The incubation period of hepatitis E genotype 1: insights from pooled analyses of travellers.

Hepatitis E virus genotype 1 (HEV G1) is an important cause of morbidity and mortality in Africa and Asia. HEV G1's natural history, including the incubation period, remains poorly understood, hindering surveillance efforts and effective control. Using individual-level data from 85 travel-related HEV G1 cases in England and Wales, we estimate the incubation period distribution using survival analysis methods, which allow for appropriate inference when only time ranges, rather than exact times are known for the exposure to HEV and symptom onset. We estimated a 29.8-day (95% confidence interval (CI) 24.1-36.0) median incubation period with 5% of people expected to develop symptoms within 14.3 days (95% CI 10.1-21.7) and 95% within 61.9 days (95% CI 47.4-74.4) of exposure. These estimates can help refine clinical case definitions and inform the design of disease burden and intervention studies.

Confirmation of specificity of reactivity in a solid phase ELISA for the detection of hepatitis E viral antigen improves utility of the assay.

Genotype 3 hepatitis E virus (HEV) can lead to persistent infections in immunocompromised hosts. A recently available commercial assay for the detection of HEV antigen (HEV-Ag ELISA, Wantai diagnostics) may enable the study of HEV-Ag dynamics in such persistent infections, however currently there is no confirmatory test available. We generated a putative neutralising reagent from a pool of four convalescent blood donor samples and explored neutralising activity against HEV antigens from clinical samples, HEV tissue-culture and virus-like particles. Using this neutralisation method we were able to differentiate true reactivity from non-specific reactivity in plasma, stool and urine samples. This could also facilitate the introduction of HEV-Ag detection as a screening assay or the study of HEV-Ag in different body fluids.

Pork products associated with human infection caused by an emerging phylotype of hepatitis E virus in England and Wales.

Since 2010, human hepatitis E infections have increased in England and Wales. Most cases are locally acquired and caused by hepatitis E virus genotype 3 (HEV G3). HEV G3 is linked to the consumption of pork products. The increase is associated with the emergence of a new phylotype, HEV G3-group 2 (G3-2, also known as G3abcdhij). Sixty individuals with confirmed hepatitis E infection and no history of travel outside the UK were recruited: 19 were infected with HEV G3-group 1 (G3-1 or G3efg) and 41 with G3-2. Epidemiological data relating to usual shopping habits and consumption of ham and sausages were analysed together with typing data to identify any associations with HEV phylotype. Study participants who purchased ham and/or sausage from a major supermarket were more likely to have HEV G3-2 infection (Relative risks 1·85, P = 0·06, CI 0·97-3·53). The HEV G3-2 phylotype has not been detected in indigenous UK pigs and it is suggested that human infections could be the result of consumption of products made from pork originating outside the UK. This does not infer blame on the supermarket but the epidemiology of HEV is dynamic and reflects complex animal husbandry practices which need to be explored further.

Hepatitis E: the current state of play.

The hepatitis E virus (HEV) is a major cause of acute hepatitis globally. Genotypes 1 and 2 (G1 and G2) are obligate human pathogens transmitted faeco-orally, leading to epidemics in developing countries. In contrast, genotypes 3 and 4 (G3 and G4) have a wider host range, including humans, but are primarily porcine viruses and are transmitted from animals to humans as a food-borne zoonosis when meat from an infected animal is consumed. HEV is increasingly recognised as a problem in developed countries, including countries in Europe. G3 HEV is now the most common cause of acute viral hepatitis in the UK and cases continue to rise. The majority of these infections are acquired within the UK and thought to be from insufficiently cooked meat, predominantly processed pork meat. Previously thought to only cause self-limiting disease, HEV infection can persist in immunosuppressed patients, which may lead to chronic hepatitis and the rapid development of cirrhosis. Of particular interest to the transfusion community has been the possibility of transfusion-transmitted HEV, which has been reported from countries classically considered HEV-endemic but also non-endemic countries in Europe and Japan. This has prompted some countries to introduce screening for HEV in blood donations.

No evidence of transfusion transmission of Adenovirus and Epstein-Barr virus infections in paediatric recipients post-bone marrow transplant.

Adenovirus and Epstein-Barr virus can cause significant morbidity and mortality in paediatric patients post-bone marrow transplant. The source of infection is thought to be either reactivation of latent viruses or primary infection. We have investigated whether transfusion of blood components from viraemic donors could provide a route of primary infection in these patients and sought the prevalence of viraemia in the blood donor population from England. In 32 linked donor/recipient samples and 300 unselected blood donors, we found no evidence to suggest that these infections in paediatric bone marrow transplant recipients had been acquired from transfused blood components.

The epidemiology of hepatitis B virus infection in HIV-infected and HIV-uninfected pregnant women in the Western Cape, South Africa.

OBJECTIVES: Persistent hepatitis B virus (HBV) infection is a major cause of morbidity and mortality in sub-Saharan Africa. The HIV epidemic has the potential to affect its biology. Immunisation protocols established in the pre-HIV era are based upon data showing predominantly horizontal infant transmission. This study aimed to determine whether HIV co-infection will change the epidemiology of HBV both by increasing infectivity and by favouring the escape of viruses bearing phenotypically altered HBsAg. METHODS: This retrospective cross-sectional study used antenatal samples from the 2008 Antenatal Sentinel HIV and Syphilis Prevalence Survey in the Western Cape, South Africa. All HIV-infected women were age and race-matched to HIV-uninfected women. Samples were tested for serological markers of HBV and HDV infection. HBV viral load, consensus sequencing and genotyping were performed. Luminex technology was used to determine HBsAg phenotype. All samples from HIV-infected women were tested for traces of antiretroviral drugs by mass spectrometry. RESULTS: This study showed a trend toward loss of immune control of HBV in HIV-infected women with 3.4% of samples containing HBsAg, 18.9% contained HBeAg. In contrast, 2.9% of samples from HIV-uninfected women contained HBsAg and 17.1% of these HBeAg. The median HBV load in the HIV-infected group was 9.72×10(7)IU/ml and in the HIV-uninfected group 1.19×10(6)IU/ml. Genotyping showed 63/68 samples belonged to genotype A and the remainder genotype D. Mutations in the precore region were found in 35% and 33% of samples from HIV-infected and HIV-uninfected respectively. Although no major epitope ablation was found, marked variation in HBsAg profiles in HIV-infected group was demonstrated. No HDV infection was detected. CONCLUSION: HIV-HBV co-infected women exhibit a degree of immune escape. One in six HBV-infected pregnant women, irrespective of HIV status is HBeAg seropositive. HBV immunization of newborns in sub-Saharan Africa should be implemented.

The rise and fall of XMRV.

Due to the relatively recent emergence of the human T-lymphotropic and the human immunodeficiency viruses, enthusiasm for the identification of novel viruses, especially retroviruses, with pathogenic potential in humans, remains high. Novel technologies are now available with the ability to search for unknown viruses, such as gene arrays and new generation sequencing of tissue and other samples. In 2006, chip technology identified a novel retrovirus in human prostate cancer (PCa) tissue samples. Due to close homology to a mouse retrovirus, the virus was named xenotropic murine leukaemia virus-related virus (XMRV). Ever since the initial disease association with PCa, XMRV has stirred a lot of attention and concern worldwide for the medical community, public health officials and in particular global transfusion services. Public response, in this new era of electronic communication and advocacy was rapid, wide and unprecedented. In this review, we outline the course of biomedical research efforts that were put forward internationally in the process of determining the risk to the human population, the response of the blood banking community and review the current state of knowledge of xenotropic murine retroviruses. Although XMRV is no longer regarded as an infection of humans, a lesson was learnt in modern virology that holds deeper implications for biomedical research, particularly stem cell generation and transplantation practices.

Therapy-induced clearance of HCV core antigen from plasma predicts an end of treatment viral response.

During viral assembly, viral proteins are released into plasma and can be used to infer viral load. The Architect hepatitis C virus (HCV) core antigen (Ag) assay is a potential alternative to HCV RNA quantification for measuring response to therapy and predicting an end of treatment viral response (EOTR). The HCVp22Ag assay was used to infer viral load in 68 window RNA-containing samples and in 284 samples from baseline to week 14 of ribavirin/interferon treatment in 23 patients with EOTR including three who relapsed, 20 not achieving EOTR and 11 controls. HCV Ag and RNA correlated well (r = 0.86) with linear dose responses on dilution. In patients on therapy and control patients, plasma HCV antigen was detected in 51 of 54 with an interpolated LOD cut off between 10(3) and 10(4) RNA IU/mL. Plasma HCV antigenaemia and plasma RNA levels were significantly different in EOTR from non-EOTR patients at 3 days after treatment start and all times thereafter. Positive and negative EOTR predictive values for HCV RNA >2 log drop and HCV Ag loss at 12 weeks were 70% and 74%, 85% and 93% respectively. HCV Ag reactivity has a linear dose response independent of genotype and correlates well with HCV RNA. The failure to clear HCV Ag is as accurate as the failure to clear HCV RNA at twelve weeks into therapy in predicting the likelihood of failure to achieve EOTR. HCV Ag potentially offers a convenient alternative to RNA measurement for defining a futility flag in HCV therapy.

Strategies for screening blood donors to source convalescent H1N1v plasma for intervention therapy.

BACKGROUND AND OBJECTIVES: During the 1918, pandemic blood components were successfully used to treat severe influenza pneumonia. A Proof of Principle trial investigating the clinical benefit of convalescent plasma was proposed in the 2009 H1N1v epidemic with the aim of screening donors for high titre antibody in order to stockpile plasma packs to be used for treatment for severe pneumonia. MATERIALS AND METHODS: Serum samples were collected from donors. IgG antibody capture format enzyme-linked immunoassays using recombinant proteins (GACELISAs) were compared with microneutralization (MN) and haemagglutination inhibition (HAI). The influence of age and history of influenza-like illness (ILI) on the detection of high titre antibody was examined. RESULTS: 1598 unselected donor sera collected in October and December 2009 were tested by HAI. The HAI and demographic data defined a possible strategy for selective donor screening. One of the GACELISAs was highly specific for recent infection but showed lower sensitivity than HAI. CONCLUSIONS: During the 2009 pandemic screening 17- to 30-year-old donors by HAI delivered around 10% with high antibody levels. The ELISA using a short recombinant H1N1v HA detected fewer reactives but was more specific for high titre antibody (≥1:256). Screening strategies are proposed based on using HAI on serum or GACELISA on plasma.

Outbreak of hepatitis A in an extended family after importation by non-immune travellers.

The incidence of hepatitis A in England has declined in recent years, but travel-related cases and imported infections remain a challenge. We report an outbreak of hepatitis A in an extended family where two primary cases were infected while in Pakistan and two secondary cases were infected in England. All four were infected by the same genotype IIIA virus. Testing of the children in the extended family by dried blood spots (DBS) determined that three had evidence of recent past infections (anti-HAV IgM positive), one had a current asymptomatic infection (anti-HAV IgM and HAV RNA positive) and one was incubating the virus (anti-HAV IgM negative, HAV RNA positive). HAV RNA from the DBS was identical to the adult cases. This outbreak demonstrates secondary spread of hepatitis A by asymptomatic children after importation from abroad and highlights the importance of preventing travel-associated hepatitis A infection.

Can plasma HHV8 viral load be used to differentiate multicentric Castleman disease from Kaposi sarcoma?

We measured plasma human herpesvirus 8 (HHV8) DNA load in consecutive patients presenting with HIV-associated multicentric Castleman disease (MCD) and in contemporaneous patients who had Kaposi sarcoma (KS), lymphoma or other diagnoses. All 11 patients with MCD had detectable plasma HHV8 DNA compared with 18 (72%) of 25 patients with KS, none with lymphoma and one of 38 patients with other diagnoses. Detectable plasma HHV8 DNA levels were higher among MCD patients, median (interquartile range [IQR]) = 43,500 (5200-150,000) copies/mL, when compared with those with KS, median (IQR) = 320 (167-822) copies/mL and those with lymphoma and other diagnoses (one-way analysis of variance; P = 0.0303). Using receiver operating characteristic analysis, a cut-off of >1000 copies HHV8 DNA/mL of plasma helped to discriminate between MCD and other diagnoses, with a specificity of 94.7% and a negative predictive value of 97.3%. The level of HHV8 viraemia, while not diagnostic, may aid discrimination between patients with MCD and those with KS and other systemic illnesses.

Is there evidence of recent hepatitis E virus infection in English and North Welsh blood donors?

BACKGROUND AND OBJECTIVE: The risk of hepatitis E virus (HEV) to blood safety remains unknown in England. Reports of persistent HEV infection with serious disease sequelae indicate that transfusion transmitted HEV is not a trivial disease in immunosuppressed patients. MATERIALS AND METHODS: Samples from unselected blood donors and donors with a history of jaundice were tested for HEV antibody and RNA. RESULTS: Overall, 10% of the donor sera were anti-HEV IgG reactive. Four of the donor samples were anti-HEV IgM reactive but HEV RNA negative. CONCLUSION: There is evidence of probable recent HEV infections in donors with a predicted attack rate of 2.8%.

An outbreak of hepatitis A affecting a nursery school and a primary school.

Between March and June 2008, 12 cases of hepatitis A were notified in Winchester. Cases were from a primary school and a nursery school with no direct linkage. Hepatitis A virus (HAV) RNA sequenced from nine cases confirmed the strain in both schools to be identical. The outbreak could have affected three other schools and a maternity unit and was controlled by immunization and screening of neonates in the maternity unit by dried blood spots. No neonates were infected and no further cases were reported until 5 months later when the index case's mother became infected with same strain of virus associated with the outbreak despite vaccination. Neither the source of the outbreak or the subsequent infection of the index case's mother was identified; however, with the timing of the cases continued transmission in the community by children with asymptomatic infection or a recurrent source cannot be ruled out.

Cytomegalovirus viral load testing of blood using quantitative polymerase chain reaction in acutely unwell HIV-1-positive patients lacks diagnostic utility.

We examined the usefulness of measuring cytomegalovirus (CMV) viral load (VL) in blood using quantitative polymerase chain reaction (qPCR) in establishing a diagnosis of CMV end-organ disease in consecutive unwell HIV-infected patients. The indication for testing for CMV, CD4 count, CMV VL and presence of CMV end-organ disease were abstracted from case-notes. During a 42-month period, 216 tests were performed in 181 patients; the majority (61%) had CD4 counts <100 cells/microL. The prevalence of detectable CMV by qPCR was 43.5% (94/216) with a prevalence of CMV end-organ disease of 7.4% (16/216). Of patients with CMV detectable by qPCR, 72 % (50/69) had CD4 counts <100 cells/microL. For patients with definite CMV end-organ disease, the positive predictive value of detectable CMV by qPCR was 10% (9/94), and the negative predictive value was 98% (119/122). In acutely unwell HIV-infected patients, detection of CMV by qPCR is a poor predictor of CMV end-organ disease.

Characterization of hepatitis B surface antigen strains circulating in the Kingdom of Cambodia.

A collection of hepatitis B surface antigen (HBsAg)-reactive serum specimens from the Pasteur Institute of Cambodia was investigated for the genotype, predicted serotype and the presence of diagnostically significant mutations in the surface protein. From a set of 794 samples, 15 were identified serologically to harbour possible HBsAg mutants and were investigated further. An additional 20 samples were included into the study for PCR and sequence analysis. Of the 22 samples which were HBV-DNA-positive, 16 were of genotype C with the remaining six being genotype B. Point mutations resulting in amino acid substitutions were noted in 10 samples. The majority of these mutations occurred outside the a determinant.

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control.

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.

Characterization of HIV type 1 clades in the Caribbean using pol gene sequences.

To date, 11 HIV-1 M group clades, A to K, have been characterized, displaying different distributions, prevalences, and biological properties. Approximately 90% of new HIV-1 infections occur in developing countries, including the Caribbean. However, information on HIV-1 subtypes from this region is limited. We report subtype characterization of viruses from 71 individuals, obtained during the period 2000-2002. RNA from the pol region was sequenced, generating data on subtype and drug resistance associated mutations for 71 specimens from 9 countries. Sixty-seven (94.4%) sequences were classified as clade B, three (4.2%) as D/B, and one (1.4%) as clade C. Numerous polymorphisms were observed, including some associated with drug resistance, but not signifying exposure to chemotherapy. This study adds to our knowledge of HIV-1 clades in the Caribbean, and indicates possibilities for monitoring HIV-1 chemotherapy.

HBV DNA levels and transmission of hepatitis B by health care workers.

BACKGROUND: Laboratory-based study funded by the Research and Development Division of the Department of Health to inform the decision making on guidelines for the conduct of exposure prone procedures (EPPs) by health care workers who are hepatitis B carriers. OBJECTIVES: Define the quantity and nature of hepatitis B virus (HBV) DNA in hepatitis carriers whose serum does not contain hepatitis B e antigen (HBeAg) and in surgeons previously cleared to conduct EPPs who have transmitted HBV to their patients. STUDY DESIGN: Cross-sectional survey using HBV DNA quantification, genotyping and sequencing comparing transmitting surgeons and asymptomatic carriers. RESULTS: HBV DNA could be detected and quantified in 64.5% (136 of 211) of carriers whose serum did not contain HBeAg with a median level 3.6 log(10) copies/ml (range of 5.7 log(10) copies). Pre-core mutation appeared not to affect the HBV DNA level, however, all surgeons carried codon 28 variants and transmitted these variants to their patients. The lowest HBV DNA level in a transmitting surgeon was 4 x 10(4) copies/ml. CONCLUSIONS: Pre-core mutations are common in carriers whose serum does not contain HBeAg and do not specifically identify carriers whose HBV DNA levels are high. It was possible to define a level of virus above which transmission of hepatitis B during conduct of EPPs could not be excluded.