Fiocruz and PAHO standardize diagnosis for cutaneous leishmaniasis in the Americas

After nine years of effort, experts have completed the development of a standardized and validated methodology for the molecular diagnosis of cutaneous leishmaniasis in the Americas. Advances in diagnosis could help control the disease, which is considered neglected by the World Health Organization (WHO). The infection causes sores on the skin, damaging patients' quality of life both due to the impact of the lesions and the stigma. The disease is present in 19 countries on the continent, with almost 38,000 cases recorded in 2021, 15,000 of them 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).

Standardized methodology identifies the DNA of the parasite that causes cutaneous leishmaniasis in samples obtained from skin lesions (photo: Ricardo Schmidt)

Experts from six countries – Brazil, Argentina, Bolivia, Mexico, Panama and Peru – collaborated in developing the methodology. The initial stage of the project also involved researchers from Colombia, Costa Rica and Spain. The Drugs for Neglected Diseases Initiative (DNDi) organization supported the initiative.

The molecular test uses the real-time PCR technique to detect the DNA of the protozoan Leishmania, the parasite that causes leishmaniasis, in samples obtained from skin lesions. Besides confirming the diagnosis, the standardized methodology makes it possible to quantify the volume of parasites present in the lesion.

"The lack of diagnostic methods is a major challenge for the control of cutaneous leishmaniasis. PCR is a very sensitive methodology that can help clarify suspected cases and increase access to treatment for the disease", said Elisa Cupolillo, head of the Leishmaniasis Research Laboratory at IOC and one of the project's coordinators.

"Real-time PCR can provide results in just a few hours. Following the COVID-19 pandemic, all Brazilian states and all countries in the Americas are equipped and trained for molecular diagnostics. It is a favorable scenario for the application of this method", said Otacílio Moreira, head of the Virology and Molecular Parasitology Laboratory at IOC/Fiocruz, who is also coordinating the initiative.

The final protocol for the exam was established at a workshop held from November 7 to 9 at Fiocruz's campus in Manguinhos, Rio de Janeiro. The meeting gathered 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 PAHO's Regional Leishmaniasis Program, Ana Nilce Maia Elkhoury, and the deputy director of Research, Technological Development and Innovation at IOC/Fiocruz, Elmo de Almeida Amaral, stressed the commitment to tackling leishmaniasis.

"We have completed an important step in making this methodology available to people affected by the disease in our region. We are going to work to ensure that this advance becomes a reality in public health practice in the Americas", said Ana Nilce, one of the initiative's coordinators.

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

DNDi's clinical project manager for leishmaniasis, Joelle Rode, highlighted the role of improving diagnosis in pursuit of the goal of controlling the disease as a public health problem by 2030, set by the WHO. "This standardized methodology is a milestone in the fight against 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, the scientists established a consensus protocol for the test and selected three molecular targets for assessment.

The molecular targets are the stretches of the genome detected by PCR. Selecting the right region is important for the test to be specific (detecting only parasites of the genus Leishmania) and sensitive (able to identify the parasite in small quantities and covering different species of Leishmania).

At IOC/Fiocruz, the methodology was developed and tested on reference Leishmania strains kept in the laboratory. Two validation stages were then carried out on clinical samples. In the first, IOC/Fiocruz scientists applied the protocol to the diagnosis of 116 samples obtained from patients in the six countries that are part of the project. In the second, the tests were repeated in seven research centers, one in each foreign country and two in Brazil. Each center applied the methodology to the diagnosis of 40 samples.

The following participated in the project: Fiocruz Rondônia and the Evandro Chagas Institute (IEC), from Brazil; the National Institute of Tropical Medicine (Inmet), from Argentina; Universidad Mayor de San Simón (UMSS), from Bolivia; Instituto Conmemorativo 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, taking into account the sensitivity and specificity indices observed.

The project coordinators emphasize 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, where there is circulation of several species of Leishmania. We observed that the results matched even with different methods of sample collection, equipment and software of analysis", said Otacílio.

Currently, 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 for it to be used in a complementary manner. For example, to clarify suspected cases of cutaneous leishmaniasis with negative microscopy results.

In the future, the methodology could also contribute to patient follow-up. "The quantification of the parasite load can function as a prognostic marker, since we have 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 in order to share the information with the academic community.

The project should continue with the development of a kit for the molecular diagnosis of cutaneous leishmaniasis, gathering all the compounds needed for the test in a single product. The aim is to make it easier and quicker to carry out the test, reducing the chances of error.

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