Detection of mixed populations of wild-type and YMDD hepatitis B variants by pyrosequencing in acutely and chronically infected patients

Abstract

BackgroundLamivudine (LAM) is associated with the highest known rate of resistance mutations among nucleotide analogs used to treat chronic hepatitis B virus (HBV) infection. Despite this, LAM continues in widespread use, especially in combination therapies. The primary LAM resistance mutation (rtM204V/I) occurs in the YMDD motif of HBV polymerase. The aim of this study was to characterize Brazilian HBV isolates from acute and chronic cases by direct sequencing, and to identify HBV quasispecies in the YMDD motif using a pyrosequencing method capable of detecting single-nucleotide polymorphisms. HBV DNA from serum samples of 20 individuals with acute HBV infection and 44 with chronic infection undergoing antiviral therapies containing LAM were analyzed by direct sequencing and pyrosequencing methods.ResultsPhylogenic analyses of direct-sequenced isolates showed the expected genotypes (A, D and F) for the Brazilian population in both acute and chronic infections. However, within genotype A isolates, subgenotype A2 was more frequently detected in acute cases than in chronic cases (P = 0.012). As expected, none of the individuals with acute hepatitis B had LAM-resistant isolates as a dominant virus population, whether detected by direct sequencing or pyrosequencing. However, pyrosequencing analyses showed that 45% of isolates (9/20) had minor subpopulations (4-17%) of LAM-resistant isolates. Among chronic patients undergoing LAM treatment, YMDD mutants were frequently found as a dominant virus population. In cases where wild-type virus was the dominant population, subpopulations of YMDD variants were usually found, demonstrating the complexity of HBV quasispecies.ConclusionsYMDD variants were frequently detected as a minor population in acute HBV infection. The occurrence of pre-existing variants may lead to a high frequency of resistant mutants during antiviral therapy in the chronic phase. In chronic infection, detection of YMDD variants before virological or biochemical breakthrough might contribute to making better therapy choices and thus improving treatment outcome.

Characterization of the dynamics of hepatitis B virus resistance to adefovir by ultra-deep pyrosequencing

“…UDPS has been used to assess the preexistence of HBV variants resistant to nucleoside/nucleotide analogs in a few studies. However, these works suffered from important methodological flaws, including lack of sensitivity, no consideration of the error rate of the method to establish reliable cutoffs and ensure specificity, too‐short genomic region analyzed, and/or no linkage studies …”

Prevelance of Common YMDD Motif Mutations in Long Term Treated Chronic HBV Infections in a Turkish Population

“…The second two substitutions were not found in the current cohort.The current hypothesis is that interactions between residues at position 204 and those at 184, 202 or 250 are important for a functional HBV RT enzyme, or perhaps that the LVDr, ADVr and/or both substitutions further decrease the replication capacity of the more impaired M204I virus to below levels required to survive. Recently, lots of reports have demonstrated that YMDD motif mutations can naturally occur in chronic HBV patients without antiviral theraphy (Ağca et al, 2012;Lee et al, 2012;Mello et al, 2012;Tan et al, 2012;Wu et al, 2012). Li et al(2013) have claimed that HBV-related HCC patients with YMDD motif mutations have no response to lamivudine therapy.…”

Comparison of Pyrosequencing, Sanger Sequencing, and Melting Curve Analysis for Detection of Low-Frequency Macrolide-Resistant Mycoplasma pneumoniae Quasispecies in Respiratory Specimens

“…The techniques that have been applied for the direct detection of MRMP include PCR followed by Sanger sequencing (11,14,16), pyrosequencing (17,18), high-resolution melting curve analysis (19)(20)(21), allele-specific PCR (22), and PCRrestriction fragment length polymorphism (23). In the context of viral infections, natural occurrences of mixed drug-sensitive and -resistant subpopulations are well documented (24)(25)(26)(27)(28). The enrichment of minor resistant subpopulations has further been demonstrated to be associated with antiviral treatment failure (25,26).…”

“…In the context of viral infections, natural occurrences of mixed drug-sensitive and -resistant subpopulations are well documented (24)(25)(26)(27)(28). The enrichment of minor resistant subpopulations has further been demonstrated to be associated with antiviral treatment failure (25,26). This has led to the term "quasispecies," which has been used widely to describe sequence variants in heterogeneous virus populations (25,26).…”

“…The enrichment of minor resistant subpopulations has further been demonstrated to be associated with antiviral treatment failure (25,26). This has led to the term "quasispecies," which has been used widely to describe sequence variants in heterogeneous virus populations (25,26). Recent studies involving Sanger sequencing suggested that mycoplasma infections might also comprise mixed populations of drug-sensitive and drug-resistant molecular mutants (i.e., quasispecies at the 23S rRNA macrolide resistance motif) (29).…”