Abstract
The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.
Highlights
According to the recent World Health Organization (WHO) report, globally, 7.1 million people with tuberculosis (TB) were reported to have been diagnosed in 2019 –a small increase from 7.0 million in 2018 but a large increase from 6.4 million in 2017 and 5.7–5.8 million annually in the period 2009–2012 [1]
While an estimated quarter of the world is latently infected with Mycobacterium tuberculosis (Mtb), active TB is caused by uncontrolled infection leading to a predominantly respiratory and transmissible disease
In this report we show that we were successful in our immobilization of proteinase K (ProK) and the immobilized strips (IPK) perform to the standard ‘add and deactivate’ procedure
Summary
According to the recent WHO report, globally, 7.1 million people with tuberculosis (TB) were reported to have been diagnosed in 2019 –a small increase from 7.0 million in 2018 but a large increase from 6.4 million in 2017 and 5.7–5.8 million annually in the period 2009–2012 [1]. In 2020, the COVID-19 pandemic has already had a negative impact on access to TB diagnosis and treatment and will continue beyond 2021 [2]. While an estimated quarter of the world is latently infected with Mycobacterium tuberculosis (Mtb), active TB is caused by uncontrolled infection leading to a predominantly respiratory and transmissible disease. To fight against TB, better vaccines, therapies, and new tools for both diagnosis and research are critical. Mtb has a unique cell wall with multiple lipid-based molecules that create a thick impermeable ‘waxy’ surface [3]. LAM is firmly but non-covalently attached to the inner membrane and extends to the exterior of the cell wall [10] where it interacts as a potent virulence factor that modulates the host immune response and plays an important role in the pathogenesis of Mtb infection [11]
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