Anne Marie Roque-Afonso

Comparison of real-time RT-PCR assays for hepatitis E virus RNA detection

BACKGROUND Hepatitis E virus (HEV) is an increasing cause of acute viral hepatitis in developed countries. Serological testing alone may fail to diagnose acute infection, especially in immunocompromised patients, which justifies the use of molecular assays for diagnosis. Few studies have compared accuracy of HEV RNA detection assays. OBJECTIVES The performances of five real-time PCR procedures for HEV RNA detection were compared. STUDY DESIGN First, RNA quantification of hepatitis E diluted standards of 3a and 3b genotypes were performed. Secondly, forty-seven clinical samples of patients with known acute HEV infection were tested using five hepatitis E RNA detection methods of assigned letters A, B, C, D and E. RESULTS Standards of HEV 3a genotype were detected in 100% of replicates with 2500 UI/ml of sensitivity by using A, B and C assays. Standards of HEV 3b genotype were more accurately detected with a sensitivity of 25 UI/ml in 100% of replicates using C assay and were detected in 100% of replicates with 2500 UI/ml of sensitivity by using A, B and E assays. Overall, B assay detected all of 250 UI/ml dilution and occasionally the 25 UI/ml dilution on both subtypes. The detection rates of clinical samples were 100%, 100% 97%, 97% and 83% for the respective A, B, C, D and E assay. Assays A and B were well correlated, independently of the subtype. However, discrepancies were observed when these techniques were compared to C, D and E assays according to the different subtypes. CONCLUSION A and B assays appear reliable for HEV RNA detection. These assays target the ORF2/3 overlapping region, described as more conserved than ORF2.

Detection of Hepatitis A Virus RNA in Saliva

ABSTRACT Hepatitis A virus (HAV) is shed in feces but also in saliva. HAV RNA was detected in saliva in five out of six acutely infected patients with HAV viremia. Serum and saliva sequences were identical. The simplicity of obtaining material allows the recommendation of the use of saliva for investigation of outbreaks.

Influence of CD4 Cell Counts on the Genetic Heterogeneity of Hepatitis C Virus in Patients Coinfected with Human Immunodeficiency Virus

To study the effects of reduced CD4 T cell activity on hepatitis C virus (HCV) genetic heterogeneity, HCV quasi-species complexity and diversification over time were analyzed for 56 human immunodeficiency virus-coinfected patients. Patients were selected retrospectively from the French Seroconverter Cohort (SEROCO) and the French Hemophilia Cohort (HEMOCO) for having stable CD4 cell counts for 3 years. HCV complexity was assessed by single-strand conformation polymorphism analysis of the envelope-coding region (HVR) and the core region at 2 time points 3 years apart. Increased HVR complexity was associated with higher CD4 cell count and HCV genotype 1 infection. Qualitative variation of HVR and core region was not related to CD4 cell count and depended on the initial complexity. Complexity of both regions remained unchanged over 3 years. Among these HCV-HIV-coinfected patients with stable CD4 cell counts, viral genotype and CD4 cell count may have influenced HVR complexity before their inclusion in the study but were not involved in HVR diversification during the 3-year follow-up period.

Rational Basis for Optimizing Short and Long-term Hepatitis B Virus Prophylaxis Post Liver Transplantation: Role of Hepatitis B Immune Globulin.

Abstract Antiviral therapy using newer nucleos(t)ide analogues with lower resistance rates, such as entecavir or tenofovir, suppress hepatitis B virus (HBV) replication, improve liver function in patients with compensated or decompensated cirrhosis, and delay or obviate the need for liver transplantation in some patients. After liver transplantation, the combination of long-term antiviral and low-dose hepatitis B Immune globulin (HBIG) can effectively prevent HBV recurrence in greater than 90% of transplant recipients. Some forms of HBV prophylaxis need to be continued indefinitely after transplantation but, in patients with a low-risk of HBV recurrence (i.e., HBV DNA levels undetectable before transplantation), it is possible to discontinue HBIG and maintain only long-term nucleos(t)ide analogue(s) therapy. A more cautious approach is necessary for those patients with high pretransplant HBV DNA levels, those with limited antiviral options if HBV recurrence occurs (i.e., HIV or hepatitis D virus coinfection, preexisting drug resistance), those with a high risk of hepatocellular carcinoma recurrence, and those at risk of noncompliance with antiviral therapy. In this group, HBIG-free prophylaxis cannot be recommended.

Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype by Abbott RealTime HCV Genotype II Assay Using the New Abbott HCV Genotype Plus RUO Test.

ABSTRACT Hepatitis C virus (HCV) genotyping continues to be relevant for therapeutic strategies. Some samples are reported as genotype 1 (gt 1) without subtype by the Abbott RealTime HCV Genotype II (GT II) test. To characterize such samples further, the Abbott HCV Genotype Plus RUO (Plus) assay, which targets the core region for gt 1a, gt 1b, and gt 6 detection, was evaluated as a reflex test in reference to NS5B or 5′-untranslated region (UTR)/core region sequencing. Of 3,626 routine samples, results of gt 1 without subtype were received for 171 samples (4.7%), accounting for 11.5% of gt 1 specimens. The Plus assay and sequencing were applied to 98 of those samples. NS5B or 5′-UTR/core region sequencing was successful for 91/98 specimens (92.9%). Plus assay and sequencing results were concordant for 87.9% of specimens (80/91 samples). Sequencing confirmed Plus assay results for 82.6%, 85.7%, 100%, and 89.3% of gt 1a, gt 1b, gt 6, and non-gt 1a/1b/6 results, respectively. Notably, 12 gt 6 samples that had been identified previously as gt 1 without subtype were assigned correctly here; for 25/28 samples reported as “not detected” by the Plus assay, sequencing identified the samples as gt 1 with subtypes other than 1a/1b. The genetic variability of HCV continues to present challenges for the current genotyping platforms regardless of the applied methodology. Samples identified by the GT II assay as gt 1 without subtype can be further resolved and reliably characterized by the new Plus assay.

Subgenotype D5, BCP and MHR mutations in hepatic complications among hepatitis B virus infected patients from Orissa, India

The study was undertaken to investigate the clinical implications of hepatitis B virus (HBV) genotypes, basal core promoter (BCP), precore (PC) and surface gene mutations in HBV infected patients from Orissa, southeastern India. HBV infections were identified by serology testing and HBV DNA amplification by polymerase chain reaction among the 152 patients. After sequencing, surface gene mutation were studied by sequence analysis as well as by using BLOSUM scores and BCP mutations were studied only by sequence analysis. A high proportion of HBV/D5 (66.0%) was found among the study samples having significant relation with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) patients (p<0.05). The BCP mutation, TA (81.4%) and C1753/TA (75.0%) was found in significant proportion (p<0.05) among HCC cases and in fact a gradual increase in these mutations were noted between inactive carriers (IC) to HCC group and also showed higher viral load. An increasing trend of major hydrophilic region (MHR) mutations in S gene was also observed from IC (56.0%) to chronic liver disease (CLD) (60.4%) to LC (72.4%) to HCC (95.0%) patients. In conclusion, our study suggests that the predominant HBV subgenotype HBV/D5 with high viral load and BCP mutations (double and triple) and high mutations in MHR region was significantly associated with advanced liver disease (LC and HCC) and might act as predictor of severe hepatic complications.

New sensitive tools for hepatitis B virus (HBV) detection in liver transplantation: what will be their impact on the prophylaxis of HBV infection?

Didier Samuel and Anne Marie Roque-Afonso Institut National de la Sante et de la Recherche Medicale (INSERM), U785, Villejuif, France; Universite Paris-Sud, UMR-S785, Villejuif, France; Assistance Publique–Hopitaux de Paris (AP-HP) Hopital Paul Brousse, Centre Hepato-Biliaire, Villejuif, France; Assistance Publique–Hopitaux de Paris (AP-HP) Hopital Paul Brousse, Service de Virologie, Villejuif, France

G-04 Cas groupés d’hépatite A d’origine nosocomiale

Introduction et objectifs Alerte : Quatre cas groupes d’hepatite A, 3 soignants et une patiente, ont ete diagnostiques dans un service hospitalier entre le 16 et le 20/01/08. Le diagnostic a ete confirme par la presence d’IgM anti-VHA. Une investigation a ete menee pour identifier la source de l’infection. Materiels et methodes L’enquete a envisage le risque de transmission alimentaire et le risque de transmission par contact : gestion des excretas, revue des dossiers cliniques des patients hospitalises dans le service dans le mois precedant les signes cliniques des cas (periode d’incubation). Les serums ont ete adresses au Centre National de Reference pour typage des souches (analyse de la region VP1/2A) et mesure de l’avidite des IgG anti-VHA. Resultats Lors de l’enquete, qui a ecarte la contamination d’origine alimentaire, 2 cas supplementaires ont ete identifies : un agent de la blanchisserie le 01/02/08 et une technicienne de laboratoire le 23/02/2008. Les 6 cas etaient infectes par une meme souche de genotype IA. Sur les 87 dossiers revus, un patient a presente une cytolyse du 4/12/07 au 13/01/08. Le diagnostic d’hepatite A a ete porte retrospectivement sur la presence d’IgM anti-VHA dans un serum du 07/02/2008 dans lequel une souche identique a celle des 6 autres cas a ete identifiee. Une avidite des IgG anti-VHA a 81 % suggerait une infection evoluant depuis plus de 2 mois, resultat coherent avec la date de debut de la cytolyse. Ce patient a ete hospitalise dans le service du 12/12/07 au 12/02/08 apres un sejour de 15 jours en reanimation suite a une noyade. Conclusion L’enquete a note des locaux vetustes, l’absence de supports muraux pour solution hydro-alcoolique, le rincage des bassins a la douchette, un souseffectif en personnel avec des patients tres dependants, contexte favorisant la transmission directe et indirecte du virus de l’hepatite A.

Hepatitis B virus cellular immunity after liver transplantation: a role in preventing hepatitis B virus recurrence?

Chronic hepatitis B virus (HBV) infection is a commoncause of advanced liver disease and hepatocellular car-cinoma and has become a worldwide public health is-sue. Liver transplantation (LT) is the most effectivetherapeutic option for HBV-infected patients who haveacute or chronic liver failure and/or primary liver can-cer. In the absence of prophylactic measures, the risk ofHBV recurrence is about 80% and is principally relatedto the viral load at the time of transplantation. Ad-vances in antiviral prophylaxis have achieved the pre-vention of clinically significant graft reinfection for themajority of patients.