Introduction and Background of the Project

Introduction

Buruli ulcer (BU), or Mycobacterium ulcerans disease, is a neglected tropical disease that has a debilitating, economic and psychosocial impact on affected individuals and families, from the deep skin ulcerations it causes. M. ulcerans produces mycolactone, a toxin responsible for necrosis in the cutaneous tissue, and is homogeneously distributed in the lesions. The disease is endemic in over 33 countries, mostly in Africa, but also Australia, French Guiana and Japan. Early diagnosis and treatment remains the only option for preventing disability due to BU. Confirmatory diagnosis of BU is by PCR, in laboratories that are far from the remote areas where patients live. Thus, point-of-care diagnostic tests for BU are urgently needed. We have targeted mycolactone as the analyte, and developed a game-changing BU (BU-MYCOLAC) RDT, that will bring diagnosis closer to patients. To enhance detection, novel magnetic gold nanoshells, mycolactone-specific monoclonal antibodies and a biotinylated probe are used in the RDT. We propose to complete prototype optimization, design-lock and transfer of the BU-MYCOLAC RDT to manufacturing, conduct validation studies, and register the test for licensing. The success of this project will enhance the attainment of WHO’s 2030 targets of reducing BU cases to <10%, 95% confirmation and 98% treatment.

Project objective

The objective of this project is to bring endorsement by WHO of the first RDT for Buruli ulcer that detects mycolactone, through field operational evaluation, registration and formal clinical trials. This will be undertaken through the following specific objectives:

・Assessment of operational characteristics of the prototype RDT, including usability and diagnostic performance, under different field conditions

・Optimization and design-lock of the RDT, and transfer to manufacturing

・Registration and CE marking

・Formal clinical evaluation

Project design

A field-ready prototype BU-MYCOLAC RDT employing mycolactone-specific monoclonal antibodies and biotinylated mycolactone probes in a competitive assay format, will be evaluated in field settings in BU-endemic areas, to assess its operational characteristics, usability and initial diagnostic performance against the reference standard IS2404 PCR. Samples will be evaluated using the IS2404 PCR, mycolactone ELISA, and the concentrations of mycolactone determined to inform performance of the final design-locked RDT. The inter- and intra-observer variability in mycolactone extraction and visual interpretation of the results will be assessed, to demonstrate reproducibility, and optimize the conditions for optimum reproducibility. The outcomes of the field evaluations will inform further optimization and design-locking of the RDT. Different lots of the RDT will be produced to validate manufacturing procedures, conduct formal stability studies and assess conformity for CE-marking. In the final stage, a prospective single arm accuracy study comparing the RDT to the IS2404 PCR will be carried out. Clinical diagnosis and other methods used at the study sites will also be considered in the analysis. Data from this trial will be submitted to WHO for consideration in a recommendation for adoption of the RDT for use in endemic countries.

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

Early diagnosis is the cornerstone of BU control and disability prevention. With oral antibiotics available, treatment at the community level is possible if diagnosis can be done close to where the patients live. Currently, many health workers struggle to clinically distinguish BU lesions from other ulcers. PCR reference facilities, for case confirmation, are far from affected communities, with a turnaround time of 2-4 weeks. During this waiting period, the lesions progress to stages that become more difficult to manage. BU-MYCOLAC RDT will fill these gaps and have a significant impact on the management of BU globally.

In the short term, the project will ensure that the RDT meets the ASSURED (Affordable, Sensitive, Specific, User-friendly, Robust and rapid, Equipment free, and Deliverable) criteria outlined by WHO, with the following outcomes.

・Enhance diagnosis at primary health facilities and at the community level, thus improving turnaround time for result confirmation (2 hours instead of 1 or more weeks)

・Improve treatment outcome and reduce disfigurement following early confirmation

・Contribute evidence towards WHO policy and adoption

In the long term, the RDT will enhance surveillance and control of BU in endemic countries, enable treatment monitoring, and help meet the WHO NTD 2030 roadmap targets.

What sort of innovation are you bringing in your project?

While Lateral Flow Immunoassay (LFIA.) are a mature technology, the detection of mycolactone presents two significant challenges. The first one is the need for monoclonal antibodies (mAbs) specific for mycolactone, and these have been notoriously difficult to obtain. The second one is the need to extract mycolactone from a swab using a large amount of solution and then concentrating it before applying to the lateral flow assay, an unusual but necessary step.

To address the first challenge, the Swiss TPH has developed mycolactone-specific mAbs and used these in a competition ELISA. The assay employs a mouse monoclonal antibody as detector antibody, and a biotinylated form of mycolactone as probe for the competitive assay. The development of the mycolactone specific mAbs, was extremely difficult and represent a breakthrough towards the development of a rapid diagnostic test for Buruli ulcer.

To address the challenge of mycolactone concentration from swabs, prior to its application to the RDT, DDTD introduced the use of magnetic gold nanoshells. These magnetic gold nanoshells contain a magnetite core, surrounded by a silica gel layer, and coated with an outside shell of gold. The magnetite provides magnetic concentration capabilities, the silica gel lowers the overall density of the nanoparticles to ensure easy resuspension in an aqueous medium and smooth flow across a test strip, and the gold shell surface absorbs light extremely efficiently due to plasmon resonance effects.

Role and Responsibility of Each Partner

Foundation for Innovative New Diagnostics (FIND) is the Designated Development Partner and will provide Consortium Management. FIND will be responsible for the overall consortium management, initiate the registration and CE marking of the RDT, and participate in clinical evaluations with TMGH-NU.

School of Tropical Medicine and Global Health, Nagasaki University (TMGH-NU) will validate the sample collection from wounds, as well as testing and evaluation of all protocols. They will be responsible for field activities in endemic country settings. TMGH-NU will also seek conditions for samples to be stored and analyzed, exploring direct testing in the field and sample transfer to central laboratory for testing.

Swiss Tropical and Public Health Institute (Swiss TPH) will establish the protocol for sample collection and mycolactone extraction. Samples will be independently tested at the Swiss TPH using the reference IS2404PCR, and the mycolactone ELISA. Swiss TPH will determine the concentration of mycolactone in samples to inform the performance of the final design-locked RDT.

Drugs and Diagnostics for Tropical Diseases (DDTD) is ISO-13485:2016 certified. They will develop and optimize the tests to be used in the field evaluation. They will also produce a design-locked RDT and prepare quality documentations according to ISO 13485 standard, for registration purposes.

Others (including references if necessary)

・Sarfo FS, Le Chevalier F, Aka N, Phillips RO, Amoako Y, et al. (2011). Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring. PLoS Negl Trop Dis. 5(7):e1237.

・Dangy JP, Scherr N, Gersbach P, Hug MN, Bieri R, et al. (2016). Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans. Plos Negl Trop Dis. 10(6):e0004808.

・Warryn L, Dangy JP, Gersbach P, Gehringer M, Schäfer A, et al. (2020). Development of an ELISA for the quantification of mycolactone, the cytotoxic macrolide toxin of Mycobacterium ulcerans. PLoS Negl Trop Dis. 14(6):e0008357.

Final Report

1. Project objective:

The objective of the project is to evaluate the performance of a rapid diagnostic test (RDT) for Buruli ulcer (BU) diagnosis in field settings. The test targets mycolactone, the toxin produced by Mycobacterium ulcerans. The goals include proving that mycolactone is present in at least 80% of PCR-positive samples, achieving at least 80% concordance between RDT and ELISA results, and demonstrating a specificity of 95% compared to PCR.

2. Project design:

 The project involves the development and evaluation of a rapid diagnostic test (RDT) for Buruli ulcer (BU) diagnosis. The test targets mycolactone, a toxin produced by Mycobacterium ulcerans. The design includes a competitive assay where the test line disappears if mycolactone is present. The test was optimized by adding heterophilic-antibody blocking agents, increasing pH buffers, and lengthening magnetic separation duration. The optimized RDT showed improved consistency and sensitivity, with a limit of detection reduced to less than 2 ng of mycolactone per sample.

3. Results, lessons learned:

Initial field studies in Cameroon and Cote d’Ivoire revealed many indeterminate results, leading to significant test optimization. The optimized RDT showed improved consistency and sensitivity, with a limit of detection reduced to less than 2 ng of mycolactone per sample.

However, performance analysis revealed poor concordance between PCR, ELISA, and RDT results, likely due to sample-to-sample variations and mycolactone degradation during transport. The optimized RDT prototypes showed an average specificity of 92% and sensitivity of 38% compared to Swiss TPH PCR data. The RDT was also evaluated on non-BU patient samples in Japan, showing specificity to M. ulcerans.

A positive outcome was the public health impact, with over 350 suspected BU patients reached and provided with treatment. The study highlighted the need for multiple diagnostic methods to confirm BU cases and the importance of strengthening laboratory capacity. The project also provided valuable lessons for developing RDTs for other skin ulcers.

Despite the improvements, the RDT's complexity and handling procedures make it more suitable for laboratory use than field settings. The current time-to-result of approximately 1.5 hours is not significantly different from molecular detection methods like LAMP and RPA, which have better performance characteristics. The team concluded that the potential challenges and pitfalls for a BU rapid test are significant and recommended discontinuing activities and focusing on more successful molecular tools.

The study also faced challenges with stabilizing mycolactone, as it degrades on exposure to light and high temperature. Unlike other studies that stored mycolactone in ethanol, this study used dry swabs or RDT buffer solution due to the RDT's incompatibility with ethanol. The discrepancies across testing methods led to uncertainty about which result should be considered the reference standard. The RDT revealed barely detectable mycolactone amounts, rarely exceeding 7 ng, which may explain the poor clinical sensitivity. Future evaluations should test the RDT immediately on swabs collected in the field or at the point of care.

In conclusion, while the RDT showed promise, its complexity and handling requirements limit its suitability for field use. The project underscored the importance of multiple diagnostic methods and highlighted the need for further research and development of more effective diagnostic tools for BU.