A New Method for Next-Generation Sequencing of the Full Hepatitis B Virus Genome from A Clinical Specimen: Impact for Virus Genotyping

Hepatitis B virus (HBV) is an enveloped virus that induces chronic liver disease. HBV has been classified into eight genotypes (A–H) according to its genome sequence by using Sanger sequencing or reverse hybridization. Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants, whereas reverse hybridization detects only known mutations. Next-generation sequencing (NGS) is a robust tool for clinical virology with different protocols available. The objective of this study was to develop a new method for the study of viral genetic polymorphisms or more accurate genotyping using genome amplification followed by NGS. Plasma obtained from five chronically infected HBV individuals was used for viral DNA isolation. HBV full-genome PCR amplification was the enrichment method for NGS. Primers were used to amplify all HBV genotypes in three overlapping amplicons, following a tagmentation step and Illumina NGS. For phylogenetic analysis, sequences were extracted from the HBVdb database. We were able to amplify a full HBV genome; further, NGS was shown to be a robust method and allowed better genotyping, mainly in patients carrying mixed genotypes, classified according to other techniques. This new method may be significant for whole genome analyses, including other viruses.

Chẩn đoán trước sinh bệnh thalassemia bằng kỹ thuật lai phân tử ngược trên màng lai

α- và β- thalassemia là các rối loạn di truyền đơn gen phổ biến nhất trên toàn thế giới cũng như tại Việt Nam. Nghiên cứu được tiến hành trên 88 trường hợp thai của các cặp vợ chồng có nguy cơ cao đẻ con mắc Thalassemia. Các thai phụ được chọc ối và xét nghiệm phân tử bệnh thalasemia nhằm phát hiện các dạng đột biến gen thalassemia ở tế bào ối bằng kỹ thuật lai phân tử ngược (Reverse hybridization) tại Trung tâm Chẩn đoán trước sinh, Bệnh viện Phụ Sản Trung ương năm 2017. Kết quả cho thấy 64/88 trường hợp mang gen đột biến chiếm 72,7%. Trong đó, có 51/64 thai bị đột biến α – thalassemia (79,7%), 8/64 thai bị đột biến β – thalassemia (12,5%) và 5/64 mang đồng thời đột biến α (--SEA)- và β – thalassemia (dị hợp tử CD41/42 hoặc dị hợp tử CD 17) hoặc CD 26 (7,8%). Đột biến (--SEA) trên gen α globulin chiếm tỉ lệ 100% thai bị đột biến α – thalassemia. Trong đó, 47% mang kiểu gen của bệnh α – thalassemia ở dạng đồng hợp tử. Chẩn đoán trước sinh bằng kỹ thuật lai phân tử ngược giúp đưa ra các quyết định về thai nhi cũng như tư vấn di truyền trước hôn nhân đối với các trường hợp phát hiện dị hợp tử bệnh thalasemia.

Comparison of a Prototype Reverse Hybridization Assay and MethyLight for Detection of SFRP2 Promotor Methylation in Fecal DNA

Background This study aimed to evaluate the diagnostic performance of a novel nonquantitative methylation-specific reverse hybridization (MSRH) assay to detect secreted frizzled-related protein 2 ( SFRP2) promotor methylation in fecal DNA. Methods SFRP2 promoter methylation was investigated in stool DNA isolated from 18 colorectal cancer (CRC) patients and 22 healthy controls using the MSRH assay based on methylation-specific DNA amplification followed by reverse hybridization of biotinylated amplicons to sequence-specific methylation detection probes, with MethyLight serving as a reference method. Results SFRP2 promotor methylation as determined by MSRH vs. MethyLight showed a sensitivity and specificity of 61.1% and 86.3% vs. 77.7% and 77.3%, respectively. Moderate agreement (κ = 0.54, 95% confidence interval [95% CI], 0.29-0.80, p<0.001) was observed between the 2 methods. However, the differences in SFRP2 promotor methylation observed between CRC patients and healthy individuals by both assays were statistically significant (p<0.001). Conclusions Our findings, although limited by the small sample size, do not support the use of the MSRH assay for CRC screening in stool.

HCV non-1b genotypes in injecting drug users from Romania

Introduction: Chronic hepatitis C cases diagnosed in Romania were mostly related to unsafe parenteral treatments and blood transfusions; HCV genotype 1b was prevalent. During the last decade, an increasing number of HCV infections was reported among people who inject drugs (PWID). The aim of the current study was to test if this epidemiological shift triggered a diversification of the circulating viral strains. Methodology: HCV genotypes were determined by reverse hybridization in 130 HCV-infected PWID (87.7% males; mean age 27.9 ± 6.7 years, injecting drugs for 8.1 ± 4.8 years). Results: HIV-HCV co-infection was diagnosed in 80.8% of the subjects and 26.9% were HIV-HCV-HBV triple infected. Active HCV viral replication was present in 104 PWID (80%), more frequently in those HIV-co-infected (91.4% vs. 52% in HCV mono-infected, and 77.148.5% in HIV-HCV-HBV triple-infected, p = 0.0001). Non-1b genotypes were prevalent (54.8%), with subtype 1a the most commonly detected (24%), followed by genotypes 3a (14.4%) and 4 (7.7%). Mixed infections with genotypes 1a and 1b were found in nine subjects (8.7%). There was no difference in the genotypes frequencies based on HIV or HBV co-infection status, length of drug usage, or associated risk factors (tattoos, piercing, detention). Conclusion: The continuous surveillance of HCV genotypes in PWID from Romania will add valuable information to the overall European epidemiological picture, with important therapeutic implications.