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Democratizing Molecular Diagnostics for the Developing World Ahmad N. Abou Tayoun, PhD,1 Paul R. Burchard,1 Imran Malik, PhD,2 Axel Scherer, PhD,2 and Gregory J. Tsongalis, PhD1 From the 1Department of Pathology, Geisel School of Medicine at Dartmouth, Hanover, NH, and 2Department of Electrical Engineering, California Institute of Technology, Pasadena, CA. Key Words: Molecular diagnostics; Nanotechnology; Developing world; PCR
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ABSTRACT Objectives: Infectious diseases that are largely treatable continue to pose a tremendous burden on the developing world despite the availability of highly potent drugs. The high mortality and morbidity rates of these diseases are largely due to a lack of affordable diagnostics that are accessible to resource-limited areas and that can deliver high-quality results. In fact, modified molecular diagnostics for infectious diseases were rated as the top biotechnology to improve health in developing countries. Methods: In this review, we describe the characteristics of accessible molecular diagnostic tools and discuss the challenges associated with implementing such tools at low infrastructure sites. Results: We highlight our experience as part of the “Grand Challenge” project supported by the Gates Foundation for addressing global health inequities and describe issues and solutions associated with developing adequate technologies or molecular assays needed for broad access in the developing world. Conclusions: We believe that sharing this knowledge will facilitate the development of new molecular technologies that are extremely valuable for improving global health.
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Upon completion of this activity you will be able to: • describe the impact of accessible molecular diagnostics in the developing world. • define the characteristics of accessible molecular diagnostics. • list the challenges of accessible test development. • discuss test modifications needed for democratizing polymerase chain reaction–based molecular diagnostics. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 140. Exam is located at www.ascp.org/ajcpcme.
Recent progress in the availability of highly efficacious therapeutic drugs has made it possible to lessen the infectious disease burden in the developing world. However, lack of diagnostics in resource-limited settings remains a major obstacle to treating such diseases. Reliable diagnostics are required not only for identifying patients in need of the relevant therapy but also for avoiding overtreatment or mistreatment, which often leads to drug resistance. Despite revolutionary advances in this field, almost all diagnostic tools remain largely inaccessible to developing countries. There is a notorious gap (referred to as the “10/90” gap) between the latter and industrialized countries, whereby 90% of the research money in genomics and related biotechnologies is focused on the health needs of only 10% of the world’s population.1 Current state-of-the-art laboratory medicine is highly dependent on complex platforms that assume advanced infrastructure,
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including reliable electricity and cold storage; it also requires a somewhat sterile environment, large sample volumes, and highly trained personnel to perform the tests. Some, if not all, of these requirements may not be available at low-infrastructure sites. High-impact diagnostic tools must somehow be tailored to such settings, a task that commands rethinking and often redesigning available technologies. In this article, we describe the characteristics and impact of broadly accessible diagnostics along with the challenges associated with implementing such tools. We do so by drawing on our experience and highlighting potential solutions to several technical and assay design–related issues.
Impact of Accessible Diagnostics in the Developing World The World Health Organization (WHO) has recently estimated the burdens imposed by specific diseases on the global population. Many treatable infectious diseases continue to pose the greatest threat to the developing world. In 2011, an estimated 1.7 million people died of AIDS worldwide; 34 million people (including 3.4 million children) were living with the disease, 70% of whom (including nearly 3 million children) were in sub-Saharan Africa.2,3 Despite the advances in our understanding of malaria and drug development, approximately 660,000 people died of this disease in 2010, mostly in developing countries, with a striking 3.3 billion people (50% of the global population) at risk of infection.4 In poor countries, diarrheal diseases account for 20% of deaths among children (younger than 5 years) compared with 1% in more economically developed countries.5 Furthermore, tuberculosis (TB) caused 1.4 million deaths in 2011, with the highest burden in the developing world.6 In addition to high mortality, infectious diseases also impose an enormous morbidity burden since such diseases largely afflict children and young adults. To capture both mortality and morbidity burdens, the WHO proposed the use of the disability-adjusted life-year (DALY),7 which takes into account years lost while also factoring in the reduction in quality of life due to chronic disability. It was estimated that the major infectious diseases account for nearly 325 million DALYs per year,8 equivalent to nearly 17 days lost annually for every person in the current global population. Several factors contribute to the devastating infectious disease burden in the developing world, including lack of vaccination and efficient drug delivery systems, proper sanitation and bioremediation, and, most important, affordable and accurate diagnostic tools. The latter are crucial for precisely identifying the presence and cause of disease (eg, bacterial vs viral respiratory infection), defining an appropriate treatment regimen, monitoring the effects of preventive or therapeutic interventions, and determining drug resistance. In fact, a panel 18 18
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of global health experts ranked modified molecular technologies for affordable, simple diagnosis of infectious diseases as the top biotechnology for improving health in the developing world.9 This was earlier recognized by the WHO in a report titled Genomics and World Health, which highlighted the potential of genomics in improving global health.10 However, the impact of such accessible diagnostics was only recently quantified by the global health diagnostics forum of the Gates Foundation in collaboration with the RAND Corporation.11-19 The forum consisted of disease-specific experts and representatives of technology development and the diagnostics industry, in addition to experts in modeling disease impact and evaluating probable health care infrastructure for relevant diagnostics. Six disease-specific working groups were formed, each focusing on one major disease: acute lower respiratory infections, human immunodeficiency virus (HIV)/ AIDS, diarrheal diseases, malaria, TB, and sexually transmitted diseases (STDs). These groups outlined the characteristics of the recommended new diagnostics and determined their impact in terms of DALYs saved per year ❚Figure 1❚. For example, affordable diagnostics for malaria and diarrheal diseases can potentially save 2.2 and 2.8 million DALYs per year, respectively. In all, 12.3 million DALYs are expected to be saved by the introduction of the appropriate diagnostics alone, assuming relevant treatment is available for the above six diseases (Figure 1). In terms of mortality burden alone, easy-to-use tests for the reliable detection of four pathogens— bacterial pneumonia, TB, malaria, and syphilis—could save up to 2.8 million lives each year.20 However, to save this many lives and DALYs, the desired diagnostic tools have to meet certain specifications.
STDs TB Malaria Diarrheal Diseases HIV ALRI 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Millions of DALYs Saved
❚Figure 1❚ Disability-adjusted life-years (DALYs) saved by introduction of accessible diagnostics. Recommended diagnostics should be accessible to limited or no-infrastructure laboratories. Estimates assume available treatment. ALRI, acute lower respiratory infections; HIV, human immunodeficiency virus; STDs, sexually transmitted diseases; TB, tuberculosis. Data are obtained from various sources.11,14-19
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The resources available at health care sites in the developing world determine the necessary characteristics for highimpact diagnostic tools. Experts from the global diagnostics forum identified three categories of infrastructure: moderate to advanced laboratory infrastructure, minimal laboratory infrastructure, and no laboratory infrastructure. In the first category, health care service is provided in hospitals with dependable electricity, clean water, cold storage, highly trained personnel, and a dedicated laboratory space. Samples difficult to obtain, such as sputum and venipuncture, can be used routinely. Regions with minimal or no infrastructure lack reliable electricity, cold storage, trained personnel, clean water, and a sterile environment. Health care service is offered at home by a family member or pharmacist with limited, if any, laboratory space. Certain sample types, such as venipuncture or sputum, are impossible to obtain.11 Diagnostics with broadest access and, therefore, highest impact in the developing world must assume a very limited health care infrastructure (Figure 1). In the aforementioned analysis, more people in the regions examined had access to minimal than to advanced infrastructure sites. For instance, 75% of the population in Africa had access to sites with scarce resources, while only 28% would have access to advanced health care facilities ❚Figure 2❚. This means that a test requiring minimal infrastructure could potentially extend access to an additional 47% of the African population. Diagnostic tests with performance characteristics (specificity and sensitivity) equivalent to tests in use today would have a tremendous impact if they could be implemented outside of advanced laboratory settings. Gains from wider implementation would far exceed those from improving the performance characteristics of current tests. As an example, improving test accuracy for bacterial pneumonia detection in advanced health care facilities led to only 119,000 more DALYs saved per year. However, if the same test was made accessible to sites with minimal resources, 263,000 more DALYs could be saved annually.20 Hence, improving accessibility of currently available tests is most essential for alleviating infectious disease burden, as shown in Figure 1. Maintaining the performance characteristics of a certain test in such settings is not trivial. In many cases, tests accessible to low-infrastructure sites have to be greatly modified or even completely redesigned if they are to have equivalent characteristics to those used in advanced sites. Previously, the WHO provided a list of the general characteristics (abbreviated using the acronym ASSURED) of a test intended for use in resource-limited areas ❚Table 1❚. On the basis of this and information about available resources, the disease-specific working groups suggested more test specifications for each of the six diseases.14-19 For instance, a diagnostic test for chlamydia and gonorrhea (with 85% sensitivity and 90% specificity) that © American Society for Clinical Pathology
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Percent of Population
Characteristics of Accessible Diagnostics
100 90 80 70 60 50 40 30 20 10 0
Africa
Asia
❚Figure 2❚ Percentage of African and Asian populations that have access to advanced (white bars) vs minimal (black bars) health care infrastructure. Data obtained from the RAND Corporation.20 ❚Table 1❚ General Characteristics of High-Impact Diagnostic Tests for the Developing World ASSURED Affordable by those at risk of infection Sensitive (few false-negative results) Specific (few false-positive results) User friendly (simple to perform by persons with minimal training) Rapid treatment at the first visit and robust use without the need for special storages Equipment free (no large electricity-dependent instruments; portable, handheld, battery-operated devices are acceptable) Delivered to those who need it
can be performed at minimal- or no-infrastructure sites in less than one hour could save 4 million DALYs per year (Figure 1 and Aledort et al19). Appropriate sample types and biomarkers were also suggested for the six diseases. For chlamydia and gonorrhea, nucleic acids extracted from a urine sample might be a suitable choice in resource-limited areas.19 Therefore, defining details, such as sample type and volume, appropriate biomarker, and turnaround time, requires overall assessment of the challenges present at resource-limited sites and their implications on all test parameters.
Challenges In this section, we discuss the key challenges and their implications for accessible test development. Current state-ofthe-art diagnostic tools are expensive, are complex, and require advanced laboratory infrastructure with well-trained personnel. Test operations, including sample processing, signal generation/amplification, detection, and analysis, are often performed on separate platforms that occupy large spaces and are capable of expansive data storage via electronic records. Such tools will not be accessible to health care sites that lack the minimal requirements for performing those tests. Understanding each
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specific barrier is indispensable for designing a new test, and solutions must be conceived early in the development process. Although we focus on molecular, polymerase chain reaction (PCR)–based diagnostic assays, several issues discussed here are still relevant to other types of testing. Electricity Perhaps the major challenge facing diagnostics at low- or no-infrastructure settings is the lack of reliable electricity, if at all available. This implies that tests should ideally be equipment free. Alternatively, devices could be battery powered, with the batteries amenable to recharging using accessible energy forms such as solar energy. Cold Storage and Temperature Control Without electricity, cold storage is not an option. This implies that samples must be processed immediately. Furthermore, since a patient’s biomarker (DNA or protein) or any related biological by-products cannot be stored appropriately, tests must run to completion with the results obtained soon after a patient sample is introduced into the test device. One possible solution would be to integrate test processes into fast sample-to-answer devices. In addition to patient samples, reagents (extraction buffers, enzymes, and other chemicals) necessary for running a certain test have to be provided in a highly stable form with an extended shelf life at temperatures reaching ~40°C. Currently, available reagents are highly thermosensitive and require very low storage temperatures (4°C or –20°C) for prolonged stability. Such reagents cannot withstand elevated temperatures. In fact, we have recently shown that most commonly used commercial PCR master mixes exhibit significant performance deterioration after four and two weeks of incubation at 40°C and 45°C, respectively.21 As such, thermosensitive reagents have to be stabilized in some way or replaced by new thermostable reagents with equivalent performance. Another important consideration is the lack of laboratory or room temperature control. Diagnostics might be performed at ambient temperatures that can be higher than the room temperature at which the test was first developed and assessed. Test performance must be evaluated at these conditions. For PCR-based assays, it is important to ensure that cycling temperature set points are higher than the ambient temperature where the test is performed. The lowest PCR annealing temperature must be well above the highest potential ambient temperature; primers for a certain assay have to be carefully designed to meet this criterion. For RNA viruses, a reverse transcription reaction must be included for complementary DNA synthesis, a process that requires low temperature incubation (37°C-50°C) for optimal performance. In such cases, a cooling system might have to be incorporated into the device; this requirement may increase complexity and cost. 20 20
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Clean Water Most tests performed in current diagnostic laboratories require clean water. In the absence of proper sanitation and bioremediation programs, purified water might not be available at diagnostic sites in many developing countries. For nucleic acid–based assays, such as PCR, DNase/RNase-free water is essential. One possible solution would be to embed purified or treated water in a disposable cartridge that also contains all other reagents required for running a certain test. Trained Personnel In most diagnostic sites in developing countries, health care personnel lack proper training. In parts of Africa, a family member or a local community pharmacist may be the only health care provider. Therefore, an ideal test would be fully automated, with a simple interface that minimizes interaction with the user. Such a test must integrate all necessary processes into a sample-to-answer system such that the end user will only need to introduce the appropriate sample into the device/cartridge before pressing a “Start” button. Results could be displayed in a very simple format, like a “YES” or “NO,” or the presence/absence of a band as in commercial pregnancy tests. Sample Type and Volume In the absence of trained personnel and a sterile environment, certain sample types, such as sputum, are impossible to obtain. Venipuncture, which is routinely used in advanced health care facilities to obtain large blood volumes (5-7 mL) with enough biomarker for reliable detection, might have to be replaced with a heel stick or a finger prick. It is also important to obtain free-flowing blood from the heel stick or finger stick since measurement of some analytes has been shown to be affected by this collection mechanism. The latter yield low blood volume and biomarker, suggesting that assays must be highly sensitive to attain a limit of detection equivalent to those that use venipuncture samples. When possible, test developers must switch to easy-to-obtain sample types that still contain the relevant biomarker. For example, testing for STDs such as chlamydia, gonorrhea, and trichomoniasis can use urine specimens instead of the more difficult-to-collect vaginal or urethral swabs. Finally, it is preferable to have mesofluidic systems that can efficiently process a range (0.1-1 mL) of sample volumes. Turnaround Time To achieve highest impact, it is best to deliver results and treatment at the first encounter in resource-limited settings. To that extent, all the disease-specific groups of the global diagnostics forum (see above) recommended sample-to-answer tests within less than one hour. Most available nucleic acid– based tests include long incubations and, therefore, results © American Society for Clinical Pathology
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require several hours to be generated. New technologies and/ or novel modifications of existing technologies must be used to significantly cut down turnaround time to meet developing world diagnostic needs. Footprint and Cost It is imperative that broadly accessible diagnostic tools are small, portable, and highly affordable. Highly expensive hardware and chemistries should be avoided whenever possible. For instance, in PCR-based assays, intercalating dyes can be used instead of the more expensive probe-based chemistry if assays are designed appropriately. Data Storage Another major challenge is the ability to adequately store and track necessary data. Most convenient would be the ability to remotely access or update each patient’s electronic health record. In fact, a recent study reported the development of a diagnostic microfluidic chip (mChip, Columbia University, New York, NY) that incorporated wireless communications to satellite (global coverage) and to local cell phone towers.22 This allows not only remote storage of data but also monitoring of patients’ medical records and any potential disease outbreaks, as well as allocation of medications and other resources. Manufacturers should strive toward such mobile diagnostic devices with high connectivity for data tracking in resource-limited sites. It is important to reemphasize that solutions for all the above issues have to be conceived as part of the initial design process. Implementing such solutions at the end of development is impractical, if at all possible.
nucleic acid extraction, albeit with novel physical formats that allow the DNA/RNA-binding silica membrane to be embedded in a horizontal bar that can slide between vertical chambers containing different necessary buffers ❚Figure 3❚. Fluids can be automatically moved through the membrane using a linear actuator and a plunger, thus eliminating the need for centrifugation. When needed, membrane filters could be installed to separate blood cells from plasma or epithelial cells from urine. The lysis chamber has access, through the device, to heat, chemical, ultrasonic, and/or magnetic-driven mechanical lysis. The latter is a novel technology that can apply powerful magnetic shearing on a small scale. This is highly important for lysing pathogens with high-molecularweight cell walls, such as TB. Therefore, lysis can be performed efficiently without the need for supporting equipments such as vortex mixers. This module enables minimally trained personnel at lowinfrastructure sites to perform the test. For a blood sample, for instance, the operator only needs to prick the patient’s finger, draw a few blood drops through a capillary tube (not shown), transfer it to the disposable cartridge, and insert the cartridge into the device (Figure 3). Using this automated module, we can reduce extraction time to 10 to 15 minutes and isolate highquality DNA/RNA extraction from different sample types (urine, plasma, swabs, and whole blood) using varied sample volumes (0.1-1 mL). In the final step, eluted DNA/RNA reconstitutes the lyophilized PCR reaction preembedded into a PCR chamber within the consumable cartridge (Figure 3).
Sample-to-Answer Diagnostics In this last section, we summarize our experience in developing molecular, PCR-based devices for the diagnosis of the major infectious diseases in resource-limited settings. PCR is a highly sensitive, well-known technology that has been the cornerstone of molecular diagnostics in laboratory medicine. We discuss the modifications introduced into the design of each test module, including sample preparation, amplification, detection, and interpretation. We hope this might represent a good example of how most available diagnostic tools must be redesigned in response to the substantial challenges mentioned above. Sample Processing As an example, one could integrate into a mesofluidic cartridge all sample processing steps, including filtration, extraction, and purification of nucleic acids from pathogens. The well-proven technology by Boom et al23 can be used for © American Society for Clinical Pathology
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❚Figure 3❚ An iPad-like, sample-to-answer prototype. An installed cartridge is shown. The polymerase chain reaction (PCR) chip of the cartridge extends inward and cannot be seen here. Instead, the PCR chip is shown separately at the bottom left. The cell phone can be used to control this device through Wi-Fi signals.
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A conventional PCR instrument requires 45 to 120 minutes to amplify a DNA target through 35 to 40 cycles from 55°C to 95°C. Our current approach uses a novel PCR chamber made of a metal substrate containing a plastic molded top with a shallow, flat liquid reservoir (Figure 3). It has high thermal conductivity compared with conventional glass or polymer sample holders. The flat surface provides a high surface area to volume ratio and solves the problem of heat transfer issues through a polymer. This reduces the overall thermal power and time required to ramp between temperatures while also minimizing thermal equilibration time at the temperature set points. In addition to this novel chamber, our device depends on instantaneous heat transfer to ensure rapid cycling between temperatures. All required PCR reagents (disease-specific primers, enzymes, buffers, intercalating dyes, and deoxynucleotides) are lyophilized and preembedded into the PCR chamber. The eluted 10- to 50-µL DNA/RNA sample reconstitutes the PCR reaction to start targeted DNA amplification. The transparent cover of the PCR chamber (Figure 3) is highly amenable to optics for detection and carefully optimized to increase optical performance, thus reducing optical readout time. Detection A small polymer prism, molded from the same material as the transparent sample cover, is carefully engineered such that it captures optical light from the LED and strictly directs it first to the cover and then into the sample well. The pump light excites the reporter fluorophores attached to the amplified DNA by intercalating dyes, labeled PCR primers, or labeled hybridization probes. The resulting fluorescence signal exits the sample holder and strikes the filtered photodetector mounted directly above the sample well. Signals
A
Fluorescence
100 80 60
HIV
Process control
40 20 0 –20 65 67 69 71 73 75 77 79 81 83 85 87 89 °C
Color A10 A11 A12 A13 A14 A15 A16
Copy Number Blank 10 50 100 500 1,000 5,000
detected can be used to monitor real-time amplification in addition to performing melt curve analysis to identify a specific product corresponding to a pathogen of interest ❚Figure 4❚. Melt curve analysis using intercalating dyes is inexpensive, is highly sensitive, and allows for simultaneous detection of multiple pathogens (multiplexing) as long as the assays are well designed to resolve melting temperatures of amplified DNA/RNA targets for each organism (Figure 4). For this detection system, a software code is implemented that transforms the presence or absence of a disease-specific melt peak into a “YES” or “NO” for that disease and displays it on the device’s screen. An alternative approach to optics would be to use a lateral flow immunoassay for detection. In this case, a specific hapten label is introduced into each amplified DNA target through differentially labeled, target-specific primers. Anti– hapten antibodies coated on a nitrocellulose membrane can then be used to identify the presence or absence of a target of interest. Although very simple, this approach does not allow quantification and has the major disadvantage of presenting a high false-positive rate due to possible primer dimer formation, especially toward the lower limit of detection. Although their formation is a possibility in any detection system, primer dimers normally have low melting temperatures and, therefore, can be easily pointed out using optics and melt curve analysis in well-designed assays. Overall, the fully automated, sample-to-answer device shown in Figure 3 integrates all test processes on disposable cartridges preembedded with the necessary stabilized reagents. The device can be used for the diagnosis of multiple diseases by running a different protocol for each diseasespecific cartridge. It is portable (small in size), is battery powered, and has a simple interface: once a cartridge, loaded with the appropriate patient sample, is installed into the device, an
B 100 Fluorescence
Amplification
80 60 40
Chlamydia
Process control
Gonorrhea Trichomonas
20 0 –20 60
70
80
Color A9 A10 A11 A12 A13 A14 A15 A16
Copy Number Blank 15 31 62 125 250 500 3,000
90
°C
❚Figure 4❚ In-house human immunodeficiency virus 1 (HIV-1) (A) and sexually transmitted diseases (B) assays. Melt curve analysis shows RNA detection from as low as 10 HIV-1 copies (A) and DNA detection from as low as 15 copies each of chlamydia, gonorrhea, and Trichomonas (B). Process control is included in every run, including Blank (blue curve), which does not contain any pathogenic nucleic acids.
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answer will be displayed on a touch screen in 30 to 60 minutes. Furthermore, the device has onboard a Wi-Fi connection, allowing it to be controlled by smart phones, tablets, and computers while it can also use Wi-Fi signals to send data to central databases autonomously. With such signals, the instrument can also be controlled and monitored over the Internet. Finally, the device has general packet radio service capability to make use of highly available cellular networks for monitoring, logging, and quality control. Assay Design and Stabilization Highly sensitive assays are developed by designing primers that target multicopy regions in each pathogen’s genome. Using this approach, the HIV-1 and STD assays shown in Figure 4 demonstrated limits of detection of 10 and 15 copies, respectively. Primers should also be carefully designed to detect all variants within a species. In the previous example, all HIV-1 subtypes and chlamydia serovars should be considered when designing primer sequences for each organism. Proper, highly stable process controls should be introduced into each sample to control for potential sample preparation and/or amplification failure. For example, a viral-like RNA molecule was spiked into each HIV-1 patient sample, while a plant-specific DNA sequence was spiked into each reaction for the STD assay (Figure 4). Both molecules can be lyophilized and embedded into their respective disposable cartridges for an extended shelf life at elevated temperatures. A major challenge is PCR assay stabilization. Almost all available PCR master mixes routinely used in advanced diagnostic laboratories require cold storage and cannot withstand elevated temperatures for prolonged periods.21 Lyophilization of PCR mixes24 is, therefore, highly desirable to deliver coldchain free molecular diagnostics in the developing world. However, such mixes contain large amounts of compounds, such as glycerol and dimethyl sulfoxide, that make them incompatible with lyophilization. Very few companies, such as GeneOn GmbH (Ludwigshafen am Rhein, Germany), produce diagnostic PCR master mixes that are amenable to freeze-drying. Alternatively, such mixes can be built in-house, but their amplification efficiency might not be equivalent to most proprietary commercial master mixes. All in all, test developers should consider using the appropriate PCR components when optimizing highly stable assays for use in lowinfrastructure sites. Finally, each disease-specific assay has to be embedded in its stabilized (most likely lyophilized) form into the PCR chip within the disposable cartridge. Once embedded, assay stability and performance should be evaluated after incubations at elevated temperatures and for extended periods. It is highly important that analytic performance, including sensitivity, specificity, precision, and accuracy, is validated using the stabilized assays in disposable cartridges. © American Society for Clinical Pathology
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Conclusions Most available molecular diagnostics require advanced infrastructure and, therefore, are largely inaccessible to populations in resource-limited areas. In addition, many settings within developed countries such as the United States could also qualify as “low-infrastructure sites” (eg, rural laboratories, many nonacademic and smaller hospital laboratories, and clinics of various sorts) for which simple and rapid molecular diagnostic testing would also be beneficial. Fast, affordable, fully automated sample-to-answer molecular diagnostic tools promise to significantly alleviate the infectious disease burden in these settings. However, development of such tools is very challenging and requires careful understanding of the limited resources at diagnostics sites in poor countries. Manufacturers and test developers have to implement several modifications in their test designs to democratize diagnostics to minimal- or no-infrastructure sites. We believe that sharing knowledge about diagnostics in the developing world will facilitate the development of new molecular technologies that are extremely valuable for improving global health. Address reprint requests to Dr Tsongalis: Dept of Pathology, Dartmouth Hitchcock Medical Center, 1 Medical Center Dr, Lebanon, NH 03756; [email protected].
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18. Keeler E, Perkins MD, Small P, et al. Reducing the global burden of tuberculosis: the contribution of improved diagnostics. Nature. 2006;444(suppl 1):49-57. 19. Aledort JE, Ronald A, Rafael ME, et al. Reducing the burden of sexually transmitted infections in resource-limited settings: the role of improved diagnostics. Nature. 2006;444(suppl 1):59-72. 20. RAND Corporation. Estimating the global health impact of improved diagnostic tools for the developing world. 2007. http://www.rand.org/pubs/research_briefs/RB9293.html. Accessed June 2, 2013. 21. Abou Tayoun AN, Ward BP, Maltezos G, et al. Evaluating the thermostability of commercial fast real-time PCR master mixes. Exp Mol Pathol. 2012;93:261-263. 22. Chin CD, Cheung YK, Laksanasopin T, et al. Mobile device for disease diagnosis and data tracking in resource-limited settings. Clin Chem. 2013;59:629-640. 23. Boom R, Sol CJ, Salimans MM, et al. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;3:495-503. 24. Klatser PR, Kuijper S, Van Ingen CW, et al. Stabilized, freeze-dried PCR mix for detection of mycobacteria. J Clin Microbiol. 1998; 36:1798-1800.
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Democratizing Molecular Diagnostics for the Developing World Ahmad N. Abou Tayoun, PhD,1 Paul R. Burchard,1 Imran Malik, PhD,2 Axel Scherer, PhD,2 and Gregory J. Tsongalis, PhD1 From the 1Department of Pathology, Geisel School of Medicine at Dartmouth, Hanover, NH, and 2Department of Electrical Engineering, California Institute of Technology, Pasadena, CA. Key Words: Molecular diagnostics; Nanotechnology; Developing world; PCR
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ABSTRACT Objectives: Infectious diseases that are largely treatable continue to pose a tremendous burden on the developing world despite the availability of highly potent drugs. The high mortality and morbidity rates of these diseases are largely due to a lack of affordable diagnostics that are accessible to resource-limited areas and that can deliver high-quality results. In fact, modified molecular diagnostics for infectious diseases were rated as the top biotechnology to improve health in developing countries. Methods: In this review, we describe the characteristics of accessible molecular diagnostic tools and discuss the challenges associated with implementing such tools at low infrastructure sites. Results: We highlight our experience as part of the “Grand Challenge” project supported by the Gates Foundation for addressing global health inequities and describe issues and solutions associated with developing adequate technologies or molecular assays needed for broad access in the developing world. Conclusions: We believe that sharing this knowledge will facilitate the development of new molecular technologies that are extremely valuable for improving global health.
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Upon completion of this activity you will be able to: • describe the impact of accessible molecular diagnostics in the developing world. • define the characteristics of accessible molecular diagnostics. • list the challenges of accessible test development. • discuss test modifications needed for democratizing polymerase chain reaction–based molecular diagnostics. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 140. Exam is located at www.ascp.org/ajcpcme.
Recent progress in the availability of highly efficacious therapeutic drugs has made it possible to lessen the infectious disease burden in the developing world. However, lack of diagnostics in resource-limited settings remains a major obstacle to treating such diseases. Reliable diagnostics are required not only for identifying patients in need of the relevant therapy but also for avoiding overtreatment or mistreatment, which often leads to drug resistance. Despite revolutionary advances in this field, almost all diagnostic tools remain largely inaccessible to developing countries. There is a notorious gap (referred to as the “10/90” gap) between the latter and industrialized countries, whereby 90% of the research money in genomics and related biotechnologies is focused on the health needs of only 10% of the world’s population.1 Current state-of-the-art laboratory medicine is highly dependent on complex platforms that assume advanced infrastructure,
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including reliable electricity and cold storage; it also requires a somewhat sterile environment, large sample volumes, and highly trained personnel to perform the tests. Some, if not all, of these requirements may not be available at low-infrastructure sites. High-impact diagnostic tools must somehow be tailored to such settings, a task that commands rethinking and often redesigning available technologies. In this article, we describe the characteristics and impact of broadly accessible diagnostics along with the challenges associated with implementing such tools. We do so by drawing on our experience and highlighting potential solutions to several technical and assay design–related issues.
Impact of Accessible Diagnostics in the Developing World The World Health Organization (WHO) has recently estimated the burdens imposed by specific diseases on the global population. Many treatable infectious diseases continue to pose the greatest threat to the developing world. In 2011, an estimated 1.7 million people died of AIDS worldwide; 34 million people (including 3.4 million children) were living with the disease, 70% of whom (including nearly 3 million children) were in sub-Saharan Africa.2,3 Despite the advances in our understanding of malaria and drug development, approximately 660,000 people died of this disease in 2010, mostly in developing countries, with a striking 3.3 billion people (50% of the global population) at risk of infection.4 In poor countries, diarrheal diseases account for 20% of deaths among children (younger than 5 years) compared with 1% in more economically developed countries.5 Furthermore, tuberculosis (TB) caused 1.4 million deaths in 2011, with the highest burden in the developing world.6 In addition to high mortality, infectious diseases also impose an enormous morbidity burden since such diseases largely afflict children and young adults. To capture both mortality and morbidity burdens, the WHO proposed the use of the disability-adjusted life-year (DALY),7 which takes into account years lost while also factoring in the reduction in quality of life due to chronic disability. It was estimated that the major infectious diseases account for nearly 325 million DALYs per year,8 equivalent to nearly 17 days lost annually for every person in the current global population. Several factors contribute to the devastating infectious disease burden in the developing world, including lack of vaccination and efficient drug delivery systems, proper sanitation and bioremediation, and, most important, affordable and accurate diagnostic tools. The latter are crucial for precisely identifying the presence and cause of disease (eg, bacterial vs viral respiratory infection), defining an appropriate treatment regimen, monitoring the effects of preventive or therapeutic interventions, and determining drug resistance. In fact, a panel 18 18
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of global health experts ranked modified molecular technologies for affordable, simple diagnosis of infectious diseases as the top biotechnology for improving health in the developing world.9 This was earlier recognized by the WHO in a report titled Genomics and World Health, which highlighted the potential of genomics in improving global health.10 However, the impact of such accessible diagnostics was only recently quantified by the global health diagnostics forum of the Gates Foundation in collaboration with the RAND Corporation.11-19 The forum consisted of disease-specific experts and representatives of technology development and the diagnostics industry, in addition to experts in modeling disease impact and evaluating probable health care infrastructure for relevant diagnostics. Six disease-specific working groups were formed, each focusing on one major disease: acute lower respiratory infections, human immunodeficiency virus (HIV)/ AIDS, diarrheal diseases, malaria, TB, and sexually transmitted diseases (STDs). These groups outlined the characteristics of the recommended new diagnostics and determined their impact in terms of DALYs saved per year ❚Figure 1❚. For example, affordable diagnostics for malaria and diarrheal diseases can potentially save 2.2 and 2.8 million DALYs per year, respectively. In all, 12.3 million DALYs are expected to be saved by the introduction of the appropriate diagnostics alone, assuming relevant treatment is available for the above six diseases (Figure 1). In terms of mortality burden alone, easy-to-use tests for the reliable detection of four pathogens— bacterial pneumonia, TB, malaria, and syphilis—could save up to 2.8 million lives each year.20 However, to save this many lives and DALYs, the desired diagnostic tools have to meet certain specifications.
STDs TB Malaria Diarrheal Diseases HIV ALRI 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Millions of DALYs Saved
❚Figure 1❚ Disability-adjusted life-years (DALYs) saved by introduction of accessible diagnostics. Recommended diagnostics should be accessible to limited or no-infrastructure laboratories. Estimates assume available treatment. ALRI, acute lower respiratory infections; HIV, human immunodeficiency virus; STDs, sexually transmitted diseases; TB, tuberculosis. Data are obtained from various sources.11,14-19
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The resources available at health care sites in the developing world determine the necessary characteristics for highimpact diagnostic tools. Experts from the global diagnostics forum identified three categories of infrastructure: moderate to advanced laboratory infrastructure, minimal laboratory infrastructure, and no laboratory infrastructure. In the first category, health care service is provided in hospitals with dependable electricity, clean water, cold storage, highly trained personnel, and a dedicated laboratory space. Samples difficult to obtain, such as sputum and venipuncture, can be used routinely. Regions with minimal or no infrastructure lack reliable electricity, cold storage, trained personnel, clean water, and a sterile environment. Health care service is offered at home by a family member or pharmacist with limited, if any, laboratory space. Certain sample types, such as venipuncture or sputum, are impossible to obtain.11 Diagnostics with broadest access and, therefore, highest impact in the developing world must assume a very limited health care infrastructure (Figure 1). In the aforementioned analysis, more people in the regions examined had access to minimal than to advanced infrastructure sites. For instance, 75% of the population in Africa had access to sites with scarce resources, while only 28% would have access to advanced health care facilities ❚Figure 2❚. This means that a test requiring minimal infrastructure could potentially extend access to an additional 47% of the African population. Diagnostic tests with performance characteristics (specificity and sensitivity) equivalent to tests in use today would have a tremendous impact if they could be implemented outside of advanced laboratory settings. Gains from wider implementation would far exceed those from improving the performance characteristics of current tests. As an example, improving test accuracy for bacterial pneumonia detection in advanced health care facilities led to only 119,000 more DALYs saved per year. However, if the same test was made accessible to sites with minimal resources, 263,000 more DALYs could be saved annually.20 Hence, improving accessibility of currently available tests is most essential for alleviating infectious disease burden, as shown in Figure 1. Maintaining the performance characteristics of a certain test in such settings is not trivial. In many cases, tests accessible to low-infrastructure sites have to be greatly modified or even completely redesigned if they are to have equivalent characteristics to those used in advanced sites. Previously, the WHO provided a list of the general characteristics (abbreviated using the acronym ASSURED) of a test intended for use in resource-limited areas ❚Table 1❚. On the basis of this and information about available resources, the disease-specific working groups suggested more test specifications for each of the six diseases.14-19 For instance, a diagnostic test for chlamydia and gonorrhea (with 85% sensitivity and 90% specificity) that © American Society for Clinical Pathology
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Percent of Population
Characteristics of Accessible Diagnostics
100 90 80 70 60 50 40 30 20 10 0
Africa
Asia
❚Figure 2❚ Percentage of African and Asian populations that have access to advanced (white bars) vs minimal (black bars) health care infrastructure. Data obtained from the RAND Corporation.20 ❚Table 1❚ General Characteristics of High-Impact Diagnostic Tests for the Developing World ASSURED Affordable by those at risk of infection Sensitive (few false-negative results) Specific (few false-positive results) User friendly (simple to perform by persons with minimal training) Rapid treatment at the first visit and robust use without the need for special storages Equipment free (no large electricity-dependent instruments; portable, handheld, battery-operated devices are acceptable) Delivered to those who need it
can be performed at minimal- or no-infrastructure sites in less than one hour could save 4 million DALYs per year (Figure 1 and Aledort et al19). Appropriate sample types and biomarkers were also suggested for the six diseases. For chlamydia and gonorrhea, nucleic acids extracted from a urine sample might be a suitable choice in resource-limited areas.19 Therefore, defining details, such as sample type and volume, appropriate biomarker, and turnaround time, requires overall assessment of the challenges present at resource-limited sites and their implications on all test parameters.
Challenges In this section, we discuss the key challenges and their implications for accessible test development. Current state-ofthe-art diagnostic tools are expensive, are complex, and require advanced laboratory infrastructure with well-trained personnel. Test operations, including sample processing, signal generation/amplification, detection, and analysis, are often performed on separate platforms that occupy large spaces and are capable of expansive data storage via electronic records. Such tools will not be accessible to health care sites that lack the minimal requirements for performing those tests. Understanding each
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specific barrier is indispensable for designing a new test, and solutions must be conceived early in the development process. Although we focus on molecular, polymerase chain reaction (PCR)–based diagnostic assays, several issues discussed here are still relevant to other types of testing. Electricity Perhaps the major challenge facing diagnostics at low- or no-infrastructure settings is the lack of reliable electricity, if at all available. This implies that tests should ideally be equipment free. Alternatively, devices could be battery powered, with the batteries amenable to recharging using accessible energy forms such as solar energy. Cold Storage and Temperature Control Without electricity, cold storage is not an option. This implies that samples must be processed immediately. Furthermore, since a patient’s biomarker (DNA or protein) or any related biological by-products cannot be stored appropriately, tests must run to completion with the results obtained soon after a patient sample is introduced into the test device. One possible solution would be to integrate test processes into fast sample-to-answer devices. In addition to patient samples, reagents (extraction buffers, enzymes, and other chemicals) necessary for running a certain test have to be provided in a highly stable form with an extended shelf life at temperatures reaching ~40°C. Currently, available reagents are highly thermosensitive and require very low storage temperatures (4°C or –20°C) for prolonged stability. Such reagents cannot withstand elevated temperatures. In fact, we have recently shown that most commonly used commercial PCR master mixes exhibit significant performance deterioration after four and two weeks of incubation at 40°C and 45°C, respectively.21 As such, thermosensitive reagents have to be stabilized in some way or replaced by new thermostable reagents with equivalent performance. Another important consideration is the lack of laboratory or room temperature control. Diagnostics might be performed at ambient temperatures that can be higher than the room temperature at which the test was first developed and assessed. Test performance must be evaluated at these conditions. For PCR-based assays, it is important to ensure that cycling temperature set points are higher than the ambient temperature where the test is performed. The lowest PCR annealing temperature must be well above the highest potential ambient temperature; primers for a certain assay have to be carefully designed to meet this criterion. For RNA viruses, a reverse transcription reaction must be included for complementary DNA synthesis, a process that requires low temperature incubation (37°C-50°C) for optimal performance. In such cases, a cooling system might have to be incorporated into the device; this requirement may increase complexity and cost. 20 20
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Clean Water Most tests performed in current diagnostic laboratories require clean water. In the absence of proper sanitation and bioremediation programs, purified water might not be available at diagnostic sites in many developing countries. For nucleic acid–based assays, such as PCR, DNase/RNase-free water is essential. One possible solution would be to embed purified or treated water in a disposable cartridge that also contains all other reagents required for running a certain test. Trained Personnel In most diagnostic sites in developing countries, health care personnel lack proper training. In parts of Africa, a family member or a local community pharmacist may be the only health care provider. Therefore, an ideal test would be fully automated, with a simple interface that minimizes interaction with the user. Such a test must integrate all necessary processes into a sample-to-answer system such that the end user will only need to introduce the appropriate sample into the device/cartridge before pressing a “Start” button. Results could be displayed in a very simple format, like a “YES” or “NO,” or the presence/absence of a band as in commercial pregnancy tests. Sample Type and Volume In the absence of trained personnel and a sterile environment, certain sample types, such as sputum, are impossible to obtain. Venipuncture, which is routinely used in advanced health care facilities to obtain large blood volumes (5-7 mL) with enough biomarker for reliable detection, might have to be replaced with a heel stick or a finger prick. It is also important to obtain free-flowing blood from the heel stick or finger stick since measurement of some analytes has been shown to be affected by this collection mechanism. The latter yield low blood volume and biomarker, suggesting that assays must be highly sensitive to attain a limit of detection equivalent to those that use venipuncture samples. When possible, test developers must switch to easy-to-obtain sample types that still contain the relevant biomarker. For example, testing for STDs such as chlamydia, gonorrhea, and trichomoniasis can use urine specimens instead of the more difficult-to-collect vaginal or urethral swabs. Finally, it is preferable to have mesofluidic systems that can efficiently process a range (0.1-1 mL) of sample volumes. Turnaround Time To achieve highest impact, it is best to deliver results and treatment at the first encounter in resource-limited settings. To that extent, all the disease-specific groups of the global diagnostics forum (see above) recommended sample-to-answer tests within less than one hour. Most available nucleic acid– based tests include long incubations and, therefore, results © American Society for Clinical Pathology
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require several hours to be generated. New technologies and/ or novel modifications of existing technologies must be used to significantly cut down turnaround time to meet developing world diagnostic needs. Footprint and Cost It is imperative that broadly accessible diagnostic tools are small, portable, and highly affordable. Highly expensive hardware and chemistries should be avoided whenever possible. For instance, in PCR-based assays, intercalating dyes can be used instead of the more expensive probe-based chemistry if assays are designed appropriately. Data Storage Another major challenge is the ability to adequately store and track necessary data. Most convenient would be the ability to remotely access or update each patient’s electronic health record. In fact, a recent study reported the development of a diagnostic microfluidic chip (mChip, Columbia University, New York, NY) that incorporated wireless communications to satellite (global coverage) and to local cell phone towers.22 This allows not only remote storage of data but also monitoring of patients’ medical records and any potential disease outbreaks, as well as allocation of medications and other resources. Manufacturers should strive toward such mobile diagnostic devices with high connectivity for data tracking in resource-limited sites. It is important to reemphasize that solutions for all the above issues have to be conceived as part of the initial design process. Implementing such solutions at the end of development is impractical, if at all possible.
nucleic acid extraction, albeit with novel physical formats that allow the DNA/RNA-binding silica membrane to be embedded in a horizontal bar that can slide between vertical chambers containing different necessary buffers ❚Figure 3❚. Fluids can be automatically moved through the membrane using a linear actuator and a plunger, thus eliminating the need for centrifugation. When needed, membrane filters could be installed to separate blood cells from plasma or epithelial cells from urine. The lysis chamber has access, through the device, to heat, chemical, ultrasonic, and/or magnetic-driven mechanical lysis. The latter is a novel technology that can apply powerful magnetic shearing on a small scale. This is highly important for lysing pathogens with high-molecularweight cell walls, such as TB. Therefore, lysis can be performed efficiently without the need for supporting equipments such as vortex mixers. This module enables minimally trained personnel at lowinfrastructure sites to perform the test. For a blood sample, for instance, the operator only needs to prick the patient’s finger, draw a few blood drops through a capillary tube (not shown), transfer it to the disposable cartridge, and insert the cartridge into the device (Figure 3). Using this automated module, we can reduce extraction time to 10 to 15 minutes and isolate highquality DNA/RNA extraction from different sample types (urine, plasma, swabs, and whole blood) using varied sample volumes (0.1-1 mL). In the final step, eluted DNA/RNA reconstitutes the lyophilized PCR reaction preembedded into a PCR chamber within the consumable cartridge (Figure 3).
Sample-to-Answer Diagnostics In this last section, we summarize our experience in developing molecular, PCR-based devices for the diagnosis of the major infectious diseases in resource-limited settings. PCR is a highly sensitive, well-known technology that has been the cornerstone of molecular diagnostics in laboratory medicine. We discuss the modifications introduced into the design of each test module, including sample preparation, amplification, detection, and interpretation. We hope this might represent a good example of how most available diagnostic tools must be redesigned in response to the substantial challenges mentioned above. Sample Processing As an example, one could integrate into a mesofluidic cartridge all sample processing steps, including filtration, extraction, and purification of nucleic acids from pathogens. The well-proven technology by Boom et al23 can be used for © American Society for Clinical Pathology
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❚Figure 3❚ An iPad-like, sample-to-answer prototype. An installed cartridge is shown. The polymerase chain reaction (PCR) chip of the cartridge extends inward and cannot be seen here. Instead, the PCR chip is shown separately at the bottom left. The cell phone can be used to control this device through Wi-Fi signals.
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A conventional PCR instrument requires 45 to 120 minutes to amplify a DNA target through 35 to 40 cycles from 55°C to 95°C. Our current approach uses a novel PCR chamber made of a metal substrate containing a plastic molded top with a shallow, flat liquid reservoir (Figure 3). It has high thermal conductivity compared with conventional glass or polymer sample holders. The flat surface provides a high surface area to volume ratio and solves the problem of heat transfer issues through a polymer. This reduces the overall thermal power and time required to ramp between temperatures while also minimizing thermal equilibration time at the temperature set points. In addition to this novel chamber, our device depends on instantaneous heat transfer to ensure rapid cycling between temperatures. All required PCR reagents (disease-specific primers, enzymes, buffers, intercalating dyes, and deoxynucleotides) are lyophilized and preembedded into the PCR chamber. The eluted 10- to 50-µL DNA/RNA sample reconstitutes the PCR reaction to start targeted DNA amplification. The transparent cover of the PCR chamber (Figure 3) is highly amenable to optics for detection and carefully optimized to increase optical performance, thus reducing optical readout time. Detection A small polymer prism, molded from the same material as the transparent sample cover, is carefully engineered such that it captures optical light from the LED and strictly directs it first to the cover and then into the sample well. The pump light excites the reporter fluorophores attached to the amplified DNA by intercalating dyes, labeled PCR primers, or labeled hybridization probes. The resulting fluorescence signal exits the sample holder and strikes the filtered photodetector mounted directly above the sample well. Signals
A
Fluorescence
100 80 60
HIV
Process control
40 20 0 –20 65 67 69 71 73 75 77 79 81 83 85 87 89 °C
Color A10 A11 A12 A13 A14 A15 A16
Copy Number Blank 10 50 100 500 1,000 5,000
detected can be used to monitor real-time amplification in addition to performing melt curve analysis to identify a specific product corresponding to a pathogen of interest ❚Figure 4❚. Melt curve analysis using intercalating dyes is inexpensive, is highly sensitive, and allows for simultaneous detection of multiple pathogens (multiplexing) as long as the assays are well designed to resolve melting temperatures of amplified DNA/RNA targets for each organism (Figure 4). For this detection system, a software code is implemented that transforms the presence or absence of a disease-specific melt peak into a “YES” or “NO” for that disease and displays it on the device’s screen. An alternative approach to optics would be to use a lateral flow immunoassay for detection. In this case, a specific hapten label is introduced into each amplified DNA target through differentially labeled, target-specific primers. Anti– hapten antibodies coated on a nitrocellulose membrane can then be used to identify the presence or absence of a target of interest. Although very simple, this approach does not allow quantification and has the major disadvantage of presenting a high false-positive rate due to possible primer dimer formation, especially toward the lower limit of detection. Although their formation is a possibility in any detection system, primer dimers normally have low melting temperatures and, therefore, can be easily pointed out using optics and melt curve analysis in well-designed assays. Overall, the fully automated, sample-to-answer device shown in Figure 3 integrates all test processes on disposable cartridges preembedded with the necessary stabilized reagents. The device can be used for the diagnosis of multiple diseases by running a different protocol for each diseasespecific cartridge. It is portable (small in size), is battery powered, and has a simple interface: once a cartridge, loaded with the appropriate patient sample, is installed into the device, an
B 100 Fluorescence
Amplification
80 60 40
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Process control
Gonorrhea Trichomonas
20 0 –20 60
70
80
Color A9 A10 A11 A12 A13 A14 A15 A16
Copy Number Blank 15 31 62 125 250 500 3,000
90
°C
❚Figure 4❚ In-house human immunodeficiency virus 1 (HIV-1) (A) and sexually transmitted diseases (B) assays. Melt curve analysis shows RNA detection from as low as 10 HIV-1 copies (A) and DNA detection from as low as 15 copies each of chlamydia, gonorrhea, and Trichomonas (B). Process control is included in every run, including Blank (blue curve), which does not contain any pathogenic nucleic acids.
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answer will be displayed on a touch screen in 30 to 60 minutes. Furthermore, the device has onboard a Wi-Fi connection, allowing it to be controlled by smart phones, tablets, and computers while it can also use Wi-Fi signals to send data to central databases autonomously. With such signals, the instrument can also be controlled and monitored over the Internet. Finally, the device has general packet radio service capability to make use of highly available cellular networks for monitoring, logging, and quality control. Assay Design and Stabilization Highly sensitive assays are developed by designing primers that target multicopy regions in each pathogen’s genome. Using this approach, the HIV-1 and STD assays shown in Figure 4 demonstrated limits of detection of 10 and 15 copies, respectively. Primers should also be carefully designed to detect all variants within a species. In the previous example, all HIV-1 subtypes and chlamydia serovars should be considered when designing primer sequences for each organism. Proper, highly stable process controls should be introduced into each sample to control for potential sample preparation and/or amplification failure. For example, a viral-like RNA molecule was spiked into each HIV-1 patient sample, while a plant-specific DNA sequence was spiked into each reaction for the STD assay (Figure 4). Both molecules can be lyophilized and embedded into their respective disposable cartridges for an extended shelf life at elevated temperatures. A major challenge is PCR assay stabilization. Almost all available PCR master mixes routinely used in advanced diagnostic laboratories require cold storage and cannot withstand elevated temperatures for prolonged periods.21 Lyophilization of PCR mixes24 is, therefore, highly desirable to deliver coldchain free molecular diagnostics in the developing world. However, such mixes contain large amounts of compounds, such as glycerol and dimethyl sulfoxide, that make them incompatible with lyophilization. Very few companies, such as GeneOn GmbH (Ludwigshafen am Rhein, Germany), produce diagnostic PCR master mixes that are amenable to freeze-drying. Alternatively, such mixes can be built in-house, but their amplification efficiency might not be equivalent to most proprietary commercial master mixes. All in all, test developers should consider using the appropriate PCR components when optimizing highly stable assays for use in lowinfrastructure sites. Finally, each disease-specific assay has to be embedded in its stabilized (most likely lyophilized) form into the PCR chip within the disposable cartridge. Once embedded, assay stability and performance should be evaluated after incubations at elevated temperatures and for extended periods. It is highly important that analytic performance, including sensitivity, specificity, precision, and accuracy, is validated using the stabilized assays in disposable cartridges. © American Society for Clinical Pathology
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Conclusions Most available molecular diagnostics require advanced infrastructure and, therefore, are largely inaccessible to populations in resource-limited areas. In addition, many settings within developed countries such as the United States could also qualify as “low-infrastructure sites” (eg, rural laboratories, many nonacademic and smaller hospital laboratories, and clinics of various sorts) for which simple and rapid molecular diagnostic testing would also be beneficial. Fast, affordable, fully automated sample-to-answer molecular diagnostic tools promise to significantly alleviate the infectious disease burden in these settings. However, development of such tools is very challenging and requires careful understanding of the limited resources at diagnostics sites in poor countries. Manufacturers and test developers have to implement several modifications in their test designs to democratize diagnostics to minimal- or no-infrastructure sites. We believe that sharing knowledge about diagnostics in the developing world will facilitate the development of new molecular technologies that are extremely valuable for improving global health. Address reprint requests to Dr Tsongalis: Dept of Pathology, Dartmouth Hitchcock Medical Center, 1 Medical Center Dr, Lebanon, NH 03756; [email protected].
References 1. Global Forum for Health Research. The 10/90 Report on Health Research 2000. Geneva, Switzerland: Global Forum for Health Research; 2000. http://announcementsfiles.cohred. org/gfhr_pub/assoc/s14791e/s14791e.pdf. Accessed June 2, 2013. 2. World Health Organization (WHO). Global Health Observatory (GHO): HIV/AIDS. Geneva, Switzerland: WHO; 2013. http://www.who.int/gho/hiv/en/index.html. Accessed June 2, 2013. 3. World Health Organization (WHO). Paediatric HIV and Treatment of Children Living With HIV. Geneva, Switzerland: WHO; 2013. http://www.who.int/hiv/topics/paediatric/en/ index.html. Accessed June 2, 2013. 4. World Health Organization (WHO). Deaths and Statistics: Malaria. Geneva, Switzerland: WHO; 2013. http://www.who. int/research/en/. Accessed June 2, 2013. 5. O’Ryan M, Prado V, Pickering LK. A millennium update on pediatric diarrheal illness in the developing world. Semin Pediatr Infect Dis. 2005;16:125-136. 6. World Health Organization (WHO). Global Health Observatory (GHO): TB. Geneva, Switzerland: WHO; 2013. http://www.who.int/gho/tb/epidemic/cases_deaths/en/index. html. Accessed June 2, 2013. 7. Murray CJL. Quantifying the burden of disease: the technical basis for disability-adjusted life years. Bull WHO. 1994;72:429-445. 8. World Health Organization (WHO). The World Health Report 2003: Shaping the Future. Geneva, Switzerland: WHO; 2003. http://www.who.int/whr/2003/en/whr03_en.pdf. Accessed June 2, 2013.
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DOI: 10.1309/AJCPA1L4KPXBJNPG
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