This article has been updated from a previous version to correct the spelling of Lakshmi Sundaram's name.
By Ben Butkus
A new molecular point-of-care test for African sleeping sickness being developed by the Foundation for Innovative New Diagnostics and Japanese clinical diagnostics firm Eiken Chemical has entered clinical field trials, FIND said this week.
The test, which is based on Eiken's loop-mediated isothermal amplification, or LAMP, technology, has the potential to dramatically improve the ability to diagnose African sleeping sickness, also known as human African trypanosomiasis, or HAT, obviating the need for the costly equipment and invasive medical procedures currently used in diagnosis.
HAT is the latest disease focus of an ongoing collaboration between FIND and Eiken to develop LAMP-based point-of-care molecular diagnostics, joining similar efforts for tuberculosis and malaria, Lakshmi Sundaram, an advocacy officer for FIND, told PCR Insider this week.
"Our idea is that the more disease tests we can have using the same technological platform, that's good for the end users," Sundaram said. "At the end of the day, it's the same people in the same clinics that have to do all of these different tests. So if there is one methodology and one [type of] kit that they need to have in the lab, that makes life so much simpler."
LAMP is promising as a core technology for point-of-care diagnostics due to its ability to amplify and detect target genes in a single step by incubating a mixture of sample, primers, a DNA polymerase with strand-displacement activity, and substrates at a constant temperature, typically in the region of 65° C.
According to Eiken, the method has a high amplification efficiency, with DNA being amplified 109- to 1010-fold in less than an hour; and high specificity, allowing users to detect the presence of a target gene sequence merely by noting the presence of amplified products.
Eiken and FIND are taking advantage of those characteristics to develop a test that will be as easy as possible for even non-trained laboratory personnel to use.
"The test kit involves a bunch of Eppendorf tubes with the reagents dried into the cap," Sundaram said. "We put some of the sample into the tubes; shake the tubes to reconstitute the reagents; put it in a heating block; and then on the front of the heating block there is a fluorescent reader, and you can visually read the output. Where parasites have been detected, it basically glows."
This protocol would be a far cry from the current process of diagnosing HAT, which involves multiple steps that necessitate expensive equipment and trained medical staff.
"There are three parts to the current method of diagnosing African sleeping sickness," Sundaram said. "The first part is this basic screening test … and that's a serological test. Usually what happens is there has to be a confirmation test, which is currently done with microscopy, to confirm there are actually parasites in the blood."
HAT typically affects impoverished rural communities in sub-Saharan Africa. It is transmitted by the bite of the tsetse fly, and about 60 million people in 36 countries are thought to be at risk, FIND said. The disease has no clinical signs, making it difficult to diagnose; and if infected patients are not treated they usually die.
Also, because HAT has two stages — a more innocuous stage 1 disease followed by parasites entering the brain — it can involve two different treatments, "and you actually have to do a lumbar puncture and do a test on the [cerebrospinal fluid] to be able to determine whether the parasite has gone into the brain," Sundaram said.
Having validated the LAMP-based HAT test in the laboratory, FIND and Eiken will now test the assay in clinical field trials in the Democratic Republic of Congo and Uganda. Researchers from the organizations will investigate whether the test can be used to confirm a diagnosis of the disease, even when parasites are present in low numbers; and whether it can be used to confirm cure after treatment for HAT, which would help reduce the follow-up period and eliminate the need for multiple lumbar punctures.
Sundaram said that the organizations would also like to develop a version of the test that can discriminate between the different stages of the disease, but added that the research team is "very far from that, quite a few years out … because we still don’t know the markers. FIND is working internally and with external partners to identify these molecular markers, she added.
If all goes well with the clinical field trials, FIND said, it hopes to have a NAT confirmatory assay available for clinical use next year. One of the bugaboos slowing commercialization efforts is sample prep, a common hurdle for developers of point-of-care molecular diagnostics.
"We're hoping that during these field trials we will be able to address sample prep," Sundaram said. "The test is currently done on blood. During the trials we will be figuring out the most optimal sample prep for blood. We think from preliminary data and the lab trials that we would be able to use dried blood spots on filter paper, but part of our protocol is also to look at different ways to prepare the sample."
Having partnered for the better part of six years, FIND and Eiken also have LAMP-based POC tests in the works for other diseases commonly found in developing nations, including tuberculosis and malaria. Those tests are similarly plagued by sample prep issues, but Sundaram said that the organizations are hoping they will also be commercially available in 2012.
FIND and Eiken have not disclosed financial terms of their various agreements, but Sundaram said that in general FIND helps fund development of the diagnostic tests and Eiken is obligated to sell it at cost to FIND-defined markets in the developing world.
"[Eiken] can't make a profit on it, but they don't lose money by making this test," Sundaram said. "It gets them into a variety of other markets that they wouldn't ordinarily get into. And they can use this to potentially [develop tests] in other markets."
Sundaram also noted that it's too early to speculate on the cost of the HAT test "because it will depend on the final configuration and everything that goes into the kits, and what types of volumes we are thinking about."
In the meantime, FIND and Eiken are also exploring other potential uses for the assay. "Because we think this technology has potential for surveillance, as well, we're also exploring the idea of developing a high-throughput version" to use in central labs for epidemiology studies, Sundaram said.
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