Development of A Molecular Point-of-Care Test for Hepatitis B Virus (HBV) Infection

Abstract

Hepatitis B (HBV) is the predominant blood-borne pathogen globally, infecting around 250 million people. It can cause a chronic infection leading to life-threatening cirrhosis and hepatocellular carcinoma (HCC). Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic tool with potential for detecting infectious microorganisms. The aim of this research project was to develop a molecular point-of-care test using LAMP technology for the rapid detection of HBV DNA that could be deployed in resource-limited regions.

In silico bioinformatics approaches were used to define the most suitable target gene in the HBV genome. Three sets of degenerate HBV primers HBV-SP-(1), (2), and (3) were designed to align to the target gene in all HBV genotypes specifically and were then screened against HBV-positive DNA samples. An inclusivity experiment was carried out to ensure that the newly designed primer sets were able to cover all HBV genotypes. Optimisation of LAMP assay conditions such as MgSO4 concentration, and primer ratios allowed the determination of optimal conditions to be 2.00 mM MgSO4 and 10:2:10 (Loop: Displacement: Internal) primer ratios for HBV-SP-(2) and 4.00 mM MgSO4 for HBV-SP-(3). The optimum LAMP reaction temperature was found to be 65C as lower temperature reactions were prone to non-specific amplification.

Sample preparation optimisation of plasma specimens was carried out as the initial phase to integrate the assay into point-of-care cartridges. Strategies such as dilution of the sample, heat treatment, and detergent treatment (digitonin, SDS, tween-20) were tested and found that 20-fold dilution combined with heating at 95C for 5 minutes was the most optimum condition, which resulted in the fastest Tp (time of positivity). Whole blood was selected as the preferred sample type for the detection of this bloodborne infection in point-of-care testing settings. This is because blood is easy to obtain, especially as a finger-prick sample, and overcomes the complex sample preparation required for obtaining plasma samples. Using whole blood as the sample type would also reduce the time to result. Therefore, the possibility of using whole blood as the sample of choice was examined. Like plasma, the optimal sample preparation method was found to be 20-fold dilution and heat treatment at 95C for 5 minutes.

Lyophilisation techniques were assessed to increase the stability of the assay reagents and prolong the shelf-life of the final test. This was successfully achieved with the adoption of a lyophilised cake format. With the test formulated into this platform, some initial analytical testing was able to be conducted. This included the initial LoD testing, which detected 15,138.6 IU/mL in a 30-minute assay and 1,513.9 IU/mL in a 60-minute assay. This is sufficient to detect chronic active HBV carriers.

The assay was then formulated into a point-of-care test platform that has been developed by Australian manufacturing company, ZiP Diagnostics Pty Ltd. The ZiP-P2 test platform comprises a dual-tube test cartridge which integrates with a dedicated instrument that also handles the workflow required for this test. The test manages all other data management requirements and is suitable for deployment in community settings, like general practice clinics and low-middle income settings. The ZiP-P2 instrument is approved for use in Europe and is currently under evaluation by the TGA for use in Australia. It offers a genuine option for deployment of this HBV test in the future.

In conclusion, the project successfully developed a fast, reliable, sensitive and highly specific molecular method to identify HBV infection without DNA extraction in clinical samples. This test is suitable for HBV testing in resource-limited regions.