The co-infection of HIV and Mycobacterium tuberculosis (Mtb) has been termed the "deadly duet" and in 2010, among the 8.8 million incident cases of tuberculosis (TB), 1.1 million were co-infected with HIV. Tuberculosis (TB) remains the number one bacterial killer as every 20 seconds a human dies of this continually forgotten pandemic. As a result of the co-infection with HIV, the rise in multidrug resistant Mtb strains and the very sparse drug pipeline, successful treatment of TB has become problematic. The current TB treatment regimen for TB is 2 months of rifampin, isoniazid, ethambutol and pyrazinamide (PZA), followed by 4 months of rifampin and isoniazid. The hallmark of this "short-course" protocol is the presence of PZA, a pro-drug that has been shown to improve sterilization when used in the initial phase of treatment. PZA has also proven beneficial in recent clinical trials with new TB drugs as "treatment arms" that include PZA sterilize more quickly and shorten the overall course of therapy. However, the clinical susceptibility testing against PZA has always been inconsistent and many laboratories in the US and in countries with a high burden of TB and HIV do not even test and treat empirically. This has proven to be a clinical nightmare as recent studies from South Africa and have reported over 65% resistance rates associated with MDR tuberculosis cases. This finding and the desire to move new TB drugs through clinical trials have elevated the urgency of developing a rapid and accurate molecular approach to determine PZA susceptibility. It is now well accepted that the target for PZA resistance is the gene pncA which encodes for pyrazinamidase, and enzyme that converts the pro- drug PZA to its active form, pyrazinoic acid. It is believed that mutation in pncA will correlate with PZA resistance. An assay to identify the pncA mutations has been proposed to predict PZA resistance, similar to the approach being used to detect rpoB mutations to predict rifampin resistance. However, this is currently not feasible, as there are over 100 non-synonymous mutations identified that span the pncA gene and we do not know which genetic changes correspond with resistance. The present challenge, and the focus of this proposal, is to overcome these hurdles by taking an innovative molecular approach to screen for the wild type pncA gene as a predictor of PZA susceptibility.
In Aim 1, in collaboration with the CDC, we will build a comprehensive M. tuberculosis clinical strain collection curated around the pncA genotype.
In Aim 2, we will use genotyped DNA from the pncA collection to both develop and rigorously compare two molecular platforms: high resolution melt curve analysis and lights-on/lights-off probes to distinguish the wild type pncA gene from those with genetic alterations and accurately determine PZA susceptibility in less than 2 hours.
Routine pyrazinamide (PZA) susceptibility assays are slow and inaccurate and molecular approaches are needed to improve the quality and speed of the testing and improve patient care. Mutations in the M. tuberculosis pncA gene are believed to correlate with resistance and similarly, the wild type pncA gene predicts a susceptible strain. We will pursue the use of high resolution melt curve analysis and lights-on / lights-off technologies, both surrogates of direct DNA sequencing, to distinguish the wild type pncA gene from those with mutations and accurately predict PZA susceptibility in <2 hours.