(Invited) Lab-in-a-Tube Design Enables a Rapid Diagnosis of Infectious Disease

Abstract

Disease diagnosis increasingly relies on molecular tests that are often limited by infrastructure constraints. For example, tuberculosis (TB) remains a leading cause of death from infectious disease, but an estimated 4.2 million (39.6%) new TB cases were not diagnosed in 2021 and only 57% were confirmed by gold-standard sputum culture or PCR-based tests, partially due to limited accessibility. New point-of-care tests are thus urgently needed to address coverage disparities for chronic, malignant, and infectious diseases.We have developed two smartphone-based assays that can address two diagnostic shortfalls: an on-chip interferon-gamma release assay (IGRA) that detects a T-cell activation response induced by pathogen antigens in fingerstick blood samples, and a lab-in-tube assay that employs CRISPR to sensitively detect pathogen DNA in sputum. Both approaches translate current diagnostic approaches to point-of-care devices that require minimal equipment, user expertise, and time.Results from our on-chip IGRA also correlated with clinical IGRA results when we employed two proteins associated with TNF-alpha receptor signaling (4-1BB and OX-40) to analyze T-cell activation, indicating this approach could detect infections in individuals with compromised interferon-gamma responses; as observed in HIV patients, who are at increased risk for TB. On-chip IGRA results from a SARS-CoV-2-specific assay also corresponded with both infection and vaccination history, suggesting this data could be employed as a high-throughput, high-capacity test to evaluate the effect of vaccination or previous infection to confer a protective immune response, evaluate its change over time, and predict the response to new variant strains to guide healthcare decisions. Mitogen-induced results could also guide immunotherapy decisions, as reduced IGRA values in patients receiving immune checkpoint inhibitors predict poor treatment response, reduced progression-free survival, and increased risk for interstitial pneumonia, while their change after adoptive T-cell therapy can be an independent predictor for overall survival. Results from our lab-in-tube TB DNA assay also had robust diagnostic performance when compared to gold-standard clinical assays but lacked their more extensive personnel, workflow, and infrastructure requirements and were performed in a closed-tube system that protected user from exposure to infectious material. This lab-in-tube approach also permits parallel multiplex analyses to be performed in a single tube, and thus can be employed to detect mutations associated with drug-resistance to guide treatment decisions. For example, we found that an assay with an optimized guide RNA accurately detected a single-nucleotide polymorphism (rpoB S450L) responsible for the majority of rifampin-resistant TB cases.Both these self-contained handheld devices can employ lyophilized reagents to limit cold-chain concerns, can be readily adapted to diagnose other disease conditions by substituting other disease-specific antibodies or guide RNAs, use rechargeable batteries to avoid infrastructure limitations, and can directly transmit results to central sites to improve disease control efforts.