The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F

Abstract

Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice.

Keywords: HBV DNA; HBV monitoring; hepatitis B virus; real-time TMA.

FIG 1

Quantification of HBV DNA levels in a commercial panel containing 5 × 10¹ (1.7 log) to 5 × 10⁷ (7.7 log) HBV DNA IU/ml (AcroMetrix HBV panel; Thermo Fisher Scientific, Fremont, CA) with the Aptima HBV Quant assay. The average measured values are shown as a function of the expected values. The dashed line represents the equality line.

FIG 2

Deming correlation and Bland-Altman plot analysis of HBV DNA levels measured by the Aptima HBV Quant assay in 158 clinical specimens (group 2) containing HBV genotypes A (n = 50), B (n = 8), C (n = 8), D (n = 52), E (n = 38), or F (n = 2). (A) Deming regression of HBV DNA levels measured by the Aptima HBV Quant and CAP/CTM HBV 2.0 assays, respectively; (B) Bland-Altman plot analysis of the Aptima HBV Quant assay versus the CAP/CTM HBV 2.0 assay; (C) Deming regression analysis of HBV DNA levels measured by the Aptima HBV Quant assay versus the Abbott RealTime HBV assay; (D) Bland-Altman plot analysis of the Aptima HBV Quant assay versus the Abbott RealTime HBV assay. In the Bland-Altman plots, the dotted and dashed lines represent mean differences ± 1.96 SD, respectively.

FIG 3

Box plot representation of the distribution of the differences between the HBV DNA levels obtained by the Aptima HBV Quant assay and the CAP/CTM HBV 2.0 assay (A) or the Aptima HBV Quant and Abbott RealTime HBV assays (B), according to the HBV genotype. The midline and the lower and upper edges of the boxes represent the median value, 25th percentile, and 75th percentile, respectively. The lower and upper error bars represent the minimum and maximum values, respectively.

FIG 4

Quantification of HBV DNA levels in 23 clinical specimens (group 4) infected with HBV bearing amino acid substitutions in the MHR of the S gene (A) and in 18 clinical specimens (group 5) infected with HBV bearing BCP and/or precore mutations (B). HBV DNA levels were measured with the Aptima HBV Quant assay (red squares), the CAP/CTM HBV 2.0 assay (black triangles), or the Abbott RealTime HBV assay (black circles). The red or black dashed lines correspond to the LODs of the Aptima HBV Quant assay (1.0 log IU/ml) and the Abbott RealTime HBV assay (1.0 log IU/ml) or the CAP/CTM HBV 2.0 assay (1.3 log IU/ml), respectively. B-1, blood donor 1; Pt-1, patient 1; gA, genotype A; gB, genotype B; gC, genotype C; gD, genotype D; gE, genotype E; gG, genotype G; nd, not determined due to PCR amplification failure or lack of hybridization to the specific probes in spite of efficient PCR amplification. Amino acid substitutions in the MHR containing the “a” determinant and BCP (AGG or TGA at positions 1762 to 1764) and precore (G or A at position 1896) mutations are indicated.

FIG 5

Kinetics of HBV DNA levels measured in patients' plasma specimens with the Aptima HBV Quant (red lines and filled circles) and CAP/CTM HBV 2.0 assays (black lines and filled circles) (A) or the Abbott RealTime HBV assay (black lines and filled circles) (B). The red or black dashed lines correspond to the LODs of the Aptima HBV Quant (1.0 log IU/ml) and CAP/CTM HBV 2.0 (1.3 log IU/ml) assays or the Abbott RealTime HBV assay (1.0 log IU/ml), respectively. HBV DNA levels are shown on the y axis. Red or black asterisks represent detectable but not quantifiable HBV DNA in the Aptima HBV Quant and CAP/CTM HBV 2.0 assays or the Abbott RealTime HBV assay, respectively. gA, genotype A; gB, genotype B; gD, genotype D; gE, genotype E; LAM, lamivudine; ETV, entecavir; ADV, adefovir; TDF, tenofovir.