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UPenn Team Receives NIAID Award to Develop LAMP-based Point-of-care Dx for Parasitic Helminths

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NEW YORK (GenomeWeb) — Parasitic worm infections remain a difficult-to-detect neglected tropical disease, in part because the most sensitive diagnostic methods require a molecular biology lab.

Now, researchers at the University of Pennsylvania have received funding to develop a point-of-care microfluidic diagnostic device that uses loop-mediated isothermal amplification to detect parasitic worms, or helminths.

The award, administered through the National Institute of Allergy and Infectious Diseases, totals almost a quarter of a million dollars for a two-year project. The end result will be a device able to detect a number of parasitic platyhelminthes and nematode species, including roundworms, hookworms, and whipworms. Initial work will focus on schistosomes, the parasites that cause schistosomiasis.

According to Robert Greenberg, lead researcher on the grant and an associate professor of pathology at the UPenn School of Veterinary Medicine, over a billion people worldwide are infected with helminth parasites. "Schistosomes alone infect about 200 to 300 million people. Soil-transmitted and filarial worms like hookworms … each constitute hundreds of millions of infected people worldwide," Greenberg said in an interview with PCR Insider.

Helminth infections cause anemia and stunted growth in children. The worms damage internal organs, and cause symptoms like elephantiasis and blindness. In addition to infecting humans, these worms also infect domestic animals and livestock.

To create the new diagnostic device, Greenberg has teamed up with a professor of mechanical engineering and applied mechanics, Hiam Bau, whose expertise is in microfluidics.

The device will use LAMP, an isothermal amplification method that generally does not require expensive instrumentation to perform, and as such is intended to help diagnose helminth infections in people residing in low-resource settings, although it could also have veterinary applications.

In an email with PCR Insider, Bau said that the components of the chip are scattered through a few papers published over the past five years. A 2011 publication in Analyst describes the core of the microfluidic component, a "multifunctional amplification reactor for nucleic acid isolation, concentration, purification, amplification, and monitoring," he said.

A 2011 paper in Lab on Chip describes an optional feature — "self heating, enabling operation without external power by supplying heat through an exothermic reaction of water with [a] magnesium oxide-iron alloy, and regulating the temperature with a phase change material," Bau said.

Meantime, a 2009 paper in Lab on Chip described "in-reactor storage with just-on-time release of the reagents and dye needed for the enzymatic amplification," while a 2013 Analytical Chemistry paper demonstrated on-site separation of plasma from whole blood without centrifugation.

What sets the proposed microfluidic device apart from others is how much of the amplification process will take place within the chip, Bau said.

"Most of the microfluidic PCR reactors described in the literature are standalone chambers that require other instruments for the sample preparation — a chip in the lab rather than lab on a chip," he said.

"Our devices differ from most others in being multi-functional [and] capable of operation without any instruments," Bau said. "Our approach is to make molecular diagnostics nearly as simple as dipsticks, with the performance of benchtop equipment, for use on site, in the clinic, in the field, and at home."

The team will initially focus on schistosomes, a parasite most prevalent in sub-Saharan Africa. Larvae in fresh water pierce human skin and take up residence in blood vessels surrounding the intestinal or uro-genital tracts. The gold standard for detection involves counting any eggs shed into the feces or urine of a human host.

"You have a microscope and you sit there and count, try to see if you can find eggs, how many there are in the stool or in bloody urine," Greenberg said. This method, called the Kato-Katz test, is a "very low tech, labor intensive, and inaccurate way to measure infection," in part because of daily fluctuations in number of excreted parasite eggs.

For example, a 2001 study in Tropical Medicine and International Health showed the Kato-Katz test can dramatically underestimate infection levels. A recent study in Parasites and Vectors showed that using Kato-Katz for liver fluke testing led to an overestimate of cure rate after drug treatment. Doing many counts on the same sample was the only way to increase the likelihood of detecting ongoing infections.

Meanwhile, a study in PLoS Neglected Tropical Diseases showed real-time PCR to be superior to counting eggs in bloody urine from 114 schistosome-infected Kenyan schoolchildren. This study also showed daily fluctuations if diagnosis were based on egg counts, while PCR remained constant. Furthermore, PCR showed greater than 90 percent sensitivity, compared to about 30 percent for microscopy, both before and after treatment with an anti-helminth drug.

It also takes about six weeks for worms to mature enough to produce eggs, Greenberg said. Standard treatment ─ a drug called praziquantel, or PZQ ─ kills mature worms, but immature ones may remain in the host and may not be detectable for weeks. However, Greenberg has preliminary results suggesting a PCR-based detection method will enable early detection of the worms, before they start producing eggs.

Antibody and antigen tests also exist, Greenberg said, but these too have problems with sensitivity, selectivity, and reliability. "They're pretty good, but not as good as what you could get with a molecular test, where you're looking for a specific gene sequence or RNA sequence that is a marker for the parasite."

Greenberg said that there have already been many molecular tests reported in the literature, and the current project is, in part, "stuffing it all together."

Greenberg is also using the new award to experiment with detecting helminth microRNAs. "It turns out that the parasites seem to be producing microRNAs that can be detected in the host as well. There's a recent paper out that showed that. So that would be a very cool way to do this [detection]," he said.

In addition, schistosomes have intermediate hosts, like snails, which could also be tested for helminth nucleic acids. This might be important for monitoring disease transmission, and could be useful in eradication programs.

Merck recently announced an effort to develop a global initiative for schistosomiasis eradication. According to a statement, it already donates $23 million dollars worth of PZQ each year, primarily for mass drug distribution programs. It plans to raise its annual donation to 250 million tablets by 2016.

Greenberg pointed out the quirky history of PZQ's development. "The only way it really got developed into a drug product was because of the veterinary market; it wasn't to treat schistosomiasis [in humans]," he said. "It is really the only drug available right now to treat this disease that affects hundreds of millions of people," Greenberg said. Combined with mass drug distributions, this raises concerns that worms could someday become drug resistant, a scenario Greenberg described as "really scary."

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