Karine Sandres-Sauné

Evidence that Clearance of Hepatitis C Virus RNA after α-Interferon Therapy in Dialysis Patients Is Sustained after Renal Transplantation

To date, there is no available treatment of hepatitis C virus (HCV) infection after renal transplantation (RT). Among 55 anti-HCV-positive/HCV RNA-positive hemodialysis patients who were treated with IFN-alpha (9 MU/wk during 6 or 12 mo), 21 of them (38%) had a sustained virologic response. Of these, 16 (76%) underwent RT 38 mo (range, 2 to 57 mo) after alpha-IFN therapy. There were 13 men and 3 women aged 46 yr (range, 27 to 68 yr). At RT, HCV serology was still positive in 15 patients, and HCV viremia was negative in all patients. Immunosuppression relied on anticalcineurin agents with or without steroids and/or antimetabolites; in addition, 12 of them received induction therapy with antithymocyte globulins. At the last follow-up after RT, at 22.5 mo (range, 2 to 88 mo), HCV viremia remained negative in all patients. Moreover, HCV RNA was not present in peripheral blood mononuclear cells when assessed in eight patients. HCV serology was found to be still positive in 13 patients. Three patients presented with acute rejection, one presented with a suppurative lymphocele, one died from a sepsis, and four presented with a cytomegalovirus infection. None of them developed posttransplant diabetes mellitus. In conclusion, hemodialysis patients waiting for a RT need to be treated with alpha-IFN because when HCV RNA clearance occurred, they experienced no relapse after transplantation despite chronic immunosuppressive treatment.

Correlation between genotypic predictions based on V3 sequences and phenotypic determination of HIV-1 tropism.

Objective:Replacing phenotypic assays with simple genotypic predictions of HIV-1 coreceptor usage would make the clinical use of CCR5 antagonists easier. Design:Paired genotypic and phenotypic determination of HIV-1 coreceptor usage was performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting. Methods:HIV-1 coreceptor usage was prospectively assessed using plasma samples from 103 patients who were candidates for treatment with a CCR5 antagonist. Direct sequencing of the V3 region and a sensitive recombinant virus phenotypic entry assay were performed in parallel for each patient from the same bulk env PCR product. Results:The 103 patients had a median CD4+ T lymphocyte count of 268 × 106 cells/l and nadirs of 98 × 106 cells/l. Paired genotypic and phenotypic data were obtained for 98 of the 103 patients. For detecting CXCR4-using viruses, the genotypic rule based on amino-acid residues at positions 11/25 and the overall net charge of V3 was 77% sensitive and 96% specific. The Geno2pheno bioinformatic tool was 88% sensitive and 87% specific. The WebPSSM tool prediction with the SI/NSI matrix was 77% sensitive and 94% specific. The global concordance between genotypic and phenotypic data was 91% with the rule combining the amino-acid residues at positions 11/25 and V3 net charge. Conclusion:Genotypic predictions performed well in paired genotypic and phenotypic assessment of HIV-1 coreceptor usage. Multicenter studies analyzing the correlations between the genotypic determination of HIV-1 tropism and clinical response to CCR5 antagonists are needed to validate this approach in clinical practice.

New Natural Intergenotypic (2/5) Recombinant of Hepatitis C Virus

ABSTRACT A 9.2-kb sequence from a hepatitis C virus (HCV) strain found in southwest France was compared to sequences from reference strains in HCV sequence databases. We found a recombinant virus with genotype 2 at the 5′ end and genotype 5 at the 3′ end. The crossover point was located between genes NS2 and NS3. Recombination between HCV genotypes must now be considered in studies on HCV epidemiology and evolution and in predictions of the virus response to antiviral therapy. Knowing the location of the recombination point may also be useful for constructing infectious chimeric viruses.

R5 to X4 switch of the predominant HIV-1 population in cellular reservoirs during effective highly active antiretroviral therapy.

Summary:HIV-1 coreceptor usage plays a critical role for virus tropism and pathogenesis. A switch from CCR5 to CXCR4-using viruses can occur in the natural course of infection and correlates with subsequent disease progression. To investigate whether HIV-1 genetic evolution might lead to changes in virus coreceptor usage during highly active antiretroviral therapy (HAART), a longitudinal genotypic analysis of the virus found in cellular reservoirs was conducted in 32 patients with undetectable viral loads on HAART for 5 years. The genotype of the 3rd variable region of the env gene predicting coreceptor usage was retrospectively determined in the plasma or in peripheral blood mononuclear cells (PBMC) at baseline and then in PBMCs at months 30 and 60 of HAART. There was a switch from R5 to X4 variants in 11 of the 23 patients who harbored a majority virus population of R5 variants at baseline. X4 variants remained predominant in the 9 patients who harbored mainly X4 variants at baseline. The patients harboring predominantly X4 variants during HAART, either from baseline or after an R5 to X4 switch, tended to have lower CD4+ T-cell counts on HAART than did patients harboring continuously a majority population of R5 variants. These results suggest that potent antiretroviral therapy produces the conditions necessary for the gradual emergence of X4 variants in cellular reservoirs.

Natural history of hepatitis C virus-related liver fibrosis after renal transplantation.

The aim of our study was to assess hepatitis C virus (HCV) evolution and long‐term liver histology outcome in anti‐HCV(+)/RNA(+) renal transplant (RT) patients. A total of 51 anti‐HCV(+)/RNA(+) RT patients underwent liver biopsies (LB) every 3–4 years after transplantation (two LBs, n = 51; three LBs, n = 42; four LBs, n = 9). The hypervariable region (HVR)‐1 of the HCV genome from all patients was characterized over time. Overall, the rate of liver fibrosis progression was 0.09 ± 0.03 Metavir units/year. We identified three groups of patients: those in whom liver fibrosis remained stable (n = 21), those with progressing liver fibrosis (n = 21) and those with a regression in liver fibrosis (n = 10). In the last two groups, the progression and the regression of liver fibrosis were gradual during follow‐up. Ferritin levels and hepatosiderosis were significantly higher in fibrosers. Initial fibrosis stage and high diversification of the HVR‐1 of HCV genome between transplantation and the first liver biopsy were independent factors associated with liver fibrosis regression. In conclusion, in the current study, more than 10 years after renal transplantation, HCV infection was not harmful upon liver histology in more than 50% of patients. The diversification of the HVR‐1 might be used to predict liver fibrosis outcome.

Hepatitis E Virus Infection without Reactivation in Solid-Organ Transplant Recipients, France

Infections with hepatitis E virus (HEV) in solid-organ transplant recipients can lead to chronic hepatitis. However, the incidence of de novo HEV infections after transplantation and risk for reactivation in patients with antibodies against HEV before transplantation are unknown. Pretransplant prevalence of these antibodies in 700 solid-organ transplant recipients at Toulouse University Hospital in France was 14.1%. We found no HEV reactivation among patients with antibodies against HEV at the first annual checkup or by measuring liver enzyme activities and HEV RNA. In contrast, we found 34 locally acquired HEV infections among patients with no antibodies against HEV, 47% of whom had a chronic infection, resulting in an incidence of 3.2/100 person-years. Independent risk factors for HEV infection were an age <52 years at transplantation and receiving a liver transplant. Effective prophylactic measures that include those for potential zoonotic infections should reduce the risk for HEV transmission in this population.

Long-Term Ribavirin Therapy in Hepatitis C Virus-Positive Renal Transplant Patients: Effects on Renal Function and Liver Histology

BACKGROUND Long-term renal transplant (RT) recipient mortality and graft loss increase significantly in hepatitis C virus positive (HCV-[+]ve) patients. Treatment with alpha-interferon in this population is associated with a high rate of acute rejection. The aims of this study were the evaluation of the efficacy and the safety of ribavirin monotherapy in 16 HCV-(+) RT patients (group A) matched to 32 HCV-(+) RT patients (group B) who did not receive ribavirin. METHODS Ribavirin was started at a daily dose of 1,000 mg and then adapted to hemoglobin level. The study was scheduled for 1 year. RESULTS Ribavirin monotherapy was associated with a decrease in liver enzymes and serum creatinine levels. When proteinuria was present, this decreased or disappeared. There were no significant changes in HCV viremia. There was a significant progression in liver fibrosis with no improvement in inflammation scores. Hemoglobin levels fall dramatically, despite an important support by recombinant erythropoeitin (median, 20,000 IU/wk). In 3 cases, ribavirin therapy had to be stopped. In the control group, after 1 year of follow-up, there was a significant increase in serum alanine aminotransferase and creatinine values. Proteinuria decreased in only 2 of 12 patients (P = 0.03 as compared with group A). CONCLUSION One year of ribavirin monotherapy seems to have, at best, no beneficial effect on liver histology, although it improves liver enzyme levels. Despite its efficiency to dramatically decrease proteinuria, its impact on renal function remains unknown.

Genotype 3 Diversity and Quantification of Hepatitis E Virus RNA

ABSTRACT Genotype 3 hepatitis E viruses (HEVs) are distributed across the world and are now considered to be an emerging public health concern in industrialized countries. At least 10 genotype 3 subtypes have been identified in humans and animals worldwide. It was recently reported that the sensitivities of HEV RNA assays differ greatly. We have assessed the influence of genotype 3 diversity on the performances of two HEV RNA assays: one targeting the ORF3 gene and the other targeting the ORF2 gene. We tested a panel of 5 HEV-positive reference samples of genotypes 3a, 3b, 3c, 3e, and 3f at 10-fold serial dilutions. The HEV RNA concentrations obtained with both reverse transcription (RT)-PCRs were correlated, but the RT-PCR based on ORF2 underestimated the HEV RNA concentrations. The mean [ORF3 − ORF2] difference was 1.41 log copies/ml. We also tested 34 clinical specimens of genotypes 3c (n = 15), 3e (n = 4), and 3f (n = 15), representing the most prevalent subtypes in Europe. The mean [ORF3 − ORF2] differences were 1.41 log copies/ml for genotype 3c, 0.96 log copies/ml for genotype 3e, and 0.70 log copies/ml for genotype 3f. The bias between the 2 RT-PCR assays was significantly greater for genotype 3c than for genotype 3f (P = 0.007). We therefore recommend the use of an RT-PCR protocol based on ORF3 to quantify HEV RNA of genotype 3 strains.

Persistence of distinct HIV-1 populations in blood monocytes and naive and memory CD4 T cells during prolonged suppressive HAART.

Objective:Reservoirs of HIV-1 are a major obstacle to virus eradication. There is therefore a need to clearly understand the molecular nature of the virus populations that persist in patients with sustained suppression of plasma viraemia on highly active antiretroviral therapy (HAART). Design:We performed a detailed analysis of the genotypes of HIV-1 quasispecies isolated from highly purified blood cell types taken from three selected patients with sustained undetectable viral loads on HAART for 7 years. Methods:We used polychromatic flow cytometry to sort naive and memory CD4 T cells, CD14 monocytes, and CD56+CD3− natural killer (NK) cells from the total peripheral blood mononuclear cells after 7 years of HAART. Clonal analysis was used to determine coreceptor use and drug-resistance genotypes of HIV-1 quasispecies in the sorted blood cell types. Results:We detected HIV-1 DNA in memory and naive CD4 T cells and in CD14 monocytes, but not in the CD56+CD3− NK cells. Phylogenetic analysis demonstrated that the various blood cells types of two of the three patients harboured genetically distinct HIV-1 quasispecies. Drug-resistance mutations were also distributed differently from one cell type to another. This compartmentalization suggests a minimal virus trafficking between blood cell types during suppressive HAART. Conclusions:We observed a cell-specific compartmentalization of the residual virus populations during prolonged suppressive HAART. The coexistence of numerous HIV-1 quasispecies with different resistance genotypes and coreceptor use in cellular reservoirs may be relevant for future antiretroviral treatment strategies.

Development and performance of a new recombinant virus phenotypic entry assay to determine HIV-1 coreceptor usage

BACKGROUND Clinical trials of CCR5 antagonists have relied on the phenotypic determination of HIV-1 coreceptor usage. Few phenotypic assays are available, with few data on their concordance, and none has been designed to determine tropism from cell-associated HIV-1 DNA. OBJECTIVES To assess the performance of the new Toulouse Tropism Test (TTT) phenotypic assay to characterize HIV-1 tropism using blood plasma and peripheral blood mononuclear cells (PBMC). STUDY DESIGN 434 plasma and 168 PBMC samples were tested with the TTT assay. We determined the correlation between our assay results on plasma samples and those of the commercial Trofile assay. RESULTS The TTT assay determined the tropism of 97% of samples after successful amplification of the env gene. It performed well on both cell samples and plasma samples with various HIV-1 loads and subtypes. It detected 0.5% of minor CXCR4-using variants in the virus population. The TTT and the Trofile assays were >90% concordant for predicting HIV-1 tropism. CONCLUSION We have validated a new recombinant virus phenotypic assay for determining HIV-1 tropism using both plasma and cell samples from patients who are candidates for treatment with CCR5 antagonists.