Measuring hepatitis B pgRNA stability using an updated automated HBV pgRNA assay with increased sensitivity.

BACKGROUND: HBV pregenomic RNA (pgRNA) is a circulating biomarker for covalently closed circular DNA activity in HBV-infected individuals and has been studied for treatment efficacy, disease staging, and off-therapy outcomes; however, data on the stability are scarce. Increasing HBV pgRNA assay sensitivity may improve its predictive value and provide additional insights at low viral levels. METHODS: Modifications to a fully automated first (v1) generation HBV pgRNA assay improved sensitivity up to 15-fold over the previous assay. Flexible sample input volumes yielded lower limits of quantitation of 10 and 22 copies/mL for 0.6 and 0.2 mL assays, respectively. Results are standardized to secondary standards that are traceable to the WHO HBV DNA standard, and internal and external controls are included. RESULTS: Comparison between v1 and modified v2 assays showed increased sensitivity from 152 copies/mL with v1 to 10 (0.6 mL) and 22 (0.2 mL) copies/mL with v2, respectively. Quantitated v2 results were indistinguishable from v1, indicating that comparisons can be made to previous studies. Single timepoint treatment-naive blood donors or longitudinal draws from patients with chronic hepatitis B on AB-729, an investigational siRNA therapy, showed improved detection and quantifiable pgRNA with v2 compared with v1. Stability testing demonstrated excellent HBV pgRNA plasma stability after 3 freeze-thaw cycles, for at least 7 days at 25-37 °C and at least 30 days at 4°C, with ≤0.25 Log U/mL decrease. CONCLUSION: HBV pgRNA v2 assays with increased sensitivity and flexible input volumes demonstrated increased detection and quantitation of low viral titer samples. Highly sensitive HBV pgRNA assays may be useful in refining predictive treatment outcomes based on this marker. HBV pgRNA was stable under multiple conditions, which increases the reliability of this marker.

Twenty-year follow-up of an outbreak of hepatitis C in a small rural town of Argentina: The O'Brien Project.

INTRODUCTION AND OBJECTIVES: In 1999, a population-based survey showed a 5.6 % (102/1832) prevalence of HCV infection in O'Brien, a small rural town of Argentina. The aim of this study was to assess the impact of screening, clinical evaluation and antiviral therapy on elimination of HCV after 20 years of follow-up. PATIENTS AND METHODS: HCV+ subjects (n=102) underwent clinical, biochemical and histological evaluation to assess the presence and severity of liver disease. Antiviral therapy included pegylated interferon + ribavirin in 2005 and direct antiviral agents from 2017. RESULTS: All viremic subjects (n=84) had genotype 1b with 90%-97.5% sequence homology scores, suggesting the existence of a common source of infection (use of unsafe injections administered by the same health professional). Liver biopsy (n=55) showed chronic hepatitis in all patients. The prevalence of cirrhosis was 28% overall (29/102) and 34.5% among viremic patients. Sustained virological response (SVR) was obtained in 20/34 (59%) patients treated with interferon. From 2005 to 2017, when oral antivirals became available 37/50 untreated patients died. Median age of this group in 2005 was 67 years. Six interferon non-responders and five naive subjects received direct antiviral agents and all developed SVR. Only 1/31 patient (3.2%) with SVR died and none developed decompensated cirrhosis or HCC. In 2019, a new population-based study showed that the prevalence of HCV in O'Brien decreased 20-fold, from 5.6% to 0.28% (3/1070). CONCLUSIONS: Despite the high mortality rate precluding timely access to direct antiviral agents, the O'Brien Project is a good example of HCV micro-elimination studies.

Resistencia a los antivirales de acción directa contra el virus de la hepatitis C / Direct acting antivirals resistance to HCV

El virus de la hepatitis C puede causar cirrosis y carcinoma hepatocelular. El tratamiento con interferón pegilado y ribavirina resulta en bajas tasas de respuesta viral sostenida y conlleva serios efectos adversos. Sin embargo, con nuevos y prometedores antivirales de acción directa contra el VHC, algunos aprobados recientemente y otros en desarrollo, las tasas de curación han mejorado significativamente. Los nuevos antivirales de acción directa incluyen inhibidores de la proteasa viral, inhibidores de la polimerasa viral e inhibidores del complejo replicativo NS5A. Una consecuencia asociada al uso de antivirales de acción directa es el desarrollo de resistencia en la mayoría de los pacientes con fallo terapéutico. El desarrollo de resistencia es más frecuente con ciertos mecanismos de acción, aunque las variantes resistentes suelen no ser detectadas en la mayoría de los pacientes tratados con inhibidores de la proteasa 1 a 2 años luego de finalizado el tratamiento. Sin embargo, queda aún por establecer si el desarrollo de resistencia acarrea consecuencias a largo plazo, en particular relacionadas al uso de estrategias de re-tratamiento

HCV can cause cirrhosis and hepatocellular carcinoma. Treatment with pegylatd interferon and ribavarin rsults in low rates of sustained virologic response and is associated with serious adverse events. however, with new promising direct acting antivirals against HCV, some recently approved and others currently in development, cures rates have significantly improved. New direct acting antivirals include protease inhibitors, polymerase inhibitors and inhibitors of the NS5A replictive complex. A consequence of the use of direct acting antivirals is the development of resistance in the majority of patients with therapeutic failure. Although resistance development is usually more frequent with certain mechanisms of action than others, resistant viral variants are usually no longer detected in the majority of patients treated with protease inhibitors 1 to 2 years after end of treatment. however, it stills remains to be determined if resistance development is associated with long term consequences, in particulr with the use retreatment strategies

The predictive value of core antigen testing for the management of hepatitis C patients receiving pegylated interferon/ribavirin treatment.

A new quantitative marker of HCV viremia based on the detection of the core antigen of the virus has recently become commercially available in Europe. The usefulness of this test was examined for the management of patients treated with pegylated interferon/ribavirin. One hundred twenty-eight pegylated interferon/ribavirin treated patients were studied. Serum samples were available at baseline, week 4 and week 12 time-points, respectively. Core antigen was quantified using the trak-C assay (Ortho Clinical Diagnostics, Raritan, NJ). For all genotypes at week 4, the positive and negative predictive values of HCV core antigen were 81.4 and 92.9%, respectively, while at week 12 they were 67.9 and 100%, respectively. These predictive values varied substantially according to viral genotype. Among patients with a negative core antigen level (<1.5 pg/ml) at week 12, only 33% of those who were positive at week 4 achieved a sustained virological response whereas 85% of those who were already negative did (P < 0.001). The core antigen assay may be used at week 4 and week 12 to distinguish patients who will achieve a sustained virological response from those who will relapse/breakthrough. This assay is a new reliable alternative for early prediction of virological non-response in patients treated with pegylated interferon/ribavirin.

Comparative evaluation of the total hepatitis C virus core antigen, branched-DNA, and amplicor monitor assays in determining viremia for patients with chronic hepatitis C during interferon plus ribavirin combination therapy.

An assay prototype designed to detect and quantify total hepatitis C virus [HCV] core antigen (HCV core Ag) protein in serum and plasma in the presence or absence of anti-HCV antibodies has been recently developed by Ortho-Clinical Diagnostics. The aim of the study was to evaluate the sensitivity, specificity, and reproducibility of the Total HCV core Ag assay in comparison with two quantitative assays for HCV RNA: Quantiplex HCV RNA 2.0 (bDNA v2.0) or Versant HCV RNA 3.0 (bDNA v3.0) assays and the Cobas Amplicor HCV Monitor version 2.0 (HCM v2.0) test. We have studied samples of a well-characterized panel and samples from patients with chronic hepatitis C treated with interferon alone or with ribavirin. We have also compared the kinetics of HCV core Ag and HCV RNA in the follow-up of treated patients. The HCV core Ag assay exhibited linear behavior across samples from different genotypes. The coefficients of variation for intra- and interassay performance were 5.11 and 9.95%, respectively. The specificity of the assay tested in blood donors was 99.5%. Samples from HCV-infected patients showed that the correlation between the HCV core Ag and the two HCV RNA quantitative assays (bDNA and HCM v2.0) was 0.8 and 0.7, respectively. This correlation was maintained across different genotypes of HCV (r(2) = 0.64 to 0.94). Baseline HCV core Ag values were significantly lower in sustained responders to interferon (IFN) than in other groups of patients (5.31 log(10) [10(4) pg/ml] versus 5.99 log(10) [10(4) pg/ml]; P < 0.001). In patients treated with IFN or combination therapy, we found an association between a decrease of more than 2 log IU/ml in viral load, undetectable HCV core Ag, and sustained response. Among sustained responders to IFN alone or combination therapy and among relapsers after IFN alone, 84 out of 101 (83.2%) had undetectable HCV core Ag, and 76 out of 96 (79.2%) had a viral load decrease of >/=2 log IU/ml, after 1 month of treatment. In conclusion, the Total HCV core Ag assay is a new useful test for the detection of HCV viremia and the monitoring of patients treated with IFN alone or in combination with ribavirin.

Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

Decreased recovery of replication-competent HIV-1 from peripheral blood mononuclear cell-derived monocyte/macrophages of HIV-positive patients after 3 years on highly active antiretroviral therapy.

We studied the release of p24 antigen from peripheral blood mononuclear cell-derived monocyte/macrophages obtained from 13 HIV-positive patients receiving highly active antiretroviral therapy (HAART). Although HIV-infected monocyte/macrophages were detected in 80% of patients after 36 months of continuous treatment, additional exposure to HAART reduced the chance of detecting HIV-releasing monocyte/macrophages. Therefore, after more than 3 years of HAART, recently infected monocytes may play a less important role as a source of emerging HIV-1 upon HAART interruption.

Clinical utility of total HCV core antigen quantification: a new indirect marker of HCV replication.

Hepatitis C virus (HCV) RNA detection, viral load quantification, and HCV genotyping are widely used in clinical practice. Recently, the availability of an anticore antigen (Ag) monoclonal antibody allowed development of an enzyme-linked immunosorbent assay (ELISA) detecting and quantifying total HCV core Ag in peripheral blood of HCV-infected patients. The aims of the present study were to investigate the biologic significance of this new marker in HCV infection, to establish the intrinsic performance of the current assay, and to determine its potential utility in the management of HCV-infected patients. A panel of infected sera calibrated to the World Health Organization International Standard and 657 serum samples from infected patients receiving antiviral treatment were studied. We showed that total HCV core Ag quantification is an accurate, precise, and specific indirect marker of HCV replication. We estimated that 1 pg/mL of total HCV core Ag is equivalent to approximately 8,000 HCV RNA international units (IU)/mL, although minor between-patient differences may exist. In conclusion, total HCV core Ag quantification can be used in the various indications of viral load monitoring, including the evaluation of baseline viral load before therapy, the assessment of the virologic response to antiviral treatment, and the study of early viral kinetics during therapy. Nevertheless, the total HCV core Ag assay cannot be used as a marker of viral replication for HCV RNA values below 20,000 IU/mL, limiting its use in the monitoring of late events during and after antiviral treatment.

Prevalencia de infección por virus de hepatitis G en una cohorte de pacientes hemofílicos HIV+ / [Prevalence of hepatitis G virus infection in a cohort of hemophilic HIV positive patients]

We analysed the prevalence of hepatitis G virus (HGV) infection in HCV+/HIV+ hemophilic patients determining HGV viremia in plasma by polymerase chain reaction (PCR). The overall prevalence of HGV infection was 13.51

. Viremia by HGV was more frequent in younger patients. Two subgroups of patients were considered taking into account prognosis of HIV disease progression. The prevalence of HGV infection was significantly higher in those with better prognosis and low risk of evolution to AIDS. The results suggest that HGV infection may slow disease progression, directly or indirectly.

Prevalence of HIV antibodies in hemophiliacs in Buenos Aires

Se estudiaron, entre junio de 1984 y marzo de 1987, 237 pacientes con hemofilia hallándose una prevalencia de anticuerpos para el virus de la inmunodeficiencia humana (HIV) del 54%. De ellos, 188 enfermos hemofílicos (156 hemofilia A y 22 hemofilia B) pudieron ser estudiados en detalle; el 54% de los receptores de factor VIII y el 59% de los de factor IX presentaron anticuerpos para HIV; en ambos grupos, la seropositividad estaba asociada a la severidad de la hemofilia. Se observó, además, que 89 de 102 (87%) pacientes hemofílicos sintomáticos presentaron anticuerpos para HIV, comparado con 9 de 26 (12%) pacientes asintomáticos. Se estudiaron 12 mujeres con contactos sexuales con pacientes hemofílicos seropositivos para HIV; en 3 (25%) de ellas se hallaron anticuerpos para el virus y una de ellas presenta linfoadenopatía y pérdida de peso. Nuestros resultados permiten inferir que un alto porcentaje de los hemofílicos estudiados, tratados con Factor VIII y Factor IX antes de 1985, presentan anticuerpos para el virus y que esta seropositividad está asociada a sintomatología y al número de transfusiones recibidas

Síndrome de inmunodeficiencia adquirida (SIDA) en pediatría: comunicación de un caso en la Argentina / Acquired immunodeficiency syndrome in pediatrics: report of a case in Argentina

Se presenta el caso de un lactante africano con desnutrición severa sin causa primaria ni gastroenterológica e infecciones reiteradas. Se investiga un déficit inmunitario diagnosticándose síndrome de inmunodeficiencia adquirida. Debe tenerse presente este diagnóstico en el estudio de pacientes de grupos de riesgo con desnutrición secundaria e infecciones reiteradas