Simple and Low-Cost Loop-Mediated Isothermal Amplification (LAMP) Assay to Diagnose High HBV DNA Levels in Resource-Limited Settings

Background: A massive scale-up of testing and treatment is indicated to globally eliminate hepatitis B virus (HBV) infection. However, access to a polymerase chain reaction (PCR), a key test to quantify HBV DNA levels and determine treatment eligibility, is limited in resource-poor countries. We developed and evaluated loop-mediated isothermal amplification (LAMP) assay to diagnose clinically important HBV DNA thresholds defined by WHO (≥20,000 and ≥200,000 IU/mL) in both resource-rich (Europe) and resource-limited (Africa) contexts. Methods: Pan-genotypic primer sets were designed on conserved HBV gene regions. Accuracy of LAMP to identify highly viraemic patients was evaluated using 400 and 550 HBV-infected samples from France and Senegal, respectively. Findings: Our primers successfully detected eight major HBV genotypes/sub-genotypes (A1/2/3/B/C/D/E/F) with a detection limit ranging between 40-400 IU/mL. In France, the area under the receiver operating characteristic curve (AUROC), sensitivity and specificity of bead-based extraction and real-time turbidimetric LAMP were 0.95 (95% CI: 0.93-0.97), 91.1% and 86.0%, respectively, to diagnose HBV DNA ≥20,000 IU/mL; and 0.98 (0.97-0.99), 98.0% and 94.6% for ≥200,000 IU/mL. The performance did not vary by viral genotypes. In Senegal, using a field-adapted method (reagent-free boil-and-spin extraction and inexpensive end-point fluorescence detection), the AUROC, sensitivity and specificity were 0.95 (0.93-0.97), 98.7% and 91.5%, respectively, to diagnose HBV DNA ≥200,000 IU/mL. Interpretation: We developed a simple, rapid (60 minutes), and inexpensive (US$ 8/assay) alternative to PCR to diagnose high viremia ≥200,000 IU/mL. HBV-LAMP may contribute to eliminating HBV mother-to-child transmission by identifying high-risk pregnant women eligible for antiviral prophylaxis in resource-limited countries. Funding Statement: ValoExpress [ANR11-CARN017-01] and Grand Challenges Africa [GCA/ISG/Round1/045]. Declaration of Interests: MS is employee of Eiken Chemical Co.,Ltd. All the other authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the Clinical Research Committee at the Institut Pasteur (n° 2016-057) and the National Ethics Committee in Senegal (SEN17/59) .All donors are informed of a potential use of their samples for research and have given their written consent.