We developed a novel real-time PCR assay that simultaneously evaluates eleven major nucleos(t)ide antiviral (NA) drug-resistance mutations (mt) in chronic hepatitis B patients (CHB), including L180M, M204I/V, and V207M (lamivudine (LMV) resistance), N/H238A/T (Adefovir (ADF) resistance) which are circulating in Vietnam; T184G/L, S202I, M250V (entecavir (ETV) resistance), and A194T (tenofovir resistance) which have been recently reported in several studies across the globe. We detected drug-resistant mt in HBV samples using our predesigned panel of allele-specific locked-nucleic acid (LNA) probes. Our assay had a high sensitivity of 5% in a low HBV DNA population of ≥5 ×10 3 IU/mL and was validated in a cohort of 130 treatment-naive children and 98 NA-experienced adults with CHB. Single-point mt for LMV- and ADF-resistance were detected in 57.7% and 54.1% of the child and adult samples, respectively, with rtV207M (42.3%, children; 36.7%, adults) and rtN238T/A (15.4%, children; 16.3, adults) being the most frequent mt in these populations. Multiple-point mt including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I were only identified in two children, resulting in LMV-ADF-resistance and reduced ETV susceptibility. In conclusion, this assay accurately identified the mt profile of children (98.4%) and adults (91.2%) with CHB, which is comparable to established methods. This fast and sensitive screening method can be used for the detection of major NA-resistant mt circulating in developing countries, as well as providing a model for the development of similar mt-detection assays, especially for use in non-hospitalised patients who need their results within half a day before starting treatment.

We investigated Nucleos(t)ide-analogue (NA)-resistance mutations (mt) in 142 treatment-naive children with Chronic Hepatitis B (CHB), using a sensitive co-amplification at lower denaturation temperature (COLD)-PCR with Sanger DNA sequencing. An NA resistance-associated mt in the hepatitis B virus (HBV) reverse transcriptase (RT) was found in 66.2% of the patients, with nonclassical mt contributing the most (64.8%). Significantly higher frequencies of Lamivudine (LMV) and Adefovir dipivoxil (ADF) resistance-associated mt were found in genotypes B and C, respectively (ORLMV/ADF: 1495.000; 95% CI: 89.800–24,889.032; p < 0.001). Single-point mt associated to LMV and ADF resistance were detected in 59.9% of the tested children with rtV207M (38.0%) and rtN238T (9.9%) being the most frequent. Multiple-point mt were found only in 8 cases (5.6%): 6 children carried double mt (rtV207M + rtL229V; rtV207M + rtI233V; rtV207I + rtV207M × 2 cases; rtV207M + rtS213T; rtN238A + rtS256G) relating to LMV or/and ADF resistance and 3 children carried triple mt (rtL180M + rtM204I + rtN238T; rtV207M + rtS213T + rtS256G) or quadruple mt (rtL180M + rtM204V + rtV207I/M) for LMV-ADF resistance and Entecavir-reduced susceptibility. Our data indicate that significantly higher frequencies of LMV and ADF-associated mutations were found in treatment-naïve children infected with HBV genotypes B and C, respectively. The developed COLD-PCR method and obtained data may contribute to the development of suitable treatments for children with CHB.