Evidence for Hepatitis A virus endemic circulation in Israel despite universal toddlers' vaccination since 1999 and low clinical incidence in all age groups.

Background: Universal toddlers vaccination (UTV) introduced in 1999, reduced hepatitis A incidence in Israel from 50.4 to <1.0/100,000. The current Hepatitis A virus (HAV) molecular epidemiology in Israel was studied 13-14y post UTV introduction.. Methods: An outbreak in Tel-Aviv with 75 cases in 2012-2013 was investigated. Real-time RT-PCR and sequencing of the VP1-2A region (1100bp) was done on: a. serum samples from patients with acute Hepatitis A (12/ 75 in Tel-Aviv and 31 patients hospitalized in 3 other major cities in 2011-2013); b. in sewage samples (27 from metropolitan Tel-Aviv, 14 from the other 3 cities and 6 from Gaza). Results: The outbreak began among intravenous drug users then spread to the general population. Patients' mean age was 33.2y, 4/75(5.3%) had been vaccinated and 58/75(77.3%) were hospitalized. No common environmental source was found. HAV was detected in sewage samples: 16/27(59.2%) from Tel-Aviv; 4/14(28.6%) collected throughout Israel and 6/6 (100%) from Gaza. Genotype IB predominated (52/53 sequenced samples) and identical strains were demonstrated in the Israeli and Palestinian populations by phylogenetic analysis. Conclusions: Despite the UTV success, HAV circulation in the Israeli population continues, apparently due to its close contacts with the endemic Palestinian population. Reassessment of vaccination policy is recommended.

Hepatitis E in Israel: A nation-wide retrospective study.

AIM: To investigate the epidemiology, risk factors and clinical course of acute hepatitis E virus (HEV) infection in Israel, an industrialized country. METHODS: A retrospective analysis of acute HEV cases diagnosed in Israel from 1993 to 2013. Acute HEV was defined by ALT/AST elevation and a positive HEV PCR test or positive anti-HEV-IgM serology. HEV RNA was tested by quantitative reverse transcription PCR. Antibodies to HEV were tested retrospectively using an ELISA assay. HEV-RNA was sequenced using RT-PCR of ORF1 and ORF2 regions to diagnose genotype of the virus. Epidemiologic and clinical data were collected by reviewing the clinical files and through a telephone interview according to a structured questionnaire. RESULTS: Acute HEV was diagnosed in 68 patients. Among the 59 patients who gave an informed consent and were interviewed, 41% of infections were autochthonous (acquired in Israel), 44% travel-related and 15% imported by foreign workers. Autochthonous patients were mainly females (62.5%), more than half of them pregnant, 26% recalled consuming food or water in areas with poor sanitation, 44% ate non-kosher meat. Fulminant hepatitis developed in 3 patients (5%), all of them were females, two of them with post-partum infection, all acquired the disease in Israel (autochthonous). Israeli travelers with imported infection were predominantly males (73%), acquired the disease in the Indian subcontinent (81%), with 100% reporting having consumed fresh vegetables and drinks with ice cubes abroad. Six patients' sera were tested for genotype and revealed HEV genotype 1 (all cases acquired in the Indian subcontinent). CONCLUSION: This is the first report which highlights the existence of hepatitis E as an autochthonous infection in Israel. Imported HEV originates mostly from the Indian subcontinent.

Acute hepatitis E virus in pregnant women in Israel and in other industrialized countries.

BACKGROUND AND OBJECTIVES: Acute hepatitis E virus (HEV) is the most common etiology of viral hepatitis in adults in developing countries. HEV is rare in industrialized countries but its incidence is rising both in returning travelers and through autochthonous infection. In developing countries HEV is associated with a high rate of fulminant hepatitis and mortality during pregnancy and contributes to poor obstetric and fetal outcomes. There are no reliable data on the outcome of HEV during pregnancy in industrialized countries. STUDY DESIGN: A retrospective analysis of acute HEV cases diagnosed in Israel were examined. The clinical course of the disease among pregnant women was retrieved. A systematic review of the literature was performed for cases of HEV and pregnancy, originating or treated in industrialized countries RESULTS: Between the years 1993-2013, 68 cases of acute HEV were diagnosed in Israel, including 9 pregnant women (13%). An additional 6 reported cases were found from a literature search. From the 15 women (10 autochthonous cases and 5 imported cases), the outcome was favorable in 10 cases, however, 5 cases (33%) resulted in fulminant hepatitis and two women underwent an urgent liver transplantation. No fatality occurred in the mothers and all babies were born alive and healthy. DISCUSSION: This is the first case series of acute HEV infection in pregnant women in industrialized countries. Acute HEV infection poses a significant risk in pregnancy, irrespective of patients' country of origin. In contrast to reports from developing countries, all babies and mothers survived.

Breastmilk hepatitis A virus RNA in nursing mothers with acute hepatitis A virus infection.

Breastmilk specimens from three women with acute hepatitis A virus (HAV) infection were studied. Anti-HAV immunoglobulin M and immunoglobulin G antibodies were detected in serum and breastmilk specimens of the three women. The three women also had serum HAV RNA. However, HAV RNA was detected only in two of the three breastmilk specimens. It is interesting that none of the three infants contracted clinical HAV infection. Furthermore, mothers with HAV infection should not be encouraged to discontinue breastfeeding.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization.

A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.

Genotype prevalence, viral load and outcome of hepatitis B virus precore mutant infection in stable patients and in patients after liver transplantation.

OBJECTIVE: The precore mutant is detectable in most Israeli patients with persistent hepatitis B virus (HBV) infection. The aim of this study was to determine the prevalence of HBV genotypes, viral load and outcome of precore mutant infection in stable patients and in patients after liver transplantation. METHODS: The prevalence of HBV genotype and viral load were investigated in 81 patients with HBV precore mutant infection. Of these, 50 patients (40 males, 10 females; mean age 43.4 +/- 11.0 yr) underwent liver transplantation and were serum HBV DNA-negative by hybridization at the time of transplantation. Patients received long-term HBV immunoprophylaxis and immunosuppression, and lamivudine in cases of graft HBV recurrence. The remaining 31 patients were stable, with serum anti-HBe-positivity. Genotypes were tested by restriction fragment length polymorphism of an S gene amplicon. Precore mutations were studied with an INNO-LiPA probe assay. RESULTS: Follow-up was 46.6 +/- 37.7 months. Most of the transplanted group was of Middle Eastern origin (53.6%); the remainder were from Eastern Europe (21.4%), Western Europe and the USA (10.8%), Africa (7.1%), and Asia (7.1%). In the transplanted group, the pre-transplant HBV genotype D was the most prevalent (96%), while genotype A was found in only 4%. Eleven patients (22%) developed recurrent HBV infection post-transplantation. There were no differences in genotype distribution between patients with graft reinfection or lamivudine resistance and patients without recurrence. Mean viral load at recurrence was 148.4 x 10(6) +/- 60.4 x 10(6) copies/mL. The stable group had a similar origin and HBV genotype prevalence, but a lower mean viral load of 12.4 x 10(6) +/- 29.4 x 10(6) copies/mL (p = 0.007). The prevalence of mutations at the precore region and codon 28 was similar in both groups. CONCLUSIONS: The chronic precore mutant HBV-infected patients were characterized as follows: (i) genotype D was the most frequent genotype, (ii) the HBV genotype distribution was similar in patients with stable infection and after liver transplantation, (iii) viral load at recurrence was significantly higher than in stable infection, and (iv) HBV genotype was unrelated to the development of recurrence or lamivudine resistance in the tested population.

Genotypic and phenotypic resistance: longitudinal and sequential analysis of hepatitis B virus polymerase mutations in patients with lamivudine resistance after liver transplantation.

OBJECTIVE: Lamivudine-resistant strains appear in 27-62.5% of liver transplant recipients treated with lamivudine for hepatitis B virus (HBV) recurrence, and may lead to failure of antiviral therapy. In an extension of our previous study, we investigated the molecular events associated with the emergence of lamivudine-resistant mutants in this population. METHODS: Sequential serum samples from 10 consecutive patients with lamivudine resistance after liver transplantation were analyzed for viral genotype, precore mutants, and viral polymerase gene mutants (L528M, M552V, M552I) using restriction fragment length polymorphism. Quantitative analysis of HBV DNA was performed using hybridization assay and polymerase chain reaction. RESULTS: Eight patients (80%) were infected with genotype D and two (20%) with genotype C. Polymerase mutants (genotypic resistance) were identified in all the patients. Phenotypic resistance (rise in serum HBV DNA titers above the detection limit of the hybridization assay) developed in five patients (50%); of the remainder, three (30%) did not have phenotypic resistance, and two were primary nonresponders. Genotypic resistance was detected earlier than phenotypic resistance (median 285 days [range 42-510] vs median 387 days [range 320-420], p = 0.055). In five patients (50%), the emergence of the YMDD mutants took over the wild type; in three (30%), the YMDD mutant took over the wild type, but the wild type re-emerged during lamivudine therapy; and in two (20%), the YMDD mutants were detected in a mixture with the wild type (in different percentages). The mean pretreatment serum ALT level was significantly lower in the patients who did not develop phenotypic resistance (p = 0.0002). The M552I pure viral population was found mainly in these patients, and all retained stable graft function (median follow-up 33 months). A high pretreatment HBV DNA level (>50 x 10(6) copies/ml) was highly statistically significantly correlated with the rapid occurrence of phenotypic resistance (r = -0.90, p = 0.04). CONCLUSIONS: We reached the following conclusions: 1) In our area, liver transplant recipients who develop resistance to lamivudine given for recurrent HBV infection seem to be mainly infected with genotype D. 2) Re-emergence of the wild type can occur during lamivudine therapy. 3) Genotypic resistance precedes phenotypic resistance, although phenotypic resistance does not always follow genotypic resistance. 4) Quantitative determination of viremia and analysis of polymerase gene mutants are recommended for monitoring antiviral therapy of liver transplant patients with HBV reinfection in the graft.

The hepatitis C virus (HCV)-Trimera mouse: a model for evaluation of agents against HCV.

The lack of small-animal models that are suitable for evaluation of agents used to treat infection with hepatitis C virus (HCV) severely hinders the assessment of potential new therapies for the disease. This study created such a model, termed the "HCV-Trimera" model. The HCV-Trimera model was developed by using lethally irradiated mice, reconstituted with SCID mouse bone marrow cells, in which human liver fragments infected ex vivo with HCV had been transplanted. Viremia (positive-strand HCV RNA levels) in HCV-Trimera mice peaked at approximately day 18 after liver transplantation, and an infection rate of 85% was reached. Viral replication in liver grafts was evidenced by the presence of specific negative-strand HCV RNA. The usefulness of this model for evaluation of anti-HCV agents was demonstrated by the ability of a small molecule (an HCV internal ribosomal entry site inhibitor) and an anti-HCV human monoclonal antibody (HCV AB(XTL)68) to reduce virus loads in HCV-Trimera mice in a dose-dependent manner.