Hepatitis E virus (HEV) is the major cause of acute viral hepatitis in several countries in Europe. HEV is acquired mainly by consumption of contaminated pork but can also be transmitted through ...blood transfusion. HEV infection is usually self-limited but can become persistent in immunocompromised persons. During the first 30 months of HEV RNA universal screening of blood donations in Catalonia, Spain, we identified 151 HEV RNA-positive donations (1/4,341 blood donations). Most infected donors reported consumption of pates and sausages, and 58% were negative for HEV IgM and IgG. All HEV isolates belonged to genotype 3. All infected donors spontaneously resolved the infection, and no neurologic symptoms and reinfections were observed after 1 year of follow-up. Since the implementation of HEV RNA universal screening, no new cases of transfusion-transmitted HEV infection were reported. Our data indicate HEV screening of blood donations provides safer blood for all recipients, especially for immunosuppressed persons.
HCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the ...impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV. We analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-Æ ELISPOT, intracellular cytokine staining (IL-2 and IFN-Æ) and CFSE-based proliferation assays. We observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-Æ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies. Our results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA's therapies.
Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide, has an incidence rate equal to mortality. Over 80% of HCC cases occur within a high‐risk population, mainly patients with both ...cirrhosis and chronic hepatitis B or C. With a 5‐year survival rate ranging from <16% for advanced HCC to >90% for early stage HCC, there is a high medical need for the early detection of HCC. In this study, we systematically evaluated biomarkers mentioned in international guidelines and peer‐reviewed literature for HCC surveillance and diagnosis with the aim of identifying combinations that display high sensitivity and specificity for early stage HCC. Fifty biomarkers were measured in the first sample panel, panel A (n = 110), and subjected to univariate analysis. Of these, 35 biomarkers (38 assays) from panel A and an additional 13 biomarkers from the literature were prioritized for subsequent multivariate evaluation with lasso regression and exhaustive search of two‐ to four‐biomarker combinations (panel B). Panel B included 1,081 samples from patients with HCC (n = 308) or with chronic liver diseases (n = 740). Among all patients, 61.0% had hepatitis B, 32.9% had hepatitis C, and 60.5% had cirrhosis; 40.6% of patients with HCC had early stage cancer. Protein induced by vitamin K absence‐II (PIVKA‐II; also known as des‐gamma‐carboxy prothrombin DCP) and alpha‐fetoprotein (AFP) demonstrated the best clinical performance, both individually and in combination, and the addition of a third biomarker (Lens culinaris agglutinin‐reactive fraction of AFP AFP‐L3, cartilage oligomeric matrix protein COMP, insulin‐like growth factor‐binding protein 3 IGFBP3, or matrix metalloproteinase 3 MMP3) further increased sensitivity for the detection of both early stage and all‐stage HCC. The addition of age and sex to the three‐biomarker panel resulted in an improved diagnostic performance. Conclusion: The combination of AFP and PIVKA‐II, with either IGFBP3, COMP or MMP3, plus age and sex, demonstrated the best performance for the detection of early‐ and all‐stage HCC. These novel panels performed similar to that of the GALAD score (sex gender, age, plus serum levels of AFP, AFP‐L3 and DCP PIVKA‐II), a promising screening tool developed for HCC detection.
Human T-cell lymphotropic virus type 1 and 2 (HTLV-1/2) screening is not mandatory in Spanish blood banks. In Catalonia, selective screening was introduced in 2008, followed by universal screening in ...2011. We present herein a 10-year experience of HTLV testing in blood donors. HTLV-1/2 selective screening was performed using Ortho-Clinical Diagnostics HTLV-I/HTLV-II Ab-Capture ELISA between February 2008 and May 2009, then Abbott Prism HTLV-I/ HTLV-II assay until December 2010. Abbott Architect rHTLV-I/II assay was then used for HTLV-1/2 universal screening in pooled samples. INNO-LIA HTLV I/II Score (Fujirebio) and in-house HTLV-1/2 proviral DNA real-time PCR were used in reactive samples. Follow-up was offered to confirm HTLV-1/2 donors in Vall d’Hebron Hospital. Between 2008 and 2017, 51 blood donors were confirmed HTLV positive (46 HTLV-1, 4 HTLV-2 and 1 HTLV) out of 2,114,891 blood donations (1 in 41,468). Sixty-nine percent were female, median age was 40 years and most were born in Latin America (69%), followed by Europe (25%), Africa (4%) and Asia (2%). Screening of relatives and partners identified 12 additional HTLV-1 cases. Lookback studies did not show any HTLV-1/2 transmission. HTLV infections found in blood donors mirror epidemiological changes in the population of Spain. Consequently, HTLV should be considered a potential risk for recipients and calls for the design of optimal strategies to ensure transfusion safety.
MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent ...due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.
Background
There is a need for new serum biomarkers for early detection of hepatocellular carcinoma (HCC). Haptoglobin (Hp) N-glycosylation is altered in HCC, but the diagnostic value of ...site-specific Hp glycobiomarkers is rarely reported. We aimed to determine the site-specific glycosylation profile of Hp for early-stage HCC diagnosis.
Method
Hp glycosylation was analyzed in the plasma of patients with liver diseases (n=57; controls), early-stage HCC (n=50) and late-stage HCC (n=32). Hp phenotype was determined by immunoblotting. Hp was immunoisolated and digested into peptides. N-glycopeptides were identified and quantified using liquid chromatography–mass spectrometry. Cohort samples were compared using Wilcoxon rank-sum (Mann-Whitney U) tests. Diagnostic performance was assessed using receiver operating characteristic (ROC) curves and area under curve (AUC).
Results
Significantly higher fucosylation, branching and sialylation of Hp glycans, and expression of high-mannose glycans, was observed as disease progressed from cirrhosis to early- and late-stage HCC. Several glycopeptides demonstrated high values for early diagnosis of HCC, with an AUC of 93% (n=1), >80% (n=3), >75% (n=13) and >70% (n=11), compared with alpha-fetoprotein (AFP; AUC of 79%). The diagnostic performance of the identified biomarkers was only slightly affected by Hp phenotype.
Conclusion
We identified a panel of Hp glycopeptides that are significantly differentially regulated in early- and late-stage HCC. Some glycobiomarkers exceeded the diagnostic value of AFP (the most commonly used biomarker for HCC diagnosis). Our findings provide evidence that glycobiomarkers can be effective in the diagnosis of early HCC – individually, as a panel of glycopeptides or combined with conventional serological biomarkers.
The pathogenic mechanisms determining the diverse clinical outcomes of HEV infection (e.g., self-limiting versus chronic or symptomatic versus asymptomatic) are not yet understood. Because specific ...microRNA signatures during viral infection inform the cellular processes involved in virus replication and pathogenesis, we investigated plasma microRNA profiles in 44 subjects, including patients with symptomatic acute (AHE,
= 7) and chronic (CHE,
= 6) hepatitis E, blood donors with asymptomatic infection (HEV BDs,
= 9), and anti-HEV IgG
IgM
(exposed BDs,
= 10) and anti-HEV IgG
IgM
(naive BDs,
= 12) healthy blood donors. By measuring the abundance of 179 microRNAs in AHE patients and naive BDs by reverse transcription-quantitative PCR (RT-qPCR), we identified 51 potential HEV-regulated microRNAs (
value adjusted for multiple testing by the Benjamini-Hochberg correction
< 0.05). Further analysis showed that HEV genotype 3 infection is associated with miR-122, miR-194, miR-885, and miR-30a upregulation and miR-221, miR-223, and miR-27a downregulation. AHE patients showed significantly higher levels of miR-122 and miR-194 and lower levels of miR-221, miR-27a, and miR-335 than HEV BDs. This specific microRNA signature in AHE could promote virus replication and reduce antiviral immune responses, contributing to the development of clinical symptoms. We found that miR-194, miR-335, and miR-221 can discriminate between asymptomatic HEV infections and those developing acute symptoms, whereas miR-335 correctly classifies AHE and CHE patients. Our data suggest that diverse outcomes of HEV infection result from different HEV-induced microRNA dysregulations. The specific microRNA signatures described offer novel information that may serve to develop biomarkers of HEV infection outcomes and improve our understanding of HEV pathogenesis, which may facilitate the identification of antiviral targets.
There is increasing evidence that viruses dysregulate the expression and/or secretion of microRNAs to promote viral replication, immune evasion, and pathogenesis. In this study, we evaluated the change in microRNA abundance in patients with acute or chronic HEV infection and asymptomatic HEV-infected blood donors. Our results suggest that different HEV-induced microRNA dysregulations may contribute to the diverse clinical manifestations of HEV infection. The specific microRNA signatures identified in this study hold potential as predictive markers of HEV infection outcomes, which would improve the clinical management of hepatitis E patients, particularly of those developing severe symptoms or chronic infections. Furthermore, this study provides new insights into HEV pathogenesis that may serve to identify antiviral targets, which would have a major impact because no effective treatments are yet available.
HBsAg proteins are useful to identify HBV inactive carriers (ICs), but data on chronic hepatitis D (CHD) are scarce. This study aimed to describe HBsAg composition in CHD, its changes during the ...evolution and the potential association with clinical outcomes. Besides, we assess the composition of HBsAg across different HBV genotype and validate previous results on HBsAg proteins in an independent HBV cohort.
Quantitative HBsAg (qHBsAg), medium and large HBsAg proteins (MHBs, LHBs) were measured in two cohorts: 1) CHD: cross-sectional study of samples from two European institutions (N=46); Outcomes were assessed in a retrospective-prospective study of those patients with follow-up (FU) >1 year (N=36), and the longitudinal evolution of HBsAg proteins in those with samples >5 years apart (N=12); 2) HBeAg-negative HBV subjects: cross-sectional study (N=141).
Forty-one (89%) CHD patients had detectable HDV-RNA, and the presence of HDV-RNA was associated with higher LHBs% (p=0.010). Baseline MHBs (p=0.051) and MHBs% (p=0.086) tended to be higher in those developing clinical outcomes (9/36, 25%) after a median FU of 5.9 years. Subjects in which HDV-RNA became spontaneously undetectable during FU (5/31, 16.1%) tended to present lower MHBs% (p=0.085). In the longitudinal study, changes in LHBs% were observed (p=0.041), whereas MHBs% remained stable (p=0.209). Regarding HBV, ICs showed lower LHBs% (p=0.027). LHBs and MHBs differed significantly according to HBV-genotype, regardless of the HBV phase.
CHD subjects with detectable HDV-RNA presented higher LHBs% than those undetectable. A trend to have higher baseline MHBs% was observed in patients who developed clinical outcomes or remained with detectable HDV-RNA. This study validates the different HBsAg composition in HBV-ICs and reveals the HBV-genotype influence in HBsAg composition.
The composition of Hepatitis B surface antigen in chronic hepatitis delta differs in subjects with detectable and undetectable HDV viral load, and may help to predict the likelihood to achieve undetectable HDV viremia and the development of clinical events such as decompensation. The composition of the surface antigen is also useful to distinguish inactive carriers of hepatitis B virus, and it varies according to HBV-genotype.
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•Chronic hepatitis D with detectable HDV-RNA showed higher HBsAg and LHBs% than those with undetectable viremia.•In chronic hepatitis D, a trend to higher baseline MHBs% was observed in patients who developed clinical outcomes.•A different HBsAg composition, with lower LHBs%, has been validated for HBV HBeAg-negative inactive carriers.•HBV-genotype has shown a significant impact in HBsAg composition in HBV-infected subjects.
Abstract An increasing number of new hepatitis C virus NS3-protease inhibitors are being evaluated for the treatment of chronic hepatitis C. Treatment-induced selection of mutants conferring ...resistance to protease inhibitors has been shown both in vivo and in vitro. A specific mutation, A156T has been shown to confer high-level resistance to several such agents (BILN2061, VX-950, SCH446211 (SCH6) and SCH503034). Here we report the presence of the A156T mutation in close to 1% of NS3 sequences within the liver quasispecies of a chronic hepatitis C patient never treated with anti-NS3-protease inhibitors.
Abstract
Background
Transfusion-transmitted hepatitis E virus (HEV) infections have raised many concerns regarding the safety of blood products. To date, enveloped HEV particles have been described ...in circulating blood, whereas nonenveloped HEV virions have only been found in feces; however, no exhaustive studies have been performed to fully characterize HEV particles in blood.
Methods
Using isopycnic ultracentrifugation, we determined the types of HEV particles in plasma of HEV-infected blood donors.
Results
Nonenveloped HEV was detected in 8 of 23 plasma samples, whereas enveloped HEV was found in all of them. No association was observed between the presence of nonenveloped HEV and viral load, gender, or age at infection. However, samples with HEV-positive serology and/or increased levels of liver injury markers contained a higher proportion of nonenveloped HEV than samples with HEV-negative serology and normal levels of liver enzymes. These results were further confirmed by analyzing paired donation and follow-up samples of 10 HEV-infected donors who were HEV seronegative at donation but had anti-HEV antibodies and/or increased levels of liver enzymes at follow up.
Conclusions
The HEV-contaminated blood products may contain nonenveloped HEV, which may pose an additional risk to blood safety by behaving differently to pathogen inactivation treatments or increasing infectivity.
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