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Inexpensive LAMP-Based Schistosome Tests Could Help Eradication Efforts

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NEW YORK – Schistosome infection currently afflicts a staggering 250 million people globally. Eradication efforts rely on mass distribution of an anti-parasitic drug, but sensitive diagnostics are needed for post-treatment surveillance and total elimination of the parasite from a community.

A handful of isothermal molecular diagnostics tailored to this task have recently been developed and are moving closer to a stage when they might be commercialized. A study published last week in Scientific Reports describes one such technology, a loop-mediated isothermal amplification (LAMP) diagnostic test developed by researchers in Salamanca, Spain that is nearing commercialization and is now at a stage incorporating lyophilized reagents.

Meanwhile, other LAMP-based schistosome tests are also being developed by researchers globally, and a number of these are also on a path to eventual commercialization.

Parasitic schistosomes spend part of their life cycle in aquatic snails, and part in human hosts. Free-swimming larvae will burrow into the skin of children playing in contaminated fresh water, for example, maturing and mating in the veins surrounding the host's intestines or bladder and releasing eggs into the stool or urine that can then hatch to infect more snails in the water.

The eggs of schistosomes cause — and exacerbate — disease in people. They can become trapped in the liver and bladder, migrate to the lungs, and they provoke an inflammatory immune response, granuloma formation, and can lead to conditions such as liver fibrosis and bladder cancer. Chronic co-infection with hepatitis B or C viruses increases damage to the liver, and an estimated 20 million women worldwide have schistosomiasis of the urogenital area, which creates lesions that are hypothesized to also enhance HIV transmission.

Approximately 250 million people are infected with schistosomes, yet there are also 700 million at risk of infection in 74 countries. Control involves treating infected people with praziquantel (Biltricide), a drug that is donated by Merck KGaA, as well as improving sanitation, according to the US Centers for Disease Control and Prevention.  

But large-scale eradication efforts are not always completely successful, in part because praziquantel only works on mature adult parasites, and in part because people constantly migrate in and out of treatment areas for employment, according to Nilanjan Lodh, a researcher at Marquette University who develops diagnostics for neglected tropical diseases.

Reinfection is also likely if water contamination persists, Lodh said. And, reinfection can happen so quickly, there are some reports suggesting it can be difficult to discern whether a patient has a new infection after treatment, or perhaps instead has a drug-resistant strain of the parasite.

According to Christina Faust, a researcher at the University of Glasgow focusing on the epidemiology of infectious diseases, more accurate diagnostics for schistosomes are very important for monitoring treatment success, to know whether the praziquantel treatment actually cured someone.

"Tests like LAMP could be important for monitoring drug efficacy, particularly if they become more cost-effective and are easy to use in remote settings," Faust noted in an email.

Stool diagnostics called Kato-Katz tests are typically used to look for schistosome eggs in stool and urine, but egg production starts late in the parasite's life cycle. "Accurate diagnostics, or at least more accurate than standard Kato-Katz [tests], will be important for areas that are moving towards elimination," Faust also said, since individuals in these localities tend to have lower levels of infection, which are difficult to detect with current methods.

There are immunoassays for the infections, with development funded in part by the Bill and Melinda Gates Foundation's support of Schistosomiasis Consortium for Operational Research and Evaluation (SCORE) at the University of Georgia, and a UK-based group called the Schistosome Control Initiative (SCI). At this time, however, these require more complex labs to run them, Lodh said.

A new consortium announced this year will also support development of a test for an antigen secreted by the parasites, called circulating anodic antigen, or CAA. Along with the Foundation for Innovative New Diagnostics, the consortium is developing two rapid CAA-based diagnostic tests. However, while previously published research notes that CAA assays are much better than the Kato-Katz test, to date they still have sensitivities in the 80 percent range compared to PCR. These tests also have the disadvantage of being unable to detect early infections or juvenile parasites, which may contribute to their overall low sensitivities.

For full eradication, molecular tests that can pick up all infections would be preferable. But because these infections are occurring in places with few financial resources and sparse networks of complex labs, the tests must also be cheap and easy to use.

Lodh noted that there are three main species of parasites — Schistosoma mansoni, S. haematobium, and S. japonicum. The S. haematobium tends to cause urogenital schistosomiasis, while S. mansoni tends to infect the intestines. The LAMP-based test his lab has developed detects both S. mansoni and S. haematobium in urine, and an evaluation of the test on 111 urine samples collected from school children in Zambia after a mass drug distribution showed a surprisingly high sensitivity — higher than PCR, in fact — that was also seen previously in a LAMP-based test for S. mansoni in snails.

In a recent cost-effectiveness analysis, Lodh and his team at Marquette estimated that LAMP-based schistosome testing could be brought down to a cost of approximately $4 per test, with another $1 to $4 required for extraction.

His group is collaborating with groups in Ghana, Gambia, and Tanzania. He also noted that the current global burden of disease estimate is from about six months ago, and the disease burden is probably closer to 300 million now, and also likely underestimated because sensitive and specific diagnostic tests are not routinely available.

Lodh wants to bring the testing to the field in Africa, building a trained team there, rather than focusing energies on commercializing the test. To meet the WHO ASSURED guidelines for rapid testing — namely, that it be affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users — the tests also have to be radically inexpensive, which Lodh suggested might not appeal to commercial vendors. That said, if a company came along that would be interested and could meet the cost estimates, the lab would work to commercialize the test, which might help reach more countries immediately.

Lodh said the call in the field for the past few years, from funders like the Gates Foundation, is for a total test cost, from extraction to data collection and including labor, of approximately $1, which is "a huge stretch for molecular testing."

His lab is looking into other technologies as well, like recombinase polymerase amplification (RPA), which can better enable multiplex detection.

Other groups are also developing molecular testing for schistosomes. For example, a group in Australia has pioneered a digital PCR test. That group is not planning to commercialize the dPCR version, but it is working on LAMP-based point-of-care diagnostics with collaborators at the University of Queensland, according to Don McManus, a researcher at the QIMR Berghofer Medical Research Institute in Brisbane.

A team at the University of Pennsylvania has been working on a LAMP-based microfluidic assay, but as of yet has not undertaken commercialization efforts. Haim Bau, one of the developers of the microfluidic technology, is currently focusing on multiplexing up to 16 targets in a single cassette, including schistosomes in blood and other body fluids, he said in an email. The technology is called RAMP, for rapid amplification, and relies on a two-stage, isothermal–isothermal, enzymatic amplification method that consists of a first-stage RPA and second-stage LAMP, as described last year in Clinical Chemistry.

A group at the Kenya Medical Research Institute (KEMRI) is also developing a LAMP-based test to detect schistosomes in stool samples. Ibrahim Ndungu, at KEMRI, said in an email that the test is part of a wider study aimed at developing a LAMP-based diagnostic kit for three common soil-transmitted helminths — Askaris lubricoides, hook worm, and Trichuris trichuiria — as well as S. mansoni, which is a commonly occurring co-infection with STHs. "The kit will have the advantage of quick, reliable, one-step diagnosis for all four co-occurring helminth infections ... carried out simultaneously [and] providing remarkable turnaround time," Ndungu said in an email.

Meanwhile, Pedro Fernández-Soto, a researcher at the Research Centre for Tropical Diseases at the University of Salamanca and co-author of the Scientific Reports study, noted in an email that his team is planning to commercialize its test soon. The test has been evaluated in urine samples from mice that had infection in the intestines, as well as in people with the strain of infection that prefers the gut, and in clinical samples in Brazil, where 1.8 million people are thought to be infected with S. mansoni

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