The PATHFAST TB LAM Ag assay: a validated protocol for quantitative tuberculosis treatment monitoring

Tuberculosis (TB) is a global health challenge and is curable, but tools for rapid treatment monitoring are limited. Current methods, like smear microscopy, lack sensitivity, while culture-based approaches require weeks for results and specialized biosafety facilities. This paper presents a comprehensive protocol for the PATHFAST TB LAM Ag assay, a rapid chemiluminescent enzyme immunoassay that quantifies lipoarabinomannan (LAM) mainly in sputum and likely other biological samples within one hour. This protocol details sample collection, heat inactivation for biosafety, and the automated measurement process. Heat inactivation at 100 °C for 20 minutes renders samples non-infectious while preserving LAM for detection. The assay demonstrates excellent analytical performance with a detection limit of 6.67 pg/mL and a linear measurement range of 10.0-50,000 pg/mL. Published clinical evaluation demonstrated 88.8% (95% CI: 80.0-94.0%) sensitivity and 100% (95% CI: 83.9-100%) specificity compared to culture, with strong correlation to bacterial load. The PATHFAST TB LAM Ag assay successfully tracks bacterial decline during treatment, with LAM concentration reductions mirroring culture results but available significantly faster. Unlike culture methods that suffer from contamination (4.2-9.6% invalid results), the PATHFAST TB LAM Ag assay consistently provides valid results. This protocol enables implementation of a rapid, quantitative TB treatment monitoring tool that enhances biosafety, reduces turnaround time, and maintains performance comparable to conventional methods, addressing critical needs in both clinical care and drug development settings.