Ultrasensitive Genotypic Detection of Antiviral Resistance in Hepatitis B Virus Clinical Isolates

Abstract

Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other L-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy.Nearly 400 million people are chronically infected with hepatitis B virus (HBV) worldwide (25). After prolonged infection, often lasting decades, patients frequently develop severe liver disease that can lead to cirrhosis and hepatocellular carcinoma (reviewed in reference 13). Current therapies for chronic HBV include regimens of type I interferons and the nucleoside/nucleotide analogs lamivudine, adefovir-dipivoxil, entecavir, telbivudine, and tenofovir-disoproxil fumarate (13). Long-term antiviral therapy with these nucleosides/nucleotides often results in the emergence of drug-resistant variants, which can result in a loss of treatment benefits and impair the effectiveness of subsequent therapies. Although resistant viruses are often growth impaired, additional changes emerging under continued drug selection enable the expansion of adapted, resistant variants, which come to dominate the viral population (20). Detection of resistance early, either prior to or during therapy, can positively impact patient management and treatment selection. For example, resistance to lamivudine, from substitutions in the HBV reverse transcriptase (RT) YMDD active site motif, results in complete in vitro cro...

Combination of allele-specific detection techniques to quantify minority resistance variants in hepatitis B infection: A novel approach

“…absolute sensitivity between 500–5000 copies) but have higher false positive and false negative rates, and also cannot detect linked mutations (Degertekin et al, 2009; Libbrecht et al, 2007). Several allele-specific quantitative PCR methods have been published for the quantitation of multiple HBV drug resistance mutations, but again cannot detect specific combinations of mutations (Fang et al, 2009; Geng et al, 2006; Punia et al, 2004; Wen and Li, 2007). Furthermore, several of these methods require initial non-allele-specific PCR amplification before a quantitative nested PCR reaction, making these assays only semi-quantitative (Fang et al, 2009; Punia et al, 2004).…”

“…Several allele-specific quantitative PCR methods have been published for the quantitation of multiple HBV drug resistance mutations, but again cannot detect specific combinations of mutations (Fang et al, 2009; Geng et al, 2006; Punia et al, 2004; Wen and Li, 2007). Furthermore, several of these methods require initial non-allele-specific PCR amplification before a quantitative nested PCR reaction, making these assays only semi-quantitative (Fang et al, 2009; Punia et al, 2004). The reported sensitivities of these assays range from about 100 to 1000 copies of mutant template in the presence of excess wild type (Fang et al, 2009; Geng et al, 2006; Wen and Li, 2007).…”

“…Furthermore, several of these methods require initial non-allele-specific PCR amplification before a quantitative nested PCR reaction, making these assays only semi-quantitative (Fang et al, 2009; Punia et al, 2004). The reported sensitivities of these assays range from about 100 to 1000 copies of mutant template in the presence of excess wild type (Fang et al, 2009; Geng et al, 2006; Wen and Li, 2007). In most cases the specificities decrease at low concentrations of mutant virus relative to wild type, yielding false-positive results (Fang et al, 2009; Geng et al, 2006; Wen and Li, 2007).…”

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

“…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. It is also important to note that the potential advantages and clinical significance of an improved capability to detect minor populations of HBV drug-resistant mutants remain to be determined, given the uncertain influence of such minor viral populations on therapeutic response (17,22,28).…”

“…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.…”