Tween 80 Improves the Acid-Fast Bacilli Quantification in the Magnetic Nanoparticle-Based Colorimetric Biosensing Assay (NCBA).

Cristina Gordillo-Marroquín,Adriana Gómez-Bustamante,Héctor J Sánchez-Pérez,Janeth Vázquez-Marcelín,Anaximandro Gómez-Velasco,Karina Guillén-Navarro,Miguel Martín,Letisia Jonapá-Gómez,Evangelyn C Alocilja

doi:10.3390/bios12010029

Abstract

Despite its reduced sensitivity, sputum smear microscopy (SSM) remains the main diagnostic test for detecting tuberculosis in many parts of the world. A new diagnostic technique, the magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was optimized by evaluating different concentrations of glycan-functionalized magnetic nanoparticles (GMNP) and Tween 80 to improve the acid-fast bacilli (AFB) count. Comparative analysis was performed on 225 sputum smears: 30 with SSM, 107 with NCBA at different GMNP concentrations, and 88 with NCBA-Tween 80 at various concentrations and incubation times. AFB quantification was performed by adding the total number of AFB in all fields per smear and classified according to standard guidelines (scanty, 1+, 2+ and 3+). Smears by NCBA with low GMNP concentrations (≤1.5 mg/mL) showed higher AFB quantification compared to SSM. Cell enrichment of sputum samples by combining NCBA-GMNP, incubated with Tween 80 (5%) for three minutes, improved capture efficiency and increased AFB detection up to 445% over SSM. NCBA with Tween 80 offers the opportunity to improve TB diagnostics, mainly in paucibacillary cases. As this method provides biosafety with a simple and inexpensive methodology that obtains results in a short time, it might be considered as a point-of-care TB diagnostic method in regions where resources are limited.

Highlights

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is a curable and preventable disease that primarily affects the lungs and remains one of the leading causes of death from a single infectious agent
In sputum smear microscopy (SSM), the median for the number of acid-fast bacilli (AFB) per field analyzed was 9 (IQR, 1–44.8), which was lower compared to nanoparticle-based colorimetric biosensing assay (NCBA) at low glycan-functionalized magnetic nanoparticles (GMNP) concentrations (0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mg/mL)
Such improvement is explained by the fact that during NCBA-GMNP assay, there is the formation of complexes with AFB that, when extracted and concentrated from the sample matrix with a common magnet, increase the proportion of AFB observed in smears [20]

Summary

Introduction

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is a curable and preventable disease that primarily affects the lungs (pulmonary tuberculosis, PTB) and remains one of the leading causes of death from a single infectious agent. Developing countries either have limited (or non-existent) health care coverage or lack access to rapid and reliable diagnoses [1,2]. In that same year (2020), only 59% of PTB cases were bacteriologically confirmed in developing countries, while 81% were reported in high-income countries, with access to more sensitive but more expensive diagnostic tests [3]. It is estimated that about 2.8 million people have no access to prevent, diagnose, and care for TB [3]. The COVID-19 pandemic has globally caused an 18% reduction in the diagnosis of new TB cases (compared to 2019) and an increase in deaths due to TB [3]. Having an accessible, sensitive, and quick diagnosis is critical for reducing TB transmission, incidence, and mortality, in settings with a high prevalence and limited resources

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