Lipoarabinomannan antigenic epitope differences in tuberculosis disease subtypes

Abstract

An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients. We measured 430 microbiologically confirmed pretreatment tuberculosis patients and controls from Peru, Guinea Bissau, Venezuela, Uganda and the United States using three monoclonal antibodies, MoAb1, CS35, and A194, which recognize distinct LAM epitopes, a one-sided immunoassay, and blinded cohorts. We evaluated sources of assay variability and comorbidities (HIV and diabetes). All antibodies successfully discriminated TB positive from TB negative patients. ROAUC from the average of three antibodies' responses was 0.90; 95% CI 0.87-0.93, 90% sensitivity, 73.5% specificity (80 pg/mL). MoAb1, recognizing the 5-methylthio-D-xylofuranose(MTX)-mannose(Man) cap epitope, performed the best, was less influenced by glycosuria and identified culture positive pediatric (N = 19) and extrapulmonary (N = 24) patients with high accuracy (ROAUC 0.87, 95% CI 0.77-0.98, 0.90 sensitivity 0.80 specificity at 80 pg/mL; ROAUC = 0.96, 95% CI 0.92-0.99, 96% sensitivity, 80% specificity at 82 pg/mL, respectively). The MoAb1 antibody, recognizing the MTX-Man cap epitope, is a novel analyte for active TB detection in pediatric and extrapulmonary disease.

Figure 1

Characteristics of LAM immunoassays. (A) Monoclonal antibodies CS-35, A194 and MoAb1 recognize different LAM epitopes. Structural epitope configurations adapted from Sigal et al., Amin et al., and De et al.. (B) Calibration curve of anti-LAM antibodies. Analytical measurement range for CS35, MoAb1, and A194 was 0.6–4 ng/mL in urine samples without dilution or concentration. (C) Example images show different mAb reactivity to the same urine sample cropped from different blots (Supplementary Figure S1).

Figure 2

Urinary LAM discriminates TB positive from TB negative patients with high accuracy. (A) In a geographically diverse cohort (N = 430), urinary LAM concentration discriminates between cases and controls (Kruskal Wallis p = 8E−37, p = 5E−27, p = 6E−34, for MoAb1, CS35, A194 respectively). (B) Urinary LAM levels are higher in HIV+/TB+ patients than in HIV−/TB+ patients (Kruskal Wallis p = 5E−05, p = 0.002 for CS35 and A194, respectively). (C) Urinary LAM distinguishes microbiologically confirmed untreated TB positives from TB negatives with 90% sensitivity and 70% specificity, at a threshold of 80 pg/mL. (D) Urinary LAM antigenicity varies across samples. 57% of patients yield a signal for all three antibodies, whereas 95% of patients yield a signal to at least one antibody (cut-off 0.080 ng/mL).

Figure 3

Urinalysis and geographical diversity influence LAM values. (A) Abnormal urine values were noted in both controls and TB positive patients. (B) Median urinary LAM concentration varies across different geographic areas (independent sample median test p = 0.030, pairwise comparison in Supplementary Figure S4). Assuming no collection and storage bias, this might be caused by different levels of disease severity at diagnosis, since access to healthcare might be delayed to a greater degree in certain countries. (C) Urinary LAM values were lower in TB patients with glycosuria (n = 22) when measured using CS-35 (p = 0.015) and A194 (p = 0.035).

Figure 4

Urinary LAM is present at detectable concentrations in extrapulmonary adult TB patients and in microbiologically confirmed pediatric TB patients. (A) Levels of urinary LAM as measured using MoAb1 are significantly higher in extrapulmonary patients with respect to pulmonary patients. (B) MoAb1 is superior in discriminating extrapulmonary patients from non TB controls. (C) Levels of urinary LAM measured using CS35 are lower in TB positive pediatric patients than in TB positive adults. (D) ROC analysis demonstrated that MoAb1 was superior to the other antibodies in discriminating pediatric TB patients from negative controls.