: 43%, sCJD case 4: 43.7%.
vCJD blood sample on EDTA and fractionated by a 10 minutes 3000 g centrifugation at 12°C . Plasma was collected and directly frozen stored. The buffy coat was collected and washed twice in NaCl 0.9% (2 min, RT) before being pelleted at 3000 g 10 min and frozen.
The sample was submitted to standard biochemical analyze and the blood formula was red cells 5.21 1012/L, hemoglobin 149 g/L, hematocrit: 48%, total white cells 17.1 109/L, lymphocytes: 27.1%, monocytes 9.3%, neutrophils: 60%, eosinophil: 1.8%, Basophils: 1.8%, Platelets:356 109/L.
Page 2 of 3
Brain and Blood Samples Handling and Bioassay
Blood was collected during the diagnostic procedures when patients were evaluated for CJD diagnosis at notifying hospital. The time between blood sampling and patients’ decease are reported in Technical Appendix Table 1.
For sCJD patients, blood was processed at the CSF reference laboratory of the National TSE Reference Center at the Department of Neurology Göttingen, Germany. Autopsy was performed by the Department of Pathology of the notifying hospital and reference material was sent to the Department of Neuropathology, Göttingen, Germany. Blood and brain samples were stored in separate department and handled by different staff in the Gottingen University hospital.
The vCJD blood sample was collected and fractionated in the Bron Hospital (France). In this hospital the department handling CSF and blood samples and the pathology department (post mortem sampling) are distinct. The vCJD reference brain sample was obtained from the UK CJD reference laboratory in Edinburgh.
All the samples were dispatched to the laboratory that performed the bioassays (UMR INRA ENVT 1225) in separated sealed containers. Samples were kept untouched and prepared only a few hours before their inoculation in mice.
The sCJD endpoint titration in tgHu mice was performed 1 year before the reception of sCJD plasma samples.
Plasma and Brain samples from the four sCJD affected patients were prepared and inoculated separately; Brain from the affected patients (text Table 2) were inoculated after the first positive transmission occurred in mice inoculated with sCJD plasma.
Similarly the vCJD endpoint titration experiment and the inoculation of the vCJD blood samples in tg Bov were performed at different dates (9 months interval).
Negative control (phosphate-buffered saline and healthy blood samples) were inoculated during the same inoculation session than the inoculation of the blood fractions from the vCJD and sCJD patients. Healthy brain controls (human and bovine) were inoculated during the same session than the endpoint titration of sCJD and vCJD brain material. Page 3 of 3
Monday, December 02, 2013
A parliamentary inquiry has been launched today into the safety of blood, tissue and organ screening following fears that vCJD – the human form of ‘mad cow’ disease – may be being spread by medical procedures
Friday, November 29, 2013
Identification of Misfolded Proteins in Body Fluids for the Diagnosis of Prion Diseases
International Journal of Cell Biology
Oral.05: Contaminated blood products induce a highly atypical prion disease devoid of PrPres in primates
Emmanuel Corney,1 Nina Jaffre,1 Jacqueline Mikol,1 Valerie Durand,1 Christelle Jas-Duval,1,2 Sophie Luccantoni-Freire,1 Evelyne Correia,1 Nathalie Lescoutra-Etcheqaray,3 Nathalie Streichenberqer,4 Stephane Haik,5 Chryslain Sumian,3 Paul Brown1 and Jean-Philippe Deslys1
1Commissariat a l'Energie Atomique; Institute of Emerging Diseases and Innovative Therapies (iMETI); Division of Prions and Related Diseases (SEPIA); Fontenay-aux- Roses, France; 2EFS·Nord de France; Lille, France; 3MacoPharm; Tourcoing, France; 4Hospices Civils de Lyon; Prion Unit; Neurobiology Department; Bron, France; 5Inserm; U 975·CNRS; UMR 7225 - Universite Pierre et Marie Curie; Paris, France
Background, Concerns about the blood-borne risk of prion infection have been confirmed by the occurrence in the UK of four transfusion-related infections of vCJD and an apparently silent infection in an hemophiliac patient. Asymptomatic incubation periods in prion diseases can extend over decades in humans. We present here unexpected results of experiments evaluating blood transmission risk in a non-human primate model.
Material and Methods, Cynomolgus macaques were inoculated with brain or blood specimens from vCJD infected humans or monkeys. Neuropathological and biochemical findings were obtained using current methods used for human patients.
Results, Thirteen out of 23 primates exposed to various human or macaque blood products exhibited a previously undescribed myelopathic syndrome, devoid of the classical features of prion disease, notably abnormal prion protein (PrPres) deposition, whereas the 14 corresponding brain-inoculated donor animals and 1 transfused animal exhibited the classical vCJD pattern. In passage experiments, plasma transfusion induced the same atypical phenotype after two years (again, with no detectable PrPres), whereas the intracerebral inoculation of spinal cord led to a typical prion disease with cerebral spongiosis and PrPres accumulation in the brain of the primate recipient. Interestingly, passage experiments in transgenic mice were largely unsuccessful.
In another experiment designed to test the efficacy of antiprion filters, three recipients of filtered red blood cells suspended in plasma are still healthy 4.5 y after transfusion whereas the recipients of unfiltered inocula died after 2.5 y with the atypical neurological profile.
Conclusion. We describe a new fatal neurological myelopathic syndrome in monkeys exposed to various vCJD/BSE-infected blood components.
Secondary transmission in primates confirms
(I) the transmissibility of this myelopathy, and
(2) its prion origin which could not be diagnosed as such in the first recipients.
This myelopathy might be compared in some respects to certain forms of human lower motor neuron disease, including neuromyelitis optica, the flail arm syndrome of amyotrophic lateral sclerosis (ALS), and the recently described FOSMN (facial onset sensory and motor neuronopathy) syndrome.
Friday, August 16, 2013
Creutzfeldt-Jakob disease (CJD) biannual update August 2013 U.K. and Contaminated blood products induce a highly atypical prion disease devoid of PrPres in primates
Sunday, August 11, 2013
Creutzfeldt-Jakob Disease CJD cases rising North America updated report August 2013
Creutzfeldt-Jakob Disease CJD cases rising North America with Canada seeing an extreme increase of 48% between 2008 and 2010
Transmission of sporadic Creutzfeldt-Jakob disease by blood transfusion: risk factor or possible biases
Singeltary submission to FDA 2001
Singeltary submission to FDA 2003
From: Terry S. Singeltary Sr. [firstname.lastname@example.org]
Sent: Monday, July 24, 2006 1:09 PM
To: FSIS RegulationsComments
Subject: [Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine Spongiform Encephalopathy (BSE) Page 1 of 98
4th CASE VCJD VIA BLOOD TRANSFUSION
Other US BSE risks: the imported products picture
24 Jul 00
Trade Statistics: UK to US Compiled by Terry S.Singeltary Sr of Bacliff, Texas
[Opinion (webmaster): The US has focused for years on tracing, containing, and eradicating live animal imports from the UK or other countries with acknowledged BSE like Belgium, including some 499 cattle and the Vermont sheep. This strategy does not acknowledge imports of rendered bovine products from England during the BSE period nor secondary products such as surgical catgut, which is to say surgical cowgut, or dairy cattle embryos, vaccines for veterinarian and human medicines. What has become of these? Mr. Singeltary, who lost his mother to CJD of unexplained origin a few years back and went on to became a well-known TSE activist, has tracked down voluminous pertinent import data through correspondence with UK officials and searches of government web sites. Imports of such products are frequently cited by Europeans in rating BSE risks in the US and in shutting out US exports.
Many people's eyes glaze over when reviewing reams of sometimes older trade statistics. There is no proof that any of the imported products was contaminated with BSE nor if so, any evidence that any BSE product lead to infection in US livestock, surgical patients, or what not. Nonetheless, the data obtained by Mr. Singeltary establish that an appalling variety and tonnage of products that were imported by the US from the UK and othr BSE-affected countries during the peak of the BSE epidemic years.]
10 January 1990 COMMERCIAL IN CONFIDENCE
NOT FOR PUBLICATION
COMMITTEE ON SAFETY OF MEDICINES WORKING PARTY ON BOVINE SPONGIFORM ENCEPHALOPATHY
SURGICAL CATGUT SUTURES
The documents below were provided by Terry S. Singeltary Sr on 8 May 2000. They are optically character read (scanned into computer) and so may contain typos and unreadable parts.
TIP740203/l 0424 CONFIDENTIAL