Chemical and Biomolecular Engineering, Department of


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A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Interdepartmental Area of Engineering (Chemical and Biomolecular Engineering) Under the Supervision of Professor Hendrik J. Viljoen. Lincoln, Nebraska: May, 2010
Copyright (c) 2010 Elsje Pienaar


Tuberculosis is an ancient and worldwide epidemic affecting millions of people in mainly the developing world, killing almost 2 million people in 2008. Current diagnostic techniques are outdated and have proven insufficient to control the disease. Smear microscopy has poor sensitivity and culture is slow to yield results. Modern diagnostic techniques are making great strides in shortening time to result but are restricted by two qualities: 1) prohibitively high costs prevent implementation in resource poor areas, and 2) equipment and technician requirements limit application to centralized laboratories. There exists a divide between new technologies and the people that need them most. Here, a novel epidemiological model of tuberculosis in an urban community confirms the importance of improved diagnostics in lowering prevalence. The model highlights the importance of sensitivity and accessibility.

This work presents the development of a nucleic acid amplification test for tuberculosis diagnosis from sputum. The prototype system consists of 1) a sputum processing unit capable of extracting DNA within 5 minutes, and 2) a rapid PCR thermocycler which amplifies Mycobacterium tuberculosis complex specific sequences (IS6110 and IS1081) in under 15 minutes and detects product in real-time. Lysis protocol development was guided by a combined theoretical/experimental analysis of the kinetics of heat lysis of Mycobacterium smegmatis. The analysis revealed the activation energy of lysis (22.1 kcal/mole) and the minimum cell wall damage that result in cell distruction (14-17%). The PCR is capable of amplifying template amounts below smear microscopy concentrations.

The test was applied to 58 clinical samples from the Steve Biko Academic Hospital in Pretoria, South Africa. Sensitivity was 95% on smear positive culture positive samples and 70% on smear negative culture positive samples. Specificity was 86%.

In summary, the test moves toward an important niche of rapid (less than 30 minutes) and affordable ($5-10) diagnosis in a peripheral setting. Sensitivity of the test is comparable to other available systems, while specificity still needs improvement. However, turnaround times and costs are far below other tests currently being developed.