Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis

Abstract

Background: Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results.

Methods and findings: In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine.

Conclusions: Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively.

Conflict of interest statement

BH is an employee of Biopromic AB. Biopromic AB and Intellectual Ventures Laboratory are developing a point-of-care TB LAM test, however, that test is not directly based on this work and this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. (a) A photographic image of fabricated PMSSQ gratings on 1 inch x 1inch glass slides and (b) a diagram of FLISA on gratings showing the sandwich structure of antibodies for detection of LAM.

Fig 2. SM FLISA standard curves and fitted data for (a) Assay #1 and (b) Assay #2 demonstrating a good agreement with each other for reproducibility of the data.

Fig 3. Combined result graph of all 20 patient samples with three measurement for each patient.

The result demonstrated good agreement with Xpert while conventional ELISA could not quantify LAM concentration.