Improving Diagnosis Of Tropical Diseases (Part 2)
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.
University of Washington
Box It Up The DxBox, a prototype diagnostic system for developing countries, combines a lab card for samples and a reader.
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.
University of Washington
Box It Up The DxBox, a prototype diagnostic system for developing countries, combines a lab card for samples and a reader.
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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