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Phase 2 (2006 Deadline)
Multiplex Immunoassays for the Detection of Tuberculosis
Paul A. Luciw, University of California, Davis
Azra Khanum, University of Arid Agriculture, Rawalpindi (UAAR)
Pakistani Funding (HEC): $173,945
US Funding: $189,500
Project Dates on US Side: February 1, 2007 - January 31, 2009
Project Overview
The high prevalence of tuberculosis (TB) in Pakistan is a significant burden to the country’s healthcare system and its economy. Methods for accurate and cost-effective diagnosis of infection with pathogenic Mycobacterium tuberculosis (M. tb.) are essential for controlling the spread of this disease in the population. It is critical to detect both the earliest stage of infection and latent infection and to monitor the efficacy of therapy, but current methods have various limitations in terms of sensitivity, accuracy, and time-to-result. Building on recent advances in knowledge of mycobacterial genomics, proteomics, and immunology and in novel multiplex diagnostic instrumentation for infectious disease, this project was intended to build healthcare capacity in Pakistan by developing new multiplex diagnostic methods for detecting TB.
Major Results
- Developed a novel antibody profile-based TB diagnostic multiplex test for clinical applications
- Used the data from the above study to obtain funding from the World Health Organization (Eastern Mediterranean Regional Office) to perform a validation study of the antibody TB diagnostic test in Pakistan
- Provided one year of training at UC Davis to two Pakistani graduate students in genetic engineering, protein expression/purificatrion, and multiplex diagnostics
- Submitted a manuscript for publication (co-authored by Pakistani and US scientists), with two additional manuscripts in preparation
- Established collaborative infrastructure with TB clinics in Pakistan
- Obtained funding from the US State Department to develop a nucleic acid-based diagnostic test for the detection of multiple drug resistant TB in Pakistan, based on the experience with the successful development of a multiplex serodiagnostic test for TB
Progress Report Summaries
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2008 Show summary || Hide summary
Ms. Nawaz and another UAAR PhD student, Mr. Mirza Imran Shahzad, arrived at UC Davis in March 2008 for one year of training on multiplex immunoassay development. During their stay, Dr. Luciw’s lab received a large number of blood samples from Dr. Khanum, which represented a major achievement in view of the large number of institutional review board approvals that had to be obtained on both sides and the complicated requirements for the international shipment of biological specimens. By the end of June 2008, Dr. Luciw reported that he and his colleagues had already generated the first set of TB detection results from these blood specimens, and further study is ongoing.
As of November 2008 about half of the samples had been transferred to UC Davis for analysis. Although the US side of the grant has ended, the Pakistani side has reportedly been extended by HEC through the summer of 2009, and Drs. Luciw and Khan have found other sources of support to allow them to continue consulting with their Pakistani partners. In the coming months, the data collected so far will be used to compare the novel multiplex immunoassay diagnostic method with conventional methods for TB diagnosis and to determine the cost effectiveness of the new method. If these comparisons prove favorable, then a full clinical validation study can be done to attain the long-term goal of significantly improved TB diagnosis through implementation of the new multiplex diagnostic technology.
Harnessing the power of collaboration between scientists in Pakistan and the United States, this project applied recombinant DNA technology and the multiplex immunoassay technology to achieve its four objectives. The results achieved on each are listed below:
- Development and optimization of the Luminex/BioPlex multiplex system for detecting antibodies and cell-based immune responses to M. tb. and detecting M. tb. antigens: The basic method has been optimized at UC Davis and the results published in 2008 (Khan et al., 2008, Clin. Vaccine Immunol. 15:433-438).
- Installation of the Luminex/Bioplex instrument at UAAR and training of technical staff in multiplex detection of TB: The equipment has been purchased and in the spring of 2009 Dr. Khan will travel to UAAR to help install and calibrate it. In addition, the two students trained at UC Davis will also be returning to Pakistan in March 2009, so they will also be able to assist in training other students and researchers.
- Application of these novel multiplex assays for cross-sectional and longitudinal studies of TB patients at the National Tuberculosis Center in Rawalpindi: About one-half of the 290 samples had been analyzed at UC Davis by the end of 2008, with the rest to be completed in the coming months. Results will be published in a peer-reviewed journal.
- Preparation of a detailed laboratory manual and other training materials that can be used in Pakistan for detecting M. tb. infection by multiplex immunoassay: This portion of the project is being completed by Dr. Khanum and her colleagues at UAAR and should be finished, with input from the UC Davis partners, by the time their side of the grant ends in the summer of 2009.
2007 Show summary || Hide summary
In the course of this collaborative research, several Pakistani graduate students, post-docs, and research technicians were trained in the novel multiplex technology for infectious disease detection. Multiplex methods developed and optimized at UC Davis were transferred to Dr. Khanum’s research laboratory at UAAR. Importantly, for evaluation of the multiplex diagnostic system in the field, clinical samples from 290 M. tb.-infected individuals were provided by Drs. S.K. Shah and Sabira Tahseen at the National Tuberculosis Center in Rawalpindi.
By October 2007 two graduate students from Dr. Khanum’s laboratory, Ms. Irum Nawaz and Mr. Kumail Rizvi, had been trained at UAAR and subsequently became actively involved in collecting blood samples at the NTBC. Dr. Khan made a two-week visit to Rawalpindi September 26 to October 10, 2007, to (1) review procedures of collection, processing, and storage of patient blood samples, (2) monitor progress on expression of M. tb. proteins in genetically engineered bacterial vectors by Dr. Khanum’s laboratory personnel, and (3) assist in the purchase of the multiplex instrument.
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