Abstract
ObjectiveWe developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest.MethodsWe first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user intervention.ResultsThe finalized system consists of a disposable injection molded and film-laminated microfluidic CARD disposable device and a portable, software controlled instrument, which together can automatically perform all steps of both assays without any user intervention after the initial loading of samples and reagents. The microfluidic CARD cartridge has multiple microchannels, valves, pumps and reservoirs, which perform the immunoassay, isolates viral RNA for detection by magnetic bead based purification, and Reverse Transcriptase loop-mediated isothermal amplification (RT-LAMP). The microfluidic system was able to detect host anti-HIV antibodies and viral RNA in either a blood or saliva sample.ConclusionThe ability to detect antibodies and simultaneously confirm a seropositive HIV-RNA result provides healthcare workers with a complete and accurate appraisal of a patient's infection status in the earliest stages of the disease and represents an important tool for the "Test and Treat" and "Treatment as Prevention" approaches for controlling the HIV epidemic.
Highlights
The global HIV/AIDS epidemic continues to be fueled by individuals who do not know they are infected and they continue to transmit the virus
We have previously reported the simultaneous detection of antibodies and viral RNA in both blood and saliva using a portable device equipped with an open architecture dual path microfluidic cartridge [16]
Reliability and throughput, we describe further improvements to the system: replacing silica-based nucleic acid purification with a magnetic bead-based process; replacing polymerase chain reaction (PCR) with Loop Mediated Isothermal Amplification (LAMP); and increasing the number of samples analyzed from one to four on a single CARD microfluidic device
Summary
The global HIV/AIDS epidemic continues to be fueled by individuals who do not know they are infected and they continue to transmit the virus. The challenges faced in low resource settings have led to the use of rapid, easy-to-use dipstick tests to screen individuals for the presence of anti-HIV antibodies. As this class of diagnostics lacks the sensitivity and specificity of more sophisticated assays, initial seropositive results are often confirmed only by use of another manufacturer’s rapid screening test. The assay should be run on an integrated diagnostic platform that can process samples from the subject through to analytical result Such a system will facilitate the “Test and Treat” approach to controlling the HIV epidemic [4,6,8,9]
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