The global burden of tuberculosis (TB) stands at ~9 million cases per year; and South Africa is the most heavily TB-burdened country globally. TB is also the most common opportunistic infection in HIV-infected persons and it is responsible for 21% of deaths in South Africa. A program of active TB case finding and drug resistance typing in both urban and rural communities is needed to disrupt TB transmission, yet we currently lack accessible point of care diagnostics to achieve this goal. Particularly in rural South Africa, where TB infection is widespread, microscopic examination of a sputum smear is the most accessible method for detection of TB. Two major challenges are 1) the more accessible microscopy methods have a current limit of detection of 10,000 bacilli per ml of sputum which prevents early detection and allows continued spread of infection, and 2) no methods using bright field microscopy are available for detecting drug resistant Mycobacterium tuberculosis genotypes. In this proposal Vanderbilt University and The University of Cape Town form a partnership to develop and test bright field microscopy methods for diagnosing TB from sputum. We propose to develop simple methods to improve the limit of detection of existing and more accessible bright field microscopy of sputum samples to 100 bacilli/ml. Our approach is based on a magnetic concentration strategy and high density deposition onto a standard microscope slide using the coffee ring phenomenon (Aim 1). Secondly, we propose to develop a dendrimer-genotyping probe followed by a silica condensation reaction to enable bright field identification of drug resistant phenotypes (Aim 2). These laboratory designs are then packaged into a simple kit format for initial clinical testing of retrospective sputum samples in a laboratory as well as in rural South Africa (Aim 3). When completed, these simple modifications to the current widely accessible microscopy methods will identify individuals with low numbers of M. tuberculosis bacilli present in their sputum and enable much earlier treatment. In addition, enabling the identification of drug resistant bacilli by bright field microscopy will provide an earlier means to begin more aggressive therapies and further prevent the spread of drug-resistant TB.

Project Narrative In many settings, inspection of a stained sputum sample under a microscope remains the primary diagnostic tool for tuberculosis. We propose a simple modification of this well-established procedure to increase its sensitivity and extend its capabilities to detect drug resistant strains. Our approach is based on a magnetic gathering of tuberculosis bacilli from the total sputum volume and, using the microfluidics of “coffee ring” formation, concentrate any tuberculosis present into a pre-selected region of a slide for inspection by standard bright field microscopy.