Pilot Evaluation of Simplified HBV DNA Extraction-LAMP Workflows for Hepatitis B Molecular Testing in...

Background/Objectives: Access to hepatitis B virus (HBV) DNA testing remains limited in resource-limited settings. The main reasons for this are the high cost of molecular assays and the need for specialized infrastructure. This pilot study aimed to preliminarily develop and evaluate simple HBV DNA extraction methods combined with the colorimetric loop-mediated isothermal amplification (LAMP) technique. Methods: Three extraction methods were evaluated: (i) boiling-based, (ii) paper-based, and (iii) an adapted magnetic bead-based extraction method. Preliminary analytical performance was assessed using serially diluted HBV DNA-positive pooled plasma. Detection performance was evaluated using 40 clinical plasma specimens, including 35 HBV DNA-positive and 5 HBV DNA-negative samples, with real-time PCR as the reference standard. Results: The preliminary analytical detection level observed under the tested conditions was 10⁵ IU/mL for the LAMP methods using boiling- or paper-based extraction, while it was 10³ IU/mL with the adapted magnetic bead-based extraction-LAMP method. Evaluation using clinical specimens showed sensitivities of 43%, 54%, and 63%, respectively. Detection was dependent on viral load, with the highest detection observed among specimens with HBV DNA concentrations above 10⁵ IU/mL. Conclusions: In this laboratory pilot, the workflows showed viral-load-dependent qualitative detection, with the adapted magnetic bead-based workflow yielding the highest observed detection rate. These findings require confirmation through formal analytical validation and larger, independent clinical studies before any clinical or implementation use can be considered.