Clinical evaluation of patterned dried plasma spot cards to support quantification of HIV viral load and reflexive genotyping

Quantifying viral load, a key measure required to achieve control and elimination of the HIV epidemic, requires cell-free plasma or serum to ensure measurements are not biased by proviral DNA contained in infected CD4 T lymphocytes. Plasma separation cards (PSC) collect and preserve a dried specimen, which makes them practical solutions for decentralized sample collection and transport in limited-resource settings. However, physiological variations in hematocrit levels can introduce significant variability in sample quality and lead to inaccurate test results. In addition to hematocrit-dependent sampling, the Roche PSC, the commercial standard for dried plasma collection, is also known to induce hemolysis, which further reduces specimen quality and impacts concordance with liquid plasma. We address these gaps with the development of a new patterned dried plasma spot (pDPS) card, which generates cell-free plasma with improved hematocrit independence and minimal hemolysis. This study directly compares pDPS cards to the Roche PSC to measure HIV viral load. Analysis of HIV RNA from 75 different donors revealed strong agreement in sensitivity, specificity, overall accuracy, and viral load band placement between collection devices, with quantitative metrics suggesting improved performance for pDPS cards. In reflexive genotyping, remnant dried blood from pDPS cards exhibited greater success in amplification and sequencing (71% v. 62%) and in detecting drug resistance mutations (63% v. 42%) than Roche PSC. These results suggest that pDPS cards could be versatile across multiple analytical platforms, can integrate seamlessly into existing clinical laboratory workflows, and aid clinicians in making informed treatment decisions.