Role of the line probe assay INNO-LiPA HBV DR and ultradeep pyrosequencing in detecting resistance mutations to nucleoside/nucleotide analogues in viral samples isolated from chronic hepatitis B patients

Meşe, Sevim; Arıkan, Muzaffer; Çakıris, Aris; Abacı, Neslihan; Gumus, Ergun; Kurşun, Olcay; Önel, Derya; Ustek, Duran; Kaymakoğlu, Sabahattin; Badur, Selim; Yenen, Osman Şadi; Bozkaya, Emel

doi:10.1099/vir.0.053041-0

Cited by 8 publications

(7 citation statements)

References 24 publications

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“…First, the fragment amplified for NGS is different from the fragment analyzed by LiPA; and second, the two techniques have methodological differences: NGS is a sequencing method and the definition of the genotype is obtained by phylogenesis, whereas LiPA is a hybridization method and therefore, an indirect measurement. Inconsistencies between LiPA and UDPS have been described in HCV genotyping [ 38 ] and HBV variant resistance detection [ 39 ].…”

Section: Discussionmentioning

confidence: 99%

Complex Genotype Mixtures Analyzed by Deep Sequencing in Two Different Regions of Hepatitis B Virus

et al. 2015

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This study assesses the presence and outcome of genotype mixtures in the polymerase/surface and X/preCore regions of the HBV genome in patients with chronic hepatitis B virus (HBV) infection. Thirty samples from ten chronic hepatitis B patients were included. The polymerase/surface and X/preCore regions were analyzed by deep sequencing (UDPS) in the first available sample at diagnosis, a pre-treatment sample, and a sample while under treatment. HBV genotype was determined by phylogenesis. Quasispecies complexity was evaluated by mutation frequency and nucleotide diversity. The polymerase/surface and X/preCore regions were validated for genotyping from 113 GenBank reference sequences. UDPS yielded a median of 10,960 sequences per sample (IQR 16,645) in the polymerase/surface region and 11,595 sequences per sample (IQR 14,682) in X/preCore. Genotype mixtures were more common in X/preCore (90%) than in polymerase/surface (30%) (p<0.001). On X/preCore genotyping, all samples were genotype A, whereas polymerase/surface yielded genotypes A (80%), D (16.7%), and F (3.3%) (p = 0.036). Genotype changes in polymerase/surface were observed in four patients during natural quasispecies dynamics and in two patients during treatment. There were no genotype changes in X/preCore. Quasispecies complexity was higher in X/preCore than in polymerase/surface (p = 0.004).The results provide evidence of genotype mixtures and differential genotype proportions in the polymerase/surface and X/preCore regions. The genotype dynamics in HBV infection and the different patterns of quasispecies complexity in the HBV genome suggest a new paradigm for HBV genotype classification.

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Section: Discussionmentioning

confidence: 99%

Complex Genotype Mixtures Analyzed by Deep Sequencing in Two Different Regions of Hepatitis B Virus

et al. 2015

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“…Several studies have demonstrated the higher relative sensitivity of ultra-deep sequencing compared with conventional direct-population sequencing. Therefore, ultra-deep sequencing is useful for detecting low-frequency drug-resistant mutations that cannot be detected with standard methods [18,19,20,21]. Conventional sequencing based on the Sanger method can only detect mutations present in >20% of viral quasispecies.…”

Section: Discussionmentioning

confidence: 99%

“…Conventional sequencing based on the Sanger method can only detect mutations present in >20% of viral quasispecies. Although the line probe assay is another common and convenient method to detect mutations, it can only detect mutations present in >15% of viral quasispecies [20]. Cloning-based sequencing methods are also commonly used, but they usually detect less than 50% of the substitutions identified with ultra-deep sequencing [21].…”

Section: Discussionmentioning

confidence: 99%

A Deep-Sequencing Method Detects Drug-Resistant Mutations in the Hepatitis B Virus in Indonesians

et al. 2014

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Objective: The long-term administration of a nucleos(t)ide analogue (NA) for the treatment of chronic hepatitis B may encourage the emergence of viral mutations associated with drug resistance. Minor populations of viruses may exist before treatment, but are difficult to detect because of technological limitations. Identifying minor viral quasispecies should be useful in the clinical management of hepatitis B virus (HBV) infection. Methods: Six treatment-naïve Indonesian patients with chronic HBV infection participated in this study. The polymerase region of the HBV genome, including regions with known drug-resistant mutations, was subjected to capillary sequencing and MiSeq sequencing (Illumina). Mutations were analyzed with Genomics Workbench software version 6.0.1 (CLC bio). Results: The mean mapping reads for the six samples was 745,654, and the mean number of amplified fragments ranged from 17,926 to 25,336 DNA reads. Several known drug-resistant mutations in the reverse transcriptase region were identified in all patients, although the frequencies were low (0.12-1.06%). The proportions of the total number of reads containing mutations I169L/M, S202R, M204I/L or N236S were >1.0%. Conclusion: Several known NA-resistant mutations were detected in treatment-naïve patients in Indonesia using deep sequencing. Careful management of such patients is essential to prevent drug-resistant mutations from spreading to other patients.

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“…Although the line probe assay INNO‐LiPA HBV DR is also capable of detecting variants down to 5% of the mixture population, it does not provide the quantitative data of the mixture sequences. Solmone et al and Mese et al reported that ultradeep pyrosequencing (UDPS) has a higher sensitivity than the direct sequencing and line probe assay. Solmone et al used UDPS to investigate the HBV quasispecies in reverse transcriptase and surface region while Mese et al did the whole‐genome analysis of HBV sequences.…”

Section: Discussionmentioning

confidence: 99%

“…Although the line probe assay INNO-LiPA HBV DR is also capable of detecting variants down to 5% of the mixture population, 22 showed that UDPS provides a high quantity of information for both known and novel mutations. However, currently the assay is costly, so it is not practical for clinical practice.…”

Section: Discussionmentioning

confidence: 99%

Pyrosequencing method for sensitive detection of HBV drug resistance mutations

Lee

Inoue

Chong

et al. 2018

Journal of Medical Virology

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Hepatitis B (HBV) drug resistance assay is important for guiding therapy after the development of virologic breakthrough for patients receiving nucleoside/-tide analog therapy. However, the existing genotyping tools are either costly or lack sensitivity to detect mixed genotypes, and an improved method of resistant mutation detection is needed. An assay protocol for clinical application using pyrosequencing method was developed, capable of detecting all known validated HBV polymerase gene mutations that impart resistance to lamivudine, adefovir, tenofovir, and entecavir. Sixty-eight serum samples with known HBV resistance genotypes, previously tested with either Sanger sequencing assay or commercial line probe assay, were used for validation. Where there were discrepancies between the two methods, clonal sequencing by Sanger's method was used for confirmation. The modified pyrosequencing method accurately identified all the cloned polymerase genotypes and was able to distinguish as little as 5% of the mutant populations. This assay can be performed on serum sample with HBV DNA as low as 13.5 IU/mL. The cost per test was less than existing commercial assay. HBV drug resistance pyrosequencing assay was accurate, more sensitive and cheaper compared with the existing methods. It can detect minor populations of drug-resistant clones earlier, before the drug resistant clones become dominant, allowing the opportunity for an earlier change of therapy.

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Role of the line probe assay INNO-LiPA HBV DR and ultradeep pyrosequencing in detecting resistance mutations to nucleoside/nucleotide analogues in viral samples isolated from chronic hepatitis B patients

Cited by 8 publications

References 24 publications

Complex Genotype Mixtures Analyzed by Deep Sequencing in Two Different Regions of Hepatitis B Virus

Complex Genotype Mixtures Analyzed by Deep Sequencing in Two Different Regions of Hepatitis B Virus

A Deep-Sequencing Method Detects Drug-Resistant Mutations in the Hepatitis B Virus in Indonesians

Pyrosequencing method for sensitive detection of HBV drug resistance mutations

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