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The study investigated the hepatitis B virus (HBV) genotypic resistance profile in 1803 nucleos(t)ide analogue (NA)-experienced Chinese patients with chronic HBV infection. Serum HBV DNA was extracted, and the reverse transcriptase region was analysed by a high-sensitive direct PCR sequencing and verified by clonal sequencing if necessary. Drug-resistant mutations were detected in 560 of the 1803 patients, including 214 of 490 patients who received lamivudine (LAM), 35 of 428 patients who received adefovir (ADV), five of 18 patients who received telbivudine and 306 of 794 patients who received various sequential/combined NA therapies. ADV-resistant mutations were detected in 36 of 381 patients who received LAM and then switched-to ADV in contrast to one of 82 patients who received ADV add-on LAM. Entecavir (ETV)-resistant mutations were detected not only in LAM- and ETV-treated patients but also in LAM-treated ETV-naïve patients. Double mutations rtM204I and rtL180M were detected more frequently in genotype C than in genotype B virus, and patients infected with this mutant had higher alanine transaminase levels than those infected with mutant containing the rtM204I substitution alone. Multidrug-resistant HBV strains were identified in eight patients, including two novel strains with mutational patterns rtL180M + A181V + S202G + M204V + N236T and rtL180M + S202G + M204V + N236T. The results provide new information on HBV genotypic resistance profiles in a large cohort of Chinese patients with chronic HBV infection and may have important clinical implication for HBV drug resistance management in China.