Introduction      

Diagnostic tools and treatments for febrile illnesses have significantly improved over the last few decades.Individuals can get prescriptions for antimalarials before traveling to endemic countries; a dengue vaccine, albeit controversial, has been approved; and a variety of tuberculosis antibiotics exist for distinctive strains (NHS,2018, 2017; WHO, n.d.). However, despite low- and middle-income countries (LMICs) carrying a vast majority of the febrile disease burden, those innovative resources are not uniformly available. Of the 1.5million deaths from tuberculosis in 2015, 95% occurred in economically developing countries (Magroet al., 2017). Meanwhile, treatment for multi-drug resistant tuberculosis can cost hundreds of thousands of dollars in the U.S. (Markset al., 2014).

A critical component in successfully treating these febrile illnesses is the ability to distinguish between viral, bacterial, and protozoan infections, as early diagnosis allows for effective treatment and decreased spread. But the nucleic acid amplification tests (NAATs)necessary for early detection and distinction are unaffordable in the countries that need them most. Despite incentives such as the Gates Foundation GrandChallenges and global organizations such as FIND, no nucleic acid diagnostic that fits the World Health Organization’s ASSURED criteria has become broadly available (Katobaet al., 2019). A major reason for this lack of availability is that a successful diagnostic must not only provide fast and cheap results but also be accompanied by an innovative production and distribution plan to ensure accessibility. The expensive importation of high-tech diagnostics is not feasible compared a solution that is integrated with existing economic systems.

This paper proposes a novel nucleic acid diagnostic tool whose design is optimized to allow for local production in countries with the greatest febrile illness burden. Local production is one aspect of the larger proposed distribution pipeline to establish an economically viable solution to the delivery of diagnostics in low- and middle-income countries.

Origami Paper Diagnostics (OPD) for Febrile Disease

To design an economically viable diagnostic for febrile illnesses, the necessary properties of a NAAT must first be considered.Any point-of-care NAAT must complete the following steps: “i) lysis of the infectious agent to release nucleic acids (NAs); (ii) purification of NAs,(iii) NA amplification, and (iv) detection of the amplified nucleic acids” (Kaurand Toley, 2018). Traditional nucleic acid amplification techniques, such as PCR, require laboratory conditions and external power, making them expensive and inaccessible to LMICs (Xuet al., 2016). A low cost-alternative to PCR that has demonstrated success in low-resource settings is loop-mediated isothermal amplification (LAMP) (Xu et al., 2016). In LAMP reactions, a DNA polymerase and four specially designed primers synthesize the DNA in a series of steps, ultimately producing a stem-loop structure (Notomi et al., 2000). Subsequent LAMP cycling builds on this loop, with an accumulation of 109copies in less than an hour (Ibid).

Paper-based tests offer an affordable platform for employing LAMP in point-of-care diagnostics. Paper tests are not a wholly novel invention: paper has been used as a diagnostic tool for decades, such as for at-home pregnancy tests (Rollandand Mourey, 2013). But a key development occurred when the invention of patterning paper demonstrated that controlling the channels in which fluids are wicked across the paper could mimic standard microfluidics—this led to the invention of micro-Paper Analytical Devices (μPAD)(Magroet al., 2017). Since then, multiple labs, including George Whitesides at Harvard, have worked on developing paper-based portable bioassays.

As it pertains to LAMP, paper’s natural wicking ability via capillary action and capacity for filtration make it an ideal candidate for a cheap testing platform. In a paper-based LAMP device, all of the NAAT steps described above occur on a single surface that takes advantage of paper’s naturally porous quality, which enables the wicking of fluids along assays without the need for pumps (Kaurand Toley, 2018). Lysis purification is accomplished via a wash buffer, then the extracted DNA is wicked via the paper to the LAMP zone where heat exposure (60-65°C) allows for primer annealing and amplification, and final detection is typically visible to the naked eye (Kaurand Toley, 2018).

Amongst the heterogeneity of paper-based LAMP tests, the Origami-assembly approach is particularly promising for enabling LMIC diagnostic access. In this approach, a large sheet of paper(typically a cellulose substrate) is specifically cut to allow for a series of folds onto a main square. The main square contains a glass-fiber disc surrounded by printed hydrophobic wax onto which a blood sample is deposited. A series of connected paper segments are folded in order onto the main segment and deliver reagents for extraction, amplification, and detection. The first fold delivers lysis buffer, and the entire paper is subsequently heated at 95 °C for 5 minutes (on a hot plate) followed by washing; the buffer is then absorbed by surrounding filter paper, and additional folds allow for the captured DNA to be eluted by rehydrated LAMP reagents that identify the presence of infectious DNA (Kaur and Toley, 2018). All NAAT materials are self-contained on the foldable sheets, and the entire process requires only forty-five minutes.

The rapid and self-contained amplification process of these Origami Paper Diagnostic already suggests feasibility for POC testing in LMICs, but additional advances on the approach further support the technology’s accessibility. Govindarajan et al. (2012) described the ease of OPD distribution when they demonstrated the ability to store dehydrated lysis reagents in the paper which could then be rehydrated at POC for DNA amplification. Garcia-Bernalt Diego et al. (2019) described the same dehydrated storage for LAMP reagents and demonstrated its success in testing for human intestinal schistosomiasis. Combining these two experiments suggests that all reagents in OPDs could be dehydrated for ease of distribution and storage across LMICs, which is particularly important for accessing rural areas.Furthermore, Xu et al. (2016) successfully incorporated multiple LAMP reagents on a single OPD to distinguish between strains of malaria (Plasmodium pan, P. falciparum, and P. vivax), confirmingOPD’s capability to screen for multiple diseases in a single test. Xu also demonstrated the addition of fluorescent tags to LAMP reagents, which allows for reading of OPD results with only a handheld UV lamp (Xu et al., 2016).

The combination of these improvements results in a single Origami-paper diagnostic with a glass mounting disc and a series of predesignated folding segments containing dehydrated buffers and LAMP reagents with fluorescent tags. With the rehydration of reagents and aUV-light, this OPD would allow for rapid nucleic acid testing for the identification of febrile diseases. Critically, because utilizing the describedOPD at POC would require only rehydration fluid, a hot plate, and a UV light, the test could be administered outside of a clinical setting by an individual with limited clinical training. This approach takes full advantage of paper’s natural suitability for dry chemical storage and potential for manipulation via printing and forming 3D structures, offering a materially basic, chemically stable diagnostic platform.  

While the inclusion of four separateLAMPs in one OPD has been successfully demonstrated, and accuracy for malaria diagnoses have been shown to be as high as 98%, the ideal test would be capable of distinguishing between multiple strains of malaria, dengue fever, Zikavirus, Chikungunya, and Typhoid (Capedinget al., 2013; University of Glasgow, 2019). The core complexity to this breadth of disease cover is the need to identify both DNA and RNA viruses. In order to amplify RNA, reverse transcriptase LAMP would need to be run on the same device.A one-step rtLAMP process has been successfully demonstrated in testing for ZikaVirus with 95% accuracy (Silvaet al., 2019). rtLAMP could feasibly be completed on a distinct amplification site, similar to having multiple LAMP pads as demonstrated by Xu et al. (2016).

Material Accessibility and Local Production

OPDs offer a promising diagnostic technology that is chemically versatile and easy to use and distribute. But a successful diagnostic must be feasible throughout the entire value chain, from production to POC, to generate real uptake in LMICs. A significant barrier to making diagnostic technology readily available is the expense. FIND, a distributor of discounted diagnostics, prices their basic LAMP diagnostic kit at $3.33 pertest (FINDdx,n.d.). But when 10% percent of the world lives on less than$1.30 per day, these costs can still be insurmountable (TheWorld Bank, 2018). One solution the WHO and others have considered for increasing affordability is local production. Producing diagnostics in the country of interest (or at least in neighboring countries)will not only support local economies but can encourage price-based competition in local markets. For example, governments in Argentina and Indonesia have begun sourcing medical procurement from local producers because of cost advantages over importation (WHO,2011a). Local production will additionally allow for exact specifications of OPDs to be catered to countries’ particular needs: for example, countries such as Bangladesh may prioritize the addition of a typhoidLAMP to the test, whereas Uganda can prioritize malaria. Finally, local production has a demonstrated record for achieving extensive distribution:Quality Chemicals in Uganda, a firm producing ARVs, has successfully managed extensive rural networks (WHO,2011a).

In order to make local production feasible, however, manufacturing must be manageable in lower-resourced labs, and distributors must be incentivized to provide raw materials at reduced costs. Due to the low cost and complexity of production as well as ease of use,OPDs are equally promising as a value chain solution. After receiving basic materials, OPDs can be assembled with only a wax printer, hot plate, and vacuum oven (Xuet al., 2016). Wax is printed to create a three-dimensional barrier for the liquids, and these printed designs can be regularly modified using a computer (Cateet al., 2015). After the wax is deposited, the sheet is heated above melting temperature to allow the wax to permeate and embed the pattern (Rollandand Mourey, 2013). The buffer is added to the rehydration pad, dried in a vacuum oven, and attached to the device using mounting tape, while the LAMP reagents are similarly dried in a vacuum oven and centrifuged at 1400 rpm, then deposited to specific storage and rehydration pads (García-Bernalt Diego et al., 2019; Govindarajan et al., 2012).

The simple design of OPDs clearly supports production in less-resourced facilities. Primarily, the lack of requirement for a clean room or a micro-fabrication facility makes production more feasible than traditional diagnostic tests (Govindarajanet al., 2012). But the ease of fabrication is only part of the process—the price of materials must additionally be feasible for local facilities. It would appear at first that many of OPD’s requisite components would be financially accessible. Paper is extremely cheap at only a few hundredths of a cent per sheet, while wax and hot plates could likely be purchased in the country of production or imported from a developing economy such as China or India (Magroet al., 2017). In 2012, Govindarajan et al.estimated the entire construction process for OPDs could be completed for less than $2.

But the hope that these costs would remain affordable is not a sustainable solution, particularly when local production facilities are required to manage distribution relationships on their own. Of particular concern is the lack of Purchasing Power Parity in gaining access to these materials. PPP refers to rates of currency conversion that attempt to equalize the purchasing power of different currencies (OECD,2019). Without PPP, the affordability of goods does not transfer equitably across currencies, meaning that, for example, raw materials that are cheap in the United States can still be prohibitively expensive in sub-Saharan Africa. To ensure the availability of raw materials for local production of OPDs, this proposal suggests the creation of a centralized, non-profit, material distribution hub that coordinates with raw material distributors, ensures a high level of material quality, and sells aggregated materials to approved manufacturers in local countries at prices adjusted forPurchasing Power Parity.

This proposed organization—theGlobal Diagnostic Materials Facility—is modeled after the Global Drug Facility and would be an arm of the WHO. The Global Drug Facility procures tuberculosis diagnostics and treatment by coordinating with suppliers and stakeholders to facilitate access, provide technical assistance, and aid in the distribution ofTB resources (UNOPS,n.d.). The proposed GDMF would perform a similar role for nucleic acid diagnostics, with the primary goal of managing raw material access and distribution. The GDMF would purchase all necessary raw materials—including paper, glass, reagents, buffers, etc.—and sell them as entire packages to verified local producers (see Tables 1 & 2). The GDMF would be able to negotiate reduced rates with suppliers because of its non-profit status and association with the WHO.

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The purpose of this raw material procurement and distribution model is to streamline the supply chain and enable local production. The proposed process—with the GDMF managing raw materials procurement and local factories completing production—aligns with the WHO methods of separating Primary and Secondary Manufacturing. PrimaryManufacturing consists of manufacturing APIs, intermediaries, and excipients, whereas Secondary Manufacturing consists of the mixing of raw materials and producing different dosage formulations (WHO,2011a). Typically, only Tertiary Manufacturing (packaging already formulated products) is undertaken in LMICs, but this new model would enable the expansion to Secondary Manufacturing. Because the manufacturing is being completed locally and the raw materials have been purchased with PPP adjustments, these local producers will be able to sell the diagnostics to governments and local healthcare players at community-sensitive costs. This removes the issue of PPP at the local distribution level, as exemplified by the cost breakdown in Table 2. While the distributed manufacturing logistics may initially appear difficult, it is critical to remember that 42% of the diagnostics market is already claimed by small companies, suggesting that the diagnostics market in particular is amenable to local manufacturing (WHO,2011a).

Technology Transfer

A critical consideration in this proposed value chain is the necessary Technology Transfer between the third-party original developers of the OPDs and verified local manufacturers. The WHO (2011a)defines Technology Transfer as: “transfer of technical information, tacit know-how and performance skills, technical materials or equipment…with the intent of enabling the technological or manufacturing capacity of the recipients.” Successful Technology Transfer has been demonstrated through partnerships such as the U.S.-based Orasure Technology licensing assembly of its OraQuick HIV device to manufacturers in Thailand, as well as the a U.S.-based company that transferred R&D know-how to a non-profit public institution, Fiocruz, in Brazil (WHO,2011b, 2011a).

To facilitate successful Technology Transfer for the proposed development, there are two components: (1) the verification process of the local manufacturers by the GDMF, and (2) the alignment of the original developer’s economic incentives. The verification process will require coordination between local governments, the GDMF, and potential producers. Governments will be able to recommend production facilities, but facilities will be able to apply independently, as well. After application, the GDMF will deploy anon-the-ground evaluator to inspect the facility and verify conditions and resources are sufficient for OPD production. In order to receive approval, the facility will sign a contract in which they commit to only manufacturing OPDs with the materials provided by the GDMF and to regular inspections of their OPD manufacturing process and final products. At this point, the GDMF will help the facility meet ISO 13485 production standards for quality assurance purposes (ISO,2016). Finally, and most relevant for component two (2),the facilities will agree to apply the know-how delivered via TechnologyTransfer exclusively for the purposes of OPD development.

Regarding component two (2), the alignment of economic incentives for OPD IP holders, the primary consideration is that the technologies employed in OPD production are not useful exclusively for LMIC diagnostics. The non-profit organization Diagnostics for All (DFA) has taken advantage of the diverse applications of paper tests by creating a for-profit subsidiary, Paper Diagnostics (Maceand Ryan, 2012). Paper Diagnostics is the sole distributor of the paper-based technology used in DFA devices, but it also has the ability to partner with other for-profit producers on its technology, thereby generating external revenue. Given the variety of applications available for paper-based sensors (including monitoring food quality, pollutant and sun exposure, chemical balance, etc.) these companies will only be sacrificing a small amount of potential market value by offering OPD development know-how to local producers (Singhet al., 2018). Particularly given the low expected cost of these devices, the local use of IP will not be a significant loss to large development companies (see Table 4 for total anticipated revenues). The GDMF will act as facilitator in the transfer of technological know-how and will assume responsibility for ongoing support for effective implementation of the technology as well as ongoing monitoring to ensure the technological know-how is only being used for approved purposes. A summary of the described incentives for all stakeholders is collected in Table 3.  

Establishing the GDMF as an intermediary player between the original developers of OPDs and local producers is critical to filling a communication gap. A 2015 workshop found that despite the desire of laboratories to collaborate with inter-continental partners, poor communication resulted in a disconnect from the actual needs of the populations the partnerships were intended to serve (Derdaet al., 2015). The result was ineffective or insubstantial technology transfer.   

Distribution Plan

The next portion of the proposal presumes successful Technology Transfer, the streamlining of raw material access and subsequent distribution to local producers, and the local production of quality assured OPDs. The final challenge facing a successful diagnostic is the distribution plan from producer to POC. While OPDs can be feasibly implemented in rural clinics given the limited usage requirements of rehydration fluid and hot plates, the actual delivery of these diagnostics to large rural and semi-rural populations, and subsequently monitoring supply and demand, is an ongoing challenge. Here again the functional design of OPDs allows for the implementation of local systems without the need for advanced technology: OPD scan be stored at room temperature for 30 days, or in a refrigerator for 75days, without losing effectiveness (Chenet al., 2019). This level of device stability allows for a two-prong approach, overseen by the GDMF: partnerships with government clinics, and support for non-profit franchises.

Partnerships with government clinics are a more traditional method of distribution in which governments will purchase from the approved local producers with the GDMF acting as facilitator for price negotiation. Many governments in sub-Saharan Africa already maintain some kind of central medical store and a government-owned transport fleet, and these central medical stores maintain relationships with regional medical stores, which distribute to districts and community centers (Yadav,2015). Local producers will likely already have experience acting within these systems, and the benefit of working within an established network is that there tends to be greater sensitivity to local supply status (WHO,2011a). Governments will purchase the tests in bulk from local producers at the prices outlined in Table 2 (a case study for total costs is outlined in Table 4). In public healthcare systems, these tests will then be delivered freely to individuals at POC.  

Government spending on healthcare differs across countries, however, and in some countries such as India, over 70% of patients are treated in the private sector (Sayed et al.,2018). Because of this large private coverage, there must be additional, non-governmental distribution networks. For this distribution, the most promising network would be a private franchise model. Greenstar, a contraceptive provider, demonstrated the success of private franchise modelsi n Pakistan, where they signed franchising agreements with over 7,500 private healthcare providers (Bhattacharyya etal., 2010). In the case of Greenstar, Bhattacharyya et al.(2010) found that the majority of partnered clinics had been founded by local entrepreneurs interested in contributing to the success of local healthcare systems. Because many of these clinics receive funding from outside the government—including partnerships, grants and donations, and user fees—they function as distinct and independent methods of distribution. This model provides multiple benefits, including reducing the number of tiers in the healthcare delivery system, empowering local entrepreneurs to invest back into their community, and further supporting the local economy by facilitating the distribution of low-cost, high-volume diagnostics (AMS,2016; Yadav, 2015). Finally, maintaining these two distinct methods of distribution will generate healthy local competition and serve to keep prices low. Unlike public healthcare systems, these local clinics may charge individuals a fee for the test; but those who are privately insured may still receive the test for free, and those who pay out-of-pocket will still benefit from the low producer’s price of $1.00, because the final sale price can still be barely over $1.00.

These two distribution models will be united by a supply monitoring system. Review studies have suggested that stock-outs of POC diagnostic tests are mainly attributed to poor inventory management and inaccurate distribution systems (Kuupielet al., 2019). The GDMF will require certified producers to implement a barcode system in which all OPDs produced have a unique identifier barcode linked to both local producers as well as the GDMF which can be scanned by different players in the supply chain as they move through the pipeline. Barcodes have been demonstrated as a successful method for monitoring the flow of diagnostics within a healthcare system and the successful delivery of those materials to rural areas, as well as offering an easy method for local producers to order additional materials when running low (Pisaand McCurdy, 2019). This digital trail of information will enable steady supply and equitable distribution, greater ensuring effective implementation.  

Anticipated Difficulties 

There are a number of anticipated difficulties that could affect this proposal’s success. The most immediate concern is funding for full clinical trials and regulatory approvals. As a NAAT, this will necessarily include preclinical studies that will likely need to be performed at field sites in the countries of interest due to the low incidence of these diseases in developed countries. These preclinical tests will have to determine Sensitivity and Specificity; for ethical deployment, it is suggested that the tests be at a minimum 85% accurate (CBER,1999). In terms of full clinical testing and regulatory approval, the WHO outlines the following preferred approvals before launch:European CE mark, FDA approval, endorsement by the WHO, and approval by local authorities (Kosacket al., 2017). All trials and approvals will require significant financial investment. While certainly developers can apply for government grants and appeal to organizations such as the WHO and Bill andMelinda Gates Foundation for support, the lack of direct financial return from the approval of OPDs will present a significant lack of incentive. As was previously discussed, however, the fundamental technologies involved in OPDs will be transferable to more economically rewarding projects. Hopefully, humanitarian incentive and NGO financial support will be enough to overcome these financial hurdles.

Another important consideration is that some countries simply do not have an established enough healthcare infrastructure to deliver diagnostics. In these most severe cases, third-party logistics providers (3PLs) could be considered. It is important to note that this would require a greater amount of financing on the part of the GDMF, as the organization would be responsible for funding all distribution costs. In this model, after local production was completed likely in a more developed neighboring country, the GDMF would outsource distribution to a 3PL, which would act as a local fleet in delivering diagnostics to high-need areas. This model has been successfully employed by a number of non-profits, includingUNICEF in distributing antiretrovirals in Malawi (Dowling,2011). The limitation of this solution is its failure to empower local systems and provide meaningful technological investment. But in the most difficult cases of delivery, it remains a powerful option. 

Conclusion

Rapid, affordable diagnostic tests are a critical component of improving outcomes of febrile disease in developing countries. Existing solutions are not feasible for large-scale deployment because they fail to integrate with existing healthcare and economic systems.By relying on technologies such as OPDs which can be viably and economically produced in local facilities, improved diagnostics serve to not only improve medical outcomes but also facilitate the ownership of LMICs over their healthcare progress.

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