Identification of a duplicated V3 domain in NS5A associated with cirrhosis and hepatocellular carcinoma in HCV-1b patients.

BACKGROUND: The NS5A protein of the hepatitis C virus has been shown to be involved in the development of hepatocellular carcinoma. OBJECTIVES: In a French multicenter study, we investigated the clinical and epidemiological features of a new HCV genotype 1b strain bearing a wide insertion into the V3 domain. STUDY DESIGN: We studied NS5A gene sequences in 821 French patients infected with genotype 1b HCV. RESULTS: We identified an uncharacterized V3 insertion without ORF disruption in 3.05% of the HCV sequences. The insertion comprised 31 amino-acids for the majority of patients; 3 patients had 27 amino-acids insertions and 1 had a 12 amino-acids insertion. Sequence identity between the 31 amino-acids insertions and the V3 domain ranged from 48 to 96% with E-values above 4e(-5), thus illustrating sequence homology and a partial gene duplication event that to our knowledge has never been reported in HCV. Moreover we showed the presence of the duplication at the time of infection and its persistence at least during 12 years in the entire quasispecies. No association was found with extrahepatic diseases. Conversely, patients with cirrhosis were two times more likely to have HCV with this genetic characteristic (p=0.04). Moreover, its prevalence increased with liver disease severity (from 3.0% in patients without cirrhosis to 9.4% in patients with both cirrhosis and HCC, p for trend=0.045). CONCLUSIONS: We identified a duplicated V3 domain in the HCV-1b NS5A protein for the first time. The duplication may be associated with unfavorable evolution of liver disease including a possible involvement in liver carcinogenesis.

[Hepatitis E virus: Blood transfusion implications]. / Virus de l'hépatite E, implications en transfusion sanguine.

Hepatitis E virus (HEV) is a non-enveloped RNA virus transmitted by the fecal-oral route. Autochthonous hepatitis E occurring in developed countries is caused by genotypes 3 and 4 and is a zoonotic infection. Humans are infected mostly after ingestion of undercooked meat from infected animals. Most HEV 3 and 4 infections are clinically inapparent. However, genotype 3 (HEV 3) can lead to chronic hepatitis in immuno-compromised patients such as organ-transplant recipients and patients with haematological malignancies. In Europe, HEV 3 is implicated in transfusion-transmitted HEV infection. In France, as observed in several European countries, prevalence of HEV RNA and specific IgG antibodies are high indicating that viral circulation is important. The systematic HEV NAT screening of blood donations used for preparation of solvent detergent plasma indicate that 1 to 2218 donation is infected by HEV RNA. The need or implementation's impacts of safety measures to prevent HEV transmission by blood transfusion are under reflexion by French's health authorities. The HEV NAT screening is the only available tool of prevention. Alternative strategies are under investigation including individual or mini pool NAT testing all or part of blood donations.

The use of a multiplex real-time PCR assay for diagnosing acute respiratory viral infections in children attending an emergency unit.

BACKGROUND: The use of a multiplex molecular technique to identify the etiological pathogen of respiratory viral infections might be a support as clinical signs are not characteristic. OBJECTIVES: The aim of the study was to evaluate a multiplex molecular real-time assay for the routine diagnosis of respiratory viruses, to analyze the symptoms associated with the pathogens detected and to determine the spread of virus during the period. STUDY DESIGN: Respiratory samples were collected from children presenting with respiratory symptoms and attending the emergency unit during the 2010-2011 winter seasons. Samples were tested with the multiplex RespiFinder(®) 15 assay (PathoFinder™) which potentially detects 15 viruses. RESULTS: 857 (88.7%) of the 966 samples collected from 914 children were positive for one (683 samples) or multiple viruses (174 samples). The most prevalent were the respiratory syncytial virus (39.5%) and the rhinovirus (24.4%). Influenza viruses were detected in 139 (14.4%) samples. Adenovirus was detected in 93 (9.6%) samples, coronaviruses in 88 (9.1%), metapneumovirus in 51 (5.3%) and parainfluenzae in 47 (4.9%). Rhinovirus (40%) was the most prevalent pathogen in upper respiratory tract infections while respiratory syncytial virus (49.9%) was the most prevalent in lower respiratory tract infections. Co-infections were associated with severe respiratory symptoms. CONCLUSION: The multiplex assay detected clinically important viruses in a single genomic test and thus will be useful for detecting several viruses causing respiratory tract disorders.

Prospective evaluation of a new automated nucleic acid extraction system using routine clinical respiratory specimens.

The aim of the study was to evaluate the MagNA Pure 96™ nucleic acid extraction system using clinical respiratory specimens for identifying viruses by qualitative real-time PCR assays. Three extraction methods were tested, that is, the MagNA Pure LC™, the COBAS Ampliprep™, and the MagNA Pure 96™ with 10-fold dilutions of an influenza A(H1N1)pdm09 sample. Two hundred thirty-nine respiratory specimens, 35 throat swabs, 164 nasopharyngeal specimens, and 40 broncho-alveolar fluids, were extracted with the MagNA Pure 96™ and the COBAS Ampliprep™ instruments. Forty COBAS Ampliprep™ positive samples were also tested. Real-time PCRs were used to identify influenza A and influenza A(H1N1)pdm09, rhinovirus, enterovirus, adenovirus, varicella zoster virus, cytomegalovirus, and herpes simplex virus. Similar results were obtained on RNA extracted from dilutions of influenza A(H1N1)pdm09 with the three systems: the MagNA Pure LC™, the COBAS Ampliprep™, and the MagNA Pure 96™. Data from clinical respiratory specimens extracted with the MagNA Pure 96™ and COBAS Ampliprep™ instruments were in 98.5% in agreement (P < 0.0001) for influenza A and influenza A(H1N1)pdm09. Data for rhinovirus were in 97.3% agreement (P < 0.0001) and in 96.8% agreement for enterovirus. They were in 100% agreement for adenovirus. Data for cytomegalovirus and HSV1-2 were in 95.2% agreement (P < 0.0001). The MagNA Pure 96™ instrument is easy-to-use, reliable, and has a high throughput for extracting total nucleic acid from respiratory specimens. These extracts are suitable for molecular diagnosis with any type of real-time PCR assay.

JC virus DNA in the peripheral blood of renal transplant patients: a 1-year prospective follow-up in France.

There is little information on JC virus (JCV) infection in renal transplant patients. A long-term prospective follow-up study was conducted to assess the incidence of JCV DNA in the blood of 103 adult renal transplant patients enrolled prospectively between 1 January and 31 December 2006. Patients were monitored until April 2008. JCV DNA was quantified by a real-time polymerase chain reaction in whole blood samples collected regularly for at least 1 year post-transplant. JCV was detected in seven patients (6.8%) (31/1,487 whole blood samples) at a median time of 139 days post-transplant. The median JC virus load of the first positive DNA blood sample was 3.4 log(10) copies/ml (1.9-5.7 log(10) copies/ml). Induction therapy were either anti-CD25 monoclonal antibodies (n = 5) or antithymocyte globulins (n = 2). Post-transplant immunosuppressive treatment included steroids with tacrolimus/mycophenolate mofetil (MMF) (n = 2), or ciclosporin/MMF (n = 1), or belatacept/MMF (n = 4). Two patients were also treated with rituximab. All seven patients infected with JCV had other viral infections(s): BK virus (3), Epstein-Barr virus (2), Cytomegalovirus (1) or both BK virus and Epstein-Barr virus (1). Three patients had BKV-associated nephropathy and decoy cells shedding. JCV infection was not associated with acute rejection episodes or nephropathy, regardless of the virus load. No patient developed progressive multifocal leukoencephalopathy during follow-up. Thus the incidence of JCV infection in renal transplant patients was low and not associated with any specific clinical manifestations. JCV replication must still be diagnosed and differentiated from BK virus infection because of its non-aggressive course.

Low interleukin-10 production by monocytes of patients with a self-limiting hepatitis C virus infection.

Host factors seem to be crucial for the spontaneous clearance of hepatitis C virus (HCV). Monocytes play a pivotal role in innate immunity and help regulate adaptive responses. This study assesses the characteristics of monocytes from patients with self-limiting HCV infections. We studied 35 consecutive patients [11 with a self-limiting HCV infection, 16 chronically infected with HCV and sustained virological responders (SVR) following antiviral therapy, and eight chronically infected HCV but untreated] and eight healthy donors (HD). The production of interleukin (IL)-10, tumour necrosis factor-alpha (TNF-alpha) and IL-12p40 by monocytes stimulated with lipopolysaccharides(LPS) or HCV Core protein was measured by enzyme-linked immunoassay. Monocyte surface markers were analysed by flow cytometry. LPS and Core protein triggered IL-10 and TNF-alpha production, but monocytes from self-limiting infection patients produced significantly less IL-10 and TNF-alpha than those of SVR, chronically infected or HD (P < 0.05), while IL-12p40 production was unchanged. This cytokine production profile did not appear to be due to expansion of the CD14(+) CD16(+) monocyte subset or to a classical or alternative activation monocyte profile. Monocytes from self-limiting infection patients had more CCR7 than those from SVR or chronically infected patients (P < 0.05). Monocytes of self-limiting infection patients appear to produce little IL-10 and TNF-alpha in response to viral or unspecific stimulation and to have a higher CCR7 expression. This profile seems to be independent to a particular monocyte subset or activation state. Low IL-10 production may help establish an effective immune response and spontaneous HCV clearance.

Comparison of two highly automated DNA extraction systems for quantifying Epstein-Barr virus in whole blood.

BACKGROUND: Optimal automated molecular methods are needed to monitor Epstein-Barr virus (EBV) infections in transplant recipients. OBJECTIVES: To compare the extraction of EBV DNA from whole blood using the COBAS Ampliprep and the MagNA Pure instruments (Roche) for quantifying EBV DNA by real-time PCR. STUDY DESIGN: EBV DNA content was determined on clinical samples extracted by both systems. RESULTS: The detection limit was 2.16log(10)copies/mL using the COBAS Ampliprep extraction system. Specificity was 100% and we saw no cross-contamination. Extraction was linear from 2.60 to 5.60log(10)copies/mL. The intra-assay variation was 1.91% for 3.60, 2% for 4.60 and 4.51% for 5.60log(10)copies/mL; inter-assay variation was 4.88%. Sixty-six samples were tested: 26 were positive and 28 were negative by both methods. One sample was MagNA Pure positive/COBAS Ampliprep negative (virus load 3.15log(10)copies/mL) and 10 samples were MagNA Pure negative/COBAS Ampliprep positive (virus loads from 1.59 to 3.51log(10)copies/mL) (P<0.0001). Both methods gave similar quantitative results (average difference 0.07log(10)copies/mL) which were well correlated (r=0.73, P<0.001). CONCLUSIONS: The COBAS Ampliprep extraction system is comparable to the MagNA Pure and offers a high reliability for extracting EBV DNA from whole blood.

Hepatitis E virus-related cirrhosis in kidney- and kidney-pancreas-transplant recipients.

Hepatitis E virus (HEV) infection was thought to be responsible for acute hepatitis that did not become chronic. However, we have recently reported that HEV infection can evolve to chronic hepatitis, at least in solid-organ transplant patients. We report on two cases of rapidly progressive of HEV-related cirrhosis that occurred in two organ-transplant patients. Case 1: A kidney-pancreas-transplant patient developed acute HEV hepatitis 60 months after transplantation, which evolved to chronicity as defined by persisting elevated liver-enzyme levels and positive serum HEV RNA. At 22 months after the acute phase, she presented with cirrhosis and portal hypertension, that is ascites and esophagus varices. Case 2: A kidney-transplant patient developed acute hepatitis 36 months after transplantation, which persisted and remained unexplained for 38 months. Then, HEV RNA was searched for in their serum and stools, and was found to be positive in both. Retrospective analysis of available stored serum, mainly the serum obtained at the acute phase, confirmed the diagnosis of chronic hepatitis E. In both cases, a liver biopsy showed cirrhosis. We conclude that HEV infection cannot only evolve to chronic hepatitis, but can also be responsible for rapidly progressing cirrhosis in organ-transplant patients.

Hepatitis C virus-related kidney disease: an overview.

Hepatitis C virus (HCV)-infection leads to chronic liver disease, but also to extra-hepatic manifestations, including kidney disease. We provide an overview of HCV-related kidney diseases in non-transplanted and in kidney transplant patients, and their therapies. Membranoproliferative glomerulonephritis, associated with Type 2 cryoglobulinemia, is the predominant Type of HCV-related glomerulonephritis. Membranous glomerulonephritis and focal segmental glomerular sclerosis are less commonly described. HCV infection seems to be linked to Type 2 diabetes mellitus, and might alter the progression of diabetic-related nephropathy. Patients infected by HCV should be annually screened for markers of kidney disease and, similarly, patients with membranoproliferative or membranous glomerulonephritis should be screened for HCV infection. After transplantation, cryoglobulinemia is frequent and is associated with HCV markers. HCV-related kidney disease requires specific treatment. In non-kidney-transplant patients, treatment relies on either only anti-HCV therapy in cases of moderate renal disease, or combined anti-viral and immunosuppressive therapies in cases of severe renal disease, i.e., nephrotic syndrome and/or progressive renal failure, and in diseases that are refractory to anti-HCV therapy. In kidney transplant patients, ribavirin monotherapy could be used cautiously, whereas rituximab might be a treatment of choice in the presence of cryoglobulinemia. In liver-transplant patients, in addition to anti-HCV therapy, rituximab might be also used.

Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay.

The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from plasma samples. The HCMV DNA from WB, PBL, and plasma was measured by real-time Light Cycler PCR. Primers and probe were located in the UL 83 region. HCMV DNA was detected more frequently in WB (88.5%) than in the PBL (65.7%) (P < 0.0001) or the plasma (55.2%) (P < 0.0001). There was a good correlation between the positive results in WB and in PBL (r = 0.68; P < 0.0001), and 3.15 log(10) genome copies in 200000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 microl of WB. WB was shown to be suitable for automated extraction and the quantitation of HCMV DNA by real-time Light Cycler PCR by analysis of serial samples from representative patients of various populations. This system may be very useful for monitoring of immune-suppressed and unsuppressed patients.

[Is there a place for ribavirin in the treatment for renal transplant patients infected by hepatitis C virus?]. / Y a-t-il une place pour la ribavirine dans le traitement des transplantés rénaux infectés par le virus de l'hépatite C?

Treatment of chronic hepatitis C in renal-transplant (RT) recipients with alpha-interferon is associated with a high rate of acute rejection. We therefore evaluated the biochemical, virological, histological efficacies, as well as the safety of one year ribavirin monotherapy in 16 HCV-(+) RNA (+) RT patients (group A) matched to 32 HCV-(+) RNA (+) RT patients (group B) who did not receive ribavirin. Ribavirin was initially started at a daily dose of 1000 mg and then adapted to hemoglobin level. Ribavirin monotherapy was associated with a significant decrease in AST, ALT and gamma glutamyl transpeptidase levels. Serum creatinine decreased as well. When proteinuria was present (n = 5), this decreased or disappeared. There was no significant changes in HCV viremia. The histological analysis of liver biopsies revealed a significant progression in liver fibrosis with no improvement in inflammation scores. There was a significant decrease in hemoglobin levels, despite an important support by recombinant erythropoeitin. However, in three cases, ribavirin therapy had to be stopped. In group B, after 1 year of follow up, there was a significant increase in serum ALT and creatinine values. Proteinuria decreased in only 2 of 12 patients. In conclusion, one year ribavirin therapy in HCV-(+) RNA (+)ve RT has no impact upon liver histology, although it improves liver enzyme levels. It impact upon renal function remains unknown. Nevertheless when proteinuria is present it disappears.

Screening for hepatitis B virus DNA in serum of organ donors and renal transplant recipients.

In order to determine the impact of screening potential organ donors for hepatitis B virus DNA using a standardized test, the serum of 145 donor candidates was tested. All of the candidates were negative for hepatitis B virus DNA, but the status of one donor was doubtful for hepatitis B virus surface antigen and seven donors tested positive for hepatitis B virus core antibody without hepatitis B virus surface antigen. Nine transplant recipients tested positive for hepatitis B virus surface antibody; they were given kidneys from the donor with a doubtful hepatitis B virus surface antigen result and from four of the seven donors who tested positive for hepatitis B core antibody. Follow-up revealed no case of hepatitis B transmission. In this study, screening for hepatitis B virus DNA was useful and did not lead to donor organ shortage. Patients with hepatitis B virus surface antibodies can safely be given kidneys from donors who are positive for hepatitis B core antibody but negative for hepatitis B virus DNA.

Determination of HCV genotype using two antibody assays and genome typing.

Two serotyping assays for hepatitis C virus (Serotyping 1-6 assay; Murex, UK and RIBA Serotyping SI; Chiron, USA) were compared to a standardized genotyping assay (Inno-LiPA HCV II; Innogenetics, Belgium) using serum samples collected from 126 patients chronically infected with hepatitis C virus. Serotyping was positive in 87% and 80% of the samples tested with Murex and Chiron, respectively, and concordant with genotyping in 93% and 88%, respectively. Sequence analysis of the NS5b region of 15 samples with discrepant typing results confirmed the Inno-LiPA finding in all instances. The Murex enzyme immunoassay serotyping method was less sensitive for identifying genotype 2 (P<0.05). The concordance with genotyping of the RIBA serotyping method was lower for genotype 2 than for the other genotypes (P<0.05).

Maintenance therapy with gradual reduction of the interferon dose over one year improves histological response in patients with chronic hepatitis C with biochemical response: results of a randomized trial.

BACKGROUND/AIMS: Our aim was to assess whether histological response was improved by continuing interferon-alpha (IFN) treatment in patients with chronic hepatitis C (HCV) with a biochemical response and no viral clearance after a usual IFN treatment. METHODS: Fifty-seven patients with normal alanine aminotransferase (ALAT) levels and positive HCV RNA at the end of a 1 year IFN treatment were randomly assigned to either group 1 (n = 28) where IFN was stopped, or group 2 (n = 29) where IFN was continued for 1 more year with gradual reduction of the dose to keep serum ALAT activity below the upper limit of normal. Liver biopsies were obtained before, and then 6 months after the end of treatment. RESULTS: Knodell's index improved between paired biopsies in group 2 (8.2+/-2.4 vs. 5.5+/-2.1), but not in group 1 (8+/-2.3 vs. 6.5+/-2). In post-treatment biopsies, the METAVIR activity score was significantly lower in group 2 than in group 1 (0.7+/-0.2 vs. 1.1+/-0.3, P < 0.05). In group 2, an improvement of the METAVIR fibrosis score was observed (1.3+/-0.4 vs. 1.1+/-0.2), whereas fibrosis progressed in group 1 (1.3+/-0.4 vs. 1.6+/-0.4). CONCLUSIONS: Maintenance therapy by the minimal dose of IFN able to maintain biochemical response prevents histological progression in the sub-group of patients without virological response.

Mother-to-child transmission of HIV infection and CTL escape through HLA-A2-SLYNTVATL epitope sequence variation.

Cytotoxic T lymphocytes (CTL) play a central role in containment of HIV infection. Evasion of the immune response by CTL escape is associated with progression to disease. It is therefore hypothesised that transmitted viruses encode escape mutations within epitopes that are required for successful control of viraemia. In order to test this hypothesis, escape through the dominant HLA-A2-restricted CTL epitope SLYNTVATL (p17 Gag residues 77-85 SL9) in the setting of mother-to-child-transmission (MTCT) was investigated. Initial data from two families in which the HIV-infected mother expressed HLA-A*0201 and had transmitted the virus to other family members were consistent with this hypothesis. In addition, analysis of the gag sequence phylogeny in one family demonstrated that CTL escape variants can be successfully transmitted both horizontally and vertically. To test the hypothesis further, a larger cohort of transmitting mothers (n=8) and non-transmitters (n=14) were studied. Variation within the SL9 epitope was associated with expression of HLA-A2 (P=0.04) but overall no clear link between variation from the SL9 consensus sequence and MTCT was established. However, the high level of background diversity within p17 Gag served to obscure any possible association between escape and MTCT. In conclusion, these studies highlighted the obstacles to demonstrating CTL escape arising at this particular epitope. Alternative strategies likely to be more definitive are discussed.

Genetic heterogeneity of hypervariable region 1 of the hepatitis C virus (HCV) genome and sensitivity of HCV to alpha interferon therapy.

Hepatitis C virus (HCV) populations persist in vivo as a mixture of heterogeneous viruses called quasispecies. The relationship between the genetic heterogeneity of these variants and their responses to antiviral treatment remains to be elucidated. We have studied 26 virus strains to determine the influence of hypervariable region 1 (HVR-1) of the HCV genome on the effectiveness of alpha interferon (IFN-alpha) therapy. Following PCR amplification, we cloned and sequenced HVR-1. Pretreatment serum samples from 13 individuals with chronic hepatitis C whose virus was subsequently eradicated by treatment were compared with samples from 13 nonresponders matched according to the major factors known to influence the response, i.e., sex, genotype, and pretreatment serum HCV RNA concentration. The degree of virus variation was assessed by analyzing 20 clones per sample and by calculating nucleotide sequence entropy (complexity) and genetic distances (diversity). Types of mutational changes were also determined by calculating nonsynonymous substitutions per nonsynonymous site (K(a)) and synonymous substitutions per synonymous site (K(s)). The paired-comparison analysis of the nucleotide sequence entropy and genetic distance showed no statistical differences between responders and nonresponders. By contrast, nonsynonymous substitutions were more frequent than synonymous substitutions (P

Prostate-specific antigen in acute hepatitis and hepatocellular carcinoma.

BACKGROUND: Prostate-specific antigen (PSA) is the most important tumor marker in prostate cancer diagnosis and follow-up. Its catabolism by the liver has not influenced its use as a prostate marker until the recent report of a significant increase in a man and a woman with acute hepatitis. In addition, PSA was detected in liver tumor extracts, which warranted its evaluation in liver cytolysis and hepatocellular carcinoma. In this study, PSA was evaluated in a cohort of both sexes presenting either acute hepatitis or hepatocellular carcinoima. METHODS: Forty-two patients with acute hepatitis (21 male patients, 21 female patients) and 54 patients with hepatocellular carcinoma (31 male patients, 23 female patients) were tested for PSA by equimolar immunoassay (Abbott AxSYM Total PSA, Abbott Diagnostics, Rungis, France) and for relevant liver biological parameters (alpha-fetoprotein, alanine aminotransferase, aspartate aminotransferase, total bilirubin, and prothrombin rate). RESULTS: PSA was undetectable in all the female patients and was consistent with age in the males (PSA median and range in acute hepatitis, 0.36 microg/l (range, 0.05-1.3); in hepatocellular carcinoma, 0.36 microg/l (range, 0.02-3.9)). It did not correlate with alpha-fetoprotein and aminotransferases. CONCLUSIONS: Our results confirm the well-established reliability of PSA, and show that PSA remains a valid prostate marker in patients with acute hepatitis and hepatocellular carcinoma.

[Long-term consequences of co-infection by hepatitis G virus in hepatitis c virus infected kidney transplant patients]. / Conséquences à long terme d'une surinfection par le virus de l'hépatite G chez des transplantés rénaux infectés par le virus de l'hépatite C.

The objectives of this retrospective study were to determine the prevalence of hepatitis G virus (HGV) infection in hepatitis C virus positive (HCV+ve) renal transplant (RT) patients and to evaluate the impact of HGV both on liver function tests, liver histology tests and renal parameters such as the prevalence of acute rejection and renal function. Seventy-one HCV+ve renal transplant patients with a functioning graft for whom a post renal transplant liver biopsy was available, were included. Serum HGV RNA was assessed by reverse transcription polymerase chain reaction before, at the time of, and after renal transplantation. A total of 21 (30%) of the HCV+ve RT patients had a positive HGV RNA (Group 1); seventeen of these patients (81%) were already HGV RNA+ve when the most recent renal transplantation was performed. The other 4 patients became HGV RNA+ve following renal transplantation. The mean duration of HGV infection was at least 119 +/- 64 months (18-240). Patients in group 1 did not statistically differ from the 50 HGV RNA-ve/HCV+ve RT patients (Group 2) according to sex ratio; time on dialysis; number of blood transfusions; HLA matching; the duration of HCV infection; duration and type of immunosuppression or levels of liver enzymes i.e. aspartate aminotransferase, alanine aminotransferase and gamma glutamyl transpeptidase; serum HCV RNA concentration; or frequency of genotype 1b. However, Group 1 patients were statistically younger (41 +/- 10 y compared to 47 +/- 10 y; p = 0.016) than Group 2 patients. Liver histology showed a significantly lower degree of fibrosis in Group 1 (0.4 +/- 0.5) than in Group 2 (1 +/- 1.2; p = 0.02); two patients from Group 2 but none of Group 1 had overt cirrhosis. Conversely, the extent of hepatic inflammation and hepatocellular necrosis was not statistically different between the two groups. The number of patients who experienced at least one acute rejection episode was significantly higher in Group 1 (76.2%) than in Group 2 (46%; p = 0.02), although the difference was no longer significant in the multivariate analysis. In conclusion, this study shows that: i) HGV infection was often present when the patients seroconverted for HCV; ii) HGV RNA+ve/HCV+ve RT patients experience acute rejection more frequently than HGV RNA-ve/HCV+ve RT patients; iii) HGV infection seems to have no detrimental effect upon liver enzymes or liver histology in HCV+ve RT patients.

Molecular evidence for nosocomial transmission of hepatitis C virus in a French hemodialysis unit.

A systematic virological follow-up of hemodialysis patients identified 11 cases of de novo hepatitis C virus (HCV) infection in the same unit that were not due to blood transfusion. There were three groups of infection, each occurring within a period of 3 months: four infections with genotype 1b, two infections with genotype 1b, and five infections, four with genotype 1a and one with genotype 5a. The possibility of patient-to-patient transmission was addressed by sequencing the first hypervariable region of the HCV genome in sera taken shortly after infection. Phylogenetic analysis indicated clustering of most of the cases of de novo infections. Sequence homologies identified potential contaminators among already infected patients. All patients who were infected with closely related HCV isolates were found to have been treated in the same area and during the same shift or on the previous one. These infections could have been due to occasional breaches of the usual hygiene measures. Strict adhesion to hygiene standards and routines, continuously supervised, remains the key rule in the management of dialysis patients. Nevertheless, the isolation of patients with HCV could reduce the risk of infection because occasional lapses of preventive hygiene measures or unpredictable accidents can always take place in a hemodialysis unit. This policy needs to be evaluated by large-scale prospective studies.

Intermittent selection pressure with zidovudine plus zalcitabine treatment reduces the emergence in vivo of zidovudine resistance HIV mutations.

The development of mutations conferring drug resistance was investigated in 49 antiretroviral-naive asymptomatic HIV-1 subjects with CD4+ cell counts of 250-500/mm3 given intermittent (6-week courses, 6 weeks apart) or continuous treatment with zidovudine (AZT) plus zalcitabine (ddC) over 54 weeks. The concentration of human immunodeficiency virus type 1 RNA in the plasma and the CD4 cell counts were measured every 6 weeks. The rate of decrease of HIV-1 RNA concentration in plasma after a 6-week course of AZT + ddC was similar for each treatment cycle (approximately 1-log reduction). The plasma HIV-1 RNA concentration returned to its initial level at each treatment interruption. The mean CD4 cell counts after 54 weeks in the two treatment groups were similar. Genotype analysis by sequencing the reverse transcriptase coding region from plasma viral RNA on treatment showed a lower frequency of AZT resistance mutations after 54 weeks in patients given intermittent treatment (18%) than in those treated continuously (79 %, P < 0.001). No mutations conferring ddC resistance or multidideoxynucleoside resistance were observed in either group. These findings may have clinical implications for long-term treatment strategies.