BACKGROUND AND OBJECTIVES: A virus population often exists as a complex mixture of genetic populations. Antiviral-resistant mutants could be circulating as minority variants in the mixed virus population that are not detected by standard sequencing methods. The role of minor drug-resistant variants and clinical outcome is slowly evolving and there is a need to employ sensitive methods for detection of minority variants that emerge as dominant species and subsequently affect the antiviral efficacy. This study was intended to develop a technique called the ligation amplification assay (LigAmp) to identify minor drug-resistant variants of hepatitis B virus (HBV). METHODS: A LigAmp HBV assay was developed and clinical samples were tested from chronic hepatitis B subjects on antiviral treatment. Nucleotide sequencing of HBV reverse transcriptase (rt) region was performed and the results were compared with LigAmp assay. The performance of LigAmp assay was validated by clonal sequencing. Virological response was measured using HBV DNA levels and the results were correlated with antiviral-resistant mutations detected by sequencing and LigAmp assays. RESULTS: A total of 80 reactions of LigAmp assay were performed for rtM204V and rtM204I (ATT) mutant detection. Samples were obtained from 40 chronic hepatitis B subjects. Among these subjects, rtM204V and rtM204I (ATT) mutations were identified by standard sequencing in 10 (25%) and 12 (30%) subjects, respectively. LigAmp detected both rtM204V and rtM204I (ATT) mutations in 13 (32.5%) subjects, rtM204I mutation in 12 (30%) subjects and rtM204V mutation in 1 (2.5%) subject, respectively. LigAmp detected primary resistant mutants in 69.4% of lamivudine non-responders while sequencing detected resistant mutations in only 55.6% subjects (p < 0.001). CONCLUSIONS: This data shows significantly higher sensitivity of LigAmp for detection of minority rtM204V and rtM204I (ATT) mutations over standard sequencing. Therefore, LigAmp has potential clinical utility for appropriate monitoring and tailoring of HBV therapy.
BACKGROUND AND OBJECTIVES: A virus population often exists as a complex mixture of genetic populations. Antiviral-resistant mutants could be circulating as minority variants in the mixed virus population that are not detected by standard sequencing methods. The role of minor drug-resistant variants and clinical outcome is slowly evolving and there is a need to employ sensitive methods for detection of minority variants that emerge as dominant species and subsequently affect the antiviral efficacy. This study was intended to develop a technique called the ligation amplification assay (LigAmp) to identify minor drug-resistant variants of hepatitis B virus (HBV). METHODS: A LigAmp HBV assay was developed and clinical samples were tested from chronic hepatitis B subjects on antiviral treatment. Nucleotide sequencing of HBV reverse transcriptase (rt) region was performed and the results were compared with LigAmp assay. The performance of LigAmp assay was validated by clonal sequencing. Virological response was measured using HBV DNA levels and the results were correlated with antiviral-resistant mutations detected by sequencing and LigAmp assays. RESULTS: A total of 80 reactions of LigAmp assay were performed for rtM204V and rtM204I (ATT) mutant detection. Samples were obtained from 40 chronic hepatitis B subjects. Among these subjects, rtM204V and rtM204I (ATT) mutations were identified by standard sequencing in 10 (25%) and 12 (30%) subjects, respectively. LigAmp detected both rtM204V and rtM204I (ATT) mutations in 13 (32.5%) subjects, rtM204I mutation in 12 (30%) subjects and rtM204V mutation in 1 (2.5%) subject, respectively. LigAmp detected primary resistant mutants in 69.4% of lamivudine non-responders while sequencing detected resistant mutations in only 55.6% subjects (p < 0.001). CONCLUSIONS: This data shows significantly higher sensitivity of LigAmp for detection of minority rtM204V and rtM204I (ATT) mutations over standard sequencing. Therefore, LigAmp has potential clinical utility for appropriate monitoring and tailoring of HBV therapy.
Authors: Ralph Stadhouders; Suzan D Pas; Jeer Anber; Jolanda Voermans; Ted H M Mes; Martin Schutten Journal: J Mol Diagn Date: 2009-11-30 Impact factor: 5.568
Authors: Richard J Colonno; Ronald Rose; Carl J Baldick; Steven Levine; Kevin Pokornowski; Cheng F Yu; Ann Walsh; Jie Fang; Mayla Hsu; Charles Mazzucco; Betsy Eggers; Sharon Zhang; Mary Plym; Kenneth Klesczewski; Daniel J Tenney Journal: Hepatology Date: 2006-12 Impact factor: 17.425
Authors: M I Allen; M Deslauriers; C W Andrews; G A Tipples; K A Walters; D L Tyrrell; N Brown; L D Condreay Journal: Hepatology Date: 1998-06 Impact factor: 17.425
Authors: F Ntziora; D Paraskevis; C Haida; E Magiorkinis; E Manesis; G Papatheodoridis; S Manolakopoulos; A Beloukas; S Chryssoy; G Magiorkinis; V Sypsa; A Hatzakis Journal: J Clin Microbiol Date: 2009-06-24 Impact factor: 5.948