A total of 620 patients consisting of 286 males and 334 females with chronic HBV infection were enrolled in the study in Beijing Ditan Hospital. The ages of these patients ranged from one month to 80 years, with a mean of 33.7±12.2 years. Among the 620 patients, 271 received NA-based therapy, 35 received IFN-based therapy, eight received the combination therapy of NA and IFN, and the other 306 were NA/IFN naïve. Informed consent was obtained from adult patients or the guardians of minor patients at the time of whole blood collection for the purpose of HBV testing. The plasma samples obtained from whole blood were used to determine the HBV DNA levels using real-time quantitative PCR. In addition, serum samples were used to determine the HBV genotypes and drug resistance-related mutations.

HBV DNA was quantified using a Cobas AmpliPrep/Cobas Taqman (CAP/CTM) platform (Roche Molecular Systems, Pleasanton, CA, USA). HBV DNA was extracted from 1-mL plasma using a Cobas AmpliPrep automated extractor according to the manufacturer's instructions. The Cobas Taqman 48 Analyzer was used for automated real-time PCR amplification and the detection of PCR products according to the manufacturer's instructions. HBV DNA levels were expressed as IU/mL.

HBV DNA was extracted from 200-μL serum samples using a Magbind CFDNA kit (KWBiotech, Beijing, China). Multiplex PCR, microarray hybridization, and microarray scanning were conducted as recommended by the manufacturer of the microarray kit (CapitalBio Corporation, Beijing, China) for the simultaneous detection of the HBV genotypes (HBV/B, C, and D) and drug-resistance-related hotspot mutations (rtL180M, rtA181T/V, rtM204I/V, rtN236T in the HBV reverse transcriptase region, and G1896A in the HBV precore region). The recommended genotype and mutation-related allele-specific primers were used for multiplex PCR. After amplification, the reaction mixture was hybridized with probes immobilized on the microarray chip at 50°C for 60min. Subsequently, the microarray chip was washed in washing buffer (0.1% SDS and 0.3× SSC) at 42°C for 2min. The chips were scanned and imaged using a microarray Scanner LuxScan™10K-B (Capitalbio). Assay results were determined based on the fluorescent hybridization signal and distribution of the microarray probes. The microarray probes corresponding to HBV genotypes and drug-resistance-related mutations are shown in Fig. 1A.

Fig. 1.

Microarray-based HBV genotyping and drug resistance-related mutations. (A) Layout of the microarray used in the current study. All probes, with the exception of QC and PC, were spotted in three replicates; QC and PC were spotted in six replicates. QC and BC were the positive and negative controls for array production, respectively. PC and NC were the positive and negative controls for hybridization, and IC1 and IC2 were the internal controls for HBV amplification, respectively. B, C, and D represent HBV genotypes B, C, and D, respectively. 180M, 181T/V, 204I/V, and 236T represent the rtL180M, rtA181T/V, rtM204I/V, and rtN236T, respectively, mutations in the HBV reverse transcriptase region. 1896A denotes the G1896A mutation in the HBV precore region. (B) Hybridization results of the classical HBV samples with genotypes B and C. (C) Hybridization results of eight classical NA-resistant samples.

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The results of HBV DNA genotyping and drug-resistance-related mutations obtained from the microarrays for a random selection of samples were further confirmed using direct DNA sequencing with the BigDye Terminator v3.1 Cycle Sequencing kit and an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA). The primer sequences and PCR amplification conditions used for direct DNA sequencing have been described elsewhere.14–16

Statistical analysis was performed using the SPSS 17.0 software package. All values in the text and figures are expressed as the mean±standard deviation (SD) of these observations.

The HBV DNA levels in the 620 enrolled samples ranged from 1.00E5 to 1.50E9IU/mL, with a mean of 9.89E6IU/mL (see Supplementary Table S1), as determined by real-time quantitative PCR. The results also confirmed that all studied clinical samples were HBV-positive.

Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bjid.2015.03.012.

Sample information and microarray-based assay results.

Two HBV genotypes (B and C) were detected among the 620 HBV DNA positive samples using the Capitalbio microarray assay, as shown in Supplementary Table S1. The microarray hybridization results for the HBV genotypes B and C are shown in Fig. 1B. Of these, 44 samples with genotype B and 151 samples with genotype C were selected randomly and the results were validated using direct DNA sequencing. As shown in Table 1, most samples were identified as genotype C, 471/620 (76.0%), followed by genotype B, 149/620 (24.0%).

By microarray assay, NA resistance-related mutational patterns of the 620 patient samples were also directly acquired from hybridization results, as shown in Supplementary Table S1. The mutation patterns of 211 patient samples were then selected and further confirmed using direct DNA sequencing. Among the 620 patient samples, 17 (17/620; 2.7%) carried NA resistance-related mutations, as showed in Table 2 and Fig. 1C. The mean age of these 17 patients was 47.7±12.5 years, which was higher than that of the 620 patients (33.7±12.2). Most of the 17 patient samples belonged to genotype C (14/17, 82.4%), and the remaining to genotype B (3/17, 17.6%). Among the 17 patients, nine carried LAM-/LdT-resistance mutations (rtL180M, rtM204I/V), and eight had ADV-resistance mutations (rtA181T/V, rtN236T). The most common mutations in the LAM-resistant patients were rtM204I and rtM204V, which were found in 35.3% (6/17) of the 17 NA-resistant patients. The compensatory mutation rtL180M was simultaneously detected with rtM204I/V in 17.6% (3/17). The ADV-resistance mutations rtA181V and rtN236T were found in 29.4% (5/17) of these patients. No patient samples with LAM and ADV or ETV-resistance mutations were detected. Among the 17 patients, 4 carried NA-resistance mutations but were NA/IFN-naïve. Ten carried NA-resistance mutations and the NA/IFN-based therapeutic strategy adopted previously was appropriate and effective. The remaining three patients also carried NA-resistance mutations but the previous NA/IFN therapy was not effective and led to treatment failure and was altered. More specifically, patients P119 and P287 both carried rtA181V but received ADV/IFN, P217 carried rtM204I but received LdT. In addition, the microarrays revealed that 397 (64.0%) of the 620 patient samples also carried the precore stop-codon mutation (G1896A), and this was confirmed in 197 samples using direct DNA sequencing.