NEW YORK – A global plan to reduce neglected tropical disease transmission by 2030 may soon get a boost by a new assay to detect Onchocerca volvulus, the parasite that causes river blindness. Researchers at the US National Institutes of Health have developed a new, more sensitive O. volvulus PCR test and are in the process of adapting it into a CRISPR-Cas12 assay with isothermal amplification for field use.
The O. volvulus parasite is transmitted by black flies that breed along fast-flowing rivers and typically thrive on cattle. After an infectious fly bites, the larva swim under the skin or into the eyes where they can live for up to 15 years in small nests. Although not fatal, they can cause an inflammatory response when they eventually die, resulting in intense itching and vision loss. The infection also has an outsized and transgenerational economic impact in small villages, as blinded adults can't work, and children typically quit school to care for them.
The NIH estimates that there are 18 million onchocerciasis infections in 31 countries with 40,000 cases each year resulting in permanent blindness.
Infection is currently diagnosed through incubating multiple skin snips in warm buffer until the worm larva swim out and can be confirmed using a cumbersome multiday lab test process involving PCR and ELISA, according to Sasisekhar Bennuru, a helminth parasite researcher at the NIH.
Bennuru and his colleagues, however, have recently created a new molecular method that targets repetitive elements in the parasite genome.
As described in a preprint published online in MedRxiv this month, they also discovered that this region can be detected in urine, that the nucleic acids can be concentrated by passing the urine through a filter coated with a polysaccharide called chitosan, and that the PCR can be ported to isothermal methods like recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP).
To make the test less invasive than skin snips, the NIH researchers first attempted to detect the repeat regions they'd discovered in plasma, but they found that the circulating cell-free DNA of this skin-dwelling organism was not so abundant in blood.
Then, the team thought to try detection from urine samples, considering that the kidneys can essentially serve to filter and concentrate substances in blood.
Using urine samples with spiked-in DNA as well as archived samples from patients, the NIH researchers showed the test had 100 percent specificity and more than 32-fold increased sensitivity compared to a gold-standard qPCR test.
They then discovered that they could enhance the sensitivity of the test by further concentrating the urine with chitosan, a polysaccharide that changes charge based on local pH. The sample prep now involves passing urine through a filter using a large syringe and drying down the filter, Bennuru said, so that punches of this filter will release DNA when incubated in water.
Unlike the gold-standard PCR, the new assay can distinguish between the O. volvulus human parasite and a closely related species, O. ochengi, that exclusively infects cattle. While a high-resolution melt PCR assay for this purpose was previously described, ease of use of the new test could be critical for so-called xenomonitoring programs that test the black fly vectors as a way to determine where to focus treatment and prevention efforts.
In an interview, Sara Lustigman, a researcher studying O. volvulus at the New York Blood Center who has collaborated with Bennuru in the past, said the new test is exciting because of its improved specificity and sensitivity and ability to differentiate the cattle from human strains.
"It can substitute the skin snips for urine, and that will be a blessing," she said, as it is less invasive.
Push for elimination
The World Health Organization recently formed a global working group called the Global Onchocerciasis Network for Elimination (GONE), and as part of a strategy to curb neglected tropical disease transmission by 2030, the WHO has emphasized that better river blindness diagnostics are critical.
The WHO's diagnostics technical advisory group for neglected tropical diseases, WHO-DTAG, also established a subgroup to develop two target product profiles for onchocerciasis diagnostics. One profile is for products needed for screening and mapping regions where infections occur, while the other is for products meant to aid in deciding to stop MDA campaigns.
For the latter, the TPP advises a portable and highly sensitive, shelf-stable product costing less than $2 with no need for capital equipment. Bennuru's team did not estimate the cost of testing in the study, but a CRISPR-based diagnostic could theoretically meet the TPP.
The US Agency for International Development (USAID) is also targeting river blindness for eradication through support of mass drug administration (MDA) of the anti-worm drug ivermectin. The drug's manufacturer, Merck, has reportedly donated more than 4.4 million doses of ivermectin for river blindness eradication MDA campaigns over the past 35 years, sparing 600,000 cases of blindness.
Unfortunately, while ivermectin effectively kills O. volvulus larva, it can also cause a fatal swelling of the brain if a person happens to be co-infected with another eye worm parasite called Loa loa. And, the drug does not kill the nests, or nodules, of adult worms under the skin, so infection control comes down to repeated MDA campaigns. Furthermore, infections may be simmering in so-called hypoendemic areas, and the parasite can be transported when people migrate or travel. The parasite has also developed resistance to ivermectin, and another drug, moxidectin, is currently in the validation process to obtain inclusion in WHO guidelines.
But, after 36 years of work, Lustigman and her colleagues have created an O. volvulus vaccine they have named TOVAx, which they hope will enter Phase I clinical trials by 2025. To effectively deliver a vaccine, one needs good diagnostic tests to know where the infections are occurring, Lustigman said. Vaccinating children is also a goal of the project, she said, as they can develop an unusual epilepsy with uncontrollable head nodding typically provoked by the sight of food that is hypothesized to be related to infection.
Going forward, Bennuru said the NIH team will work to adapt the test to a workflow that uses CRISPR-Cas12 combined with RPA or LAMP reactions and dipstick detection to increase sensitivity of the isothermal reactions, a technique also used by Sherlock Biosciences.
He and his NIH colleagues have also developed an assay for L. loa, he said, and have managed to multiplex it with the O. volvulus test. Both tests will be publicly available through NIH's Open Access policy, Bennuru said.