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The National Academies
500 5th St NW - KWS 502
Washington, DC 20001
Tel: (202) 334-2800
Fax: (202) 334-2139
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)
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.
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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2011 Show summary || Hide summary || Download full report
Dr. Khan and his team at UC Davis continued to optimize the multiplex microbead assay they previously developed to detect mutations in the rpoB and katG genes, which are involved in drug resistance to two frontline TB drugs, rifampicin and isoniazid, respectively. They have initiated the work on developing multiplex assays for genes associated with resistance to streptomycin and ethambutol. During the last quarter they received the first shipment of DNA from 30 patient isolates of drug-resistant M. tb. collected by their colleagues in Pakistan. These samples will be analyzed over the coming months, and work will also focus on developing tests for mutations in the gene pncA, associated with resistance to pyrazinamide. Standard testing protocols are being shared with the Pakistani team as they are developed. Further shipments of DNA from patient samples are also expected as they are collected and processed by the Pakistani researchers. The rates of sample collection has been slower than expected due to delays on the Pakistani side that pushed back the schedule for hiring technical staff. The procurement process associated with upgrading the Pakistani lab has also moved more slowly than the participants would have liked, so several pieces of equipment are still on order, but the researchers are nevertheless proceeding with their work as best they can with the existing lab infrastructure.
2012 Show summary || Hide summary || Download full report
In November 2011, Dr. Imran Khan made his third visit to Pakistan on the project, during which he had the opportunity to meet with his Pakistani counterparts and work with them in selecting three hospitals for additional collection of TB samples. He and his colleagues visited two of those collection sites—Jinnah Hospital and Gulab Devi Hospital—to review their standard operating procedures and make any necessary modifications. The team also conducted a day-long national seminar on TB at the University of Punjab. The event was attended by scientists and clinicians from many leading institutes and hospitals such as the Aga Khan Medical University Karachi, the Punjab provincial TB program, and TB hospitals in Lahore.
Following Dr. Khan's visit, he and his team at UC Davis have continued working on multiplex PCR and multiplex mutation detection for rpoB and katG genes. Basic assays for the multiplex PCR for additional genes involved in resistance against the TB drugs ethambutol and pyrazinamide have been developed, and PCR for streptomycin resistance genes is under optimization. Previously, PCR assays for rpoB and katG have also been developed and optimized at UC Davis. On the Pakistani side, the rates of sample collection has been slower than expected due to bureaucratic delays that pushed back the schedule for hiring technical staff. However, development of new protocols and modification of the existing ones at the partner TB hospitals and research laboratories in Pakistan have helped to streamline the process. For the next few months, the Pakistani researchers will continue ship additional drug resistant samples of M. tb. patient isolates that will be analyzed by the U.S. team. Additional tests for the detection of mutations in pncA for resistance against the drug pyrazinamide will be developed. Dr. Khan and his colleagues at UC Davis will also continue to supply standard protocols to the Pakistani PIs as new methodologies are developed. Dr. Akhtar is expected to visit California in July, and Dr. Khan plans to make another visit to Pakistan in August.
The UC Davis team then continued working to optimize microbead multiplex detection of MDR mutations in genes conferring resistance to streptomycin and ethambutol. Control samples (extracted DNA) from drug sensitive M. tb. patient isolates have been shipped from Dr. Akhtar’s lab and have been analyzed, with the results matching with those of culture experiments. DNA samples isolated from MDR patients have also arrived recently and will be analyzed in the coming months. Recently, Dr. Akhtar visited UC Davis for a week to work with Dr. Khan. The PIs discussed the project status, current and future activities. A female graduate student from Dr. Akhtar’s lab is expected to arrive at UC Davis later this year to work with Dr. Khan for several months.
The rate of sample collection in Pakistan has been slow but steady, and additional shipments of DNA samples (more than 100) are expected to arrive at UC Davis. The US team will continue to supply standard protocols to the Pakistani PIs as new methodologies are developed. Dr. Khan is planning a visit to Pakistan in September 2012, during which he will coordinate the arrival and installation of the two new multiplex instruments recently ordered by Dr. Akhtar. Thanks to a recent decrease in the price of this equipment, he is able to afford two instruments for the same price that had been budgeted for just one. This will make it possible to expand the impact of the project significantly, as one of the instruments will be installed in Dr. Akhtar’s lab and the other will be rotated among high-volume TB hospitals to perform clinical diagnostic studies. Dr. Khan’s visit will also enable the implementation of new protocols and modifications of the existing ones in Pakistan. The new equipment plus the training to be provided by Dr. Khan should help to expedite the project.
2013 Show summary || Hide summary || Download full report
During spring 2013, DNA samples from over 50 Mycobacterium tuberculosis (M.tb) isolates from TB patients with multi-drug resistance (MDR) have been analyzed at UC Davis. The results from the analysis have shown that the new technology developed by Dr. Khan’s laboratory is accurate and efficient. In Jan 2013, the PIs attended the first program symposium on Economic Growth through Technology Transfer at the National University of Science and Technology (NUST). This project was featured and presented at the symposium. After the symposium, Dr. Khan visited Lahore after the symposium and helped with the placement and installation of two new instruments that were recently purchased under this program – one in Dr. Akhtar’s laboratory and the other for field study in Gulab Devi TB Hospital for Chest Diseases. During his visit, Dr. Khan also provided basic training to additional laboratory personnel.
Although the rate of sample collection in Pakistan is slow, it has been slightly improved and steady. Dr. Akhtar’s laboratory will continue collecting MDR samples and shipping these samples to UC Davis for further analysis. In May, Dr. Khan’s lab is expecting a female graduate student (Ms. Asia Khaliq) from Dr. Akhtar’s lab to arrive at UC Davis for training. Research work will continue on both sides.
After obtaining US visa, Ms. Aasiq Khaliq, a female graduate student from Dr. Waheed Akhtar’s laboratory, arrived at UC Davis at the beginning of June and started to be trained on the use of multiplex methodologies for the detection of MDR causing mutations by Dr. Khan. She is currently conducting analysis on the DNA samples from additional Mycobacterium tuberculosis (M. tb.) isolates from TB patients with multi-drug resistance (MDR) from Pakistan. Results of the analysis show that the new technology developed by Dr. Khan’s lab at UC Davis is accurate and efficient. Meantime, the US team is continuing the optimization of microbead multiplex detection of MDR mutations in genes conferring resistance to pyrazinamide and ethambutol; and the work on optimization will continue during the upcoming months. Additional MDR samples are being collected by Dr. Akhtar’s team and those samples will be shipped to UC Davis in the coming weeks and months.
In early August, Ms. Khaliq will complete her training in the US and return to Pakistan to practice what she learnt at UC Davis. Before she leaves UC Davis, she will start writing her Ph.D. thesis under Dr. Khan’s supervision. Upon her return to Pakistan, Ms. Khaliq will actively engage in teaching other students and lab technicians at Punjab University. The US team will continue to supply multiplex reagents and standard protocols to Dr. Akhtar’s team as new methodologies are developed, and as needed, for various technical aspects of the project.