COLD-PCR Method for Early Detection of Antiviral Drug-Resistance Mutations in Treatment-Naive Children with Chronic Hepatitis B

• Main Authors: Thuy Thi Bich Phung, Son Van Chu, Son Thien Vu, Hanh Thi Pham, Hang Minh Nguyen, Hoan Dang Nguyen, Ngan Thi Le, Dung Van Nguyen, Phuong Thai Truong, Van Thi Tuong Vu, Anh Thi Van Nguyen
• Format: Article
• Language: English
• Published: MDPI AG 2020-07-01
• Series: Diagnostics
• Subjects: COLD-PCR method; hepatitis B virus (HBV); drug-resistance mutation; nucleos(t)ides analogues; treatment-naive children
• Online Access: https://www.mdpi.com/2075-4418/10/7/491

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 (OR_LMV/ADF: 1495.000; 95% CI: 89.800–24,889.032; <i>p</i> < 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.