Véronique Venard

Low Genetic Diversity despite Hyperendemicity of Hepatitis B Virus Genotype E throughout West Africa

Sub-Saharan Africa suffers from an excessively high endemicity of hepatitis B virus (HBV), but little is known about the prevalent genotypes. In this study, we investigated the PreS1/PreS2/S genes of 127 viruses obtained from 12 locations in Mali, Burkina Faso, Togo, Benin, Nigeria, Cameroon, and the Democratic Republic of Congo. Except for those obtained from the Cameroon HIV cohort (18/22 HBV genotype A), 96 of 105 sequences belonged to HBV genotype E (HBV/E), and viral DNA was very similar (1.67% diversity) throughout this vast HBV/E crescent, which spans 6000 km across Africa. The low diversity suggests that HBV/E may have a short evolutionary history. Considering a typical mutation rate of DNA viruses, it would take only 200 years for the strain diversity of HBV/E viruses to develop from a single introductory event. The relatively recent introduction of HBV/E into humans would also explain its conspicuous absence in the Americas, despite the forced immigration of slaves from west Africa, until the early 19th century. Infection during infancy is mostly associated with chronic carrier status, and this combination can account for the explosive spread of virtually identical viruses within a community, but whether other routes of long-range transmissions must be considered becomes an important question.

Prevalence of hepatitis B and C and risk factors for nonvaccination in inflammatory bowel disease patients in Northeast France.

Background: Data regarding the prevalence of hepatitis C (HCV) and hepatitis B (HBV) in inflammatory bowel disease (IBD) patients are conflicting. Methods: In all, 315 IBD (252 Crohns disease [CD] and 63 ulcerative colitis [UC]) patients were consecutively recruited between June 2005 and May 2009. Results: The median age was 33 years (interquartile range [IQR]: 24–43) and median disease duration was 5 years (IQR: 2–11). Present and/or past HBV and HCV infection was found in 2.86% of 315 patients (CD: HBsAg 0.79%, anti‐HBc 2.78%, anti‐HCV 0.79%; UC: HBsAg 1.59%, anti‐HBc 1.59%, anti‐HCV 1.59%). Effective vaccination (anti‐HBs without anti‐HBc) was present in 48.9% of 315 patients. In multivariate analysis, age at diagnosis over 31 years (odds ratio [OR] 0.29; 95% confidence interval [CI] 0.15–0.58; P = 0.005), disease duration over 7 years (OR 0.43; 95% CI 0.23–0.83; P = 0.005), age at inclusion over 33 years (OR 0.44; 95% CI 0.20–0.94; P = 0.005), and CD (OR 0.29; 95% CI 0.15–0.58; P = 0.005) were associated with the lack of effective vaccination. Two HBsAg‐positive patients, including 1 under curative nucleoside/nucleotide analog treatment, had received 6 and 7 infliximab infusions, and 1 HCV RNA‐positive subject had been receiving corticosteroid and azathioprine therapies for 12 and 33 months, respectively. No viral reactivation occurred in these patients. Conclusions: The prevalence of HBV and HCV infection in French IBD patients is similar to that of the general population. While the ECCO recommends an effective HBV vaccination in IBD, half of the patients were not vaccinated. The nonvaccination risk factors identified in our study may allow targeted vaccination coverage. (Inflamm Bowel Dis 2009;)

Human herpesvirus 6 infection after allogeneic stem cell transplantation: incidence, outcome, and factors associated with HHV-6 reactivation.

Background Human herpesvirus 6 (HHV-6) is susceptible to latency and reactivation in hematopoietic stem cell transplant (HSCT) recipients. We investigated the incidence of HHV-6 DNAemia and factors related to HHV-6 DNAemia and death after allogeneic stem cell transplantations. We also explored the relationship between HHV-6 viral load and the presence of clinical signs. Methods Data concerning age, sex, transplantation conditions, graft-versus-host disease (GVHD), treatments, clinical signs, outcome, HHV-6, and other infections were collected for a historical cohort of 390 HSCT performed between 1999 and 2008 in the Transplant Unit of Nancy University Hospital Center. Univariate analysis was used to evaluate influences between the different parameters. Results The study included 220 of the 390 allogeneic HSCTs. For the analyzed period, 44 patients (n=44/220, 20%) presented HHV-6 DNAemia in whole blood, including three integrated forms. Fifteen percent (7/41) of HHV-6–positive patients presented clinical signs not related to higher viral load (P=0.164). The factors associated with HHV-6 DNAemia were as follows: cord blood transplantation (P<0.001), conditioning regimen (P=0.030), acute GVHD (P=0.003), and the type of prophylactic treatment for GVHD (P=0.001). HHV-6 DNAemia was not significantly associated with cytomegalovirus infection (P=0.937). HHV-6 DNAemia was not associated with death (P=0.151). Conclusions HHV-6 DNAemia was not so frequent after allogeneic transplantation. Factors associated with HHV-6 DNAemia were similar to those for other infections. No abnormally high death rate was observed in the HHV-6 positive population. The presence of clinical signs did not appear to be statistically related to HHV-6 viral load.

Rapid generation of full clinical-grade human antiadenovirus cytotoxic T cells for adoptive immunotherapy.

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-γ (IFN-γ) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01±0.84×106 total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56%±20.8%, yield=51%±32.4%) but also CD8+ (mean=42%±27%, yield=56%±39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-γ and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

Comparison of In-House Real-Time Quantitative PCR to the Adenovirus R-Gene Kit for Determination of Adenovirus Load in Clinical Samples

ABSTRACT In the context of hematopoietic stem cell transplantation, adenovirus infections are associated with relevant mortality and morbidity. Detection of adenovirus DNA by quantitative PCR is the “gold standard” for these patients. A total of 150 samples, namely, 78 whole-blood, 22 cerebrospinal fluid, 24 digestive biopsy, and 26 stool samples, from 29 patients, including 24 hematopoietic stem cell transplant recipients, were tested for the detection of adenovirus using an in-house real-time quantitative PCR assay (A. Heim, C. Ebnet, G. Harste, and P. Pring-Akerblom, J. Med. Virol. 70:228-239, 2003) and the commercially available Adenovirus R-Gene kit. Adenovirus DNA was automatically isolated from whole-blood samples (Magna Pure LC system; Roche) or was manually extracted from other specimens (QIAamp; Qiagen) using the appropriate kit. The intra- and interassay reproducibilities and sensitivities were evaluated with cell culture supernatant dilutions. Of the 150 samples tested, 86 were found to be positive and 55 were found to be negative using both techniques. Nine (6%) discordant results were obtained. In most cases, discrepant results concerned samples with low viral loads. Quantitative results for all concordant positive samples were analyzed using the Spearman correlation test. A good correlation between the results of the in-house assay and those of the kit assay was obtained (r = 0.95; P < 0.001). Regarding the threshold cycle value for internal control spiked samples, none of the 150 samples tested contained a PCR inhibitor. In conclusion, a relevant correlation of results between the in-house assay and the kit assay, as well as the high-quality reproducibility and sensitivity of the kit assay, warranted its use for follow-up of hematopoietic stem cell transplantation recipients.

Contribution of human herpesvirus 6 (HHV-6) viral load in whole blood and serum to investigate integrated HHV-6 transmission after haematopoietic stem cell transplantation.

BACKGROUND Human herpesvirus 6 (HHV-6) is susceptible to latency and recurrence. A less-frequent form of HHV-6 persistence is the integration of viral DNA into host chromosomes. OBJECTIVES To investigate HHV-6 viral load after haematopoietic stem cell transplantation (HSCT) in whole blood (WB) and serum with regard to integrated HHV-6 transmission diagnosis. STUDY DESIGN HHV-6 DNA quantitation in serum and WB was performed using quantitative polymerase chain reaction for the follow-up of a 16-year-old girl after HSCT. In whole blood, results were expressed as HHV-6 genomic equivalent copies (gec) per milliliter of WB or per million cells. RESULTS HHV-6 viral load (undetectable before HSCT) increased up to 3.05 x 10(7)gec/10(6)cells. HHV-6 viral load in the donor sample (3.44 x 10(6)gec/10(6)cells) was in favor of viral transmission through HSCT. The correlation between viral load in WB and serum was significant (p=0.0005). Viral load results expressed as gec/10(6)cells in WB was more reliable than results expressed as gec/ml of whole blood. CONCLUSION These findings indicate that HHV-6 may be transmitted during HSCT as integrated virus contained in the graft. This reiterates that in the setting of HSCT, HHV-6 viral load must be correctly interpreted. Using HHV-6 viral load expressed as gec/10(6) cells may be more suitable for the follow-up of patients with integrated HHV-6.

Effective ribavirin concentration in mice brain using cyclodextrin as a drug carrier: Evaluation in a measles encephalitis model

Ribavirin (RBV) is a water-soluble synthetic nucleoside with broad spectrum antiviral properties, but it is ineffective against major viral encephalitis because of a failure to cross the blood-brain barrier (BBB). The antiviral activity of the complex ribavirin/alpha-cyclodextrin was previously demonstrated to be stronger than free ribavirin, in an in vivo model of measles virus (MV) encephalitis in mice. The role of cyclodextrin (CD) on ribavirin uptake into the brain needs to be defined. Ribavirin specific extraction from brain tissue was developed, based on a solid phase extraction. It was quantified by high performance liquid chromatography at different time points after intraperitoneal injection of single or multiple doses of free ribavirin or of the complex ribavirin/alpha-cyclodextrin. Whatever the tested dose (40 or 100mg/kg), the amount of ribavirin in the brain was significantly higher (p<0.001) when the drug was injected as a complex with alpha-cyclodextrin, in healthy or measles virus-infected mice.

In vitro antiviral efficacy of the ganciclovir complexed with β-cyclodextrin on human cytomegalovirus clinical strains

The toxicity of the compounds currently used in the treatment of human cytomegalovirus (HCMV) infections in immunocompromised hosts may force the treatment to be discontinued. The aim of this study was to improve the antiviral activity of ganciclovir (GCV), one the most widely used drug, by complexing it with beta-cyclodextrin. Cyclodextrins (cds) have the property to form inclusion complexes with a great number of molecules and to enhance bioavailability and biological properties of these molecules. In this study, we investigated the in vitro antiviral activity of complexed GCV against several strains of HCMV: AD169, a reference strain, RCL-1, a laboratory mutant resistant to GCV, and four clinical isolates. The complexed GCV was more effective than free GCV against all HCMV strains tested. Cds as carriers for antiviral drugs would represent a useful adjunct to classical treatment procedures. They may make it possible to administer lower doses, thus reducing the toxic side effects of the drugs.

The clinical value of concomitant Epstein Barr virus (EBV)-DNA load and specific immune reconstitution monitoring after allogeneic hematopoietic stem cell transplantation.

BACKGROUND Monitoring of EBV DNAemia after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary, but not sufficient, to identify patients at risk of EBV-induced post-transplantation lymphoproliferative disorders (PTLD). Combining this with quantifying EBV-specific cellular immunity was shown to be helpful. In this study, we evaluated the value of IFNγ-Elispot assay in monitoring EBV DNAemia after HSCT. METHODS EBV-DNA load in whole blood was monitored at least weekly using real-time PCR in 40 recipients of HSCT. Quantitative and qualitative T-cell recoveries, including EBV-specific T-cell quantification by Elispot assay, were studied 60, 100, 180 and 360 days after HSCT. RESULTS Among the 35 evaluable patients, 14 (35%) presented EBV DNAemia, only 2/14 (14%) needing pre-emptive treatment with rituximab. The greatest risk factor for EBV DNAemia was the presence of anti-thymocyte globulin (ATG) (p=0.005). EBV-specific cellular immune recovery was monitored by IFNγ-Elispot assay. Using multivariate analysis, four factors were found to significantly influence IFNγ-Elispot results at defined times post-HSCT: EBV DNAemia, young age, global T-cell recovery and severe acute GVHD. In those cases where EBV DNAemia occurred and cleared spontaneously, Elispot results gave more than 1000 spot-forming cells (SFC)/10(6)PBMC. CONCLUSION Elispot assay may be usefully combined with EBV-DNA load monitoring to determine when a patient should receive pre-emptive treatment, or when the clinician should avoid Rituximab use which severely immunocompromises patients.

Hepatitis B virus X gene variability in French-born patients with chronic hepatitis and hepatocellular carcinoma

The polymorphism of the hepatitis B virus (HBV) X gene from patients born in Lorraine has been studied in serum samples from 22 HBV infected patients, 14 presenting with chronic hepatitis and 8 with hepatocellular carcinoma (HCC). Subtypes adw and ayw represented 21 of the 22 sequenced isolates. The sequence of the X gene of HBV strains from these patients differed from the ones of Far East origin by A to T1678 and G to A1759 changes for subtype ayw and C to T1792 for adw.