Environmentally Resilient Microfluidic Point-of-Care Immunoassay Enables Rapid Diagnosis of Talaromycosis

Point-of-care tests (POCTs) are increasingly being used in field settings, particularly outdoors. The performance of current POCTs-most commonly the lateral flow immunoassay-can be adversely affected by ambient temperature and humidity. We developed a self-contained immunoassay platform-the D4 POCT-that can be conducted at the POC by integrating all reagents in a capillary-driven passive microfluidic cassette that minimizes user intervention. The assay can be imaged and analyzed on a portable fluorescence reader-the D4Scope-and provide quantitative outputs. Here, we systematically investigated the resilience of our D4 POCT to varied temperature and humidity and to physiologically diverse human whole blood samples that span a wide range of physiological hematocrit (30-65%). For all conditions, we showed that the platform maintained high sensitivity (0.05-0.41 ng/mL limits of detection). The platform also demonstrated good accuracy in reporting true analyte concentration across environmental extremes when compared to the manually operated format of the same test to detect a model analyte-ovalbumin. Additionally, we engineered an improved version of the microfluidic cassette that improved the ease-of-use of the device and shortened the time-to-result. We implemented this new cassette to create a rapid diagnostic test to detect talaromycosis infection in patients with advanced HIV disease at the POC, demonstrating comparable sensitivity and specificity to the laboratory test for the disease.