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Background While HBV DNA has served as a primary biomarker of HBV infection, newer biomarkers including pre-genomic HBV RNA (pgRNA) and quantitative HBV surface antigen (qHBsAg) may prognosticate treatment responses better. We analyzed the changes in these biomarkers over time following initiation of antiviral therapy in persons living with HIV (PLWH) to determine the relationship to ALT, a key marker of liver injury. FigureBiomarker changes Methods NIH AIDS Clinical Trials Group Study 5127 enrolled HBV/HIV PLWH into a Phase 2 treatment trial that randomized them to receive 48 weeks of either tenofovir (TDF) or adefovir (ADV). ALT and HBV DNA were evaluated as measures of treatment response. Stored samples were tested for HBV pgRNA and qHBsAg at baseline, 4, 12, 24, 36, and 48 weeks. HBV pgRNA was isolated using RNA-selective extraction chemistry followed by a multiplex RT-qPCR that targets the HBV X and core targets on the m2000 system (Abbott Molecular, Des Plaines, Illinois). A sensitive second-generation assay with a lower limit of quantification (LLOQ) of 10 copies/mL was utilized. Quantitative HBsAg was determined by Abbott ARCHITECT (List# 6C36) with a LLOQ of 0.05 IU/mL. Data were analyzed using SAS® version 9.4. Results Overall, 47 study participants had sufficient samples for biomarker testing. Participants were predominantly male (93.6%) and identified as white, non-Hispanic (55.3%) or black (34%). Groups were evenly randomized to receive either TDF (n=23) or ADV (n=24). Baseline ALT mean was 64.9 U/L, and HBV DNA was 9.1 (+1.6) copies/mL. Pre-treatment pgRNA serum concentration was 7.0 (+1.3) log U/mL, and qHBsAg was 195,574 (+272,190) IU/mL. Over the course of treatment, pgRNA was significantly correlated with the change in serum ALT levels (r=0.48; p=0.0035) but not HBV DNA (r=0.21; p=0.226). ALT was negatively correlated with qHBsAg (r=-0.52; p=0.02). pgRNA was also highly negatively correlated with qHBsAg (r=-0.49l; p=0.03) but not HBV DNA (r=0.13; p=0.59). (Figure). Conclusion HBV pgRNA is a better predictor of ALT change – a key measure of hepatocyte injury – than HBV DNA in PLWH who are initiated on nucleotide/nucleoside therapy. Quantitative HBsAg is inversely associated with ALT levels, supporting the hypothesis that HBsAg inhibits immune mediated injury to hepatocytes. Disclosures Michael E. Stec, MSc, Abbott Laboratories: Employee of Abbott|Abbott Laboratories: Stocks/Bonds mark C. Anderson, PhD, Abbott Laboratories: Employment|Abbott Laboratories: Stocks/Bonds Marion G. Peters, MD, Aligos: Advisor/Consultant|antios: Advisor/Consultant|Excision Biotherapeutics: DSMB Gavin Cloherty, PhD, Abbott: Employee|Abbott: Stocks/Bonds|Abbott Labs: Employee.

Persistent immune activation is a hallmark of human immunodeficiency virus (HIV) infection and thought to play a role on chronic diseases in people with HIV (PWH). Food insecurity is disproportionately prevalent in PWH and is associated with adverse health outcomes. We determined whether food insecurity was associated with increased plasma levels of soluble CD14, CD27, and CD163 in 323 antiretroviral-treated PWH from the Miami Adult Studies on HIV cohort. Nearly half (42.7%) of participants were food insecure, and 85.5% were virally suppressed (

Background: Hepatitis B virus (HBV) infection is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Due to their shared routes of transmission, approximately 10% of HIV-infected patients worldwide are chronically coinfected with HBV. Additionally, liver disease has become a major cause of morbidity and mortality in HBV/HIV coinfected patients due to prolonged survival with the success of antiretroviral therapy. The relationship between immune exhaustion markers (PD-1/PD-L1) and apoptotic markers such as Fas/FasL, TGFβ1, TNF-α, and Th1/Th2 cytokines are not clearly delineated in HBV/HIV coinfection.Methods: Levels of soluble Fas/FasL, TGFβ1, TNF-α, and sPD-1/sPD-L1 as well as Th1 and Th2 cytokines were evaluated in the sera of HBV-monoinfected (n=30) and HBV/HIV-coinfected (n=15) patients and compared to levels in healthy controls (n=20).Results: HBV-monoinfected patients had significantly lower levels of the anti-inflammatory cytokine IL-4 (P

Hepatitis E virus (HEV) is thought to be common in the United States with increased prevalence in those with concomitant hepatitis C virus (HCV) or HCV/HIV coinfection. Little is known regarding tr...

Background Liver disease remains a significant cause of morbidity and mortality in HIV-infected persons. Soluble CD163 is a marker of Kupffer cell activation that is highly associated with development of hepatic fibrosis. The relative contributions of HIV-associated systemic immune activation vs other etiologies of injury are poorly characterized. Methods We utilized subjects in the Miami Adult Studies on HIV (MASH) cohort to evaluate 464 participants including 361 people with HIV (PWH) and 103 hepatitis C virus (HCV)/HIV-uninfected controls. Subjects underwent testing for hepatic fibrosis using both magnetic resonance elastography and the Enhanced Liver Fibrosis Index. Steatosis was evaluated by magnetic resonance imaging–derived proton density fat fraction. Immune activation markers and cytokines were quantitated using Luminex methodologies. Results Participants with HIV with or without HCV coinfection had higher levels of sCD163 than uninfected controls (P Conclusions Injury and resultant hepatic fibrosis occur by distinct though overlapping mechanistic pathways. In PWH, sCD163 is highly associated with both injury and fibrosis, suggesting that persistent systemic immune activation is a major contributor to long-term outcomes, adding to damage caused by alcohol, steatosis, and other hepatotoxic drug effects.

OBJECTIVES: Immune activation is central to developing insulin resistance and is implicated in the pathophysiology of liver steatosis. People living with HIV (PLWH) have elevated biomarkers of immune activation, which may play a role in faster development of insulin resistance and liver steatosis. The objective of this study was to examine if HIV status and immune activation are related to insulin resistance and liver steatosis. METHODS: Demographic and anthropometric data on MASH cohort participants were obtained. Insulin resistance was estimated using the triglyceride-glucose (TyG) index from fasting blood. HIV status was abstracted from participants’ medical records. Immune activation biomarkers soluble CD163 (sCD163), sCD27, sCD14, and monocyte chemoattractant protein (MCP-1) measured with multiplex flow cytometry in Dr. Sherman's laboratory. High sensitivity C-Reactive Protein (hsCRP), measure of inflammation, was determined by LabCorp. Liver steatosis was defined as liver fat >3.5% obtained with magnetic resonance elastography scans. Statistics included descriptive analysis, Mann-Whitney tests, multivariate linear, and logistic regressions, controlled for age, sex, and BMI. RESULTS: Of the 712 participants (age 54.24 ± 7.48 years), 336 were PLWH with suppressed viral load, and 376 were uninfected healthy participants. PLWH had higher levels of sCD27 (P = 0.003) and MCP-1 (P = 0.034) than uninfected participants. Multiple linear regressions showed HIV status and sCD163 were independently associated with higher insulin resistance (HIV status b = 0.130, P = 0.014, sCD163 Multiple logistic regressions showed higher levels of sCD163 (OR = 1.097, 95% CI: 1.01–1.19, P = 0.032) and insulin resistance (OR = 7.126, 95% CI: 2.59–19.58, P

Background Accurate noninvasive biomarkers of fibrotic progression are important for hepatitis C virus (HCV) management, but commonly used modalities may have decreased efficacy in human immunodeficiency virus (HIV)/HCV-coinfected persons. The enhanced liver fibrosis (ELF) index is a highly sensitive noninvasive marker of hepatic fibrosis that has had limited assessment in the HIV/HCV population. We compared ELF index performance to FIB4 and aspartate to platelet ratio index (APRI) at different stages of liver fibrosis as determined by liver histology, and validated the efficacy of the three noninvasive biomarkers in HIV/HCV-coinfected versus HCV-monoinfected. Methods The ELF index was determined in 147 HIV/HCV-coinfected and 98 HCV-monoinfected persons using commercial ELISA assays for the component elements of the index. Area under the receiver-operator curve was used to validate ELF and to compare its performance to liver histology as well as to other noninvasive biomarkers of liver fibrosis, FIB4, and APRI. Results The ELF index increased with histological stage of liver fibrosis and exhibited a linear relationship with Metavir score in all subjects. ELF performance was comparable between HIV/HCV and HCV with advanced liver fibrosis/cirrhosis. In the HIV/HCV cohort ELF cutoffs of 8.45 and 9.23 predicted mild and moderate fibrosis with 85% sensitivity, whereas the ELF cutoff of 9.8 had the highest specificity for advanced fibrosis and the cutoff of 10.4 was 99% specific for cirrhosis. ELF performance was superior to FIB4 and APRI in all subjects regardless of HIV status. Conclusions ELF index demonstrated excellent characteristics toward accurate prediction of liver fibrosis and cirrhosis with superior performance to APRI and FIB4 in HIV/HCV coinfection. Applying this noninvasive biomarker index for diagnosis of liver fibrosis and progression in HIV/HCV is warranted.

BACKGROUND AND AIMS: The patatin-like phospholipase domain containing 3 (PNPLA3) gene has been associated with development of alcoholic and non-alcoholic steatohepatitis. Using a newly developed and validated assay for PNPLA3, we explored the prevalence of gene polymorphisms in a cohort of HCV/HIV coinfected individuals to determine if there was an association with insulin resistance or hepatic fibrosis. METHODS: A high-resolution melting point (HRM) assay was developed and validated. The assay was used to evaluate samples obtained in the context of a clinical trial performed at ACTG sites across the U.S. in HIV-infected patients. Clinical features and treatment outcomes were assessed in relation to the PNPLA3 genotype. RESULTS: The HRM methodology demonstrated 100% concordance with results obtained by Sanger sequencing. Among 241 participants tested, 66.0% had the wildtype allele (CC) and the remainder had the aberrant PNPLA3 gene polymorphism in the homozygotic (GG) or heterozygotic (CG) form. Race and ethnicity were associated with PNPLA3 genotype but fibrosis stage, HOMA (Homeostatic Model Assessment of Insulin Resistance), and HCV treatment outcome was not. CONCLUSION: The HRM method is an effective, rapid technique for characterizing PNPLA3 genotype. In those with HCV/HIV infection, nearly 40% carry gene polymorphisms associated with development of NASH or ASH. Prospective studies should focus on this group to determine if they represent a subset of HIV-infected persons at increased risk of fibrotic progression.

OBJECTIVE: To determine the prevalence and epidemiologic associations of Zika Virus (ZIKV) in HIV-infected patients in Ghana, West Africa. METHODS: We examined the seroprevalence of ZIKV in HIV/HBV co-infected persons in Ghana from sera samples collected from 2012 to 2014 using ELISA assays and plaque reduction neutralization tests (PRNT). RESULTS: Overall, ZIKV antibody was detected in 12.9% of 236 tested samples, though the true estimate of exposure is probably less due cross-reactions with other related viruses. PRNTs were performed on a subset to provide an estimate of the frequency of false positive reaction. Dengue virus testing was also performed and antibody prevalence was 87.2%. The median CD4 count was 436 (range 2–1781 cell/mm(3)) and did not affect antibody results. Regional geographic ethnicity was associated with ZIKV exposure. DISCUSSION: Overall, these data suggest that ZIKV infection is a relatively prevalent infection in HIV-positive persons in Ghana though not as common as dengue. Further evaluation of the effect of ZIKV and HIV co-infection is warranted given the large geographical overlap of populations exposed to both viruses.

The prevalence of naturally occurring HCV-NS5A resistance-associated substitutions (RAS) to DAA drugs might affect the response to treatment in HCV/HIV coinfected subjects. There are limited data on the frequency of HCV-NS5A naturally occurring drug-RAS at baseline in HCV/HIV coinfected patients when ultra-deep sequencing methodologies are applied. HCV-NS5A-RAS were evaluated among 25 subjects in each group. Patients were matched by age, gender, and hepatic fibrosis stage category to control for selection bias. Within subtype 1a, RAS were observed in 28% of HCV monoinfected and 48% of HCV/HIV coinfected subjects. More patients in the HCV/HIV coinfected group had clinically relevant mutations to DAA directed at NS5A. While the clinical significance of this observation may be limited in highly drug adherent populations, some HCV/HIV coinfected persons may be at greater risk of viral resistance if suboptimal dosing occurs.

Background and aimThe hepatitis C virus (HCV) is a single-strand RNA virus that infects millions of people worldwide. Recent advances in therapy have led to viral cure using two- and three- drug combinations of direct acting inhibitors of viral replication. CCR5 is a chemokine receptor that is expressed on hepatocytes and represents a key co-receptor for HIV. We evaluated the effect of CCR5 blockade or knockdown on HCV replication in Huh7.5JFH1 cells.MethodsCells were exposed to varying concentrations of maraviroc (CCR5 inhibitor), cenicriviroc (CCR2/CCR5 inhibitor), sofosbuvir (nucleotide polymerase inhibitor), or raltegravir (HIV integrase inhibitor).ResultsHCV RNA was detected utilizing two qualitative strand-specific RT-PCR assays. HCV core antigen and NS3 protein was quantified in the supernatant and cell lysate, respectively. siRNA was utilized to knockdown CCR5 gene expression in hepatocytes. Alternatively, anti-CCR5 antibodies were employed to block the receptor. Supernatant levels of HCV RNA (expressed as fold change) were not reduced in the presence of raltegravir but were reduced 8.55-fold and 12.42-fold with cenicriviroc and maraviroc, respectively. Sofosbuvir resulted in a 16.20-fold change in HCV RNA levels. HCV core and NS3 protein production was also reduced in a dose-dependent manner. Two distinct anti-CCR5 antibodies also resulted in a significant reduction in HCV protein expression, as did siRNA knockdown of CCR5 gene expression.ConclusionsThese data provide evidence that CCR5 modulation could have a significant effect on HCV replication in an in vitro system. Further evaluation of the role of CCR5 inhibition in clinical settings may be warranted.

Noninvasive fibrosis markers are routinely used in patients with liver disease. Magnetic resonance elastography (MRE) is recognized as a highly accurate methodology, but a reliable blood test for fibrosis would be useful. We examined performance characteristics of the Enhanced Liver Fibrosis (ELF) Index compared to MRE in a cohort including those with HCV, HIV, and HCV/HIV. Subjects enrolled in the Miami Adult Studies on HIV (MASH) cohort underwent MRE and blood sampling. The ELF Index was scored and receiver–operator curves constructed to determine optimal cutoff levels relative to performance characteristics. Cytokine testing was performed to identify new markers to enhance noninvasive marker development. The ELF Index was determined in 459 subjects; more than half were male, non-white, and HIV-infected. MRE was obtained on a subset of 283 subjects and the group that had both studies served as the basis of the receiver–operator curve analysis. At an ELF Index of > 10.633, the area under the curve for cirrhosis (Metavir F4, MRE > 4.62 kPa) was 0.986 (95% CI 0.994–0.996; p

The IL28B gene is associated with spontaneous or treatment-induced HCV viral clearance. However, the mechanism by which the IL28B single nucleotide polymorphism (SNP) affects the extra-hepatic HCV immune responses and its relationship to HCV pathogenesis have not been thoroughly investigated. To examine the mechanism by which IL28B affects HCV clearance. Forty Egyptian patients with chronic HCV infection receiving an Interferon/ribavirin treatment regimen were enrolled into this study. There were two groups: non-responders (NR; n = 20) and sustained virologic responders (SVR; n = 20). The initial plasma HCV viral loads prior to treatment and IL28B genotypes were determined by quantitative RT-PCR and sequencing, respectively. Liver biopsies were examined to determine the inflammatory score and the stage of fibrosis. Colonic regulatory T cell (Treg) frequency was estimated by immunohistochemistry. No significant association between IL28B genotypes and response to therapy was identified, despite an odds ratio of 3.4 to have the TT genotype in NR compared to SVR (95 % confidence interval 0.3-35.3, p = 0.3). Patients with the TT-IL28Brs12979860 genotype (unfavorable genotype) have significantly higher frequencies of colonic Treg compared to the CT (p = 0.04) and CC (p = 0.03) genotypes. The frequency of colonic Treg cells in HCV-infected patients had a strong association with the IL-28B genotype and may have a significant impact on HCV clearance.

The efficacy of protease inhibitor drugs in hepatitis C virus (HCV) treatment is limited by the selection and expansion of drug-resistant mutations. HCV replication is error-prone and genetic variability within the dominant epitopes ensures its persistence. The aims of this study are to evaluate the role of cellular immune response in the emergence of HCV protease resistance mutations and its effects on treatment outcome. Ten chronically HCV-infected subjects were treated with boceprevir (BOC)-based triple therapy. HCV-RNA was tested for BOC resistance-associated viral variants. HCV protease resistance mutations were investigated pretreatment and 24 weeks post-treatment. Synthetic peptides representing the wild-type and the potential nonstructural (NS)3 variants were used to evaluate T cell responses and human leukocyte antigen binding. Sustained viral response was achieved in 70% of patients, two patients were treatment nonresponders (NRs) and one was classified as a relapse. Pretreatment, the proportion of drug-resistant variants within individuals was higher in sustained viral responders (SVRs) than in NR patients. However, resistance-associated variants increased in NRs after BOC combined triple therapy. In contrast to NR patients, significant stronger cell-mediated immune responses were observed at the baseline among those who achieved sustained viral response for all T cell epitopes tested. Despite the increase in cell-mediated immune responses at week 24 in NRs, they failed to control the virus replication, leading to development of overt drug-resistant variants. Our data suggest that strong NS3-specific T cell immune responses at the baseline may predict a positive outcome of directly acting antiviral-based therapy, and the presence of pre-existent resistance mutations does not play a significant role in the outcome of anti-HCV combined therapy.

Zika virus (ZIKV) has emerged globally as an important pathogen, since it has been recognized as a cause of microcephaly and other neurologic processes and sequalae in newborns. The virus shares homology with Hepaciviruses and therefore may be a cause of hepatitis. We sought to characterize ZIKV replication in hepatocyte-derived cell lines. Huh7.5 and HepG2 cells were infected with ZIKV and replication potential was evaluated by multiple methods including plaque assay, qRT-PCR, negative-strand ZIKV RNA production, and ZIKV NS1 protein production. Growth curves in cells and supernatant were compared to replicative capacity in Vero cells. Overall, viral replication in both hepatocyte lines approximated that observed in the Vero cells. Cell cytopathology was observed after 3 days of infection and apoptosis markers increased. Transmission electron microscopy revealed evidence of viral capsids in cells and negative staining revealed ZIKV particles in the supernatant. Conclusions: Hepatocyte-derived cell lines are permissive for ZIKV replication and produce an overt cytopathic effect consistent with development of an acute viral hepatitis. Further evaluation of replication and injury is warranted.

Background The C-C chemokine receptor Type 5 (CCR5) is a key receptor for human immunodeficiency virus type 1 (HIV-1) entry into T-cells and a variant allele, CCR5 delta-32, is associated with decreased viral replication and disease progression. Active HIV-1 replication is highly associated with accelerated rates of hepatic fibrosis. We postulated that CCR5 plays a role in the development of hepatic fibrosis and evaluated the longitudinal effect of natural or drug-induced CCR5 mutation and blockade on biomarkers of liver fibrosis in HIV-1 patients. Methods To accomplish this goal, we examined 2 distinct cohorts. First, we evaluated fibrosis markers in the Multicenter Hemophilia Cohort Studies (MHCS), which included subjects with HIV and hepatitis C virus (HCV) coinfection with the CCR5 delta-32 allele. We also evaluated an HIV-1 infected cohort that was treated with a dual CCR5/CCR2 antagonist, cenicriviroc. The enhanced liver fibrosis (ELF) index was validated against liver histology obtained from HCV/HIV and HCV patients and demonstrated strong correlation with fibrosis stage. Results In both the MHCS patients and patients treated with cenicriviroc, CCR5 mutation or blockade was associated with a significant decrease in the ELF index. Among the patients with the delta-32 allele, the ELF index rate significantly decreased in sequential samples as compared to CCR5 wild-type patients (P = .043). This was not observed in control subjects treated with efavirenz nor with a lower dose of 100 mg cenicriviroc. Conclusion These findings suggest that hepatic fibrosis in HIV-1 infected patients can be modulated by the mutation of CCR5 and/or use of CCR5/CCR2 blockade agents. Clinical Trials Registration NCT01338883.

Background Hepatic fibrosis in individuals with HCV/HIV coinfection or HCV mono-infection may be modulated by a variety of host factors. In this study, we investigated the role of gene polymorphisms of putative genes that might influence fibrosis progression including MICA (rs2596542), interferon-stimulated gene OAS2 (rs1293762) and the pathogen recognition receptors TLR7 (rs179009) and TLR9 (rs187084). Effect on a cytokine panel was evaluated. Methods Longitudinal samples were obtained from subjects enrolled in the NCI Multicenter Hemophilia Cohort Study. Within the cohort, a subset of subjects were included based upon presence or absence of the CCR5 delta-32 mutation which was previously shown to influence the rate of fibrosis progression. Hepatic fibrosis change was determined using the serum-derived Enhanced Liver Fibrosis (ELF) Index. Four putative genes with polymorphisms that have been previously associated with the development or progression of hepatic fibrosis were evaluated using Taqman SNP genotyping assays. Cytokine assays were performed using Luminex chipsets. Samples were analyzed using Statistix 10.0 using ANOVA and least square regression models. Results 58 unique subjects were evaluated. The mean age was 38 years, and all were male. 74% were HIV infected and 97% were HCV infected (76.8% coinfection). Controlling for the effect of CCR5, only the TLR7 A -> G polymorphism was predictive of change in the ELF Index. There was no statistically significant predictive difference between genotypes in the other three polymorphisms. Subjects with the TLR7 A allele (n = 47) had an average increase in ELF of 0.79 units, while the G allele (n = 11) had an increase in ELF of 2.1 units (P = 0.008). A regression model identified TLR7 as a key factor in ELF change, as well as HCV/HIV coinfection. Interferon alfa-2 levels were highly associated (increased, P = 0.0007) with the TLR7 A -> G polymorphism, while RANTES levels were inversely associated (decreased, P = 0.0443) with it. Conclusion Of the gene polymorphisms investigated, only TLR7 (rs179009) is an independent predictor of development of hepatic fibrosis in HCV/HIV coinfected subjects. The mechanism may involve modulation of inflammatory response pathways. Disclosures All authors: No reported disclosures.

Background Treatment of HCV/HIV coinfection is now largely based on utilization of direct acting agents. Pretreatment viral resistant-associated variants (RAVs) and host liver condition may affect the sustained virological response. In this study, we explored relative prevalence of protease resistance-associated mutations, the evolution of those RAVs after 12 weeks of pegylated interferon alfa exposure, and the role hepatic fibrosis might have on RAV display. Methods Thirty nonresponder HCV/HIV-coinfected subjects were evaluated before and after 12 weeks of PegIFN treatment. Ultra-deep sequence analysis of NS3 RAVs was performed. Hepatic fibrosis was determined by sensitive computer-assisted histomorphometry determination. Results At baseline, protease inhibitor RAVs were present in 73.3% of patients and expanded to 83.3% of patients after 12 weeks of PegIFN exposure. Q80K showed the highest prevalence before and after treatment at 46.7% and 56.7%, respectively. The presence of Q80K is positively correlated with percent collagen content of the liver tissue. Conclusions Key RAVs for HCV protease inhibitors are present in a major portion of the HCV/HIV-coinfected population before therapy. Some variants get selected after exposure. Correlation of Q80K with collagen content of the liver suggests that compartmentalization within the liver may contribute to persistence of mutations less fit than wildtype.

Hepatitis E virus (HEV) has been reported to cause acute and chronic hepatitis in those with HIV infection and among solid organ transplant recipients in Europe. Limited data indicate that HEV is endemic in the United States, but the prevalence and significance of HEV infection among those with HIV and awaiting solid organ transplantation is unknown. We evaluated anti-HEV IgM and IgG antibodies and HEV RNA in 166 HIV-infected solid organ transplant candidates enrolled in the NIH HIV-Transplant Cohort. Overall prevalence of anti-HEV IgG approached 20% in both liver and renal transplant candidates. Evidence of recent infection was present in approximately 2% of liver transplant candidates and none of the kidney transplant candidates. HEV RNA was not detected in any patient. We conclude that markers of HEV infection are frequent among candidates for transplantation, but active, ongoing viremia is not seen. Evidence of recent infection (acute on chronic) liver disease was present in liver but not kidney recipients.

OBJECTIVES: Antiretroviral therapy has increased life expectancy for HIV infected patients; however, this population is developing chronic illnesses associated with aging. Liver disease is a major cause of non-AIDS mortality, characterized by progressive fibrosis. Infection with HIV and with Hepatitis C Virus (HCV) promotes liver fibrogenesis. Tissue inhibitor of metalloproteinase-1 (TIMP1), inhibits fibrosis regression and is profibrogenic. Association between TIMP1 and liver disease progression in an aging population of HIV/HCV co-infected, HIV mono-infected, HCV mono-infected, and healthy groups from the Miami Adult Studies on HIV (MASH) cohort in Miami, Florida, was investigated. METHODS: Serum TIMP1 levels were determined by ELISA. A non-invasive estimate of liver fibrosis, FIB-4 score was calculated. Liver fibrosis was defined as FIB-4: Low 3.25. ANOVA with Tukey's test assessed the mean differences of FIB-4 score and TIMP1 level between groups, TIMP1 levels between 3 FIB-4 categories, and the effect of age on FIB-4 and TIMP1. Linear regression predicted the association of FIB-4 score and TIMP-1 level. RESULTS: Mean age of the cohort was 54.3 ± 8.1 years with no difference between groups. Mean FIB-4 for HIV/HCV co-infected group was the highest among the 4 groups (P

Aim Chronic hepatitis C virus (HCV) infection is an important source of morbidity and mortality among haemophiliacs. Limited data are available regarding treatment intervention using direct-acting antivirals (DAAs) and theoretical concerns regarding accumulation of drug-associated resistance variants (RAVs) remain. We conducted a pilot study of treatment with telaprevir/pegylated interferon-alfa/ribavirin to evaluate treatment response and the role of lead-in DAA therapy on mutational selection of resistance variants. Methods Ultra-deep sequence analysis was performed at baseline, 48 hours and 168 hours after treatment initiation. Results No dominant RAVs were identified at baseline, but low-level RAVs were noted at baseline in all subjects. Viral dynamic models were used to assess treatment responses. The efficacy parameter (Ɛ) for lead-in ranged from 0 to 0.9745 (mean = 0.514). Subsequent addition of telaprevir resulted in a mean efficacy of more than 0.999. This was comparable to subjects who started all three medications simultaneously. A total of 80% achieved SVR. While rapid shifts in the RAV population following DAA initiation were observed, treatment failure associated with A156V was observed in only one patient. Adverse event profiles were similar to that observed in non-haemophilia cohorts. There was no evidence of factor inhibitor formation. There was no evidence that lead-in provided benefit in terms of response efficacy. Conclusion These data support DAA-based therapy in those with inherited bleeding disorders.

Background Targeted hepatitis C virus (HCV) screening is recommended. Implementation of screening in emergency department (ED) settings is challenging and controversial. Understanding HCV epidemiology in EDs could motivate and guide screening efforts. We characterized the prevalence of diagnosed and undiagnosed HCV in a Midwestern, urban ED. Methods This was a cross-sectional seroprevalence study using de-identified blood samples and self-reported health information obtained from consecutively approached ED patients aged 18-64 years. Subjects consented to a "study of diseases of public health importance" and were compensated for participation. The Biochain ELISA kit for Human Hepatitis C Virus was used for antibody assay. Viral RNA was isolated using the Qiagen QIAamp UltraSens Virus kit, followed by real-time reverse transcription polymerase chain reaction using a Bio-Rad CFX96 SYBR Green UltraFast program with melt-curve analysis. Results HCV antibody was detected in 128 of 924 (14%; 95% confidence interval [CI], 12%-16%) samples. Of these, 44 (34%) self-reported a history of HCV or hepatitis of unknown type and 103 (81%; 95% CI, 73%-87%) were RNA positive. Two additional patients were antibody negative but RNA positive. Fully implemented birth cohort screening for HCV antibody would have missed 36 of 128 (28%) of cases with detectable antibody and 26 of 105 (25%) of those with replicative HCV infection. Conclusions HCV infection is highly prevalent in EDs. Emergency departments are likely to be uniquely important for HCV screening, and logistical challenges to ED screening should be overcome. Birth cohort screening would have missed many patients, suggesting the need for complementary screening strategies applied to an expanded age range.

Occult hepatitis B virus (HBV) infection is characterized by the absence of detectable hepatitis B surface antigen (HBsAg) in the serum, despite detectable HBV DNA. Investigations of the mechanisms underlying the development of occult HBV infection are lacking in the current literature, although viral mutations in the surface region, resulting in decreased HBsAg expression or secretion, represent one potential mechanism. Wild-type HBsAg expression vectors were constructed from genotype-matched chronic HBV sequences. Site-directed mutagenesis was then utilized to introduce three genotype A mutations - M103I, K122R and G145A - associated with occult HBV infection in vivo, alone and in combination, into the wild-type HBsAg vectors. Transfection of Huh7 and HepG2 cell lines was performed, and cell culture supernatants and cell lysates were collected over 7 days to assess the effects of these mutations on extracellular and intracellular HBsAg levels. The G145A mutation resulted in significantly decreased extracellular and intracellular HBsAg expression in vitro. The most pronounced reduction in HBsAg expression was observed when all three mutations were present. The mutations evaluated in vitro in the current study resulted in decreased HBsAg expression and potentially increased hepatic retention and/or decreased hepatic secretion of synthesized HBsAg, which could explain the lack of HBsAg detection that is characteristic of occult HBV infection in vivo.

Occult hepatitis B virus (O-HBV) infection is characterized by the presence of HBV DNA without detectable hepatitis B surface antigen (HBV DNA+/HBsAg-) in the serum. Although O-HBV is more prevalent during HBV/HIV co-infection, analysis of HBV mutations in co-infected patients is limited. In this preliminary study, HBV PreSurface (PreS) and surface (S) regions were amplified from 33 HIV-positive patient serum samples - 27 chronic HBV (C-HBV) and six O-HBV infections. HBV genotype was determined by phylogenetic analysis, while quasispecies diversity was quantified for the PreS, S and overlapping polymerase regions. C-HBV infections harboured genotypes A, D and G, compared to A, E, G and one mixed A/G infection for O-HBV. Interestingly, nonsynonymous-synonymous mutation values indicated positive immune selection in three regions for O-HBV vs one for C-HBV. Sequence analysis further identified new O-HBV mutations, in addition to several previously reported mutations within the HBsAg antigenic determinant. Several of these O-HBV mutations likely contribute to the lack of detectable HBsAg in O-HBV infection by interfering with detection in serologic assays, altering antigen secretion and/or decreasing replicative fitness.

Hepatitis C virus (HCV) treatment response rates remain low in HCV/HIV-1-coinfected individuals compared with those with HCV alone. Persons with inherited coagulation disorders have high rates of HCV and HIV-1 infection, but HCV treatment trials in this patient population are scarce. We hypothesized that differences by infection status in HCV viral kinetics would be associated with differences in HCV quasispecies complexity over time and with treatment response disparities. Coinfected and monoinfected patients were enrolled in a treatment trial for pegylated-interferon alpha-2a (peg-IFN) + ribavirin. Patients were treated for 48 weeks and followed for an additional 24. Quantitative HCV RNA was tested at multiple times during and after treatment. Viral kinetic parameters associated with response were estimated with a mathematical model. Quasispecies emergence was determined via heteroduplex complexity assay. Twenty-two patients were HCV RNA-positive at baseline, with no significant demographic or virological differences by infection status. Five of eleven (45%) of monoinfected and 3 of 11 (27%) of coinfected patients achieved sustained viral response (SVR). Peg-IFN efficacy (epsilon) of 90% or greater was associated with probability of end-of-treatment response (ETR) (P = .001) and SVR (P = .06). Patients with SVR had lower baseline quasispecies complexity than those without SVR (P = .07). Those with epsilon of 90% or greater also had lower baseline complexity (P = .07). Coinfection status mediated changes in complexity over time (P = .04). In conclusion, low pretreatment quasispecies complexity may predict peg-IFN response; early peg-IFN response is critical for sustained HCV clearance and is altered in coinfection. Further studies are warranted.

Background & Aims: Clostridium difficile is a common cause of diarrhea in hospitalized patients and is associated with significant morbidity and cost. The current diagnostic standard, enzyme immunoassay (EIA), has low sensitivity, leading to duplicate testing and empiric treatment. We sought to show the usefulness and potential cost effectiveness of polymerase chain reaction (PCR) amplification of toxin B gene for diagnosis of C. difficile —induced diarrhea. Methods: A total of 148 stool samples from academic and community-based hospitals were sent for EIA testing and were evaluated prospectively for the presence of toxin B gene by PCR. Results were compared with EIA regarding sensitivity, specificity, and predictive values. Medical charts were reviewed to determine the following: (1) number of EIAs sent per admission, (2) number sent within a 24-hour time period, and (3) how caregivers practiced based on EIA results. Results: The mean age of 130 patients was 55 years. EIA and PCR were positive in 6.8% and 13.6% of patients, respectively. EIA sensitivity was 40%, specificity was 98%, and positive and negative predictive values were 80% and 91%, respectively. The cost of the PCR was $22/sample. Empiric treatment for C. difficile was given unnecessarily in 42% of EIA-negative results. Thirty percent of patients had 3 or more EIAs sent during their hospital admission. Of patients with multiple samples sent, 57% had more than 1 sample sent in a 24-hour period. Conclusions: Many physicians do not conform to practice guidelines regarding recommended diagnosis and empiric treatment of C. difficile . Toxin B gene PCR represents a more sensitive and potentially cost-effective method to diagnose C. difficile —induced diarrhea than EIA and should be considered for use as an alternative diagnostic standard.

African Americans infected with hepatitis C virus (HCV) are less responsive than whites to interferon-based therapy. HCV quasi species have been implicated. Quasi-species complexity and diversity were evaluated in matched African American and white individuals. Complexity and diversity in the first hypervariable region were similar in the 2 groups. Both the frequency of nonsynonymous amino acid substitutions and the mean ratio of nonsynonymous mutations to synonymous mutations were greater in clones derived from white patients. Racial differences in amino acid usage at otherwise conserved positions were observed. Differences in amino acid representation at key positions are suggestive of immunologic and functional selection along racial lines.

Prevalence of hepatitis B virus (HBV) markers, including occult HBV, has not been described in diverse cohorts among HIV-infected patients. The objective of this study was to assess prevalence and significance of active and occult HBV infection in an HIV-positive US cohort. A random sample was taken from 2 prospective multicenter treatment intervention cohorts. The sample population (n = 240) was HIV-1 infected and highly active antiretroviral therapy-naive. Prevalence of HBV serologic markers and quantitative HBV DNA were determined. Serum alanine aminotransferase (ALT) levels were measured to evaluate correlates of hepatocyte injury. A total of 64.6% of subjects demonstrated reactivity for any marker of current or past HBV infection or prior vaccination. Chronic HBV infection characterized by hepatitis B surface antigen (HBsAg) reactivity was present in 7.1% while 15.8% exhibited HB anticore IgG only. Approximately 10% of the latter group was HBV DNA positive by a polymerase chain reaction-based assay. Only patients with a serologic pattern of HBsAg or HB anticore alone reactivity had HBV DNA. Occult HBV was observed in approximately 10% of HIV-infected patients with HB anticore IgG antibody in a geographically representative national cohort. Though viral titers and serum ALT levels were low, screening of this subset of HIV-infected patients may have implications in terms of antiretroviral therapy and risk of immune reconstitution-associated flares.

Quantification of hepatitis C virus (HCV) RNA is important in the assessment of HCV-associated liver disease in patients coinfected with HCV and human immunodeficiency virus (HIV). To investigate whether the standard integrity of competing test methodologies might be compromised by higher HCV titers in coinfected patients, 2 technologies (a polymerase chain reaction-based assay [COBAS Amplicor 2.0 assay; Roche Diagnostics] and a branched-chain DNA assay [Versant 3.0; Bayer]) were evaluated by testing paired serum samples from 68 coinfected patients and 137 HCV-monoinfected patients. Although the correlation was highly significant (r=0.81; P

The hepatitis C virus (HCV) is an important contributor to morbidity and mortality in patients coinfected with human immunodeficiency virus (HIV). Coinfection results in increased HCV replication and more rapid rates of liver disease progression. The effect of HIV combination antiretroviral therapy (cART) on HCV replication has not been studied in depth. To address this issue, we enrolled a small cohort of HCV/HIV coinfected patients into a cART initiation trial, and used dynamic modeling combined with evaluation of immune responses and microarray profiles to determine how effective treatment of HIV affects HCV. Treatment with cART resulted in HCV flare and alanine aminotransferase (ALT) increase (2× or more increase from baseline) in a subset of treated patients. Subjects with evidence of hepatic injury (increased ALT) were more likely to have HCV-specific immune responses directed against HCV epitopes. Over time, HCV viral loads declined. Reproducible and biologically important gene expression changes occurred in patients who underwent successful cART, particularly with respect to downregulation of genes with known antiviral roles. Our findings suggest that the effective suppression of HIV by cART initiates a cascade of early and late events in treated patients with HCV. Early events involving downregulation of interferon-stimulated genes may lead to transiently increased viral replication and hepatic injury. At later time points, HCV viral load declines to levels comparable to those seen in the setting of HCV monoinfection. These findings support early antiretroviral therapy in those with HCV/HIV coinfection.

Summary Haemophiliacs have high hepatitis C virus (HCV) exposure risk from blood products that did not undergo heat inactivation or disease-specific screening prior to 1987. Repeated exposure to infected factor concentrates predisposes haemophiliacs to higher likelihood of HCV from multiple sources. HIV coinfection could result in impaired clearance of less fit variants resulting in enrichment of quasispecies carrying resistance mutations. We postulated that haemophiliacs demonstrate increased prevalence of baseline signature mutations in the HCV NS3/4 serine protease coding domain. We examined the prevalence of putative HCV protease inhibitor mutations, mutations, subclassified into dominant mutations if changes conferred resistance, and minor variants not associated with drug resistance, in patients with haemophilia A or B, infected with HCV or HCV/HIV, prior to HCV PI exposure. A total of 151 subjects were evaluated, including 22 haemophiliacs and 129 non-haemophilic controls. Of the 58 mutations detected, 55 (95%) were resistance mutations and three (5%) were minor variants. Dominant mutations were detected in 10 (45.5%) haemophiliacs and in 43 (33.3%) controls (OR 1.67, 95% CI 0.67–4.16). There was no statistical difference in proportion of dominant mutations (P = 0.27) or minor variants (P = 0.47) between groups, despite adjustment for HIV status (P = 0.44). No significant differences in dominant or minor resistance mutations between haemophiliacs and non-haemophiliacs were observed. HIV presence or prior HAART exposure did not affect baseline distribution. We conclude that haemophiliacs are not at higher risk for pre-existing HCV PI mutations, and prospective studies of response to PI-based regimens with HCV activity are indicated.

Indinavir is a viral protease inhibitor used for the treatment of HIV infection. Unconjugated hyperbilirubinemia develops in up to 25% of patients receiving indinavir, prompting drug discontinuation and further clinical evaluation in some instances. We postulated that this side-effect is due to indinavir-mediated impairment of bilirubin UDP-glucuronosyltransferase (UGT) activity and would be most pronounced in individuals with reduced hepatic enzyme levels, as occurs in ≈10% of the population manifesting Gilbert's syndrome. This hypothesis was tested in vitro , in the Gunn rat model of UGT deficiency, and in HIV-infected patients with and without the Gilbert's polymorphism. Indinavir was found to competitively inhibit UGT enzymatic activity ( K I = 183 μM) while concomitantly inducing hepatic bilirubin UGT mRNA and protein expression. Although oral indinavir increased plasma bilirubin levels in wild-type and heterozygous Gunn rats, the mean rise was significantly greater in the latter group of animals. Similarly, serum bilirubin increased by a mean of 0.34 mg/dl in indinavir-treated HIV patients lacking the Gilbert's polymorphism versus 1.45 mg/dl in those who were either heterozygous or homozygous for the mutant allele. Whereas saquinavir also competitively inhibits UGT activity, this drug has not been associated with hyperbilirubinemia, most likely because of the higher K I (360 μM) and substantially lower therapeutic levels as compared with indinavir. Taken together, these findings indicate that elevations in serum-unconjugated bilirubin associated with indinavir treatment result from direct inhibition of bilirubin-conjugating activity.

TT virus is a small, circular DNA virus, that has been associated with transfusion hepatitis. We sought to determine the prevalence of TT virus (TTV) in patients with human immunodeficiency virus (HIV) infection and to characterize the virus in terms of genotypic variability and in the relationship to CD4+, HIV viral loads, HCV/HIV coinfection, and ALT abnormalities. A cross-sectional analysis of HIV-infected patients in the United States, including 86 HIV-positive subjects and 118 HIV-negative controls was performed. TTV was detected using a seminested PCR technique. Samples underwent cloning and sequence analysis and/or RFLP to determine genotype. Thirty-eight percent of HIV-positive patients had TTV infection versus 14.4% of patients within the matching cohort (P = 0.0009). The highest rate of TTV infection was in patients with concurrent HCV/HIV infection (54% vs 30%, P = 0.038). HIV-infected subjects with TTV had lower ALT levels than those without TTV (P = 0.036). Intravenous drug use was the leading factor associated with TTV positivity among HIV-positive subjects. Mixed genotypes were more common in those with HIV. Therefore, TTV prevalence, ALT levels, and genomic heterogeneity of TTV all seem to be altered in patients with HIV.

In the United States, the seroprevalence rate for hepatitis E virus (HEV) is approximately 20%. This study examined HEV seroprevalence in persons with and without chronic liver disease. Our data indicate that HEV seropositivity is high in patients with chronic liver disease and that HEV seroprevalence increases significantly with age.

Alcohol abuse is described as a major cofactor in the development of hepatitis C (HCV) associated liver disease and may play a role in the outcome of interferon-based treatment interventions. The association between HCV viral heterogeneity and alcohol has not been previously described. This study was designed to evaluate the quasispecies nature of the HCV population in patients with compensated and decompensated alcoholic liver disease, to test the hypothesis that alcoholics have greater complexity than matched nonalcoholic controls. A nonisotopic heteroduplex complexity assay (HCA) was first validated by comparison with results of quasispecies complexity determined by subcloning and sequencing of amplicon products from the E2/NS1 hypervariable coding region (HVR). Subsequently, this methodology was applied to comparison of 20 compensated (Child's-Pugh A) and decompensated (Child's-Pugh B/C) alcoholic and 20 nonalcoholic controls. The complexity of the HVR, as well as the 5' Untranslated (5'UT) and the NS5b coding domains were evaluated. The HCA methodology provided a reasonable semiquantitative measure of HCV RNA quasispecies variability compared with subclone sequence homology comparison. Overall, alcoholic patients had greater quasispecies complexity (2.65 bands) than nonalcoholic controls (1.6 bands; P = .01). Subset analysis revealed that compensated alcoholic patients had a mean of 3.1 homo/heteroduplex bands per sample whereas Child's-Pugh B/C alcoholics showed intermediate complexity. A similar quasispecies complexity difference was seen in the 5'UTR, but not in the NS5b coding domain. Quasispecies complexity was not associated with viral titer, complementary DNA concentration, or genotype. The differences in quasispecies complexity may help explain reports of poor interferon responsiveness in alcoholic patients.

LI, X., C. J. GROSSMAN, C. L. MENDENHALL, P. HURTUBISE, S. D. ROUSTER, G. A. ROSELLE AND P. GARTSIDE. Host response to mycobacterial infection in the alcoholic rat: Male and female dimorphism . ALCOHOL 16 (3) 207–212, 1998.—Increased susceptibility to tuberculosis occurs in the alcoholic. One explanation for the altered susceptibility is a change in T-lymphocyte modulation. To evaluate this, 24 male and 24 female Sprague–Dawley rats were treated with either a Lieber-type liquid ethanol diet (LED) or an isocaloric control (LCD). After 2 weeks, half the subjects were infected with BCG (10 8 colony-forming units) and sacrificed after 42 days. Splenic helper (CD4) and suppressor/cytoxic (CD8) cells were quantitated by flow cytometry. By three-way analysis of variance, splenic cellularity was significantly increased by infection ( p p = 0.0002). There was a marginal sexual difference ( p = 0.065) with females exhibiting a 35% lower response while on alcohol. Examining lymphocyte subsets, the most significant changes were observed after infection (BCG) and alcohol treatment (LED). CD4 levels were deminished by LED ( p = 0.0002) but markedly increased by infection ( p p p = 0.0078). CD8 was influenced only by infection ( p p = 0.0032). Splenic T-lymphocytes, predominately CD4, are involved in the host response to BCG hepatitis and are adversely influenced by LED, which may contribute to increased susceptibility. Published by Elsevier Science Inc.

A series of experiments was performed to assess the alterations in immune status in vivo that are associated with differences in the amount and duration of ethanol intake. Using a nonspecific delayed cutaneous hypersensitivity-like response to the intradermal injection of phytohemagglutinin, the area of induration (skin test response) was significantly enhanced (p = 0.008) after low-dose ethanol (0.5 g/kg) administered daily by gastric gavage for 5 days. High-dose ethanol (6.0 g/kg) significantly diminished this response (p = 0.03). Using an experimental model of Mycobacterium bovis hepatitis, the host immune response was also altered in a biphasic manner after chronic, 28-day ethanol consumption. With this model 0.43 +/- 0.03 g/kg/day (mean +/- SEM) of ethanol (low dose) was associated with a 40% improvement in the removal of the organisms from liver tissue (p = 0.002). High dose (12.1 +/- 0.5 g/kg/day) impaired removal, resulting in a 55% increase in the number of viable organisms (p = 0.001). The levels of three cytokines, MIF, TNF-alpha, and IL-2, known to be involved in the modulation of the host response to mycobacterial infections, were measured in sera after the infection. The serum levels of these cytokines in response to infection did not correlate with this biphasic response to different alcohol dose levels.

To evaluate the hepatic regenerative response in patients with alcoholic liver disease, sera from 263 patients with severe alcoholic hepatitis and/or cirrhosis were analyzed for hepatocyte growth factor (HGF) and alpha-fetoprotein (AFP). HGF concentration was elevated above healthy controls in 95% of the patients (median level = 2.4 ng/ml), whereas AFP tended to be depressed below controls (median level = 4.1 ng/ml). Correlations with parameters of liver injury (i.e., ascites, encephalopathy, AST bilirubin, and protime) all showed a more significant correlation with HGF concentrations than those of AFP. Patients with HGF levels below the mean (4 ng/ml) exhibited significantly better survival (median survival = 35 months vs. 8.5 months for those with HGF > or = 4 ng/ml; p = 0.007). Serum HGF levels were associated with various specific histologic features of alcoholic hepatitis that included, but were not exclusively related to, necrosis.