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Dutch Group Develops RPA-based Assay for Leptospira

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NEW YORK (GenomeWeb) — Researchers at The Royal Tropical Institute in Amsterdam have developed a novel isothermal recombinase polymerase amplification assay for pathogenic Leptospira, the spirochete bacterium that causes leptospirosis.

The new assay uses TwistAmp chemistry from Alere subsidiary TwistDx, and detects fewer than two genome copies per reaction with high sensitivity and specificity in as little as 25 minutes.

Leptospirosis is a zoonotic disease estimated to affect up to 10 million people each year. Work on the new assay, published this month in The International Journal of Environmental Research and Public Health, was also supported by a collaboration between the World Health Organization, the Food and Agriculture Organization of the United Nations, and the World Organization for Animal Health. The WHO/FAO/OIE tripartite partnership was created to focus on the unique health risks of the human-animal-ecosystem interface.

Leptospira tend to inhabit the kidneys of infected wild and domestic animals. From this reservoir, the spirochetes pass via animal urine into water or soil, and can infect people through contact with open wounds, drinking water, or splashing contaminated water into the eyes or mouth. Flooding tends to drive outbreaks in endemic regions. Leptospira infection initially has a mild febrile phase, followed later by potentially severe inflammatory phase. In the most serious cases, this results in jaundice, kidney and liver failure, and hemorrhaging in the eyes, skin, and lungs.

In a case study in PLoS Neglected Tropical Diseases published in January, lead researcher on the current study, Ahmed Ahmed, and a team in Nigeria described the process of diagnosing a young man with leptospirosis. This instance highlighted the standard of care and the obstacles clinics in low-resource settings face. Because the symptoms of leptospirosis are common to many tropical illnesses, such as dengue fever and malaria, there are a number of differential diagnoses to rule out. Diagnosis of Leptospira infection is based on culturing the bacteria from patient blood or urine, which can take several weeks, and antibody tests, which tend to be inconclusive until late stages of the illness.

Animal vectors can also be monitored to gauge the potential for an outbreak in a community. For example, Ahmed recently co-authored a study in which researchers captured and tested mice, rats, and shrews in the city of Cotonou, Benin. Using real-time PCR, they were able to amplify Leptospira DNA from the kidneys of nearly 20 percent of these animals. Ahmed also recently authored a vector sampling study in Kenya with similar results.

In the current study, the authors emphasized that early detection could result in patients receiving appropriate antibiotic treatment, and improved outcomes. PCR-based methods are the best way to show the presence of Leptospira in patient samples, but these usually require expensive labs and expert technicians.

Intended to enable molecular assays in low-resources settings, the new RPA test was first validated on a set of 45 Leptospira strains. These included pathogenic, non-pathogenic, and intermediate species. The assay's probe and forward and reverse primers were designed to align to a 90bp conserved sequence from pathogenic species.

The researchers found a temperature of 38 °C was optimal for the RPA reaction, resulting in a limit of detection of fewer than two genome equivalents. Importantly, the assay did not cross-react with non-pathogenic or intermediate species.

The researchers next tested 63 clinical samples from confirmed cases, and determined the new assay to be around 95 percent sensitive and similarly specific.

To extract DNA, researchers used either a QiaAmp kit from Qiagen or Roche's MagNa Pure system. The RPA assay was also compared to two previously published real-time PCR assays, and shown to have a similar limit of detection. The authors emphasized that the assay can now be modified to a lateral flow device, and that the digital nature of the readout suggests the potential for generating diagnostic data clouds to help track cases.

The new assay was developed using TwistAmp, a product of TwistDx. As covered previously in PCR Insider, numerous diagnostic assays relying on this chemistry have recently been reported. Adding to the growing number, last week a high school student won a Sanofi-sponsored Canadian biology challenge for developing a point-of-care HIV test using RPA. A paper published this week in Analytical Chemistry also showed that RPA can be used to not only detect but also to quantify HIV levels.

As TwistDx CEO Niall Armes explained previously, "This upswing reflects ... 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.

TwistDx was acquired by Alere in 2010. In order to develop assays using TwistDx products, Armes explained, users must license the rights to the RPA technology from the Alere group. He added that the company encourages "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 added that 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.

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