Direct Detection of Drug-Resistant Hepatitis B Virus in Serum Using a Dendron-Modified Microarray

BACKGROUND/AIMS: Direct sequencing is the gold standard for the detection of drug-resistance mutations in hepatitis B virus (HBV); however, this procedure is time-consuming, labor-intensive, and difficult to adapt to high-throughput screening. In this study, we aimed to develop a dendron-modified DNA microarray for the detection of genotypic resistance mutations and evaluate its efficiency. METHODS: The specificity, sensitivity, and selectivity of dendron-modified slides for the detection of representative drug-resistance mutations were evaluated and compared to those of conventional slides. The diagnostic accuracy was validated using sera obtained from 13 patients who developed viral breakthrough during lamivudine, adefovir, or entecavir therapy and compared with the accuracy of restriction fragment mass polymorphism and direct sequencing data. RESULTS: The dendron-modified slides significantly outperformed the conventional microarray slides and were able to detect HBV DNA at a very low level (1 copy/μL). Notably, HBV mutants could be detected in the chronic hepatitis B patient sera without virus purification. The validation of our data revealed that this technique is fully compatible with sequencing data of drug-resistant HBV. CONCLUSIONS: We developed a novel diagnostic technique for the simultaneous detection of several drug-resistance mutations using a dendron-modified DNA microarray. This technique can be directly applied to sera from chronic hepatitis B patients who show resistance to several nucleos(t)ide analogues.

Associations of HLA Alleles with Chronic Infection and Prophylaxis in Vertical Transmission of Hepatitis B Virus

Perinatal transmission and infection of hepatitis B virus (HBV) in early childhood were observed in the offsprings of hepatitis B surface antigen (HBsAg)-positive mothers who had been vaccinated against HBV immediately after giving birth. This prophylaxis failure of perinatal HBV infection is likely due to the interplay of the virus and host immune response. To investigate whether the HLA polymorphism affected the outcome of the perinatal prophylaxis, HLA class I (HLA-A, B and Cw) and class II (HLA-DRB1, DQA1, DQB1 and DPB1) were typed using serology, PCR-SSOP (polymerase chain reaction-sequence specific oligonucleotide probe), and PCR-ARMS (amplification refractory modification system) methods in 22 HBeAg-positive mothers and their 10 prophylaxis-succeeded and 12 prophylaxis- failed children. The HLA types of the mothers and their children were compared with 198 HBsAg-negative healthy controls in a Korean population. HLA-B35 (relative risk=4.2, p<0.01), B51 (relative risk=3.2, p<0.02), DRB1*07 (relative risk=3.8, p<0.03), and DQA1*02 (relative risk=3.8, p<0.03) alleles were more frequent in HBeAg-positive mothers than in the controls. Also, HLA-DRB1*13 (relative risk=0.1, p<0.02) and DPB1*0401 (relative risk=0.1, p<0.02) alleles were less frequent in HBeAg-positive mothers. However, HLA alleles did not affect the outcome of the perinatal prophylaxis against HBV. These results suggest that the reported influences of some HLA alleles on the natural chronic HBV infections may not operate in the HBV infections in children received perinatal prophylaxis.

Distribution of HLA-DQA1*01, *03, *05 and DQB1*02 Subtypes and the Associated Haplotypes in the Korean Population

BACKGROUND: As all HLA class II genes, the DQ genes show their polymorphic variation mainly in the second exon, which encodes the first extracellular domain of the molecule. PCR-SSOP (Polymerase chain reaction-Sequence specific oligonucleotide probe) techniques were frequently used for HLA-DQA1 and DQB1 typing but certain alleles, DQA1*0101/0104/0105, *0302/0303, *0501/0505 and DQB1*0201/*0202, which differ from each other in segment other than exon 2, could not be unequivocally assigned. METHODS: To overcome this problem, we applied additional PCR-SSP (PCR-Sequence specific primer) method to analyze DQA1 exons 1, 3 and 4 and DQB1 exon 3. And we investigated the distributions and haplotypes of HLA-DRB1, DQA1 and DQB1 alleles in 406 unrelated Korean healthy individuals. RESULTS: Using this method the indistinguishable alleles of DQA1 and DQB1 in PCR-SSOP were typed definitively. We also found several important associations between DQA1 and DQB1 alleles in the Korean population; DQA1*0101-DQB1*0501, DQA1*0104-DQB1*0502 or -*0503, DQA1 *0105-DQB1*0501, DQA1*0302-DQB1*0303, DQA1*0303-DQB1*0401 or -*0402, DQA1 *0501-DQB1*0201, DQA1*0505-DQB1*0301, and DQA1*0201-DQB1*0202. The haplotypes of DRB1-DQA1-DQB1 associated with DQA1*01, *03, *05, and DQB1*02 subtypes were investigated. Several haplotypes associated with these alleles were observed in the Korean population. CONCLUSION: Our results can be helpful to find potential unrelated donors for bone marrow registries and study the HLA-associated disease and anthropology at high-resolution allelic level.