The effects of needle-sharing and opioid substitution therapy on incidence of hepatitis C virus infection and reinfection in people who inject drugs.

Although high hepatitis C virus (HCV) prevalence has been observed in people who inject drugs (PWID) for decades, research suggests incidence is falling. We examined whether PWIDs' use of opioid substitution therapy (OST) and their needle-and-syringe sharing behaviour explained HCV incidence. We assessed HCV incidence in 235 PWID in Melbourne, Australia, and performed discrete-time survival with needle-sharing and OST status as independent variables. HCV infection, reinfection and combined infection/reinfection incidences were 7·6 [95% confidence interval (CI) 4·8-11·9], 12·4 (95% CI 9·1-17·0) and 9·7 (95% CI 7·4-12·6) per 100 person-years, respectively. Needle-sharing was significantly associated with higher incidence of naive HCV infection [hazard ratio (HR) 4·9, 95% CI 1·3-17·7] but not reinfection (HR 1·85, 95% CI 0·79-4·32); however, a cross-model test suggested this difference was sample specific. Past month use of OST had non-significant protective effects against naive HCV infection and reinfection. Our data confirm previous evidence of greatly reduced HCV incidence in PWID, but not the significant protective effect of OST on HCV incidence detected in recent studies. Our findings reinforce the need for greater access to HCV testing and prevention services to accelerate the decline in incidence, and HCV treatment, management and support to limit reinfection.

The relationships between IFNL4 genotype, intrahepatic interferon-stimulated gene expression and interferon treatment response differs in HCV-1 compared with HCV-3.

BACKGROUND: The biological mechanism underlying the association between IFNL4/IFNL3 polymorphism and peginterferon/ribavirin (PR) response in HCV-1 is thought to involve differential intrahepatic interferon-stimulated gene expression. HCV-3 is more sensitive to PR, but there are no studies of the association between IFNL4 polymorphism, PR treatment response and liver interferon-stimulated gene expression in HCV-3. AIM: We evaluated the association between IFNL4/IFNL3 genotypes, PR treatment outcomes and intrahepatic interferon-stimulated gene expression, according to HCV genotype. METHODS: HCV-1 and HCV-3 patients who received PR therapy were identified. IFNL3 (rs12979860) and IFNL4 genotype (rs368234815) were determined. A second cohort with stored liver specimens was identified. Expression of ISGs was measured by rt-PCR. RESULTS: Two hundred and fifty-nine patients were identified: 55% HCV-1, 45% HCV-3. IFNL4 genotype frequency was TT/TT 44%, TT/ΔG 42% andΔG/ΔG 14%. Linkage disequilibrium with IFNL3 genotype was high (r(2) = 0.98). The association between IFNL4 genotype and PR response was attenuated in HCV-3 vs. HCV-1 (HCV-3: SVR 89% vs. 76% vs. 72% for TT/TT vs. TT/ΔG vs. ΔG/ΔG, P = 0.09; HCV-1: SVR: 82% vs. 29% vs. 24%, P < 0.001). Intrahepatic ISG expression was evaluated in 92 patients; 61% HCV-1. The association between IFNL4 genotype and liver ISG expression was significantly different for HCV-3 vs. HCV-1 (P-value for interaction = 0.046), with levels of interferon-stimulated gene expression being highest in HCV-1 patients who carried a poor-response IFNL4 genotype. CONCLUSIONS: The relationship between IFNL4 genotype and PR treatment response as well as intrahepatic interferon-stimulated gene expression differs between HCV-1 and HCV-3. These data suggest fundamental differences in host-virus interactions according to HCV genotype.

Short duration of lamivudine for the prevention of hepatitis B virus transmission in pregnancy: lack of potency and selection of resistance mutations.

This study sought to assess the antiviral efficacy of lamivudine (LMV) administered during third trimester to reduce maternal viraemia and to identify the emergence of LMV resistance. A prospective observational analysis was performed on 26 mothers with high viral load (>107 IU/mL). Twenty-one women received LMV (treated group) for an average of 53 days (range 22-88 days), and the remaining five formed the untreated control group. Serum samples from two time points were used to measure HBV DNA levels and antiviral drug resistance. The LMV-treated women achieved a median HBV DNA reduction of 2.6-log10 IU/mL. Although end-of-treatment (EOT) HBV DNA in four (18%) LMV-treated women remained at >10(7) IU/mL (± 0.5 log IU/mL), no mother-to-baby transmission was observed. In contrast, a baby from the untreated mother was HBsAg positive at 9 months postpartum. Four technologies were used for drug resistance testing. Only ultra-deep pyrosequencing (UDPS) was sufficiently sensitive to detect minor viral variants down to <1%. UDPS showed that LMV therapy resulted in increased viral quasispecies diversity and positive selection of HBV variants with reverse transcriptase amino acid substitutions at sites associated with primary LMV resistance (rtM204I/V and rtA181T) in four (19%) women. These viral variants were detected mostly at low frequencies (0.63-5.92%) at EOT, but one LMV-treated mother had an rtA181T variant that increased from 2.2% pretherapy to 25.59% at EOT. This mother was also infected with the vaccine escape variant (sG145R), which was inhibited by LMV treatment. LMV therapy during late pregnancy only reduced maternal viraemia moderately, and drug-resistant viral variants emerged.

Hepatic TLR2 & TLR4 expression correlates with hepatic inflammation and TNF-α in HCV & HCV/HIV infection.

Signalling activated by Toll-like receptors (TLRs) can result in the production of tumour necrosis factor alpha (TNF-α) which is implicated in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infection. No study has examined or compared hepatic expression of TLRs in both HCV and HCV/HIV. Liver and peripheral blood mononuclear cells (PBMCs) were obtained from HCV & HCV/HIV-infected patients and PBMCs from HIV-infected patients. Liver RNA was analysed by microarray and reverse transcription quantitative PCR (RT-qPCR). PBMCs were analysed by flow cytometry. Associations with hepatic histology and infection type were sought. Forty-six HCV, 20 HIV and 27 HCV/HIV-infected patients were recruited. Increasing Metavir inflammatory activity score was associated with increased hepatic TLR mRNA by RT-qPCR: TLR2 (P ≤ 0.001), TLR4 (P = 0.008) and TNF-α (P ≤ 0.001). A high degree of correlation was seen between hepatic mRNA expression of TNF-αvs TLR2 (r(2) = 0.66, P < 0.0001) and TLR4 (r(2) = 0.60, P < 0.0001). No differences in TLR gene or protein expression was observed between HCV, HCV/HIV- or HIV-infected groups. Hepatic TLR2, TLR4 and TNF-α mRNA are associated with hepatic inflammation in both HCV and HCV/HIV infection. High correlation between TNF-α and TLR2/TLR4 suggests a role for the innate immune response in TNF-α production. Activation of the innate immune response appears to be independent of infection type.

Evidence of patient-to-patient transmission of hepatitis C virus through contaminated intravenous anaesthetic ampoules.

Two separate cases of acute hepatitis C virus (HCV) infection following medical procedures, arthroscopy and colonoscopy, are reported. In both episodes, patient risk factors were reviewed, and staff and other patients' sera were tested for HCV antibodies and RNA. HCV RNA positive samples were genotyped, sequenced, and subjected to phylogenetic analysis. No risk factors for HCV infection were identified for either case except for medical procedures. HCV RNA positive patients were identified preceding both cases on the respective theatre lists. HCV infection in a second low risk patient was also identified. Nucleic acid sequencing and phylogenetic analysis of HCV from the two putative source patients and the three recipient patients demonstrated a high degree of relatedness respectively. The results suggest that patient-to-patient transmission occurred in both episodes via contamination of intravenous anaesthetic ampoules with HCV used on multiple patients. Injectable medication ampoules should not be used for more than one patient.

Rubella virus replication and links to teratogenicity.

Rubella virus (RV) is the causative agent of the disease known more popularly as German measles. Rubella is predominantly a childhood disease and is endemic throughout the world. Natural infections of rubella occur only in humans and are generally mild. Complications of rubella infection, most commonly polyarthralgia in adult women, do exist; occasionally more serious sequelae occur. However, the primary public health concern of RV infection is its teratogenicity. RV infection of women during the first trimester of pregnancy can induce a spectrum of congenital defects in the newborn, known as congenital rubella syndrome (CRS). The development of vaccines and implementation of vaccination strategies have substantially reduced the incidence of disease and in turn of CRS in developed countries. The pathway whereby RV infection leads to teratogenesis has not been elucidated, but the cytopathology in infected fetal tissues suggests necrosis and/or apoptosis as well as inhibition of cell division of critical precursor cells involved in organogenesis. In cell culture, a number of unusual features of RV replication have been observed, including mitochondrial abnormalities, and disruption of the cytoskeleton; these manifestations are most probably linked and play some role in RV teratogenesis. Further understanding of the mechanism of RV teratogenesis will be brought about by the investigation of RV replication and virus-host interactions.

Molecular analysis of human immunodeficiency virus strains associated with a case of criminal transmission of the virus.

An investigation was done of the evidence for transmission of human immunodeficiency virus (HIV) from an HIV-positive man to several male and female sex contacts. Phylogenetic analysis of sequences from the gag and env genes showed a close relationship between the predominant virus strains from the source and 2 contacts. However, the likelihood that a female contact was infected by the source could not be determined, despite contact tracing indicating that this may have occurred. One male, shown by contact tracing and molecular evidence to have been infected by the source, subsequently transmitted HIV to his female sex partner. HIV sequence from a plasma sample used as a control in the phylogenetic analysis contained env and gag sequences that were closely related to those from the source. An epidemiologic link between these 2 individuals was subsequently confirmed by contact tracing.

Localization of rubella virus core particles in vero cells.

Rubella virus (RV) infection induces a variety of morphological changes in the host cell including the modification of lysosomes to produce "replication complexes" and the alteration of mitochondrial morphology and distribution. The morphogenesis of RV was further characterized with particular emphasis on the localization of RV core particles. Thin-section electron microscopy (TSEM) studies indicated that RV core-like particles, measuring approximately 33 nm in diameter, were found associated with RV replication complexes. Immunogold-labeling electron microscopy (EM) using monoclonal antibodies to RV capsid proteins confirmed that these particles were viral cores. RV core particles were also detected in association with mitochondria as observed by TSEM and immunogold-labeling EM using monoclonal antibodies to capsid or polyclonal antibodies to RV virions. The results of this study indicate that the localization of RV core particles in relation to replication complexes is similar to that found for the alphaviruses. However, the association of RV core particles with mitochondria appears unique within the family Togaviridae.

Hepatitis G: viroprevalence and seroconversion in a high-risk group of children.

Hepatitis G virus (HGV), a recently discovered flavivirus, is parenterally transmitted and significantly associated with hepatitis C viraemia. Data on the viroprevalence of this agent in children is scant and its seroprevalence is unknown. The aim of this study was to determine the viroprevalence and seroprevalence of HGV in paediatric patients at risk of parenterally transmitted virus infection. Sera from 35 patients, previously tested for hepatitis C virus (HCV) infection, were analysed for the presence of HGV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and for antibody to the E2 envelope protein (anti-E2) of HGV using the HGV-env kit. The mean age of the patients was 9.4 years (range 1-17 years), and risk factors included multiple transfusions and maternal HCV infection. Co-infection with HCV and HGV was a relatively common occurrence (31%). The prevalence of anti-E2, a marker of recovery from infection, was low (5%) when compared with overall viroprevalence (20%). This study highlights the significant association of HGV with HCV in children. The novel finding of a low ratio of anti-E2:HGV RNA contrasts with the pattern seen in adults and may reflect a higher risk of long-term carriage with acquisition of HGV infection at an early age.

Inactivation of feline calicivirus, a Norwalk virus surrogate.

Norwalk and Norwalk virus-like particles (NVLPs) [also known as small round structured viruses (SRSVs)] are members of the family Caliciviridae and are important causes of gastroenteritis in humans. Little is known about their survival in the environment or the disinfection procedures necessary to remove them from contaminated settings. As NVLPs cannot be grown in tissue culture, survival studies require the use of a closely related cultivable virus. This study assesses the survival of the surrogate feline calicivirus (FCV) after exposure to commercially available disinfectants and a range of environmental conditions. Disinfectants tested included glutaraldehyde, iodine, hypochlorite, a quaternary ammonium-based product, an anionic detergent and ethanol. Complete inactivation of FCV required exposure to 1000 ppm freshly reconstituted granular hypochlorite, or 5000 ppm pre-reconstituted hypochlorite solution. Glutaraldehyde and the iodine-based product effectively inactivated FCV whereas the quaternary ammonium product, detergent and ethanol failed to completely inactivate the virus. The stability of FCV in suspension and in a dried state was assessed after exposure to 4 degrees C, room temperature (20 degrees C) and 37 degrees C. With increasing temperature, the stability of FCV was found to diminish both in suspension and in the dried state. FCV in the dried state did not survive for one day at 37 degrees C. This study provides a basis for establishing guidelines for disinfection protocols to decrease the spread of NVLPs in a community setting.

Combined infection of Norwalk-like virus and verotoxin-producing bacteria associated with a gastroenteritis outbreak.

Detection of multiple pathogens, particularly a combination of viruses and bacteria, is infrequently documented in outbreaks of gastroenteritis. This paper reports the presence of Norwalk-like virus (NLV) and enterohaemorrhagic verotoxin-producing Escherichia coli in one individual, and NLV and verotoxin-producing Aeromonas sobria in another individual, both part of a large gastroenteritis outbreak. The causes of gastroenteritis in such outbreaks may be more complex than previously thought.

Rubella virus replication complexes are virus-modified lysosomes.

Replication complexes are membrane-bound cytoplasmic vacuoles involved in rubella virus (RV) replication. These structures can be identified by their characteristic morphology at the electron microscopy (EM) level and by their association with double-stranded (ds) RNA in immunogold labeling EM studies. Although these virus-induced structures bear some resemblance to lysosomes, their exact nature and origin are unknown. In this study, the localization of two lysosomal markers, lysosomal-associated membrane protein (Lamp-1) and acid phosphatase, relative to the replication complexes was examined by light and electron microscopy. Confocal microscopy using antibodies to dsRNA and Lamp-1 showed colocalization of these two markers in the cytoplasm of RV-infected cells. Immunogold labeling EM studies using antibodies to Lamp-1 confirmed that Lamp-1 was associated with RV replication complexes. EM histochemical studies demonstrated the presence of acid phosphatase in the vacuoles of RV replication complexes. Taken together, these studies show that RV replication complexes are virus-modified lysosomes.

Hepatitis G virus and fulminant hepatic failure: evidence for transfusion-related infection.

BACKGROUND/AIMS: In the majority of cases of fulminant "viral" hepatitis in Australia, no known aetiological agent can be isolated. We have examined the possible role of the recently discovered hepatitis G virus (HGV) in such cases. METHODS: An HGV specific reverse transcription polymerase chain reaction (RT-PCR) was performed on pre- and post-liver transplant serum from 14 patients who were referred for transplantation at our unit between 1989 and 1995 for unexplained fulminant hepatic failure. Eleven patients successfully underwent transplantation and three died while waiting for a suitable donor organ. Hepatitis viruses A-E were excluded by standard serological and PCR based testing. HGV RT-PCR was also performed on 21 other, randomly selected, liver transplant recipients ("controls"). RESULTS: The 14 fulminant cases were HGV RT-PCR negative prior to transplantation while five of 21 controls were positive. Post-transplant, eight of the 11 fulminant patients were found to be HGV RT-PCR positive and the same five controls remained HGV RT-PCR positive. In three of the eight fulminant patients the HGV infection resolved. CONCLUSIONS: Our data indicate that HGV infection is unlikely to be responsible for fulminant hepatitis and that it is probably acquired from blood and/or blood products during the transplantation process. Furthermore, long-term carriage of HGV post-transplant is not associated with clinically apparent liver disease.

Hepatitis C virus genotypes in Australia.

The relative distribution of Australian hepatitis C virus (HCV) genotypes was determined for 500 isolates. Genotyping was performed using a commercial reverse phase hybridization assay after amplification of the 5' untranslated region of HCV by the polymerase chain reaction. Australian isolates comprised, predominantly, genotype 1 (55%) and genotype 3 (38%) with genotype 2 accounting for only 7%. Genotype 3a was the most common subtype. When the major risk groups of injecting drug users or transfusion-acquired hepatitis C were compared, there was a significantly higher incidence of genotype 1b in the transfusion-acquired group (P < 0.03). When the age of the patients was analysed, genotype 3a was more prevalent in the 21-40-year age group than the 41-60-year age group (P < 0.05). There was no significant difference in genotype distribution between males and females. HCV genotypes 1, 2 and 3 are most often found in developed countries but the relatively high prevalence of genotype 3a in Australia is unusual.

Mutations in the hepatitis B virus precore/core gene and core promoter in patients with severe recurrent disease following liver transplantation.

Recurrent hepatitis B virus (HBV) infection is a major problem in patients undergoing liver transplantation. Previously, we reported that infection with HBV strains containing a mutation in the precore region (G-to-A at nucleotide 1896) was associated with severe recurrent disease posttransplantation. In this study we investigated other mutations in the precore/core gene and core promoter which may be associated with this severe recurrence. The precore/core gene and core promoter of HBV from pre and posttransplantation sera of 15 patients with HBV recurrence were amplified by polymerase chain reaction (PCR) and sequenced. Pre and posttransplant sequences were very similar for each patient. HBV from patients who developed severe recurrence had significantly more mutations in both the nucleotide (P < .05) and predicted amino acid (P < .05) sequences of the precore/core gene, but not in the core promoter, than virus from patients with mild recurrence. There was also an apparent link between severe disease and HBV strains of genotype D (P < .05). The number of nucleotide and amino acid mutations in the precore/core gene was strongly associated with the presence of the precore mutation (P < .01). Mutations were found throughout the entire gene, however, at the amino acid level clustering was observed in the B- and helper T-cell epitopes as well as nuclear localization signals. In the encapsidation signal, nucleotide mutations were found that were predicted to increase the stability of the stem-loop structure. Overall, our data shows that genotype D and accumulated mutations throughout the HBV precore/core gene, but not core promoter, were associated with severe recurrent disease posttransplantation. These mutations were strongly linked to the presence of the precore mutation at nucleotide position 1896 and may contribute to the poor outcome in these patients.

Chronic hepatitis C: effect of alcohol on hepatitic activity and viral titre.

BACKGROUND/AIMS: Alcohol and the hepatitis C virus have been postulated to interact to adversely affect the natural history of patients with chronic liver disease. The aim of this study was to examine the effect of alcohol on hepatitic activity and serum HCV RNA levels in patients with chronic hepatitis C. METHODS: Forty-five consecutive patients with chronic hepatitis C were classified according to alcohol intake over the 3-month period preceding study entry: group 1 (n = 23), > 10 g alcohol/day; group 2 (n = 22), < or = 10 g alcohol/day. Hepatitic activity and alcohol intake were assessed at study entry and, following moderation of alcohol intake, after a mean follow-up period of 4.4 +/- 0.2 months. RESULTS: Hepatitic activity was significantly greater in the patients who consumed > 10 g of alcohol/day. Moderation of alcohol consumption in patients consuming > 10 g/day resulted in a significant decrease in both disease activity (p = 0.0002) and viral RNA titre (p = 0.018); there was no change over the study period in patients with a consistently low alcohol intake. CONCLUSION: The results support the hypotheses that, in patients with chronic hepatitis C, alcohol aggravates hepatic injury, increases viral load and adversely affects the natural history of the associated liver disease.