Awarded Amount$1,382,696DiseaseMalariaInterventionDiagnosticDevelopment StageProduct DevelopmentCollaboration PartnersCellFree Sciences Co. Ltd. , Frontier Institute Co., Ltd. , Oasis Diagnostics Corp. , ERADA Technology Alliance, Ltd. , The University of Florida
Introduction and Background of the Project
Malaria remains a major global heath burden with as much as a third of the global population at risk of disease. Importantly, it is the leading cause of death for children under five, especially in Sub-Saharan Africa. The malaria elimination and eradication effort has stalled in recent years and the World Health Organization Strategic Advisory Group on Malaria Eradication, has renewed the call for new, innovative tools to overcome the current limitations of our current tools. Malaria rapid diagnostic tests (RDTs) have been a critical component of the eradication arsenal but recent studies suggest that the Plasmodium falciparum parasite has developed mutations that effectively limit the diagnostic capacity of current RDTs to confirm parasitic infection in the field. Our project addresses this limitation by producing a commercial RDT that can either use saliva or blood as input material, have high enough sensitivity to diagnose asymptomatic cases with high reliability, and greater acceptability, designed especially for easier diagnostic screening of children.
The University of Florida identified a new P. falciparum protein marker PSSP17 that could replace presently used parasite markers like the Plasmodium histidine-rich protein-2 (HRP-2), which has become increasingly ineffective due to a growing prevalence of parasite mutations. The PSSP17 protein is present in infected red blood cells and is also present as a soluble molecule in the saliva of individuals with clinical and subclinical infections. Our recent data using a prototype test demonstrated that this marker allows the identification of children with subclinical malaria infection with high precision. Based on these promising results, we are developing and validating a commercial PSSP17-based RDT for highly sensitive detection of malaria infection at the point of care (clinic) and point of need (villages).
We propose three objectives for the development and validation of Saliva-based Malaria Asymptomatic and Asexual Rapid Test (SMAART-1):
Objective 1 – Design and small-scale production of 2,000 SMAART-1 kits, which contains a saliva-collection device and a lateral flow immunoassay cassette.
Objective 2 – Quality-assured medical device production conditions of heat-stable, high-affinity recombinant humanized monoclonal antibodies for capture and detection of PSSP17 on a lateral flow test.
Objective 3 – Thorough testing and validation of the commercial SMAART-1 test in the Democratic Republic of Congo (DRC), a priority malaria endemic country, or in Uganda as an alternative trial site. During these studies, the acceptability, usability, sensitivity and reliability of the SMAART-1 RDT will be compared to the most sensitive molecular laboratory tests used in malaria detection. The overall outcome of the project is to fulfill all the criteria for obtaining CE marking and subsequent WHO prequalification of the SMAART-1 kit.
How can your partnership (project) address global health challenges?
Detection of malaria at the point of need is essential for making appropriate treatment decisions, for expanded surveillance of malaria transmission and for assessing the efficacy of other malaria control interventions across malaria-endemic regions of the globe. Current malaria tests in the field, either RDTs or classical blood tests using microscopes, cannot rapidly detect often sub-microscopic, asymptomatic malaria infections in individuals. While these individuals do not have any symptoms for the disease and thus do not seek medical support, they are still an important reservoir for the parasite, playing a key role in malaria transmission through mosquitoes. A successful malaria elimination strategy will critically depend on improved means of uncovering these hidden parasite reservoirs and ensuring timely treatment and better health outcomes. It is envisioned that the SMAART-1 RDT will provide the necessary sensitivity and specificity to reliably detect all individuals infected by P. falciparum malaria, the most common and deadliest of the malaria parasite species. However, this RDT also has potential utility in diagnosing infections with P. vivax parasites since these parasites also express the PSSP17 protein marker. Therefore, this test will address key objectives for the elimination of malaria as expressed by the WHO in their WHO Malaria Report 2018: “Prompt diagnosis and treatment is the most effective way to prevent a mild case of malaria from developing into severe disease and death”. In addition, this test may also prove to be an enabling technology toward achieving the goal expressed in the WHO global technical strategy pillar for malaria 2016-2030: “Transform malaria surveillance into a core intervention”, since the test is portable, non-invasive, highly sensitive and does not need clinical or medical training for its use.
What sort of innovation are you bringing in your project?
New markers for the detection of malaria are required to overcome limitations facing the current panel of available RDTs, which can no longer detect certain P. falciparum strains that appear to be spreading throughout Sub-Saharan Africa. This development adds to principle problems of current malaria RDTs that have high false negative rates due to their overall low sensitivity. Our project addresses the limitations of standard malaria RDTs through the non-molecular detection of a new marker for P. falciparum malaria infection with exceptional sensitivity in the saliva of individuals infected with the parasite. Using accessible fluorescent detection technologies that do not rely on advanced instrumentation, this project will develop and validate a new malaria RDT with thus far unmatched sensitivity and reliability. In addition, this test will for the very first time enable non-invasive testing of malaria parasite infections from saliva; avoiding the needs for collecting blood. We anticipate that this new test format will increase significantly the testing of children.
Role and Responsibility of Each Partner
University of Florida: Overall project oversight and management, lateral flow test design, development and pre-testing of the RDT, conducting performance and usability evaluation field studies in the DRC or Uganda.
CellFree Sciences Co., Ltd.: Production of recombinant PSSP17 protein marker for use in lateral flow test optimization and finally as a positive control in each commercial SMAART-1 Kit that is produced.
Frontier Institute, Co. Ltd.: Process development and scaled-up production of recombinant humanized monoclonal antibodies for capture and detection of PSSP 17 on the lateral flow cassette.
Oasis Diagnostics Corp.: Design and production of saliva collection device, lateral flow strip and lateral flow strip cassettes and for field evaluation and WHO prequalification.
ERADA Technology Alliance, Ltd.: Provision of co-funding support to ensure the provision of stabilized cell lines expressing the two humanized monoclonal antibodies for capture and detection of PSSP17 on the lateral flow cassette. ERADA holds the exclusive license to the SMAART-1 technology and will take responsibility to ensure that the RDT will meet requirements for compliance with CE marking and the WHO prequalification.
Others (including references if necessary)
WHO Malaria Report 2018: https://www.who.int/malaria/publications/world-malaria-report-2018/en/
Malaria eradication: benefits, future scenarios and feasibility. Executive summary of the report of the WHO Strategic Advisory Group on Malaria Eradication: https://www.who.int/publications-detail/strategic-advisory-group-malaria-eradication-executive-summary
A saliva-based rapid test to quantify the infectious subclinical malaria parasite reservoir.
Tao D, McGill B, Hamerly T, Kobayashi T, Khare P, Dziedzic A, Leski T, Holtz A, Shull B, Jedlicka AE, Walzer A, Slowey PD, Slowey CC, Nsango SE, Stenger DA, Chaponda M, Mulenga M, Jacobsen KH, Sullivan DJ, Ryan SJ, Ansumana R, Moss WJ, Morlais I, Dinglasan RR.: Sci Transl Med. 2019 Jan 2;11(473). - Free access as PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441545/
1. Project objective
The primary objective of this project is to produce a commercial version of the Saliva-based Malaria Asymptomatic and Asexual Rapid Test, aka SMAART-1. SMAART-1 test is non-invasive, using saliva collected from individuals 5 years old and above. The test takes advantage of a familiar lateral flow format but leverages fluorescence for enhanced detection sensitivity of the Plasmodium falciparum PSSP17 biomarker at the point of need, enabling the early identification of individuals with subclinical malaria infections and the provision of antimalarial treatment prior to the onset of life-threating disease.
2. Project design
During the two-year project and no cost extension phase (due to the ongoing COVID-19 pandemic), we sought to design and small-scale manufacture a lateral flow immunoassay cassette, produce recombinant humanized monoclonal (capture/detection) antibodies in CHO cells, and determine the acceptability of the SMAART-1 test (secondary objective) in the Democratic Republic of Congo (DRC), a priority malaria endemic country, including evaluation testing of the kit and instruction pamphlet.
3. Results, lessons learned
We established a manufacturing and validation workflow, and a functioning, SMAART-1 commercial lateral flow assay (LFA) for PSSP17, produced stable, recombinant human-mouse chimeric monoclonal antibodies for use in rapid test construction in a scalable manufacturing process, and determined the wide acceptability of the test by the envisioned end-users in the DRC. In addition, SMAART-1 was found to be stable at 1-8 °C and 52 °C for 0 days, 7 days, 14 days, and 21 days, with no resulting false positivity observed. However, despite these successes the COVID-19 pandemic proved to be a significant obstacle, preventing the completion of the study during the no cost extension period. Although we produced 200 commercial tests (without the saliva collection device), we observed a marked reduction in sensitivity of the PSSP17 biomarker antigen in the final dry test format, as compared to the limit of detection (LOD) that we originally reported for the non-commercial, wet-format for the rapid test. We have identified the potential issue lies with the capture and detection monoclonal antibodies, wherein the independent epitopes are too close together, preventing the antibody capture-detection complex to form in a dry test format. We have since proposed to develop a new recombinant chimeric monoclonal antibodies to more spatially distant epitopes on PSSP17, allowing the dry test format to achieve the envisioned LOD.
Commercial Development of a Saliva-based Malaria Asymptomatic and Asexual Rapid Test (SMAART-1)