Rapid diagnosis of onchocerciasis using urinary biomarkers

Onchocerciasis or river blindness is a parasitic disease affecting 17 million in sub-Saharan Africa. It causes visual impairment, sometimes leading to irreversible blindness, and intense itching of the skin. The symptoms of the disease can be controlled by annual treatment with the drug ivermectin, which kills the larval stages in the skin, but the adult worms are not eliminated and can survive for more than 10 years.
Diagnosis of the disease has been dependent for many decades on microscopic examination of skin snips for the larval stage. This is a painful procedure that, not surprisingly, is being met with reduced compliance in communities that have been sampled repeatedly over many years.
In this proposal, we aim to radically improve the diagnosis of onchocerciasis by testing for the presence of adult worms using a patient's urine sample. We have discovered that the worms release proteins and small RNA molecules into urine that can be detected by mass spectrometers and sequencing technology. The molecules are associated with small "packets" called extracellular vesicles. We will isolate these vesicles from urine samples to identify the best markers for the disease. To facilitate the process, we will also test urine samples from African cattle, which are infected with a parasite very closely related to that which causes human onchocerciasis. By treating the cattle with drugs that kill the adult parasites, we will test whether the molecules released in urine are a good indicator of whether live worms remain in the host's body. This is a particularly important at the present time, as several drugs with potential activity against adult worms are being tested in clinical trials in humans, and currently the only way to know whether the worms have been killed is to perform surgery on the volunteers.
In the final stages of the proposal, we will investigate methods to capture parasite molecules from urine in an efficient manner. This will be necessary to move away from a diagnostic test that is dependent on expensive laboratory equipment to a kit that ultimately could be used in rural Africa.

Onchocerciasis or river blindness is a parasitic disease affecting 17 million in sub-Saharan Africa. It causes visual impairment and severe dermatitis. The symptoms of the disease can be controlled by annual treatment with ivermectin, which kills the larval stages in the skin, but the adult worms are not eliminated and can survive for more than 10 years.
For decades, diagnosis of onchocerciasis has relied on microscopic examination of skin biopsies for the first-stage larvae (microfilariae) of Onchocerca volvulus. This is an invasive procedure that is increasingly being met by non-compliance in endemic communities. Moreover, annual ivermectin treatments that are administered to prevent disease symptoms kill the microfilariae but do not eliminate the long-lived adult worms. Thus, skin snips are becoming less sensitive for diagnosis, and a non-invasive diagnostic test that could detect a single viable adult female worm would revolutionise the field.
Applying tandem mass spectrometry and next-generation sequencing, we have recently identified proteins and small RNAs released by O. volvulus in patient urine specimens. These molecules appear to be associated with extracellular vesicles. In this proposal, we will investigate the biomarker potential of these molecules by treating cattle infected with the closest relative of O. volvulus, O. ochengi, with drugs targeting the adult worms. We will isolate extracellular vesicles, characterise the protein and small RNA content of parasite-derived material, and explore methods to enrich for biomarkers as a prelude towards a field-deployable urine test for human use. Through our overseas partners, we will also examine human urine specimens (both from infected patients from endemic communities, and from participants in clinical trials for drugs targeting the adult worms) for O. volvulus-derived biomarkers. The cattle samples will serve as validated controls for loss of the biomarkers from urine once the adult worms are killed.

In addition to academic beneficiaries in the filariasis fields and other helminthologists in both the medical and veterinary parasitology disciplines, our findings will be of great interest to the World Health Organisation, NGOs, non-profit organisations, small or medium enterprises, and public-private partnerships in the neglected tropical diseases field.

Currently, the World Health Organization Africa Region (WHO-AFRO) co-ordinates onchocerciasis control, alongside that for other diseases where preventive chemotherapy is the main tool, through a network of endemic country government-led programmes called the Expanded Special Project for Elimination of NTDs. Diagnosis of onchocerciasis in the field is still largely dependent on invasive skin snips, and although antibody assays are becoming more widely used, they are of limited use for determining if adults are free of infection. This is because people can remain antibody-positive for many years after an infection has been cleared.

Other organisations committed to supporting WHO-AFRO in improving diagnostic options for onchocerciasis include the Bill & Melinda Gates Foundation (BMGF) and a network supported by BMGF and other organisations called the Coalition for Operational Research on NTDs (COR-NTD). As drugs that can target the adult worms rather than just the microfilariae (first-stage larvae) are currently being evaluated in clinical trials, the Drugs for Neglected Diseases Initiative is also committed to identifying biomarkers that can indicate if all adult worms have been killed without recourse to surgery.

Finally, international non-profit organisations (such as PATH headquartered in Seattle) and some small companies have invested in new technologies for the diagnosis of NTDs, leading to new products becoming available in just the past few years.

All of these organisations will be represented at the American Society of Tropical Medicine & Hygiene Meeting in New Orleans in 2018, where our findings will be presented. To ensure that our data can be discussed in detail with key stakeholders, we will also make presentations at the Macrofilaricide Drug Accelerator Program and COR-NTD meetings that run in parallel with the ASTMH Meeting. Here, we will solicit detailed feedback on how to develop a practicable field assay in the longer term.

Of course, the ultimate stakeholders are the onchocerciasis patients themselves. At this early stage of diagnostics development, we do not intend to engage directly with patients in rural communities in Africa in case expectations are raised prematurely. However, we are committed to a culturally acceptable, easy-to-use test in the longer term and would include social anthropologists from endemic countries in any follow-up programme to this GCRF Foundation Award.