The accuracy of urine-based mycobacterial antigens to detect childhood tuberculosis using an ultrasensitive immunoassay

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ABSTRACT Background Urine-based testing offers a promising non-sputum approach for diagnosing paediatric tuberculosis. However, the currently available lipoarabinomannan (LAM) assay shows limited sensitivity in children and is primarily indicated for those living with HIV. Co-detection of LAM with Mycobacterium tuberculosis ( Mtb ) proteins in urine could provide complementary pathogen-derived biomarkers that improve diagnostic performance. Methods We developed an ultrasensitive multiplex electrochemiluminescence (ECL) immunoassay to measure Ag85B, CFP-10, ESAT-6, MPT32, and MPT64 in urine. We determined the analytical limits of detection and evaluated the diagnostic performance of individual proteins and LAM using urine samples from children with Confirmed, Unconfirmed, and Unlikely pulmonary tuberculosis enrolled across five high-burden countries (The Gambia, India, Peru, South Africa, and Uganda). Performance was assessed overall, by HIV and nutritional status, and across biomarker combinations. Findings Urine samples from 630 children were analysed (median age was 4 years [IQR 2-8]; 44% female, 15% living with HIV, 19% underweight, 24% with Confirmed tuberculosis). The ECL assay achieved femtomolar limits of detection (1·5 to 4·0 fM). The sensitivity and specificity of individual Mtb proteins were 12-33% and 98-100%, respectively. Ag85B had the highest sensitivity (33%, 95% CI 26-41) for Confirmed tuberculosis and was similar to LAM. A four-antigen signature (Ag85B, MPT64, MPT32, LAM) was 50% sensitive (95% CI 42-58) and 94% specific (95% CI 90-96), and was significantly more sensitive than LAM alone, in particular among those without HIV. An additional sixteen (10%) of children with Unconfirmed TB had at least one Mtb protein or LAM detected. Interpretation Multiple Mtb proteins are detectable in paediatric urine with high specificity, and multi-antigen signatures can augment sensitivity versus LAM alone. These findings demonstrate the potential of multi-antigen urine detection for childhood TB and define analytical targets for the development of future point-of-care diagnostics. Funding National Institutes of Health. RESEARCH IN CONTEXT Evidence before this study We examined the literature for peer-reviewed research articles on the accuracy of biomarker- based urine tests for pulmonary tuberculosis in children <15 years old. We used PubMed and Google Scholar, with the search terms “child”, “tuberculosis”, “urine”, and “diagnosis” regardless of language from July 2016 to July 2026. We excluded articles on host-based markers and extrapulmonary tuberculosis. Molecular urine assays, including Xpert MTB/RIF, have limited sensitivity to detect childhood pulmonary tuberculosis. Most of the research on urine-based diagnostics has focused on detection of lipoarabinomannan (LAM), which has had variable sensitivity and specificity in children against a microbiological reference standard. Accuracy is higher in those with HIV, and current guidelines only recommend LAM for adults and children with HIV. Added value of this study We developed an ultrasensitive multiplex immunoassay to detect and measure Mtb- specific proteins in urine samples from children with presumptive tuberculosis in five high-burden countries. We found that Mtb proteins could be detected in paediatric urine samples with high specificity, and Ag85B had similar sensitivity as LAM. A four-marker panel (Ag85B, MPT32, MPT64, and LAM) improved sensitivity over LAM alone without a significant loss of specificity, in particular among children without HIV. Implications of all the available evidence Multi-antigen urine tests can improve sensitivity over single marker assays, and have the potential to provide non-sputum, point-of-care tuberculosis detection in children regardless of HIV status.