NEW YORK (360Dx) — Researchers in Scotland are using the art of paper folding as an inspiration to better detect malaria.
This week, University of Glasgow researchers initiated a first-in-human clinical study to validate a mobile phone-based, origami-inspired microfluidic test using loop-mediated isothermal amplification (LAMP) to detect the DNA from malarial and parasitic flatworms called schistosomes in 220 children in Uganda.
The microfluidic technology that they are developing is unusual in its combination of an origami paper structure for preparing a blood sample and the integration of isothermal amplification of DNA with lateral flow detection, Jonathan Cooper, one of the test developers and chair of bioengineering at the University of Glasgow, said in a phone interview from Uganda.
The lateral flow detection format is familiar to community users and easy to interpret, he said, adding that making available an affordable diagnostic test that can differentiate malaria from other diseases that are often mistaken for malaria, such as brucellosis, is vital for administering the most targeted treatment for patients in such settings.
He and his colleague Julien Reboud, both of whom are conducting field testing this week in Tororo, an underserved and resource-limited region of Uganda, were among the authors of a paper published earlier this month in Proceedings of the National Academy of the Sciences. In it, they described their development of paper microfluidics that enable low-cost, multiplexed DNA-based diagnostics for malaria, delivered, in an earlier first-in-human study, in schools in rural Uganda.
In the paper-based microfluidic device, the sample-processing steps included DNA extraction, LAMP isothermal amplification, and lateral flow antibody-based detection of DNA amplicons. The test was designed as a multiplexed assay to identify Plasmodium falciparum, the endemic malaria-causing parasitic species in the region of study, as well as all other Plasmodium species that cause malaria, which might be present for various reasons such as human migration.
The University of Glasgow researchers said that the results of the study demonstrate that paper-based microfluidic devices can deliver precision diagnostics for malaria in low-resource, underserved settings with a sensitivity that is higher than that of the current malaria diagnostic tests used in the field, such as microscopy and rapid immunoassay-based diagnostic tests, and with performance that is similar to that of a laboratory-based real-time PCR test.
The paper-based microfluidic test under development "represents another demonstration of the potential of low-cost materials to carry out complex bioassays near the patient," Paul Yager, a professor in the department of bioengineering at the University of Washington, said in an interview.
Yager, who is not involved in the Glasgow team's efforts, and his colleagues are developing a low-cost paper-based diagnostic platform that integrates quantum dots, a cellphone camera, and an ultraviolet light-emitting diode to detect infectious diseases at the point of care, and he favors the use of lateral flow immunoassays for malaria testing.
"If there is a disease for which there is a plethora of valid, simple, and inexpensive rapid protein-based lateral flow tests, it is malaria," Yager said. Lateral flow immunoassays are widely used in low-resource areas and don't require instrumentation or electric power. They can detect the presence of species-specific antigens derived from the malarial parasites from a finger prick of blood in about 15 minutes.
However, the performance of lateral flow immunoassays in detecting malaria has been limited by their generation of false-positive results, which occur when nonspecific biomolecules present in the blood react with the test antigens, the University of Glasgow researchers said, citing a study published in the Journal of Clinical Microbiology.
The sensitivity of lateral flow immunoassay tests is less than the accepted target of 97 percent sensitivity as a minimal requirement set by the World Health Organization and Foundation for Innovative Diagnostics, they noted.
Current field testing for the diagnosis of malaria also relies on the use of microscopy that can lead to false positives and false negatives, Gerald Kost, director of the point-of-care testing center for teaching and research at the University of California Davis School of Medicine, said in an interview.
These performance limitations — revealed by field inspections of performance and quality control at testing sites in limited-resource countries — "decrease corresponding post-hoc predictive values," he said.
The University of Glasgow researchers "offer a practical and valuable alternative and document high sensitivity," said Kost, who is not involved in the test's development.
Such a high-sensitivity assay could be effectively applied in testing that requires ruling out specific indications. For example, the microfluidic device in development could prove to be useful for ruling out malaria when patients suspected of having the Ebola virus present with fever, Kost added.
The University of Glasgow test consists of origami paper-based microfluidic sample preparation using hot wax printing to form channels that either repel or attract blood moving through the structure by capillary force prior to detecting DNA that is specific to malaria.
Almost all the cost for the platform in its current form is for the freeze-dried enzymes and reagents that are used to trigger an isothermal amplification event that makes the device sensitive enough to differentiate between disease pathogens, even when they are present at such low abundance that the individual is asymptomatic, Cooper said.
In an earlier, published study, the research group used the test in village schools and were able to complete individual diagnoses from a finger prick of blood in less than 50 minutes.
They evaluated the performance of the device in 67 children ages 6 to 12 in primary schools in St. Kizito and Mayuge districts, Uganda. They compared the effectiveness of the device against two standard field-based techniques — rapid lateral flow immunoassay diagnostic testing and light microscopy — and against a laboratory-based, real-time PCR assay conducted retrospectively and developed in collaboration with Colin Sutherland at the London School of Hygiene and Tropical Medicine.
The test enabled the diagnosis of malaria species in whole blood. The microfluidic device proved to be highly sensitive and specific, detecting malaria in over 98 percent of infected individuals in a double-blind, first-in-human study. The analytical sensitivity of the Plasmodium pan assay, which detects several Plasmodium species (including P. falciparum, P. malariae, P. vivax, and P. ovale), was 105 IU/mL after 45 minutes of amplification. The P. falciparum assay detected this species alone with a similar level of sensitivity as the Plasmodium pan assay.
The method was more sensitive than other field-based, benchmark techniques, including optical microscopy and rapid immunoassay diagnostic tests, both performed by an experienced local healthcare team and which detected malaria in 86 percent and 83 percent of cases, respectively, according to the researchers.
The innovations that they described demonstrate the potential for such tests, either in the precise diagnosis of disease, enabling informed species-specific therapy, and longer term, for global surveillance or screening, Reboud said in an interview.
People living in remote rural communities would benefit from rapid, highly sensitive molecular, DNA-based diagnostics to inform the correct and timely treatment of infectious diseases, he said.
Healthcare workers currently face practical and logistical problems in the implementation of such tests, which often involve complex instrumentation and centralized laboratories.
By performing sample preparation using paper folding, the researchers were able to determine when fluid from one paper fold moves onto into the next sample processing step. The use of filter paper from commercial sources and printing of wax to create a contrast between hydrophobic and hydrophilic surfaces could be done almost anywhere in the world, Reboud said, adding that the test that they are developing could consequently be manufactured almost anywhere with the use of these minimal resources.
To prepare the design of their microfluidic device for scale-up, the research group is considering implementing screen printing and possibly 3D printing of a cartridge into which the microfluidic test would be inserted. In Uganda, they are working with Makerere University to determine whether they can manufacture the device at its Industrial Research Institute and simultaneously test the concept and potential to manufacture the tests near communities in need.
Low resources, high prevalence
Malaria is a leading cause of morbidity and mortality worldwide with more than 219 million cases in 90 countries and more than 435,000 deaths in 2017, according to the World Health Organization.
The 2018 WHO malaria report noted that malaria cases have significantly risen in 13 countries after almost two decades of decline. Healthcare workers require low-cost, rapid field tests that are sensitive and robust in order to help combat the spread of infectious diseases including malaria. However, many existing tests have proven unsuitable for remote, rural communities that lack refrigeration and laboratory equipment.
To address such issues, researchers are developing and deploying advanced diagnostic tests. In addition to the work by the University of Glasgow, researchers led by a team from Washington University in St. Louis recently received National Institutes of Health funding for a malaria breathalyzer project.
Additionally, the nonprofit Global Good Fund is partnering with companies such as QuantuMDx to develop diagnostic technologies for low-resource setting targeting diseases including malaria.
Sight Diagnostics and Abbott also have efforts aimed at malaria.
Cooper noted that while the prevalence of infectious diseases is on the rise, access to tests for them in low-resource communities is limited.
When schistosomiasis testing was previously completed a few years ago in the Tororo district, the results showed "an extremely high prevalence that was treated with mass drug administration a few years ago," Cooper said. His team has added schistosomiasis detection to its platform to enable access to testing for the condition, and it is currently exploring the potential for a resurgence in the Tororo district.
Since the work described in PNAS, they have translated their technology to operate with a portable smart-phone based platform, using its battery to provide the heat for the LAMP assay. The mobile phone also controls the thermal distribution and displays real-time results. Their initial data collected in Tororo this week looks very promising, according to Cooper.
He noted that their multiplexed tests are also being used in One Health scenarios, which recognize that human health is connected to animal health and the environment. The tests simultaneously test DNA from several species from a single sample.
The work, being codeveloped with the University of Makerere in Uganda, is focused on helping mitigate the issue of disease transmission from animals — such as dogs and livestock — to humans and includes the detection of brucellosis DNA. The disease is often transmitted in underserved settings to young children through drinking of milk that hasn't been sterilized because of the lack of an energy infrastructure to boil the milk.
Their research and development work is being funded by the UK's Global Challenges Research Fund, the Engineering and Physical Sciences Research Council (EPSRC), National Institute for Health Research (NIHR), the Scottish Funding Council, and Biotechnology and Biological Sciences Research Council (BBSRC).