Integration of a Reverse Transcription Loop Isothermal Amplification of HCV RNA and a Lateral Flow Test for true point-of-care detection of Hepatitis C

Introduction: According to the World Health Organization (WHO), Hepatitis C Virus (HCV) has become a major public health problem with 350 million infections worldwide and 58 million of them being chronic. In 2019, 290,000 patients died from the progression of an HCV infection to liver cancer and cirrhosis that could’ve been avoided with proper treatment. Although the newer treatment options have demonstrated a cure rate of over 95% of infections, the big challenge relies on making timely diagnosis to prevent transmission and integrate infected people into the care continuum. This bloodborne pathogen’s most predominant mode of transmission is through the sharing of drug-injection equipment, thus predisposing people who use drugs (PWUD) to a high-risk category. Current testing paradigms involve a two-tiered testing protocol that makes it challenging for PWUD and even patients at clinics in low- and middle-income countries (LMICs), to have complete HCV testing and integration to care. The HCV prevalence in the U.S. on average is only 1% but is 60% among PWUD. This disparity can be attributed to the lack of HCV testing and linkage to care for this marginalized population. Alternative screening technologies lack the necessary characteristics to test these populations at the true point- of- care (POC) and give a same visit diagnosis. Therefore, rapid and more effective POC approaches are urgently needed for current HCV infection (HCV viremia) detection and treatment in order to close this gap.

Materials and

Methods: We developed a feasibility and acceptability study to learn and translate user requirements into new design specifications. This consists of phone interviews to our stakeholders: the providers, harm reduction organizations, and HCV testers; in addition to the PWUD community, as they’re the ones getting tested. In order to ensure the device’s widespread adoption and usage we’re conducting a usability study. Providers and HCV testers will be assigned into focus groups where they will answer survey questions, run a mock test, and provide feedback. Video recordings of the participants interacting with the platform and audio recordings will be collected. The responses from the interviews will be audio-recorded and transcribed with Otter.ai, the interviews will be coded in NVivo to identify themes and patterns for the data analysis. Additionally, we’ll analyze the quantitative System Usability Scale questionnaire to look at the score of the device and learn about the potential areas of opportunity. Using a Human Centered Design with the data collected from the stakeholders we will adjust the test to ensure we tackle current barriers. Currently we’re developing a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP), and visualizing the amplification products in a Milenia Biotec Lateral Flow Test (LFT) from plasma samples. This technology will later be integrated to the MicroRAAD, which consists of the reagents needed for the RT-LAMP reaction, a circuit that provides low power heating for the reaction to take place, and thermally controlled valves to allow fluid flow into the LFT.

Results, Conclusions, and Discussions: Our collaborator, Dr. Eddy Odari, who was the person that started this project, decided to focus the primer design across the highly conserved region 5’NTR whose length is 340 nucleotides. That initial design was tested with different HCV genotypes and concentrations. From the experiments run we concluded that genotype 2 was not being properly targeted by the primers as it had very late amplification and only with high viral load. On the other hand, when there is a low viral load of genotype 3 amplification takes on average > 60 minutes, making the original primer design not completely optimal for our needs. However, we can see the correlation that across all the genotypes the lower the viral load the higher the amplification time (Fig. 1). Because of this, Dr. Odari did an in-silico and redesigned the Loop Forward (LF) primer. With the newly designed LF primer we ran RT- LAMP again on high (E4) concentrations to validate the new design. To validate the results, we performed gel electrophoresis. We did the experiments with an n=3 and none of the genotypes amplified. Unfortunately, that data got corrupted thus we ran another experiment, setting us at n=4. When this last set was performed, we saw amplification which we hypothesized to be primer dimerization, but when we imaged the gel, we saw actual amplification (Fig. 2). By performing these experiments, we can conclude that the new primer design is not optimal and very unreliable. Currently in the process of performing further in-silico analysis and redesigning the primers to ensure a specific and sensitive amplification of the HCV genotypes across a range of concentrations within the time requested by the stakeholders.

Acknowledgements (Optional): Dr. Lee | Post-doc at Purdue University
Logan Lee | Health Educator, IU Health Positive Link