Dados do Trabalho

Título

Rapid Detection of Leishmania spp. in small mammals from Corumbá – MS by point-of-care molecular Diagnosis

Introdução

Several species of wild and domestic mammals are important reservoirs of Leishmania spp. Although leishmaniasis cases have been widely reported in Mato Grosso do Sul, its prevalence in wild hosts in rural settlements in Corumbá is still unknown. Studies on the occurrence of this parasite in different hosts and geographic regions are essential for the development of surveillance and control strategies for visceral and tegumentary leishmaniasis, mainly in border areas.

Objetivo (s)

To determine the occurrence of Leishmania spp. in small mammals by isothermal amplification mediated by loop (LAMP), using the 18S rRNA region as the target, in an endemic area for leishmaniasis.

Material e Métodos

In July 2022, we used live-trap to capture small mammals, placed on trails with a final effort of 800 traps per night, in the Paraguay sub-region of Pantanal, in the municipality of Corumbá, in a rural settlement that borders Bolívia. The individuals were euthanized and spleen samples were collected and subjected to DNA extraction with the DNeasy Blood and Tissue kit (QIAGEN, Maryland, USA). Detection of Leishmania spp. 18S rRNA gene by LAMP was performed with a set of primers described by Sriworarat et al., 2015, with changes. The reactions were performed in a final volume of 25µL, containing 1X isothermal buffer; 6mM MgSO4; 1.4mM dNTP, 40 pmol FIP and BIP; 5 pmol of F3 and B3 and 8U of Bst DNA polymerase, incubated for 60 minutes at 65°C. Colorimetric detection was performed by adding 1µL SybrGreen® I (1:10) to the reaction after amplification. To confirm the presence of Leishmania DNA, conventional PCR was performed with F3 and B3 primers, and the products were sent for sequencing.

Resultados e Conclusão

Fourteen animals were captured, divided into three species of marsupials: Gracilinanus agilis (06/14), Marmosa micoureus budini (06/14), Marmosops sp. (01/14) and a rodent, Callomys callosus (01/14). The occurrence of infected animals was 12/14 (85%) positive for Leishmania spp. via LAMP. The amplified products were sequenced and showed 100% identity with 18SrDNA sequences from L. infantum. These results suggest a high prevalence of Leishmania spp. in this area and suggest that LAMP can be used as a good point-of-care technique for diagnosing and monitoring leishmaniasis, in addition to being readily applicable for screening populations of wild animals infected by Leishmania in endemic areas.

Palavras-chave

Diagnosis; Isothermal Amplification; Monitoring

Agradecimentos

CAPES; CNPq(407856/2021-8); FAPERJ-JCNE(E-26/201.287/2022); Centelha 2/FUNDECT-MS/FINEP