Fast and Sensitive Real-Time PCR Detection of Major Antiviral-Drug Resistance Mutations in Chronic Hepatitis B Patients by Use of a Predesigned Panel of Locked-Nucleic-Acid TaqMan Probes

• Published in: Journal of clinical microbiology 2021-09, Vol.59 (10), p.e0093621-e0093621
• Main Authors: Chu, Son V, Vu, Son T, Nguyen, Hang M, Le, Ngan T, Truong, Phuong T, Vu, Van T T, Phung, Thuy T B, Nguyen, Anh T V
• Format: Article
• Language: English
• Subjects: Adult; Antiviral Agents - pharmacology; Antiviral Agents - therapeutic use; Child; Clinical Microbiology; DNA, Viral - genetics; Drug Resistance, Viral; Hepatitis B virus - genetics; Hepatitis B, Chronic - drug therapy; Humans; Lamivudine - therapeutic use; Mutation; Real-Time Polymerase Chain Reaction; Virology
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.00936-21

We developed a novel real-time PCR assay that simultaneously evaluates 11 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; and T184G/L, S202I, and 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 hepatitis B virus (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  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 (children, 42.3%; adults, 36.7%) and rtN238T/A (children, 15.4%; adults, 16.3%) being the most frequent mt in these populations. Multiple-point mt, including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I, were identified in only 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 nonhospitalized patients who need their results within half a day, before starting treatment.