Solar Powered Field Kit for the Diagnosis of Tuberculosis - Research Paper Example

? Solar powered field kit for the diagnosis of tuberculosis July 17, Approximately one third of the world population is infected with the bacterium Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), and millions die each year from the dreaded disease. Much of this population is in remote locations in Africa and South America. Current methodologies for the diagnosis of TB rely on labor intensive and delayed laboratory analysis of blood samples. A field kit is needed to diagnose patients in these remote locales that does not rely on extensive laboratory analysis and the utility of electrical power. It is proposed that a field kit be developed for the diagnosis of TB that utilizes a hand-held fluorometer powered by energy from the sun. The biochemical principal for the kit will be based on antibody capture of M. tuberculosis membrane constituents isolated from patient blood samples and detection of a secondary fluorescently-labeled molecule. Blood samples will be analyzed in a microtiter dish and processed by a series of simple salt solution washes in the field. The same microtiter dish will be placed in the solar-powered, hand-held fluorometer. Diagnosis of the presence of M. tuberculosis will take merely minutes instead of weeks, and an appropriate antibiotic regime can be initiated by a field physician immediately. This will save valuable time in combating the infection, and will significantly reduce costs associated with the diagnosis of TB. The company profiled is Genzyme Worldwide, a biotechnology giant specializing in prenatal diagnostics, therapeutics, cancer, and infectious disease. Genzyme has the financial backing to develop and market the product on a global scale. Introduction. The company in profile is Genzyme Corporation, one of the leading biotechnology companies in the world. Genzyme is headquartered in Cambridge, MA, USA, but also has operations in Europe and other locations across the world. Genzyme has over 12,000 employees and generated revenue in excess of 4.5 Billion dollars in 2009. Genzyme specializes in diagnostics, therapeutics, cancer research, and infectious disease. The diagnostics division of Genzyme has facilities which specialize in prenatal diagnostics, with tests offered in fetal genetic screens for Downs Syndrome (Trisomy 21), Tay Sach’s Disease, and Gaucher’s Disease. Genzyme also conducts national and international clinical trials for novel diagnostic tests, which enable the corporation to continually analyze and validate new technology on the cutting edge of life science research. The biochemical principals for how the kit will work are straight forward. A monoclonal antibody specific for a unique membrane component of M. tuberculosis will be developed and utilized as a “capture” molecule lining the plastic wells of the microtiter dish. The antibody is the key to sensitivity and signal amplification of the assay. The antibody must also be stable as a freeze dried powder subsequently sterilely packaged and transported at ambient temperature to the field. M. tuberculosis has a myriad of “sugar-lipids” in the bacterial membrane (Stanier et al, Springer et al, Master et al, Zahrt et al), contributing to the specific nature of the kit. As the antibody molecules attach to the bacterial membrane component, they will be unable to bind to a secondary antibody specific to the capture antibody. The secondary antibody will be conjugated to a fluorescent marker which will be detected by the fluorometer (www.RnDsystems.com). The more bacteria present in the microtiter dish, the more reduction of the fluorescent signal. Thus, a bright signal indicates the absence of the bacteria, and a dim signal is indicative of a high M. tuberculosis load. The solar powered fluorometer will also be simple in design. The instrument will run on solar batteries similar to those present in a calculator. The energy will be used to generate a specific wavelength of visible light to excite the fluorophores in the assay. The fluorescence will be detected in a “light tight” chamber and converted into a readout number. Fluorescence is much more sensitive than a colorometric reaction, as a few hundred photons of light can be easily detected (Hect, E.), indicative of a large number of bacteria present. The fluorometer will be about the size of a shoebox, and will be easily transported in a common backpack. Development of the monoclonal antibody will be as follows. Laboratory cultures of M. tuberculosis will be utilized to isolate and purify the lipopolysaccharide that elicits the most pronounced immune response in mice. Cultures of M. tuberculosis will be lysed and lipids will be fractionated and isolated utilizing standard liquid chromatography. The molecule of choice will be injected into mice, and the mice will be euthanized after an incubation period to isolate a lymphocyte cell line that produces the monoclonal antibody. Cell cultures will be stored at -70? C, and a subset will be cultured in mass to isolate and purify the antibody. Antibodies will be tested for robustness to freeze drying and subsequent hydration and binding to M. tuberculosis lipopolysaccharide. The antibody of choice will be purified by affinity column filtration in bulk, and subsequently utilized to coat plastic microtiter dishes for the kits. Field physicians will draw venous blood from patients and transfer the whole blood to a plastic test tube. The blood will be lysed with a sodium hydroxide-SDS solution and transferred to wells in the M. tuberculosis microtiter plate to allow binding of the bacterial membranes to the capture molecules (Fig 1). After incubation to promote binding of bacterial membranes to capture molecules, microtiter plates will be rinsed with saline with a manual micropipetor to remove constituents from the wells. Blood from a total of nine patients can be simultaneously analyzed in one plate. The plates are eight wells by twelve wells for a total of ninety-six wells. The first row of wells will be utilized for background noise, the second row of wells will be used for positive control, and the third row of wells will be designated for negative control. The rest of the wells will be devoted to patient samples, each patient consisting of eight well replicates. The readout from the fluorometer will be an average of the eight wells minus background noise. Each plate will contain results for positive and negative controls to ensure the assay is working properly. The positive control will consist of lipopolysaccharide purified from laboratory M. tuberculosis cultures. All reagents, plates, and blood tubes will be disposed of in a biohazard bag and incinerated at a later date. A training class will be offered in combination with the kits to demonstrate to field physicians how to use the kits. Basic principles of ELISA technology and laboratory practice utilizing the kits for diagnosis will be included in the course. Physicians will use laboratory cultures of M. tuberculosis in human blood to learn how to lyse biological samples, process the samples in the kits, obtain data, and use data to determine infection. The course will be one week in duration, with lectures in the mornings on microbiology, biochemistry, and fluorescence, and laboratory exercises in the afternoons. The cost of the kits will be kept as low as possible to insure world-wide distribution. The fluorometer will cost approximately $1000 US dollars, and each kit will sell for about $100 US dollars. The fluorometer will be a one-time purchase, and the kits will be a disposable and renewable resource. The large scale distribution of the kits will result in a sizable and sustainable profit for Genzyme (www.genzyme.com), as well as saving millions of lives per year in isolated regions of the world. Initial distribution of the kits will be targeted to agencies including the Peace Corp, the American Red Cross, the US military, and other world health organizations, but distribution of the kits will eventually be available for any trained physician regardless of location. It is anticipated that the combination of solar power and specificity and sensitivity of the assay will generate a large demand for the kits, and will be key elements in battling the fight against tuberculosis. It is the hope of this project that TB will be a disease of the past, and that efficient and accurate administration of antibiotics will eradicate the dreaded disease from the human population (www.who.com). References Stanier, R., Ingraham, J., Wheelis, M., Painter, P. The microbial world. 1986. 689 pp. Prentice-Hall, New Jersey. Springer, B.; Master, S.; Sander, P.; Zahrt, T.; McFalone, M.; Song, J.; Papavinasasundaram, K. G.; Colston, M. J.; Boettger, E.; Deretic, V. Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation; Infect. Immun 2001. 69, p5967-5973. Master, S.; Zahrt, T.C.; Song, J.; Deretic, V. Mapping of Mycobacterium tuberculosis KatG promoters and their differential expression in infected macrophages; J. Bacteriol.; 2001. 183, p4033-4039. Zahrt, T. C.; Song, J.; Siple, J.; Deretic, V. Mycobacterial FurA is a negative regulator of catalase-peroxidase gene katG; Mol. Microbiol.; 2001. 39, p1174-1185. www.RnDsystems.com Hect, E. Physics. 1994. 1164 pp. Brooks/Cole, California. www.genzyme.com www.who.org Read More