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TOWARDS QUANTITATIVE MOLECULAR ISOTHERMAL AMPLIFICATION FOR POINT-OF-CARE HIV VIRAL LOAD MONITORING

thesis
posted on 2024-04-22, 18:15 authored by Emeka NwanochieEmeka Nwanochie

Since the beginning of the HIV/AIDS epidemic, 85.6 million people worldwide have become infected with HIV; more than half of whom have died from AIDS-related complications.[1] Sustained viral suppression below the clinically relevant threshold (1000 copies per mL) with highly active antiretroviral therapy (HAART) has proven effective at managing and prolonging the life expectancy of people living with HIV (PLHIV). However, in 2022, 11.3 million PLHIV had still not achieved viral suppression and may become susceptible to both HIV transmission and a variety of opportunistic infections. Of particular importance is the complex issue of patient non-compliance in global HIV management due to social, economic, behavioral, and healthcare access barriers, potentially disconnecting many PLHIV from the HIV care continuum. Therefore, to boost patient engagement in clinical care and to improve overall patient outcomes, new approaches to viral load monitoring practices need to be developed to increase access, particularly in regions of high HIV prevalence.

Nucleic acid amplification tests (NAATs) have emerged as potent tools for monitoring viral load, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) being recognized as the benchmark due to its sensitivity and ability for real-time quantification enabled by fluorescence signal emission. Nevertheless, RT-qPCR is burdened by drawbacks including extended processing times, high operational costs, and the requirement for specialized laboratory facilities. In this study, we propose a novel method for HIV-1 viral load monitoring by integrating reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) with real-time particle diffusometry (PD). This approach allows for the continuous monitoring of changes in the diffusion of 400 nm fluorescent particles during RT-LAMP amplification, targeting the p24 gene region of HIV-1 RNA. This enables the real-time detection of amplification curves, achieving a detection sensitivity in water samples as low as 25 virus particles per μL within a short duration of 30 minutes. Additionally, to address challenges related to amplification inhibition in complex human specimens, we developed a power-free sample processing system specifically designed for extracting HIV-1 RNA from both whole blood and plasma.Top of FormBottom of FormThis system modifies a commercially available spin-column protocol by integrating a syringe device and handheld bulb dryer, thus eliminating the requirement for a centrifuge. The adaptation allows for the completion of the entire extraction procedure, encompassing viral lysis, RNA capture, washing, and elution of purified HIV-1 RNA, within a timeframe of less than 16 minutes. Subsequent analyses, including RT-LAMP and RT-qPCR, demonstrate a limit of detection of 100 copies per μL and an average RNA recovery of 32% (for blood) and 70% (for plasma) in the elution fraction. Further investigations emphasize the significant presence of purified RNA in the spin column volume (termed as dead volume), and the cumulative recovered RNA copies align with those obtained using the gold standard centrifugation extraction method. Ultimately, we incorporated the real-time quantitative PD-RT-LAMP assay onto a field-compatible handheld portable platform suitable for field use, featuring built-in quality control measures. This platform enables sample-to-answer viral load testing near the point of care (POC). Subsequently, we undertook essential preparatory steps, such as reagent drying to obviate the need for cold storage, initial device calibration, and hands-on training of laboratory personnel regarding device operation, to validate device performance within a cohort of individuals living with HIV (PLHIV). These innovations facilitate quick and comprehensive viral load determination, offering promise for enhanced HIV management and patient care

Funding

NIH NIAID R33AI140474

NIH NIAID DP2DA051910

History

Degree Type

  • Doctor of Philosophy

Department

  • Biomedical Engineering

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Jacqueline C. Linnes

Advisor/Supervisor/Committee co-chair

Tamara L. Kinzer-Ursem

Additional Committee Member 2

J. Paul Robinson

Additional Committee Member 3

Aman Russom