| dc.description.abstract | Tuberculosis (TB) remains a major cause of infectious disease death worldwide, particularly in developing countries. One major barrier to TB elimination is a lack of effective diagnostic tools available for use at the point-of-care (POC). Despite its poor diagnostic sensitivity, century-old microscopic examination of sputum smears remains the most common diagnostic method. Furthermore, increases in multi-drug resistant TB cases highlight the urgent need for improved diagnostic tools available at POC.
To begin addressing the lack of molecular diagnostics available at POC, a magnetic bead-based nucleic acid extraction (NAE) assay enabled by high-gradient magnetic separation was developed, performing comparably to two commercial NAE kits for detection of cell-free TB DNA from urine and sputum. This method also worked with large-volume samples, which is universally a limiting factor of commercial kits. Additionally, this technique was featured in a total NAE challenge using pathogen-spiked blood, sputum, and stool with the Gates Foundation, demonstrating applicability with other infectious diseases.
To address the poor diagnostic sensitivity of sputum smear microscopy, I designed an assay using microfluidic flows in evaporating droplets to spatially concentrate M. bovis BCG following volumetric magnetic enrichment, concentrating samples over 100-fold. Optical coherence tomography confirmed that inward-oriented Marangoni flows were responsible for spatial enrichment. This combined enrichment method improved the limit-of-detection for sputum smear microscopy by 10-fold compared to direct smear while using inexpensive reagents already available at POC.
Diagnostic development begins with creating artificial samples derived from pathogens grown in vitro. Currently there is no information on the impact these required preparation methods have on surface marker accessibility of M.tb before use in assay development. To address this, novel molecular recognition elements that bind different structural features of M.tb. surface antigen lipoarabinomannan (LAM) were characterized and then used to investigate the impact of mechanical or enzymatic processing on LAM accessibility. Mechanical dissociation recovered more M.tb bacilli from clumps formed during bacterial culture, and yielded greater LAM accessibility than unprocessed or enzymatically digested bacilli.
While there is still significant work to be done, the advancements made here for POC TB diagnosis in low-resource settings are an important step towards global TB elimination. | |
