Differences in tetracycline resistance determinant carriage among Shigella flexneri and Shigella sonnei are not related to different plasmid Inc-type carriage.

OBJECTIVES: The aim of this study was to establish the prevalence of the most common molecular mechanisms involved in tetracycline resistance as well as their relationship with plasmid incompatibility (Inc) groups in a collection of Shigella spp. causing traveller's diarrhoea. METHODS: Tetracycline susceptibility was established in 187 Shigella spp. (74 Shigella flexneri and 113 Shigella sonnei), of which 153 isolates were recovered as a confirmed cause of traveller's diarrhoea. The prevalence of the tet(A), tet(B) and tet(G) genes was analysed by PCR. Eighteen plasmid Inc groups was determined in a subset of 59 isolates. RESULTS: Among 154 tetracycline-resistant isolates, 122 (79.2%) harboured at least tet(A) or tet(B). The tet(B) gene was the most frequently detected, being present in 70 isolates (45.5%), whilst tet(A) was detected in 57 isolates (37.0%). The tet(G) gene was present in only 11 (7.2%) isolates. Moreover, the tet(A) gene was more frequent in S. sonnei (P=0.0007), whilst the tet(B) gene was more frequent in S. flexneri (P<0.0001). Plasmids belonging to Inc group B (P<0.05) were significantly more frequent among S. flexneri, whilst those belonging to groups K, FIC and FIIA (P<0.05) were preferentially detected among S. sonnei. CONCLUSION: The prevalence of the tet(A) and tet(B) genes differed between S. sonnei and S. flexneri. Moreover, the prevalence of plasmid Inc groups in S. flexneri and S. sonnei differed. However, no relationship was found between the two phenomena.

HEV infection in two referral centers in Spain: epidemiology and clinical outcomes.

BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is one of the major causes of icteric hepatitis worldwide. In industrialized countries it is considered an emerging disease, as a growing number of autochthonous cases have been reported in recent years. Occasional extrahepatic manifestations have been described in the setting of HEV infection. STUDY DESIGN: To characterize the epidemiological pattern and clinical outcomes of new cases of HEV infection diagnosed in two referral centers during the period 2011-2013. RESULTS: During the study period, four cases of self-limited acute hepatitis E after travel to endemic areas were recorded, as well as five cases of HEV infection after solid organ transplantation. Four patients failed to spontaneously clear the virus and received ribavirin monotherapy; all of them had HEV genotype-3. Ribavirin was effective in inhibiting HEV replication, although in one patient a virological relapse occurred after the end of therapy. Finally, we report a case of HEV-genotype-3 related agranulocytosis in an immunocompetent patient, resulting in a fatal outcome; this is the first case reported of its kind. CONCLUSION: Diagnosis of HEV infection needs to be taken into consideration in patients with acute or chronic hepatitis in whom other etiologies have been excluded. Although hematological complications related to acute HEV infection are infrequent, these may affect any of the bone marrow series, even after viral clearance.

Hepatitis C virus infection inhibits P-body granule formation in human livers.

BACKGROUND & AIMS: Decoding the myriad of interactions that hepatitis C virus (HCV) establishes with infected cells is mandatory to obtain a complete understanding of HCV biology and its associated pathogenesis. We and others have previously found that HCV infection disrupts the formation of P-bodies in cell culture. These are cytoplasmic RNA granules with key roles in post-transcriptional regulation of gene expression. Therefore, P-body disruption might have consequences beyond viral propagation. However, whether P-body disruption occurs also in vivo is unknown. Aim of this study was to address this important issue. METHODS: Formalin-fixed paraffin-embedded liver biopsies from four groups of patients (healthy donors, patients with non-virus related liver inflammation, HCV- and HBV-infected patients) were immunostained to detect DDX6 and Dcp1, two core P-body components. Changes in the localization of these proteins were assessed by confocal microscopy. RESULTS: HCV specifically inhibited P-body formation in hepatocytes from human livers regardless of viral genotype, inflammation grade or whether the infection was recent or long established. Importantly, this alteration was reversed once HCV was eliminated by therapy. Furthermore, we observed in vivo an unexpected heterogeneity in P-body composition, which might reflect functional specializations. CONCLUSIONS: This is the first comprehensive in vivo P-body analysis that links a pathogenic condition to P-body alterations. Because of their role in gene expression, the alteration of P-bodies should be further studied to understand fully complex HCV-associated pathologies.

IFNL4 polymorphism predicts response to hepatitis C treatment after liver transplantation.

BACKGROUND: Recent studies in chronic hepatitis C patients have shown that rs368234815 polymorphism nearby IL28B is a better predictor of response to antiviral treatment with pegylated interferon and ribavirin than IL28B polymorphisms (rs12979860 and rs8099917). Its effect could be related to interferon lambda 4 (IFNL4), a protein which seems to confer some paradoxical disadvantages in hepatitis C virus (HCV) immune response. OBJECTIVES: To assess the role of IFNL4 rs368234815 polymorphism on the response to antiviral treatment after liver transplantation (LT). STUDY DESIGN: IFNL4 and IL28B polymorphisms were genotyped in 86 HCV-infected LT recipients and in their donors; all patients had undergone antiviral treatment with pegylated interferon and ribavirin after LT. RESULTS: IFNL4 polymorphism strongly correlated with IL28B ones (p < 0.001). The favorable IFNL4 genotype (TT/TT) was significantly more frequent among donors than recipients (60% donors vs. 22% recipients, p <0.001). Recipient TT/TT genotype was associated with a higher sustained virological response rate after LT (p = 0.024). Nevertheless, the highest sustained virological response frequency was found when both donors and recipients had favorable genotypes (73% vs. 25%, p = 0.002), suggesting a role for donor genotype. CONCLUSIONS: Our study demonstrates that IFNL4 rs368234815 polymorphism is an important predictor of response to antiviral treatment in the LT setting. These findings warrant further studies on IFNL4 role in immune response against HCV.

Early periportal sinusoidal fibrosis is an accurate marker of accelerated HCV recurrence after liver transplantation.

BACKGROUND & AIMS: Significant liver fibrosis (F ⩾ 2) and portal hypertension (hepatic venous pressure gradient [HVPG] ⩾ 6 mmHg) 1 year after liver transplantation (LT) are predictors of severe hepatitis C recurrence. Periportal sinusoidal fibrosis (SF) is an early expression of the fibrogenic process in response to liver injury. We aimed to evaluate whether SF in early liver biopsies represents an early and accurate marker for identifying patients with severe HCV recurrence after LT. METHODS: A total of 101 HCV LT patients with early biopsy (<6 months), and HVPG measurement and/or liver biopsy one year after LT were included. Early biopsies were stained with Sirius Red and SF was graded semi-quantitatively. Results were compared between groups (significant SF vs. non-significant SF) and correlated with the development of severe HCV recurrence one year after LT. RESULTS: Patients with early significant SF had older donor age and higher necroinflammatory activity (NIA). The presence of early significant SF enabled identification of 78.9% and 90.6% of patients with F ⩾ 2 and HVPG ⩾ 6 mmHg, respectively, one year after LT. Donor age and NIA were independent predictors of significant fibrosis (F ⩾ 2) one year after LT, whereas donor age, ALT (3 months), NIA, and SF grade were independent predictors of portal hypertension (HVPG ⩾ 6). CONCLUSIONS: Significant SF in early biopsies is a good predictor of severe hepatitis C recurrence. This histological finding, when combined with simple variables, may be useful to select the best candidates for early antiviral therapy after LT.

Imaging of hepatitis C virus infection in liver grafts after liver transplantation.

BACKGROUND & AIMS: The detection of native hepatitis C virus (HCV) antigens in liver tissue may be relevant to diagnostic purposes and to better understand the pathogenesis of HCV infection. The aim of our study was to characterize HCV antigens in liver grafts. METHODS: We selected 32 liver transplant (LT) recipients with recurrent hepatitis C. HCV core and NS5A antigens were detected in formalin-fixed, paraffin-embedded (FFPE) liver biopsies obtained immediately after graft reperfusion (negative controls), during the acute phase of HCV infection (1-6 months) and during follow-up (7-74 months) after LT. Viral antigens were assessed by immunohistochemistry and confocal microscopy. RESULTS: All reperfusion biopsies were negative for both antigens. Core protein was detected in 75% and 33% of acute phase and follow-up biopsies, respectively. HCV antigens were not detected in any of the 10 samples from patients who cleared HCV after antiviral treatment. Immunostaining was hepatocellular, with a granular cytoplasmic pattern and a wide spectrum of intensity. We found a significant association between viral load and the presence of HCV core-positive hepatocytes (p=0.004). NS5A colocalized strongly with core (66%) and adipophilin (36%), supporting the localization of core and NS5A around lipid droplets. A detailed three-dimensional analysis showed that NS5A surrounded the core and adipophilin-positive areas. CONCLUSIONS: HCV antigens can be detected in FFPE liver biopsies by immunohistochemistry. The in vivo colocalization of core and NS5A proteins around the lipid droplets supports that the latter may play a role in virus particle production, similar to what reported in vitro.

Interplay between basic residues of hepatitis C virus glycoprotein E2 with viral receptors, neutralizing antibodies and lipoproteins.

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H³86 and R4°8), and the two highly conserved basic residues H488 and R648 contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.

Fitness and molecular mechanisms of resistance to rifaximin in in vitro selected Escherichia coli mutants.

AIMS: This study sought to analyze the molecular mechanisms contributing to the development of rifaximin (Rfx) resistance in vitro in Escherichia coli. Twenty-eight Rfx-resistant mutants as well as four clinical isolates of E. coli were analyzed. The results obtained show that mutations in the rpoB gene and overexpression of Phe-Arg-ß-naphthylamide (PAßN)-inhibitible efflux pump were implicated in Rfx resistance. RESULTS: Amino acid substitutions at position 516 of the ß-subunit of RNA polymerases were the most frequently obtained (53.6% of the mutants). The efflux pump inhibitor decreased the minimal inhibitory concentration (MIC) of 71.43% (20/28) of the mutant strains. CONCLUSIONS: Mutations studied in the rpoB gene and overexpression of PAßN-inhibitible efflux pumps contribute to Rfx resistance (together or not), whereas alterations in porin levels do not seem to have a relevant role in the acquisition of Rfx resistance.

Cell culture replication of a genotype 1b hepatitis C virus isolate cloned from a patient who underwent liver transplantation.

The introduction of the genotype 2a isolate JFH1 was a major breakthrough in the field of hepatitis C virus (HCV), allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancing mutations. However, such mutations may interfere with viral assembly, as occurs in the case of the genotype 1b isolate Con1. In this study, we show that a clinical serum carrying a genotype 1b virus with an exceptionally high viral load was able to infect Huh7.5 cells. Similar to previous reports, inoculation of Huh7.5 cells by natural virus is very inefficient compared to infection by cell culture HCV. A consensus sequence of a new genotype 1b HCV isolate was cloned from the clinical serum (designated Barcelona HCV1), and then subjected to replication studies. This virus replicated poorly in a transient fashion in Huh7.5 cells after electroporation with in vitro transcribed RNA. Nonetheless, approximately 3 weeks post electroporation and thereafter, core protein-positive cells were detected by immunofluorescence. Surprisingly, small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles might be assembled and released. Our findings not only enhance the current method of cloning in vitro HCV replication-competent isolates, but also offer valuable insights for the realization of fully competent culture systems for HCV.