Research Project
7483737
[unreadable] DESCRIPTION (provided by applicant): Preventing the spread of multidrug-resistant and extreme multidrug-resistant strains of Mycobacterium tuberculosis (MDR/XDR) is an important public health problem. Because of the slow growth of Mycobacterium tuberculosis, it is currently impossible to obtain timely and accurate antibiotic susceptibility data for up to several months after a positive diagnosis has been made. This delay substantially increases the risk of spreading the disease. Ironically, the mechanisms of resistance to the most important drugs to treat Mycobacterium tuberculosis are well understood and should be amenable to determination by rapid molecular methods. However, development of a rapid and accurate molecular test for drug resistance in Mycobacterium tuberculosis has proven to be elusive because 1.) Mutations that mediate drug resistance are dispersed across several regions of over a dozen genes. 2.) Molecular determination of drug resistance must be nearly 95% accurate in order to be useful, therefore the entire "resistance space" must be covered, and 3.) Emerging new drug resistance mutations occur more quickly than molecular methods can be developed, validated, approved, and marketed. In our Phase I program, we successfully demonstrated that these hurdles can be overcome using a powerful new methodology that couples multiplexed PCR reactions with electrospray ionization mass spectrometry (PCR/ESI-MS) and high resolution base composition analysis of the amplicons. This technology, previously known as TIGER and currently sold commercially as the Ibis T5000, offers a high throughput and cost effective solution to the conundrum of keeping up with the drug resistance of an evolving microbe. In Phase I we proved this principle by analyzing several key "hot spots" in drug-resistance genes. In this analysis we successfully identified approximately 95% of the mutations known to correlate with drug resistance. In the Phase II effort we propose to expand this paradigm to achieve the theoretical maximum sensitivity for a molecular method for determining the drug-resistance for all important TB drugs. We solved the conundrum of keeping up with evolving mutations by adopting an "inside/outside" strategy. In the base assay, we comprehensively cover the area inside the hot spots so that new mutations within these regions are automatically detected without having to anticipate them and change the assay. New drug-resistance mutations that occur outside the hot spots will be covered by leaving expansion space in the assay so that new primer pairs can be rapidly added without disturbing the rest of the assay. We also propose to develop an inexpensive and simple sample preparation protocol and will validate the expanded assay by screening approximately one year of TB samples (about 750 isolates) from the New York City Department of Health, where drug resistance will be confirmed by conventional culture methods. The deliverable of this Phase II application will be a validated product that identifies drug resistant and MDR/XDR TB at a level suitable for public health action and ready for a prospective human clinical trial. PUBLIC HEALTH RELEVANCE: Preventing the spread of multidrug-resistant and extreme multidrug-resistant strains of Mycobacterium tuberculosis (MDR/XDR) is an important public health problem. We propose to develop and validate a product that will determine the drug-resistance profile of a Mycobacterium tuberculosis strain within hours of obtaining the culture, as opposed to months with the current technology. The product will be validated at a level suitable for immediate use by public health officials, and it will be ready to be tested in a human clinical trial to prove that physicians can reliably use this product to direct appropriate antibiotic treatment for TB patients. [unreadable] [unreadable] [unreadable]
