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

PLoS One. 2019 Mar 26;14(3):e0214161. doi: 10.1371/journal.pone.0214161. eCollection 2019.

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.

Publication types

  • Clinical Trial
  • Multicenter Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Biosensing Techniques*
  • Female
  • HIV Seronegativity*
  • HIV-1*
  • Humans
  • Lipopolysaccharides / urine*
  • Middle Aged
  • Tuberculosis / urine*

Substances

  • Lipopolysaccharides
  • lipoarabinomannan

Grant support

Funding was provided by the Bill and Melinda Gates Foundation Trust through The Global Good Fund I, LLC www.globalgood.com) at Intellectual Ventures. The Global Good Fund I, LLC provided support in the form of salaries for authors AB, KM, AS, JC, and BW as well as financial support for the study, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.