Dados do Trabalho

Título

Multiplex Real-Time Taqman PCR differentiates the three principal subgenera of Leishmania

Introdução

Protozoan parasites of the Leishmaninae subfamily, transmitted by sandflies of the Phebotominae subfamily, cause the tegumentary (TL – subgenera Viannia and Leishmania in America) and visceral (VL – Leishmania in Old World) forms of leishmaniasis, posing significant public health concerns globally. Accurate diagnosis of Leishmania species is necessary to identify vertebrate reservoirs, invertebrate vectors, and affected humans, to establish control strategies, and to treat the disease correctly. Although several Leishmania diagnostic methods have been developed, including serological, microscopic observation, and molecular tests such as PCR, there still is no gold standard available. The GH18 chitinase encoding gene has been proven to be useful in identifying Leishmania through PCR, which is very conserved in the genus Leishmania and absent in the genus Trypanosoma, whose co-circulation results in diagnosis cross-reactions.

Objetivo (s)

To develop a multiplex qPCR assay targeting the GH18 Leishmania chitinase gene for Subgenera differential diagnosis.

Material e Métodos

Recombinant clones of GH18 Leishmania chitinase from three species endemic in Brazil (L. infantum, L. mexicana and L. braziliensis) were utilized as controls, and chitinase primers and probes were designed for TaqMan qPCR assays to differentiate three Leishmania subgroups: VL Leishmania (InfDo), New World Leishmania Leishmania (MexAm) and Leishmania Viannia. Sensibility tests were performed by serial dilution of the recombinant clones. For specificity tests, DNA from several species of Leishmania, humans, dogs, and T. cruzi, were employed.  

Resultados e Conclusão

The qPCR assays demonstrated successful amplification of the chitinase gene with high specificity from species of Leishmania, and sensibility, allowing for accurate differentiation among the three principal Leishmania Subgenera affecting humans.The developed multiplex qPCR assay offers a promising approach for differential Subgenera diagnosis of Leishmania infections, particularly in regions with diverse Leishmania and Trypanosoma genera species coexistence. Implementation of this assay could significantly contribute to the control and management of leishmaniasis in endemic regions, including the complex Amazonian environment where multiple species coexist, facilitating targeted interventions and treatment strategies.

Palavras Chave

Leishmania; leishmaniosis; chitinase; qPCR; diagnosis