Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis

Abstract

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection.We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/l. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Hepatitis B is caused by the hepatitis B virus (HBV), an enveloped DNA virus that infects the liver, causing hepatocellular necrosis and inflammation (1). Chronic hepatitis B (CHB) infection affects approximately 248 million people worldwide and is a leading cause of liver-related morbidity and mortality, particularly in low-and middle-income countries (LMICs) (2). Patients with CHB can be successfully treated using nucleos(t)ide analogs (NAs), but drug-resistant HBV mutants frequently arise, leading to treatment failure and progression to liver disease (3). The development of drug resistance begins with mutations in the HBV polymerase gene, followed by an increase in the viral load and serum alanine aminotransferase levels several weeks to months later (4). Detection of drug resistance mutations is thus critical in prompt decision making for new therapeutic regimes (5, 6). A method enabling detection of NA resistance mutations should be reliable, rapid, and, in particular, easy to use and cost-effective when the aim is for it to be used in high-CHB-burden countries, which are often undeveloped a...

Hepatitis B Virus: From Diagnosis to Treatment

“…Real-time PCR reduces the risk of contamination due to its applicability and speed. Therefore, it is widely used to detect drug resistance mutations ( Mou et al 2016 ).…”

A novel multiplex PCR for virus detection by melting curve analysis

“…Recent studies have shown that the TaqMan probes can be used not only for monitoring PCR in real time but also for melting curve analysis under asymmetric PCR conditions (Huang et al, 2011). Multicolor melting curve analysis using the TaqMan probe has been successfully applied in mutant gene detection and genotyping (Botezatu et al, 2017;Mou et al, 2016;Wang et al, 2017;Xia et al, 2016;Huang et al, 2016;Gao et al, 2015;Huang et al, 2017). Nevertheless, the application to multiple virus detection has not yet been reported.…”