Patient 5 (Fig. 2D) exhibited a simple resistance pattern, characterized by a suboptimal response to adefovir and virological breakthrough at month 37, resulting from outgrowth of a resistant variant bearing two amino acid substitutions (rtA181V(sL173F) plus rtN236T). Patient 6 (Fig. 2E) responded to adefovir, but low-level viremia persisted, with transient selection of a variant bearing an rtN238E substitution. Subsequently, a variant bearing the double rtN236T plus rtN238E substitution took over and persisted at a low level (approximately 102 IU/mL). Finally, in C646 patient 7, who initially responded and subsequently broke through (Fig. 2F), the virological breakthrough was related to the selection
of a major viral population bearing the rtA181V(sL173F) substitution and a minor population bearing the double rtA181V(sL173F) plus rtF221Y substitution. Both variants SAHA HDAC datasheet were partially inhibited,
but remained dominant when lamivudine was added to adefovir. Drug resistance is the principal cause of antiviral treatment failure, which may result in clinical disease progression. Next-generation sequencing technologies, such as UDPS, have the capacity to generate thousands of sequences from complex genomic mixtures, including sequences from dominant, intermediate, and minor viral populations.[24, 25] UDPS-based GS FLX technology provides sequence reads of sufficient length to span the region of interest when studying HBV resistance to nucleoside/nucleotide analogs. To interpret the data generated with this method, we developed an original package of four complementary software programs capable of analyzing large numbers of sequences (nearly 500,000 in this study) in the specific
find more context of viral resistance and used it in this study. The HBV DNA level in the starting sample may theoretically have an effect on the sensitivity of detection of minor quasispecies variants by UDPS. This did not influence our description of baseline distributions of HBV variants because all patients had high viral levels in the absence of therapy. On adefovir or adefovir-lamivudine treatment, we were able to generate sequence information by UDPS down to HBV DNA levels of the order of 2-3 Log10 IU/mL. To avoid a bias linked to differences in the HBV DNA levels in different blood samples, we chose to express the results as absolute amounts of viral variants in each sample (in IU/mL), rather than proportions of the full quasispecies. In addition, the use of PyroDyn and PyroLink software allowed us to avoid a bias related to the HBV DNA amount in the sample, because only viral populations that were exponentially growing or decreasing over time in serial blood samples were detected and described, regardless of HBV DNA content. Preexistence of resistant HBV variants in patients never exposed to nucleoside/nucleotide analogs is an accepted concept.