Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1. h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)-mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.
Bibliographical noteFunding Information:
We are indebted to Kazue Hashiguchi (Centro de Biomedicina, Universidad Central del Ecuador), Flavio-Valeriano Zambrano C. (Servicio Nacional de Erradicacion de la Malaria, Guayaquil, Ecuador) and Roberto Sud A. (Ministerio de Salud Publica y Asistencia Social, Guayaquil, Ecuador) for their technical assistance during the field phase of the present study. This study was financially supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Grant nos. 25257501 and 23256002 ), the Program for Leading Graduate Schools “Fostering Global Leaders in Veterinary Science for Contributing to One Health” (FO1) , MEXT, Japan , and the Prometeo Project of the Secretaria Nacional de Educacion Superior, Ciencia, Tecnologia e Innovacion (SENESCYT), Ecuador .
- Loop-mediated isothermal amplification (LAMP)
- Malachite green
- Sand fly