Establishment of a Mass Screening Method of Sand Fly Vectors for Leishmania Infection by Molecular Biological Methods

Abstract

Surveillance of the prevalence of Leishmania and its vector, sand fly species, in endemic and surrounding areas is important for prediction of the risk and expansion of leishmaniasis. In this study, a method for the mass screening of sand flies for Leishmania infection was established. This method was applied to 319 field-captured specimens, and 5 positive sand flies were detected. Sand fly species were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the18S rRNA gene, and all the positive flies were Lu. hartmanni. Furthermore, cytochrome b (Cyt b) gene sequence analyses identified all the parasites as Endotrypanum species including a probable novel species. Because the method requires minimum effort and can process a large number of samples at once, it will be a powerful tool for studying the epidemiology of leishmaniasis.

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

“…DNA from Leishmania-related parasites capable of infecting sand flies, such as Endotrypanum (Kato et al, 2007) and anuran Trypanosoma species (Ferreira et al, 2008), and also DNA from the sand flies Phlebotomus papatasi and Lutzomyia ayacuchensis, were included to evaluate the present LAMP primer specificity.…”

“…Conventional PCR was performed with previously described primers specific for Leishmania minicircle kinetoplast DNA (mkDNA) (Kato et al, 2005(Kato et al, , 2007. The primer sequences used for amplification were 5´ -CTRGGGGTTGGTGTAAAATAG-3´ (L.MC-1S) and 5´ -TWTGAACGGGRTTTCTG-3´ (L.MC-1R) (Kato et al, 2005).…”

“…To identify sand fly by PCR-restriction fragment length polymorphism (PCR-RFLP), PCR amplification with Lutzomyia 18S rRNA gene-specific primers and subsequent digestion with the Afa I enzyme were performed as previously described (Kato et al, 2007. …”

“…The LAMP products were purified using a PCR purification kit (Qiagen, Valencia, CA) and then sequenced with FIP and BIP primers (Table 1) CTACAATAAACAAATCATAATATRCAATT-3´ using Ampdirect Plus (Shimadzu Biotech, Tsukuba, Japan) as previously described (Kato et al 2005(Kato et al , 2007. The products were cloned into the pGEM-T Easy Vector System (Promega, Madison, WI) and sequences were determined by the dideoxy chain termination method using a BigDye Terminator Cycle-Sequencing Kit (Applied Biosystems).…”

“…LAMP amplicons were directly detected in 8 samples reports from the same areas (Kato et al, 2005. In addition, based on PCR-RFLP analysis of sand fly 18S rRNA with Afa I endonuclease digestion (Kato et al, 2007, all the LAMPpositive flies were identified as Lutzomyia ayacuchensis (Table 2), a proven and sole vector of L.…”