A public-private consortium formed to investigate multi-drug resistance in malaria this week reached the end of a five-year, €2.8 million (about $3.6 million) project having identified a number of genetic markers related to drug resistance and developed a highly sensitive and specific direct-from-blood, lateral flow strip-based PCR assay for Plasmodium, the family of parasites that causes the disease.
The consortium, which comprises universities, research institutions, and companies from several countries in Europe and Africa, has now been awarded follow-on funding of approximately €3 million over the next three years to further refine the assay by pairing it with a portable and inexpensive thermal cycler and enabling it to discriminate between Plasmodium species, among other goals.
"Ultimately the aim is to bring this to the market," Henk Schallig, parasitology research coordinator at Amsterdam's Royal Tropical Institute (KIT) and a principal investigator on the project, told PCR Insider this week. "It will be one of the first molecular diagnostics for malaria that will go through all evaluation phases comparable to phase I, II, [and] III testing with medicines."
The consortium, called the Malactres project, began in 2008 with the goal of assessing specific genetic markers for Plasmodium resistance to combination therapies based on the anti-viral artemisinin. Consortium members include KIT and Wageningen University in the Netherlands; the London School of Hygiene and Tropical Medicine and Forsite Diagnostics in the UK; the Institute of Tropical Medicine in Antwerp, Belgium; the Kilimanjaro Christian Medical Centre in Moshi, Tanzania; the University of Benin City in Nigeria; and Centre Muzaz in Bobo Dioulasso, Burkina Faso.
Schalling explained that artemisinin-based combination therapy, or ACT, is currently the most widely used treatment for malaria worldwide. It is generally efficacious, but this efficacy is at risk due to emerging resistance. As such, one of the consortium's main goals was to develop innovative, rapid, and simple molecular diagnostics for the parasite and ACT resistance.
The gold standard for malaria diagnosis has long been smear microscopy, which is accurate but expensive, labor-intensive, and often not conducive to use in resource-limited settings. Many research groups are developing molecular assays based on PCR or isothermal amplification because of their accuracy and relative speed, but those technologies present their own challenges.
"To apply PCR methods in resource-poor conditions, you need to make it as simple as possible," Schallig said. "The methodology that [we are] currently exploiting is circumventing at least two major steps — first, DNA extraction; and second, the readout system is much simpler, thereby circumventing the use of gel electrophoresis, which needs laboratory equipment and generates toxic waste due to the use of ethidium bromide."
The researchers have obviated the need for DNA extraction by developing a method to conduct PCR amplification of specific Plasmodium DNA targets directly from human blood, which is known to contain several potent PCR inhibitors.
The researchers achieved this capability somewhat serendipitously, Schallig said. Their PCR cocktail uses a combination of the Phusion PCR buffer and Phire Hotstart II DNA polymerase from Finnzymes, a subsidiary of Thermo Fisher Scientific. The buffer is a component of Thermo's Phusion blood direct PCR kit, while the polymerase is a component of the company's Phire Animal Tissue Direct PCR kit.
"We don't know exactly why it works, because blood is a strong inhibitor for PCR reactions," Schallig said. "It was just by chance that one of our technicians was able to … combine some of those [components]."
Schallig noted that because the group is currently using buffer from one commercial kit and polymerase from another, it is "a bit of a waste of money," and as such it would like to work with Thermo to create customized kits for their specific assay.
Thermo Fisher did not respond to a request for comment prior to publication.
Meantime, the Malactres consortium has simplified assay readout by using a nucleic acid lateral flow immunoassay, or NALFIA — a rapid immunochromatographic test in which labeled amplicon products are deposited on a nitrocellulose stick coated with specific antibodies and can be essentially detected with the naked eye.
"We just put a drop of blood in the PCR tube, add the reagents, and put it in the PCR machine," Schallig said. "After about 45 minutes we add the [amplicon-containing solution] … to these NALFIA strips. Within about an hour we have a result, and you can run several samples in one go."
The simplicity of the assay currently lends itself particularly well to malaria control and elimination programs, in which you need to scan a lot of people with low parasitemias," Schalling noted. "Normally if you do microscopy, which is the only alternative, at a sensitivity about 20 times less than PCR it may take you up to 20 or 30 minutes to screen one person. With this method you can run 96 patients in an hour or so."
In a paper published in November in the Journal of Clinical Microbiology, Schallig and other consortium scientists described their assay in detail and compared it to a Plasmodium lactate dehydrogenase-based rapid diagnostic test and microscopy using samples from confirmed Sudanese malaria patients, returning travelers, the Dutch Blood Bank, and suspected malaria cases in the field in Burkina Faso and Thailand.
The sensitivity and specificity of the direct-from-blood, NALFIA-based PCR assay compared to initial diagnosis in the laboratory were 94.4 percent and 97.4 percent, respectively. In Burkina Faso, the assay achieved sensitivity of 94.8 percent and specificity of 82.4 percent compared to microscopy and 93.3 percent and 91.4 percent compared to RDT. Finally, in Thailand, the sensitivity and specificity were 93.4 percent and 90.9 percent, respectively, compared to microscopy, and 95.6 percent and 87.1 percent compared to RDT.
The assay still has several shortcomings before it can be successfully deployed for regular malaria diagnosis or control programs in the developing world. Chief among these is the fact that it still requires a thermal cycler — a standard piece of equipment in almost any laboratory worldwide, but one that still necessitates electricity.
In addition, the direct-from-blood, NALFIA-based PCR assay is highly sensitive for the detection of P. vivax and P. falciparum — the two most relevant disease-causing species — but cannot differentiate between them. In addition, the researchers have not yet assessed the ability of the assay to detect other human Plasmodium species.
The consortium is now looking to address these issues, and to further evaluate their assay in the field, using a new €3 million funding infusion from the EU, which has not yet been formally announced, Schallig said. In addition, the Malactres project will look to commercial entities for help.
Besides Forsite Diagnostics, which develops and manufactures the lateral flow immunoassay components of the assay and has been involved in the project from the start, the consortium has brought on board German firm Q Bioanalytic, which specializes in PCR and real-time PCR systems for detecting pathogenic organisms in food and feed; and Finland's Global Innovation Network, which specializes in laboratory automation.
In addition, "there are all kinds of inventions emerging to make PCR simpler using less equipment," Schallig said. For instance, the consortium has already worked with Korean company Ahram Biosciences to potentially marry its handheld, battery-powered Palm PCR system (PCR Insider, 4/21/2013) with the NALFIA-based PCR assay.
So far, that marriage has "not been very successful, because the capillary tubes inside [Palm PCR] are very small, and because we are working with blood, it gets sticky and you don't get very good movement in the tubes," Schallig said.
Furthermore, Schallig said he has been in contact with some founders of Dutch startup company Amplino, which has been developing a mobile, cloud-connected, cartridge-based diagnostic device specific for malaria field testing. "They are trying to build a very simple PCR machine that will cost about €250 [and] fits in a shoebox," Schallig said.
"And then you can start to think about PCR systems that are battery-operated or operated on a solar panel," he added. "By combining this direct-from-blood method with hopefully a simplified PCR machine, and a simple readout system, then we can really bring this closer to the field."
Schallig estimated that the consortium will now spend about a year optimizing its PCR reaction and enabling it to discriminate between Plasmodium species. In parallel, the group will also attempt to optimize a closed format of its assay to reduce the risk of contamination that exists from having to transfer amplicons from the reaction tube to the NALFIA strip.
"Then I assume we'll have about one to one-and-a-half years for field testing … in a variety of malaria-endemic settings," Schallig said. "And of course there are some pre-requisites from the [World Health Organization] pre-qualification that we still need to fulfill. We will try to do that in parallel."