Development of ‘all-in-one’ diagnostic kit for Buruli ulcer using lateral flow DNA-chromatography
Project Completed
Please click to see the final report.
  • RFP Year
  • Awarded Amount
  • Disease
    NTD (Buruli ulcer)
  • Intervention
  • Development Stage
    Concept Development
  • Collaboration Partners
    Keio University School of Medicine ,  TBA Co., Ltd. ,  Fasmac Co., Ltd. ,  Raoul Follereau Institute Côte d’Ivoire ,  Institut Pasteur de Côte d'Ivoire ,  Hope Commission International ,  Nagasaki University ,  Teikyo University

Introduction and Background of the Project


Buruli ulcer is a cutaneous mycobacterial infection, which is reported in about 2,000 new patients annually worldwide, mainly in West Africa. Children are mainly affected due to infection with the acid-fast bacteria Mycobacterium ulcerans (M. ulcerans) that exists in the water system of the environment such as ponds and rivers. When diagnosis or treatment is delayed, a wide range of intractable ulcers will form on the skin, leaving aftereffects such as limited joint movement after healing. It is often difficult to distinguish Buruli ulcers from other skin ulcers of various causes, and the only way to make a reliable diagnosis is to take a sample from the ulcer and confirm it by PCR. However, it is difficult to perform such a test that requires such advanced technology and expensive equipment in the West Africa where the number of patients is high. We have established a method for amplifying M. ulcerans DNA without using expensive equipment and a determination method by DNA chromatography. We will combine these techniques to develop a new diagnostic method for Buruli ulcer.


Project objective

As one of the skin NTDs, there is no point-of-care diagnostic method for Buruli ulcer, which results in delay in diagnosis and treatment and many patients have to live with serious sequelae. The number of new cases of Buruli ulcer reported to the WHO is about 2,000 per year, which is due to the fact that the diagnosis itself has not been made because the disease itself is not well recognized. This indicates that there are many cases in which the patient is left without being properly diagnosed due to lack of proper diagnostic method, and wrong treatment might be given. In order to perform PCR, which is currently the only diagnostic method recommended by WHO, a specialized laboratory with expensive equipment and technologists with a high level of knowledge and skills are required. Therefore, it is not practical to perform PCR in endemic area of Buruli ulcer. In this project, we will develop a diagnostic kit for Buruli ulcer that can be tested by anyone in anywhere.


Project design

In order to perform DNA isothermal amplification methods such as the LAMP method, it is necessary to mix special enzymes and various reagents with the sample in the correct proportions. In this project, we will develop a small kit in which these reagents are fixed and dried in a stabilized state, and the reaction takes place simply by adding a sample. By adding the solution after the DNA amplification reaction is completed, the amplified DNA will be developed by DNA chromatography, and if M. ulcerans DNA is present, a visually recognizable line will appear. The detection sensitivity is about the same as the PCR method, and we aim to develop a kit that greatly reduces the cost and time required for testing. The kit developed in Japan will be used by local medical staff in Côte d'Ivoire, further improved to produce the final kit.

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

The availability of a simple diagnostic kit for Buruli ulcer will enable early diagnosis of patients with suspicious skin lesions that enables appropriate treatment with antibiotics recommended by WHO. This will allow proper examination and treatment of Buruli ulcer, one of the NTDs, which will reduce the number of children with sequelae on joints. In addition, even if Buruli ulcer is denied by this diagnostic kit, the accuracy of diagnosis and treatment can be improved by examining other possibilities. Such a kit will help reduce the number of patients with other skin disorders in addition to Buruli ulcers.

What sort of innovation are you bringing in your project?

This project will provide the first diagnostic kit for Buruli ulcer. The method is simple and requires no special equipment or technology, and the kit can be stored at room temperature for a long period of time without the need for a freezer or refrigerator. This has been made possible by combining techniques for stably fixing and drying reagents, as well as techniques for performing DNA amplification without a special device, and DNA chromatography that allows easy visual detection of its detection. The combination of the advantages of these technologies has created a completely new and innovative diagnostic kit.

Role and Responsibility of Each Partner

Teikyo University plays a central role in developing the kit in cooperation with Keio University. TBA Co., Ltd. and Fasmac Co., Ltd. manufacture DNA chromatography strips and primers used for LAMP method, and make necessary improvements. Hope Commission International will work with Raoul Follereau Institute Côte d'Ivoire and Pasteur Institute Côte d'Ivoire to collect clinical specimens and evaluate kits in Côte d'Ivoire. Nagasaki University will provide necessary cooperation and support for these activities in Côte d'Ivoire.

Others (including references if necessary)

Suzuki K, Luo Y, Miyamoto Y, Murase C, Mikami-Sugawara M, Yotsu RR and Ishii N. Buruli Ulcer in Japan. In: Pluschke G., Röltgen K. (eds) Buruli Ulcer. Springer, Cham, 87-105, 2019. 

Yotsu RR, Suzuki K, Simmonds RE, Bedimo R, Ablordey A, Yeboah-Manu D, Phillips R, Asiedu K. Buruli ulcer: a review of the current knowledge. Curr Trop Med R 5(4), 1-10, 2018.

Scherr N, Bieri R, Thomas S, Chauffour A, Kalia NP, Schneide P, Ruf M-T, Lamelas A, Malathy SSM, Gruber G, Ishii N, Suzuki K, Tanner M,. Moraski GC, Miller MJ, Witschel M, Jarlier V, Pluschke G, Pethe K. Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer. Nat Commun 9, 5370, 2018.

Yotsu RR, Murase C, Sugawara M, Suzuki K, Nakanaga K, Ishii N, Asiedu K: Revisiting Buruli ulcer. J Dermatol 42, 1-9, 2015.

Final Report

1. Project objective

Buruli ulcer, caused by Mycobacterium ulcerans infection, is a skin NTD mainly encountered in West Africa. Early case detection and adequate treatment are essential to prevent the formation of large skin ulcers, avoid loss of joint mobility, and ensure that patients are not subject to stigma. Although PCR is recommended for definitive diagnosis, it is difficult to perform routinely in most endemic countries. To address this issue, we have developed a rapid diagnostic kit that can be easily tested by anyone, anywhere.


2. Project design

In developing the POCT kit for Buruli ulcer, we took into account three important requirements: 1) easy of use by anyone without the need for special equipment or advanced knowledge and skills, 2) long-term storage at room temperature and quick and easy-to-understand test results, and 3) prevention of cross-contamination, ensuring that amplified bacterial DNA does not leak and affect the results of other samples.


3. Results, lessons learned

We have developed a simplified kit that can detect the DNA of Mycobacterium ulcerans, the causative agent of Buruli ulcer. In an earlier project supported by the Medical Research and Development Agency (AMED) of Japan, we developed a technique using Loop-Mediated Isothermal Amplification (LAMP) to achieve isothermal DNA amplification, which was then visualised on DNA chromatography strip. However, this method required 1.5 mL tubes to conduct the reaction and needed specific facilities and techniques. Additionally, it is well known that contamination by aerosols generated by opening the tube’s lid after amplification by the LAMP method is a significant problem.

To address these issues, we aimed to develop an all-in-one type small-sized Point-of-Care Testing (POCT) kit, in which all processes from DNA amplification to DNA chromatography detection are completed in a single closed device. We developed a technique to immobilise all reagents for the LAMP reaction on carriers and confirmed that they could be stored at 37°C for at least one year. After verifying the kit’s capabilities using multiple 3D-printed models, we found that DNA amplification and detection were completed within one hour, and the sensitivity was sufficient to detect DNA derived from a single bacterial cell.

Although we had planned to evaluate the kit using clinical samples in Côte d'Ivoire, we were unable to travel due to the ongoing COVID-19 pandemic. Instead, we collaborated with the Ministry of Health and local medical institutions and trained healthcare workers to collect swab samples from skin ulcer lesions in remote rural areas, which were then transported to Japan for analysis. Analysis of these samples confirmed that the kit was able to detect the target DNA with the same sensitivity as pre-study evaluations.

In the future, with further research support, we aim to establish a framework for distributing this kit free of charge to countries in need. We also plan to optimise the internal structure of the kit and reaction conditions for mass production and expand the kit's capabilities to include multiplex testing for other skin NTDs.