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

Ouneissa R, Bahri O, Ben Yahia A, Touzi H, Azouz MM, Ben Mami N, Triki H - Hepat Mon (2013)

Bottom Line: Obtained results were confirmed by partial sequencing in the same region.Atypical profiles were significantly associated with advanced liver disease (P = 0.001) as well as older age (P < 0.05).So, we recommended completing the investigation by partial sequencing to confirm obtained results.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Virology, Institute Pasteur de Tunis, Tunis, Tunisia.

ABSTRACT

Background: Hepatitis 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.

Objectives: This study was conducted to evaluate the efficiency of restriction fragment length polymorphism (RFLP) analysis for HBV genotyping.

Patients and methods: We 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.

Results: Two 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).

Conclusions: Several 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.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic Tree Based on the Analysis of a 328-bp Fragment in pre-S GeneThe tree includes eight reference sequences representative of genotype (A-H) and 95 Tunisian sequences from those studied in this work. Sequences of this work were indicated by the laboratory code followed by the country code (TUN) and the year of isolation. The reference sequences were indicated by their GenBank Accession number followed by the genotype designation.
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fig6554: Phylogenetic Tree Based on the Analysis of a 328-bp Fragment in pre-S GeneThe tree includes eight reference sequences representative of genotype (A-H) and 95 Tunisian sequences from those studied in this work. Sequences of this work were indicated by the laboratory code followed by the country code (TUN) and the year of isolation. The reference sequences were indicated by their GenBank Accession number followed by the genotype designation.

Mentions: Partial sequencing was performed for all samples to verify obtained results by PCR-RFLP; it confirmed infection by genotype D for 74 samples having typical profiles by RFLP (samples characterized by D1, D2, and D-del pattern). For 24 remaining isolates which were un-typeable isolates, genotype D and A were observed in 20 and 4 cases, respectively. Overall, prevalence of two detected genotypes was 96% (Genotype D) and 4% (Genotype A). Figure 1 shows a phylogenetic tree obtained from GenBank after comparison with selected sequences.


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

Ouneissa R, Bahri O, Ben Yahia A, Touzi H, Azouz MM, Ben Mami N, Triki H - Hepat Mon (2013)

Phylogenetic Tree Based on the Analysis of a 328-bp Fragment in pre-S GeneThe tree includes eight reference sequences representative of genotype (A-H) and 95 Tunisian sequences from those studied in this work. Sequences of this work were indicated by the laboratory code followed by the country code (TUN) and the year of isolation. The reference sequences were indicated by their GenBank Accession number followed by the genotype designation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842526&req=5

fig6554: Phylogenetic Tree Based on the Analysis of a 328-bp Fragment in pre-S GeneThe tree includes eight reference sequences representative of genotype (A-H) and 95 Tunisian sequences from those studied in this work. Sequences of this work were indicated by the laboratory code followed by the country code (TUN) and the year of isolation. The reference sequences were indicated by their GenBank Accession number followed by the genotype designation.
Mentions: Partial sequencing was performed for all samples to verify obtained results by PCR-RFLP; it confirmed infection by genotype D for 74 samples having typical profiles by RFLP (samples characterized by D1, D2, and D-del pattern). For 24 remaining isolates which were un-typeable isolates, genotype D and A were observed in 20 and 4 cases, respectively. Overall, prevalence of two detected genotypes was 96% (Genotype D) and 4% (Genotype A). Figure 1 shows a phylogenetic tree obtained from GenBank after comparison with selected sequences.

Bottom Line: Obtained results were confirmed by partial sequencing in the same region.Atypical profiles were significantly associated with advanced liver disease (P = 0.001) as well as older age (P < 0.05).So, we recommended completing the investigation by partial sequencing to confirm obtained results.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Virology, Institute Pasteur de Tunis, Tunis, Tunisia.

ABSTRACT

Background: Hepatitis 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.

Objectives: This study was conducted to evaluate the efficiency of restriction fragment length polymorphism (RFLP) analysis for HBV genotyping.

Patients and methods: We 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.

Results: Two 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).

Conclusions: Several 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.

No MeSH data available.


Related in: MedlinePlus