Fiocruz creates a 100% national ingredient for use in the rapid test for visceral leishmaniasis

Fiocruz Pernambuco has created a 100% national ingredient to be added to the rapid screening test for the diagnosis of visceral leishmaniasis (VL). The product can be used for both the human and canine populations, being the first with this dual efficacy. The product has already been assessed in laboratories and will soon be implemented in the field. VL is an infectious disease transmitted by the drain fly that can affect both humans and animals, with domestic dogs being the main reservoirs of the disease. Lack of treatment in humans can lead to death in 90% of cases, according to the Ministry of Health.

The protein produced at Fiocruz PE is sent at a specific temperature, to guarantee its stability, to Bio-Manguinhos, in Rio de Janeiro, which adds the input to the rapid serological test device (photo Fiocruz Pernambuco)

For the new stage, in addition to qualifying the technology, the research was approved in the Inova Ceis RFP, a partnership between the MS and Fiocruz. More than R$800,000 will be secured for the work over the next three years. The results of the RFP were published last March and funding has already started in April.

The Fiocruz team has managed to create different versions of a synthetic protein in the laboratory, generated by combining fragments of various proteins from the protozoan that causes visceral leishmaniasis, Leishmania infantum. With this new protein (or recombinant antigen) made in the laboratory, it can interact with the antibody present in humans or animals positive for VL, identifying whether that sample is infected with the disease-causing agent.

The protein produced at Fiocruz PE is sent at a specific temperature, to ensure its stability, to the Immunobiological Technology Institute (Bio-Manguinhos/Fiocruz) in Rio de Janeiro, which adds it to the rapid serological test device. The Institute also took part in the preliminary stages of development and assessment of the proposed test.

Another innovation of the new antigen is that it showed efficacy in both symptomatic and asymptomatic dogs positive for visceral leishmaniasis, improving the applicability of the test compared to those already on the market. The expectation is that the new antigen produced can be added to the rapid serological test used in the Unified Health System (SUS) once all the necessary stages have been completed. Currently, there is no national product with this efficacy, and it is necessary to import one product that is used in the human test and another for the dog test.

This finding is the result of more than two decades of work at the institution, which began in collaboration with Fiocruz Bahia. The efforts involved graduate students and post-doctoral fellows. In the most recent stages, biologist Hemilly Silva and biomedical researcher Adalucia da Silva, currently post-doctoral students at Fiocruz PE, and biomedical researcher Wagner Tenório dos Santos, from Pernambuco and who continues to collaborate at Bio-Manguinhos, played a critical role. The project is coordinated by biologist Osvaldo Pompílio de Melo Neto, a researcher at the institution and advisor to the Fiocruz PE Graduate Program in Biosciences and Biotechnology in Health, and to the UFPE Graduate Program in Genetics.

"The availability of a new rapid test that is efficient, accessible and uses technology produced in the country, responds to a public health demand in Brazil and should make a positive contribution to strategies to control visceral leishmaniasis, a disease that is still neglected and which has Fiocruz as an institution that seeks understanding and creating solutions to tackle and treat it. This work is part of the efforts of a team that has been dedicated to the subject for years and it is gratifying to see that, despite the setbacks in science in our country, we have managed to have quality production that can benefit our population," says biologist Hemilly Silva.

Osvaldo Pompílio ratifies that, "so far, the diagnostic screening methods for visceral leishmaniasis raise some questions. The best ones are more complicated, difficult to make and expensive. Others, which are cheaper, are more likely to give inconsistent results, with false positives and false negatives. Additionally, imported technologies make it expensive for the country to acquire them. Our method is cheaper and faster, with the same logic as the COVID-19 rapid test. It does not need any infrastructure. We have also achieved an effective method for human and canine screening. There's no point in a person being healthy if there's an infected and undiagnosed dog nearby, making it possible for the mosquito to bite the animal and transmit it to humans. The idea is to diagnose both".

Inova Ceis

A partnership between Fiocruz and the Ministry of Health, through the Secretariat for Science, Technology, Innovation and the Health Complex, this RFP supports proposals that are at advanced levels of technological maturity and preferably, but not exclusively, protected by patents. The call encourages the development of innovative products to be potentially incorporated into the Unified Health System. In total, more than R$50 million will be used to support the selected projects.

In addition to the funding from Inova CEIS, Fiocruz PE will be taking the necessary steps to register the product with the Brazilian Health Regulatory Agency (Anvisa). At the same time, a patent for the new technology has already been applied for.

The effectiveness of the new antigen was proven during the doctoral work of Wagner Santos and Hemilly Silva. The applicability of the protein in the diagnosis of human and canine VL was assessed. Due to the absence of a rapid screening test that uses antigens produced with national ingredients, it was decided to proceed with the assessment on this type of platform.

The latest version of the proposed test, based on Hemilly's doctoral work, investigated 108 dogs positive for visceral leishmaniasis. Of the 44 symptomatic animals, 42 reacted positively with the new antigen, showing a sensitivity of 95%. Of the 64 asymptomatic dogs, i.e. those with the disease but no symptoms, 58 also tested positive using the new antigen – a sensitivity of 91%. Materials from 44 healthy dogs were also analyzed, achieving 100% specificity for the diagnosis of VL. There was also no cross-reaction with other parasites.

A total of 75 samples from humans confirmed to have visceral leishmaniasis were also tested, with 69 reacting positively to the test, a sensitivity of 92%. Another 55 healthy cases without visceral leishmaniasis were also tested and 100% were negative, attesting to the effectiveness and specificity of the test. Additionally, no cross-reactivity was observed with other diseases, such as tegumentary leishmaniasis, Chagas disease, dengue and Zika. All the results, both in dogs and humans, were equivalent or superior to the serological diagnostic tests currently available in Brazil. It should be noted that, as the rapid test is used as a screening test, another confirmatory test will be carried out in positive cases.

After this laboratory stage, the aim is to produce new batches of the rapid test based on the new protein and begin field-testing, to analyze the effectiveness of the test in humans and dogs that may be infected with Leishmania infantum.

Post-doctoral student Hemilly Silva confirms that local production of the antigen could prevent the need to import it, making logistics cheaper and faster. "The dependence of technology on imported ingredients prevents any effort to increase its efficiency and applicability to adapt it to the conditions and specificities of the Brazilian population. By meeting a demand from the SUS [Unified Health System], this type of test will bring a sustainable return to the public health system. It should be remembered that, today, there is an antigen for humans and another for dogs, and ours is efficient for both populations," she said.

The disease

Visceral leishmaniasis is transmitted by the bite of an insect called phlebotomine, popularly known as drain fly. The female mosquito needs to bite an infected dog, which is the main reservoir of the protozoan Leishmania infantum in urban areas, and then bite a human, transmitting the disease. In humans, the disease is considered chronic and systemic and can affect several organs. It can cause anemia, weight loss, weakness, long-term fever, and enlargement of the liver and spleen. Early diagnosis and timely treatment are essential to avoid worsening and complications, including death. The treatment is provided free of charge by the SUS.