Hepatitis B virus genotyping based on cluster analysis of the region involved in lamivudine resistance

Rodrı́guez-Nóvoa, Sonia; Gómez-Tato, Antonio; Aguilera, Antonio; Castroagudı́n, J.F.; González‐Quintela, Arturo; García-Riestra, Carlos; Regueiro, Benito J.

doi:10.1016/j.jviromet.2003.08.011

Cited by 10 publications

(8 citation statements)

References 23 publications

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“…This restriction site resulted, in our study, in an additional fragment of 160 pb with a RFLP pattern of genotype B but it did not interfere with results of other studies relying on sequencing methods and which succeeded in identifying genotype D [13-17]. For this reason, results obtained by PCR-RFLP should be carefully analyzed because introduction of new restriction site in targeted sequences can lead to erroneous results with this technique [21]. The genotyping based on this RFLP method is largely used for epidemiological studies because it is easy to perform but this approach suffers some limits especially in area with high prevalence of genotype D [22-25].…”

Section: Discussionmentioning

confidence: 68%

Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

Hannachi

Fredj

Bahri

et al. 2010

Virol J

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BackgroundIn Tunisia, country of intermediate endemicity for Hepatitis B virus (HBV) infection, most molecular studies on the virus have been carried out in the North of the country and little is known about other regions. The aim of this study was to determine HBV genotype and subgenotypes in Central-East Tunisia. A total of 217 HBs antigen positive patients were enrolled and determination of genotype was investigated in 130 patients with detectable HBV DNA. HBV genotyping methods were: PCR-RFLP on the pre-S region, a PCR using type-specific primers in the S region (TSP-PCR) and partial sequencing in the pre-S region.ResultsThree genotypes (D, B and A) were detected by the PCR-RFLP method and two (D and A) with the TSP-PCR method, the concordance between the two methods was 93%. Sequencing and phylogenetic analysis of 32 strains, retrieved the same genotype (D and A) for samples with concordant results and genotype D for samples with discordant results. The sequences of discordant genotypes had a restriction site in the pre-S gene which led to erroneous result by the PCR-RFLP method. Thus, prevalence of genotype D and A was 96% and 4%, respectively. Phylogenetic analysis showed the predominance of two subgenotypes D1 (55%) and D7 (41%). Only one strain clustered with D3 subgenotype (3%).ConclusionsPredominance of subgenotype D7 appears to occur in northern regions of Africa with transition to subgenotype D1 in the East of the continent. HBV genetic variability may lead to wrong results in rapid genotyping methods and sequence analysis is needed to clarify atypical results.

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Section: Discussionmentioning

confidence: 68%

Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

Hannachi

Fredj

Bahri

et al. 2010

Virol J

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“…Despite these advantages, the test is fairly expensive for resource-poor countries. Direct DNA sequencing of PCR generated amplicons corresponding to genotype-specific regions of HBV genome also yields accurate genotype assignments and is the method of choice for patients infected with recombinant genotypes [9,10]. However, DNA sequencing is still considered as technically demanding, time consuming and costly in most of the developing countries.…”

Section: Resultsmentioning

confidence: 99%

“…The LiPA results were interpreted as instructed by the kit's manufacturer. The genotype-specific segment of the HBV polymerase gene was amplified and sequenced for DNA sequence-based determination of specific HBV genotypes exactly as described previously [10]. The subtractive PCR-RFLP-based genotyping was performed by semi-nested PCR amplification of a 485 bp fragment which was successively digested with 5 restriction enzymes (in separate tubes) to identify genotype-specific sequences exactly as described earlier [8].…”

Section: Resultsmentioning

confidence: 99%

Comparative evaluation of INNO-LiPA HBV assay, direct DNA sequencing and subtractive PCR-RFLP for genotyping of clinical HBV isolates

Ali

Hasan

Ahmad

et al. 2010

Virol J

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Genotypes (A to H) of hepatitis B virus (HBV) influence liver disease progression and response to antiviral therapy in HBV-infected patients. Several methods have been developed for rapid genotyping of HBV strains. However, some of these methods may not be suitable for developing countries. The performance of INNO-LiPA HBV Genotyping assay (LiPA), direct DNA sequencing and subtractive PCR-RFLP of genotype-specific HBV genome regions were evaluated for accurately determining the HBV genotypes by analyzing sera (n = 80) samples from chronic HBV patients. Both, LiPA and DNA sequencing identified 63, 4 and 13 HBV strains as belonging to genotype D, genotype A and mixed genotype A and D, respectively. On the contrary, the PCR-RFLP-based method correctly identified all 4 genotype A but only 56 of 63 genotype D strains. Seven genotype D strains yielded indeterminate results. DNA sequence comparisons showed that a single nucleotide change in the target region generated an additional restriction site for Nla IV that compromised the accuracy of this method. Furthermore, all the mixed genotype A and D strains were identified only as genotype A strains. The data show that the PCR-RFLP-based method incorrectly identified some genotype D strains and failed to identify mixed genotype infections while LiPA and DNA sequencing yielded accurate results.

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“…This disadvantage was also reported for other methods such as PCR-hybridization or type-specific primers amplification, and also for other HBV genotypes (29, 30, 35-40). The limit to genotyping in all these cases was probably due to high variability of HBV with modification in expected restriction profiles for used enzymes (41). Current results confirmed that hypothesis since 24 atypical profiles were detected; they were explained by either adding one or more restriction sites or disappearing at least one restriction site (41).…”

Section: Discussionmentioning

confidence: 99%

“…The limit to genotyping in all these cases was probably due to high variability of HBV with modification in expected restriction profiles for used enzymes (41). Current results confirmed that hypothesis since 24 atypical profiles were detected; they were explained by either adding one or more restriction sites or disappearing at least one restriction site (41). These atypical profiles were observed especially after a long evolution of the disease, since they were significantly associated with advanced age and PLD rather than groups of CAH and IC.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of PCR-RFLP in the Pre-S Region as Molecular Method for Hepatitis B Virus Genotyping

et al. 2013

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BackgroundHepatitis B virus (HBV) infection is a public health problem in developing countries. HBV genotypes play major role in the evolution of infection since they were involved in different clinical presentations and response to treatment.ObjectivesThis study was conducted to evaluate the efficiency of restriction fragment length polymorphism (RFLP) analysis for HBV genotyping.Patients and MethodsWe investigated 98 samples collected from patients chronically infected with HBV. HBV genotypes were determined by analysis of patterns obtained after amplification in Pre-S region and digestion of the amplicon by two endonucleases AvaII and DpnII. Obtained results were confirmed by partial sequencing in the same region.ResultsTwo different HBV genotypes were detected in this study, Genotype D (in 95. 9%) and Genotype A (in 4.1%). Seventy-four samples (75.5%) were successfully genotyped with RFLP analysis and all classified as genotype D. The remaining 24 samples (24.5%) which were un-genotyped by RFLP analysis, were classified by partial sequencing of the pre-S region as HBV genotype D (20 samples, 20.4%) and genotype A (4 samples, 4.1%). Atypical profiles were significantly associated with advanced liver disease (P = 0.001) as well as older age (P < 0.05).ConclusionsSeveral previous studies used PCR-RFLP to genotype HBV; however, we showed the high risk to obtain atypical profiles, especially in advanced stages of chronic infection, with as results difficulties to genotype the virus. These profiles resulted from the accumulation of mutations during natural course of infection resulting in a modification in restriction sites for enzymes. So, we recommended completing the investigation by partial sequencing to confirm obtained results.

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Hepatitis B virus genotyping based on cluster analysis of the region involved in lamivudine resistance

Cited by 10 publications

References 23 publications

Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

Comparative evaluation of INNO-LiPA HBV assay, direct DNA sequencing and subtractive PCR-RFLP for genotyping of clinical HBV isolates

Evaluation of PCR-RFLP in the Pre-S Region as Molecular Method for Hepatitis B Virus Genotyping

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