A team at the University of Potsdam and the Fraunhofer Institute for Biomedical Engineering (IBMT) in Germany has developed a lateral flow assay to detect P. falciparum parasite, the most deadly of the four Plasmodium species that cause malaria.
The assay, published last week in Malaria Journal, uses isothermal recombinase polymerase amplification, or RPA, and a lateral flow detection method. It runs at near-ambient temperature, produces a simple visual readout, and could potentially be developed into a point-of-care malaria test for low-resource settings. It was shown to be 100 percent specific for P. falciparum and detected as few as 8 parasites per 100 microliters of sample.
In an email interview with PCR Insider, lead author Sebastian Kersting of IBMT said the reaction "works quite efficiently in a broad range of temperature, from 30-45 °C." The lateral flow analysis gives a clear yes/no answer, he said, by a colored band visible to the naked eye. It is also an advantage over traditional PCR or microscopy in that it does not require additional instrumentation and can be performed by personnel with minimal training, which reduces overall cost.
To develop the assay, the group first needed to design appropriate primers. RPA requires longer ones, typically between 30 and 35 nucleotides, and databases aren't set up for this kind of search. "Using Primer-BLAST was only the final step after a long period of literature research and bioinformatic alignment of many sequences available in the Genbank database," Kersting said. His group focused on multi-copy genes, and ultimately targeted the "classical" 18S rRNA gene.
Kersting's group used RPA technology supplied by UK-based TwistDx, a company acquired by Alere in 2010. Alere has since used RPA in its HIV and influenza diagnostics.
Isothermal RPA combined with lateral flow for POC testing is currently being developed by a number of other groups as well. At least five RPA-based assays have been published so far this year. These include ones for tularemia and yellow fever, as well as cryptosporidium and Group B streptococcal infections. Late last year, an Estonian group developed an RPA test for chlamydia.
TwistDx supplied the RPA reagents for all of these assays. In an email with PCR Insider, TwistDx's CEO Niall Armes concurred that there seems to be a recent boom in publications using the company's technology. He said TwistDx began selling its R&D product four and a half years ago, and attributed the number of recent publications to the fruition of long-term research projects.
"This upswing reflects, we believe, a success in providing general reagents which customers can easily make work ‘out of the box,’ and also that the RPA technology is unique among isothermals in being applicable in as simple and potentially broad-application manner as PCR," he said.
In RPA, recombinases pair primers with homologous duplex DNA, and this kicks off amplification to detectable levels within 10 minutes, according to TwistDx's company website. The reagents do not require refrigeration. "In the RPA TwistDx-kit all reaction components come in a convenient freeze-dried pellet. The solution is then simply resuspended with buffer and the primer/template-mix," Kersting explained.
The lateral flow element used in the malaria assay was a Milenia Biotec Hybridetect 2 T dipstick. This strip was used to "keep it as simple as possible," Kersting said. The readout mechanism uses dried anti-carboxyfluorescein to detect the RPA products, but Kersting said any test strip with antibody detection could work, as could biotin-based readouts.
One obstacle for deploying the malaria test is a possible requirement for electricity. The authors found the test worked best if incubated on a shaker. Kersting said "a complete omission of shaking leads to around 100-fold reduction in sensitivity and a generally less efficient amplification with low amounts of template DNA." He said that he was aware TwistDx has developed a portable battery-powered mixing incubator "which is rather low cost and which might allow a standardization of the assay in field use." He also added that, for low-resource settings, it might be possible to conduct the test by heating the sample using body temperature and occasional shaking, although this would require further testing.
The published assay was not run using patient samples. Instead, the authors spiked varying concentrations of Plasmodium DNA into whole blood or plasma, and included common PCR inhibitors likely to be encountered in the field. While the assay performed well in most conditions, whole blood appeared to inhibit the reaction. Thus, centrifugation of blood samples could also be required to use this test in its current configuration.
Kersting said sample prep using different separating membranes, chemical lysis, dried blood spots, or sample boiling could be alternatives. "We are quite convinced that the LF-RPA is robust enough to deal with many different sample materials," he said. "Even a direct amplification from blood samples may be possible." He added that his group has not yet found the substance in blood that affects the RPA, but hypothesized it might be the anticoagulant present in the blood samples they tested. "If discovered, a potential extraction method could then eliminate this substance and allow efficient testing with minimal sample preparation," he said.
Since clinical diagnostics and healthcare company Alere acquired TwistDx four years ago, it has since used RPA to develop its own HIV and influenza assays. The flu test was launched in Europe in January. Alere also recently received $21 million in funding from The Bill and Melinda Gates Foundation, part of which was dedicated to developing a tuberculosis assay using a "previously acquired isothermal amplification technology," as reported in GWDN.
"The rights to the RPA technology reside with the Alere group, and commercial out-licensing must be approved by the company accordingly," TwistDx's Armes said. "That said, generally, we wish to encourage a broad uptake of the technology which we believe offers unique potential in many areas well beyond the scope of Alere’s interests, and of course simultaneously respect the key goals of the group."
He said TwistDx is "capable of supplying materials in customized, stabilized formats" and that it holds ISO13485 status for manufacturing RPA pellet reagents. "Thus, R&D customers who express an interest in going further and commercializing a home-grown test, or have a need for RPA in any other commercial product, are encouraged to contact TwistDx or Alere to discuss potential access, partnering and supply," Armes said.
Kersting said his group will soon publish work demonstrating multiplexing using lateral flow RPA, and that it is exploring integrating the test into a handheld, lab-on-chip diagnostic, an area in which IBMT has expertise. They are also working on parallel detection and differentiation of Plasmodium species in a single assay. "We are currently searching for a strong partnership with an institution that is experienced in malaria testing and would like to further develop and test the described method in collaboration," he said.
"In my personal opinion, a test which could significantly contribute to malaria eradication should be made widely available at the lowest possible cost," he added. "Nonetheless, further tests integrated into one of our platform technologies may also be distributed under license of the enzyme manufacturer and with an additional CE marking."
Kersting said he has no doubt that "RPA will be useful for many applications in the point-of-care test setting" and might also become a vital tool to conduct sophisticated molecular tests in a doctor’s office.