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Studies Indicate Effectiveness of New LAMP-based Malaria Test in Modern Labs, Endemic Countries

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A pair of recently published peer-reviewed studies has vetted the performance of a new test based on loop-mediated isothermal amplification, or LAMP, as a tool for the quick and easy diagnosis of malaria in both modernized laboratories and in the field in areas of the world where the disease is endemic.

According to researchers involved in the studies, the publications may help drive widespread adoption of the new test, which was developed by Japan's Eiken Chemical and the non-profit Foundation for Innovative New Diagnostics, and is now commercially available with CE marking.

In addition, the assay may prove particularly useful as a surveillance tool to assist in tracking infections and curbing transmission, particularly if a higher-throughput version of the assay being developed in collaboration with UK-based molecular diagnostics shop Lumora eventually reaches the market, the scientists said.

FIND and Eiken, which owns the intellectual property around the LAMP method, began collaborating on infectious disease test development in 2005 and began feasibility studies specifically for the malaria test in 2008.

The main advantage of LAMP over gold-standard diagnostic methods such as smear microscopy, as well as newer, more sensitive methods such as PCR, is that it generally requires no specialized equipment to perform, making it an ideal testing platform in the developing world.

"Using this test requires only a room with very basic laboratory facilities, i.e. electricity," Iveth Gonzalez, a scientific officer and project manager of LAMP projects for malaria and Chagas disease at FIND, and an author on the studies, told PCR Insider in an email. "This test can be used in any microscopy-level clinic in resource-poor nations and doesn’t need standard infrastructure for PCR."

In addition, the assay scan be run by relatively unskilled technicians, provides results in less than an hour, and uses reagents that can by lyophilized and shipped and stored without refrigeration.

Even though multiple studies have demonstrated that LAMP-based assays are at least as accurate for molecular diagnosis as PCR for a variety of infectious disease targets, the specific malaria test from Eiken and FIND had not yet been rigorously evaluated in a clinical study — until last month, when the new studies were published online at the same time in the Journal of Infectious Diseases.

In the first study, a team led by researchers at London's Hospital for Tropical Diseases, the London School of Hygiene & Tropical Medicine, and FIND compared LAMP to existing laboratory diagnostic methods on 705 blood samples of suspected imported malaria cases in the UK.

The LAMP-based test correctly identified every malaria patient out of 705 malaria tests performed. The test was also faster than PCR and more accurate than microscopic examination of blood smears, long considered the gold standard for malaria diagnosis.

In the second study, researchers from HTD, FIND, LSHTM, and the Uganda Ministry of Health in Kampala examined the accuracy of the test at a rural clinic in Uganda.

The researchers tested blood samples from 272 patients with suspected malaria using LAMP along with a simple generator to provide electrical current, then compared the results with expert microscopy and PCR performed at central reference laboratories. The LAMP assay detected cases of low-level malaria parasite infection that were missed by expert microscopy and was essentially as accurate as PCR down to very low levels.

Scientists at the Malaria Reference Laboratory at LSHTM now plan to use the LAMP malaria test to help identify imported cases of malaria in the UK, and the partners are hoping to begin deploying the assay in the field in malaria-endemic countries.

"What's different about this compared to any other evaluation I've done of a technique is that it actually has been commercially produced and CE marked, and the company is now set to sell this to people," Colin Sutherland, clinical scientist at HTD, a reader in parasitology at the Malaria Reference Laboratory at LSHTM, and an author on the studies, told PCR Insider.

"Now, in our own practice I can just order up this kit and fire away," he added. "Because it's CE marked and supported by peer-reviewed publications, I can use it with confidence to help diagnose patients in the UK health system, for example. So it now fits all the criteria of a front-line test that can be used for diagnosis. And that's not a trivial thing."

FIND's Gonzalez underscored the importance of the LAMP test's ability to detect very low parasite densities in blood that are not detected by microscopy or rapid tests.

"Detecting these low parasitemias allows early treatment and prevention of severe malaria," she said. "LAMP can also be used in population surveys to detect and treat asymptomatic carriers with the final goal of stopping transmission."

Sutherland did note that in malaria-endemic countries, there are still operational issues to work through in laboratories, but the new test should still be a boon for malaria diagnosis and control.

"Lab technology is currently set up at low throughput, and the kit is quite low-throughput, but it's still a lot faster than doing [microscopy] slide readings," he said.

Sutherland added that although microscopy is still used at the level of district hospitals in endemic countries, rapid immunoassays are still favored in more remote areas. Here, too, LAMP holds an advantage.

"The antigen tests have benefits in that they are very quick and easy, and if they are positive, it means that someone has malaria or has had it in the last couple of weeks," he said. "But they have two main disadvantages: they're expensive, a few bucks for each patient; and they are not as sensitive as microscopy, and are nowhere near as sensitive as LAMP."

As such, "for frontline diagnoses LAMP has advantages over [antigen testing], but there is another application in Africa, Asia, and Latin American countries … and that's for malaria-control programs," Sutherland added. "We are quite excited about the prospect of LAMP as a surveillance tool, because you can take samples from a whole village or school, and determine with very good sensitivity which of those individuals have got malaria parasites. So you can start the control program even when people aren't coming to the clinic sick."

This application may be advanced by the development of a higher throughput version of the assay currently being developed by Lumora and FIND (PCR Insider, 4/18/2013). This version, which will combine Lumora's bioluminescent assay in real-time, or BART, technology, with the LAMP assay, may greatly increase testing throughput.

"The current LAMP kit has been developed for case management, and small groups of samples can be tested simultaneously," Gonzalez said. "FIND is working with Lumora [to develop] the next generation of the assay which is a high-throughput format that will allow testing of bigger groups of samples. The application of this assay will mainly be for population screening in elimination campaigns."

Challenges remain, particularly in the area of sample prep. The researchers demonstrated in their paper the use of both a relatively rudimentary but inexpensive "boil and spin" method, and an Eiken-developed Loopamp PURE DNA extraction kit to purify DNA for downstream analysis. Both worked well, but prepping nucleic acids from blood samples can still be a technical hurdle, especially in less-well-equipped laboratories.

"One of the big advantages of LAMP over PCR is that the enzyme involved is not very fussy about the odd bit of hemoglobin or other contaminant coming through in the mix," Sutherland said. "There is a level of purity below what is required for PCR amplification. So the boiling [method] is a real option here for the endemic countries and will be attractive because it's cheap."

In terms of pricing for the LAMP assay itself, FIND's Gonzalez said that the kit is produced and commercialized by Eiken through FIND, and that one kit to test 48 samples for both the presence of Plasmodium species in general and specifically Plasmodium falciparum costs around $380, a figure she estimated from Japanese Yen pricing. That comes out to a little less than $8 per sample. PCR-based tests are priced similarly, Sutherland said, although antigen-based testing is likely a little cheaper.

"It's hard to tell because the kits will be made only in very small numbers, but if it becomes widely used, prices go down," he said.

Through their partnership, Eiken and FIND have also commercialized CE-marked LAMP kits for tuberculosis and human African trypanosomiasis, and they are developing kits for leishmaniasis and Chagas disease.

The recently published studies on the LAMP malaria test were funded by the Bill and Melinda Gates Foundation, the Ministry of Foreign Affairs of the Government of The Netherlands, and the UK Department for International Development.

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