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Pakistan-US Science and Technology Cooperation Program                                                            
Phase 4 (2009 Deadline)

Rapid Detection of Infection and Drug Resistance in Tuberculosis Patients by Multiplex Analysis 

Imran Khan, University of California, Davis
Waheed Akhtar, Punjab University and Allama Iqbal Medical College
Pakistani Funding (Department of State): $262,342
US Funding (Department of State): $267,360
Project Dates on US Side: November 15, 2010 - November 14, 2013 (Extended through September 30, 2014)
 
Project Overview
 
According to a World Health Organization study in 2009, Pakistan is ranked eighth in tuberculosis (TB) prevalence worldwide. Over the last decade the country has seen a substantial increase in drug resistant cases, which does not bode well for Pakistan’s already overburdened healthcare system and its economy in general. Methods for accurate and cost-effective diagnosis of infection with Mycobacterium tuberculosis (M. tb.), as well as rapid detection of multiple-drug resistance (MDR) strains, are essential for controlling the spread of TB in the population.It is critical to detect both infection and drug susceptibility of M. tb. in new and retreatment cases as early as possible for effective treatment. Current methods have various limitations in terms of sensitivity, accuracy, cost-effectiveness, and time-to-result. The main goal of this project is to build healthcare capacity in Pakistan by developing new multiplex microbead diagnostic methods for detecting TB infection and drug resistance. Importantly, this research project aims to develop procedures to detect M. tb. infection and MDR strains in one day, as opposed to several weeks required using conventional methods. The researchers involved in this project estimate that the proposed new test will cost about $1, in contrast to culture-based tests, which cost $50-90 per patient in Pakistan. During the course of this collaborative research, several Pakistani graduate students, researchers, and technicians will receive training in the multiplex diagnostic methods to be developed and optimized at UC Davis and subsequently transferred to Punjab University. The project team will produce a detailed laboratory manual for standardized implementation of multiplex M. tb. detection in TB clinics in Pakistan to ensure that their findings are widely disseminated and applied.
 
Quarterly Update
 
During the past year, the research work focused on collecting DNA samples from patients with MDR TB in Pakistan with increased sample collection from Gulab Devi Chest Hospital, Lahore. These samples were analyzed using the newly developed molecular methods in Dr. Khan’s lab. Ms. Aasiq Khaliq, a female graduate student from Dr. Waheed Akhtar’s lab, successfully completed her training in the new molecular methods at UC Davis. She returned to Pakistan in August 2013 and is now actively involved in establishing the methodologies in Dr. Akhtar’s lab.
 
Dr. Khan has made two trips to Lahore, Pakistan to meet with the Pakistani team. He visited the hospitals where sample collections are ongoing to meet with the TB doctors and discuss the project. During his trips, Dr. Khan also worked with graduate students and staff in the laboratories of Drs. Waheed Akhtar and Noshin Yusuf for their scientific and technical education. His visits to Pakistan have enabled assessment of the project, planning for the next phases and training of personnel. In addition, these visits have enabled designing and development of standard protocols for sample collection, preparation, storage and shipping to UCD. These protocols will continue to evolve in the light of future experience and future visits by Dr. Khan. The PIs report that the experimental work being done under this project shall lead to the establishment of procedures for multiplex PCR and detection of polymorphism in the specific genes related with first line drugs (i.e. rifampicin and Isoniazid). PhD students were involved and trained in the project, which improved their skills in molecular diagnostics. The multiplex microbead immune assay based on protein-protein and DNA homologies has been introduced in the institution of Lahore for the first time through this project. Two pieces of multiplex equipment were purchased and installed successfully in Pakistan: at the School of Biological Sciences and at Gulab Devi Hospital. Development of this technology shall be helpful in the analysis of wide variety of the proteins and DNA sequences that can act as diagnostic biomarkers for the disease. At the same time, the team is working hard to develop linkages with industry to explore commercialization of the molecular diagnostic tests developed in the project. In September 2013, the Pakistani PI Dr. Akhtar visited Washington, DC, to attend a conference on “Biomarkers for Tuberculosis”, and the UC Davis laboratory. During the Washington visit, NAS program staff met with both PIs (a rare opportunity) to discuss project progress and other programmatic issues. 
 
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