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
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
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