Formes circulantes du virus de l’hépatite B et nouveaux marqueurs viraux

Chronic hepatitis B remains a major public health problem. Knowledge about the complex morphogenesis of hepatitis B virus (HBV) has been evolving since its discovery. Classical viral markers used in clinical practice for the diagnosis and management of patients are only partially informative. New viral markers are being proposed and their clinical utility is under investigation. We focused on developing a system to study the different viral forms circulating in patient plasma according to the genotype and HBeAg status. In parallel, we studied the contribution of new markers such as ultrasensitive HBsAg, HBcrAg, and HBV-RNA in different clinical settings. The results confirm the presence of enveloped RNA-containing particles in plasmas and reveal an abundant circulation of genome-free capsids enveloped with or without HBs proteins depending on the HBeAg status. Concerning viral markers, the use of an ultrasensitive HBsAg assay on samples previously identified as HBsAg negative with conventional assays led to a change of the serological profile interpretation in nearly a quarter of the cases. HBcrAg and HBV-RNA tested on samples at inclusion in the CirVir cohort seem not predictive of a clinical event. This work allowed to characterize HBV particles and markers whose roles and clinical impact remain to be determined.L’hépatite B chronique demeure un problème de santé public majeur. Les connaissances sur la morphogenèse complexe du virus de l’hépatite B (VHB) ne cessent d’évoluer depuis sa découverte. Les marqueurs virologiques classiques utilisés en pratique clinique ne sont que partiellement informatifs pour le diagnostic et le suivi des patients. Des marqueurs émergents sont proposés dont l’utilité clinique est à l’étude. Nos travaux de recherche se sont attachés à développer un système pour étudier les différentes formes virales circulantes au niveau plasmatique, en fonction du génotype et du statut vis-à-vis de l’AgHBe. En parallèle, nous avons étudié l’apport des nouveaux marqueurs tels que l’AgHBs ultrasensible, l’HBcrAg, et l’ARN-VHB dans différentes situations cliniques. Les résultats confirment la présence de particules enveloppées contenant de l’ARN dans les plasmas et révèlent une circulation abondante de capsides sans acide nucléique enveloppées avec ou sans protéines HBs selon le statut de l’AgHBe. Concernant l’étude des marqueurs viraux, l’utilisation d’un test AgHBs dit ultrasensible, sur des échantillons initialement testés négatifs par les tests AgHBs courants, a conduit à un changement d’interprétation du profil sérologique dans près d’un quart des cas. Les marqueurs HBcrAg et ARN-VHB, testés sur le prélèvement à l’inclusion dans la cohorte CirVir, ne semblent pas prédictifs de la survenue d’un évènement clinique. En alliant une approche novatrice et des tests standardisés, ce travail a permis de mieux caractériser les formes virales et les nouveaux marqueurs dont le rôle et l’impact clinique restent à déterminer

Circulating hepatitis B virus forms and new viral markers

L’hépatite B chronique demeure un problème de santé public majeur. Les connaissances sur la morphogenèse complexe du virus de l’hépatite B (VHB) ne cessent d’évoluer depuis sa découverte. Les marqueurs virologiques classiques utilisés en pratique clinique ne sont que partiellement informatifs pour le diagnostic et le suivi des patients. Des marqueurs émergents sont proposés dont l’utilité clinique est à l’étude. Nos travaux de recherche se sont attachés à développer un système pour étudier les différentes formes virales circulantes au niveau plasmatique, en fonction du génotype et du statut vis-à-vis de l’AgHBe. En parallèle, nous avons étudié l’apport des nouveaux marqueurs tels que l’AgHBs ultrasensible, l’HBcrAg, et l’ARN-VHB dans différentes situations cliniques. Les résultats confirment la présence de particules enveloppées contenant de l’ARN dans les plasmas et révèlent une circulation abondante de capsides sans acide nucléique enveloppées avec ou sans protéines HBs selon le statut de l’AgHBe. Concernant l’étude des marqueurs viraux, l’utilisation d’un test AgHBs dit ultrasensible, sur des échantillons initialement testés négatifs par les tests AgHBs courants, a conduit à un changement d’interprétation du profil sérologique dans près d’un quart des cas. Les marqueurs HBcrAg et ARN-VHB, testés sur le prélèvement à l’inclusion dans la cohorte CirVir, ne semblent pas prédictifs de la survenue d’un évènement clinique. En alliant une approche novatrice et des tests standardisés, ce travail a permis de mieux caractériser les formes virales et les nouveaux marqueurs dont le rôle et l’impact clinique restent à déterminer.Chronic hepatitis B remains a major public health problem. Knowledge about the complex morphogenesis of hepatitis B virus (HBV) has been evolving since its discovery. Classical viral markers used in clinical practice for the diagnosis and management of patients are only partially informative. New viral markers are being proposed and their clinical utility is under investigation. We focused on developing a system to study the different viral forms circulating in patient plasma according to the genotype and HBeAg status. In parallel, we studied the contribution of new markers such as ultrasensitive HBsAg, HBcrAg, and HBV-RNA in different clinical settings. The results confirm the presence of enveloped RNA-containing particles in plasmas and reveal an abundant circulation of genome-free capsids enveloped with or without HBs proteins depending on the HBeAg status. Concerning viral markers, the use of an ultrasensitive HBsAg assay on samples previously identified as HBsAg negative with conventional assays led to a change of the serological profile interpretation in nearly a quarter of the cases. HBcrAg and HBV-RNA tested on samples at inclusion in the CirVir cohort seem not predictive of a clinical event. This work allowed to characterize HBV particles and markers whose roles and clinical impact remain to be determined

Diagnostic et suivi de l'infection par le VIH-1 (groupe M) (évaluation du support buvard pour la réalisation des techniques sérologiques et de biologie moléculaire)

Malgré d'importants progrès réalisés dans l'accès au traitement antirétroviral dans les pays à ressources limitées, l'absence de mesure périodique de charge virale empêche une prise en charge optimisée. Le support buvard (DBS) apparaît comme une alternative au plasma pour le diagnostic de l'infection et le suivi sous traitement des patients. Dans une étude prospective menée chez 59 patients infectés par le VIH-1, nous avons évalué ce support pour les tests de dépistage, la quantification de l'ARN, la détection de l'ADN proviral et les génotypiques de résistance aux antirétroviraux. Les résultats obtenus sont satisfaisants mais confirment la moindre sensibilité du support DBS en cas de faibles charges virales. L'optimisation des protocoles opératoires semble nécessaire mais l'utilisation des DBS pour les pays du Sud reste une option prometteuse.Despite important progress in access to antiretroviral therapy in resource-limited countries, the lack of periodic viral load measurements prevents an optimised monitoring. Dried blood spots (DBS) could serve as an interesting alternative to plasma for infection diagnosis and treatment monitoring. In a prospective study in 59 patients infected with HIV-1, we evaluated DBS for screening tests, RNA quantification, detection of proviral DNA and PCR amplification for drug resistance testing. The results are satisfying but confirm the lower sensitivity of DBS in low loads. The optimization of operating protocols seems necessary but the use of DBS for the South remains a promising option.LYON1-BU Santé (693882101) / SudocRENNES1-BU Santé (352382103) / SudocSudocFranceF

Using a commercial diagnostic assay requires compliance with the manufacturer’s recommendations

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Combined use of dried blood spot and rapid molecular systems: A robust solution to monitor hepatitis B virus infection with potential for resource-limited countries

International audienceChronic hepatitis B (CHB) particularly affects resource-limited countries. CHB management in these areas faces many obstacles for optimal care of patients, including poor access to HBV-DNA quantification, a key marker. This study aims to evaluate the quantification of HBV-DNA on dried blood spots (DBS) using rapid, standardized and fully automated on-demand systems. After a simple and rapid DBS elution protocol, HBV-DNA was simply and accurately quantified on this matrix using two different systems. Limit of quantification was estimated at 400 IU/mL. DBS and plasma HBV-DNA quantification provided comparable results. HBV-DNA stability for up to one month was demonstrated on DBS stored at room temperature, a condition compatible for preservation or transport before analysis. The combined use of DBS and commercially available automated molecular on-demand systems for HBV-DNA quantification could represent a reliable alternative in resource-limited countries to reach remote populations. The good sensitivity of this approach makes it attractive for mother-to-child transmission prevention, treatment decision and follow-up. Costs can be limited if such systems are also validated for other molecular markers

Influence of preanalytical parameters on influenza and respiratory syncytial virus molecular detection

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Contribution of quantitative viral markers to document hepatitis B virus compartmentalization in cerebrospinal fluid during hepatitis B with neuropathies

International audienceExtrahepatic manifestations linked to hepatitis B Virus (HBV) are usually indirect consequences of immune-mediated mechanisms triggered by the virus replication. Strong evidence of brain HBV replication is missing and direct involvement of HBV in nervous system symptoms has been rarely reported. We report two cases of neurological manifestations contemporary to HBV infection. In both cases, HBV-DNA and HBsAg could be quantified in the cerebrospinal fluid (CSF) at relatively high levels. Differential quantification of HBsAg and HBV viral load both in CSF and in blood as well as phylogenic studies on HBV genomic sequences obtained from blood and CSF provided strong arguments for central nervous system viral replication in both cases. Direct causality of HBV replication in the central nervous system in these clinical situations is certainly not demonstrated but these findings could expand the list of hepatitis viruses possibly involved in neurological disorders. Further studies should be promoted to better document possible HBV replication in the brain tissues and its consequences

Macrophage activation syndrome with acute hepatitis E during tocilizumab treatment for rheumatoid arthritis.

International audienceTocilizumab is a humanized antibody against the membrane and soluble receptors for interleukin-6. Tocilizumab is among the disease-modifying antirheumatic drugs (DMARDs) used to treat moderate-to-severe active rheumatoid arthritis (RA) refractory to conventional DMARDs. We report a case of macrophage activation syndrome that complicated acute hepatitis E and started within 24hours after the fourth tocilizumab infusion in a patient with RA

Genetic diversity of genotype 6 HCV infections in France: Epidemiology and consequences for treatment strategy

International audienceGenotype-6 hepatitis C virus (GT6-HCV) exhibits a high genetic diversity. GT6 prevalence, diversity and real-life response to treatment were studied among 14 603 HCV mono-infected patients from the French ANRS-CO22-Hepather cohort. NS3, NS5A and NS5B-HCV genes were amplified and sequenced for all GT6-infections identified in the database. Following phylogenic characterization, resistance-associated substitution polymorphisms were identified. GT6-infected patients (n = 36; 0.25%) did not differ from patients infected with other genotypes with regard to gender, age or liver fibrosis. GT6e was the most prevalent (27.8%), followed by 6a (22.2%), 6q (11.1%) and 6o (8.3%). Each subtype p and xc were found in two patients (5.6%) and subtypes f/h/r and t were each detected in one patient. Four strains (11.1%) clustered with unclassified reference sequences. Concordant genotype determination throughout NS3, NS5A and NS5B-genes is consistent with lack of recombination within this genomic region. All, but three patients were born in Asia, Cambodia (44.4%), Vietnam (38.9%) or Laos (8.3%). GT6a were found in 42.8% of Vietnamese and 6e in 37.5% of Cambodian. GT6q, 6p and 6r were only found in Cambodian patients. Resistance-associated polymorphisms for each DAA classes were identified in baseline sequences. Twenty-seven patients were treated with sofosbuvir-based combinations and 3 with glecaprevir/pibrentasvir. All treated patients, whether naive or previously treated, achieved a sustained viral response. In conclusion, GT6-infections are uncommon in France and their genetic diversity likely reflects infection within the country of origin. Despite residue variability at DAA resistance-associated positions, sustained viral response was obtained in all treated patients