Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection
Graphical abstract
Loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection.
Introduction
Leishmaniasis is a vector-borne parasitic disease caused by an obligate intracellular protozoan of the genus Leishmania. It is one of the most neglected diseases worldwide, distributed especially in tropical and subtropical areas, and it has strong and complex associations with poverty (Alvar et al., 2006). Leishmania parasites cause three forms of leishmaniases (cutaneous, mucocutaneous and visceral leishmaniasis) depending on the infecting species. The protozoan is transmitted by the bite of an infected female sand fly of the genus Phlebotomus in the Old World and Lutzomyia in the New World (Munstermann, 2004, Kato et al., 2010). The spread of leishmaniasis largely depends on the distribution of sand fly vectors. Approximately 800 sand fly species have been described, but only a few are medically important (Munstermann, 2004, Kato et al., 2010).
The monitoring of natural Leishmania infection in sand fly populations is an important epidemiological parameter for predicting the risk and prevalence of disease, the estimation of which depends on the reliable identification of infected sand flies. Estimation of Leishmania infection rates in the vector could serve as an indicator of a change in transmission intensity at a given endemic area. Such studies have been conducted either by dissecting individual insects and detecting the parasites under a microscope, or polymerase chain reaction (PCR)-based techniques to detect Leishmania DNA in sand flies (Kato et al., 2005). The former is laborious and time-consuming due to the low Leishmania infection rate in sand flies in most endemic areas (Aransay et al., 2000, Kato et al., 2005). The latter method has been proved to have both high sensitivity and specificity; however, the PCR methods require specialized instruments and take several hours, which make their use in resource-limited countries and under field conditions unfeasible. There is therefore a need for a simplified method of amplification and amplicon detection that can complement the available surveillance tools and generate information on the distribution or expansion of the disease.
As an alternative, the loop-mediated isothermal amplification (LAMP) technique may provide an answer to vector monitoring needs. This technique has been proven to be an accurate, rapid and simple method, which amplifies the target nucleic acid under isothermal conditions (Notomi et al., 2000). Recently, wide applicability of LAMP in the detection of parasitic protozoa such as Babesia, Plasmodium, Leishmania and Trypanosoma in clinical samples has been demonstrated (Ikadai et al., 2004, Poon et al., 2006, Njiru et al., 2008, Takagi et al., 2009, Thekisoe et al., 2007, Laohasinnarong et al., 2011). Studies have also shown the application of LAMP to survey vectors of infectious diseases (Aonuma et al., 2009, Thekisoe et al., 2010, Nakao et al., 2010); however, these studies used purified DNA as a template for the LAMP assays. The use of a DNA extraction kit and the time-consuming DNA purification process in template preparation reduces the utility of LAMP technique as a surveillance tool for mass screening of infected vectors with pathogens in endemic regions. To date, no reports have been available on the use of the LAMP method for detection of Leishmania-infected sand flies. Considering these points, we developed and validated a novel LAMP assay for mass screening of sand fly vectors for Leishmania infection. In this study, a newly developed LAMP method for rapid detection of the Leishmania DNA within sand fly vectors is described, in addition to the use of a non-fluorescent cationic dye, namely, malachite green (MG) for the first time as a simpler colorimetric assay for the detection of LAMP reactions. The present method was evaluated with field-captured sand fly samples in order to demonstrate its efficacy and reliability as an important potential tool in assessing Leishmania infection and/or transmission intensity in endemic areas of Ecuador.
Section snippets
Sand fly collection
Wild sand flies were collected in March, July and August 2012 in Andean areas of Ecuador; Alausi, Chanchan and Huigra (Province of Chimborazo), where Andean-type cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana is endemic (Kato et al., 2005, Kato et al., 2008). Sand flies were caught using protected human bait, CDC light traps and Shannon traps. The collected sand flies were dissected and then identified at the species level mainly based on the morphology of their spermathecae
Sensitivity and specificity of LAMP
Successful amplification reactions with I8S rRNA Leishmania LAMP primers (Table 1) were achieved in sample tubes containing each target DNA. The LAMP results were identical when a thermal-cycler, water bath and the heating block were used as the source of heat. The sensitivities of the broad range LAMP assay were assessed with serial dilutions of L. (L.) mexicana and L. (L.) major-like DNA (104 to 0.01 parasites). The LAMP assay was able to detect all the broad range concentrations tested,
Discussion
Monitoring of infection in the vector sand fly population is an essential tool for surveillance of infections and identification of the vector species. The mass screening of sand flies for Leishmania infection has the potential to provide insight into the current transmission of the infection in endemic areas. In this study, we demonstrated the detection of Leishmania DNA within sand fly vectors using a newly developed LAMP method based on the 18S rRNA gene. The LAMP method is rapid, reliable
Acknowledgments
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.
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