Abstract
Viral load measurements are an essential tool for the long-term clinical care of hum an immunodeficiency virus (HIV)-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification (RT-LAMP) on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a ~ 60 nL RT-LAMP droplet, corresponding to a whole blood concentration of 670 viruses per µL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation.
Highlights
Human immunodeficiency virus (HIV) affects 36.9 million people worldwide [1]
Viral load instruments are traditionally reverse-transcription polymerase chain reaction (RT-PCR), nucleic acid sequence-based amplification (NASBA), or branched DNA assays; these can be capable of detecting fewer than ten viral RNA copies per mL of blood plasma, these instruments require a laboratory setting, extensive sample handling, and sophisticated processing [6, 8,9,10]
reverse-transcription loopmediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) fluorescence curves for the first method are shown in Comparison with lysed whole blood—The experiment compared the threshold time and fluorescence intensity of RT-LAMP containing 9380 whole virus particles of HIV-1 IIIB in lysed blood to the corresponding amount of purified HIV-1 RNA in water as an initial test of the feasibility of RT-LAMP in lysed whole blood
Summary
Human immunodeficiency virus (HIV) affects 36.9 million people worldwide [1]. During the course of nearly four decades since the emergence of HIV on a pandemic scale, advances in antiretroviral therapy have transformed HIV infection from a death sentence into a chronic illness that can have little impact on life expectancy for those in whom the infection is properly managed [2]. The lack of availability of the appropriate diagnostic technologies essential to informing treatment in routine HIV care is still among the chief barriers preventing access to the standard of care for millions of HIV-positive individuals worldwide, in resource-limited settings. CD4+ T lymphocyte counts and blood plasma viral load are the two core diagnostic measurements broadly considered essential to HIV care, as they both guide the initiation of therapy and indicate the efficacy of each individual’s treatment regimen [3]. CD4 counts, traditionally performed by flow cytometry, are increasingly available in remote settings due to the introduction of new portable platforms [4,5,6,7]. Viral load instruments are traditionally reverse-transcription polymerase chain reaction (RT-PCR), nucleic acid sequence-based amplification (NASBA), or branched DNA (bDNA) assays; these can be capable of detecting fewer than ten viral RNA copies per mL of blood plasma, these instruments require a laboratory setting, extensive sample handling, and sophisticated processing [6, 8,9,10]
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