title: Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection.
abstract: 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 1h 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.
authors.affiliation: Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
authors.forename: Chukwunonso O
authors.initials: CO
authors.lastname: Nzelu
authors.affiliation: Servicio Nacional de Erradicacion de la Malaria (SNEM), Ministerio de Salud Publica, Guayaquil, Ecuador.
authors.forename: Eduardo A
authors.initials: EA
authors.lastname: Gomez
authors.affiliation: Departamento Académico de Microbiología Médica, Facultad de Medicina Humana, Universidad Nacional Mayor de San Marcos, Lima, Peru; Laboratorio de Entomología, Instituto Nacional de Salud, Lima, Peru.
authors.forename: Abraham G
authors.initials: AG
authors.lastname: Cáceres
authors.affiliation: Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: Departamento de Parasitologia, Instituto Nacional de Higiene y Medicina Tropical 'Leopoldo Izquieta Perez', Guayaquil, Ecuador.
authors.forename: Luiggi
authors.initials: L
authors.lastname: Martini-Robles
authors.affiliation: Department of Dermatology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uezato
authors.affiliation: Department of Microbiology, Faculty of Life Sciences, Graduate School of Health Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Tatsuyuki
authors.initials: T
authors.lastname: Mimori
authors.affiliation: Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Katakura
authors.affiliation: Department of Parasitology, Kochi Medical School, Kochi University, Kochi, Japan; Centro de Biomedicina, Universidad Central del Ecuador, Quito, Ecuador; Prometeo, Secretaría Nacional de Educacion Superior, Ciencia, Tecnologia e Innovacion (SENESCYT), Guayaquil, Ecuador.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Hashiguchi
authors.affiliation: Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan. Electronic address: hkato@vetmed.hokudai.ac.jp.
authors.forename: Hirotomo
authors.initials: H
authors.lastname: Kato
mesh_terms: D000818:Animals; D003124:Colorimetry / Q000379:methods; D004484:Ecuador; D004772:Entomology / Q000379:methods*; D007891:Leishmania / Q000235:genetics / Q000302:isolation & purification*; D008403:Mass Screening / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011576:Psychodidae / Q000469:parasitology*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012394:Rosaniline Dyes / Q000032:analysis; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D012337:RNA, Ribosomal, 18S; D012394:Rosaniline Dyes; C005095:malachite green
keywords: Leishmania; Loop-mediated isothermal amplification (LAMP); Malachite green; Sand fly