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NGS-Based Pipeline Used to Develop Improved Parasitic Worm MDx

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NEW YORK (GenomeWeb) – The global burden of soil-transmitted helminths (STHs) may be decreasing due to large-scale treatment programs, but improved diagnostics are needed to support eradication efforts.

Scientists at Smith College have now developed a pipeline using next-generation sequencing and a Galaxy-based web server called RepeatExplorer to uncover non-coding repetitive sequences in five species of STH and develop a qPCR assay with improved specificity and sensitivity.

The method and results of an assessment using field samples were published last week in PLoS Neglected Tropical Diseases.

The new method, which was funded by a sub-grant from the Bill and Melinda Gates Foundation through the Task Force for Global Health, uses a previously-described TaqMan-based technique called multi-parallel qPCR that allows users to mix and match assays in small-volume reactions.

"If there's only one worm present in a region, you can run a small-volume reaction to test for that one worm and you don't have to worry about wasting reagents looking for the others in an optimized multiplex assay that isn't necessary," Nils Pilotte, first author on the study, told GenomeWeb.

Importantly, the study describes a unique assay development process, using next-generation sequencing of worm species followed by RepeatExplorer informatics to detect the region of the worm genome with the highest possible number of repeats.

Local control and elimination of STHs could improve the lives of an estimated one in six people globally. Children bear the greatest burden of infections, with worms competing for scarce nutrition in some regions and causing developmental problems, Pilotte said.

Pilotte, a doctoral student in the lab of co-author Steven Williams at Smith, noted programs to eliminate a similar infectious disease, lymphatic filariasis, commonly known as elephantiasis, have been incredibly successful. Modeled on those programs, a global STH eradication program could be a feasible long-term goal.

However, helminthic infections are also considered neglected tropical diseases. "Generally speaking they're not fatal, and they're co-endemic in so many locations with terrible things like malaria, so there's competition for funding that may have led to a lot of the neglect," Pilotte said.

As with lymphatic filariasis, STHs can be treated with large-scale blanket treatment of populations. Inexpensive drugs, akin to those used to treat heartworm, are administered annually to entire communities for about six years, and this can eliminate the worms from the soil and people.

The gold-standard historically has been microscopic examination of stool samples for the presence of eggs of worms or larva. When infection levels are really high, this strategy can provide a good grasp of levels. Even though its not very sensitive or specific to species of worm, "if there's a ton of it you're going to see it."

But, "as elimination and treatment programs have started to recognize some successes and worm burden within individuals and populations is declining, there's really a need for more sensitive diagnostics," Pilotte said. With reduced numbers of worms, the likelihood of missing them is greater. "That becomes very important for making programmatic decisions about treatment efforts, such as when treatment should be continued or discontinued, and in determining what the actual levels of infection are on a more global level in the community."

Fortunately, it turns out that multicellular eukaryotic organisms possess a large number of non-coding, repetitive sequences within their DNA.

The group, which also included researchers at University of Massachusetts Amherst, and QMIR Berghofer Medical Research Institute in Brisbane, Australia, first used Illumina MiSeq to do de novo sequencing of five species of STH.

"We're looking for these high copy number repeats, and historically that sort of a thing has been kind of a guess-and-check method and a tedious process where you have to do restriction digest and look for banding," Pilotte said. "If you find a band then that means it was something that was probably prevalent in the genome, and then you can sequence it and try to figure out what it is."

Now, however, for a cost of about $3,000 and a week or two of work, the researchers were able to get sequencing results they could then evaluate with informatics to find better targets for qPCR.

The RepeatExplorer software is free, and Pilotte said it was quite simple to use and did not require specialized informatics expertise.

"It's almost like BLASTing — you just take a read and you BLAST it against everything else to see what is most commonly matched ... to find the sequences within the overall set of reads that are most abundant."

The group is now adapting the technique to other infections. "Because these are individual small-volume reactions you can develop new assays as you go and roll them into the pick-and-choose format," Pilotte said.

The researchers don't have plans to pursue any IP. "It's out there now, so we would love it if people used it." Including assay validation, Pilotte suggested interested groups could "start with a worm and end with a diagnostic within a two-month time frame."

The group hopes to ultimately see the new assays applied in the field with "field-friendly" portable PCR platforms, such as the Amplyus miniPCR, Pilotte said. He imagines that funding and global health organizations are also interested in potentially adapting portable qPCR devices for their work.

He further noted that, while there is definitely a place for other molecular technologies, researchers and clinicians are familiar and comfortable with traditional qPCR and large global programs may be hesitant to take on new technologies before they are well-validated.

The Smith group is now expanding the method to other diseases and conditions, including veterinary and marine mammal parasites. "We're also taking a second look at existing assays for things like loa loa, Schistosoma, and potentially other hookworm species — in some cases there are assays out there already but we're betting they aren't optimal and could be improved upon."

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