TB-UNMASK: SEQUENCING THE AIR AT THE POINT-OF-CARE TO DIAGNOSE TUBERCULOSIS

TUBERCULOSIS (TB) IS AMONG THE TOP TEN CAUSES OF DEATH WORLDWIDE. ACCURATE DIAGNOSTICS THAT LEAD TO PROPER DRUG TREATMENT REGIMES PLAY, ALONG WITH A THOROUGH SURVEILLANCE, A KEY ROLE IN THE CONTROL OF THE DISEASE. WHOLE GENOME SEQUENCING (WGS) HAS DEMONSTRATED TO BE A PROMISING TOOL FOR THE CONTROL OF TUBERCULOSIS, BEING CAPABLE OF SIMULTANEOUSLY PREDICTING DRUG RESISTANT PHENOTYPES AND ESTABLISHING DETAILED EPIDEMIOLOGICAL RELATIONSHIPS. IN THIS PROJECT, WE AIM TO CAPTURE AND SEQUENCE TUBERCULOSIS FROM AIR DROPLETS RELEASED BY TB INFECTED PATIENTS, INSTEAD OF THE TRADITIONAL SPUTUM SAMPLE. BACILLARY LOAD IS EXPECTED TO BE LOWER IN AIR DROPLETS THAN IN SPUTUM, YET IT PRESENTS SEVERAL ADVANTAGES, CONTAINING MUCH LESS AMOUNT OF HUMAN DNA, INHIBITORS, AND BEING MUCH MORE HOMOGENEOUS AND CLEAN SAMPLES. THIS, COUPLED TO TB ENRICHMENT PROTOCOLS DEVELOPED IN OUR LABORATORY DURING SAF2013, WILL ALLOW US TO ACHIEVE UNPRECEDENTED SENSITIVITY PERFORMING WGS IN RESPIRATORY SAMPLES. REMARKABLY, SEQUENCING TB FROM AIR DROPLETS, WILL ALLOW ACCESS TO THE GENOME OF THE INFECTING AGENT THAT IS BEING TRULY TRANSMITTED, OPENING NEW PATHS TO THE RESEARCH AND UNDERSTANDING OF TB EPIDEMIOLOGY. PARTICULARLY, THIS WILL MAKE A GREAT DIFFERENCE REGARDING PATIENTS WITH LIGHT SYMPTOMS, FOR WHOM OBTAINING A SPUTUM IS NOT POSSIBLE. WE WILL ACHIEVE THIS BY USING A SIMPLE AND AFFORDABLE DEVICE, WHICH CONSISTS IN A MODIFIED MASK WITH RETENTION FILTERS THAT WILL CAPTURE THE EXHALED BACILLI DURING PATIENT BREATHING AND COUGHING. AFTER APPLYING OUR TB ENRICHMENT METHODS, WE WILL DECIDE TO EITHER, PROCEED WITH DIRECT WGS, OR FIRST PERFORM A WHOLE GENOME AMPLIFICATION. IN ORDER TO MAKE THIS DECISION, WE WILL DEVELOP A MULTIPLEX QPCR ASSAY, CAPABLE OF QUANTIFYING BACTERIAL, HUMAN AND TB DNA IN A SINGLE REACTION. IF SUCCESSFUL, THIS DEVICE AND MOLECULAR PROTOCOLS WOULD BE EASILY IMPROVED AND DEVELOPED FOR A PATENT, ALLOWING THEREFORE AN EASY TRANSFERENCE TO DEVELOPING COUNTRIES DUE TO ITS MINIMAL COSTS