Urine lipoarabinomannan glycan in HIV-negative patients with pulmonary tuberculosis correlates with disease severity

Abstract

An accurate urine test for pulmonary tuberculosis (TB), affecting 9.6 million patients worldwide, is critically needed for surveillance and treatment management. Past attempts failed to reliably detect the mycobacterial glycan antigen lipoarabinomannan (LAM), a marker of active TB, in HIV-negative, pulmonary TB-infected patients' urine (85% of 9.6 million patients). We apply a copper complex dye within a hydrogel nanocage that captures LAM with very high affinity, displacing interfering urine proteins. The technology was applied to study pretreatment urine from 48 Peruvian patients, all negative for HIV, with microbiologically confirmed active pulmonary TB. LAM was quantitatively measured in the urine with a sensitivity of >95% and a specificity of >80% (n = 101) in a concentration range of 14 to 2000 picograms per milliliter, as compared to non-TB, healthy and diseased, age-matched controls (evaluated by receiver operating characteristic analysis; area under the curve, 0.95; 95% confidence interval, 0.9005 to 0.9957). Urinary LAM was elevated in patients with a higher mycobacterial burden (n = 42), a higher proportion of weight loss (n = 37), or cough (n = 50). The technology can be configured in a variety of formats to detect a panel of previously undetectable very-low-abundance TB urinary analytes. Eight of nine patients who were smear-negative and culture-positive for TB tested positive for urinary LAM. This technology has broad implications for pulmonary TB screening, transmission control, and treatment management for HIV-negative patients.

Conflict of interest statement

L.L., A.L., and E.F.P. are inventors on U.S. patents 9,012,240 and 8,497,137 related to the nanocage particles. Ceres Nanosciences licensed the rights of these patents that are owned by George Mason University. L.L., E.F.P., and A.L. own shares of Ceres Nanosciences. All other authors declare that they have no competing interests.

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Fig. 1

Nanocages that were covalently functionalized with copper complex dye Reactive Blue 221 sequestered and concentrated lipoarabinomannan from urine. (A) Schematic depicting high internal/external surface area ratio and binding capacity of nanocages. Affinity ligands covalently immobilized in the inner volume establish high-affinity noncovalent interaction with tuberculosis (TB) antigens. (B) Schematic showing the concentration factor given by the volumetric ratio between the initial urine volume and the final testing volume. Structures within the urine sample are nanocages. (C) Molecular structure of lipoarabinomannan (LAM) (right) and affinity probe Reactive Blue 221 (RB221) {cuprate(4-),[2-[[[[3-[[4-chloro-6-[ethyl[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5- sulfophenyl]azo]phenylmethyl]azo]-4-sulfobenzoato(6-)]-,tetrahydrogen} (left). (D) Western blot, glycan staining, and image analysis of protein macroarray assay of LAM. C, LAM control (50 ng); IS, initial solution (50 ng of LAM spiked in 50 µl of human urine); S, supernatant; E, eluate from the nanocages; P, nanocages; AU, arbitrary units. Mean and SD, n = 3 replicates.

Fig. 2

LAMantigenwas detected in the urine of HIV-negative/TB-positive patients using RB221 nanocages for diseased and control patients listed in Table 1. (A) Image of a quantitative immunomacroarray for LAMdetection, incorporating (B) a dilution curve in every membrane. Neg, negative; BKG, background. (C) Example immunomacroarray comparing urine samples from a set of true-positive and known TB-negative samples using nanocage preprocessing. (D) Bar plot of the intensities of LAM determined via immunomacroarray and ImageJ analysis from urine samples from healthy TB-negative, TB-negative diseased, and TB-positive patients shown in Table 1 (mean ± SD, n = 4 patient replicates).

Fig. 3

Urinary LAM concentration predicted pulmonary TB and correlated to mycobacterial burden and weight loss. (A) Box plot of the intensities of LAM in the urine of HIV-negative/TB-positive patients versus controls collected in endemic areas (Wilcoxon signed-rank test). (B) Box plot of the intensities of LAM in the urine of HIV-negative/TB-positive patients stratified on the basis of the auramine staining (low amount of microorganism, scores 0 and 1; high amount of microorganism, scores 2 and 3; Wilcoxon signed-rank test; n = 42). (C) Receiver operating characteristic analysis of the LAM intensity data. AUC, area under the curve. (D) Ordinal regression analysis shows statically significant correlation between the concentration of urinary LAM and the loss of body mass (P = 0.038, n = 37).

Fig. 4

Nanocages captured multiple TB-related analytes. (A) SDS– polyacrylamide gel electrophoresis (PAGE) analysis; chemical bait incorporated in the nanocages (NP1, blue 3G-A; NP2, pigment red 177; NP3, disperse yellow 3). P, nanocage eluate. (B) Affinity probes (affinity probe 1, pigment red 177; affinity probe 2, blue 3G-A; affinity probe 3, trypan blue). (C) Nanocages effectively captured TB-related analytes from human urine (Western blot). U, negative control; C, recombinant protein (positive control, 75 ng). (D) Nanocage detection of TB antigen ESAT6 in the urine of untreated HIV-negative/TB-positive patients (Western blot).

Fig. 5

Magnetic hydrogel nanocages. (A) Schematic of magnetization. (B) Western blot analysis of ESAT6 and CFP10 expression in eluates of centrifugation-separated nanocages (top), in eluates of magnetic-separated nanocages (middle), and in supernatants after magnetic separation of nanocages from urine samples (bottom). Top and middle: Lane 1, positive control (recombinant protein; 10 ng); lanes 2 to 7, two to six eluates from nanocages incubated with 1 ml of urine containing ESAT6 (10, 5, 2.5, 1.2, 0.6, and 0.3 ng/ml) and CFP10 (10, 5, 2.5, 1.2, and 0.6 ng/ml). Bottom: Lane 1, positive control (recombinant protein; 10 ng); lanes 2 to 7, two to six supernatants after nanocage processing of 1 ml of urine containing ESAT6 (10, 5, 2.5, 1.2, 0.6, and 0.3 ng/ml) and CFP10 (10, 5, 2.5, 1.2, and 0.6 ng/ml).

Fig. 6

Partially dissolvable nanocages captured antigen for antibody binding in a high-sensitivity sandwich immunoassay. (A) Schematic demonstrating nanocage cross-link degradation in an oxidative environment. (B) Change in hydrodynamic diameter after nanocage oxidation [t test, n = 10; mean and SD of nanocage hydrodynamic diameter before (a) and after (b) oxidative degradation]. (C) SDS-PAGEanalysis comparing N,N′-(1,2-dihydroxyethylene) bisacrylamide (DHEA) cross-linked nanocages mixed with a solution of monoclonal antibody (Ab) (0.05 mg/ml) with pores open (lanes 2 and 3) and closed (lanes 4 and 5). (D) Immunomacroarray demonstrating that antigen bound to the chemical bait retains its capability to bind to the antibody. a, nanocages deposited on polyvinylidene difluoride (PVDF) membrane after incubation with urine containing ESAT6 (1 ml, 10 ng/ml) andDHEAcross-linkdegradation; b, nanocages deposited on PVDF membrane after incubation with urine containing ESAT6 (1 ml, 10 ng/ml) in the absence of DHEA cross-link degradation; c, ESAT6 deposited on PVDF membrane (starting amount, 1 ng); d, DHEA nanocages deposited on PVDF membrane after incubation with urine in the absence of ESAT6. (E) Plot of immunoassay signal intensity as a function of bait capture affinity. High-affinity chemical baits achieve >2 log increased sensitivity for antigen capture compared to conventional antibody, as mathematically demonstrated in Supplementary Materials and Methods. (F) Schematic depicting direct, nonelution sandwich immunoassay using partially degradable nanocages. Inset shows an enzyme-linked antibody interacting with TB antigens captured inside the nanocage. (G) Calibration curve of a direct nanocage immunoassay for ESAT6 showing linearity in the 1- to 0.03-ng range. (H) Schematic of a lateral flow immunoassay using one antibody. Nanocages capture and preserve antigen in solution, migrate through the filter membrane, and provide colorimetric detection. (I) Lateral flow immunoassay for ESAT6 detection in urine. Positive signal for 10 ng of ESAT6 in 10ml of human urine both visually (blue line, a) andwith chemiluminescence (black line, b). Negative control urine in the absence of ESAT6 yields no signal (c and d).