FLIPP-NAAT: A paper-based LAMP assay for point-of-care TB detection

Abstract

Molecular diagnostic techniques like polymerase chain reaction (PCR) revolutionized the world of healthcare. Instead of waiting for infectious agents to physically grow as in conventional culture-based tests, genetic material of pathogens could be used as a biomarker to detect their presence. Though PCR has deeply penetrated and greatly benefited the field of disease diagnosis, infrastructure required to perform PCR testing makes it unaffordable and hence inaccessible for majority of world population. The COVID-19 pandemic has strongly highlighted many such grave limitations of existing molecular diagnostic systems, which are rampant in all kinds of healthcare settings. More importantly, obstacles in timely, accurate, and affordable diagnosis of infections make infectious diseases the leading cause of mortality in low-income economies.

The aim of my research has been to develop an ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment free and deliverable to end users) molecular diagnostic tool for infectious disease diagnosis that fits well in the purview of WHO prescribed criteria for developing point-of-care (POC) diagnostic tools. While the diagnostic tool is a platform technology, adaptable to different infectious diseases, the first application has been demonstrated for tuberculosis (TB) diagnosis because it is the deadliest infectious disease today. The developed prototype called fluorescent isothermal paper-and-plastic nucleic acid amplification test (FLIPP-NAAT) uses loop-mediated isothermal amplification (LAMP) for DNA amplification and the only ancillary equipment required for testing is a laboratory incubator. A very low-cost imaging box for filter-free end-point fluorescence detection of amplified DNA using a cell phone has also been developed. To best of our knowledge, this work is the first demonstration of a paper-based NAAT for TB starting from gDNA as template and for testing clinical patient samples using a paper-based NAAT. TB testing for 30 clinical samples demonstrated a clinical sensitivity of 100% and clinical specificity of 68.75%. Material cost for a 12-test zone device is $0.88 and reagent cost per reaction is $0.43.

Since clinical specificity of FLIPP-NAAT required improvement, a new detection strategy has hence been proposed where lateral flow detection replaces fluorescence-based detection. Proof-of-concept demonstrations have been made to confirm correct diagnosis of patient samples which were detected as false positives with FLIPP-NAAT. Consistent efforts were also made to enable dry storage of NAAT chemistry in paper pads to develop a shelf-stable product. In addition to developing FLIPP-NAAT, a first of its kind stoichiometric and pseudo-kinetic model has also been developed for LAMP. This is the first model which can predict concentrations of different amplicon types and help researchers design informed experiments for sequence-specific DNA detection strategies.

In conclusion, my PhD research reports significant advancement in development of a paper-based ASSURED diagnostic tool and demonstrates its successful application for TB diagnosis. Furthermore, contributions have been made to improve the fundamental understanding of technologies used in building ASSURED molecular diagnostics.