Development and validation of a hepatitis B virus DNA sequencing assay for assessment of antiviral resistance, viral genotype and surface antigen mutation status

Mallory, Melanie A.; Page, Sam; Hillyard, David R.

doi:10.1016/j.jviromet.2011.06.009

Cited by 31 publications

(35 citation statements)

References 41 publications

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“…We extracted HBV DNA from serum samples using the QIAamp DNA mini kit (Qiagen, Hilden, Germany), amplified the polymerase and surface antigen genes using publically available PCR primer sets, 10,11 and purified it using QIAquick PCR purification kit (Qiagen, Hilden, Germany). Sanger sequencing was performed using an AB3130 genetic analyzer (Life Technologies, California, USA) and nucleotide sequences were analyzed with Sequencher version 5.0 (Gene Codes Corporation, Michigan, USA).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B viral load in dried blood spots: A validation study in Zambia

Vinikoor

Zürcher

Musukuma

et al. 2015

Journal of Clinical Virology

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Background Access to hepatitis B viral load (VL) testing is poor in sub-Saharan Africa (SSA) due to economic and logistical reasons. Objectives To demonstrate the feasibility of testing dried blood spots (DBS) for hepatitis B virus (HBV) VL in a laboratory in Lusaka, Zambia, and to compare HBV VLs between DBS and plasma samples. Study design Paired plasma and DBS samples from HIV-HBV co-infected Zambian adults were analyzed for HBV VL using the COBAS AmpliPrep/COBAS TaqMan HBV test (Version 2.0) and for genotype by direct sequencing. We used Bland-Altman analysis to compare VLs between sample types and by HBV genotype. Logistic regression analysis was conducted to assess the probability of an undetectable DBS result by plasma VL. Results Among 68 participants, median age was 34 years, 61.8% were men, and median plasma HBV VL was 3.98 log IU/ml (interquartile range, 2.04–5.95). Among sequenced viruses, 28 were genotype A1 and 27 were genotype E. Bland-Altman plots suggested strong agreement between DBS and plasma VLs. DBS VLs were on average 1.59 log IU/ml lower compared to plasma with 95% limits of agreement of −2.40 to −0.83 log IU/ml. At a plasma VL ≥2,000 IU/ml, the probability of an undetectable DBS result was 1.8% (95% CI: 0.5–6.6). At plasma VL ≥20,000 IU/ml this probability reduced to 0.2% (95% CI: 0.03–1.7). Conclusions In a Zambian laboratory, we observed strong agreement between DBS and plasma VLs and high sensitivity in DBS at plasma VL ≥2,000 IU/ml. As HBV treatment expands, DBS could increase access to HBV VL testing in SSA settings.

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Section: Methodsmentioning

confidence: 99%

Hepatitis B viral load in dried blood spots: A validation study in Zambia

Vinikoor

Zürcher

Musukuma

et al. 2015

Journal of Clinical Virology

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“…Thus, sustained complete virologic suppression with prior LAM treatment failed to ensure developing no ETV-R. HBV may exist in the form of quasispecies in CHB patients (25), and an antiviral-resistant strain(s) sometimes cannot be detected in time due to the limitation of test sensitivity, especially when its proportion is less than 20% in the pool of viral quasispecies (13,26). The sensitivity of direct sequencing is reported as 43.2% to 66.7% (27)(28)(29). Thus, theoretically, there might have been a small number of LAM-resistant strains, although tests failed to detect them at the time of initiating ETV treatment.…”

Section: Fig 2 Breakthrough With Etv Therapy (A)mentioning

confidence: 99%

Prior Exposure to Lamivudine Increases Entecavir Resistance Risk in Chronic Hepatitis B Patients without Detectable Lamivudine Resistance

Lee

Cho

Lee

et al. 2014

Antimicrob Agents Chemother

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The efficacy of entecavir (ETV) treatment in chronic hepatitis B (CHB) patients who were exposed to lamivudine (LAM) but had no detectable LAM resistance (LAM-R) is not well evaluated. In this study, we aimed to evaluate whether the probability of developing genotypic resistance to ETV in LAM-exposed patients with or without LAM-R is comparable to that in antiviral-naive patients. This retrospective cohort study included 500 consecutive patients with CHB who started ETV monotherapy at a single tertiary hospital in Korea. The patients were divided into three groups: nucleos(t)ide analogue (NA)-naive patients (group 1, n ‫؍‬ 142), patients who were previously exposed to LAM and had no currently or previously detected LAM-R (group 2, n ‫؍‬ 233), and patients with LAM-R when starting ETV (group 3, n ‫؍‬ 125). The overall median ETV treatment duration was 48.7 months. The probabilities of virologic breakthrough were significantly increased not only in group 3 (hazard ratio [HR] ‫؍‬ 14.4, P < 0.001) but also in group 2 (HR ‫؍‬ 5.0, P < 0.001) compared to group 1. Genotypic ETV resistance (ETV-R) developed more frequently in group 2 (HR ‫؍‬ 13.0, P ‫؍‬ 0.013) as well as group 3 (HR ‫؍‬ 43.9, P < 0.001) than in group 1: the probabilities of developing ETV-R in groups 1, 2, and 3 were <1.0%, 8.0%, and 28.2%, respectively, at month 48. The results of this study indicate that ETV-R occurred more frequently in LAM-exposed patients, even though they had no detectable LAM-R, than in NA-naive patients. Therefore, LAM-exposed CHB patients, regardless of the presence or absence of LAM-R, should be monitored more cautiously for the development of ETV-R during ETV monotherapy. E ntecavir (ETV) is an orally administered guanosine analogue that has been approved for treatment of chronic hepatitis B (CHB). In antiviral-naive CHB patients, ETV has shown excellent antiviral efficacy with remarkably low probabilities of genotype resistance (1.2%) and virologic breakthrough (0.8%) for up to 5 years of treatment (1). In contrast to antiviral-naive patients, the rates of genotypic resistance to ETV are much higher in patients with lamivudine (LAM) resistance (LAM-R) (i.e., 51% 5-year cumulative probability) (1). Consequently, ETV is now recommended as one of the first-line therapeutic regimens for antiviralnaive patients with CHB but not for patients who had developed LAM-R (2-4).The emergence of LAM-R variants has been relatively frequent even in antiviral-naive patients with CHB: approximately 20% of patients treated with LAM develop LAM-R at 1 year and 70% to 80% at 5 years of treatment (5-7). Although some selected cases that have succeeded in achieving treatment endpoints (i.e., hepatitis B e antigen [HBeAg] seroconversion and/or maintenance of complete virologic suppression) were able to discontinue LAM without developing LAM-R (8, 9), the rates of durable response after cessation of LAM have been low (10, 11). Unfortunately, the retreatment strategies for virologic relapse in those cases are still indefinite. Since LAM was the first...

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“…The only notable discrepancy among the results was the failure of the Trugene HBV Genotyping kit to detect the NA resistance-associated mutations rt80V and rt80I in 2 of these 7 specimens. This discrepancy was the direct result of a limitation in assay design (coverage from codons rt99 to rt280 only) associated with the Trugene HBV Genotyping kit that has previously been noted by other investigators (19,22).…”

Section: Resultsmentioning

confidence: 93%

“…However, our admixture experiments did present a significant challenge to the assay by testing various dilutions of wild-type HBV containing a subpopulation of a drug-resistant mutant present at a very low level (i.e., 500 IU/ ml). Although population-based sequencing is capable of detecting mutant HBV subpopulations present at a minimum of 10% to 50% of the total population in a given clinical specimen (19,27), one should be aware that detectability is dependent on both the relative abundance of the subpopulation and the total HBV viral load. A minor variant present at 30% of a total viral population of 1,500 IU/ml may not be detectable, while a variant present at 25% of a total population of 5,000 IU/ml may be detectable.…”

Section: Discussionmentioning

confidence: 99%

“…Examples of some of the methods used in these laboratory-developed assays include direct sequencing, pyrosequencing, allele-specific realtime PCR, PCR combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), Invader technology, and microchip-based sequencing (17)(18)(19)(20)(21)(22). Several research-use-only assays based on either direct sequencing (such as the Trugene HBV Genoytping kit; Siemens Healthcare Diagnostics Inc., Tarrytown, NY) or reverse hybridization (such as the INNO-LiPA HBV Multi-DR and INNO-LiPA HBV Genotyping kits; Innogenetics NV, Ghent, Belgium) are also commercially available.…”

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confidence: 99%

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Evaluation of the Abbott HBV RUO Sequencing Assay Combined with Laboratory-Modified Interpretive Software

et al. 2013

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The Abbott HBV RUO Sequencing assay (Abbott Molecular Inc., Des Plaines, IL), which combines automated sample processing, real-time PCR, and bidirectional DNA sequencing, was evaluated for detection of nucleos(t)ide analogue (NA) resistanceassociated mutations located in the hepatitis B virus (HBV) polymerase (Pol) gene. Interpretive software from the assay manufacturer was modified to allow interrogation of the overlapping HBV surface (S) gene sequence for HBV genotype determination and detection of immune escape mutations. Analytical sensitivity (detection and sequencing) of the assay was determined to be 103.9 IU/ml (95% confidence interval [CI], 80.0 to 173.3) for HBV genotype A. Testing of commercially available HBV genotype panels consisting of 23 individual members yielded complete agreement between expected results and results obtained from the laboratory-developed HBV genotype library. Excellent specificity was observed among clinical specimens with serologic or molecular markers for various unrelated blood-borne viruses (n ‫؍‬ 6) and sera obtained from healthy, HBV-negative blood donors (n ‫؍‬ 20). Retrospectively selected clinical specimens tested by a commercial reference laboratory HBV sequencing assay (n ‫؍‬ 54) or the Trugene HBV Genotyping kit (n ‫؍‬ 7) and the Abbott HBV RUO Sequencing assay showed minor differences in detection and reporting of NA resistance-associated mutations in 7 of 61 (11.5%) specimens but complete agreement of genotype results. The Abbott HBV RUO Sequencing assay provided a convenient and efficient assay workflow suitable for routine clinical laboratory use, with the flexibility to be modified for customized detection of NA resistance-associated mutations, HBV genotype determination, and detection of immune escape mutations from a single contiguous HBV sequence. G lobally, over 400 million individuals are chronically infected with hepatitis B virus (HBV) (1, 2). Ongoing HBV replication in these chronic carriers has been shown to increase their risk for disease progression, including the development of cirrhosis and hepatocellular carcinoma (3, 4), while infection with HBV genotype C has also been independently associated with a potential for more-severe liver disease (5-7). Treatment options for chronic hepatitis B have continued to expand and currently include the use of standard or pegylated interferon or nucleos(t)ide analogues (NA). However, recent reports of reduced effectiveness of interferon-based therapies in patients infected with HBV genotypes C or D compared to genotypes A or B (8-10), along with the welldocumented potential for NA resistance, can complicate treatment decisions.Among orthotopic liver transplant (OLT) recipients treated for end-stage liver disease due to chronic hepatitis B or at risk for de novo HBV infection, hepatitis B immunoglobulin (HBIG) has been used together with long-term NA therapy to prevent HBV reinfection of the graft (11, 12). Unfortunately, in addition to the potential for development of NA resistance with long-term use, extended...

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Development and validation of a hepatitis B virus DNA sequencing assay for assessment of antiviral resistance, viral genotype and surface antigen mutation status

Cited by 31 publications

References 41 publications

Hepatitis B viral load in dried blood spots: A validation study in Zambia

Hepatitis B viral load in dried blood spots: A validation study in Zambia

Prior Exposure to Lamivudine Increases Entecavir Resistance Risk in Chronic Hepatitis B Patients without Detectable Lamivudine Resistance

Evaluation of the Abbott HBV RUO Sequencing Assay Combined with Laboratory-Modified Interpretive Software

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