Development of a protocol for the quantitative determination of HBeAg using the Elecsys® HBeAg immunoassay.

Loss of hepatitis 'e' antigen (HBeAg) in patients with HBeAg-positive chronic hepatitis B is associated with improved long-term clinical outcome and is defined as a goal of antiviral treatment by clinical practice guidelines. Recent studies suggest that baseline levels and on-treatment monitoring of HBeAg levels may identify patients most likely to respond to therapy. The aim of this study was the development of a protocol for the quantitative determination of HBeAg using the Elecsys® HBeAg immunoassay. The linear range of the Elecsys® HBeAg immunoassay was established using recombinant HBeAg and five different diluents. The assay was validated against the Paul Ehrlich Institute (PEI) international standard serum. Linearity was demonstrated up to a cut-off index (COI) of 1000, independent of the diluent used. Optimal linearity was obtained using the Elecsys® Universal Diluent. Using the PEI reference standard, conversion factors were established as 4.50 COI for 1 PEIU/mL corresponding to 0.222 PEIU/mL for a COI of 1. Based on the results from these analyses, a simple algorithm for the quantitative measurement of HBeAg using the Elecsys® HBeAg immunoassay was developed. Using a simple algorithm with an initial 1:40 dilution, the Elecsys® HBeAg assay provides robust quantification of serum HBeAg in an easy-to-use and rapid system. The use of a commercially available, standardized diluent improves comparability between laboratories.

Fulminant hepatic failure due to chemotherapy-induced hepatitis B reactivation: role of rituximab.

Hepatitis B virus reactivation during immunosuppressive therapies can lead to liver failure with very limited treatment options available. We report here on two cases of severe hepatitis B reactivation during chemotherapy including rituximab for B cell lymphoma which were treated with liver or liver-cell transplantation. Liver function was normal and HBV infection was unknown in both patients before chemotherapy was started. Impaired liver function became apparent after 4 and 6 courses of chemotherapy, respectively, and both patients experienced fulminant hepatic failure despite antiviral treatment with lamivudine or entecavir. Patient A underwent liver transplantation after documentation of complete remission of the lymphoma and survived without any evidence for hepatitis B recurrence. Patient B received 4 courses of hepatocyte transplantation but did not survive. These cases underline the importance of anti-HBc screening in patients receiving immunosuppressive treatments in particular when rituximab is given. Pre-emptive antiviral treatments should be administered since delayed antiviral treatment is frequently unable to prevent liver failure.

[Interferon-alfa induced reactivation of sarcoidosis in a patient with chronic hepatitis C]. / Interferon-alpha-induzierte Reaktivierung einer Sarkoidose bei einer Patientin mit chronischer Hepatitis C.

The data on reactivation of sarcoidosis during treatment with interferon-alfa is unsatisfactory. We here report the case of a female patient with a history of sarcoidosis more than 20 years ago. Due to chronic hepatitis C the patient was treated with pegylated interferon-alfa and ribavirin. Beginning in the 32(nd) week, the patient developed a reactivation of sarcoidosis. The antiviral therapy was continued until week 48 without administration of systemic steroids. Six month after the end of antiviral treatment the sarcoidosis was in remission and the hepatitis C was cured.

Using secondary structure predictions and site-directed mutagenesis to identify and probe the role of potential active site motifs in the RT6 mono(ADP-ribosyl)transferases.

The RT6 T cell mono(ADP-ribosyl)transferases are expressed as GPI-anchored membrane proteins by mature T lymphocytes. We performed secondary structure prediction analyses of RT6 with a profile based neural network system based on multiple alignments of RT6 with other vertebrate mono(ADP-ribosyl)transferases (mADPRTs). The results reveal a linear order of predicted beta sheets/alpha helix in RT6 that are quite similar to those in the catalytic subunit of the four known crystal structures of mono-ADP-ribosylating bacterial toxins. Recognizable amino acid similarities occur throughout the region of predicted structural homology to the bacterial toxins. Three residues which have been shown to be important for catalysis in bacterial toxins (e.g. R9, S52 and E129 in pertussis toxin) occur in a similar context also in RT6 (R126, S147 and E189). We have mutated these residues in RT6 by site-directed mutagenesis. The RT6 mutants exhibit remarkably similar alterations in enzymatic phenotype as those reported for mutations of the proposed analagous residues in bacterial toxins. These results support the hypothesis that eu- and procaryotic mADPRTs share a common fold and have a common ancestry.

Multicenter evaluation of the Elecsys hepatitis B surface antigen quantitative assay.

The Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a new quantitative electrochemiluminescence immunoassay which uses onboard dilution and a simple algorithm to determine HBsAg levels expressed in international units (IU)/ml (standardized against the World Health Organization [WHO] Second International Standard). This study evaluated its performance using routine serum samples from a wide range of HBsAg carriers and patients with chronic hepatitis B (CHB). HBsAg levels were measured in serum samples collected independently by five centers in Europe, Australia, and Asia. Serial dilution analyses were performed to assess the recommended dilution algorithm and determine the assay range free of hook effect. Assay precision was also established. Following assessment of serial dilutions (1:100 to 1:1,000,000) of the 611 samples analyzed, 70.0% and 85.6% of samples tested with analyzers incorporating 1:100 (Elecsys 2010 and cobas e 411) and 1:400 (Modular Analytics E170) onboard dilution, respectively, fell within the linear range of the assay, providing a final result on the first test. No high-dose hook effect was seen up to the maximum HBsAg serum level tested (870,000 IU/ml) using the dilution algorithm. HBsAg levels were reliably determined across all hepatitis B virus (HBV) genotypes, phases of HBV infection, and stages of disease tested. Precision was high across all analyzers (% coefficient of variation [CV], 1.4 to 9.6; HBsAg concentrations, 0.1 to 37,300 IU/ml). The Elecsys HBsAg II quantitative assay accurately and reliably quantifies HBsAg in routine clinical samples. Onboard dilution minimizes retesting and reduces the potential for error.

Prevalence, incidence, and clinical relevance of the reverse transcriptase V207I mutation outside the YMDD motif of the hepatitis B virus polymerase during lamivudine therapy.

The reverse transcriptase V207I mutation within the hepatitis B virus (HBV) polymerase is associated with resistance to lamivudine in vitro. The prevalence of this mutation in treatment-naive patients was 1% (1/96). A follow-up of the patient carrying this mutation prior to treatment revealed no loss of sensitivity of HBV to lamivudine in vivo.