The accuracy of urine-based mycobacterial antigens to detect childhood tuberculosis using an ultrasensitive immunoassay
- 1Meso Scale Diagnostics, LLC.
Abstract
BackgroundUrine-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.
MethodsWe 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.
FindingsUrine 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{middle dot}5 to 4{middle dot}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.
InterpretationMultiple 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.
FundingNational Institutes of Health.
RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSWe 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 studyWe 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 evidenceMulti-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.
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BibTeX
@misc{nkereuwem2026accuracy,
title = {{The accuracy of urine-based mycobacterial antigens to detect childhood tuberculosis using an ultrasensitive immunoassay}},
author = {E. Nkereuwem and S. Misaghian and D. Jaganath and R. I. Calderon and J. Luiz and M. Paradkar and P. Wambi and R. Castro and R. Nerurkar and M. Wang and J. Wohlstadter and M. F. Franke and B. Kampmann and A. Kinikar and H. J. Zar and M. Segal and M. Kato-Maeda and J. M. Collins and D. Swaney and A. Cattamanchi and J. D. Ernst and E. Wobudeya and G. Sigal and The Combo Study},
howpublished = {medRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.28.26361530},
url = {https://www.medrxiv.org/content/10.64898/2026.08.28.26361530v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - The accuracy of urine-based mycobacterial antigens to detect childhood tuberculosis using an ultrasensitive immunoassay
AU - E. Nkereuwem
AU - S. Misaghian
AU - D. Jaganath
AU - R. I. Calderon
AU - J. Luiz
AU - M. Paradkar
AU - P. Wambi
AU - R. Castro
AU - R. Nerurkar
AU - M. Wang
AU - J. Wohlstadter
AU - M. F. Franke
AU - B. Kampmann
AU - A. Kinikar
AU - H. J. Zar
AU - M. Segal
AU - M. Kato-Maeda
AU - J. M. Collins
AU - D. Swaney
AU - A. Cattamanchi
AU - J. D. Ernst
AU - E. Wobudeya
AU - G. Sigal
AU - The Combo Study
PY - 2026
DA - 2026/08/28
PB - medRxiv
DO - 10.64898/2026.08.28.26361530
UR - https://www.medrxiv.org/content/10.64898/2026.08.28.26361530v1
AB - BackgroundUrine-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. MethodsWe 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. FindingsUrine 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{middle dot}5 to 4{middle dot}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. InterpretationMultiple 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. FundingNational Institutes of Health. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSWe 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 studyWe 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 evidenceMulti-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.
ER -