Non-invasive volatiles test for canine leishmaniasis diagnosis

Project Information

CANLEISH

Grant agreement ID: 101007653

DOI

10.3030/101007653

Project closed

EC signature date 23 October 2020

Start date 1 May 2021

End date 30 April 2025

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 1 881 400,00

EU contribution

€ 1 881 400,00

Coordinated by

UNIVERSITATEA SPIRU HARET
Romania

Periodic Reporting for period 2 - CANLEISH (Non-invasive volatiles test for canine leishmaniasis diagnosis)

Reporting period: 2023-05-01 to 2025-04-30

Summary of the context and overall objectives of the project

The EU-funded CANLEISH project focused on the non-invasive diagnosis of canine leishmaniasis, one of the most important vector-borne parasitic diseases of dogs. Researchers from nine partner countries in Europe, South America, Africa and Asia (electronic engineers, biochemists, veterinarians, human doctors, biologists, technicians, IT specialists) developed an innovative, non-invasive diagnostic method that detects specific volatile compounds (VOCs) emitted by dogs' breath and hair.
Leishmaniasis is a disease caused by the Leishmania intracellular parasites that are transmitted through the bite of certain female sandflies. It is found in the tropics, subtropics and southern Europe, and diagnosis takes place through tissue examination, parasitological diagnosis and serological diagnosis.
We defined the following scientific objectives for the present project:
- Identification of CanL volatile biomarkers in dogs’ breath and hair
- Fabrication of a chemical gas sensor system for CanL diagnosis from analysis of breath and hair volatiles
- Fabrication of a portable e-nose system for CanL diagnosis
- On-site testing in real-world environments of the portable e-nose system for CanL diagnosis
Conclusion :The importance of this research for society is major: in zoonotic visceral leishmaniasis, dogs are the main carriers, and their rapid identification allows reducing the risk of disease transmission to humans.
VOCs analysis and the production of equipment that can exploit the results provided by these compounds represent a future technology in the detection and control of leishmaniasis, providing rapid, non-invasive diagnosis. By reducing the animal reservoir, optimizing public interventions and reducing costs, they can generate a major impact on human and community health. The CANLEISH partnership has developed and is further researching an AI-based decision support system capable of interpreting sensor outputs and distinguishing between healthy and infected dogs.
The research has also developed a strong partnership between multidisciplinary research teams from Europe, Africa, Asia and Latin America.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The following work was carried out during this project:
- Carrying out activities in accordance with the ethical approvals initially proposed and those added during the course of the project.
- Elaboration of the protocol for the collection, storing and shipment of the breath and hair volatile organic compounds (VOCs) emitted by dogs.
- Collection of the breath and hair VOCs samples from dogs diagnosed with canine leishmaniasis and negative dogs for this disease in Romania, Colombia, Tunisia and Algeria, as well as of environmental air samples. Patient-friendly protocol for breath samples collection from dogs was tested.
- Design and development of the thermal desorption unit for the release of the VOCs stored in Tenax sorbent tubes.
- Optimisation of GC-MS operation parameters for the analysis of the VOCs samples acquired in this project.
- GC-MS measurement of the samples from all dogs included in this study and of the environmental air samples.
- Statistical analysis of the GC-MS data and identification of the volatile biomarkers of canine leishmaniasis released by the breath and hair of dogs.
Artificial intelligence-based algorithm for CanL diagnosis was developed.
- Fabrication of an array of chemical gas sensors having different sensing nanomaterials for the electronic nose system.
- Sensors characterisation upon exposure to different volatiles.
- Design and fabrication of the first version of the electronic nose (e-nose) prototype
- Preliminary sensing measurements with the e-nose prototype, and redesign of the sensors chamber.
- On-site validation tests of the electronic nose.
For the final testing of the prototype, Romania was chosen, a country where very few cases of leishmaniasis in dogs are reported, and the cases encountered are asymptomatic, which was an objective pursued since the beginning of this project: how to identify these infected animals early, through rapid and non-invasive methods.

- Realisation of the following network-wide training events: seven (out of seven) training courses, three (out of three) workshops, and two (out of two) conference.
- Publication of peer-reviewed research articles in international journals
- Participation at conferences
- Launch of the project website
- Results presentation at specialised fairs
- Project communication in mass media; press releases; radio interview; video interview at USH (Romania) with team members from Romania, Colombia and Estonia; TV show DIGI Animal Club (https://www.youtube.com/watch?v=AZ1mWofO0R0) social media (Facebook posts).
- Communication to target groups: Universities community; Meeting with health representatives; Visits at schools; Meeting with public authorities.
As part of the communication and dissemination activities of the project results, two meetings were organized with health authorities in Colombia. During these events, the scientific advances of the CANLEISH project, i.e. the use of e-nose technology for the rapid and non-invasive diagnosis of canine leishmaniasis, were presented and the potential usefulness of this method for public health campaigns was emphasized. The ethical issues raised by the CANLEISH project, which focused on minimizing animal suffering and avoiding unnecessary euthanasia, were highlighted. The meeting also addressed the issues of governance and good practices in the management of canine leishmaniasis. The importance of collaboration between health authorities, academic institutions and animal welfare organizations was emphasized to ensure the efficiency, effectiveness and sustainability of OneHealth interventions that combine animal welfare, human health and ecosystem dynamics.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Identification of CanL volatile biomarkers in dogs’ breath and hair and development of an non-invasive method for detecting CanL infection to limit the spread of the disease and facilitate the adoption and transport of dogs between countries.
The electronic nose utilized in this study has been configured and consists of three primary components: the sensor detection system, the airflow system, and the data analysis system. The sensor detection system features an array of gas sensors, which includes sixteen gas sensors. Gas sensors are the most employed type in electronic nose devices due to their sensitivity, stability, and effectiveness across a broad spectrum of volatile compounds.
Trainees from Romania (USH), Thailand (ENCL), Colombia (UP) and Algeria (UCBET) participated in the on-site testing of the e-nose prototype that were carried out in Romania, under the coordination of the USH team. The trainees had the opportunity to observe good research practices and ethical standards aimed at the protection of animals involved in research.
The research conducted in this project has contributed to the expected impacts through: enhancing the potential and future career prospects of the staff members by organizing scientific events and sharing activities to provide to the staff involved in the project with multilateral training in the broad research areas covered by this project and developing new and lasting research collaborations, achieving transfer of knowledge
between participating organisations and contribution to improving research and innovation potential at the European and global levels.

Prototype setup

Additional laboratory analyses (PCR technique)_UP

The development and implementation of the portable e-nose is almost complete. The program for opera'

Start of the last stage of the project - e-nose tested at USH

GC-MS measurement of the samples collected

Collection of blood samples

Collection of breath samples

Chemical gas sensors measurement_UP

Development of a lab-made e-nose system. UP together with EMU and IPT secondees. A lab-made e-nose

E-nose testing within secondments ENCL-UP-USH

Collection of breath and hair samples

Collection of breath and hair samples. Transfer of VoC to Tenax tube

Additional laboratory analyses_UP

E-nose is presented by the team of secondee from ENCL to secondee from USH and UCBET