Development of a point-of-care test to detect hepatitis B virus DNA threshold relevant for treatment indication

Abstract

Background Hepatitis B virus (HBV) has been the most prevalent blood-borne pathogen wherein utero transmission has still not been properly managed. Recent practice guidelines suggested that an antiviral drug should be administered to third-trimester pregnancies with significant viremia (>2 × 105 IU/mL). Objectives To develop a novel turbidity-based loop-mediated isothermal amplification (LAMP) coupled with heat treatment DNA extraction method that is a rapid, cost-effective, and feasible viral load assessment and could be applied to antenatal screening. Methods Primers and reagents were designed, turbidity-based platform and heat treatment method were added, and evaluated for optimal efficiency. Assay sensitivity was tested from serially diluted standard HBV DNA. Assay specificity was tested with six standard viral DNAs. Clinical samples were analyzed and the results were compared with those of quantitative polymerase chain reaction (qPCR) diagnostic records. Results The optimized condition was 60°C with no betaine, 1.4 mM deoxyribonucleotide triphosphates (dNTPs) and 6 mM of MgSO4 for 60 min. The assay accurately detected samples with standard HBV DNA at >2 × 105 IU/mL in both distilled water and spiked serum. Results can be interpreted within 31.48 ± 1.41 min in real-time turbidimeter. The amplification is exclusively specific to HBV, but not with the other six human-specific viruses. Moreover, the assay showed comparable performance within 95% confidence interval to the previously developed HBV LAMP toward clinical specimens. Conclusions This newly developed method was accurate, affordable, and flexible to further implementation to large-scale third-trimester pregnancy screening.

Loop-Mediated Isothermal Amplification assays for on-site detection of the main sweetpotato infecting viruses

“… Esmatabadi et al (2015) listed several platforms that could be incorporated into LAMP detection system: Lateral flow dipstick, enzyme-linked immunosorbent assay (ELISA), and microfluidic chip using antibody-labeled streptavidin–biotin, fluorescent-labeled probes, giant magnetoresistive (GMR) sensors, probe-functionalized nanoparticles, magnetic nanoclusters (MNCs), and line probe assay (LiPA). Some of these platforms have the potential to increase the sensitivity and reduce the cost per reaction ( Pankaew et al, 2019 ). Furthermore, we recommend the pooling of samples and multiplexing LAMP primers when screening to reduce the cost of LAMP.…”

“…LAMP is versatile and has the potential to be deployed on-site. Its products can be detected by a number of methods: colorimetric - visualized by color indicators (SYBR Green and hydroxyl naphthol blue – HNB) ( Wastling et al, 2010 ); turbidity of magnesium pyrophosphate formed during the reaction (precipitate) ( Wastling et al, 2010 ); display of ladder-like patterns on an agarose gel electrophoresis ( Goto et al, 2009 ; Pankaew et al, 2019 ) and recently in real-time, based on intercalating fluorescent dyes ( Armson et al, 2019 ).…”

Point-of-care test to detect hepatitis B virus DNA: Is it useful?

“…The paper by Pankaew et al in this volume reported a point-of-care test to detect hepatitis B viral DNA threshold relevant for treatment indications [10]. This can be an important tool for screening pregnant mothers to identify those with significant hepatitis B viral DNA and thus can link the infected individuals to facilities that can provide further desirable optimal care for mothers and infants and thus help curb chronic hepatitis B infections in the population.…”