Detection of Infectivity in Blood of Persons with Variant and Sporadic Creutzfeldt-Jakob Disease
>>>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.<<<
>>>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.<<<
>>>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.
Contact: Press Relations INRA News Office – Jeremy Zuber +33 1 42 75 91 69
presse@inra.fr Olivier Andreoletti Joint Research Unit “Interactions
Hosts-Pathogens” (INRA/ENVT) o.andreoletti@envt.fr
Notice: Art in Science: Selections from Emerging Infectious Diseases, a
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### U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICESExternal Web Site Icon
Volume 20, Number 1—January 2014
Dispatch
Detection of Infectivity in Blood of Persons with Variant and Sporadic Creutzfeldt-Jakob Disease
[31 KB - 3
pages]
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
Abstract
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.
The Study
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).
Figure
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.
Conclusions
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.
Acknowledgment
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).
References
snip...
Article DOI: http://dx.doi.org/10.3201/eid2001.130353
Detection of Infectivity in Blood of Persons with Variant and Sporadic
Creutzfeldt-Jakob Disease
Technical Appendix
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. [flounder9@verizon.net]
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
TSS
posted by Terry S. Singeltary Sr. at
4:02 PM
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