44 Molecular Diagnosis of Entecavir Resistance genotypic mutations generally have occurred in patients who have developed virologic breakthrough, defined as a ≥1 log10 increase in serum HBV DNA above nadir, on two or more occasions 1 month apart while receiving treatment, but genotypic mutations can also emerge in patients with persistent viraemia and without virologic breakthrough (Fig 1.) (13). The testing for genotypic resistance can not be recommended prior to initiation of therapy unless the patient is undergoing treatment for CHB (20); but, despite advances in HBV genotypic resistance testing, an ETV mutant virus population can be detected at present, in the absence of ETV treatment (in naïve patients and during LAM therapy) (21-25). Molecular Assays in the Diagnosis of ETV Resistance Four types of molecular assays are available for the diagnosis and managment of ETV drug resistance: direct sequencing, line probe assay, clonal analysis, and RFLP analysis. Applications of other assays are currently more specialized, and their use is more limited. The utility of these assays and their performance characteristics are defined in Table 2. However, the assays more widely used are reviewed below. Direct Sequencing of the HBV pol Gene Region The reference method for the detection of resistance-conferring mutations is population-based sequencing (ie, a direct sequence analysis of the HBV pol gene). Genotypic resistance assays use DNA sequencing methods to examine the pol region of the HBV genome for recognizable resistance-associated mutations (26-28). Sequence analysis is considered the gold standard for characterizing HBV DNA isolates (29, 30). However, this assay is time-consuming for a large number of clinical samples, but is suitable for high-throughput screening in a large viral-genome region (Table 2). The in-vitro phenotyping of HBV mutations associated with antiviral resistance in a database program such as geno2pheno (http:// coreceptor.bioinf.mpi-inf.) and SEQHEPB tools (http://www.seqvirology.com) (work in the Fasta format of the HBV genomic sequence) is a convenient approach (31). These tools have great applicability to the interpretation of amino - acid substitutions. The results between amino acid positions 80 - 250 of the HBV pol gene were similarly detected in manual and geno2pheno tool analysis. But, for reliable predictions the sequences must contain the motif “TCCCATCCCATC” starting ateast with codon 159 in the pol frame (24, 25). Line-Probe Assay Line-probe assays, using probes for individual mutations, are specific and reproducible and significantly more sensitive in detecting resistance mutations than population-based sequencing. This technology is a useful tool for the rapid and accurate detection of mutants, which make up as little as 5% of Fig 1. Serial changes in serum HBV DNA and ALT levels in association with emergence of antiviral- resistant HBV mutants. The first manifestation of resistance is the detection of resistance-conferring mutations (ie, genotypic resistance) (5, 20). ALT: alanine aminotransferase; ULN: upper limit of normal. Hepatitis Monthly, Winter 2010; 10(1): 42-47