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

Thuy Thi Bich Phung,Anh Thi Van Nguyen,Hoan Dang Nguyen,Hanh Thi Pham,Hang Minh Nguyen,Son Thien Vu,Ngan Thi Le,Phuong Thai Truong,Dung Van Nguyen,Van Thi Tuong Vu,Son Van Chu

doi:10.3390/diagnostics10070491

Abstract

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.

Highlights

Hepatitis B virus (HBV), which could be life threatening by causing liver infection, is a challenging public-health problem worldwide
None of the methods could detect 1% mt in a. These results revealed that the optimized co-amplification at lower denaturation temperature (COLD)-PCR method was more sensitive than conventional mixed mt:wt population
These results revealed that the optimized COLD-PCR method was more for detecting minor mt PCR

Summary

Introduction

Hepatitis B virus (HBV), which could be life threatening by causing liver infection, is a challenging public-health problem worldwide. Diagnostics 2020, 10, 491; doi:10.3390/diagnostics10070491 www.mdpi.com/journal/diagnostics (ETV), Telbivudine (LdT), and Tenofovir disoproxil fumarate (TDF), have been recommended by international guidelines for suppressing HBV replication in patients with Chronic Hepatitis B (CHB). In terms of resistance development, the NA can be classified as drugs with low (LMV, ADF, and LdT) and high genetic barriers (ETV and TDF) [13,14]. A number of studies have shown that natural HBV RT mt exists even in treatment-naive adult patients with CHB [15,16,17,18]; to the best of our knowledge, detection of NA-drug-resistance mt in treatment-naive HBV-infected children has not yet been or is rarely reported

Objectives

Methods

Results

Conclusion