Tuberculosis is a devastating disease of mankind resulting in 3 million deaths per year. There are 10 million new cases of tuberculosis worldwide, and after a 32- year decline, the number of new cases is now increasing dramatically in the United States with a nearly 10 percent increase in the number of AIDS patients since HIV both reduces immunity against Mycobacteria tuberculosis infection and activates existing, latent infections. Although there are effective chemotherapeutic approaches for tuberculosis, rapid diagnosis is important for effective treatment and current methods take from two to six weeks. Moreover,there are increasing reports of the apparent appearance of drug resistantstrains of M. tuberculosis and the determination of drug susceptibilitiessometimes takes too long to be of use to the patient. The goal of this project is to develop a rapid, sensitive and specific tool for the diagnosis of tuberculosis and rapid assessment of drug sensitivities, using diagnostic reporter bacteriophages. Diagnostic reporter phages will be constructed by insertion of genes encoding luciferase enzymes expressed from mycobacterial-specific signals into mycobacteriophage genomes. When these recombinant phage infect mycobacteria, the luciferase enzyme is expressed and can be detected with an extremely sensitive assay for the production of photons in a luminometer. This assay is very sensitive and should be able to detect fewer than 100 live M. tuberculosis bacilli in a clinical sample. The need for cultivation is thus eliminated and diagnosis can be performed in a few hours rather than a few weeks. Moreover, the response of antibiotics can be monitored at the same time. Specificity of the assay will be generated through the construction of several reporter phages that have different, but well defined host ranges. The phages used will include the well-characterized L5, TM4, and a number of novel uncharacterized phages. Phage L5 offers particular advantages as a model for gene expression following phage infection and for the construction of alternative cosmid vectors.
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