Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

Anita G Amin,Delphi Chatterjee,Prithwiraj De,Roger I Calderon,Barbara Graham,Molly F Franke

doi:10.1038/s41598-021-82445-4

Anita G Amin, Delphi Chatterjee + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-82445-4

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Abstract

Our study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

Highlights

Tuberculosis (TB) remains one of the leading causes of death worldwide
The specificity of the FujiLAM test was lower than the AlereLAM test, maybe due to the antibodies used for the test as the presentation of antigenic determinants in urinary LAM in structural organization and increased/reduced multiplicity may have direct consequences on LAM immunoassay
Earlier we reported that using CS35/A194-01 as the antibody pair for capture and detection respectively, after Pro K pretreatment followed by fivefold sample concentration, we achieved a sensitivity of 98% and specificity of 92%9

Summary

Introduction

Tuberculosis (TB) remains one of the leading causes of death worldwide. The number of new cases of TB has been declining steadily in recent years. The detection of LAM spiked in urine from a healthy volunteer with capture ELISA has consistently been shown to have an LoD at ~ 100 pg/mL after pretreatment with Pro K followed by fivefold concentration prior to ELISA9. Earlier we reported that using CS35/A194-01 as the antibody pair for capture and detection respectively, after Pro K pretreatment followed by fivefold sample concentration, we achieved a sensitivity of 98% and specificity of 92%9.

Results

Conclusion