Transfusion‐transmitted vCJD and its consequences for the Asia‐Pacific region

The epidemic of bovine spongiform encephalopathy (BSE) in cattle in the United Kingdom, that began in 1980 and the subsequent outbreak of variant Creutzfeldt-Jakob Disease (vCJD) in humans that began in 1996 heralded a new fear for the safety of the blood supply, not only in the UK but throughout the world [1,2]. The disease, which had originally appeared in cattle, is caused by a prion (a misfolded protein). The normal protein PrPC, a membrane sialoglycoprotein, converts from an alpha helical conformation to a beta sheet structure (PrPSC or PrPTSE), forming abnormal aggregates resistant to protease digestion. Prion propagation probably occurs through template-induced conversion of further PrPC to PrPTSE. The aggregates of PrPTSE accumulate in the central nervous system, and these aggregates and associated abnormal cellular functions cause irreversible brain damage [3]. Fears that the disease might be transmissible by transfusion were confirmed by early studies in rodent animal models [4]. Sheep experimentally infected with BSE and scrapie, a transmissible prion encephalopathy in sheep, confirmed transmission by whole blood and buffy coat [5]. In a longitudinal study of transmission of prions by transfusion in sheep, the reported transmission rates were 36% for BSE and 43% for scrapie [6]. Three of the eight affected transfusion recipient sheep were alive for up to 7 years without showing signs of clinical disease. The majority of transmissions resulted from blood collected from donors at greater than 50% of the estimated incubation period. The authors concluded that infectivity titres in blood were substantial and/or that blood transfusion is a particularly efficient means of transmission. The epidemic of vCJD in humans beginning in 1996 was caused by the ingestion of abnormal BSE prions in beef. The disease is a progressive encephalopathy manifested by spongioform degeneration of the brain with florid amyloid plaques and neuronal loss. Early symptoms are neuropsychiatric and include anxiety, depression, dysaesthesia and ataxia. Patients develop progressive dementia, myoclonus and choreoathetosis with an average clinical course to death of 6 months to 2 years (median 14 months). The disease predominantly affects young adults and is invariably fatal. The first cases of vCJD were reported in the UK in 1996. To date, there have been a total of 211 cases in humans, with the majority occurring in the United Kingdom (168 cases) and France (25 cases), several cases in other European countries and one case in Saudi Arabia [7]. The USA has reported three cases and Canada one case, but Japan is the only country in Asia to have reported a case to date – these cases appear to be related to residence in the United Kingdom during the risk years (Table 1). All confirmed clinical cases to date have been homozygous for methionine at codon 129 of the PRNP gene. The first putative transfusion transmitted case in humans occurred in the UK in 2004. The risks of prion transmission associated with transfusion have been extensively reviewed by Lefrère and Hewitt [8]. Four cases associated with transfusion of fresh components, namely non-leucodepleted red cells, have now been reported although one of the cases was subclinical with vCJD detected in lymphoreticular tissues at post-mortem. None of the cases in France have been associated with transfusion, even though three of the reported cases had donated blood [9]. This year, UK Health authorities reported a patient with haemophilia who showed pathological signs of abnormal prion protein (vCJD) in the spleen after dying of unrelated causes. Although he had received 14 units of red cells, none of the donors had gone on to develop vCJD. A risk analysis showed that there is a 99% probability of the infection being caused by clotting concentrates [10]. Although the number of vCJD cases in the United Kingdom has decreased since 2000, the number of asymptomatic subclinical or preclinical cases is essentially unknown. In the absence of a specific and sensitive screening test it is impossible to know the extent of the disease in its subclinical or preclinical form. One retrospective study of 14 674 tonsillectomy and appendicectomy specimens in 2004 revealed that three appendicectomy specimens were positive for vCJD with an estimated prevalence of 237 carriers per million population [11]. Although all clinical cases of vCJD described to date have been methionine/methionine (M/M) homozygous at the PRNP codon 129 of the PrPC gene it is noteworthy that two of the three asymptomatic appendicectomy cases were valine/valine (V/V) homozygous at codon 129. Furthermore, while the three clinical transfusion transmitted cases were M/M homozygous, the fourth subclinical case was methionine/valine (M/V) heterozygous at codon 129 [12]. A more recent cross-sectional opportunistic survey of anonymous tonsil specimens in Britain, examined 63 007 samples using two enzyme immunoassays [13]. None of the samples tested were unequivocally reactive in both assays. The authors concluded that the observed prevalence of PrPCJD in tonsils in the 1961–1995 birth cohort (when the risk of exposure to BSE was high) was 0/32 661 (95% confidence interval 0–113 per million). In the subset 1961–1985 birth cohort the prevalence was still zero (95% confidence interval of 0–289 per million which was consistent with the previous survey). Two further studies may provide further information about prevalence, a large scale survey of appendices due to commence this year and a suggested study of post-mortem spleen samples in an older population. The declining number of cases of vCJD since 2000 has not led to confidence that the outbreak is over. On the contrary, there is concern that a second wave may occur in the future in heterozygous methionine/valine (M/V) individuals (50% of the population) and in valine/valine (V/V) homozygotes (11% of the population). This is supported not only by the heterozygous patient who died from a non-vCJD related disease 5 years after receiving the contaminated red cell transfusion and the two asymptomatic valine homozygous patients identified as positive for the prion protein, but also by the recently reported suspected vCJD heterozygous patient in the UK [12–14]. One possibility is that while it is possible that individuals heterozygous for methionine and valine or homozygous for valine at codon 129 of the PRNP gene may remain asymptomatic, they may act as sources of infection for others. Thus, as subclinical carriers such individuals could act as sources of infection either via contaminated surgical instruments or through blood transfusion for other persons homozygous for methionine at codon 129. On the other hand, using a simulation approach the number of future cases of vCJD in France, which has the second highest number of cases of vCJD, has been estimated at 33. The modelling suggests that a large vCJD epidemic in France is very unlikely. The authors further suggest that as France was the recipient of 60% of the total British exports of bovine carcasses, their results should be reassuring for most countries worldwide [15]. From experimental studies, it is known that scrapie and BSE induced in sheep can be transmitted by blood transfusion, and observational studies in humans confirm the transmission of vCJD in humans. Rodent study data suggest that the infectivity level via the intravenous route may be in the region of 10 infectious doses per ml (10 ID/ml), and prions are partitioned between plasma and leucocytes with 40–70% associated with leucocytes and negligible levels associated with red cells and platelets [16,17]. The United Kingdom, experiencing the greatest number of cases of vCJD, implemented a number of strategies to minimize the risk of transfusion transmission of prions [3]. These strategies were designed to reduce exposure to donor plasma; reduce exposure to donor leucocytes; reduce the number of door exposures; defer previously transfused donors; encourage appropriate transfusion practices; and encourage use of alternatives to transfusions. These strategies were achieved by non-use of UK plasma for fractionation and importing all plasma for fractionation from the USA, by importation of clinical fresh frozen plasma for younger transfusion recipients who had not been exposed to BSE, by universal leucodepletion of fresh blood products, by increasing the proportion of apheresis platelets, by deferral of previously transfused donors and by vigorous promotion of good transfusion practice. Other countries adopted some of these measures according to the level of risk in their individual countries (Table 2). Several countries adopted donor deferral strategies. For example, France deferred donors who had spent a cumulative period of 1 year or more in the UK between 1980 and 1996. Australia deferred donors who had spent a cumulative period of 6 months of more in the UK between 1 January 1980 and 31 December 1996, the USA deferred donors who had spent 3 months or more in the UK or who had resided in Europe for 5 years or longer since 1980. Japan deferred donors who had spent any time in the UK between 1980 and 1996, as well as those who had spent 6 months or more in the UK between 1997 and 2004. Several countries also deferred donors who had been transfused in the UK (and some also in Europe) between 1980 and the present time. The Food and Drug Administration issued draft guidelines in 2006 proposing the indefinite deferral of donors who have been transfused in France since 1980. Some countries also deferred donors who had been treated with European-derived bovine insulin. The variety of measures adopted by individual countries to reduce the risk of transfusion transmitted vCJD were precautionary in nature and, in several countries (in the absence of sensitive and specific tests for screening potential donors), were based on risk modelling. In countries such as Australia, where up to 30% of the population had travelled to or resided in the UK between 1980 and 1996, the level of risk reduction had to be balanced with maintenance of the sufficiency of the blood supply. The loss of donors (5% of regular donors in Australia) also had to be balanced against the risk of a potential increase in other transfusion transmitted infections such as HIV, HCV and HBV, which have a higher prevalence in new compared with established repeat donors [18]. While most screening tests aim to detect PrPSC, there is no reliable information on correlation of detection of PrPSC with infectivity nor any certainty about the implications of a positive test for the donor [3]. It has been estimated that infectivity in blood might be four or even six orders of magnitude lower than that in brain, with 1 ID/ml of blood obtained from scrapie-infected hamsters to be equivalent to 1 pg/ml of PrPSC. While a multitude of techniques for detection of prions both in tissues and blood have been described, none has yet been licensed as a screening assay for human use. These tests include immunocapillary electrophoresis using a competitive inhibition, conformation dependant immunoassay using the binding differences of the 3F4 monoclonal antibody to PrPC and PrPTSE, a system for detecting multimers in plasma and tests using PrPTSE. Other assays such as the protein misfolding cyclic amplification (PMCA) are based on the principle that small amounts of PrPTSE can convert excess amounts of PrPC into PrPTSE [19]. With repeated cycles of template-based conversion and sonication to fragment the aggregated abnormal prion, PrPTSE can be amplified to levels capable of detection in immunoassays. A variation of this assay using recombinant PrP as substrate and automated shaking instead of sonication is called the quaking-induced conversion (QIC) assay. Perhaps, the most promising assay at the present time is the assay developed by Amorfix1. This so called epitope protection assay (EP-vCJD) uses peroxynitrite to selectively mask PrPC epitopes. Aggregated PrPTSE is protected, but when disaggregated these epitopes can be detected in a standard immunoassay. This assay has now been used to screen brain-spiked donor samples and has a high sensitivity. Further studies are proceeding to determine the true sensitivity and specificity of the assay. One of the problems standing in the way of validation of candidate tests for vCJD in blood is the lack of appropriate vCJD positive plasma panels stored in a format suitable to assess potential screening assays. Spiked samples using brain and spleen homogenates are used, although doubts remain about the physical state of prions in such spiked samples and their relevance to natural infection. A second problem relates to the development of reliable confirmatory assays. It has been estimated that in the UK, which collects approximately 2.5 million donation a year and with a possible prevalence of 1 in 10 000 of subclinical vCJD, an assay with 99% specificity and sensitivity would result in approximately 25 000 false-positive tests per year [20]. Such a high number of false-positive tests in the absence of robust confirmatory tests would generate impossible donor management scenarios for transfusion services. Even a test with 99.5% specificity would produce 12 500 false reactive results in the first year of testing, and a specificity of 99.9% would still produce roughly 2500 false reactives. Specificity becomes even more of a problem in those countries expected to have much lower rates of subclinical infection in blood donors such as Asia-Pacific countries. The ethical, legal social, medical and economic issues associated with the application of tests designed to screen the blood supply are addressed in a very comprehensive recent UK Department of Health report [21]. The UK is expected to lead the way in validation of a screening test when one becomes available, and it has been proposed that there should be a study of 50 000 blood donors using 5000 USA plasma samples as a negative control panel. However, before such a study is undertaken it will be necessary for tiered preclinical studies, firstly by detection of blinded samples from dilutions of plasma pools spiked with infected brain and spleen homogenates, and if successful screening infected animal plasma samples and then available endogenously infected human plasma samples. The issues relating to the potential contamination of the blood supply by vCJD in the Asia-Pacific region are different from those affecting Europe, with its proximity to the UK particularly and its extensive import, export and travel networks which may have facilitated spread of BSE before its significance as an infectious disease was realized. Australia and New Zealand, for example, are considered to be BSE-free and they are also scrapie-free. Testing programs continue to assure freedom from BSE. Regulatory agencies in Australia have assured that products potentially contaminated by BSE are not imported into Australia. Furthermore, the National Health and Medical Research Council (NHMRC) established a Special Expert Committee on Transmissible Spongiform Encephalopathies (now called the TSE Advisory Committee) in 2002, and this committee has monitored all aspects of animal, food, medical, transfusion and transplantation issues arising from the BSE and vCJD epidemics. New Zealand has a similar committee with the same responsibilities (the TSE Steering Committee). Information about exposure to BSE in many non-European countries is not definitely known. Although the extent of spread of subclinical infection is currently unknown, as described above, risk assessments suggest that the major risk to the blood supply in any individual country is related to the extent of residency of blood donors in the UK and, to a lesser extent, residency in other affected European countries (especially France) between 1980 and 1996. It has also been suggested that the major risk of exposure to BSE contaminated beef and bovine products in the UK was between 1988 and 1993. Deferral of donors who have lived in the UK for a cumulative arbitrary period in the risk years for BSE should reduce the risk of contamination of the blood supply, although other routes of infection such as contaminated neurosurgical, ophthalmological and endoscopic biopsy instruments as well as transplantation may pose additional risks if donors subject to these procedures are not deferred. Risk modelling shows that the added policy of deferral of blood donors who have been previously transfused in the UK (with or without the addition of deferral for previous transfusion in France) at any time since 1980 should further limit the risk of transmission by blood transfusion. In the absence of a sensitive screening test, an alternative approach has been the development of filters capable of removing prions from blood. Such prion filtration steps have been developed for plasma pools during the manufacture of fractionated products. Several companies are now in the process of developing prion removal filters for whole blood and blood components. Pall2 has developed a polyester fibre filter (Leucotrap Affinity Reduction Filter) whose surface binds prions in leucodepleted red cells. One study used human red cells spiked with brain homogenate from scrapie-infected hamsters with the removal of 3.7 log ID/ml by Bioassay [22]. The second study used endogenously infected hamster blood during the clinical phase of the disease. Six of 187 animals who received non-filtered red cells injected into their brain developed clinical symptoms caused by PrPSC, but one recipient of filtered red cells also became infected. The filter has been redesigned as a combined leucodepletion and prion filter and is undergoing further evaluation. have also developed a the filter which a by The filter binds with high abnormal prions from both and variant and from hamster red cells spiked with scrapie-infected brain homogenate and filtered infectivity the level of detection by and by log ID/ml in of scrapie-infected brain-spiked red cells into hamsters showed a reduction of ID/ml of blood infectivity with no infected hamsters up to months has also developed a combined prion reduction filter by of the filter surface with a results a reduction of prion by more than with substantial reduction of although early results are the results of clinical studies such as of of red cells as well as on plasma and during the filtration process when to human blood are which has been to be against a variety of infectious such as and is not against the many still prion disease in and vCJD in the issues associated with the of transfusion transmitted vCJD are the experimental in animal models and the human cases of putative transfusion transmission by non-leucodepleted red cells and more recently by a fractionated plasma concern for the safety of the blood supply is Although there has been only one case in the Asia-Pacific region to date, the incubation period of the disease, the potential for subclinical and preclinical infections in methionine homozygous and the implications for the precautionary approach by many countries is The prevalence of vCJD in individual countries may on a number of These the importation of infected cattle and beef products from the UK and to a lesser extent Europe in the years before BSE was and the proportion of the population who may have been exposed during travel to or residence in UK and other risk the number of imported blood and blood products from BSE risk and the proportion of the population who might have been transfused in the UK or other European countries. For blood transfusion a risk based on the available information on the potential exposure of blood donors to prions is the approach to the blood supply. Risk reduction measures can then be based on safety balanced against the sufficiency of the blood supply in In the absence of sensitive and specific screening donor deferral strategies are the of a number of measures to sufficiency is not and safety is not further by the of new donors who may be at higher risk of other infectious is also a risk reduction but at the additional unrelated to reduction of Prion filters validation that there are no on red cells, platelets or plasma may be in some but will at the of sensitive and specific screening tests will a more approach to the transfusion will be to without a standard confirmatory The ethical, and economic will be and it is that countries in the Asia-Pacific region will not procedures many of these issues have been addressed in the UK and France, which have the greatest potential of disease at the present time. It is that in many countries the problems of other infectious disease and may be of much greater significance to the safety of the blood supply than vCJD at the present and currently much more in the of and in the for example, by in studies, may all Asia-Pacific countries to a through the of prion disease and to the safety of the blood supply in

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