Detection of malarial DNA in blood donors--evidence of persistent infection.

BACKGROUND AND OBJECTIVES: The English transfusion service has screened donations from malaria-risk donors for malarial antibodies for over 10 years. The donor population includes migrants from many malaria-endemic countries and, from our experiences with post-transfusion malaria, some of these may remain parasitaemic and need clinical review. MATERIALS AND METHODS: Malarial antibody screen-reactive donations with serological evidence of malaria identified by the reference laboratory were further investigated for the presence of malarial DNA. RESULTS: Malarial DNA was found in 14 of 1955 samples investigated; three P. falciparum, five P. vivax, three P. ovale, two P. malariae and one dual parasitaemia P. falciparum/P. malariae. All of these were donors whose malaria risk was residency rather than travel. CONCLUSION: Malarial parasitaemia in healthy donors occurs, and donor malaria-risk strategies must take into account the possibility of such donors presenting. Countries not utilizing malarial antibody screening should consider carefully the collection of donations from donors previously resident in endemic countries; temporary deferral is insufficient.

Effective serological and molecular screening of deceased tissue donors.

A comprehensive and effective screening programme is essential to support the banking of tissues from deceased donors. However, the overall quality of the samples obtained from deceased donors, quantity and condition, is often not ideal, and this may lead to problems in achieving accurate and reliable results. Additionally a significant percentage of referrals are still rejected upon receipt as unsuitable for screening. We are actively involved in improving the overall quality of deceased donor screening outcomes, and have specifically evaluated and validated both serological and molecular assays for this purpose, as well as developing a specific screening strategy to minimise the specificity issues associated with serological screening. Here we review the nature and effectiveness of the deceased donor screening programme implemented by National Health Service Blood and Transplant (NHSBT), the organisation with overall responsibility for the supply of tissue products within England. Deceased donor screening data, serological and molecular, from August 2007 until May 2012 have been collated and analysed. Of 10,225 samples referred for serology screening, 5.5 % were reported as reactive; of 2,862 samples referred for molecular screening, 0.1 % were reported as reactive/inhibitory. Overall 20 % of the serological and 100 % of the molecular screen reactivity was confirmed as reflecting true infection. The use of a sequential serology screening algorithm has resulted in a marked reduction of tissues lost unnecessarily due to non-specific screen reactivity. The approach taken by NHSBT has resulted in the development of an effective and specific approach to the screening of deceased tissue donors.

Qualification of serological infectious disease assays for the screening of samples from deceased tissue donors.

Whilst some of the assays used for serological screening of post-mortem blood samples from deceased tissue donors in some countries have been specifically validated by the manufacturer for this purpose, a significant number of those currently in use globally have not. Although specificity has previously been considered a problem in the screening of such samples, we believe that ensuring sensitivity is more important. The aim of this study was to validate a broader range of assays for the screening of post-mortem blood samples from deceased tissue donors. Six microplate immunoassays currently in use within National Health Service Blood and Transplant (NHSBT) for the screening of blood, tissue and stem cell donations were included. Representative samples from confirmed positive donors were titrated in screen negative post-mortem samples in parallel with normal pooled negative serum to determine if there was any inhibition with the post-mortem samples. There were no significant differences seen (P < 0.005) between the dilution curves obtained for the positive samples diluted in post-mortem samples and normal pooled sera. Although small numbers of samples were studied, it can be surmised that the post-mortem blood samples from deceased tissue donors, collected according to United Kingdom guidelines, are a suitable substrate for the assays evaluated. No diminution of reactivity was seen when dilution with sera from deceased donors was compared to dilution using pooled serum from live donors. In the absence of genuine low titre positive post-mortem samples, the use of samples spiked with various levels of target material provides a means of qualifying serological screening assays used by NHSBT for the screening of post-mortem blood samples from deceased tissue donors.

Lot release testing of serological infectious disease assays used for donor and donation screening.

BACKGROUND AND OBJECTIVES: Monitoring of the ongoing performance of infectious disease screening assays is a critical part of any donation screening programme. Although assay sensitivity is formally evaluated prior to implementation, it is essential that this level of performance is maintained from lot-to-lot. In 2002, National Health Service Blood and Transplant developed and implemented a formal system for the lot release testing of serology infectious disease screening assays. MATERIALS AND METHODS: Lot release panels were prepared for each of the serological screening markers. They each comprise 10-15 members and include both genuine low-titre and diluted high-titre materials. For each panel member, a minimum reactivity is expected, based upon the formal sensitivity evaluation of each assay. All new lots of the screening assays used are assessed prior to supply of the lot to the organization. RESULTS: Since 2002, a total of 887 different lots of the serology screening assays used have been supplied. Of these, 876 (98.8%) passed lot release and were authorized for supply to the organization. Eleven lots (1.2%) were failed because the lots did not meet the release criteria or were unsuitable for some other reason. CONCLUSION: The lot release system has proved to be effective in objectively assessing assay performance to ensure that there is no significant lot-to-lot variation such that the performance of the assay may fall below that originally determined at evaluation. The few assays that have failed lot release did have proven performance issues that were subsequently accepted by the manufacturers. CONTENTS SUMMARY: Description of the Lot Release Testing system for serology infectious disease screening assays in use within NHSBT with a critical analysis and review of the data generated in the 7 years that the system has been in use.

The serological screening of deceased tissue donors within the English Blood Service for infectious agents--a review of current outcomes and a more effective strategy for the future.

BACKGROUND AND OBJECTIVES: The overall effectiveness of the NHSBT screening programme for infectious agents in deceased tissue donors is examined and evaluated in terms of current outcomes and how to improve upon these outcomes. MATERIALS AND METHODS: The screening results and any subsequent confirmatory results from a total of 1659 samples from NHSBT deceased donors referred to NTMRL for screening for infectious agents were included in the analysis. RESULTS: Overall 1566/1659 (94.4%) of the samples were screen negative. A total of 93 were repeat reactive on screening for one or more of the mandatory markers screened for, of which only 12 (13%) were subsequently confirmed to be positive on confirmatory testing. The majority of the repeat reactive samples were demonstrating non-specific reactivity with the screening assays in use. CONCLUSION: Overall, the NHSBT screening programme for infectious agents in deceased tissue donors is very effective with a relatively low overall loss of donors because of non-specific reactivity. However, unnecessary loss of tissue products is not acceptable, and although this programme compares favourably with the outcomes of other such programmes, the confirmatory results obtained demonstrate both the need and the potential for improving the outcomes. This is particularly important as one donor may donate more than one product, and can be achieved very easily with a change to the screening algorithm followed, using the confirmatory data obtained to support and validate this change. CONTENTS SUMMARY: Critical analysis of the NHSBT screening programme for infectious agents in deceased tissue donors and a strategy involving the design and use of a different screening algorithm to improve these outcomes.

HIV screening reactivity due to donor participation in HIV vaccine trials.

We report two instances of human immunodeficiency virus (HIV) serological screening reactivity in blood donations which were subsequently determined to be due to donor participation in HIV vaccine trials. Both donations were screen reactive with atypical patterns on confirmation; no definitive conclusion could be given for either donor. Subsequent questioning identified that both donors had been involved in HIV vaccine trials. In both cases the screening and confirmation identified the presence of HIV antibodies, although vaccine induced. While clinical trials of vaccines are important, the implications of some need careful consideration if they are not to adversely impact other areas of healthcare.

Clomipramine treatment of trichotillomania: a follow-up report on four cases.

Four consecutive patients treated for trichotillomania (hair pulling) with clomipramine reported initially dramatic reductions in symptoms. However, three of the four patients had relapsed completely at 3-month follow-up, although all four were still taking previously effective levels of the drug. The fourth patient relapsed for about 2 weeks but regained initial treatment benefits. Implications for the treatment of trichotillomania are discussed.

Malaria and blood transfusion.

The transmission of malaria by blood transfusion was one of the first recorded incidents of transfusion-transmitted infection. Although a number of different infections have been reported to be transmitted by transfusion since then, on a global scale malaria remains one of the most common transfusion-transmitted infections. Transfusion-transmitted malaria can have serious consequences, as infection with Plasmodium falciparum may prove rapidly fatal. Ensuring that, in non-endemic countries, the blood supply is free from malaria is problematical, especially as travel to malarious areas is increasing and there is some spread of the disease into new areas, as well as a resurgence of malaria in areas where previously it had been eradicated. In non-endemic countries, donor deferral can be effective, but clear guidelines are needed. In endemic countries the problem is far greater as the majority of donors may be potentially infected with malaria parasites. In both situations, the simple deferral of donors may be wasteful and can eventually erode the donor base. Thus, other strategies are needed to ensure safety with sufficiency. However, the screening of donations for evidence of malaria is not without its problems. Although the examination of blood films is still the basis for diagnosing acute malaria, in most situations it is not sufficiently sensitive for blood bank screening. In non-endemic countries, donor deferral in combination with screening for specific antimalarial immunoglobulin provides an effective means of minimizing the risk of transmission. In endemic countries, more specific donor questioning, consideration of seasonal variation and geographical distribution may help to identify the population of donors who are most likely to be infected. In addition, the administration of antimalarials to transfusion recipients may help to prevent transmission. Nonetheless, no matter what strategy is adopted, it is likely that cases of transfusion-transmitted malaria may still occur, so malaria must always be considered in any patient with a febrile illness post-transfusion.

Documented cases of post-transfusion malaria occurring in England: a review in relation to current and proposed donor-selection guidelines.

BACKGROUND AND OBJECTIVES: Although uncommon, five cases of transfusion-transmitted malaria have been documented in England over the last 20 years. With the reappearance onto the market of high-quality malaria antibody assays, and by utilizing the results of analysis of these five cases, it has been possible to review the donor malaria-deferral guidelines. MATERIALS AND METHODS: Details of the five cases of post-transfusion malaria were reviewed against the proposed new donor-deferral guidelines for malaria. RESULTS: Three of the five cases of post-transfusion malaria were directly attributable to the deferral guidelines, allowing infectious donors to be bled. CONCLUSIONS: The proposed new guidelines will prevent further cases of transmission from semi-immune individuals.

Which agents threaten blood safety in the future?

The safety of the blood supply is critical to many parts of modern medicine. In a time when prescriber's and the public's expectations are increasing, it is essential that transfusion services globally ensure the safety of the blood supply. There are, however, many threats to this safety, one being the appearance of new infectious agents. Such agents may be truly 'novel', or may be existing agents, known but not routinely screened for, posing a new or increased threat. However, before an agent is considered to be a true threat to blood safety it must be well characterized, and evidence must be presented that (i) transfusion transmission is a significant route of spread, and (ii) the agent causes significant clinical disease. If either of these criteria are not met, the question has to be asked as to whether the agent is truly a threat to blood safety.

The specificity of anti-HCV supplementary assays.

A total of 920 samples from blood donors screened as anti-HCV negative by two third-generation screening assays (Ortho Diagnostics v3.0 and Murex Diagnostics VK48) were tested by two supplementary anti-HCV assays, RIBA III (Ortho Diagnostics) and Western blot (Murex Diagnostics VK68). Six (0.65%) of the samples gave scorable bands by RIBA III and 5 (0.54%) by Western blot. Only single-band reactivity was seen and there was no consensus reactivity between the two assays. Single-band reactivity in supplementary assays, especially in samples from low-risk populations, needs to be interpreted with care before being taken as indication of genuine previous or current HCV infection.

Donor-to-donor and donor-to-patient transmission of hepatitis C virus.

A patient was reported with suspected acute post-transfusion hepatitis C virus (HCV) infection, 5 months after the transfusion of 2 units of red cells. The archived serum samples of the two implicated donations were retested by the original 3rd-generation assay together with another 3rd-generation assay and RI-BA III, and were tested for HCV RNA using the PCR. Both donations were anti-HCV negative but one was found to be PCR positive. This donor was a regular donor and was identified as the husband of a donor identified 18 months earlier as being HCV positive. This case is an example of transmission of HCV in the window period of infection, and a probable example of the transmission of HCV from wife to husband through intimate contact.

Evaluation of a malarial antibody assay for use in the screening of blood and tissue products for clinical use.

BACKGROUND AND OBJECTIVES: A new recombinant Plasmodium antigen enzyme immunoassay (EIA) for the detection of malarial antibodies was evaluated for the screening of 'malaria-risk' blood and tissue donations. MATERIALS AND METHODS: A total of 13,269 donor and patient samples were tested by both the EIA and the standard diagnostic antibody immunofluorescence test (IFAT). RESULTS: A total of 114/138 (82.6%) samples from patients with P. falciparum and 11/13 (84.6%) samples from patients with P. vivax tested positive. A total of 714/13,053 (5.47%) samples from donors identified as 'malaria risk', owing to residency or travel, were reactive in the EIA. CONCLUSIONS: The assay is more sensitive than a previously implemented malarial antibody EIA (73% in acute P. falciparum and 56% in acute P. vivax infections). The sensitivity of this new EIA is comparable to that of the IFAT, and the specificity is sufficient to screen 'malaria-risk' donors.

Viral release from HIV-I-induced syncytia of CD4+ C8166 cells.

Infection of the permissive C8166 CD4+ lymphoid cell line with human immunodeficiency virus (HIV)-I rapidly leads to syncytium formation. Ultrastructural features of medium-sized (40 to 60 microns) syncytia and larger syncytia are presented, with emphasis upon HIV-I release from the syncytial surface and into intrasyncytial vacuoles. Although surface release of HIV-I is shown to diminish with increasing syncytial size, it does not totally stop. Massive HIV-I release and entrapment within intrasyncytial vacuoles is clearly apparent within the "foamy" multivacuolated zones within medium and large syncytia. Numerous multicored aberrant viruses are routinely detected within the intrasyncytial vacuoles, and double-budding events likewise are readily detectable at the vacuolar membranes, but less so at the syncytial surface. It is suggested that this observation may be a reflection of the more restricted availability of vacuolar membrane area for excessive viral budding as compared with that at the syncytial surface.

Effect of gamma irradiation on the human immunodeficiency virus and human coagulation proteins.

The effect of gamma irradiation on HIV and plasma coagulation factors F VIII:C, F VIII:vWF and FIX was studied. Donor plasma was harvested from single donations, frozen and irradiated in the frozen state at target doses from 0 to 40 kGy (0-4 mRad). HIV was inoculated into human plasma and irradiated in a similar manner. A range of other viruses, not suspended in plasma, were also irradiated to establish viral inactivation. An inactivation rate of 0.164 TCID50 dose/ml/kGy was demonstrated for HIV compared to rates of 0.00173, 0.00526 and 0.00286 log10 units/ml/kGy for F VIII:C,F VIII:vWF and FIX respectively. The use of gamma irradiation to inactivate infectious agents present in human plasma may eliminate the need for any post-production viral inactivation methods and provide a means of assuring the safety of as yet untreated products such as cryoprecipitate and fresh frozen plasma.

Feasibility and usefulness of an efficient anti-HBc screening programme in blood donors.

Post-transfusion hepatitis B remains a risk for recipients of hepatitis B surface antigen (HBsAg) screened blood. Anti-hepatitis B core antibody (anti-HBc) screening may help reduce this risk. To evaluate its usefulness, 9,238 East Anglian blood donors were screened for anti-HBc. Those with isolated anti-HBc were identified with two confirmatory anti-HBc and anti-HB surface antibody (anti-HBs) assays. The prevalence of anti-HBc reactions in screening and confirmatory assays was 1.29% and 0.35%, respectively. The level of reactivity was significantly higher when two anti-HBc assays gave concordant results or, being concordant, were anti-HBs positive. All isolated anti-HBc-positive units (0.04%) were negative for additional HBV markers including DNA tested with nested polymerase chain reaction (PCR). A 0.31% prevalence of past HBV infection was found in this population, all carrying both anti-HBc and anti-HBs antibody, most above the protective level (0.1 IU/ml). The proposed screening schemes would limit the number of deferred donors and discarded units and keep the testing time within the remit of routine blood banking practices for an additional cost of approximately 1 pound per unit. However, no evidence was found in this donor population to suggest that anti-HBc screening would significantly reduce the incidence of post-transfusion hepatitis B.

Detection of human cytomegalovirus in peripheral mononuclear cells and urine samples using PCR.

Samples of peripheral blood lymphocytes from 105 different blood donors were investigated for the presence of human cytomegalovirus (HCMV) DNA using the polymerase chain reaction (PCR) with primers specific for the Pst I w fragment (IE region). Viral DNA sequences were detected in 53 samples, a fifth of which had been previously serotyped as HCMV negative. In the latter cases, Western blot analysis re-determined two out of three individuals that were resampled as seropositive. PCR could therefore be used to extend existing methods employed for the identification of HCMV infected blood samples prior to transfusion to individuals in high risk groups. In addition, the value of PCR as a diagnostic test was evaluated in a small pilot study by comparing the results obtained with urine samples from babies suffering congenital infection and from other high risk patients, with data obtained by isolation of infectious virus or through the detection of immediate early antigens in infected cultures. Data from this study indicated that PCR is at least as sensitive as the other methods used in HCMV diagnosis.

Incidence and significance of hepatitis B core antibody in a healthy blood donor population.

To determine the current incidence of hepatitis B core antibody (anti-HBc) in a healthy blood donor population, 1,893 donors were screened for anti-HBc. Forty-one (2.16%) were found to be initially positive and 35 (1.85%) repeatably positive. Sera from the repeatably positive donors were further screened for hepatitis B surface antibody (anti-HBs), and hepatitis B virus DNA (HBV DNA) by dot hybridisation. The repeatably positive donors were subsequently recalled for further investigation, and their peripheral blood lymphocytes were also screened for HBV DNA by dot hybridisation. Eighteen (51.4%) of the anti-HBc-positive donors were also anti-HBs-positive. HBV DNA was not detected in the serum or the lymphocytes of any of the anti-HBc-positive donors.