Inexpensive multiplexed ligation probe assay to genotype drug resistant M.tubberc

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DESCRIPTION (provided by applicant): Diagnosis of tuberculosis (TB) is the weakest link in global TB control and drug resistance complicates the selection of an appropriate treatment course. The most widely used diagnostic test, sputum smear microscopy, is cumbersome, insensitive, involves multiple patient visits and cannot detect multi drug or extensively drug resistant tuberculosis (MDR/XDR-TB). Sputum culture is costly and takes 4-8 weeks to detect TB and up to 16 weeks to detect MDR/XDR-TB. In mid-2011, the WHO issued a report that contains strong cautions on the use of commercial serodiagnostic test for the diagnosis of tuberculosis (WHO, 2011). There is an urgent need for a diagnostic test to simultaneously detect the presence of Mycobacterium tuberculosis (MTB) in patient samples and identify many mutations involved in MDR/XDR-TB. Our objective is to develop a multiplex ligation probe assay (MLPA)-based MTB detection and MDR/XDR mutation identification assay that can be used in moderately equipped clinical labs (the MLPA-XDR). This assay would be rapid, sensitive, specific and user-friendly, use accessible equipment and provide a simple to understand drug resistance genotype. The steps in the assay would be: (1) addition of an internal process control (such as quantified spores for extraction control) followed by standard sputum treatment and DNA isolation, (2) division of isolated DNA into MLPA reaction chambers containing prepackaged hybridization probe cocktail and process controls, (3) post- hybridization addition of ligase, (4) post-ligation PCR amplification after the addition of polymerase and two common PCR primers, and (5) imaging and image interpretation for the detection of MTB and mutations. Investigen has developed: (1) sputum processing protocols, (2) ultra-specific signature regions for MTB detection and regions of interest in drug resistance, and (3) a small set of MLPA hybridization probes that discriminate single base changes in rpoB. There is a high likelihood of success as key components have been demonstrated and the pieces simply need to be put together. This will be accomplished in three specific objectives: (1) Design MLPA LHS and RHS probes. (2) Test probe sets. (3) Test on clinical MTB and sputum samples. Investigen and collaborators have domain knowledge in MTB detection and MDR/XDR - TB mutation analysis, instrumentation/consumables prototyping, assay development, developing world product placement and global TB research agenda and treatment goals. A fuller, inexpensive MDR/XDR-TB genotyping system would greatly improve MDR/XDR-TB diagnosis and treatment. In the Phase I we intend to develop the MLPA-XDR with probes for 100 mutations. Eventually, this may be divided into a two-step approach. First an inexpensive survey of 30 or 60 common drug resistant mutations followed by an "as needed" further survey of 360 less common drug resistant mutations. Accordingly, the project is relevant to the NIH mission of reducing the burden of illness through supporting research for the improved diagnosis, prevention and cure of human diseases. The development of technologies for the easy/rapid detection of MDR/XDR-TB is a NIAID biodefense research priority. NIAID goals for TB include the development of new and improved diagnostic tools to more accurately diagnose early TB disease, help optimize therapy by identifying drug-resistant strains, and track the spread of TB in a community. If feasibility is shown (milestones in specific aims), the phase II objectives are anticipated to include; (1) instrument and consumables concepts that are geared to automation (lab on a chip), (2) inclusion of dUTP for potential amplicon contamination control, (3) determining final format and mutant panel, (4) further performance validation, (5) assay volume reduction, and (6) and assay performance and guard band optimization/characterization. PUBLIC HEALTH RELEVANCE: The feasibility of a robust method to identify multidrug resistant and extensively drug resistant tuberculosis will be explored. If successful, this can hel the global effort to control TB and prevent the spread of drug resistance.