Rapid Detection of Multiple Drug resistant TB from Sputum

DESCRIPTION provided by applicant Tuberculosis TB is a leading cause of mortality from an infectious disease in the world second only to HIV AIDS The WHO reports that in there were M deaths from TB When a case of TB does not respond to two or more of the first line drugs available the infection is considered to be multidrug resistant MDR TB The rising incidence of MDR TB is leading to poor patient outcomes since the second line drugs are introduced at a late stage of the disease These drugs are expensive require injection and have serious side effects therefore cannot be administered before a diagnosis of MDR TB occurs The majority of TB cases are seen at peripheral health clinics with no access to current diagnosis technologies Early diagnosis before the failure of the first line drugs is needed for effective treatment as well as for isolation of infected individuals We propose to develop a test that will detect the known Mycobacteria tuberculosis alleles that are found in of resistance to the main first line drugs Rifampicin and Isoniazid using our hand held battery powered TangenDx Point of Care POC diagnosis system TangenDx consists of a sample preparation device which is followed by mycobacterial capture via filtration at which point lysing and amplification solutions are introduced Lysis is performed by sonication after which the sample is introduced into a disposable assay disk for DNA extraction and amplification using isothermal amplification and real time detection via fluorescent intercalating dyes The assay amplifies the hspx gene which is unique to MTB A built in precise temperature controller enables the use of the highly robust and versatile loop mediated isothermal amplification LAMP STEM primers are used with the LAMP technology to accelerate reactions We were able to detect CFUs of TB per disk assay or genomic copies with andgt confidence We propose to add DST to our TangenDx TB Assay to enable detection of of all known MDR TB strains by following these specific aims In Specific Aim LAMP primer sets specific to the three genetic locations associated with resistance to rifampicin and isoniazid will be designed and screened STEM primers to each of the mutant alleles will be designed and tested for their ability to selectively accelerate LAMP reactions using synthetic DNA targets Finally we will determine the conditions that allow amplification and multiplexing of the three MDR TB genetic regions rpoB katG and inhA in the central chamber of the Tangen Dx Disk prior to STEM specific acceleration of specific LAMP reactions in the detection chambers In Specific Aim the optimized primer sets and conditions of Aim will be tested with the whole TangenDx TB Assay system using characterized drug resistant TB strains spiked into healthy human sputum samples The proposed system would make quick MDR TB diagnosis accessible and feasible in regions of the world where such technologies are lacking and where TB is most widely spread A timely diagnosis with an accurate DST evaluation could significantly lower TB associated mortality and costs of care worldwide PUBLIC HEALTH RELEVANCE Tuberculosis TB is a leading cause of mortality from an infectious disease in the world second only to HIV AIDS Since the second line drugs are introduced at a late stage of the disease the rising incidence of drug resistant TB is leading to poor patient outcomes Early diagnosis before the failure of the first line drugs is needed for effective treatment as well as for isolation of infected individuals We propose a test that will detect TB drug resistance using our hand held battery powered TangenDx Point of Care POC diagnosis system