Issue Date: March 19, 2007
Improving Diagnosis Of Tropical Diseases
THE FIRST STEP in treating a disease is diagnosing it. For tropical diseases in developing countries, such diagnoses aren't as easy as they should be. Many of these countries lack the trained personnel or reliable infrastructure to support complicated analyses. Simplified methods and equipment could improve such diagnoses. A symposium at Pittcon, held last month in Chicago, focused on efforts to develop better, cheaper methods for diagnosing tropical diseases. The American Chemical Society's Division of Analytical Chemistry organized and sponsored the symposium.
Helen Lee of the department of hematology at the University of Cambridge presented surprising statistics about the availability of basic supplies and infrastructure in developing countries. In a survey of African facilities, a small yet significant fraction lacked even the most basic resources, such as a reliable source of electricity and tap water (17% and 7%, respectively), she said. The availability of other supplies was worse, and 40% of facilities do not have incinerators to deal with medical waste.
Beyond the availability of basic resources, cost is a big issue in developing countries. For example, Lee said, the blood banks in Kumasi, Ghana, have a total annual budget of approximately $70,000, with 15% allocated to testing and 17% to consumables. In light of such meager funds, Bernhard Weigl, the group leader for the diagnostics development team at the Seattle-based nonprofit organization Program for Appropriate Technology in Health (PATH), said that his organization's goal is to lower the cost of each infectious disease test to no more than $1.50.
Infectious disease testing is important not only for diagnosing and treating patients but also for ensuring the safety of the emergency blood supply. African hospitals and clinics can't rely on a large volunteer donor pool like that available in many developed countries, Lee said. Approximately half of the donors are older family members with a higher prevalence of transfusion-transmitted viruses. In a study of more than 1,000 blood donors at a hospital in Ghana, nearly 20% were infected with one or more of HIV, hepatitis B, and hepatitis C, Lee said. Such findings demonstrate the magnitude of the challenge, because among developing nations "Ghana is a well-managed country with good infrastructure," she said.
The testing is often carried out under less than ideal circumstances. Lee described open-air pre-donation testing of volunteer blood donors recruited by FM radio in Kumasi. Access to refrigeration is often limited, so assays and reagents in tropical climates must be able to withstand temperatures that can soar past 35 oC (95 oF).
Given such challenges, scientists must make sure they are developing diagnostic tests that health care providers in developing countries want and need and, most important, can actually use. "The last thing" Western scientists "want to do is build something and find that no one wants to use it," said Paul Yager, a professor of bioengineering at the University of Washington, Seattle. For his projects, his team has done an initial assessment of the needs in India, their first target country. They are working on similar assessments in Brazil and sub-Saharan Africa.
For example, Yager said, tests need to be fast enough for patients to receive treatment. If the analysis takes too much time, patients might leave without appropriate treatment. Each sample test should ideally be completed in 15 minutes, he noted.
Diagnosis of tropical infectious diseases generally involves two types of assays. Nucleic acid analyses measure the presence of the pathogen's genetic material—either DNA or RNA—in the patient; immunoassays measure the patient's antibody response to the pathogen. Which assay provides better diagnosis depends on the stage of the infection. At early stages, nucleic acid analysis is better, but at later stages, the immunoassay is preferable. Because health care workers don't know at what stage of infection they are testing, they ideally will do both assays.
Nucleic acid testing involves three unavoidable steps, Lee said: sample preparation, amplification, and detection. Of those three, sample prep is always the one that will "stump" practitioners, she said. "There's no sense in using a simple back end if you don't have a simple front end," she said.
MICROFLUIDIC SYSTEMS are attractive for diagnostics in resource-limited settings, Weigl said, because they allow all three steps to be integrated in a single device that can be used by minimally trained personnel. "We can assume some training but not Ph.D. chemists," Yager said. Normally an instrument is required to drive the fluid through the microfluidic system, and sometimes for other functions such as heat cycling and detection, but manual methods also are available that allow microfluidics to be used without an instrument, Weigl said.
Yager leads a team that is developing a microfluidics-based diagnostic system called the DxBox. The name is a sly nod to Microsoft's Xbox game system, acknowledging funding from the Bill & Melinda Gates Foundation through its Grand Challenges for Global Health initiative. The University of Washington team includes PATH and the diagnostics companies Micronics and Nanogen. Although U.S. companies are involved in the project, Yager doubts that all components of the final system will be manufactured in the U.S. "It must be produced at a cost that is appropriate for the end users," he said.
The researchers are focusing on a panel of fever-causing pathogens, such as those that cause dengue fever, measles, malaria, and typhoid, as the first application of the DxBox. To run the analysis, a blood sample is injected onto a disposable polymeric microfluidic card that is inserted into a reader. After the sample is injected, the microfluidic card takes over via computer control. The researchers will do nucleic acid assays and immunoassays on a single microfluidic card.
One type of immunoassay being pursued in the DxBox system uses a porous membrane that supports an antibody that captures antigens from the sample. The researchers then add labeled antibodies that can be detected by optical imaging of the membrane.
Weigl, who collaborates with Yager on other projects, described a different project the team is working on—a disposable microfluidic card to diagnose enteric (intestinal) pathogens. This project is slightly easier than the one to diagnose fever-causing pathogens, Weigl said, because of the higher levels of enteric pathogen in stool samples compared with the levels of fever-causing pathogens in blood.
The current state of the art in enteric pathogen identification is bacterial culture that takes one to four days and costs $200-$500 per sample. The goal is to reduce that to one day and $1.00-$5.00 per sample. With the microfluidic card, the entire process, from feces swab to polymerase chain reaction amplification of pathogen DNA to visual readout, takes less than 30 minutes, Weigl said. The researchers read the samples by integrating colored particles into the nucleic acid as it is amplified and then capturing the particles at specific places on the bottom of the microfluidic card, generating colored bands that can be read visually. The device required to read the microfluidic card is still fairly complex, he said, with a footprint the size of a laptop computer.
In another example of tropical disease detection, Antje J. Baeumner, an associate professor of bioengineering at Cornell University, described work to analyze dengue virus, which is an RNA flavivirus. Dengue virus has four different serotypes that can cause three different diseases. Unfortunately, they don't provide cross-immunity, so someone who has been infected with one serotype doesn't have immunity against the others. Knowing the serotype is important for receiving proper treatment.
Baeumner applies a method called NASBA (for nucleic acid sequence-based amplification), which uses the enzymes reverse transcriptase, RNase H, and RNA polymerase to amplify single-stranded RNA from the different types of dengue virus. The amplified nucleic acid is then combined with two sets of DNA probes in a sandwich assay. One set of probes is immobilized to a solid support and pulls the target dengue virus sequences out of the sample. A second probe, which also binds the captured nucleic acid, is tagged to dye-containing liposomes. Lysing the liposomes releases the dye and increases the signal, thereby improving the sensitivity of the assay. Baeumner has demonstrated the detection method in lateral flow assays and microfluidic systems.
Baeumner is working with New York-based Innovative Biotechnologies International to commercialize the assay technology. The assay takes only 10 to 15 minutes. The team is working on an integrated microfluidic system that can do the entire analysis from sample preparation to final detection in 30 minutes.
EVEN BETTER than tests with simple instruments would be tests that require no instruments at all. Lee's team is developing a dipstick test that can visually detect nucleic acid targets via a capture probe and reporter molecule. If a target DNA sequence is present, a colored line appears on the dipstick. Lee lamented the slow development process. "It took two years to develop the visual chemistry and another two years to make it sensitive enough," she said. Lee, who worked at Abbott Laboratories for 10 years, said that if she had been working on the project while in industry, "I would have been fired. In fact, I would have fired myself if it took that long."
These groups and others are taking steps toward affordable diagnostic methods for the developing world, but there's still a long way to go. "You never trip on boulders; you trip on pebbles," Lee said. "There are many pebbles," she said, along the way to developing simple, affordable diagnostic methods.
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