Sabrina Bagaglio

Hepatitis C virus populations in the plasma, peripheral blood mononuclear cells and cerebrospinal fluid of HIV/hepatitis C virus-co-infected patients

Background:Chronic hepatitis C virus (HCV) infection has occasionally been associated with diseases of the central nervous system, thus suggesting that the virus has a direct effect on cerebral function. Objective:To investigate the presence and heterogeneity of HCV populations in the cerebrospinal fluid (CSF), plasma and peripheral blood mononuclear cells (PBMC) of HIV-infected and anti-HCV-positive patients. Methods:Reverse transcriptase–polymerase chain reaction was used to detect HCV RNA in 21 paired CSF/plasma samples and 20 PBMC samples from HIV/HCV-positive patients. HCV was genotyped by means of a hybridization assay, and its compartmental heterogeneity was analysed by cloning the related amplicons. Results:All of the plasma samples, 14 out of 20 PBMC samples and five out of 21 CSF samples were positive for HCV RNA. Of the five patients with HCV-positive CSF, two had the same genotype in the plasma/CSF/PBMC samples, two had a different genotype in the CSF from that in plasma and PBMC, and one had the same genotype in paired CSF/plasma samples. An analysis of the HCV populations showed that two patients seemingly infected with the same genotype in different compartments harbored mixed infection in the CSF but not in the plasma and PBMC samples. Conclusions:These findings of different HCV genetic diversification in different compartments suggest that CSF is an independent site of viral replication and persistence.

Viral interference between hepatitis B, C, and D viruses in dual and triple infections in HIV-positive patients.

Objective:To investigate the reciprocal inhibitory effects of hepatitis B virus (HBV)/hepatitis C virus (HCV)/hepatitis D virus (HDV) infections in naive and previously antiretroviral-experienced HIV-positive patients. Design:This retrospective study involved 72 consecutive patients of the Italian Cohort Naive Antiretroviral cohort: 21 coinfected with HBV/HCV (group 1BC), 18 infected with HBV (group 2B), and 33 infected with HCV (group 3C). Methods:Viral interference between HBV and HCV was assessed by means of the qualitative detection, quantification, and genotyping of each virus; HDV infection was assessed by means of genomic amplification. Results:Univariate analysis showed that HBV DNA was less frequently detected in group 1BC than in group 2B (16 of 21 vs 18 of 18; P = 0.02), their HBV load was significantly lower (median 3.9 vs 5.4 log10 HBV DNA copies/mL; P = 0.002), and they more frequently carried HBV genotype D (12 of 13 vs 4 of 11; P = 0.0071). HCV RNA was less frequently detected in group 1BC than in group 3C (12 of 21 vs 33 of 33; P < 0.0001), and HDV RNA was more frequently detected in group 1BC than in group 2B (9 of 21 vs 2 of 18; P = 0.028). Multivariate analysis of the HBV-infected subjects showed that the risk of HCV coinfection was associated with older age [relative risk 0.28, 95% confidence interval (CI): 0.09 to 0.90; P = 0.033 for every 10 years older] and intravenous drug use (relative risk 73, 95% CI: 2.4 to >999.999; P = 0.013). The only predictor of HBV coinfection in HCV-infected individuals was a lower HCV load (relative risk 0.30, 95% CI: 0.11 to 0.79 for every additional log10 HCV RNA; P = 0.015). Conclusion:HBV and HCV showed alternative dominant replication in the I.Co.N.A. cohort, with HBV having a more unfavorable effect on HCV replication.

Prevalence of wild-type in NS5A-PKR protein kinase binding domain in HCV-related hepatocellular carcinoma

BACKGROUND/AIMS Experimental studies have demonstrated that the wild-type PKR-NS5A strain of hepatitis C virus (HCV) may have oncogenic potential through the binding and functional repression of PKR protein kinase. To assess whether the NS5A-PKR-binding domain may be involved in HCV-related liver carcinogenesis, its sequence was analyzed in the sera of 85 patients with hepatocellular carcinoma (HCC) and in 51 patients with chronic active hepatitis (CAH). In 13 HCC cases sequence analysis was also performed in tumor and nontumor liver tissues. METHODS The nucleotide sequences of the PKR-binding domain were inferred by direct sequencing of the amplified HCV products and deduced amino acids were compared with the sequence of HCV-J. RESULTS A wild-type or single mutated strain which retains PKR-binding activity was found in 88% of HCC and 69% of CAH cases (P=0.0096). All but three HCC cases showed no divergences in amino acid changes between serum and liver tissues. The wild-type strains were equally distributed between the HCC with or without cirrhosis. CONCLUSIONS The prevalance of the wild-type NS5A-PKR strain is significantly higher in HCC than in CAH. These data suggest that inhibition of PKR activity by HCV might represent a potential mechanism of HCV-related carcinogenesis.

Mother-to-child transmission of TT virus: sequence analysis of non-coding region of TT virus in infected mother-infant pairs ∗

Summary. To investigate vertical transmission of TT virus, TTV-DNA was looked for in serum samples taken from 22 mothers and their 22 infants at birth and during nine months of follow-up. Sixteen mothers at delivery and six infants within nine months of age had TTV-DNA detected by the amplification of the non coding (NC) region. Two of these newborns had positive viremia at birth. Sequence analysis of the NC region of five mother-infant pairs revealed that the TTV strains detected at three and six months of age in two of the infants were closely related to that of their mothers, whereas two that became TTV-DNA positive at three moths had a different nucleotide sequence from that of their mothers. One of the two infants with detectable viremia at birth also had a different nucleotide sequence from her mother. These findings suggest that both in utero and perinatal transmission of TT virus may occur, and that the strain detected in the infants was not invariably dominant in the mothers at delivery.

Virological analysis, genotypes and mutational patterns of the HBV precore/core gene in HBV/HCV-related hepatocellular carcinoma.

Summary.  We investigated the replicative profile of hepatitis B (HBV) and hepatitis C (HCV) viruses and the mutational pattern of the HBV precore/core (pre‐C/C) domain in hepatocellular carcinoma (HCC). Thirty‐eight consecutive patients with HCC were included in the study – 18 of them with HBV/HCV co‐infection and 20 with HBV single infection. Twenty‐three additional patients with co‐infection, without HCC were recruited as the control group. Replication activity was evaluated by detecting and quantitating both HBV and HCV genomes. The HBV pre‐C/C region, encompassing the pregenome encapsidation signal involved in viral replication, was analysed by direct sequencing. HBV viraemia levels were significantly lower (P = 0.04) in patients with co‐infection in comparison with single‐infected HCC, whereas two different HBV viraemia profiles were detected in co‐infection with or without circulating HCV. HBV genotype D was prevalent in the three groups and HCV genotype 1b was found to be the infecting strain in all patients. Lower variability in the pre‐C/C region was found in co‐infection in comparison with HBV single infection (P = 0.0004). A synonymous T1936C mutation was found in all co‐infected HCC cases not related to the presence or absence of circulating HCV, and a hypermutated pre‐C strain, characterized by the same mutational pattern, was identified in three HCC cases. The mutational pattern of the pre‐C/C region was closely related to HBV replication efficiency, and specific HBV mutations selectively associated with HCV co‐infection could be linked with accelerated HBV/HCV‐related disease progression.

Frequency of natural resistance within NS5a replication complex domain in hepatitis C genotypes 1a, 1b: Possible implication of subtype-specific resistance selection in multiple direct acting antivirals drugs combination treatment

Different HCV subtypes may naturally harbor different resistance selection to anti-NS5a inhibitors. 2761 sequences retrieved from the Los Alamos HCV database were analyzed in the NS5a domain 1, the target of NS5a inhibitors. The NS5a resistance-associated polymorphisms (RAPs) were more frequently detected in HCV G1b compared to G1a. The prevalence of polymorphisms associated with cross-resistance to compounds in clinical use (daclatasvir, DCV, ledipasvir, LDV, ombitasvir, and OMV) or scheduled to come into clinical use in the near future (IDX719, elbasvir, and ELV) was higher in G1b compared to G1a (37/1552 (2.4%) in 1b sequences and 15/1209 (1.2%) in 1a isolates, p = 0.040). Interestingly, on the basis of the genotype-specific resistance pattern, 95 (6.1%) G1b sequences had L31M RAP to DCV/IDX719, while 6 sequences of G1a (0.5%) harbored L31M RAP, conferring resistance to DCV/LDV/IDX719/ELV (p < 0.0001). Finally, 28 (2.3%) G1a and none of G1b isolates harbored M28V RAP to OMV (p < 0.0001). In conclusion, the pattern of subtype-specific resistance selection in the naturally occurring strains may guide the treatment option in association with direct acting antivirals (DAAs) targeting different regions, particularly in patients that are difficult to cure, such as those with advanced liver disease or individuals who have failed previous DAAs.

A novel stop codon mutation within the hepatitis B surface gene is detected in the liver but not in the peripheral blood mononuclear cells of HIV-infected individuals with occult HBV infection

Summary.  To characterize occult HBV infection (OHB) in different compartments of HIV+ individuals. This retrospective study involved 38 consecutive HIV+ patients; 24 HBsAg negative (HBV−) and 14 HBsAg positive (HBV+). OHB was assessed in serum samples, liver tissue (LT) and peripheral blood mononuclear cells (PBMC) by genomic amplification of the partial S, X and precore/core regions. HBV genomic analysis was inferred by direct sequencing of PCR products. The intracellular HBV‐DNA was measured by a quantitative real‐time PCR. HBV+ patients were used as a control for HBV replication and genomic profile. In HBV− patients, HBV‐DNA was undetectable in all serum samples, while it was found positive in 7/24 (29%) LT in which genotype D prevailed (57%). HBV‐DNA was found in 6/7 (86%) PBMC of occult‐positive and none of occult‐negative LT. Significantly lower HBV‐DNA load was present in both compartments in OHB+ with respect to the HBV+ group (LT: P = 0.002; PBMC: P = 0.026). In the occult‐positive cases, HBV replication was significantly higher in LT than in PBMC (P = 0.028). A hyper‐mutated S gene in PBMC and a nucleotide mutation at position C695 in LT that produces a translational stop codon at amino acid 181 of the HBs gene characterized OHB. In this group of HIV+ persons, OHB is frequent and exhibits lower replication levels than chronic HBV in the different compartments examined. HBV‐DNA detection in PBMC may offer a useful tool to identify OHB in serum‐negative cases. The novel HBs gene stop codon found in LT could be responsible for reduced production leading to undetectability of HBsAg.

Virological pattern of hepatitis B infection in an HIV-positive man with fatal fulminant hepatitis B: a case report

IntroductionThere seem to be no published data concerning the clinical impact of populations of hepatitis B virus (HBV) in the hepatic and extrahepatic compartments of HIV-infected people with severe acute hepatitis.Case presentationA 26-year-old Caucasian man presenting to our hospital with clinical symptoms suggesting acute hepatitis was found to have an acute hepatitis B profile upon admission. He developed fatal fulminant hepatitis and was found to be heavily immunocompromised due to HIV-1 infection. He had a high plasma HBV and HIV load, and analysis of the partial pre-S1/pre-S2 domain showed the presence of mixed infection with D and F genotypes. Analysis of the point mutations within this region revealed the presence of HBV strains with amino acid substitutions at the immunodominant epitopes involved in B or T cell recognition. A homogeneous population of a pre-core mutant strain harbouring the A1896G and A1899G affecting HBeAg expression was invariably found in the liver tissue, plasma and peripheral blood mononuclear cells despite active HBeAg secretion; it was the dominant strain in the liver only, and was characterised by the presence of two point mutations in the direct repeat 1 domain involved in HBV replication activity. Taken together, these mutations are indicative of a highly replicative virus capable of evading immune responses.ConclusionThis case report provides clinical evidence of a possible association between the rapid spread of highly replicative escape mutants and the development of fulminant hepatitis in a heavily immunocompromised patient. Virological surveillance of severe acute hepatitis B may be important in establishing an early treatment strategy involving antiviral drugs capable of preventing liver failure, especially in individuals for whom liver transplantation is not accepted as a standard indication.

Non-invasive fibrosis biomarkers - APRI and Forns - are associated with liver stiffness in HIV-monoinfected patients receiving antiretroviral drugs.

HIV‐monoinfected patients are susceptible to liver injury by different factors and may develop liver fibrosis, which requires adequate clinical management in terms of therapy and disease monitoring. We aimed to evaluate the presence of liver fibrosis identified by transient elastography (TE), its relationships with indirect biochemical markers [the aspartate aminotransferase/platelet ratio index (APRI), the Forns index and FIB‐4] and its predictive factors in HIV‐monoinfected patients receiving antiretroviral therapy (ART).

Evolution of hepatitis C virus non-structural 5A gene in the progression of liver disease to hepatocellular carcinoma.

Background: The interaction between the hepatitis C virus (HCV) non‐structural 5A (NS5A) protein of HCV and the protein kinase R (PKR), which is an effector of the cellular antiviral response and has been defined as a tumour suppressor, may affect the control of protein synthesis and cell growth.