Accuracy of a history of blood donation from surrogate witnesses: data from the UK TMER study.

Look-back studies of blood transfusion in Creutzfeldt-Jakob disease commonly rely on reported history from surrogate witnesses. Data from the UK Transfusion Medicine Epidemiology Review have been analysed to determine the accuracy of the blood donation history provided by the relatives of cases. Our results show that only a small percentage of cases were found to be registered as donors on UK Blood Service (UKBS) databases when there was no family report of blood donation. In contrast, a history of reported donation was less accurate.

Ten-year follow-up of two cohorts with an increased risk of variant CJD: donors to individuals who later developed variant CJD and other recipients of these at-risk donors.

BACKGROUND: Transmission of variant Creutzfeldt-Jakob disease (vCJD) through blood transfusion is implicated in three deaths and one asymptomatic infection. Based on this evidence, individuals assessed to be at increased risk of vCJD through donating blood transfused to individuals who later developed vCJD, or through being other recipients of such donors, are followed up to further understand the risks of vCJD transmission through blood. OBJECTIVES: To provide a ten-year follow-up of these at-risk cohorts. METHODS: Blood donors to patients who later died from vCJD were identified by the Transfusion Medicine Epidemiological Review (TMER) study. A reverse risk probability assessment quantified the risk of blood transfusion or exposure through diet as the source of vCJD in the recipients. Donors to these recipients, and these donors' other recipients, with a probability risk above 1%, are classified as at increased risk of vCJD for public health purposes. These cohorts are monitored for any vCJD occurrences. RESULTS: A total of 112 donors and 33 other recipients of their donated blood have been classified as at increased risk. After 2397 and 492 vCJD-free years of follow-up, respectively, no deaths in either at-risk cohort were of vCJD-related causes. CONCLUSIONS: The at-risk cohorts have survived disease-free far longer than the estimated incubation time for dietary-acquired vCJD (donors) and transfusion-acquired disease (other recipients). However, due to our still limited understanding of, and a lack of a reliable test for, asymptomatic vCJD infection, public health follow-up is necessary for continued monitoring of at-risk cohorts.

Creutzfeldt-Jakob disease and blood transfusion: updated results of the UK Transfusion Medicine Epidemiology Review Study.

BACKGROUND AND OBJECTIVES: This paper reports the results to 31 May 2015 of an ongoing UK study to look for additional cases of variant Creutzfeldt-Jakob disease (vCJD) transmission by blood transfusion, and to seek evidence whether other subtypes of Creutzfeldt-Jakob disease (CJD) may be transmissible via blood components. MATERIALS AND METHODS: All vCJD cases of appropriate age and any sporadic CJD (sCJD) or familial CJD (fCJD) cases with a history of blood donation or transfusion are notified to the UKBS. Donation records are sought and the usage of all donations is determined by look back. Death certificates are obtained for all donors to patients with CJD and recipients of transfused components from patients with CJD who are deceased. RESULTS: The study identified 29 sCJD blood donors, of 370 reported, with transfusion to 211 recipients. Five of these recipients were reported to have died with or of dementia, but were not believed to be cases of CJD. The vCJD arm found 18 vCJD blood donors who had donated blood which was issued for clinical usage, of 24 traced donors from 177 UK vCJD cases. To date, 3 cases of vCJD have occurred in 67 recipients identified in this recipient group, and one recipient had post-mortem confirmation of abnormal prion protein deposition in the spleen (all previously reported). CONCLUSION: The results of the ongoing TMER study show no new cases of transfusion-associated vCJD since 2007 and no evidence of transfusion transmission of sCJD.

Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation.

The West Nile virus strain Kunjin virus (WNVKUN) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNVKUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNVKUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication.

Variant CJD and blood transfusion: are there additional cases?

BACKGROUND AND OBJECTIVES: In this study, we compare variant Creutzfeldt-Jakob disease (vCJD) cases definitely linked to blood transfusion, those with a history of blood transfusion in which no donor has developed vCJD and primary cases with no history of blood transfusion. The aim is to determine whether there are any differences in the demographics or clinical phenotype in these groups that might suggest additional cases of transfusion transmission of vCJD. MATERIALS AND METHODS: All cases of vCJD who are old enough to donate blood (i.e. >17 years old) are notified to the UKBTS at diagnosis, regardless of whether they are known to have a blood donation history. A search is then made for donor records and, if found, all components produced and issued to hospitals are identified and their fate determined. Recipient details are then checked against the NCJDRSU register to establish whether there is a match between these individuals and patients who have been diagnosed with vCJD. In the reverse study, attempts are made to trace the donors to all cases reported to have received a blood transfusion and donors' details are checked against the register to determine if any have developed vCJD. RESULTS: Of the 177 cases of vCJD diagnosed in the UK as of 1 February 2014, the TMER study identified 15 cases reported to have received a blood transfusion. Transfusion records were unavailable for 4 of these cases, all pre-1980, and in one other case there was no transfusion recorded in the medical notes. Transfusion records were found for 10 cases. One case transfused at symptom onset was excluded from this analysis. The mean age at onset of symptoms of the remaining nine transfusion recipients (four female and five male) was 42·9 years; 57·6 years in the three known transfusion-transmitted cases and 35·5 years in the six not linked cases. In one of these cases, details of components transfused were unavailable, and the remaining five cases received a total of 116 donor exposures with 112 donors identified, none of whom is known to have developed clinical vCJD. To date, five of the 112 identified donors have died and none was certified as dying of vCJD or any other neurological disorder. Two of the transfusion-transmitted cases did not fulfil diagnostic criteria for probable vCJD during life but were confirmed at post-mortem. Both cases were in the older age range (68 and 74 years, respectively), and neither had a positive MRI brain scan. The remaining cases all fulfilled the criteria for the diagnosis of vCJD in life, but two of these had atypical features and were older than the expected age at onset for vCJD. CONCLUSION: In conclusion, it is possible that one or more of the vCJD cases that received a blood transfusion derived from an individual not known to have vCJD were infected by the blood transfusion. However, the evidence for this is weak, and the absence of a past history of transfusion in most cases of vCJD excludes a large number of unrecognised transfusion-transmitted cases.

A conserved peptide in West Nile virus NS4A protein contributes to proteolytic processing and is essential for replication.

The West Nile virus strain Kunjin virus (WNV(KUN)) NS4A protein is a multifunctional protein involved in membrane proliferation, stimulation of cellular pathways, and evasion of host defense and is a major component of the WNV(KUN) RNA replication complex. We identified a highly conserved region ((120)P-E-P-E(123)) upstream of the viral protease dibasic cleavage site and investigated whether this motif was required for WNV(KUN) replication. Single point mutations to alanine and a PEPE deletion mutation were created in a full-length infectious WNV(KUN) molecular clone. All mutations drastically impaired viral replication and virion production, except that of the P122A mutant, which was slightly attenuated. These mutations were subsequently transferred to a WNV(KUN) replicon to specifically assess effects on RNA replication alone. Again, all mutants, except P122A, showed severely reduced negative-sense RNA production as well as decreased viral protein production. Correspondingly, immunofluorescence analyses showed a lack of double-stranded RNA (dsRNA) labeling and a dispersed localization of the WNV(KUN) proteins, suggesting that replication complex formation was additionally impaired. Attempts to rescue replication via conservative mutants largely failed except for substitution of Asp at E121, suggesting that a negative charge at this residue is equally important. Analysis of viral protein processing suggested that cleavage of the 2K peptide from NS4A did not occur with the mutant constructs. These observations imply that the combined effects of proline and negatively charged residues within the PEPE peptide are essential to promote the cleavage of 2K from NS4A, which is a prerequisite for efficient WNV replication.

The role of cerebrospinal fluid 14-3-3 and other proteins in the diagnosis of sporadic Creutzfeldt-Jakob disease in the UK: a 10-year review.

BACKGROUND: It is 10 years since the detection of cerebrospinal fluid (CSF) 14-3-3 was included in the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD) by the WHO. Since that time, other CSF proteins, such as S100b and tau protein, have been proposed as surrogate markers for sCJD. The authors aimed to investigate the diagnostic value of each of these three proteins. METHODS: CSF samples collected from patients who were referred to the National CJD Surveillance Unit as suspected cases of sCJD during the period 1997-2007 were analysed for 14-3-3, S100b and tau protein. The sensitivity, specificity, positive predictive value and negative predictive value of each of these markers, either alone or in combination for the diagnosis of sCJD, were assessed. The impact of CSF 14-3-3 analysis on the case classification of sCJD was investigated. RESULTS AND DISCUSSION: CSF 14-3-3 had the greatest sensitivity (86%) when compared with tau protein (81%) and S100b (65%). The combination of a positive CSF 14-3-3 or an elevated tau protein with a raised S100b had the highest positive predictive power for sCJD. During the study period, 100 patients were classified as probable sCJD solely on the basis of the clinical features and a positive CSF 14-3-3. The most sensitive marker for sCJD was a positive CSF 14-3-3. The analysis of CSF 14-3-3 plays a crucial role in the case classification of sCJD.

Variant Creutzfeldt-Jakob disease and exposure to fractionated plasma products.

BACKGROUND: The risk to public health of onward transmission of variant Creutzfeldt-Jakob disease (vCJD) via blood transfusion and plasma product administration is of on-going concern, particularly with the recent reported detection of abnormal prion protein in a person with haemophilia. OBJECTIVES: To describe the history of fractionated plasma product exposure in clinical cases of vCJD in the UK. METHODS: Through examination of records held at the National CJD Surveillance Unit (from relatives, general practices and hospitals). RESULTS: Nine out of 168 UK vCJD cases had a history of receipt of fractionated plasma products on 12 different occasions (1 pre-vCJD risk in 1970, the remaining between 1989-1998). According to the UK CJD Incident Panel risk assessment criteria, 11 were low-risk products and one was low or medium risk. CONCLUSION: It is unlikely that any of the UK vCJD clinical cases to date were infected through exposure to fractionated plasma products. However, the possibility that such transmission may result in vCJD cases in the future cannot be excluded.

Hepatitis C virus p7 protein is localized in the endoplasmic reticulum when it is encoded by a replication-competent genome.

p7 protein is a small protein encoded by Hepatitis C virus (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny in vitro) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcripts. Confocal microscopy revealed that the tagged p7 protein was localized in the endoplasmic reticulum (ER) but not associated with mitochondria. Immunoelectron microscopy confirmed the p7 localization data and, moreover, showed that intracellular virus-like particles formed in the cells transfected with the wild-type, but not the recombinant, transcripts. Following a few passages of the transfected cells, the recombinant genome with the HA tag reverted to wild-type and the entire tag was deleted. Therefore, in this study, it has been demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation.

The Baby Friendly Hospital Initiative and breast feeding rates in Scotland.

OBJECTIVE: To examine the effect of the Baby Friendly Hospital Initiative on breast feeding rates in Scotland. DESIGN: Observational study using an annual survey of progress towards the WHO/UNICEF Baby Friendly Hospital Initiative and routinely collected breast feeding rates gathered on the Guthrie Inborn Errors Screening card at 7 days of postnatal age. SETTING: Scotland, UK, population 5.1 million, with about 53 000 births a year. PARTICIPANTS: All 33 maternity units with over 50 births per annum and 464,246 infants born in Scotland between 1995 and 2002. MAIN OUTCOME MEASURES: Baby Friendly status of each maternity unit at the time of an infant's birth: certificate of commitment, UK standard award, and breast feeding at 7 days postnatal age. RESULTS: Babies born in a hospital with the UK Baby Friendly Hospital Initiative standard award were 28% (p<0.001) more likely to be exclusively breast fed at 7 days of postnatal age than those born in other maternity units after adjustment for mother's age, deprivation, hospital size, and year of birth. From 1995, breast feeding rates had increased significantly faster in hospitals with Baby Friendly status by 2002: 11.39% (95% confidence interval 10.35 to 12.43) v 7.97% (95% confidence interval 7.21 to 8.73). CONCLUSION: Being born in a hospital that held the award increased the chance of being breast fed. All maternity units should be encouraged to undertake the significant strategic and practical changes required to achieve UK Baby Friendly Hospital Initiative standard status.

Ataxia in the setting of complicated enteropathy: double jeopardy.

The differential diagnosis of subacute onset ataxia in the setting of enteropathy is wide. A 54 year old patient with a pancerebellar syndrome and known ulcerative jejunoileitis is described. Small bowel biopsy showed evidence of enteropathy associated T cell lymphoma and subsequent neuropathological analysis and immunophenotyping confirmed metastasis of this tumour to the cerebellum. The presence of anti-gliadin antibodies and MRI evidence of a more longstanding process suggested additional immunologically mediated cerebellar dysfunction. Lymphomatous involvement of the CNS is rare in patients with complicated enteropathies, and has not been previously reported to involve the cerebellar parenchyma. This diagnostic possibility should be borne in mind before attributing cerebellar dysfunction in patients with the coeliac related enteropathies to nutritional compromise or immunological dysfunction (gluten ataxia) alone.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection.

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles, but at times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

A leiomyosarcoma of the oesophagus presenting incidentally without dysphagia.

Leiomyosarcoma is a rare tumour that accounts for 0.5% oesophageal sarcomas. The most common presenting symptom is dysphagia. This report presents a case of oesophageal leiomyosarcoma in a 56-year-old Caucasian man found incidentally while being investigated for refractory cough. There was no history of dysphagia in spite of tumour mass occupying most of the oesophageal lumen. The leiomyosarcoma was managed successfully with surgical resection and adjuvant radiotherapy. The patient remains disease free after 15 months after surgical intervention. The unusual case presentation is discussed and the surgical management of this rare condition reviewed.

Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively.

The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.