Hepatitis E virus in blood donors in England, 2016 to 2017: from selective to universal screening.

IntroductionHepatitis E virus (HEV), the most common cause of acute hepatitis in many European countries, is transmitted through consumption of processed pork but also via blood transfusion and transplantation. HEV infection can become persistent in immunocompromised individuals.AimWe aimed to determine the incidence and epidemiology of HEV infection in English blood donors since the introduction of donation screening in 2016.MethodsBetween March 2016 and December 2017, 1,838,747 blood donations were screened for HEV RNA. Donations containing HEV RNA were further tested for serological markers, RNA quantification and viral phylogeny. Demographics, travel and diet history were analysed for all infected donors.ResultsWe identified 480 HEV RNA-positive blood donations during the 22-month period, most (319/480; 66%) donors were seronegative. Viral loads ranged from 1 to 3,230,000 IU/ml. All sequences belonged to genotype 3, except one which likely represents a new genotype. Most viraemic donors were over 45 years of age (279/480; 58%), donors aged between 17 and 24 years had a seven-times higher incidence of HEV infection than other donors between March and June 2016 (1:544 donations vs 1:3,830). HEV-infected blood donors were evenly distributed throughout England. Screening prevented 480 HEV RNA-positive blood donations from reaching clinical supply.ConclusionHEV screening of blood donations is a vital step in order to provide safer blood for all recipients, but especially for the immunosuppressed. The unusually high rates of HEV infection in young blood donors may provide some insight into specific risks associated with HEV infection in England.

Hepatitis E risks: pigs or blood-that is the question.

BACKGROUND: Infection with hepatitis E virus (HEV) Genotype 3 is recognized as a food-borne zoonosis in developed countries where it usually causes a mild self-limited acute hepatitis. It may cause a persistent infection in the immunosuppressed human that can progress to cirrhosis. To protect the patient from transfusion-acquired HEV infection, steps have been taken in the United Kingdom to provide for at-risk patients only components from donors screened for HEV viremia. This strategy does not protect from dietary exposure and calls into question estimation of relative risk between blood transfusion and diet. STUDY DESIGN AND METHODS: Using data on HEV viremia, component exposure, residual plasma volume, and resulting transmission, the dose of virus administered and subsequent transmission rates were determined and used to populate a model that can infer the relationship between blood and dietary exposure. RESULTS: The annual attack rate of a population, defined as seroconversion, provides an estimate of the risk of receiving a component containing HEV from a viremic donor. The lowest viral dose that resulted in infection was 2 × 104 IUs and 55% of components containing this dose transmitted infection. The transfusion risk of infection only exceeds the annual dietary risk when more than 13 individual donor components are transfused. CONCLUSION: For many solid organ transplant patients dietary exposure far exceeds the risk of transfusion from unscreened donors. It is only in the immunosuppressed patient requiring extensive blood component support that transfusion risk dominates. This understanding should inform policy decisions on HEV RNA screening of blood donations.

Virology, serology, and demography of hepatitis E viremic blood donors in South East England.

BACKGROUND: Hepatitis E virus (HEV) Genotype 3 (G3) in England comprises two principal phylogenetic groups (Group 1 and Group 2) and can be transmitted by transfusion. Unselected screening identified 79 viremic donors; 76 participated in a follow-up study. STUDY DESIGN AND METHODS: Viral RNA dynamics, phylogenetics, and seroconversion were characterized in the donors. Detailed demographic, travel, clinical, and lifestyle questionnaires were undertaken. RESULTS: The majority of viremic individuals (57/79) were seronegative at time of donation but all seroconverted. Viremia was short-lived, with a median of 6.5 weeks to confirmed viral clearance. All infections were acquired in the United Kingdom and were G3, with Group 2 viruses predominating (43/54; 80%). Infection was associated with some clinical symptoms both at and after donation (8/77; 10%). Viral loads and symptoms were more pronounced in Group 1 infections. There was no serologic evidence of reinfection. Donors were more commonly male (p = 0.002); both male and female donors were older than comparator donors. Animal contact was unlikely to be the source of infection. Consumption of chicken and pig meat was common to all infected donors; processed pig meat was most commonly purchased from one particular retail chain. CONCLUSION: Viremic donors represent primary infection in older members of the community and reflect a widespread zoonotic in the United Kingdom. The two phylogenetic groups of HEV G3 display different pathogenicity and the more common Group 2 appears less adapted to humans. There are no objective demographic criteria that can identify donors at enhanced HEV risk.

Hepatitis E virus in blood components: a prevalence and transmission study in southeast England.

BACKGROUND: The prevalence of hepatitis E virus (HEV) genotype 3 infections in the English population (including blood donors) is unknown, but is probably widespread, and the virus has been detected in pooled plasma products. HEV-infected donors have been retrospectively identified through investigation of reported cases of possible transfusion-transmitted hepatitis E. The frequency of HEV transmission by transfusion and its outcome remains unknown. We report the prevalence of HEV RNA in blood donations, the transmission of the virus through a range of blood components, and describe the resulting morbidity in the recipients. METHODS: From Oct 8, 2012, to Sept 30, 2013, 225,000 blood donations that were collected in southeast England were screened retrospectively for HEV RNA. Donations containing HEV were characterised by use of serology and genomic phylogeny. Recipients, who received any blood components from these donations, were identified and the outcome of exposure was ascertained. FINDINGS: 79 donors were viraemic with genotype 3 HEV, giving an RNA prevalence of one in 2848. Most viraemic donors were seronegative at the time of donation. The 79 donations had been used to prepare 129 blood components, 62 of which had been transfused before identification of the infected donation. Follow-up of 43 recipients showed 18 (42%) had evidence of infection. Absence of detectable antibody and high viral load in the donation rendered infection more likely. Recipient immunosuppression delayed or prevented seroconversion and extended the duration of viraemia. Three recipients cleared longstanding infection after intervention with ribavirin or alteration in immunosuppressive therapy. Ten recipients developed prolonged or persistent infection. Transaminitis was common, but short-term morbidity was rare; only one recipient developed apparent but clinically mild post-transfusion hepatitis. INTERPRETATION: Our findings suggest that HEV genotype 3 infections are widespread in the English population and in blood donors. Transfusion-transmitted infections rarely caused acute morbidity, but in some immunosuppressed patients became persistent. Although at present blood donations are not screened, an agreed policy is needed for the identification of patients with persistent HEV infection, irrespective of origin, so that they can be offered antiviral therapy. FUNDING: Public Health England and National Health Service Blood and Transplant.

Evaluation of an ethidium monoazide-enhanced 16S rDNA real-time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture.

BACKGROUND: Culture-based systems are currently the preferred means for bacterial screening of platelet (PLT) concentrates. Alternative bacterial detection techniques based on nucleic acid amplification have also been developed but these have yet to be fully evaluated. In this study we evaluate a novel 16S rDNA polymerase chain reaction (PCR) assay and compare its performance with automated culture. STUDY DESIGN AND METHODS: A total of 2050 time-expired, 176 fresh, and 400 initial-reactive PLT packs were tested by real-time PCR using broadly reactive 16S primers and a "universal" probe (TaqMan, Invitrogen). PLTs were also tested using a microbial detection system (BacT/ALERT, bioMérieux) under aerobic and anaerobic conditions. RESULTS: Seven of 2050 (0.34%) time-expired PLTs were found repeat reactive by PCR on the initial nucleic acid extract but none of these was confirmed positive on testing frozen second aliquots. BacT/ALERT testing also failed to confirm any time-expired PLTs positive on repeat testing, although 0.24% were reactive on the first test. Three of the 400 "initial-reactive" PLT packs were found by both PCR and BacT/ALERT to be contaminated (Escherichia coli, Listeria monocytogenes, and Streptococcus vestibularis identified) and 14 additional packs were confirmed positive by BacT/ALERT only. In 13 of these cases the contaminating organisms were identified as anaerobic skin or oral commensals and the remaining pack was contaminated with Streptococcus pneumoniae. CONCLUSION: These results demonstrate that the 16S PCR assay is less sensitive than BacT/ALERT and inappropriate for early testing of concentrates. However, rapid PCR assays such as this may be suitable for a strategy of late or prerelease testing.

Acute hepatitis B in blood donors over a 5-year period in England and North Wales: who is getting infected?

BACKGROUND: Hepatitis B virus (HBV) remains the infection most frequently recognized by donation testing in blood donors. It is usually a persistent infection and mostly reflects the country of origin of the donor or the donor's family. There are, however, a minority of acute infections and this study undertook their phylogenetic analysis to determine the likely source of infection. STUDY DESIGN AND METHODS: Plasma samples from 11 donors donating between July 2005 and June 2010, whose test results revealed recent infection with hepatitis B, were available for further analysis. Plasma DNA was extracted, amplified, sequenced, and analyzed phylogenetically. Donor and virus characteristics were compared with the overall demography of all hepatitis B-infected donors attending over the same period. RESULTS: Three of the 11 individuals were first-time donors. Nine were male, of whom eight were white British. All had serum markers of very recent infection. Only two indicated known HBV exclusion risk factors at postdonation discussion not declared previously. Genotype A was present in seven, Genotype B in two, and Genotype C in two, contrasting with the pattern in persistently infected persons in the United Kingdom. A single A2 strain was identified in six of the white British male donors, suggesting epidemiologic linkage. CONCLUSION: Phylogenetic analysis of HBV-infected donors performed in real time can potentially lead to identification of undeclared risk factors that donor health questionnaires may fail to identify.

A re-assessment of the epidemiology and patient characteristics of hepatitis D virus infection in inner city London.

OBJECTIVES: To re-assess the prevalence and patient characteristics of hepatitis D virus (HDV) infection among hepatitis B patients in inner city London. METHODS: All hepatitis B patients attending clinics over a 52 months period were tested for HDV antibody. All reactive samples were also tested for anti-HDV IgM and RNA. The characteristics of HDV seronegative patients first seen in the calendar year 2008 were compared with all HDV seropositive patients in the cohort. RESULTS: Of 1048 hepatitis B patients, 11 had equivocal anti-HDV serology (1%) and 22 were HDV seropositive (2.1%, 95%CI 1.39-3.16%); 12 were anti-HDV IgM positive and 15 HDV RNA positive. No patient with equivocal anti-HDV serology had detectable HDV RNA. Five HDV seropositive patients were intravenous drug users (22.7%); 17/22 were from abroad with 11/22 (50%) from sub-Saharan Africa. HDV seropositive patients had poorer laboratory parameters and were more likely to have evidence of cirrhosis. Triple infected (HIV/HBV/HDV) patients were also more likely to have cirrhosis than HIV/HBV dually infected patients. CONCLUSIONS: The prevalence of HDV in hepatitis B patients in inner city London was about 2%. The role of migration from endemic countries should be recognised.

The diversity of chronic hepatitis B virus infections within blood donors in England and North Wales 2005 through 2010.

BACKGROUND: In 2010 hepatitis B virus (HBV) was the most frequently detected infection in UK blood donation screening, typically found in first-time, male, chronically infected donors born abroad. To date there has been no comprehensive characterization of the virologic profile of these infections. STUDY DESIGN AND METHODS: Epidemiologic and serologic data were collected retrospectively for 344 chronically HBV-infected blood donors identified from July 2005 to June 2010. Additional laboratory testing was carried out to determine the HBV genotype, viral load, and prevalence of clinically significant mutations and to detect hepatitis delta virus (HDV) coinfection. RESULTS: Five HBV genotypes (A-E) were found, Genotypes D (45%), A (20%), and E (20%) were the most prevalent. A strong association was seen between genotype and donor ethnicity (p < 0.001) and between genotype and place of residence (p = 0.006). Clinically significant mutations were observed across hepatitis B surface antigen (17%), basal core promoter (25%) and precore (78%) regions. An antiviral resistance profile was identified in one donor. Evidence of HDV coinfection was found in 2% of donors. CONCLUSION: The data show the diversity of HBV in asymptomatic chronic infections detected in blood donors in England and North Wales and demonstrates the presence of mutations which may impact on disease. The global nature of these infections and the inability to identify chronically infected donors before donation highlights the importance of using screening assays capable of detecting a broad range of genotypes and mutations. Furthermore, the integration of the virologic and demographic data allows us to more accurately construct a profile of our chronically HBV-infected blood donors.

Human papillomavirus antibody reference reagents for use in postvaccination surveillance serology.

Suitably controlled serosurveillance surveys are essential for evaluating human papillomavirus (HPV) immunization programs. A panel of plasma samples from 18-year-old females was assembled, the majority of the samples being from recipients of the bivalent HPV vaccine. Antibody specificities were evaluated by three independent laboratories, and 3 pools that displayed no antibodies to any HPV type tested or intermediate or high levels of antibody to HPV16, HPV18, HPV31, and HPV45 were created. These pools will be useful as control reagents for HPV serology.

Development of an ethidium monoazide-enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates.

BACKGROUND: Bacterial contamination of platelet (PLT) concentrates remains a problem for blood transfusion services. Culture-based bacterial screening techniques are available but offer inadequate speed and sensitivity. Alternative techniques based on polymerase chain reaction (PCR) amplification have been described but their performance is often compromised by traces of bacterial DNA in reagents. STUDY DESIGN AND METHODS: Universal 16S rDNA primers were used to develop a real-time PCR assay (TaqMan, Applied Biosystems) and various reagent decontamination strategies were explored. Detection sensitivity was assessed by spiking PLT concentrates with known concentrations of 13 different organisms. RESULTS: Restriction enzyme digestion, master mix ultrafiltration, and use of alternative Taq polymerases all reduced the level of reagent DNA contamination to some extent but all proved unreliable. In contrast, ethidium monoazide (EMA) treatment of the PCR master mix followed by photoactivation was reliable and effective, permitting a full 40 amplification cycles, and totally eliminated contamination without compromising assay sensitivity. All 13 organisms were efficiently detected and the limit of detection for Escherichia coli-spiked PLTs was approximately 1 colony-forming unit/mL. Coamplification of human mitochondrial DNA served to confirm efficient nucleic acid extraction and the absence of PCR inhibition in each sample. One of five automated extraction platforms evaluated was found to be contamination free and capable of high-throughput processing. CONCLUSION: Cross-linking of EMA to DNA via photoactivation solved the previously intractable problem of reagent contamination and permitted the development of a high-sensitivity universal bacterial detection system. Trials are ongoing to assess the suitability of the system for high-throughput screening of PLT concentrates.

PCR master mixes harbour murine DNA sequences. Caveat emptor!

BACKGROUND: XMRV is the most recently described retrovirus to be found in Man, firstly in patients with prostate cancer (PC) and secondly in 67% of patients with chronic fatigue syndrome (CFS) and 3.7% of controls. Both disease associations remain contentious. Indeed, a recent publication has concluded that "XMRV is unlikely to be a human pathogen". Subsequently related but different polytropic MLV (pMLV) sequences were also reported from the blood of 86.5% of patients with CFS. and 6.8% of controls. Consequently we decided to investigate blood donors for evidence of XMRV/pMLV. METHODOLOGY/PRINCIPAL FINDINGS: Testing of cDNA prepared from the whole blood of 80 random blood donors, generated gag PCR signals from two samples (7C and 9C). These had previously tested negative for XMRV by two other PCR based techniques. To test whether the PCR mix was the source of these sequences 88 replicates of water were amplified using Invitrogen Platinum Taq (IPT) and Applied Biosystems Taq Gold LD (ABTG). Four gag sequences (2D, 3F, 7H, 12C) were generated with the IPT, a further sequence (12D) by ABTG re-amplification of an IPT first round product. Sequence comparisons revealed remarkable similarities between these sequences, endogeous MLVs and the pMLV sequences reported in patients with CFS. CONCLUSIONS/SIGNIFICANCE: Methodologies for the detection of viruses highly homologous to endogenous murine viruses require special caution as the very reagents used in the detection process can be a source of contamination and at a level where it is not immediately apparent. It is suggested that such contamination is likely to explain the apparent presence of pMLV in CFS.

No Evidence of XMRV or MuLV Sequences in Prostate Cancer, Diffuse Large B-Cell Lymphoma, or the UK Blood Donor Population.

Xenotropic murine leukaemia virus-related virus (XMRV) is a recently described retrovirus which has been claimed to infect humans and cause associated pathology. Initially identified in the US in patients with prostate cancer and subsequently in patients with chronic fatigue syndrome, doubt now exists that XMRV is a human pathogen. We studied the prevalence of genetic sequences of XMRV and related MuLV sequences in human prostate cancer, from B cell lymphoma patients and from UK blood donors. Nucleic acid was extracted from fresh prostate tissue biopsies, formalin-fixed paraffin-embedded (FFPE) prostate tissue and FFPE B-cell lymphoma. The presence of XMRV-specific LTR or MuLV generic gag-like sequences was investigated by nested PCR. To control for mouse DNA contamination, a PCR that detected intracisternal A-type particle (IAP) sequences was included. In addition, DNA and RNA were extracted from whole blood taken from UK blood donors and screened for XMRV sequences by real-time PCR. XMRV or MuLV-like sequences were not amplified from tissue samples. Occasionally MuLV gag and XMRV-LTR sequences were amplified from Indian prostate cancer samples, but were always detected in conjunction with contaminating murine genomic DNA. We found no evidence of XMRV or MuLV infection in the UK blood donors.