Liver protein profiling in chronic hepatitis C: identification of potential predictive markers for interferon therapy outcome.

The current anti-hepatitis C virus (HCV) therapy, based on pegylated-interferon alpha and ribavirin, has limited success rate and is accompanied by several side effects. The aim of this study was to identify protein profiles in pretreatment liver biopsies of HCV patients correlating with the outcome of antiviral therapy. Cytosolic or membrane/organelle-enriched protein extracts from liver biopsies of eight HCV patients were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Overall, this analysis identified 21 proteins whose expression levels correlate with therapy response. These factors are involved in interferon-mediated antiviral activity, stress response, and energy metabolism. Moreover, we found that post-translational modifications of dihydroxyacetone kinase were also associated with therapy outcome. Differential expression of the five best performing markers (STAT1, Mx1, DD4, DAK, and PD-ECGF) was confirmed by immunoblotting assays in an independent group of HCV patients. Finally, we showed that a prediction model based on the expression levels of these markers classifies responder and nonresponder patients with an accuracy of 85.7%. These results provide evidence that the analysis of pretreatment liver protein profiles is valuable for discriminating between responder and nonresponder HCV patients, and may contribute to reduce the number of nonresponder patients exposed to therapy-associated risks.

High HIV prevalence in male patients with acute hepatitis A in the Rome metropolitan area, Italy 2002-2008.

BACKGROUND & AIMS: Several outbreaks of hepatitis A affecting homosexual men have been reported in Europe. However, the prevalence of HIV infection in patients affected by hepatitis A has not been extensively studied and hepatitis A is not considered as an indicator disease for routine HIV testing. METHODS: We retrospectively analyzed all adult cases of acute hepatitis A, reported by the National Institute of Infectious Disease "L. Spallanzani", Rome-Italy, in 2002-2008. Data on HIV infection were obtained by chart review and cross-linkage with laboratory. Information on exposure to risk factors were collected from the standard questionnaire of the Local Health Unit. RESULTS: We analyzed a total of 473 cases of hepatitis A, 368 (77.2%) males that accounted for 75% of all reported cases in Rome, aged 25-64 years (same gender distribution). During the study period, we diagnosed a high proportion of cases among male individuals (78%). Among the male patients, HIV serology was available for 203/368 (55.2%). The overall HIV prevalence was 15.2% (56/368); it was significantly associated with same gender sex and was significantly higher than that observed among patients with hepatitis B (4.0%). CONCLUSIONS: We found a high HIV prevalence, associated with same gender sex, among adult male patients diagnosed with hepatitis A in the period 2002-2008, except for 2006. Our data suggest that in a low incidence area for hepatitis A, with a constant high proportion of cases among male individuals, all individuals with acute hepatitis A should be routinely offered an HIV test.

gp120 modulates the biology of human hepatic stellate cells: a link between HIV infection and liver fibrogenesis.

OBJECTIVE: In patients with hepatitis C virus (HCV)/HIV co-infection, a faster progression of liver fibrosis to cirrhosis has been reported. In this study, an investigation was carried out to determine whether gp120, an HIV envelope protein, modulates the biology of human hepatic stellate cells (HSCs), key cell types in the pathogenesis of fibrosis. METHODS: Myofibroblastic HSCs were isolated from normal human liver tissue. Gene expression was measured by real-time PCR. Cell migration was assessed in Boyden chambers. Intracellular signalling pathways were evaluated using phosphorylation-specific antibodies or by transfection of a reporter plasmid. RESULTS: Transcripts for the chemokine receptors CCR5 and CXCR4, which bind gp120, were detectable in human HSCs. Upon exposure to M-tropic recombinant gp120, which binds CCR5, a significant increase in HSC chemotaxis was observed (1.6+/-0.3-fold, p=0.03). The effects of gp120 were prevented by protein inactivation. gp120 also resulted in a significant increase in secretion (1.5+/-0.3-fold, p=0.03) and gene expression (1.47+/-0.13-fold, p=0.02) of the proinflammatory chemokine monocyte chemoattractant protein-1, and in increased gene expression of tissue inhibitor of metalloprotease-1 and interleukin-6 (2.03+/-0.57-fold, p=0.02). gp120-induced migration required Akt activation. gp120 also induced activation of nuclear factor-kappaB (NF-kappaB) and p38(MAPK). Preincubation of HSCs with TAK779, a CCR5 receptor antagonist, prevented gp120-mediated chemotaxis and monocyte chemoattractant protein-1 secretion. Expression of CCR5 was detectable in areas of inflammation and fibrogenesis in liver biopsies of patients with HCV/HIV co-infection. CONCLUSIONS: This study shows that HIV gp120 modulates different aspects of HSC biology, including directional cell movement and expression of proinflammatory cytokines. These results identify a direct pathway possibly linking HIV infection with liver fibrogenesis via envelope proteins.

Patterns of chronic hepatitis B in Central Italy: a cross-sectional study.

We investigated the patterns of chronic hepatitis B virus (HBV)-related disease in a large cohort of HBsAg-positive patients, in Central Italy, by collecting a screening form with demographic, clinical and laboratory data. Overall, 737 HBsAg-positive cases were included (70% male; median age 52 years): 30% were inactive HBsAg carriers, 51% had chronic hepatitis B (CHB) and 19% had HBV-related cirrhosis. Patients from non-European Union (EU) countries (n = 65) were significantly younger, had a higher prevalence of HBeAg-positive infection and hepatitis delta virus (HDV) co-infection than patients of Italian origin. Therefore, as immigration from non-EU countries continues to grow, we can expect a change in the landscape of HBV-related disease in our area.

Effect of HIV co-infection on mutation patterns of HBV in patients with lamivudine-resistant chronic hepatitis B.

A retrospective review was performed comparing lamivudine-resistance mutation patterns between patients infected with hepatitis B virus (HBV) with or without human immunodeficiency virus (HIV) co-infection. Medical records that included a genotypic test of patients infected with HBV and treated with lamivudine as the only anti-HBV drug were reviewed. Pol gene mutations were assessed by direct sequencing of the reverse transcriptase fragment 125-213 aa. Eighty-nine patients infected with HBV (29 co-infected with HIV) with rtM204V or rtM204I mutations were included. Multiple mutations associated with the YMDD motif were observed in 33 (55%) of 60 patients infected with HBV only and in 28 (96.6%) of patients co-infected with HIV/HBV. In this latter group, the prevalence of the rtV173L + rtL180M + rtM204V triple mutation was 31% versus a prevalence of 3.4% observed among patients infected with HBV only. All patients with the triple mutational pattern showed sE164D + sI195M changes in the envelope gene. Multivariate analysis demonstrated that HIV co-infection (adjusted OR 11.2, 95% CI 2.0-61.0) and HBV genotype A (adjusted OR 7.2, 95% CI 1.5-34.8) were the only independent variables associated with the chance of harboring rtM204V. Patients with HBV genotype A or HIV co-infection were more likely to harbor the rtM204V mutation. Patients co-infected with HIV showed multiple mutations more frequently, including the triple mutation that may elicit a vaccine escape phenotype. Among patients co-infected with HIV/HBV, strict HBV DNA monitoring is essential to detect treatment failure and adapt therapy to avoid limitations of future therapeutic options or the emergence of a public health threat.

Interferon may prevent HIV viral rebound after HAART interruption in HIV patients.

In the pre-highly active antiretroviral therapy (HAART) era, clinical trials showed that interferon (IFN) treatment was able to delay AIDS progression and prolong survival. Along with HAART, ancillary use of IFN during primary infection and before HAART therapy initiation has been effective. Also endogenous IFN may positively affect the progression of HIV infection, as suggested in GB virus type C (GBV-C) coinfected patients. In this pilot study, we tried to prevent rebound of HIV replication in patients who interrupted HAART by covering the drug-free time with administration of pegylated IFN (PEGIFN). Twenty-four HIV-hepatitis C virus (HCV) patients who started IFN treatment for liver disease, after variable time from having interrupted HAART, were enrolled. HIV RNA was determined during a 2-month period. In patients who interrupted HAART at variable times before initiating IFN and, therefore, had experienced a complete viral rebound, IFN caused a significant reduction of viral load lasting at least 4 weeks. Moreover, 3 of the 4 patients who started IFN concomitantly with the HAART discontinuation showed complete control of viral rebound, delaying the resumption of viral replication for more than 2 weeks. These preliminary findings suggest that a structured therapy interruptions (STI) strategy may be feasible provided that IFN is administered during the drug-free times, possibly delaying drug resistance, lessening toxicity, reducing costs, and prolonging survival.

Is the increased risk of liver enzyme elevation in patients co-infected with HIV and hepatitis virus greater in those taking antiretroviral therapy?

OBJECTIVES: To investigate if the risk of liver enzyme elevation (LEE) in HIV/hepatitis B or C (HBV, HCV) co-infection is altered by HAART (two or more drugs). METHODS: Analysis comprised HIV-positive patients in the ICoNA study without acute hepatitis who had >or= 1 positive HCV antibody test and > 1 positive HBV surface antigen test. LEE was defined as > 5x baseline alanine aminotransferase (ALT) or > 3.5x baseline if the baseline was > 40 IU/l. Analysis used Poisson regression with generalized estimating equation correction to examine HBV or HCV co-infection, use of HAART, baseline ALT and demographics as LEE predictors. RESULTS: Of the 5272 patients, 47.6% were co-infected with HCV/HBV; 29.9% were female and 39% were intravenous drug users. There were 275 episodes of LEE during 18 259 person-years follow up. Taking HAART did not significantly increase risk of LEE [adjusted relative risk (RR), 1.19; 95% confidence interval (CI), 0.81-1.75; P = 0.37]. Co-infection increased the risk of LEE (adjusted RR, 5.07; 95% CI, 3.47-7.48; P < 0.001), with no significant differences if taking HAART (adjusted RR, 4.99; 95% CI, 3.38-7.37) or not (adjusted RR, 6.02; 95% CI, 2.02-17.98) (P = 0.74 for interaction). Females were at lower risk of LEE than males (adjusted RR, 0.59; 95% CI, 0.42-0.83; P = 0.02). CONCLUSIONS: HIV and HBV/HCV co-infection per se is associated with increased risk of LEE that is not modified by HAART. The recommendation for caution in HAART use in co-infected patients, simply based on a high rate of LEE in people on therapy, may be questionable.

Analysis of occult hepatitis B virus infection in liver tissue of HIV patients with chronic hepatitis C.

OBJECTIVE: Current data on the prevalence of occult hepatitis B virus (HBV) infection in HIV-positive individuals conflict. As occult HBV infection could have an impact on the outcome of liver disease in HIV-positive patients, we investigated a large number of HIV-positive/HBV-surface-antigen (HBsAg) negative subjects with hepatitis C virus (HCV) infection by using the 'gold standard' approach for occult HBV detection--analysis of liver DNA extracts. METHODS: The presence or absence of HBV DNA was determined by PCR testing of four different viral genomic regions in DNA extracts of needle liver biopsy specimens of 101 HBsAg negative individuals with HIV/HCV co-infection. HBV genotyping was performed by sequencing analysis of the preS-S gene in occult HBV isolates from 18 cases. RESULTS: Occult HBV infection was diagnosed in 42 of the 101 cases (41%). No clinically relevant difference was found between occult HBV-positive and -negative patients. HBV genotype D and A were detected, respectively, in 11 (61%) and 7 (39%) of 18 cases analysed. CONCLUSIONS: Occult HBV infection frequently occurs in HIV/HCV co-infected patients indicating the importance of performing prospective studies able to clarify its clinical impact in these patients. HBV genotype A is highly prevalent in HIV-infected subjects with occult HBV infection in a similar way to HBsAg/HIV-positive individuals.

Evolution of HVR-1 quasispecies after 1-year treatment in HIV/HCV-coinfected patients according to the pattern of response to highly active antiretroviral therapy.

Hepatitis C virus (HCV) variability is mainly attributed to the ability of the virus to respond to host immune pressure, acting as a driving force for the evolution of quasispecies. This study was aimed at studying the changes in HVR-1 heterogeneity and the evolution of HCV quasispecies in HIV/HCV-coinfected patients according to the pattern of response to highly active antiretroviral therapy (HAART). Sixteen HIV/HCV-coinfected patients harbouring HCV genotype 1 and who had been on HAART for at least 1 year, 8 showing increasing CD4+ T-cell counts (immunological responders) and 8 showing a stable or decreasing CD4+ T-cell counts (immunological nonresponders), were selected from a prospective cohort study. After 1 year of HAART, 11 patients showed HIV viral load <2.6 log10 cp/ml (virological responders), and 5 showed HIV viral load above this value (virological non-responders). Plasma samples, collected before starting therapy and after 1 year of HAART, underwent clonal sequence analysis for HVR-1 region of HCV. Nonsynonymous/synonymous substitutions ratio (Ka/Ks), aminoacidic complexity (normalized Shannon entropy) and diversity (p-distance), were considered as parameters of quasispecies heterogeneity. After 1 year of HAART, heterogeneity of HVR-1 quasispecies significantly decreased in virological non-responders, whereas the heterogeneity tended to increase in virological responders. The differences in the evolution were less stringent, when considering immunological response. On the other hand, profound qualitative modifications of HVR-1 quasispecies were observed only in patients with both immunological and virological HAART response. On the whole, these findings suggest that, in patients undergoing HAART, the extent of HCV variability and the evolution of HVR-1 quasispecies is influenced by the pattern of response to antiretroviral therapy.

Role of hepatitis C virus (HCV) viremia and HCV genotype in the immune recovery from highly active antiretroviral therapy in a cohort of antiretroviral-naive HIV-infected individuals.

BACKGROUND: The roles of hepatitis C virus (HCV) viremia and HCV genotype in the immune response to highly active antiretroviral therapy (HAART) are poorly understood. Our aim was to assess the CD4+ cell count recovery after HAART in human immunodeficiency virus (HIV)-infected patients with HCV viremia and HIV-infected patients who tested negative for HCV antibody (HCV-Ab). We also aimed to assess whether the response to HAART in these patients varied according to HCV genotype. METHODS: The analysis focused on 1219 HCV-Ab-negative patients and 284 HCV-viremic patients from a cohort of HIV-infected subjects that includes persons who were antiretroviral naive before initiating HAART after cohort enrollment. HCV RNA load and HCV genotype were determined in plasma specimens obtained and stored during the 6-month period preceding the initiation of HAART. RESULTS: The chance of achieving a CD4+ cell count increase of > or = 100 cells/microL from the pre-HAART level tended to be poorer in HCV-viremic patients than in patients who tested negative for HCV-Ab (adjusted relative hazard [RH], 0.82; 95% confidence interval [CI], 0.66-1.01; P = .06). In contrast, a comparison of patients who had a HCV RNA load >1 x 10(6) IU/mL with patients who had a HCV RNA load of 5-1 x 10(6) IU/mL revealed no significant association between HCV RNA load and achievement of an increased CD4+ cell count (adjusted RH, 0.97; 95% CI, 0.75-1.27; P = .83). There was no clear association between HCV genotype and the probability of achieving a CD4+ cell count increase. CONCLUSIONS: An association between the presence of HCV-Ab and immune reconstitution after HAART has been shown elsewhere. Results of our large, prospective study support a direct role of HCV viremia in the CD4+ cell count response to HAART. Moreover, our results underline the fact that, in individuals coinfected with HIV and HCV, the goal of treating HCV infection is to eradicate HCV, to both slow the rate of HCV progression and limit potential interference with the response to HAART.

Response to HAART and GB virus type C coinfection in a cohort of antiretroviral-naive HIV-infected individuals.

The prognostic role of GB virus type C (GBV-C) viraemia in HIV-infected subjects treated with highly active antiretroviral therapy (HAART) is still undefined. The aim of this analysis is to assess the relationship between GBV-C infection and response to antiretroviral therapy among HIV-infected subjects initiating HAART when antiretroviral-naive. A prospective, observational study of 400 HIV-infected patients with measurements of GBV-C RNA, hepatitis C virus (HCV) antibodies and HCV RNA determined from plasma stored prior to HAART initiation. Time to virological (achieving HIV RNA < or =500 copies/ml) and immunological success (a CD4+ count increase of > or =200 cells/microl), and the time to virological relapse (confirmed HIV RNA >500 copies/ml) were assessed by Kaplan-Meier methods and Cox proportional hazard regression model. Of the subjects, 117 (29.3%) were GBV-C positive and, overall, 351 (87.8%) patients achieved virological success. After controlling for a number of confounders including HCV RNA, GBV-C viraemic patients experienced a significantly lower risk of HIV rebound than those who were GBV-C negative [relative hazard (RH)=0.56, 95% CI: 0.34-0.93, P=0.03]. Conversely, the probability of achieving initial virological success or CD4+ count response after HAART did not differ between GBV-C-negative and -positive subjects. These results suggest that GBV-C coinfection may play a role in determining the rate of HIV rebound possibly by competing with HIV replication after HIV load has been successfully suppressed by HAART.

Incidence of adipose tissue alterations in first-line antiretroviral therapy: the LipoICoNa Study.

BACKGROUND: Adipose tissue alterations (ATAs) are a frequent untoward effect of antiretroviral therapy, the causes of which remain incompletely explained. OBJECTIVES: To assess the incidence of ATAs and to identify the associated risk factors in patients infected with human immunodeficiency virus type 1 starting their first-line antiretroviral treatment. METHODS: In a multicenter investigation designed to study issues related to the treatment of patients starting antiretroviral therapy, physicians were requested to assess the presence of ATAs at enrollment and every 6 months thereafter. The ATAs were considered altogether and grouped as fat loss (lipoatrophy), adipose tissue accumulation (lipohypertrophy), and combined forms. RESULTS: A total of 655 patients were followed up for a median of 86 weeks; 128 patients (19.6%) were diagnosed as having at least 1 morphologic alteration during the study. Female gender and positivity for hepatitis C virus were independently linked to an increased risk of developing morphologic alterations. Age was another independent correlate of risk of developing ATAs. To have been infected through drug injection was a correlate of reduced risk of ATAs. Stavudine exposure was predictive at borderline statistical significance of lipoatrophy (but not of the other forms), and indinavir exposure was associated with a significantly higher risk of developing combined forms. Patients who started therapy with 2 nucleoside reverse transcriptase inhibitors and subsequently added a protease inhibitor during the follow-up had a significantly higher risk of having ATAs compared with patients who continued taking 2 nucleoside reverse transcriptase inhibitors up to the end of follow-up. CONCLUSIONS: Different types of ATAs might derive from distinct pathways and multifactorial causes. Adipose tissue alterations are a frequent and relatively early finding during first-line antiretroviral therapy.

ISDR pattern and evolution in patients with chronic hepatitis C treated with standard or PEG-IFN plus ribavirin.

The aim of the study was to characterize the interferon sensitivity determining region (ISDR) mutation pattern and its changes at 4 weeks of treatment in a population of patients infected with hepatitis C virus (HCV) genotype 1b receiving standard or PEG-IFN plus ribavirin (RBV), to find possible early correlates of therapy outcome. Forty-five patients with chronic hepatitis due to HCV 1b were treated by PEG-IFN-alpha2b (n=23) or IFN-alpha2b (n=22) plus RBV 1000-1200 mg/day. They were classified 24 weeks after stopping therapy as sustained responders (SR), relapsers (REL) or non-responders (NR). Sixteen patients were SR, 12 REL and 17 NR. ISDR mutations were evaluated by direct sequencing at baseline in all and after 4 weeks in patients with detectable viraemia (n=30). The frequency of the three ISDR types was 26.7% wild-type, 64.4% intermediate-type and 8.9% mutant-type, without significant difference in their frequency in SR, REL and NR, independent of IFN formulation. Average numbers of mutations in SR, REL and NR were 1.88 +/- 0.54, 1.33 +/- 0.33 and 0.94 +/- 0.25, respectively, P>0.05. The baseline number of ISDR mutations was not related to the extent of viral load decline in the first month of therapy. Sequence analysis of ISDR region performed 4 weeks after starting therapy revealed qualitative or quantitative changes of ISDR sequence in only seven patients, without correlation with response. Thus, in our patients the baseline pattern of ISDR was unrelated to treatment outcome. Selection towards a dominant IFN-resistant strain did not occur under standard or PEG-IFN plus RBV.

Access to treatment for HBV infection and its consistency with 2008 European guidelines in a multicentre cross-sectional study of HIV/HBV co-infected patients in Italy.

BACKGROUND: A survey was performed in 2008 to evaluate the profiles of patients with chronic hepatitis B cared for by Italian Infectious Diseases Centers (IDCs). This analysis describes: i) factors associated with access to the anti-HBV treatment in a cohort of HIV/HBV co-infected patients cared for in tertiary centers of a developed country with comprehensive coverage under the National Health System (NHS); ii) consistency of current anti-HBV regimens with specific European guidelines in force at the time of the study and factors associated with the receipt of sub-optimal regimens. METHODS: The study focuses on 374 (87.6%) treated patients at some point in their life out of the 427 tested HIV/HBV positive. It is multicentre, cross-sectional in the design. To account for missing values, a Multiple Imputation method is used. RESULTS: Three hundred and thirty-four (89.3%) patients were currently treated. The most common current regimen was combination therapy of tenofovir (TDF) plus LAM/FTC (lamivudine/emtricitabine) (n = 235, 70.4%), as part of antiretroviral treatment. In the multivariate analysis, an increased chance of getting treated was independently associated with increasing years since HBV diagnosis (2-10 years, p <0.001; >10 years, p <0.001). Patients consistently treated with European AIDS Clinical Society (EACS) 2008 guidelines were 255 (76.6%), of whom 202 (79.2%) with an indication to an anti-HIV treatment, 30 (11.8%)without an indication, and 21 (8.2%) with cirrhosis. Among the 78 not-consistent patients, LAM mono-therapy (n = 60, 76.9%) was the most common regimen, 34 (56.7%) of them showing HBV DNA load below 1x10(3) IU/mL. Previous anti-HBV treatment (p = 0.01) and a triple HDV co-infection (p = 0.03) reduced the chance of not-consistent regimens. Conversely, HCV co-infection was independently associated with an increased odds ratio of being inconsistently treated (p = 0.004). CONCLUSION: Our study shows that Italian IDCs treat for HBV infection the vast majority of HIV/HBV co-infected patients with no disparities limiting access to antiviral therapy. In approximately two-thirds of the patients on treatment, anti-HBV regimens are consistent with 2008 EACS guidelines. Finally, our study identifies scenarios in which clinical practice deviates from recommendations, as in case of sub-optimal regimens with effective anti-HBV response.

A randomized controlled trial of pegylated interferon-alpha2a plus adefovir dipivoxil for hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND: Pegylated interferon (PEG-IFN)-alpha monotherapy is the current standard of care for short-term antiviral treatment of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB). We aimed to assess the safety and efficacy of PEG-IFN-alpha plus adefovir dipivoxil (ADV) versus PEG-IFN-alpha monotherapy for compensated HBeAg-negative CHB. METHODS: A multicentre randomized controlled trial was performed in eight outpatient hepatology/infectious disease clinics in central Italy. A total of 60 patients (67% male and median age 48 years) with biopsy-proven HBeAg-negative compensated CHB (mean alanine aminotranferase [ALT] levels 3.3 +/-3x the upper normal limit and serum hepatitis B virus [HBV] DNA 5.8 +/-0.9 log(10) IU/ml) were randomized at baseline to receive PEG-IFN-alpha2a 180 microg/week plus ADV 10 mg/day or PEG-IFN-alpha2a monotherapy for 48 weeks. Post-treatment follow-up was for 24 additional weeks. The primary end point was sustained HBV DNA suppression defined as serum HBV DNA<2,000 IU/ml after 24 weeks of post-treatment follow-up. The secondary end point was ALT normalization at the end of follow-up. RESULTS: At week 48, HBV DNA was undetectable in 20/30 (67%) in the combination group versus 11/30 (37%) patients in the monotherapy group (P=0.02). ALT normalization was achieved in 17/30 (57%) versus 10/30 (30%) patients, respectively (P=0.03). At week 72, sustained virological response was achieved in 7/30 (23.3%) in the combination group versus 6/30 (20%) patients in the monotherapy group (P=0.75); 5 (16%) patients in each group dropped out because of adverse events or non-compliance. CONCLUSIONS: In HBeAg-negative CHB, combination PEG-IFN-alpha2a plus ADV for 48 weeks is safe and resulted in greater on-treatment efficacy than PEG-IFN-alpha2a monotherapy. No difference in sustained virological and biochemical response rates were observed between the two treatment regimens.