Application of RFLP-PCR-Based Identification for Sand Fly Surveillance in an Area Endemic for Kala-Azar in Mymensingh, Bangladesh

Mymensingh is the most endemic district for kala-azar in Bangladesh. Phlebotomus argentipes remains the only known vector although a number of sand fly species are prevalent in this area. Genotyping of sand flies distributed in a VL endemic area was developed by a PCR and restriction-fragment-length polymorphism (RFLP) of 18S rRNA gene of sand fly species. Using the RFLP-PCR analysis with AfaI and HinfI restriction enzymes, P. argentipes, P. papatasi, and Sergentomyia species could be identified. Among 1,055 female sand flies successfully analyzed for the species identification individually, 64.4% flies was classified as Sergentomyia species, whereas 35.6% was identified as P. argentipes and no P. papatasi was found. Although infection of Leishmania within the sand flies was individually examined targeting leishmanial minicircle DNA, none of the 1,055 sand flies examined were positive for Leishmania infection. The RFLP-PCR could be useful tools for taxonomic identification and Leishmania infection monitoring in endemic areas of Bangladesh. 1. Introduction Phlebotomine sand flies are blood-sucking insects belonging to the family Psychodidae in the order Diptera [1]. Hitherto, approximately 800 sand fly species have been recorded; however, the majority of the species play no part in the transmission of leishmaniasis in nature, and less than 10% of sand flies has been incriminated as vector species of leishmaniasis [1–3]. In addition, each vector species can only support the development of specific Leishmania species and consequently can only transmit certain species of the genus [2]. Thus, surveillance of prevalent sand fly species and Leishmania infection within sand flies in each endemic area is important for prediction of the risk of transmission and expansion of the disease. Kala-azar (visceral leishmaniasis: VL) caused by Leishmania donovani complex is the most severe form of leishmaniasis and the second-largest parasitic killer in the world with an estimated 500,000 new cases and more than 50,000 deaths each year. More than 90% of the world’s cases of VL is in India, Bangladesh, Nepal, Sudan, and Brazil, affecting the poorest of poor people [4]. Phlebotomus argentipes has been implicated as the only vector for VL in the north-east Indian subcontinent (part of India, Bangladesh, Nepal) [5, 6]. In Bangladesh, Mymensingh district is the most highly endemic areas of VL and accounted for more than 50% of the total cases reported from 2000 to 2004 [7]. However, except for one particular instance [8], information on distributing sand fly species and vector