GLOBAL DIAGNOSTICS
Although major progress has been made in diagnosing diseases that affect people living in affluent societies, diseases that annually kill millions in poor countries tend to go undiagnosed. There is an urgent and unmet need for more-accurate, cost-effective diagnostic technologies that would enable healthcare providers serving populations in underdeveloped areas to determine the cause of a patient's affliction and make an informed decision about treatment.
Bringing medical diagnostic tools to the developing world is not so simple a matter as supplying technologies that are already in use in the developed world. The high-tech, high-cost centralized-laboratory model found throughout the developed world has limited utility in developing nations. Where such centralized laboratories exist at all in poorer countries, most are situated in urban areas and cater primarily to the more affluent segments of society. The majority of the population does not have access to the services they provide. Healthcare facilities in the countryside, in contrast, commonly have only very basic equipment. Healthcare workers may have little training, and there are limited resources for maintaining complex equipment or handling fragile reagents.
To overcome these obstacles requires new or adapted diagnostic tools. These tools must be cost-effective and also affordable for public-sector programs. They must be simple, requiring minimal training and instrumentation. They should be rapid, yielding results quickly enough that the patient does not have to make a return visit. Also, these diagnostics have to be sufficiently stable to be used in areas lacking reliable refrigeration. And, of course, they must provide accurate diagnosis.
A number of diagnostic technologies that are appropriate for use in low-resource settings are being developed and introduced by the nonprofit Program for Appropriate Technology in Health (PATH; Seattle) in partnership with public- and private-sector collaborators. These products are designed to solve the problem of making reliable diagnostic tools accessible in regions where limitations in resources and infrastructure are obstacles to high-quality healthcare. This article describes the progress that PATH and its partners have made.
It should be of interest to the diagnostic industry worldwide that these technologies may well allow the developing world to leap ahead of the industrial world in a fashion. A move toward point-of-care (POC) diagnostics and away from centralized laboratories has long been expected in the developed countries but has been slow to materialize.
Imunochromatographic Strip Tests
Immunochromatographic strip (ICS) tests have been, for the past decade, one of the very few diagnostic technologies that are used successfully in the developing world. These lateral-flow strips provide POC diagnosis in areas without access to well-equipped and well-staffed clinical laboratories. They rely on relatively inexpensive off-the-shelf components and reagents, and thus are affordable. ICS tests, in many cases, cost the end-user less than two dollars. They can be formatted for detection of antigens or antibodies and are suitable for a wide range of specimens, making them useful for just as wide a range of applications. Most are stable for more than a year and can be shipped without refrigeration. ICS tests require relatively little, and sometimes no, sample processing, and they do not call for an external instrument.
Despite their many very positive attributes, lateral-flow test strips frequently offer only limited sensitivity, and sometimes their specificity is less than ideal. Although some lateral-flow assays can achieve sensitivities and selectivities in the range of 90% or higher, others may have a sensitivity as low as 70%. Those performance values, considered unacceptable in most high-resource settings, are a huge improvement over the purely syndromic diagnosis common in developing countries, however. Nevertheless, better tests are needed.
PATH both works to improve the sensitivity and selectivity of existing lateral-flow assays and investigates new platforms that offer better performance without an increase in either cost or complexity. ICS tests so far developed by the nonprofit—with support from the U.S. Agency for International Development (USAID) under PATH's HealthTech program—and transferred to commercial partners in the developing world include assays for malaria, hepatitis B, human immunodeficiency virus (HIV), pregnancy, and diphtheria.
ICS Tests for STDs. Sexually transmitted diseases (STDs) are epidemic in the developing world. The economic cost of untreated infections, in terms of healthcare burden and lost productivity, is high. STDs play a role in the epidemic of acquired immune deficiency syndrome (AIDS) by increasing the risk of HIV transmission. The early and accurate diagnosis capability offered by strip tests is essential for effectively controlling the spread of these diseases. With that in mind, PATH has recently developed ICS tests for gonorrhea (Neisseria gonorrhoeae), syphilis (Treponema pallidum), and chlamydia (Chlamydia trachomatis).
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Figure 1. (click to enlarge) Schematic of the immunochromato-
graphic strip test for gonorrhea (a), with examples of positive and negative test readings (b). The test results were read after 20 minutes. The positive reaction was generated using N. gonorrhoeae strain Mel and the negative reaction using extraction buffer control. |
PATH's strip test for gonorrhea is formatted to identify specific gonococcal antigens obtained directly from clinical specimens (see Figure 1). The ICS test for chlamydia can identify specific chlamydial antigens from cervical swabs. After an extraction step is performed on the specimen, the test can be completed in 15 to 20 minutes by technicians having minimal training. This allows healthcare workers to diagnose disease at the point of care so that patients can receive counseling and treatment during the same visit. Such efficiency is vital in regions where people may have to travel for days to reach the nearest clinic.
The strips are stable for many months at ambient temperatures. This property is critical in healthcare settings in which the electrical supply is inconsistent and temperature control is difficult. Because of their low cost and stability, the tests may also be used by epidemiological surveillance teams in the field, to gather baseline data or to assess the effect of public health interventions.
Technology Transfer. One indicator of the potential for widespread use of the ICS tests that PATH has developed is manufacturers' sales of the products. In many cases, the manufacturers, most of which are located in developing countries, have improved the tests' performance since the original transfer of the technology from the development program. For example, PATH's technology transfers have contributed significantly to the establishment of a vibrant diagnostics industry in India.
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Table I. (click to enlarge) Sales of rapid immunochromatographic strip tests developed by PATH and transferred to private-sector manufacturers in developing countries. All tests were distributed worldwide.
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Recent reports from companies receiving these technology transfers suggest that millions of these tests are reaching target populations (see Table I). However, although PATH's intention with the ICS tests has been to make POC testing possible in developing countries, the tests now are used more often in these countries' hospital laboratories, where they replace in vitro assays that are more complicated and expensive.
PATH has transferred the technical know-how needed to produce the gonorrhea test to a commercial manufacturer in India The chlamydia test is still in the research and development phase; in 2005, PATH conducted a large-scale field evaluation in Bolivia, in collaboration with the New York City–based Population Council, a nongovernmental reproductive health organization, and the Bolivian Ministry of Health. In addition, several technologies for signal enhancement have been investigated that would increase the sensitivity of this IVD.
Semiquantitative T-Cell Count Assay
As HIV testing becomes more common in developing countries while the AIDS epidemic continues to accelerate, the numbers of individuals who know that they are HIV positive and of those who seek treatment are rapidly increasing, especially in Africa and Asia. A bit of good news here is that the decreasing cost and increasing availability of drugs makes retroviral treatment of these patient populations progressively more feasible. However, existing methods to monitor CD4+ T-cell counts, a commonly used marker for the initiation of antiretroviral therapy, are too expensive and too complex to employ in low-resource settings.
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Figure 2. (click to enlarge) The semiquantitative CD4 assay from PATH (Seattle) is a c ombination of two technologies: magnetic bead-based CD4+ cell separation in whole blood samples (a), followed by colorimetric white blood cell determination in a flow-through device (b).
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A rapid flow-through CD4+ T-cell test being developed by PATH and PortaScience Inc. (Moorestown, NJ), a private company, is designed to offer affordable POC diagnosis. The development work is cofunded by USAID and the Doris Duke Charitable Foundation in New York City. The test, which requires no instrument, combines commercially available CD4+ purification technologies and proprietary flow-through cassettes to measure cell count status (see Figure 2). It will semiquantitatively assess CD4+ T-cell numbers as insufficient (0–200 cells per microliter), borderline (201–500 cells per microliter), or sufficient (501 or more cells per microliter) by means of colorimetric changes in the flow-through membrane.
Results from this test should allow clinicians in low-resource settings to make decisions about whether and when to provide available antiretroviral therapy to HIV-positive individuals. The test can be completed in less than two hours and can be performed by laboratory technicians with low levels of training.
PATH has finished conducting proof-of-principle experiments for this test. Its researchers have evaluated the test with blood taken from 37 HIV-negative and 18 HIV-positive individuals, checking for concordance of results with those obtained by means of flow cytometry, the reference method for CD4+ cell count determination. Upcoming development steps include optimizing the test for use with a simple quantitative reader, reducing the time and complexity of the purification steps for CD4+ T cells separated from whole blood, and evaluating the utility of the test through clinic-based trials in the developing world.
Microfluidic Platforms
Although strip tests are inexpensive and easy to use, these advantages can be offset by the challenge of achieving the necessary sensitivity. Most ICS tests rely on the detection of antibodies produced by the patient as a response to the infection. This method can be problematic in three ways. First, there is often a lag time between the occasion of infection and the patient's immune response. A patient may therefore test negative for a pathogen even though he or she is in fact infected. Second, patients with compromised immunity, such as those who are HIV-positive, often do not mount an immune response sufficiently strong to allow detection by strip tests. Finally, in certain infections, such as tuberculosis, ICS tests cannot differentiate between active and latent infection.
Assays with high sensitivity that are able to detect the pathogen directly, on the other hand, such as those involving molecular diagnostics and organism culture, are too expensive and complex for most low-resource healthcare settings. However, PATH is working with partners to develop molecular assays that use microfluidic platforms. This technology provides a way to make molecular diagnostics both affordable and simple enough to be used by health workers who have received little training.
Diagnostic tools based on microfluidic technology offer advantages in size, cost, durability, and adaptability. They can test for multiple pathogens in a single sample and can return results in less than 30 minutes. Ideal for use in low-resource settings, when available they will lower the cost of testing, accelerate diagnosis, and reduce reliance on centralized laboratory facilities. PATH and partners have two microfluidics-based tools in development, one specialized for enteric disease and the other for rapid-onset fever. The development of a third microfluidics-based diagnostic for sexually transmitted diseases is now also under way.
Enteric Card
Enteric infections are the second-leading cause of morbidity and mortality worldwide, accounting for an estimated 3.1 million deaths annually. Most of the burden of this health disaster is borne by the developing world. Outbreaks of enteric disease can be controlled with rapid diagnosis and appropriate treatment, which can also reduce the severity of disease in those afflicted. However, the industrial-world standards for diagnosis of infectious diarrhea—culture, enzyme immunoassay, and polymerase chain reaction (PCR)—are too slow, too nonspecific, or too expensive for developing-world purposes.
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Figure 3. (click to enlarge) Disposable microfluidic cartridge for the enteric card system.
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Supported with funding from the National Institutes of Health, PATH has since 2003 partnered with Micronics Inc. (Redmond, WA), Washington University (St. Louis), and the University of Washington (Seattle) in developing a device called the disposable enteric card (DEC), a lab-on-a-card, or lab card, platform that can identify any of the four pathogens—Shigella dysenteriae type 1, Shiga toxin–producing Escherichia coli (E. coli O157:H7), Campylobacter jejuni, and Salmonella—that commonly cause enteric disease (see Figure 3).
The DEC is an automated, rapid, easy-to-use POC assay for distinguishing between multiple enteric pathogens that all cause disease states having similar symptoms. The final test will be able to identify enteric bacteria in patients presenting to primary healthcare settings or public health laboratories with symptoms of acute diarrhea. With only minimal equipment, such as the swab needed for sample collection, the card can be used outside the clinical setting, as well. Component functions include multiplexed nucleic acid amplification and detection, sample processing to support direct use of clinical specimens, and dry-reagent storage and handling.
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Figure 4. (click to enlarge) Schematic of the disposable enteric card approach, showing the sequence of whole-pathogen immunocapture (immunocapture agents for Salmonella, S. dysenteriae, and C. jejuni are grouped together), nucleic acid (NA) extraction, polymerase chain reaction (PCR), and lateral-flow-strip (LFS) detection of amplicons.
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The design has two components: an assay card and a processor to carry out lateral-flow-strip detection (see Figure 4). The user injects a stool sample at one end of the card and then places the card in the processor. A combination of capillary action and positive-displacement pumping draws the sample via microchannels through a series of stations on the card. A positive control (E. coli, present in any stool sample) demonstrates that the sample was properly processed even if results for all pathogens are negative.
On the disposable card, target pathogens in the stool sample are captured and lysed, and nucleic acid is extracted onto a silica membrane and amplified using PCR. The PCR products are detected visually using a method similar to lateral-flow analysis.
The entire DEC system sequence will take less than 20 minutes. By contrast, identification of the target pathogen in a standard laboratory is generally performed by culturing in incubators, a process that requires skill and expensive equipment, and that takes between 24 hours and several days to complete.
The prototype card reader is portable; the design target is a handheld device. The assay card uses dry, heat-stable reagents and is disposable. Therefore, there is no risk of sample contamination, even when testing is carried out under less than ideal conditions. And because no manual intervention is required other than connecting the card to the reader, the user needs little training. Almost no risk of user error exists. Sensitivity and specificity should be comparable to the results achieved with conventional microbiological or PCR assays, and the cost should be much lower.
The enteric card has been tested extensively on samples obtained from individuals in the states of Washington and Missouri, where most project partners are located. Field tests involving approximately 1000 patients from several sites in northern Brazil are planned for 2007. These will constitute the final step in the technology's testing phase.
The DEC team was funded originally to achieve the goal of producing a tool for biothreat detection. Many enteric pathogens, because they spread so quickly and easily, have the potential to be used in bioterrorism. Early diagnosis would be key in halting the spread of disease after an attack with a weapon of this sort. In large part because of PATH's public-sector orientation, it was natural, as well as possible, to apply the technology to a developing-world need also.
Fever Panel
Rapid progress made with the enteric card paved the way for PATH to join a second team working on microfluidics-based diagnostic technology. This collective initiative is led by the University of Washington and supported by a Grand Challenges grant from the Bill and Melinda Gates Foundation. The team includes two private-sector companies, Nanogen Inc. (San Diego) and Micronics. It is designing another system based on Micronics' lab card platform, this one to identify organisms that cause rapid-onset fever.
In many developing countries, people who have very high fever are most likely to be treated for malaria. Other diseases also manifest as fever, however, and often are missed at diagnosis. Some of these can cause death in a very short period of time—too short to allow for multiple courses of treatment if the first diagnosis of malaria or something else turns out to be wrong. The project team's goal here is a prototype diagnostic that can identify six of the most common causes of rapid-onset fever: infection with influenza/parainfluenza viruses; malaria; typhoid/paratyphoid; dengue; rickettsial infection; and the measles.
Using microfluidic technology to build a diagnostic tool for fever presents new challenges. Several types of pathogens may cause fever—bacteria, RNA viruses, and parasites. Because these pathogens are so diverse and have radically different pathogenic profiles during the onset of fever, any diagnostic tool must be able to perform complex assays and make fine distinctions between target pathogens.
For example, in the case of dengue, a nucleic acid test will detect the virus only in the early stage of the infection. Tests for dengue-specific immunogloblulin M are the most effective in the fever's later stages, but these tests are inappropriate and inaccessible in the regions where dengue is endemic.
Also, the new diagnostic tool must analyze blood samples rather than stool. Blood is in some ways easier to accommodate on microfluidic cards; its consistency and components are much more predictable. However, pathogens are present in blood at much lower levels than in stool. Therefore, the tool must be much more sensitive.
Once these challenges are met, the fever panel can be adapted to many other types of infectious disease, for use where each is geographically prevalent, as well as to different sample formats, such as throat or vaginal swabs.
For the fever panel, PATH is actively identifying sources of samples for all target pathogens and setting up prospective study sites for some. The nonprofit is also leading efforts to conduct user-needs assessment studies to help inform product specifications. This is part of a commercialization strategy coordinated with Micronics, which has commercial rights in the developing world. PATH plays a major role in immunoassay development and selection of immunological reagents. In parallel, Nanogen is developing the nucleic acid assays and Micronics is designing the integrated microfluidics card on which the assays are optimized for detection by the reader.
Bringing IVDs to the Developing World
The obstacles that impede access by the world's neediest populations to diagnostic technologies appropriate for them go beyond resource limitations. The lack of money, training, and laboratory facilities is an obvious and major barrier, but it is not insurmountable—the industrial world has the ability to fill in these gaps while developing countries work to build the infrastructure necessary for future innovation, research, and commercialization.
However, it takes more than need to bring private-sector resources to bear on developing-world problems, many of which are unique or are low priorities when occurring in the industrial world. Diagnostic tools that represent the standard of practice in areas with strong infrastructure—countries or localities with high-tech laboratories, sterile facilities, ample access to highly trained health workers, and quick and reliable systems for communicating results—do not necessarily translate to low-resource settings. Commonly, they must be adapted, or a new technology altogether must be produced. And private-sector companies are often reluctant to invest in technologies appropriate for the developing world. They may not be able to perceive opportunities that lie outside the profit box in which they usually work.
Over the past 29 years, PATH has worked with more than 80 private-sector companies and 50 public-sector organizations. Its success stems from its ability to provide value to both the public health arena and the companies' commercial interests. PATH helps companies adapt their products to the financial and cultural realities of developing nations in order to achieve the maximum sustainable benefit for public health in those countries. In cases where appropriate technology does not exist, the nonprofit engages the private sector through collaborative research and development.
On the commercial side, PATH helps lower the risks that companies face when introducing products in places that are conventionally deemed unattractive markets. The organization navigates a tricky product introduction by conducting market studies on users' needs and potential manufacturing and distributing options, by partnering with other public-sector organizations to guarantee demand for the products, by facilitating demonstration projects and clinical evaluations, and by brokering relationships with decision makers and other gatekeepers to encourage product uptake. In collaborating with a company, it recognizes and supports the company's legitimate need to pursue profit in order to ensure a sustainable supply of the product. This catering to the capitalistic sensibilities of diagnostic device manufacturers results in developing countries being provided ultimately with a much-needed product at a price that is affordable and makes it accessible to resource-poor end-users.
Conclusion
A diagnostic assay alone cannot produce a successful outcome. Appropriate therapeutic options must be available. In many cases, however, when treatment does exist, it is not, or cannot be, appropriately administered without usable diagnostics. For example, antiretroviral therapy may be withheld, or may be started too early, if no tool is available for accurate identification of CD4+ T-cell conversion. Also, antimalarial agents and antibiotics are commonly overprescribed in treating cases of illness where the true etiologic agent cannot be identified.
For diagnostic assays to be successful in the markets of poorer nations, they must be appropriate to the need. This means adapting technologies or developing new ones, rather than simply transferring old technologies to a new setting. The process is complicated further by the greater difficulty of marketing assays to developing countries. Nevertheless, both the potential benefit to populations in the developing world and the opportunities for private-sector companies that are willing to develop and market appropriate assays are too great to ignore. The strategies modeled by PATH in the examples presented in this article provide a framework for others to use in tailoring technologies to developing-world needs and for establishing mutually beneficial partnerships between entities in the private and public sectors.
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Bernard H. Weigl, PhD, is group leader, Tala de los Santos is a commercialization officer, Matt Steele is a clinical and field research coordinator, and Gonzalo Domingo is a research scientist in the diagnostics development team of the Technology Solutions Strategic Program at PATH (Seattle). The authors can be reached respectively at bweigl@path.org, jbauertdelossantos@path.org, msteele@path.org, and gdomingo@path.org.
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