Lamivudine Resistance of Hepatitis B Virus Masked by Coemergence of Mutations in Probe Region of the COBAS AMPLICOR Assay

Abstract

The COBAS AMPLICOR hepatitis B virus assay targets a conserved region of the genome and is widely used to monitor treatment of hepatitis B in order to identify emerging resistance. However, the assay failed to recognize increasing viremia levels when YMDD mutations were paralleled by mutations in the segment targeted by the COBAS AMPLICOR probe.The aim of treating chronic hepatitis B virus (HBV) infection is to prevent the emergence of liver cirrhosis or hepatocellular carcinoma. Currently, treatment is given either as injections of pegylated interferon or as oral doses of antivirals, such as lamivudine or adefovir. Lamivudine therapy has the advantages of documented efficiency, lower cost, and minimal side effects but carries an annual 15 to 20% risk of emergence of resistant mutants, which may cause severe exacerbations of hepatitis. Lamivudine resistance is typically due to a mutation in the YMDD motif of the polymerase (M204I or M204V) (7) and is recognized as increases in HBV DNA levels (by more than 1 log).Here we describe a case where the COBAS AMPLICOR assay (Roche Diagnostics, Branchburg, NJ) failed to reveal resistance in a 56-year-old male Vietnamese patient with HBV cirrhosis.The patient had arrived in Sweden in 1999, and his chronic HBV infection was discovered in 2001 by a general practitioner (positive HBsAg and anti-HBc results obtained by use of AxSYM [Abbott, Illinois]). The patient was admitted to the infectious disease clinic, where he presented signs of active cirrhosis with a low platelet count (52 ϫ 10 9 /liter), increased prothrombin time (international normalized ratio, 1.7), and an aspartate aminotransferase level of 2.5 times the upper limit of normal. HBeAg (tested by use of AxSYM [Abbott]) was not detected in serum, but the HBV DNA level was high, above 10 million copies/ml, as detected by use of COBAS AMPLICOR. Hepatitis C antibodies (tested for with AxSYM [Abbott]) were not detected. The patient was put on long-term lamivudine therapy (100 mg daily), which resulted in a 3-log reduction in HBV DNA to around 20,000 copies/ml as measured by COBAS AMPLICOR after 3 months. Thereafter the HBV DNA levels did not decrease further but appeared to remain at levels between 20,000 and 50,000 copies/ml. The aminotransferase levels also remained moderately elevated. A sample drawn after 21 months of treatment was included (by chance) in a study comparing COBAS AMPLICOR and COBAS TaqMan (Roche) (4), which revealed a 2-log underestimation of viremia by COBAS AMPLICOR. This finding led us to further investigate the emergence of mutations in the YMDD motif as well as in the region targeted by the COBAS AMPLICOR assay.Viremia was measured by two different systems, the COBAS AMPLICOR Monitor (Roche) (5) and an in-house real-time PCR targeting a conserved region of the HBsAg gene. COBAS AMPLICOR testing was performed as instructed by the manufacturer. The S-region real-time PCR assay included DNA extraction by use of Magnapure LC (Roche) with a total nucleic acid isolation kit and 45 cycles of two-step...

Quantitation of Hepatitis B Virus Dna in Plasma Using a Sensitive Cost-Effective “In-House” Real-Time PCR Assay

“…This study reported that the COBAS AMPLICOR monitor assay failed to recognize increasing levels of viraemia in individuals on treatment due to core mutations that develop during treatment. [9] This assay, which uses the manual nucleic acid extraction protocol and the real-time Taqman chemistry, is very sensitive and can potentially be used to detect occult HBV infection. The total assay duration is 5.5-6 hours, including the extraction protocol.…”

Long-term survival of occult hepatitis B associated hepatocellular carcinoma following surgery and antiviral therapy

“…Additionally, the presence of HBV DNA in the liver tumor and the more sensitive HBV serum DNA assay suggested a case of OBI. Indeed, the failure of commercial HBV assays to detect HBV DNA has been reported, particularly in patients harboring treatment-resistant mutations [20] . This highlights the need for a more rigorous HBV DNA assay test and reinforces the need to target at least three different locations in the HBV genome [2] .…”

Characterization of a New Sensitive PCR Assay for Quantification of Viral DNA Isolated from Patients with Hepatitis B Virus Infections

“…Compared with the former, the Abbott RealTime assay offers the advantage of a much wider dynamic range (38 to 38,000 IU/ml for the Amplicor assay) and improved probe selectivity. The probe of the Cobas Amplicor HBV Monitor test targets a sequence in the viral DNA core region, and the sensitivity of the assay may be compromised with respect to viral strains bearing mutations in this region (17) and genotype F variant strains (12,31). In contrast, the Abbott RealTime assay probe hybridizes within a highly conserved region of the S gene, allowing accurate detection of all viral strains evaluated.…”