In house ELISA based on recombinant ORF2 protein underline high prevalence of IgG anti-hepatitis E virus amongst blood donors in south Brazil.

Hepatitis E Virus (HEV) is a zoonotic pathogen responsible for causing acute hepatitis in human, especially in developing countries. Diagnosis of HEV usually relies on the detection of antibodies mostly by enzyme-linked immunosorbent assay (ELISA). In the present study, we designed a new indirect ELISA (iELISA) based on a short recombinant peptide derived from the capsid protein (ORF2p) and demonstrated its potential for detecting human IgG against HEV genotype 3. The best polystyrene plate (Maxisorp®), optimal ORF2p coating antigen concentration (0,67µg/well) and primary antibody dilution (1:100) were determined. This iELISA showed a sensitivity of 91.4% and specificity of 95.9%. The comparison of our in house iELISA with a commercial assay (RecomWell, Mikrogen®) showed 94.25% of agreement and a kappa index of 0.88. The ORF2 recombinant ELISA was used to screen 780 blood donors for anti-HEV IgG and we found that 314 (40,25%) of these donors were IgG positive. This high prevalence of antibodies suggests, for the first time, that the Southern Brazil region might be endemic to Hepatitis E Virus genotype 3.

Production and characterization of a Brazilian candidate antigen for Hepatitis E Virus genotype 3 diagnosis.

Hepatitis E, caused by hepatitis E virus (HEV), is a viral infectious pathology of great importance in the public health. Hepatitis E outbreaks were registered in developing countries with poor or no sanitation, where drinking water was contaminated with fecal material, but also in many industrialized countries probably due to consumption of HEV-positive swine meat. In this study, we present the development and characterization of a recombinant antigen from ORF2 HEV genotype 3. Viral RNA was extracted from swine feces infected with the native virus. A total of 267 residues from the C-terminal ORF2((394-661)) coding sequence were cloned into the pET20a vector and expressed in Escherichia coli ER2566. Recombinant protein was purified by liquid chromatography and the fragment obtained a 98% homology against other human or swine HEV genotype 3 ORF2 sequences. Wistar rats were inoculated with ORF2p, developing antibodies able to recognize both the homologous antigen and the native HEV genotype 3 ORF2 present in infected stool. In parallel, HEV-negative swine were experimentally challenged with HEV genotype 3. ORF2 was detected by PCR 14 days post-inoculation in three-fourth piglets' feces and one week later by dot blot. In conclusion, this study proved the immunogenic and antigenic properties of the recombinant protein ORF2p.

Prospective follow-up of patients with acute hepatitis C virus infection in Brazil.

BACKGROUND: The natural outcome of infection with hepatitis C virus (HCV) varies substantially among individuals. However, little is known about host and viral factors associated with a self-limiting or chronic evolution of HCV infection. METHODS: From 1 January 2001 through 31 December 2008, a consecutive series of 65 patients from Rio de Janeiro, Brazil, with a well-documented diagnosis of acute HCV infection, acquired via various routes, were enrolled in this study. Patients were prospectively followed up for a median of 40 months after the estimated date of HCV infection with serial measurements of serum alanine aminotransferase, HCV RNA, and anti-HCV antibodies. Spontaneous viral clearance (SVC) was defined as undetectable levels of HCV RNA in serum, in the absence of treatment, for 3 consecutive HCV polymerase chain reaction tests within the first 6 months of follow-up. Cox proportional hazards regression was used to identify host and viral predictors of SVC. RESULTS: The cumulative rate of SVC was 44.6% (95% confidence interval, 32.3%-57.5%). Compared with chronic HCV evolution, patients with self-limiting disease had significantly lower peak levels of anti-HCV antibodies (median, 109.0 vs 86.7 optical density-to-cutoff ratio [od/co]; P<.02), experienced disease symptoms more frequently (69.4% vs 100%; P<.001), and had lower viral load at first clinical presentation (median, 4.3 vs 0.0 log copies; P=.01). In multivariate analyses, low peak anti-HCV level (<93.5 od/co) was the only independent predictor for SVC; the hazard ratio compared with high anti-HCV levels (> or =93.5 od/co) was 2.62 (95% confidence interval, 1.11-6.19; P=.03). CONCLUSION: Our data suggest that low levels of anti-HCV antibodies during the acute phase of HCV infection are independently related to spontaneous viral clearance.

Eosinophils involved in fulminant hepatic failure are associated with high interleukin-6 expression and absence of interleukin-5 in liver and peripheral blood.

BACKGROUND/AIMS: Although eosinophils are considered to play an important role in the pathogenesis of various parasitic, allergic and autoimmune digestive diseases, their role in fulminant hepatic failure (FHF) is unknown. Our contribution was to identify and quantify eosinophils and cytokine levels [interleukin (IL)-6, IL-5 and macrophage inflammatory protein (MIP)-1alpha] in liver parenchyma and peripheral blood from FHF patients at pre- and post-transplantation steps. METHODS: Histochemical methods were used to identify/quantify eosinophils in liver samples. Liver and plasma cytokine levels were quantified using immunofluorescence methods. RESULTS: Fulminant hepatic failure patients showed a high number of intrahepatic eosinophils concomitant with an increased expression of IL-6, besides the IL-6-positive eosinophils associated with the lack of IL-5. Also, an increased number of eosinophils and soluble IL-6 and MIP-1alpha with a low expression of IL-5 in peripheral blood at the pretransplantation step was observed. CONCLUSIONS: The increased number of intrahepatic eosinophils, besides the high production of IL-6, may be involved in liver dysfunction. In addition, the low presence of IL-5 in liver and peripheral blood may represent a particular pattern of eosinophil behaviour in human liver failure, which may also involve MIP-1alpha. Further ex vivo studies are necessary to evaluate the specific role of eosinophils in FHF.