Point-of-Care Diagnostics for the Third World

The Gates Foundation provides extensive funding for programs to improve health care for the poor with a special emphasis on resource-constrained or third-world countries. A small nonprofit in Cambridge, MA, Diagnostics For All (DFA) (www.dfa.org) receives funding for some of its programs from the Bill and Melinda Gates Foundation. DFA is developing point-of-care testing devices (POC diagnostics) that are inexpensive (<$0.10 per sample) and do not require infrastructure such as electricity; special reagents, including pure water; or skilled professional operators. The readout must be read by eye, unambiguous, and diagnostically useful in guiding therapy.

Paper microfluidics

Figure 1 – Dr. Una Ryan holding a sheet with printed diagnostic assays. The paper is laminated, then the chips are cut to size. (Photograph by Steffen Thalemann; courtesy of DFA.)

The technology behind DFA is based on paper microfluidics invented in the laboratory of Prof. George Whitesides at Harvard University (Cambridge, MA). DFA has exclusively licensed the technology from Harvard. Una Ryan, O.B.E., Ph.D., is DFA’s first CEO (see Figure 1), and she has developed an innovative business strategy that incorporates both nonprofit and for-profit funding streams. Recently, I had the opportunity to interview Dr. Ryan.

RS: What is the charter of DFA?

UR: DFA is a nonprofit enterprise fusing biotechnology and microfluidics, dedicated to creating low-cost, easy-to-use, point-of-care diagnostics designed specifically for the 60% of the developing world that lives beyond the reach of urban hospitals and medical infrastructures.

DFA is funded by program grants from several organizations, including the Bill and Melinda Gates Foundation, U.S. Agency for International Development (USAID), U.K. Department for International Development (DFID), and Defense Advanced Research Projects Agency (DARPA). Since DFA is a 501(c)(3) “not for profit” organization, it also receives support through direct charitable donations. DFA technology is based on prototypes developed in the Whitesides Laboratory. DFA has modified and developed the technology and its application to clinical chemistry, immunoassay, electrochemistry, and molecular diagnostics. DFA is currently conducting field tests on a liver function test at an AIDS clinic in Vietnam.

We work closely with all stakeholders to understand the particular need—whether it is screening, diagnosis, drug management, etc. This is a new endeavor, so we can be very creative in solving the problems, but we do keep our vision of providing improved health for all, especially the underserved.

Expanding the basic assay format to other nonclinical applications is also a major desire. We see many potential applications where “point-of-care” tests (such as in agricultural use) can improve livestock management, milk, and food safety.

RS: Please describe the basic Whitesides technology.

UR: The Whitesides Lab and others patented microfluidics technology using capillary action to move samples through a short path to a detection zone. Detection is usually colorimetric. As the sample travels to the detection zone, interferences may be selectively removed by filtration, absorption, etc. The substrate may be paper or any absorptive surface. Paper formats are ideal for high-volume production by printing; [this] is potentially a very low-cost technology capable of producing high-quality products in quantity.

DFA assay development

RS: Do you have products in the field?

UR: We are already piloting in-country programs to manufacture and distribute liver function tests for differential diagnosis in patients on antiretroviral drugs for AIDS or other medications that may damage the liver. With our assay, one has results in real time (~15 min) and the patient is a witness to the entire process. This improves confidence and patient compliance.

RS: Please tell me more about the assay.

Figure 2 – DFA liver function test. The test measures liver damage as indicated by the release of two enzymes: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood from a fingerstick (T); negative (–) and positive (+) controls are included on every chip. No instrument, power, or skilled professional is required. The test can be read by eye within 15 min. The color change can also be recorded on a camera phone. (Image courtesy of DFA.)

UR: The assay consists of a paper square (Figure 2) that has been printed with reagents specific for colorimetric detection of liver damage. Each reagent spot is isolated from the others and localized by printing the barriers on the paper with hydrophobic wax. Hydrophilic pathways connect the center of the paper to the reagent spots.

The top lamination sheet has a 5.2-mm hole in the center. A plasma separation membrane fills the hole. The paper is cold laminated between the top and bottom lamination sheet.

A drop of blood from a fingerstick is applied to the plasma membrane. Plasma flows through the membrane, but red blood cells are retained. The plasma wicks to the reagent areas where the reaction occurs. The total process is complete in 15 minutes.

The color can be read by eye, or transmitted to an image reader with a cell phone for telemedicine. Interestingly, cell phones are often available even in remote and impoverished locations.

The cost of the test is typically less than $0.10. Results conducted in the U.S. show better than 95% correlation coefficient to gold standard blood tests performed in a traditional laboratory.

RS: Are there other assays?

UR: Another set of assays that we are particularly focused on is for detection of complications of pregnancy, such as preeclampsia, leading to preterm birth. We are anxious to provide these tests while the woman is still able to walk.1 We have developed a simple urine test for detection of protein in urine and simple blood tests for glucose (for diabetes of pregnancy) and anemia. For agriculture, we are developing assays for bovine pregnancy and milk spoilage, and aflatoxin contamination of grain.

Regulatory compliance for point-of-care tests

RS: What about regulatory compliance?

UR: Regulatory compliance varies in the various regions of the world. DFA is planning to submit for the European CE mark initially. Subsequently, depending on need and with the help of our corporate partners, we may obtain FDA approval. Both the CE mark and U.S. 510K approval are well regarded in other countries. A few do ask for a small clinical trial to assure themselves that our assays work for them. After all, diets do differ, and interferences might occur.

RS: Americans are also interested in improving affordability and access to health care. So, what is the status in the U.S.A.?

UR: After obtaining 510K approval, we would work to obtain a Clinical Laboratory Improvement Amendments (CLIA) waiver. Our tests are simple and should be useful for point-of-care and in-home use. I should point out that these tests involve complex design and testing to automate manufacturing and reach required quality control standards. However, once made, the testing protocols avoid many of the problems associated with current assays, including small samples and avoidance of sample preparation.

In addition to significantly lower cost/test, we expect that our tests will improve the work flow for the patient and health-care provider. This will also lower cost and reduce the need for second or more visits.

RS: What about diagnostics for other applications such as food safety?

UR: Food safety is a particularly attractive opportunity for our technology since it is a global concern for food companies, stores, and consumers. Plus, the consumers are motivated and able to test the food before purchase, or just prior to preparation. Our testing technology with simple, direct sampling; short assay time; and emphasis on visual readout integrates the entire process, in real time. However, we are not yet funded to develop these ideas on a large scale.

The future of POC testing

RS: What is the future of POC at DFA?

UR: DFA is eager to work on projects with appropriate funding and in line with our mission of affordable diagnostics. Although DFA is a nonprofit, our platform clearly has utility and value to commercial enterprises. Toward[s] this end, DFA has a wholly owned for-profit subsidiary, Paper Diagnostics, which can engage in license agreements with for-profit companies or can form other commercial relationships with industry.2 Royalty revenues from these commercial endeavors will be subject to taxes and payments to Harvard University, but will allow DFA to remain true to its mission.1 Personally, I expect that innovative and low cost-of-goods technologies that work for the patient and provider will find a receptive position in the global market. Yes, if we are successful, this technology will someday challenge the existing structure in first-world countries, but it is the only route for improving the delivery of health care worldwide.

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While preparing for the interview, I found several relevant articles. Prof. Silvana Andreescu and colleagues at Clarkson University (Potsdam, NY) immobilized ceria nanoparticles and glucose oxidase on paper strips to create a reusable glucose sensor.2 The peroxide from the enzymatic oxidation of glucose induces a change in color from pale yellow to dark orange that is concentration indicating, yet reversible. The sensor responds over the range of 0.5–100 mM.

A comprehensive review of POC technology3 compares the drivers for POC in resource-rich and poor settings. The driver of the former is improving work flow and corresponding reduction of cost. These can justify sophisticated instrumentation to support the program, which is in stark contrast to the approach of DFA.

Yole Développement (Lyon, France) issued a market research study on POC products.4 It was forecast that the global POC market will be $38 billion in 2017. The microfluidics segment will total $16 billion. Of course, measuring the market by dollars probably favors the developed world. As DFA rolls out its products, a large fraction of the world’s 7 billion people will certainly benefit. Thus, head count is probably a better measure of the success of this innovative technology.

References

  1. Mace, C.R.; Ryan, U. A unique approach to business strategy as a means to enable change in global healthcare: a case study. Clin. Chem. [online early access]; doi:10.1373/clinchem.2012.186890; published online Apr 24, 2012; www. clinchem.org/content/early/2012/04/24/ clinchem.2012.186890.full.pdf+html.
  2. Ornatska, M.; Sharpe, E. et al. Anal. Chem.2011, 83, 4273–80.
  3. Gubala, V.; Harris, L.F. et al. Anal. Chem.2012, 84, 487–515.
  4. Breussin, F.; Roussel, B. Point of Care Testing: Applications of Microfluidic Technologies. Technology, Applications, and Market Report, May 2012; Yole Développement, Lyon, France; www.imicronews. com/upload/Rapports/Yole_ Point_of_Care_Testing_May_2012_Flyer_ launch.pdf.

Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: rlsteven@comcast.net.

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