Rapid Diagnostic Test for Hepatitis B Virus Viral Load Based on Recombinase Polymerase Amplification Combined with a Lateral Flow Read-Out

Abstract

Hepatitis B (HBV) infection is a major public health concern. Perinatal transmission of HBV from mother to child represents the main mode of transmission. Despite the existence of effective immunoprophylaxis, the preventive strategy is inefficient in neonates born to mothers with HBV viral loads above 2 × 105 IU/mL. To prevent mother-to-child transmission, it is important to identify highly viremic pregnant women and initiate antiviral therapy to decrease their viral load. We developed a simple innovative molecular approach avoiding the use of automatic devices to screen highly viremic pregnant women. This method includes rapid DNA extraction coupled with an isothermal recombinase polymerase amplification (RPA) combined with direct visual detection on a lateral flow assay (LFA). We applied our RPA-LFA approach to HBV DNA-positive plasma samples with various loads and genotypes. We designed a triage test by adapting the analytical sensitivity to the recommended therapeutic decision threshold of 2 × 105 IU/mL. The sensitivity and specificity were 98.6% (95% CI: 92.7−99.9%) and 88.2% (95% CI: 73.4−95.3%), respectively. This assay performed excellently, with an area under the ROC curve value of 0.99 (95% CI: 0.99−1.00, p < 0.001). This simple method will open new perspectives in the development of point-of-care testing to prevent HBV perinatal transmission.

Keywords: Chelex extraction; hepatitis B virus; immunochromatographic strip; lateral flow; mother to child transmission; recombinase polymerase amplification.

Figure 1

Real-time HBV RPA Exo test for primer screening. Extraction of the HBV standard sample with a viral load of 1 × 10⁶ IU/mL was carried out with 5% Chelex 100. Green line: HBV-Fc/P1R-HBV primer combination; blue: P1F-HBV/P1R_HBV; grey: HBV-Fc/HBV-R1; orange: P1F-HBV/R1-HBV; purple: non-template control; yellow: dH₂O. Rn corresponds to the ratio of FAM (6-carboxyfluorescein) to ROX (carboxyrhodamine) fluorescence.

Figure 2

Detection limit of the HBV RPA Exo test. Amplification curves for five-fold serial dilutions ranging from viral loads of 1.46 × 10⁶ IU/mL to 4.6 × 10² IU/mL. Each point of the curve represents the mean of two replicates. Rn corresponds to the ratio of FAM to ROX fluorescence. NTC refers to non-template control.

Figure 3

Detection limit of the HBV RPA-LFA (50 µL). To determine the detection limit of the RPA-LFA (lateral flow assay) in the format of extraction using 50 µL 5% Chelex 100, five-fold serial dilutions of the standard control ranging from viral loads of 1.46 × 10⁶ IU to 4.6 × 10² IU/mL were tested. Each dilution was tested in duplicate. NTC refers to non-template control.

Figure 4

Detection limit of the HBV RPA-LFA (500 µL). To determine the detection limit of RPA-LFA in the format of extraction using 500 µL 5% Chelex 100, five-fold serial dilutions of the HBV standard control ranging from viral loads of 1 × 10⁶ IU/mL to 4 × 10⁴ IU/mL were tested. Each dilution was tested in duplicate. NTC refers to non-template control.

Figure 5

ROC (receiver operating characteristic) curve of the HBV RPA-LFA test. (A): 89 HBV-positive plasma samples alongside 19 HBV-negative plasma samples extracted with 50 µL 5% Chelex 100 solution, (B): 89 HBV-positive plasma samples alongside 19 HBV-negative plasma samples extracted with 500 µL 5% Chelex 100 solution.