Evaluation of a test for its suitability in the diagnosis of variant Creutzfeldt-Jakob disease.

BACKGROUND AND OBJECTIVES: Evaluation of variant Creutzfeldt-Jakob disease (vCJD) diagnostic/donor screening tests is made complicated by the very limited supply of blood samples from clinically confirmed cases of vCJD. To determine appropriate access for test developers to rare Creutzfeldt-Jakob disease (CJD) blood samples, the oversight committee of the NIBSC CJD Resource Centre has developed a process and protocols detailing minimum requirements for both test sensitivity and specificity. This protocol is broadly similar to that outlined in the common technical specification (European Directive 98/79/EC). MATERIALS AND METHODS: Tests are subjected to a stepwise evaluation (step 1). vCJD tissue homogenates spiked into pooled human plasma (step 2). Blood samples from animals known to be incubating (Transmissible spongiform encephalopathy) TSE disease (scrapie/Bovine Spongiform encephalopathy (BSE)-infected sheep, BSE-infected primates) and appropriate controls (step 3). Fresh or frozen plasma from normal UK blood donors and (step 4). Plasma samples from individuals with confirmed clinical stage variant CJD (transfusion transmission) or sporadic CJD (no evidence of blood transmission). RESULTS: The assay evaluated performed with good sensitivity with vCJD-spiked tissue homogenates, poor sensitivity for ovine TSE-infected blood samples and failed with plasma from BSE-infected non-human primates and with true vCJD clinical samples. CONCLUSIONS: The test evaluated here is currently unsuitable for use in blood donor screening or diagnosis using blood.

Reference materials for the evaluation of pre-mortem variant Creutzfeldt-Jakob disease diagnostic assays.

BACKGROUND AND OBJECTIVES: A standard panel of materials is needed for the evaluation of assays being developed for the diagnosis of variant Creutzfeldt-Jakob disease. MATERIALS AND METHODS: Tissues from human and animals incubating transmissible spongiform encephalopathy disease have been prepared, aliquoted and where possible characterized by in vitro methods. RESULTS: A standardized preparation of materials has been generated. CONCLUSIONS: Large-scale preparations of tissues and blood fractions can be used to directly compare the sensitivities of assays using different formats.

Absence of detectable measles virus genome sequence in blood of autistic children who have had their MMR vaccination during the routine childhood immunization schedule of UK.

Leukocyte preparations from children with documented evidence of MMR vaccination and confirmed diagnosis of autism were examined by several assays designed to target multiple regions of the measles virus genome sequence. No sample was found positive by any method. The assays applied were highly sensitive, specific and robust in nature, and were based on the amplification of measles virus RNA transcripts by real-time quantitative RT-PCR (QRT-PCR) as well as by conventional RT-PCR-nested PCR. The assays applied were potentially able to detect measles virus RNA down to single figure copy numbers per reaction. The amount of total nucleic acid extract of leukocytes subjected to various measles virus-specific investigations was several fold higher than minimally required of a sample where measles virus persistence is well documented. This study failed to substantiate reports of the persistence of measles virus in autistic children with development regression.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

Effect of different immunisation schedules on the excretion and reversion of oral poliovaccine strains.

Excretion of live oral poliovaccine and molecular markers of increased virulence in the viruses isolated were examined in children who were either previously immunised with inactivated or live vaccine or were unimmunised. There appeared to be some effect of previous immunisation with either live or killed vaccine at the second dose of live vaccine. Where an effect was seen it took the form of reduced rates of excretion, shorter time periods of excretion and more rapid and complete reversion of the excreted virus. The data are consistent with the view that poliovirus is able to escape the immune pressure in the gut to some extent by improving its general fitness rather than direct evasion of immunity.

Isolation of SV40 from the environment of a colony of cynomolgus monkeys naturally infected with the virus.

The presence of SV40 viral particles in the environment of cynomolgus monkeys naturally infected with this virus has been analyzed by testing waste of the cage samples. SV40 was detected in 2/4 cages tested where mixed infection of SV40 and adenoviruses was observed after inoculation of virions concentrated from cage waste in CV-1 cells. The detected SV40 strains were identical in the regions studied to strain W17, isolated at National Institute for Biological Standards and Control, UK (NIBSC) from a (1/19) monkey kidney biopsy and contains an archetypal regulatory region. The recovery of infectious SV40 virions from the cages provides information about the potential mechanism of transmission of this virus.

Technical aspects of the development and validation of tests for variant Creutzfeldt-Jakob disease in blood transfusion.

The development of tests for variant Creutzfeldt-Jakob disease in the context of blood transfusion is technically complicated by a number of factors, including the long asymptomatic period and uncertainty as to whether infectivity is present in human blood at all. The specific needs of a donor test impose constraints. It is argued that the only possible analyte will be blood, and that while the initial work will involve animal studies, these will provide only an approximate guide. A rapid infectivity assay is key to confirming positives in human samples, and dilutions of infected human brain will probably provide adequate routine control samples to ensure the correct performance of the test.

Comparative evaluation of measles virus specific TaqMan PCR and conventional PCR using synthetic and natural RNA templates.

Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation.

Virological issues in the use of cell therapies.

The testing of cells and starting materials for biological medicinal products made from human sources has tended to focus on blood-borne viruses and those with a tropism for B lymphocytes, probably arising from the existing guidance on human monoclonal antibodies and experience with proteins fractionated from human blood. This may not be the best course although blood may contaminate starting materials. Other agents may be more likely contaminants, and it is essential to apply sound virological principles to ensure product safety, particularly in instances such as cell therapy where the product is a living cell which is intended to persist in the recipient for some time.

Beyond HIV, HBV and HCV--how to deal with other viruses?

Transmission of viruses such as HBV, HIV and HCV by blood components and protein fractions are well documented and precautions based on donor selection and screening as well as processing are well established and effective. Other viruses, including small non-enveloped viruses, pose a greater challenge for removal, but are considered less hazardous clinically. While cell associated viruses such as HTLVI and CMV pose a risk to certain kinds of recipient of blood components the biggest single current issue is that of vCJD following the BSE epidemic in the United Kingdom.

Eradication and cessation of programmes.

Eradication of disease requires the eradication of the causative agent. Smallpox is the only human disease knowingly eradicated so far and, while poliomyelitis is likely to be the second, they present very different problems. All smallpox infections were apparent when significantly infectious, the vaccine was very easy to deliver and its effects easy to monitor, and it was incapable of causing the disease it was intended to prevent. In contrast, most poliovirus infections are silent, vaccination leaves no outward sign, and there is a low, but real, incidence of vaccine-associated poliomyelitis. While smallpox was eradicated by containment of infections by quarantine and vaccination, poliomyelitis requires mass vaccination campaigns to break virus transmission. Moreover, in contrast to smallpox, the strategies for stopping polio vaccination are still under discussion. The polio eradication programme on the other hand has made and continues to make strides towards its goal, and is a major triumph of public health interventions.

Functional formation of domain V of the poliovirus noncoding region: significance of unpaired bases.

Previously we have shown that polioviruses with mutations that disrupt the predicted secondary structure of the 5' noncoding region of domain V are temperature sensitive for growth. Non-temperature-sensitive revertant viruses had mutations that re-formed secondary structure by a direct back mutation of changes in the opposite strand. We mutated unpaired regions and selected revertants of viruses with single base deletions, where no obvious back mutation was available in order to gain information on secondary structure. Results indicated that conservation of length of a three base loop between two double-stranded stems was essential for a functional domain V to form. The requirement for the unpaired "hinge" base at 484 which is implicated in the attenuation of Sabin 2 was also confirmed. Results also underline the necessity for functional folding over local secondary structure stability.

Detection and growth kinetics of SV40 preparations with different enhancer element copy number.

It has been reported that the rate of growth of SV40 in certain cell culture systems is affected by the number of copies of the enhancer element, and that this might in turn affect the ability of manufacturers of polio vaccines to detect SV40 contamination in tests for adventitious agents. Eleven strains of SV40 of which three were primary isolates, three were control strains used in different laboratories and five were well characterised were examined for their enhancer copy number. It was found that the three primary isolates contained a single copy while three preparations used as controls in different laboratories were mixtures of single and multiple copy strains. The remaining five strains consisted of one known double enhancer and four known single enhancer strains. All eleven strains were titrated in BSC-1 cells. There was no correlation between the number of copies of the enhancer element and the rate of growth or the final titre reached, although there was variation between the strains.

Are recombinant products really infection risk free?

Biological medicinal products can be manufactured from a variety of sources. Recombinant products are currently produced from extremely well characterised cells grown in vitro and priori pose the lowest risk of transmitting infection. Validated production processes make a major contribution to product safety however.

Live-attenuated strains of improved genetic stability.

The current live-attenuated vaccine strains of poliovirus are genetically unstable and capable of rapid evolution in human hosts, resulting in reversion to neurovirulence and, occasionally, disease. They can also be shed by recipients for a considerable time after vaccination. This raises questions about how and when to stop vaccination after wild-type viruses have been eliminated. Persistence of vaccine revertant viruses in the population would present a risk to new cohorts of unvaccinated children and threaten the success of the eradication programme. A number of Sabin vaccine strain derivatives have been described that are, in theory, genetically more stable than the present vaccines and therefore less likely to revert to virulence. The approaches used in their derivation are outlined here and data presented for two strains showing a significant improvement in genetic stability. These strains were designed according to our understanding of the molecular basis of attenuation and incorporate changes in the sequence of an RNA structural domain that plays a key role in attenuation. They may also be less transmissible than the current type 3 vaccine strain and are potentially useful in the strategically difficult final stages of poliomyelitis eradication.