After nine years of efforts, experts completed the development of a standardized and validated methodology for the molecular diagnosis of cutaneous leishmaniasis in the Americas. Advances in diagnosis can contribute to controlling the disease, considered neglected by the World Health Organization (WHO). 

The infection causes wounds on the skin, harming patients' quality of life, both due to the impact of the injuries and the stigma. The disease occurs in 19 countries on the continent, with almost 38 thousand cases registered in 2021, 15 thousand of which in Brazil.

The development of the standardized molecular test was led by the Oswaldo Cruz Institute (IOC/Fiocruz) in partnership with the Pan American Health Organization (PAHO). 

Experts from six countries – Brazil, Argentina, Bolivia, Mexico, Panama and Peru – collaborated in developing the methodology. The initial stage of the project also included the participation of researchers from Colombia, Costa Rica and Spain. 

The organization Medicines for Neglected Diseases (DNDi) supported the initiative.

The molecular test uses the real-time PCR technique to detect the DNA of the Leishmania protozoan, the parasite that causes leishmaniasis, in samples obtained from skin lesions. 

In addition to confirming the diagnosis, the standardized methodology allows quantifying the volume of parasites present in the lesion.

“The lack of diagnostic methods is a major challenge for controlling cutaneous leishmaniasis. PCR is a very sensitive methodology, which can help clarify suspected cases and expand access to treatment of the disease”, highlighted the head of the Leishmaniasis Research Laboratory at the IOC, Elisa Cupolillo, one of the project coordinators.

“Real-time PCR can provide results in just a few hours. After the Covid-19 pandemic, all Brazilian states and all countries in the Americas are equipped and qualified for molecular diagnosis. It is a favorable scenario for the application of this method”, pointed out the head of the Laboratory of Virology and Molecular Parasitology at the IOC, Otacílio Moreira, also coordinator of the initiative.

The final exam protocol was established in a workshop held from November 7th to 9th on the F campusiocRuz, in Manguinhos, Rio de Janeiro. The meeting brought together 16 experts, including researchers from the six countries that are part of the initiative and representatives from PAHO and DNDi.

At the opening of the event, the coordinator of the PAHO Regional Leishmaniasis Program, Ana Nilce Maia Elkhoury, and the deputy director of Research, Technological Development and Innovation of the IOC, Elmo de Almeida Amaral, highlighted the commitment to combating leishmaniasis.

“We have completed an important stage to make this methodology available to people affected by the disease in our region. We will work to make this progress effective in the practice of public health in the Americas”, stated Ana Nilce, who is coordinating the initiative. 

“I would like to thank everyone for their commitment to developing this methodology over almost ten years. This project shows the importance of partnerships between researchers in the Americas and, especially, between the IOC and Opas in the context of leishmaniasis”, said Elmo.

The clinical project manager for leishmaniasis at DNDi, Joelle Rode, highlighted the role of improving diagnosis in pursuit of the goal of controlling the disease as a public health problem by 2030, established by the WHO.

“This standardized methodology is a milestone in combating cutaneous leishmaniasis and could open doors for the development of rapid tests in the future”, commented Joelle.

International validation

The project began in 2014, bringing together experts from different countries who were already using PCR to diagnose cutaneous leishmaniasis with different protocols.

The project's first workshop was held in 2016. Together, scientists established a consensus protocol for the examination and selected three molecular targets for evaluation. 

The molecular targets are the stretches of the genome detected in PCR. Selecting the correct region is important so that the exam is specific (detecting only parasites of the Leishmania genus) and sensitive (capable of identifying the parasite in small quantities and covering different species of Leishmania).

No IOC, the methodology was developed and tested on reference Leishmania strains, maintained in the laboratory. Then, two validation steps were conducted on clinical samples. 

Firstly, scientists from IOC applied the protocol to diagnose 116 samples obtained from patients from the six countries that are part of the project.

On Monday, the tests were repeated in seven research centers, one in each foreign country and two in Brazil. Each center applied the methodology to diagnose 40 samples.

Participated in the project: Fiocruz Rondônia and Instituto Evandro Chagas (IEC), from Brazil; National Institute of Tropical Medicine (Inmet), from Argentina; Universidad Mayor de San Simón (UMSS), Bolivia; Instituto Commemorativo Gorgas de Estudios de la Salud (IGorgas), from Panama; and Universidad Peruana Cayetano Heredia (UPHC), from Peru.

The results of the validation tests were presented and discussed at the workshop held at Fiocruz. Based on the results, the researchers selected the best molecular target for the standardized PCR methodology, considering the sensitivity and specificity rates observed.

The project coordinators highlight the importance of international validation of the methodology.

“The multicenter validation covered a wide area of ​​the endemic zone for leishmaniasis in the Americas, from Mexico to Argentina, in which several Leishmania species circulate. We observed agreement between the results even with different sample collection methods, equipment and analysis software”, said Otacílio.

Currently, the laboratory diagnosis of cutaneous leishmaniasis is carried out through direct observation of the parasite by microscopy in samples obtained from skin lesions.

Considering the greater sensitivity and higher cost of the molecular test, the idea is that the test is used in a complementary way. For example, to clarify suspected cases of cutaneous leishmaniasis with a negative result in microscopy analysis.

In the future, the methodology can also contribute to patient monitoring.

“Quantifying the parasite load can function as a prognostic marker, since we observed that this load is related to the outcome of the disease. This information can help guide treatment”, says Elisa.

The technical report presenting the methodology should soon be sent to PAHO for dissemination in the region. 

The researchers also plan to submit a scientific article for publication in a journal to share the information with the academic community.

The project must continue with the development of a kit for the molecular diagnosis of cutaneous leishmaniasis, bringing together all the compounds necessary for the test in a single product. The objective is to make the exam easier and faster, reducing the chances of error. 

In recent years, the IOC contributed to the development of kits for molecular diagnosis of various diseases, including neglected infections with high impact in Brazil such as Chagas disease e leprosy.