ABSTRACT
METHODS: We applied multiparametric MRI to assess changes in liver composition, perfusion and blood flow in 17 patients before direct-acting antiviral (DAA) therapy and after treatment completion (within 12 weeks of last DAA tablet swallowed). RESULTS: We observed changes in hepatic composition indicated by a reduction in both liver longitudinal relaxation time (T1, 35 ± 4 ms), transverse relaxation time (T2, 2.5 ± 0.8 ms; T2* 3.0 ± 0.7 ms), and liver perfusion (28.1 ± 19.7 ml/100 g/min) which we suggest are linked to reduced pro-inflammatory milieu, including interstitial oedema, within the liver. No changes were observed in liver or spleen blood flow, splenic perfusion, or superior mesenteric artery blood flow. CONCLUSION: For the first time, our study has shown that treatment of HCV with DAAs in patients with cirrhosis leads to an acute reduction in liver T1, T2 and T2* and an increase in liver perfusion measured using MR parameters. The ability of MRI to characterise changes in the angio-architecture of patients with cirrhosis after intervention in the short term will enhance our understanding of the natural history of regression of liver disease and potentially influence clinical decision algorithms. KEY POINTS: ⢠DAAs have revolutionised the treatment of hepatitis C and achieve sustained virological response in over 95% of patients, even with liver cirrhosis. ⢠Currently available non-invasive measures of liver fibrosis are not accurate after HCV treatment with DAAs, this prospective single-centre study has shown that MRI can sensitively measure changes within the liver, which could reflect the reduction in inflammation with viral clearance. ⢠The ability of MRI to characterise changes in structural and haemodynamic MRI measures in the liver after intervention will enhance our understanding of the progression/regression of liver disease and could potentially influence clinical decision algorithms.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis C, Chronic/diagnostic imaging , Hepatitis C, Chronic/drug therapy , Liver/diagnostic imaging , Adult , Disease Progression , Female , Hepacivirus/isolation & purification , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/virology , Humans , Liver Circulation , Liver Cirrhosis/virology , Magnetic Resonance Imaging , Male , Middle Aged , Prospective Studies , Sustained Virologic ResponseABSTRACT
Hepatitis C virus (HCV)-infected patients are at risk of developing hepatocellular carcinoma (HCC). Individuals at heightened risk could be targeted by intensive follow-up surveillance. We have conducted a systematic review of the literature to identify host genetic predisposition to HCC in HCV-infected patients. A comprehensive search of Medline and Embase databases was performed, and the strength of evidence of associations for each gene on development of HCC was evaluated. We identified 166 relevant studies, relating to 137 different genes, or combinations thereof. Seventeen genes were classified as having "good" evidence of an association, a significant association was observed for 37 genes but this finding had not yet been replicated, 56 genes had mixed or limited evidence of an association, and 27 genes showed no association. IFNL3/4, TNF-α and PNPLA3 genes had the most evidence of an association. There was, however, considerable heterogeneity in study design and data quality. In conclusion, we identified a number of genes with evidence of association with HCC, but also a need for more standardized approaches to address this clinically critical question. It is important to consider the underlying mechanism of these relationships and which are confounded by the presence of other HCC risk factors and response to therapy. We also identified many genes where the evidence of association is contradictory or requires replication, as well as a number where associations have been studied but no evidence found. These findings should help to direct future studies on host genetic predisposition to HCC in HCV-infected patients.
Subject(s)
Carcinoma, Hepatocellular/genetics , Genetic Predisposition to Disease , Hepatitis C, Chronic/complications , Liver Neoplasms/genetics , Genetic Association Studies , HumansABSTRACT
Antiviral therapy has been shown to reduce the risk of disease progression, liver damage and death in patients with chronic hepatitis C virus (HCV) infection. While interferon labels recommend that patients with platelet counts below 50 × 10(3) /µL not receive interferon-based therapy, it is unknown to what extent thrombocytopaenia influences treatment decisions in practice. This study profiles the reasons for withholding antiviral treatment in HCV patients with thrombocytopaenia in five European countries. Medical records of 466 patients who had HCV infection and thrombocytopaenia (platelet count <100 × 10(3) /µL) in 2006 were retrospectively reviewed for clinical characteristics. Collected data included use of antiviral therapy and reasons for withholding therapy. In total 184 of 466 patients (39.5%) did not receive interferon-based therapy during the study period, with treatment withheld most frequently due to multiple clinical characteristics including hepatic cirrhosis (16.3%), thrombocytopaenia (16.3%) and age >60 years (10.9%). The reasons for lack of treatment varied among countries, with thrombocytopaenia as a reason being more common in Italy (10.9%) and Spain (20.0%), and less common in France, Germany and the UK (3.2-7.1%). Overall, thrombocytopaenia was reported as the only reason for withholding treatment in 4.9% of untreated patients. This study demonstrates that thrombocytopaenia is one of many factors, indicative of the poor clinical state of the patient, that contributes to withholding antiviral treatment. In 4.9% of untreated patients, thrombocytopaenia can be considered as a modifiable factor to enable more HCV patients to receive guideline-recommended therapy and thus improved clinical outcomes.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/drug therapy , Thrombocytopenia , Withholding Treatment/statistics & numerical data , Adolescent , Adult , Aged , Aged, 80 and over , Antiviral Agents/adverse effects , Europe , Female , Humans , Longitudinal Studies , Male , Middle Aged , Retrospective Studies , Young AdultABSTRACT
Chronic hepatitis C virus (HCV) infection places a considerable economic burden on health services. Cost-effectiveness analyses of antiviral treatment for patients with chronic HCV infection are dependent on assumptions about cost reductions following sustained virological response (SVR) to therapy. This study quantified the medium-term difference in health resource usage and costs depending on treatment outcome. Retrospective chart review of patients with HCV genotype 1 infection who had received at least 2 months pegylated interferon and ribavirin therapy, with known treatment outcome was conducted. Disease status was categorized as chronic hepatitis, cirrhosis or decompensated liver disease. Health resource use was documented for each patient in each disease state. Unit costs were from the NHS 'Payment by Results' database and the British National Formulary. One hundred and ninety three patients (108 SVR, 85 non-SVR) with mean follow-up of 3.5 (SVR) and 4.9 (non-SVR) years were enrolled. No SVR patient progressed to a more severe liver disease state. Annual transition rates for non-SVR patients were 7.4% (chronic hepatitis to cirrhosis) and 4.9% (cirrhosis to decompensated liver disease). By extrapolation of modelled data over a 5-year post-treatment period, failure of patients with chronic hepatitis to achieve SVR was associated with a 13-fold increase (roughly £2300) in costs, whilst for patients who were retreated, the increase was 56-fold, equating to more than £10 000. Achievement of an SVR has significant effects on health service usage and costs. This work provides real-life data for future cost-effectiveness analyses related to the treatment for chronic HCV infection.
Subject(s)
Antiviral Agents/economics , Genotype , Hepacivirus/genetics , Hepatitis C, Chronic/economics , Adult , Antiviral Agents/therapeutic use , Cost-Benefit Analysis , Female , Health Care Costs , Health Resources/economics , Hepatitis C, Chronic/drug therapy , Humans , Male , Middle Aged , Retreatment/economics , Retrospective Studies , Risk Factors , Treatment FailureABSTRACT
IL28B polymorphisms strongly predict spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infection. A recent study proposed a 32-base pair deletion in the CC-chemokine receptor 5 (CCR5) gene (CCR5-Δ32) interacting with the IL28B polymorphisms to influence spontaneous HCV clearance. The aim of this study was to clarify the role of CCR5-Δ32 in treatment-induced clearance of chronic hepatitis C (CHC). A cross-sectional cohort of 813 Caucasian patients with CHC genotype 1 (365 responders and 448 non-responders) who had received standard of care dual therapy with interferon (IFN)-α and ribavirin (RBV) was genotyped for the CCR5-Δ32 and IL28B polymorphisms to examine their interaction with respect to treatment response. CCR5-Δ32 did not influence treatment-induced recovery to IFN-α/RBV in CHC, and did not improve prediction of sustained virological response in the context of the IL28B polymorphisms in a multivariate model. CCR5-Δ32 homozygotes were significantly more frequent in those with CHC than healthy controls in the European cohorts (2.9% vs 0.4%, P<0.0001), but not in Australians of European ancestry. In conclusion, CCR5-Δ32 does not influence treatment response in the context of IL28B polymorphisms. Although CCR5-Δ32 may affect viral clearance within closely controlled geographical and genetic environments, we found no effect in larger cohorts treated with dual therapy.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis C, Chronic/drug therapy , Interleukins/genetics , Polymorphism, Single Nucleotide , Receptors, CCR5/genetics , Sequence Deletion , Adult , Australia , Base Sequence , Cohort Studies , Cross-Sectional Studies , Drug Therapy, Combination , Epistasis, Genetic , Europe , Female , Genotype , Hepatitis C, Chronic/ethnology , Hepatitis C, Chronic/genetics , Humans , Interferon-alpha/therapeutic use , Interferons , Male , Middle Aged , Multivariate Analysis , Prognosis , Ribavirin/therapeutic use , Treatment Outcome , White People/geneticsABSTRACT
We studied a cross-sectional sample of the population of Kech, a small rural town in Pakistan to determine the prevalence and risk factors for hepatitis C infection. The prevalence of hepatitis C was 110 out of 2000 persons (5·5%, 95% confidence interval 4·5-6·5). Higher rates were identified in males. Independent risk factors identified were age ≥75 years, being a healthcare worker, and injecting drug use. There was a high prevalence of many potential routes of transmission of bloodborne viruses and most people reported at least one potential risk factor.
Subject(s)
Hepacivirus , Hepatitis C/epidemiology , Adolescent , Adult , Age Factors , Aged , Cross-Sectional Studies , Female , Health Personnel/statistics & numerical data , Hepatitis C/etiology , Humans , Male , Middle Aged , Multivariate Analysis , Odds Ratio , Prevalence , Risk Factors , Sex Factors , Substance Abuse, Intravenous/epidemiology , Tattooing/adverse effects , Young AdultABSTRACT
OBJECTIVES: We aimed to prospectively describe the incidence and clinical spectrum of SARS-CoV-2 infection in immunocompromised paediatric patients in the UK. METHODS: From March 2020 to 2021 weekly questionnaires were sent to immunocompromised paediatric patients or their parents. Information, including symptom presentation and SARS-CoV-2 PCR test results, was collected from 1527 participants from 46 hospitals. Cross-sectional serology was investigated in February and March 2021. RESULTS: Until the end of September 2020, no cases were reported. From September 28th 2020 to March 2021 a total of 38 PCR-detected SARS-CoV-2 infections were reported. Of these, four children were admitted to hospital but none had acute severe COVID-19. Increasing age in association with immunodeficiency increased reporting of SARS-CoV-2 infection. Worsening of fever, cough, and sore throat were associated with participants reporting SARS-CoV-2 infection. Serology data included 452 unvaccinated participants. In those reporting prior positive SARS-CoV-2 PCR, there were detectable antibodies in 9 of 18 (50%). In those with no prior report of infection, antibodies were detected in 32 of 434 (7â¢4%). CONCLUSIONS: This study shows SARS-CoV-2 infections have occurred in immunocompromised children and young people with no increased risk of severe disease. No children died.
Subject(s)
COVID-19 , Adolescent , Child , Cross-Sectional Studies , Hospitalization , Humans , Immunocompromised Host , SARS-CoV-2Subject(s)
Antiviral Agents/administration & dosage , Antiviral Agents/adverse effects , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/drug therapy , Thrombocytopenia/chemically induced , Withholding Treatment , Drug Therapy, Combination/adverse effects , Drug Therapy, Combination/methods , Humans , Interferon-alpha/adverse effects , Interferon-alpha/therapeutic use , Ribavirin/adverse effects , Ribavirin/therapeutic useABSTRACT
Autoantibodies are commonly detected in chronic hepatitis C (HCV) but their significance remains uncertain. We assessed the prevalence of anti-nuclear (ANA) and anti-smooth muscle (ASM) antibodies within a cohort of 963 treatment-naïve HCV patients. We also assessed for differences between autoantibody-positive and autoantibody-negative patients in demographics, markers of disease activity and response to anti-viral treatment. One hundred and seventy-two patients (17.9%) had at least one autoantibody, of which were 104 (10.8%) ASM, 54 (5.6%) ANA and 14 (1.5%) positive for both. Autoantibody-positive patients were older (43 vs 39 years, P = 0.001) caused by an age-related increase in ANA (but not ASM). There were no differences in gender, alcohol intake, ethnicity or viral genotype. The presence of autoantibodies, and specifically ASM, was associated with an increase in interface hepatitis score amongst men (1.1 vs 0.8, P = 0.005) but no difference in other necroinflammatory measures, liver function tests or immunoglobulins (Ig). There was no difference in initial fibrosis stage or rate of fibrosis progression. Autoantibodies did not affect response to anti-viral treatment. We conclude that autoantibodies are frequent in HCV infection. Anti-nuclear antibodies increase with age, whereas ASM antibodies are associated with interface hepatitis in men. Neither autoantibody carries increased risk of fibrosis progression or failure of therapy.
Subject(s)
Autoantibodies/blood , Autoantibodies/immunology , Hepatitis C, Chronic/immunology , Adolescent , Adult , Age Factors , Aged , Alcohol Drinking , Antibodies, Antinuclear/blood , Antibodies, Antinuclear/immunology , Child , Cohort Studies , Ethnicity , Female , Hepatitis C, Chronic/pathology , Hepatitis C, Chronic/physiopathology , Humans , Male , Middle Aged , Muscle, Smooth/immunology , Risk Factors , Sex Factors , Young AdultABSTRACT
Hepatitis C virus (HCV) causes acute and chronic liver diseases in humans. Its two envelope glycoproteins, E1 and E2, provide a target for host immune recognition. HCV genotypes are classified into six genetic groups. To study the role of anti-HCV E1 and E2 (anti-E1E2) in HCV disease, the correlation between antibody level and viral load, genotype, disease severity and response to treatment was investigated. The levels of antibodies to HCV glycoproteins E1 and E2 antibodies were evaluated in 230 sera of patients with chronic hepatitis C by enzyme-linked immunosorbent assay. The antigens used were recombinant HCV glycoproteins derived from genotype 1 (H77c) and genotype 3 (UKN3A1.28). Seroreactivity was greater when sera were tested against antigen derived from their homologous genotype than against heterologous antigen. Reactivity against UKN3A1.28 in sera from patients infected with genotype 3 was significantly higher than corresponding reactivity between patients infected with genotype 1 and H77c. The seroreactivity was inversely proportional to the viral load and to the degree of liver fibrosis. The pre-treatment level of anti-E1E2 was higher in sustained responders to combination therapy. These results demonstrate that seroreactivity against E1E2 depends upon the genotypic origin of the E1E2 antigens and the infecting genotype, and suggest a possible protective effect of anti-E1E2 against disease progression.
Subject(s)
Antibodies, Viral/blood , Hepacivirus/immunology , Hepatitis C, Chronic/immunology , Hepatitis C, Chronic/pathology , Viral Envelope Proteins/immunology , Antiviral Agents/therapeutic use , Enzyme-Linked Immunosorbent Assay , Genotype , Hepacivirus/classification , Hepacivirus/genetics , Hepatitis C, Chronic/drug therapy , Humans , Liver Cirrhosis/pathology , Severity of Illness Index , Statistics as Topic , Treatment Outcome , Viral LoadABSTRACT
The diagnosis of acute hepatitis C virus (HCV) infection is not straightforward; few people exhibit clinical symptoms and genome/antigen detection techniques do not indicate when infection had occurred. Here, a strategy to detect HCV RNA in the absence of antibody ('window-period') for diagnosis of acute infection is assessed. The sentinel surveillance of hepatitis testing study was used to retrospectively identify anti-HCV negative samples from high-risk individuals (2002-2003), for testing singly for HCV RNA. Additional samples were identified prospectively (2005) and tested in pools for HCV RNA. Positive samples were genotyped. Incidence and costs of adopting the pooling strategy were estimated. In the retrospective study, 8/390 (2.1%) samples were confirmed HCV RNA positive, anti-HCV negative. Prospectively, 3237 samples were tested in 325 pools. Five positive pools identified four confirmed HCV RNA positive patients (one false positive). Estimated incidence was 12.9 per 100 person-years in injecting drug users (IDUs) (retrospective study) and 3.7 per 100 person-years among drug/alcohol services and prison attendees (prospective study). Estimated costs were pound 850 per positive sample, in areas of higher risk. The yield from a window-period strategy depends upon the population tested. Pooled HCV RNA testing of anti-HCV negative samples from the current IDUs is realistic and relatively inexpensive to identify recently infected individuals.
Subject(s)
Hepacivirus/genetics , Hepatitis C/diagnosis , Hepatitis C/epidemiology , Acute Disease/epidemiology , Adolescent , Adult , Antibodies, Viral/blood , Drug Users , England/epidemiology , Female , Hepacivirus/immunology , Hepacivirus/isolation & purification , Hepatitis C/immunology , Hepatitis C/virology , Humans , Incidence , Male , Molecular Diagnostic Techniques/economics , Prospective Studies , RNA, Viral/genetics , Retrospective Studies , Risk Factors , White PeopleABSTRACT
Evidence for efficacy of established treatment guidelines for chronic hepatitis C virus (HCV) disease is based on multinational randomized controlled trials (RCTs). Strategies for managing HCV, however, require an assessment of the effectiveness of intervention in routine clinical practice. We report the outcomes of combination therapy in a large cohort of HCV-infected individuals in the UK. A total of 347 (113 genotype 1, 234 genotype non-1) patients were treated with pegylated interferon and ribavirin according to current guidelines. Forty-two (37.2%) of those with genotype 1 infection and 164 (70.1%) with genotype non-1 infection achieved sustained viral response (SVR). Thirty-nine (11%) patients withdrew from treatment. In addition to viral genotype, factors predictive of a response to therapy were age at start of treatment and disease stage on pretreatment liver biopsy. Multivariate regression analysis demonstrated that the effects of age [odds ratio 0.5; 95% confidence interval (0.31-0.82) per 10-year increment (P = 0.006)] were confined to genotype 1 disease. In order to further inform the management of the individual patient, a multivariate logistic model was used to predict the probability of SVR for subgroups defined by disease stage, genotype and age at commencement of therapy. This model revealed striking differences in predicted response rates between subgroups and provided a strong rationale for early treatment, particularly for those with genotype 1 disease. Our study demonstrates that results comparable with those of RCTs can be achieved in clinical practice, and suggests that prediction of response rates based on probability modelling will provide a valuable adjunct to individual patient management.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis C, Chronic/drug therapy , Interferon-alpha/therapeutic use , Polyethylene Glycols/therapeutic use , Ribavirin/therapeutic use , Adult , Age Factors , Drug Therapy, Combination , Female , Forecasting , Genotype , Hepacivirus/classification , Hepacivirus/genetics , Hepacivirus/isolation & purification , Hepatitis C, Chronic/pathology , Hepatitis C, Chronic/virology , Humans , Interferon alpha-2 , Liver/pathology , Male , Middle Aged , Recombinant Proteins , Treatment Outcome , United Kingdom , ViremiaABSTRACT
Around 25% of people infected with hepatitis C virus (HCV) are able to clear the infection spontaneously, while the majority become chronically infected, with a subsequent risk for the individual patient of progressive inflammatory liver disease, cirrhosis, hepatocellular carcinoma and liver-related death (Figure 1). Much is known about the epidemiology, pathogenesis, diagnosis and management of chronic HCV infection. In comparison, knowledge about acute HCV infection is patchy. In this article, we will highlight concerns relating to acute HCV infection and suggest that public health bodies responsible for managing the HCV epidemic should redirect at least some of their resources to dealing with these issues.
Subject(s)
Disease Outbreaks/statistics & numerical data , Hepatitis C/epidemiology , Hepatitis C/prevention & control , Population Surveillance , Risk Assessment/methods , Acute Disease , Europe/epidemiology , Hepatitis C/diagnosis , Humans , Incidence , Risk FactorsABSTRACT
Acute hepatitis C virus (HCV) infections are frequently seen worldwide in certain risk groups, with an annual incidence rate varying between 0.08% and 66%. Although this incidence is substantial, a delayed diagnosis during chronic infection is most often made in the absence of clinical symptoms in the acute phase of the infection. Currently used methods to diagnose acute HCV infection are IgG antibody seroconversion and repeated HCV RNA measurements, although no definitive diagnostic test is currently available. Progress in the field of adaptive and innate immune responses has aided both advances in the field of HCV vaccine development and a more basic understanding of viral persistence. The rapid changes in the treatment of chronic HCV infection will affect therapeutic regimens for acute HCV infection in the coming years, leading to shorter treatment courses and pegylated interferon-free modalities. This review gives an overview of the current knowledge and uncertainties, together with some future perspectives on acute hepatitis C epidemiology, virology, immunology, and treatment.
Subject(s)
Hepatitis C Antibodies/blood , Hepatitis C/diagnosis , Hepatitis C/drug therapy , RNA, Viral/blood , Antiviral Agents/therapeutic use , Biomedical Research/trends , Hepatitis C/prevention & control , Humans , Viral Vaccines/immunology , Viral Vaccines/isolation & purificationABSTRACT
The immunosuppressive properties of the fungal metabolite Cyclosporin A (CsA) on the human lymphocyte response to the polyclonal B cell activator Epstein-Barr virus (EBV) in vitro were assessed. CsA showed both stimulatory and inhibitory effects on EBV-induced IgM secretion, the net effect being dependent on the dose of virus used. T cells were required for both these effects to occur. A model is proposed to account for the complex interactions of CsA in this system.
Subject(s)
B-Lymphocytes/immunology , Cyclosporins/pharmacology , Herpesvirus 4, Human/immunology , T-Lymphocytes/immunology , Antibody Formation/drug effects , B-Lymphocytes/drug effects , Cells, Cultured , Humans , Immunoglobulin M/analysis , Immunosuppression Therapy , Kinetics , T-Lymphocytes/drug effectsABSTRACT
To explain the variation in the percentage of mouse erythrocyte rosette-forming cells (MERFC) during culture of Epstein-Barr virus (EBV)-induced B-cell lines, we provide evidence that (i) there is an altered expression of mouse red blood cell (MRBC) receptors on cell line cells during the mitotic cycle, and (ii) putative receptor-negative cells are capable of de novo synthesis of the receptor, and passively adsorbing receptor shed from receptor-positive cells.
Subject(s)
B-Lymphocytes/metabolism , Erythrocytes/immunology , Receptors, Antigen, B-Cell/metabolism , Adsorption , Animals , B-Lymphocytes/immunology , Cell Cycle , Cell Line , Cell Transformation, Viral , Female , Herpesvirus 4, Human/physiology , Humans , Male , Mice , Mice, Inbred CBA , Protein Biosynthesis , Rosette FormationABSTRACT
The antiviral activity of podophyllotoxin against herpes simplex type 1 virus (HSV-1) grown in Vero cells was studied by a simple microtitration assay. Antiviral effects were induced at similar concentrations as direct cellular toxicity, as characterised by a time-dependent loss of cell monolayer. Podophyllotoxin-mediated toxicity arises from cytoplasmic microtubular, and hence cytoskeletal, decay. Some degree of selectivity was seen for inhibition of virus replication over direct cellular toxicity. Podophyllotoxin acted against an early viral process, as an antiviral effect was still seen if drug was removed 2 h after infection. Similar effects were seen with colchicine, a classical tubulin-binding compound, but not with bromovinyldeoxyuridine. Podophyllotoxin was capable of inducing a cytoprotective effect in Vero cells, as pre-treatment of cells abrogated virus growth for up to 90 min after removal of drug. This is coincident with the repolymerisation of cellular microtubules and re-formation of the cytoskeleton. We conclude that HSV-1 relies upon a functional cellular cytoskeleton for efficient completion of an early replicative event. Such a process may be the transport of viral material to the nucleus or inhibition of the formation of intranuclear viral 'replication factories', bodies containing cytoskeletal fragments constructed after viral infection.
Subject(s)
Antiviral Agents/pharmacology , Herpes Simplex/drug therapy , Microtubules/virology , Podophyllotoxin/pharmacology , Simplexvirus/drug effects , Animals , Antimetabolites/pharmacology , Chlorocebus aethiops , Colchicine/pharmacology , Deoxyuridine/pharmacology , Microtubules/drug effects , Microtubules/physiology , Simplexvirus/growth & development , Simplexvirus/physiology , Vero Cells , Virus Replication/drug effectsABSTRACT
The recent development of tagged RT-PCR and rTth RT-PCR has greatly improved strand-specific detection of hepatitis C virus (HCV) RNA but these assays are still prone to some false detection of the incorrect strand of RNA. In this study we aimed to address additional factors which contribute towards false detection of HCV RNA. Firstly the benefits of both tagged primers and the thermostable reverse transcriptase rTth during cDNA synthesis were combined and it was found that strand specificity was greatly improved without compromising sensitivity. The reliability of the assay was then optimised by addressing the following issues: control synthetic transcripts should be free of contaminating plasmid DNA, residual RT activity should be minimised in the presence of PCR primers and cDNA should be free of unincorporated tagged RT primer prior to PCR amplification. The alterations made to the assay eliminated completely false detection of the incorrect strand of RNA in the control assay whilst the correct strand was consistently detected at a cDNA dilution of 10(-3)-10(-4). Negative strand was not detected in RNA isolated from serum but was detected, at a ten-fold lower level than positive strand, in RNA isolated from liver tissue.
Subject(s)
Hepacivirus/genetics , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction , Virus Replication , DNA Repair Enzymes , Exodeoxyribonucleases/metabolism , Hepacivirus/isolation & purification , Hepatitis C/virology , Humans , Liver/pathology , Liver/virology , Plasmids , Sensitivity and SpecificityABSTRACT
The reliability and limitations of the currently used routine tests for herpes simplex virus type 2 (HSV-2) antibody in Australia are reviewed. Six case reports illustrate the clinical dangers of overinterpretation of the currently available kits and the need for a readily available specific HSV-2 antibody test. In Sydney, HSV-2 causes approximately 85% of primary genital herpes and > 95% of recurrent genital herpes. Due to the extensive serological cross-reactivity between HSV-1 and HSV-2, currently available "type specific" commercial assays cannot reliably distinguish between the 2. Isolation of herpes simplex virus (HSV) or detection of HSV antigen in vesicle fluid is the preferred diagnostic test but may be overlooked or patients may have no visible lesions. The only accurate techniques for detecting HSV-2 specific antibody are the Western blot assay and an enzymatic immunoassay using glycoprotein G (gG-2), a component of the HSV-2 envelope. These tests, which still are restricted to research laboratories can be used to accurately identify people with previous exposure to HSV-2 (IgG) or to diagnose primary infection where virus isolation has not been performed or is impossible. Current commercially available antibody tests may have extensive cross-reactivity.
Subject(s)
Herpes Genitalis/diagnosis , Reagent Kits, Diagnostic , Simplexvirus/isolation & purification , Adolescent , Adult , Blotting, Western , Diseases in Twins , Enzyme-Linked Immunosorbent Assay , Female , Humans , Infant, Newborn , Male , Middle Aged , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
OBJECTIVE: To identify clinical consequences of acute human herpesvirus type 6 infection by hypothesising that the virus will induce similar clinical syndromes to cytomegalovirus. DESIGN: Examination of consecutive serum samples from patients with illnesses compatible with acute cytomegalovirus infection or exanthem subitum by indirect immunofluorescence for the presence of antibodies to human herpesvirus type 6. An IgG absorption step was included to avoid false positive and negative results for IgM. The criterion standard for diagnosis of human herpesvirus type 6 infection was the presence of IgM human herpesvirus type 6 antibody (titre greater than 20) and a rising titre of IgG human herpesvirus type 6 antibody without serological evidence of alternative infection. SETTING: Routine viral diagnostic and reference laboratory in the largest teaching hospital in Sydney. PATIENTS: 341 Consecutive serum samples were analysed from patients with hepatitis (147 samples); infectious mononucleosis-like illness (106); screens for toxoplasma, other viruses, rubella, cytomegalovirus, and herpesvirus (38); fever in an immunocompromised patient (eight); unusual neurological (nine) or haematological syndromes (14); splenomegaly (six); and rash in a child (13). RESULTS: Three cases of acute human herpesvirus type 6 infection were identified: in one patient aged 65 with a previous diagnosis of acute non-A non-B hepatitis, one aged 25 with a glandular fever-like illness, and one aged 6 with a glandular fever-like illness. All three illnesses resolved completely. 15 Further serum samples were positive for human herpesvirus type 6 antibody but were also diagnostic for acute infection with other viruses (cytomegalovirus (nine), Epstein-Barr virus (three), and HIV (one] or had a titre of IgM human herpesvirus type 6 antibody less than 20 (two). CONCLUSIONS: Acute human herpesvirus type 6 infection in immunocompetent patients may result in a mononucleosis-like illness or an acute but self limiting hepatitis.