Large-scale survey of naturally occurring HBV polymerase mutations associated with anti-HBV drug resistance in untreated patients with chronic hepatitis B S. Mirandola, 1,2 D. Campagnolo, 1 G. Bortoletto, 1 L. Franceschini, 1 M. Marcolongo, 1,2 and A. Alberti 1,2 1 Venetian Institute of Molecular Medicine, Padova, Italy; and 2 Department of Histology, Microbiology and Experimental Medicine, University of Padova, Padova, Italy Received October 2010; accepted for publication December 2010 SUMMARY. Drug resistance is a major limitation for the long-term efficacy of antiviral therapy with nucleos(t)ide analogues (NAs) in chronic hepatitis B (CHB). Antiviral resistance mutations may pre-exist in the overall viral population of untreated patients. We aimed to assess the prevalence of such hepatitis B virus (HBV) variants in a large cohort of NAs-naı ¨ve patients with CHB and to explore possible association with viral and host variables. Serum samples from 286 NAs-naı ¨ve consecutive patients with CHB were tested for serum HBV-DNA, and 255 of them having HBV-DNA > 1000 IU/mL were further analysed for drug resistance mutations by INNO-LiPA HBV DRv2/v3. NAs- naı ¨ve patients analysed were mainly men (73%), Caucasians (85%), hepatitis B e Antigen (HBeAg) negative (79%) and genotype D (69%), with a mean age of 43.2 ± 13.4 years. HBV mutations associated with antiviral drug resistance were detected in 13 (5%) patients: three patients infected with HBV genotype C had the rtM204V + rtL180M muta- tions associated with lamivudine (LMV) resistance. Four patients had the rtI233V mutation that may reduce sensi- tivity to adefovir, and three patients had the rtM250L/V mutation typical of entecavir resistance. LMV compensatory mutations rtL80V and rtV173L were seen in two and one patients, respectively. No relationship was seen between presence of resistant or compensatory mutations and HBV- DNA levels, HBeAg/anti-HBe status or previous IFN therapy. These results confirm that HBV mutations, which confer resistance against currently available anti-HBV NAs, may already exist in patients who have never received the drug. Keywords: genotype, hepatitis B virus, mutation, nucle- os(t)ide analogue naı ¨ve, resistance. INTRODUCTION Chronic hepatitis B virus (HBV) infection remains a major cause of hepatocellular carcinoma and of liver-related death worldwide [1–3]. Progression of liver disease towards end- stage complications is associated with high and persistent virus replication and can be efficaciously prevented by antiviral therapy [4,5]. Several oral nucleos(t)ide analogues (NAs) with anti-HBV activity are currently available and widely used for treating chronic hepatitis B (CHB) and cirrhosis [6]. However, these compounds are still mainly used as monotherapies, increas- ing the risk of drug resistance. Resistance is currently less frequent among patients with CHB because of the imple- mentation of more potent new-generation NAs with high genetic barrier, such as entecavir (ETV) and tenofovir [7–11]. However, the risk of resistance is not completely abolished, and close monitoring of the virological response, with prompt treatment adaptation, is strongly recommended by most recent Guidelines [4,5]. The mechanisms of drug resistance in HBV therapy have been well characterized. Under the selec- tive pressure of NA therapy, pre-existing or new mutations conferring a selective advantage to a resistant variant will generate a progeny virus that is more fit and can spread more rapidly in the liver becoming the dominant species in the viral population. After withdrawal of the anti-HBV drug, these variants often persist, thus negatively influencing the efficacy of any subsequent oral treatment with cross-resistance [7–11]. One typical example is ETV whose efficacy and genetic barrier are both significantly reduced in the presence of lamivudine (LMV) resistance mutations [12–15]. Recently, resistance mutations associated with NA therapy have been described in the overall viral population of drug-naı ¨ve patients. Indeed, a number of reports indicated that YMDD mutations may be present in a subgroup of NA-naı ¨ve patients Abbreviations: ADV, adefovir-dipivoxil; CHB, chronic hepatitis B; ETV, entecavir; HBeAg, hepatitis B e Antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HDV, hepatitis D virus; HIV, human immu- nodeficiency virus; LMV, lamivudine; NAs, nucleos(t)ide analogues; RT, reverse transcriptase. Correspondence: Alfredo Alberti, VIMM, via Orus 2, 35129 Padova, Italy. E-mail: alfredo.alberti@unipd.it Journal of Viral Hepatitis, 2011, 18, e212–e216 doi:10.1111/j.1365-2893.2011.01435.x Ó 2011 Blackwell Publishing Ltd