Rapid diagnosis of onchocerciasis using urinary biomarkers

Lead Research Organisation: University of Liverpool
Department Name: Institute of Infection and Global Health

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description The original goal of the project was to develop methods to enable diagnosis of onchocerciasis (river blindness) through analysis of parasite-derived biomarkers (proteins and small RNAs) in urine samples. An analogous natural infection in animals (cattle) was used to validate this approach. The project objectives included both improvement of diagnosis of onchocerciasis for people harbouring adult worms who may not have larvae (microfilariae) in the skin, and also the monitoring of clinical trials in which drugs targeting adult worms are being evaluated.

Some of the underlying assumptions behind the original proposal were found to be incorrect; most importantly, the parasite biomarkers do not appear to be enriched in extracellular vesicles. This means that the parasite biomarker signal in urine may be too low and inconsistent for reliable diagnostics, although we continue to refine alternative methods to enhance the signal.

A key finding from this study is that bovine host small RNAs are influenced by Onchocerca infection. We do not yet know if this is also the case in humans, but if so, it could provide a non-invasive approach for onchocerciasis diagnosis. Another output from the study is the generation of an Onchocerca ochengi genome from Kenyan cattle, the first from East Africa, which is currently undergoing annotation.
Exploitation Route Despite some of the challenges outlined above, diagnosis of onchocerciasis using a urine test remains a promising avenue of research in the field and we are continuing to refine methods to achieve a prototype assay. We remain in contact with the NGO stakeholders described in the original Pathways to Impact as well as the principal funders of Neglected Tropical Diseases research so that new findings can be capitalized upon in a timely manner, potentially through a programme of contract research. The new O. ochengi genome from Kenya will greatly enhance the utility of this model parasite in cattle for any studies requiring data on gene targets/genome regulation etc. It is expected to be published early next year.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Evaluation of a novel diagnostic screening tool for detection of Onchocerca in blackfly populations
Amount £28,693 (GBP)
Funding ID COM_E_JG 
Organisation The Pirbright Institute 
Sector Academic/University
Country United Kingdom
Start 12/2019 
End 09/2020
 
Description Bayer macrofilaricidal drug trial 
Organisation Bayer
Department Bayer Schering Pharma
Country Germany 
Sector Private 
PI Contribution We provided expertise on the biology of the Onchocerca ochengi parasite system in Cameroon and the logistics of running large-animal experiments in West Africa. We also provided histological data on drug activity against the parasite.
Collaborator Contribution Our collaborators at L'Institut de Recherche Agricole pour le Développement in Cameroon performed the efficacy trial of a potentially macrofilaricidal drug in cattle naturally infected with O. ochengi. This included the parasitological readouts of drug activity.
Impact No outputs yet. Not multidisciplinary.
Start Year 2017
 
Description Buea 
Organisation University of Buea
Country Cameroon 
Sector Academic/University 
PI Contribution We have provided quality-control training at the partner's laboratories to ensure sterile production of parasite material, as well as access to cattle infected with Onchocerca ochengi.
Collaborator Contribution The Research Foundation for Tropical Diseases and Environment has provided large quantities of infective larvae of O. ochengi for experimental infection of cattle and proteomic studies. It has also supplied sera and urine samples from defined filariasis patient cohorts in Cameroon.
Impact This collaboration has generated three manuscripts to date: doi: 10.1074/mcp.M115.055640, doi: 10.1186/s13071-015-0656-1 and doi: 10.1371/journal.pntd.0000217.
Start Year 2014
 
Description Edinburgh 
Organisation University of Edinburgh
Department School of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided material from the Onchocerca ochengi system (bovine model of onchocerciasis) to collaborators in Edinburgh, enabling sequencing of the O. ochengi genome. We have also undertaken proteomic analysis of excretory-secretory molecules from a filarial model system in rodents, Litomosoides sigmodontis, providing novel vaccine targets for filarial diseases. We supplied bovine nodule fluid to Edinburgh, leading to the identification of Onchocerca miRNAs in host body fluids; data that were incorporated into the application for this MRC GCRF project.
Collaborator Contribution The University of Edinburgh has provided multiple rounds of EC funding that have supported the University of Liverpool as a partner. This facilitated development of the O. ochengi model in cattle and the growth of filarial research at the University of Liverpool, as well as the launch of The Onchocerciasis Vaccine for Africa Initiative in 2015. Sequencing resources at the University of Edinburgh were utilized for the sequencing of the O. ochengi genome and are being used in the current MRC GCRF project for characterization and quantification of parasite small RNAs in host body fluids.
Impact The collaboration has led to numerous publications on vaccination, chemotherapy and diagnostics of filarial diseases, including the following: doi:10.1101/gr.138420.112; doi:10.1093/gbe/evt125; doi:10.1371/journal.pgen.1004397; doi:10.1074/mcp.M114.038539; doi:10.1186/s13071-015-0656-1; doi:10.1098/rsob.150099; doi:10.1074/mcp.M115.055640; doi:10.1586/14760584.2015.1059281; doi:10.1371/journal.pntd.0003422; doi:10.1371/journal.pntd.0000217. In addition, it has led to filing of a patent application for a filarial vaccine candidate (WO2015121646A1).
 
Description IRAD 
Organisation Research Institute Agricultural For Development
Country Cameroon 
Sector Private 
PI Contribution We have trained IRAD scientists in Liverpool in molecular and immunological methods and three IRAD personnel have obtained PhDs from the University of Liverpool. We have led several internationally-funded projects in partnership with IRAD that have contributed equipment and training at the field laboratories in Cameroon (IRAD Regional Centre of Wakwa).
Collaborator Contribution The University of Liverpool, together with Eberhard Karls University of Tübingen, developed the Onchocerca ochengi bovine model of onchocerciasis with IRAD scientists in the early 1990s. It remains the only widely-accepted natural model for the human disease and enables sequential analyses of drug or vaccine effects on adult worms and microfilariae over time. The IRAD field site in northern Cameroon, near Ngaoundere, is the only location in the world where field studies on Onchocerca ochengi take place. The senior collaborator at IRAD, Dr Vincent Tanya, is an honorary visiting professor at the Institute of Infection & Global Health, University of Liverpool, and a co-supervisor of a currently registered PhD student.
Impact This collaboration has been highly productive in terms of publications in high-ranking and strong discipline-specific journals (12 in the past 10 years alone: doi:10.1016/j.ijpara.2017.06.001; doi: 10.1016/j.pt.2016.08.013; doi: 10.1074/mcp.M115.055640; doi: 10.1016/j.vetpar.2015.06.005; doi: 10.1186/s13071-015-0656-1; doi:10.1128/AAC.01995-13; doi: 10.1101/gr.138420.112; doi: 10.1098/rspb.2010.2367; doi: 10.1016/j.vetpar.2010.08.031; doi: 10.1371/journal.pntd.0000544; doi: 10.1186/1756-3305-1-18; doi: 10.1371/journal.pntd.0000217). It the past two decades, it has been funded through three EC Framework Programme grants and a Bill & Melinda Gates Foundation award. In addition to the current MRC GCRF award, it receives current funding from the National Institutes of Health (NIH) for onchocerciasis vaccine development and from Bayer Pharma AG for macrofilaricidal drug screening. A further grant application involving this collaboration is under review with NIH.
 
Description Kumasi 
Organisation Kwame Nkrumah University of Science and Technology (KNUST)
Country Ghana 
Sector Academic/University 
PI Contribution We have supplied preliminary data on potential diagnostic targets for onchocerciasis.
Collaborator Contribution KNUST has supplied body fluids from defined patient cohorts affected by filarial diseases and from control subjects.
Impact One publication on diagnostic biomarkers for onchocerciasis to date: doi: 10.1186/s13071-015-0656-1.
Start Year 2014
 
Description NIAID 
Organisation National Institute of Allergy and Infectious Diseases (NIAID)
Country United States 
Sector Public 
PI Contribution Through the MRC GCRF project, we are supplying bovine serum from animals infected with Onchocerca ochengi in Cameroon for biomarker evaluation.
Collaborator Contribution NIAID is testing the bovine sera to evaluate the sensitivity and specificity of biomarker candidates (both antibodies and antigens) for potential human use in onchocerciasis diagnostics.
Impact None to date. Not multidisciplinary.
Start Year 2018
 
Description New York Blood Center 
Organisation New York Blood Center
Country United States 
Sector Charity/Non Profit 
PI Contribution We supply access to the bovine model of onchocerciasis, Onchocerca ochengi, in Cameroon. This is a natural host-parasite system with very close parallels to the human infection. The model is being used in the MRC GCRF project for biomarker discovery and evaluation, which has extended into a collaboration with New York Blood Center (NYBC). The model is also being used to evaluate vaccine candidates for onchocerciasis by NYBC.
Collaborator Contribution NYBC is contributing substantial grant funding in the form of a subcontract from the National Institutes of Health. This is funding the cattle work and an expatriate postdoc based in Cameroon, as well as several members of a local team.
Impact Publications: doi: 10.1016/j.pt.2017.08.011; doi: 10.1038/s41541-020-00276-2 Not multidisciplinary.
Start Year 2017
 
Description Catch The Biomarker If You Can! - Meet the Scientists, World Museum, Liverpool 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We ran a stall-based activity to directly engage with the public and was designed to educate children and the general public about Onchocerciasis. We aimed to teach people about how the disease is spread, the problems it causes within communities, and the limited diagnostics that were available. The stall was run by one of the PDRAs on the project, two PDRAs from the tick-cell biobank group, and a PDRA from another group.
We initially talked to attendees about the clinical symptoms of the disease and described how debilitating it can be. We then described the current diagnostic tests available and showed them through a small activity. We prepared a model skin snip test where attendees had to try and pick out "skin biopsies" with microfilariae on them - usually this would take at least five attempts to find. We then asked them to imagine how painful it would be to have this many skin tests taken.
We then described the aim of our research to find a new biomarker collected from a non-invasive urine sample. We described them what a biomarker is and asked them to match up "worm biomarkers" to a species of worm. We then asked them to find our biomarkers in our "urine pot" by using a magnetic fishing rod to pull out the magnetic biomarkers. We then asked them to tell us which worm infection they had based on the biomarker they had found.
We were asked questions on the day, mainly by the older children and the adult accompanying them. Many of them had not heard of the disease before and were keen to find out more about it. They were surprised to know how many people this disease affects. We also had plenty of discussions about the usefulness of biomarkers for new diagnostic tests.
General feedback for the Meet the Scientist event was positive; "I loved it. It made me more interested in science", "Best day ever!", "It was very fun and exciting and I learned new things".
Year(s) Of Engagement Activity 2018
URL https://news.liverpool.ac.uk/2018/11/23/new-series-of-meet-the-scientists-begins-this-weekend