(I-323) HIV Quantitative Viral Load Self-Testing From Fingerpick Blood For ART Treatment Rebound Monitoring

Introduction:: WHO reports HIV as a major global public health issue that has claimed millions of lives. ART has greatly reduced morbidity and mortality in people with HIV-1. Viral suppression through ART lowers transmission risk but isn't a cure and viral persistence, inflammation, and metabolic issues persist. Regular viral load testing monitors treatment efficacy in patients is necessary.

To address this challenge, we have developed an HIV semi-quantitative self-detection NAT kit. This device comprises three components, namely a plasma separation card, a power-free RNA extraction kit, and an RT-LAMP-based ultra-compact analyzer. Our study shows that this kit reduces the volume of whole blood by 100 µL compared to the traditional method that requires around 10 ml of venous blood. The test involves simple steps, such as dropping the finger-prick blood sample into the plasma separation card, dropping the plasma-laden membrane into the lysate, using a manual syringe pump for RNA extraction, and finally mixing the extracted and lyophilized RNA with the RT-LAMP for amplification to obtain semi-quantitative virus detection results. The test results are easily readable on the mobile app. We have developed a novel manual syringe pump with a "lab on tube" approach that greatly simplifies the operation. This highly portable analyzer has a Bluetooth communication and integrates collaborative subsystems into an ultra-compact form factor. With these innovations, we anticipate that rapid, low-cost, easy-to-use HIV quantitative self-test kits could be especially useful for patients seeking private, periodic testing of ART treatment efficacy at home to prevent rebound.


Materials and Methods:: We developed a simple, power-free plasma separation technology for point-of-care (POC) applications using a Vivid plasma separation membrane and absorbent material. Our technology is more efficient than many other plasma separation technologies and can handle larger amounts of blood. Figure 1 shows our plasma separation apparatus with two layers: the absorber layer and the filter layer. Figure 2 displays the schematic diagram of our power-free RNA extraction kits, which use a syringe as a power source. After collecting the plasma absorbent into the lysis tube, the user can shake it to promote mixing. The RNA extraction silica column and 10 ml syringe are assembled using the connecting device. After transferring the solution to the RNA extraction silica column, the lysate passes through the silica membrane into the syringe. The RNA extraction kit includes wash buffers and an elution buffer. After elution, the RNA can be injected into the PCR reaction tube.

Our analyzer has a compact footprint of 3 in. (W) x 2 in. (H) x 4 in. (L) (Figure 4a) and automatically aligns with the temperature module, and fluorescence sensor module (Figure 4b). We designed a bespoke MCU-based circuit and software for automation and used Bluetooth for data transmission. The internal battery supports up to 900 mA at 5V for a maximum power output of 4.5 W.


Results, Conclusions, and Discussions:: Figure 1b shows the measurement results of hemoglobin content. It can be seen that the hemoglobin content in plasma separated by the plasma separation module is much lower than in the whole blood, and the effect is similar to the traditional centrifugal separation method. At the same time, the microscope image confirms that the separated plasma almost does not contain RBC and other residues. It can be seen from Figure 1c that the filtration efficiency brought by the 16mm diameter filter membrane is about 80 %. At the same time, the diameter of the 16mm diameter filter membrane extract is less affected by different hematocrit.

Figure 3 shows RNA extraction efficiency for different concentrations by using our extraction module compared with the lab-based protocol. From figure 3, we can see that although our extraction module efficiency is lower than the laboratory protocol, we can still recover the major RNA. For high -concentration samples, our extraction module can reach about 60% of the extraction efficiency. For a sample of 1000 copies/ml, our extraction module can reach about 40% of the extraction efficiency. Based on existing data, it can prove that our RNA extraction module can complete the RNA extraction without using power, which is very suitable for field testing or self-testing.

As shown from figure 5, a total of 15 HIV-infected patients with three levels of VL (1,000-10,000; 10,001-100,000; >100,000; ideally, 15 patients in each VL level) were recruited from Penn State Hershey Medical Center HIV/AIDS outpatient clinics, and 15 HIV-negative people (confirmed by Roche COBAS® AmpliPrep) have been recruited from catchment areas. These 30 test samples were prepared and analyzed with our HIV testing system. As shown in Figure 4, the test has a limit of detection of at least 1000 copies/ml and can establish a threshold detection time for reporting yes/no for a viral load of 1000 copies/ml, as well as a semi-quantitative function.

These results demonstrate the ability of our device to prepare samples from complex mediums at the point of care, with a limit of detection of at least 1000 copies/ml.


Acknowledgements (Optional): : This work was supported by the National Institutes of Health (R61AI147419), and National Science Foundation (1912410 & 1902503). Any opinions, findings, and conclusions or recommendations expressed in this work are those of the authors and do not necessarily reflect the views of the National Science Foundation and National Institutes of Health.


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