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 (similar to 9 log(10)). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R-2 = 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. (c) 2005 Elsevier B.V. All rights reserved

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

Oral fluid testing facilitates understanding of hepatitis A virus household transmission

The public health response to sporadic hepatitis A virus (HAV) infection, hepatitis A, can be complex especially when the index case is a child and no obvious source is identified. Identifying an infection source may avoid mass immunisation within schools when transmission is found to have occurred within the household. Screening of asymptomatic contacts via venepuncture can be challenging and unacceptable, as a result non-invasive methods may facilitate public health intervention. Enzyme-linked immunoassays were developed to detect HAV immunoglobulin M (IgM) and immunoglobulin G (IgG) in oral fluid (ORF). A validation panel of ORF samples from 30 confirmed acute HAV infections were all reactive for HAV IgM and IgG when tested. A panel of 40 ORF samples from persons known to have been uninfected were all unreactive. Two hundred and eighty household contacts of 72 index cases were screened by ORF to identify HAV transmission within the family and factors associated with household transmission. Almost half of households (35/72) revealed evidence of recent infection, which was significantly associated with the presence of children ⩽11 years of age (odds ratio 9.84, 95% confidence interval: 2.74–35.37). These HAV IgM and IgG immunoassays are easy to perform, rapid and sensitive and have been integrated into national guidance on the management of hepatitis A cases

Asymptomatic Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in a rehabilitation facility: evolution of the presence of nasopharyngeal SARS-CoV-2 and serological antibody responses.

At the start of the UK coronavirus disease 2019 epidemic, this rare point prevalence study revealed that one-third of patients (15 of 45) in a London inpatient rehabilitation unit were found to be infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but asymptomatic. We report on 8 patients in detail, including their clinical stability, the evolution of their nasopharyngeal viral reverse-transcription polymerase chain reaction (RT-PCR) burden, and their antibody levels over time, revealing the infection dynamics by RT-PCR and serology during the acute phase. Notably, a novel serological test for antibodies against the receptor binding domain of SARS-CoV-2 showed that 100% of our asymptomatic cohort remained seropositive 3-6 weeks after diagnosis

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

Variability and pathogenicity of hepatitis E virus genotype 3 variants

Infection with hepatitis E virus (HEV) can be clinically inapparent or produce symptoms and signs of hepatitis of varying severity and occasional fatality. This variability in clinical outcomes may reflect differences in host susceptibility or the presence of virally encoded determinants of pathogenicity. Analysis of complete genome sequences supports the division of HEV genotype 3 (HEV-3) variants into three major clades: 3ra comprising HEV isolates from rabbits, and 3efg and 3abchij comprising the corresponding named subtypes derived from humans and pigs. Using this framework, we investigated associations between viral genetic variability of HEV-3 in symptomatic and asymptomatic infections by comparing HEV-3 subgenomic sequences previously obtained from blood donors with those from patients presenting with hepatitis in the UK (54 blood donors, 148 hepatitis patients), the Netherlands (38 blood donors, 119 hepatitis patients), France (24 blood donors, 55 hepatitis patients) and Germany (14 blood donors, 36 hepatitis patients). In none of these countries was evidence found for a significant association between virus variants and patient group (P>0.05 Fisher's exact test). Furthermore, within a group of 123 patients in Scotland with clinically apparent HEV infections, we found no evidence for an association between variants of HEV-3 and disease severity or alanine aminotransferase level. The lack of detectable virally encoded determinants of disease outcomes in HEV-3 infection implies a more important role for host factors in its clinical phenotype

CD20 and CD19 targeted vectors induce minimal activation of resting B lymphocytes

B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction

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 identifi ed 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 identifi ed 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 identifi cation 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 fi ndings 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 identifi cation of patients with persistent HEV infection, irrespective of origin, so that they can be off ered antiviral therapy

Illness perceptions and explanatory models of viral hepatitis B & C among immigrants and refugees: a narrative systematic review.

© 2015 Owiti et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.BACKGROUND: Hepatitis B and C (HBV, HCV) infections are associated with high morbidity and mortality. Many countries with traditionally low prevalence (such as UK) are now planning interventions (screening, vaccination, and treatment) of high-risk immigrants from countries with high prevalence. This review aimed to synthesise the evidence on immigrants' knowledge of HBV and HCV that might influence the uptake of clinical interventions. The review was also used to inform the design and successful delivery of a randomised controlled trial of targeted screening and treatment. METHODS: Five databases (PubMed, CINHAL, SOCIOFILE, PsycINFO & Web of Science) were systematically searched, supplemented by reference tracking, searches of selected journals, and of relevant websites. We aimed to identify qualitative and quantitative studies that investigated knowledge of HBV and HCV among immigrants from high endemic areas to low endemic areas. Evidence, extracted according to a conceptual framework of Kleinman's explanatory model, was subjected to narrative synthesis. We adapted the PEN-3 model to categorise and analyse themes, and recommend strategies for interventions to influence help-seeking behaviour. RESULTS: We identified 51 publications including quantitative (n = 39), qualitative (n = 11), and mixed methods (n = 1) designs. Most of the quantitative studies included small samples and had heterogeneous methods and outcomes. The studies mainly concentrated on hepatitis B and ethnic groups of South East Asian immigrants residing in USA, Canada, and Australia. Many immigrants lacked adequate knowledge of aetiology, symptoms, transmission risk factors, prevention strategies, and treatment, of hepatitis HBV and HCV. Ethnicity, gender, better education, higher income, and English proficiency influenced variations in levels and forms of knowledge. CONCLUSION: Immigrants are vulnerable to HBV and HCV, and risk life-threatening complications from these infections because of poor knowledge and help-seeking behaviour. Primary studies in this area are extremely diverse and of variable quality precluding meta-analysis. Further research is needed outside North America and Australia