Introduction and Background of the Project

Introduction

Schistosomiasis is the second most important human parasitic disease in tropical and subtropical areas: estimates show that at least 218 million people required treatment in 2015 [1]. The global strategy for schistosomiasis control is focused on eliminating disease through periodic, large-scale population treatment with praziquantel [2, 3]. To monitor progress towards the WHO Roadmap and the London Declaration goals, periodic testing is required to certify areas currently free from schistosomiasis transmission and to red-flag locations where extra effort or alternative interventions are needed. To guide control programs and to enable post-elimination surveillance, there is a critical need for novel sensitive, affordable and user-friendly diagnostics [4]. The current proof-of-concept study will analyze the potential of schistosome antigen-specific antibodies (Abs) in urine and blood samples for the development of such diagnostics. The envisioned test(s) should be able to a) discriminate between current and previous Schistosoma infections in low-intensity settings AND/OR b) detect very light infections in near elimination settings.

Using the platforms established by each partner, DTECT-Schisto will screen serum and urine sample sets from high-quality cohorts, to provide a shortlist of validated protein and/or glycan antigens with diagnostic performance in serum and urine. This will form the basis for the overall aim: to develop highly sensitive, field-applicable diagnostics for detection of schistosomiasis Abs in blood and/or urine.

Project objective

The overall aim of the project, DTECT-Schisto, is to develop a novel rapid test for evaluation of schistosomiasis control and elimination programs. At the end of this 2-year concept development study, we will provide a shortlist of validated protein and/or glycan antigens with diagnostic performance in serum and urine. This will be the starting point for technical feasibility studies.

Current diagnostics are inadequate for detection of low worm burdens. The need for more sensitive diagnostics is evident, with the progress towards control and elimination of schistosomiasis by using mass drug administration (MDA) strategies and with the advent of vaccination trials. To instruct MDA programs and assess reinfection, diagnostics that can monitor the dynamics of schistosomiasis transmission are required. To support ‘reducing or stopping MDA’, a sensitive antibody (Ab) test should be developed that can discriminate between current and past infections in low-intensity settings. This will require the use of defined (single or multiplexed) antigen(s) and specific Ab isotype or IgG subclass detection in blood or urine. A number of pre-selected antigens (protein and glycan) are proposed for evaluation in the project. To support ‘post-elimination surveillance’, an Ab test should be developed that can sensitively detect very light infections in (near) elimination settings, preferably in urine. Non-invasiveness is important for schistosomiasis testing in the end game settings, as people will not be very keen to suffer or let their children suffer from surveys of a disease that they don’t really regard as important.

Main characteristics of rapid diagnostic methods required for monitoring control and the so-called “end game” of schistosomiasis are affordable, sensitive/specific, user-friendly, rapid/robust, equipment-free, and deliverable (ASSURED). Ab detection platforms are ideally suited for instance in the form of simple lateral flow devices. An assay allowing the non-invasive detection of diagnostic Abs in urine would be optimal

Project design

To achieve the project objectives, we will build a sustainable partnership by:

A) Working with credible and experienced partners from the start brings invaluable knowledge and resources: NUITM for its expertise with protein antigens and access to samples from Schistosoma endemic areas, and for its expertise in detecting Abs in serum and urine; LUMC for its expertise with glycan antigens, diagnostic assay development, and access to samples from a controlled human infection model, and biobanked samples from Schistosoma-endemic areas; and Lygature for its expertise in managing multi-stakeholder consortia.

B) Providing a solid governance structure, with a Joint Steering Committee (JSC) comprising all partners, that acts as the decision-making body. The JSC will be supported by Lygature.

C) Organizing the program into work packages (WPs) with WP leads, to ensure timely execution and reporting:

i. WP1: protein antigen selection, will be led by NUITM. In this WP, a library of protein antigens will be screened by (multiplex) ELISA using serum and urine. Isotype and subclass-specific response.

ii. WP2: glycan antigen selection, will be led by LUMC. In this WP, a library of glycans representing major antigenic glycan motifs from S. mansoni will be screened as neoglycoconjugate and nanoparticle using ELISA for detection of Abs in serum and urine. Isotype and subclass-specific response.

iii. WP3: sample management, will be led by NUITM and supported by LUMC. Samples managed in this WP are critical. Cohorts (existing + new) allow pre- post-treatment comparisons and infection intensity comparisons.

iv. WP4: project management, will be led by Lygature.

D) Early identification of and continuous communication with key stakeholders and future partners to ensure that the project has the expertise on board required to move from concept development to technical feasibility and development feasibility.

E) Timely identification of funding opportunities for technical and development feasibility studies.

How can your partnership (project) address global health challenges?

In the short-term, the Consortium aims to validate antigens (proteins and glycans) that can a) discriminate between current and previous Schistosoma infections AND/OR b) detect very light infections in serum and/or urine samples. Subsequently, the team will develop rapid POC diagnostic test(s) for schistosomiasis, preferably in minimally-invasive body fluids. The test should be affordable, sensitive/specific, user-friendly, rapid/robust, equipment-free, and deliverable (ASSURED).

The new Ab test(s) will be of value for monitoring and instructing MDA programs and/or for post-elimination surveillance. In areas where both S. mansoni and S. haematobium infections are co-endemic, a sensitive species-specific test is eligible to monitor the efficacy of MDA on each infection and to survey any possible re-emergence of each infection during post-MDA.

To be useful for successful control and elimination programs, the new test(s) will need to have a high sensitivity in order to identify any remaining infected individuals and also a high specificity so that few, if any, false positive results occur suggesting that program effectiveness is poorer than it actually is. While a point-of-care test for urinary antigen detection (POC-CCA) is currently available for S. mansoni surveying and monitoring, an Ab test is expected to have superior sensitivity and specificity in low intensity and elimination settings. In addition, the CCA test is not capable of species discrimination. Using specific, defined antigen(s) allowing superior sensitivity/specificity, in combination with a field applicable Ab detection format, the proposed Ab detection test will form a high-impact schistosomiasis control tool.

What sort of innovation are you bringing in your project?

We aim to develop highly sensitive, field-applicable and affordable test(s) for detecting schistosomiasis, based on the following innovations:

• Detection of antibodies against single, defined antigens (proteins and/or glycans)
• Detection of antibodies in urine
• A unique set of high-quality cohorts for validation of diagnostic performance
  1. Newly collected samples from Health Demo-graphic Surveillance System (HDSS) in Kenya
  2. Biobanked samples from various cohorts in Senegal, Cameroon, Brazil and Gabon
  3. Samples from the controlled human schistosome infection model

Role and Responsibility of Each Partner

Lygature is the Designated Development Partner and will provide Consortium Management. It provides governance in terms of progress, finance, and collaborations, and will coordinate and ensure timely execution of the project. Nagasaki University Institute of Tropical Medicine will evaluate previously identified serum reactive to S. mansoni antigens, for reactivity with urine Abs, and extend antigen selection studies using available protein libraries. In addition to previously stored sera, new samples (serum and urine) will be collected from established sites in Kenya where S. mansoni and S. haematobium are endemic. NUITM will also seek conditions for urine to be stored and analyzed for their reactivity to single/multiple glycan and protein antigens. They are also responsible for field activities including collection and management of samples. Leiden University Medical Center (LUMC) will conduct all glycan antigen selection and evaluation studies. In addition, they will provide and manage existing sample cohorts. Their expertise in developing lateral flow – POC tests will contribute to the overall project. The partners will form a dedicated Development Team that comprises at least one representative of each partner and which will include all critical functions. The Development Team is responsible for the overall and day-to-day management of the collaboration and will meet regularly to discuss the progress, issues, planning of the project and decide on the next steps. The overall project is steered by a Joint Steering Committee, which will act as the highest strategic decision-making body. Details of the overall governance and decision-making process will be included in a consortium agreement between the partners.

Others (including references if necessary)

[1] World Health Organization: http://www.who.int/schistosomiasis/en/, accessed 6 September 2017

[2] World Health Organization .A Roadmap for Implementation: accelerating work to overcome the global impact of neglected tropical diseases. Geneva: World Health Organization; 2012.

[3] World Health Organization: http://www.who.int/schistosomiasis/strategy/en/, accessed 6 September 2017

[4] Stotthard et al Parasitology 2014, 141(14):1947-61.

Final Report

1. DTECT-Schisto project objective

The overall goal of DTECT-Schisto is to develop a highly accurate diagnostic test that detects schistosome-specific antibodies in blood and/or urine of Schistosoma-infected individuals. The test is aimed for use in low endemic, near- and post-elimination settings.

The specific objectives of this concept development project focused on investigation of the diagnostic potential of an exisiting library of defined protein and glycan candidate antigens to measure schistosome-specific antibodies, both in blood and urine as well as pre- and post-treatment. Assessments were done in a broad set of archived samples as well as in newly-obtained unique DTECT-Schisto cohort samples.

2. Project design

To achieve delivery of a shortlist of validated protein and/or glycan antigens for future   technical feasibility phase, the project was organized in four work packages (WPs):

WP1 Protein antigen selection: ELISA-based screening of plasma and urine samples for evaluation of recombinant schistosome protein candidate antigens

WP2 Glycan antigen selection: Microarray-based screening of plasma and urine samples for evaluation of glycan library representing all major antigenic glycans present in schistosomes

WP3 Sample management: coordination of well-defined samples from existing and new project specific cohorts allowing for pre-/post-treatment and infection intensity assessments

WP4 Project management & communication: facilitated professionally by Lygature

3. Results, lessons learned:

Unique paired plasma and urine samples were obtained from two cohorts of Kenyan children before and after multiple praziquantel treatments in both S. mansoni (Mbita) and S. haematobium (Kwale) low prevalence endemic areas. These samples were evaluated as appropriate for schistosome infection status by presence of eggs in stool or urine, DNA in stool and circulating cathodic antigen in urine by POC-CCA. Furthermore, infection negative status was evaluated by the highly sensitive up-converting phosphor-lateral flow laboratory assay detecting circulating anodic antigen (UCP-LF CAA).

ELISA- or microarray-based assessment of perfomance of all protein and glycan antigens in the library was done on these newly obtained samples as well as on other existing well-characterised cohort samples for both IgG and IgM. This process allowed us to narrow the candidate antigen library from more than 80 to a set of three candidate antigens.

Specifically for urine samples, we found that detection of IgG specific for a combination of recombinant antigens (RP26/Serpin) performed with high sensitivity for S. haematobium infection on the ELISA platform. In general, an important lesson learned is that combinations of defined antigens (protein and/or glycan) is important for diagnostic sensitivity as not all individuals infected with schistosomes have antibodies to the same antigens. Overall, we have gained essential information on IgM and IgG (including subclass) antibody responses to the selected candidate antigens in terms of association to current schistosome infection, in pre-/post-treatment context and of longevity of antibody responses. This knowledge enables future optimisation of sensitivity and specificity of antigen combinations and importantly informs the use case for development of a highly accurate diagnostic test.

We envision that a future highly accurate schistosomiasis antibody-detection diagnostic tool will have particular impact in low endemic as well as near- and post-elimination setting for monitoring purposes. Such a tool is in line with current WHO 2030 roadmap and sustainable development goal 3 ambitions for achieving progress towards better and equitable health for all.