Antigen heterogeneity in the development and clinical validation of a multiplexed urine test for tuberculosis.

BACKGROUND: Tuberculosis (TB) is one of the leading causes of death worldwide, even though it is curable using antibiotics. Most people who die of TB never begin treatment because diagnostics are insufficiently sensitive and accessible. We aimed to measure low-abundance biomarkers and diagnose TB in urine.

METHODS: We developed and clinically validated a multiplex Single Molecule Array (Simoa) assay to detect TB in urine by measuring two TB biomarkers: lipoarabinomannan (LAM) and antigen 85B (Ag85B). Using antibodies that recognize different epitopes of LAM in a four-plex assay with three LAM and one Ag85B antibody pairs, we trained a model and demonstrated its performance in retrospective cohorts totaling 576 individuals from South Africa, Peru, Vietnam, and Cambodia, including a blinded test cohort (n = 215).

RESULTS: Here we present an assay that classifies samples with 98% specificity, 45% sensitivity overall, and 58% sensitivity among people living with the human immunodeficiency virus (HIV).

CONCLUSIONS: Different antibody pairs detecting different epitopes on LAM report diverging concentrations. We do not find that adding antibody pairs to detect different epitopes on LAM improves the assay's accuracy. Our assay is more sensitive than the existing AlereLAM lateral flow test for TB in HIV-positive individuals, uses safe and accessible urine samples, and represents a step towards an adjunctive diagnostic test to aid clinicians in starting treatment.