Visceral leishmaniasis: Test identifies coinfection – 16/10/2023 – Health

In recent years, doctors and scientists in regions of Brazil where visceral leishmaniasis (VL) is endemic have come across more and more cases of co-infection by different protozoa: Leishmania infantum It is Crithidia. Accurate diagnosis, however, is hampered by the lack of simple and specific tests.

With the aim of accelerating and facilitating this distinction and, consequently, the definition of treatment, researchers from the Federal University of São Carlos (UFSCar) developed a PCR-type test (which analyzes the genetic material contained in the sample) that takes less than two hours to be completed. The results of the study were published in the journal Tropical Medicine and Infectious Disease.

The new method represents an advance in the diagnosis of VL – the most severe form of leishmaniasis that affects organs such as the spleen, bone marrow, liver and lymph nodes.

Neglected, the disease is considered a public health problem, with more than 3,500 cases every year in the country, according to the Ministry of Health. This number represents 93% of all cases in Latin America. In 2020 alone, there were 165 deaths nationwide.

The study – funded by Fapesp through nine projects – showed that the method was highly accurate in identifying and quantifying L. infantum It is Crithidia in samples obtained in vitro and in vivo or collected from hosts (humans, dogs, cats and vectors), for example, through skin biopsies or bone marrow aspirates.

“Although other molecular methods of species identification already exist, they usually involve sequencing the sample’s DNA, a more laborious, slow and costly process”, explains Sandra Regina Costa Maruyama, professor of the Postgraduate Program in Evolutionary Genetics and Biology Molecular (PPGGEv) at UFSCar and coordinator of the study.

“Our test evaluates the genetic material of parasites directly from the vectors and in the tissues of people and animals [gatos e cachorros].”

Unlike rapid tests used in the healthcare system, which do not directly detect the parasite but rather the antibodies produced against it, the new quantitative dye-based PCR (qPCR) assay was designed and standardized with species-specific target sequences Leishmania infantum It is Crithidia in experimental and clinical samples.

The new targets proved to be specific, however it is necessary to initially carry out an assay to detect the first parasite and, subsequently, a second one to identify Crithidia.

“We selected these new targets through analyzes of the genomes of the two species. Now that we have shown that they present specificity in different types of samples, it is necessary to optimize the assay so that in a single qPCR reaction we know whether the infecting species in question is Leishmania infantum, Crithidia or both”, says Maruyama.

Currently, the research group has been testing the samples to detect L. infantum and, in a second test, detects Crithidia.

According to the researcher, any diagnostic laboratory equipped with qPCR equipment – ​​which became more accessible during the Covid-19 pandemic – could perform this test. The technique can be useful for epidemiological studies, parasite load monitoring and therapeutic monitoring.

Coinfection cases

During the work, tests were carried out with 62 parasites isolated from the tissues of patients with visceral leishmaniasis. Of these, 51 tested positive for Crithidia. Furthermore, co-infection of Leishmania infantum It is Crithidia in bone marrow was identified in two new cases of VL in Brazil. In May this year, the group published a report of a serious case of VL in which both species of parasites were detected.

According to Maruyama, in addition to the effectiveness of the test, the results indicate that infection by Crithidia It is more common than previously imagined and co-infection by the two protozoa seems to occur mainly in the most severe cases.

“We still don’t know what the clinical implications of the presence of Crithidia in cases of VL, but we suspect that co-infection may exacerbate the disease or even hinder the response to recommended treatment for Leishmania infantum“, says the researcher.

“Identifying the species of parasite correctly allows measures to be taken quickly to prevent the worsening and progression of the clinical condition, reducing mortality. Furthermore, it opens up space for more specific medications and treatments to be discovered in the future.”

The article Parasite Detection in Visceral Leishmaniasis Samples by Dye-Based qPCR Using New Gene Targets of Leishmania infantum and Crithidia can be read at www.mdpi.com/2414-6366/8/8/405.