Dados do Trabalho

Título

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

Introdução

<p>Protozoan parasites of the Leishmaninae subfamily, transmitted by sandflies of the Phebotominae subfamily, cause the tegumentary (TL – subgenera <em>Viannia </em>and <em>Leishmania </em>in America) and visceral (VL – <em>Leishmania </em>in Old World) forms of leishmaniasis, posing significant public health concerns globally. Accurate diagnosis of <em>Leishmania </em>species is necessary to identify vertebrate reservoirs, invertebrate vectors, and affected humans, to establish control strategies, and to treat the disease correctly. Although several <em>Leishmania </em>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 <em>Leishmania </em>through PCR, which is very conserved in the genus <em>Leishmania </em>and absent in the genus <em>Trypanosoma</em>, whose co-circulation results in diagnosis cross-reactions.</p>

Objetivo (s)

<p>To develop a multiplex qPCR assay targeting the GH18 <em>Leishmania </em>chitinase gene for Subgenera differential diagnosis.</p>

Material e Métodos

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

Resultados e Conclusão

<p>The qPCR assays demonstrated successful amplification of the chitinase gene with high specificity from species of <em>Leishmania</em>, and sensibility, allowing for accurate differentiation among the three principal <em>Leishmania </em>Subgenera affecting humans.The developed multiplex qPCR assay offers a promising approach for differential Subgenera diagnosis of <em>Leishmania </em>infections, particularly in regions with diverse <em>Leishmania </em>and <em>Trypanosoma </em>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.</p>

Palavras Chave

Leishmania; leishmaniosis; chitinase; qPCR; diagnosis