CYTOMETRIC TUBERCULOSIS DETECTION AND SENSITIVITY TESTS

DESCRIPTION (provided by applicant): Tuberculosis (TB) remains one of the deadliest diseases, with more than 8 million new cases and approximately 3 million deaths worldwide annually. Since the 1980s, increased incidence of active TB in HIV-infected individuals and the emergence of drug-resistant strains of Mycobacterium tuberculosis have intensified the need for rapid methods to detect the organism in clinical specimens and determine whether it is susceptible to front-line drugs. In order for such methods to be usable in developing countries, it is desirable that the technology involved be as simple and inexpensive as possible. Until recently, neither flow nor image cytometry has been inexpensive enough to be considered as an option for implementing TB diagnosis and susceptibility testing in resource-poor environments. However, recent advances in electro-optic technology, notably high-intensity light-emitting diodes (LEDs) and efficient charge-coupled device cameras (CCDs), now make it feasible to produce simple automated fluorescence image cytometers that could be sold for well under $10,000. These instruments are applicable not only to TB detection and susceptibility testing, but also to such assays as CD4+ lymphocyte counting in HIV-infected patients and diagnosis and monitoring of malaria. This project will demonstrate the capacity of fluorescence cytometers to detect and count stained mycobacteria in the presence of eukaryotic cells and debris and other microorganisms, determine the effects of first-line drugs on growth kinetics, membrane permeability, membrane potential, and redox metabolism in sensitive M. bovis, M. smegmatis, and M. tuberculosis H37Ra, and evaluate the suitability of fluorescent nitrate reductase substrates for detection and quantification of mycobacterial strains expressing that enzyme.