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Int J Environ Res Public Health. 2010 March; 7(3): 814–826.
Published online 2010 March 5. doi:  10.3390/ijerph7030814
PMCID: PMC2872317

Molecular Epidemiology for Vector Research on Leishmaniasis

Abstract

Leishmaniasis is a protozoan disease caused by the genus Leishmania transmitted by female phlebotomine sand flies. Surveillance of the prevalence of Leishmania and responsive vector species in endemic and surrounding areas is important for predicting the risk and expansion of the disease. Molecular biological methods are now widely applied to epidemiological studies of infectious diseases including leishmaniasis. These techniques are used to detect natural infections of sand fly vectors with Leishmania protozoa and are becoming powerful tools due to their sensitivity and specificity. Recently, genetic analyses have been performed on sand fly species and genotyping using PCR-RFLP has been applied to the sand fly taxonomy. In addition, a molecular mass screening method has been established that enables both sand fly species and natural leishmanial infections to be identified simultaneously in hundreds of sand flies with limited effort. This paper reviews recent advances in the study of sand flies, vectors of leishmaniasis, using molecular biological approaches.

Keywords: Leishmania, sand fly, epidemiology, mass screening

1. Leishmaniasis

Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania, which is further divided into two subgenera, Leishmania and Viannia. Leishmania protozoa are transmitted by the bite of the female phlebotomine sand fly [1,2]. The disease is widely distributed around the world especially in tropical and subtropical areas, affecting at least 12 million people in 88 countries, with another 350 million people at risk [1,2]. Approximately 20 Leishmania species are known to be pathogenic to humans, and the species is the major determinant of clinical outcome (cutaneous, mucocutaneous and visceral forms) [13].

Cutaneous leishmaniasis (CL), known as “Oriental Sores” and “Baghdad Boils” in the Old World and “Chiclero’s Ulcer” and “Uta” in the New World, is caused by various Leishmania species including Leishmania (Leishmania) major and L. (L.) tropica in the Old World and L. (L.) mexicana, L. (L.) amazonensis, L. (Viannia) braziliensis, L. (V.) guyanensis and L. (V.) peruviana in the New World [1,2,4]. Typical CL is characterized by localized refractory skin ulcers or nodules at sites of infection that heal spontaneously leaving life-long scars [1,2]. Other unusual types of CL include leishmaniasis recidiva cutis (LRC) characterized by the development of satellite nodules in or around the scar of a clinically healed lesion after a period of time, diffuse cutaneous leishmaniasis (DCL) producing non-ulcerating chronic nodules over the entire body resembling skin lesions of lepromatous leprosy, and post kala-azar dermal leishmaniasis (PKDL), which can appear on the skin of individuals who have recovered from visceral leishmaniasis (VL) [1,2]. Some 90% of CL cases are reported to occur in just seven countries; Afghanistan, Algeria, Iran, Saudi Arabia, Syria, Brazil and Peru [2].

Mucocutaneous leishmaniasis (MCL), also known as “Espundia”, is endemic in Central and South America, and characterized by destructive metastatic lesions in the mucous membranes of the nose, mouth and throat cavities and surrounding tissues that occur months or years after the onset of the primary cutaneous infection [5]. Although the principal causative agent of MCL is L. (V.) braziliensis, other Leishmania species such as L. (V.) guyanensis, L. (V.) panamensis and L. (L.) amazonensis have been reported to affect mucosal tissues [68].

VL, caused by L. (L.) donovani complex, is also known as “Kala azar” or “Dum dum fever”. VL is the most severe form of leishmaniasis, and the clinical symptoms include irregular bouts of fever, substantial weight loss, fatigue, anemia and substantial swelling of the liver and spleen [9]. The disease is usually fatal if untreated, and is the second-largest parasitic killer in the world with an estimated 500,000 new cases and more than 50,000 deaths each year [2]. Some 90% of VL cases occur in just five countries; Bangladesh, India, Nepal, Sudan and Brazil [2].

Most leishmaniases are zoonotic diseases that include animal reservoir hosts in the transmission cycle [1]. Humans are considered to be an accidental host, although anthroponotic transmission without animal reservoirs is reported in some Leishmania species [1] (Figure 1).

Figure 1.
Schematic life cycle of Leishmania parasites. 1. The sand fly ingests amastigotes during blood feeding. 2. Amastigotes transform into promastigotes. 3. Promastigotes colonize and multiply at the hindgut and midgut of the sand fly. 4. Infective promastigotes ...

The life cycle of Leishmania species involves two stages; in mammalian hosts, the parasites are observed as an intracellular amastigote with a round or ovoid-shaped and immotile form, but when taken up into the gut of sand flies, they become an extracellular promastigote characterized by a spindle-shaped and motile form with an external flagellum [10]. The transformation is triggered by a change in conditions such as temperature and pH [11,12]. The main target of the parasite in mammalian hosts is macrophages, and infections are known to occur in three ways; (1) direct infection, (2) phagocytosis of infected neutrophils by macrophages (the Trojan horse model), and 3) silent infection by parasites released from apoptotic neutrophils [13]. Neutrophils, which are recruited to sites of tissue damage caused by sand fly bites in the initial phase, are considered to ingest the majority of parasites and play a central role in the establishment of the Leishmania infection [1318] (Figure 1).

2. Phlebotomine Sand Flies as a Vector of Leishmaniasis

Phlebotomine sand flies are tiny blood-feeding insects of the family Psychodidae in the order Diptera with a body length of approximately 2–3 mm [19]. They vary in color from silver-gray to almost black, and fold their wings into a characteristic V-shape when at rest (Figure 2). They are active nocturnally and rest in houses, cellars, caves and gaps among rocks in the daytime. Unlike mosquitoes, they fly feebly and silently, and sneak up on a host using a peculiar hopping motion [20]. Only female flies suck blood for egg production, and the bites are typically painful. To date, approximately 800 sand fly species have been recorded in five major genera; Phlebotomus (94 species) and Sergentomyia (258 species) in the Old World, and Lutzomyia (379 species), Brumptomyia (23 species) and Warileya (5 species) in the New World, and of these, proven vector species of Leishmania protozoa are classified into the genus Phlebotomus and Lutzomyia [20]. The majority of the species play no part in the transmission of leishmaniasis in nature for several reasons; they may not feed on blood from humans and potential reservoir animals, and/or they may be incapable of supporting the development of Leishmania species [20,21]. Less than 10% of sand flies have been incriminated as vector species of leishmaniasis, and only about 30 species have been demonstrated to have a vectorial capacity [10]. In addition, each vector species can only support the development of and consequently transmit certain species of Leishmania [10,19]. In the susceptible vector, the promastigotes of Leishmania attach to the epithelium of the gut, multiply, and differentiate into the infective metacyclic form, which is transmitted to the mammalian host [21,22] (Figure 1). The attachment of the promastigotes to the insect midgut is crucial to the completion of their life cycle in order to avoid excretion when the sand fly defecates [21,22]. The attachment is mediated by lipophosphoglycan (LPG), the major surface glycoconjugate of promastigotes, and the structures are polymorphic among species, suggesting that LPG is the major determinant specifying a vector species [22,23]. The only midgut protein of sand flies shown to interact with Leishmania is PpGalec, a β-galactoside-binding lectin, found in Phlebotomus (P.) papatasi, a principal vector of L. (L.) major in the Old World [24]. Genomic DNA hybridized with PpGalec was present in P. papatasi and P. duboscqi, both of which transmit L. (L.) major in nature, but absent in P. sergenti and P. argentipes, vector species of L. (L.) tropica and L. (L.) donovani, respectively, in the Old World, and Lutzomyia (Lu.) longipalpis and Lu. verrucarum which transmit L. (L.) infantum (previously called L. (L.) chagasi) and L.(V.) peruviana, respectively, in the New World [24]. The result strongly suggests that midgut molecules of sand flies are the key determinant of vectorial competency.

Figure 2.
A blood-sucking phlebotomine sand fly.

3. Advances in Sand Fly Taxonomy

As described, only some of the approximately 800 sand fly species are medically important, and certain sand fly species can transmit only certain species of Leishmania [21,22]. Since the spread of leishmaniases largely depends on the distribution of the vectors, the identification of circulating sand fly species in endemic and surrounding areas is important for predictions of the risk and expansion of the disease. Sand flies are generally identified as adults based on morphologic characteristics, mainly internal structures such as the spermatheca, cibarium and pharynx in females, and terminal genitalia in males [19,20]. Other characteristics include the location and intensity of pigmentation of the thorax, and the length ratios of wing veins, and antennal and leg segments [19,20]. Approximately 90 characteristics have been demonstrated as effective descriptors, and these characters are examined and measured on each specimen under a microscope after appropriate preservation and mounting [19,20]. Thus, the morphological classification requires considerable skill as well as taxonomic expertise. In addition, the presence of intraspecific variation and cryptic species frequently complicates classifications based on morphological features [25]. Furthermore, damage caused by improper storage and mounting makes the process more difficult or can cause misidentification. Therefore, other characteristics like molecular markers have been explored for the development of simpler and more accurate ways to identify sand flies. Several genetic markers have been used to examine the systematics, relationships and evolution among sand fly species and for population analyses within species [2652]. Most results of genetic analyses support the generally accepted classification based on morphological characteristics, however, discrepancies exist between the two classifications in several groupings, suggesting the necessity for careful reconsideration of the sand fly taxonomy [42,51,52]. With the accumulation of genetic data on sand flies, attempts have been made to establish methods of identification using simple techniques such as the PCR-restriction fragment length polymorphism (RFLP) analysis of 18S rRNA genes [33,4547,50,51]. Although genetic diversity affecting RFLP-patterns was found in some species, the genotyping method was shown to be accurate and easy-to-use for the identification of sand fly species, requiring less expertise and with less risk of different interpretations among researchers than the conventional morphology-based classification [33,4547,50,51]. It is important to note that this DNA-based technique doesn’t require special storage conditions for the specimens, and different methods of preservation such as drying and the use of 70% ethanol or liquid nitrogen did not affect the quality of the results [33]. In addition, damage to samples, which affects the morphologic classification in many cases, does not affect the PCR-RFLP analysis. To date, approximately 400 Lutzomyia and 100 Phlebotomus species have been recorded, and it is impossible to distinguish them all with a PCR-RFLP-based method. Usually, however, not many species coexist in an endemic area. Therefore, the PCR-RFLP-based method targeting several different genes will be a powerful tool for sand fly research as well as studying taxonomy in given leishmaniasis-endemic areas.

Interestingly, several studies suggested differences in vectorial capacity among populations within vector species such as mosquitoes [53,54]. The population structure has been analyzed in the Old World species P. papatasi and P. sergenti [38,39] and in the New World species Lu. longipalpis and recently Lu. hartmanni and Lu. ayacuchensis [25,52,5560]. Heterogeneity of transcribed spacer (ITS) 2 sequences among populations was reported in P. sergenti, the main vector of L. (L.) tropica, and an association with vectorial capacity was hypothesized [38]. In addition, remarkable genetic variation among populations was also found in Lu. longipalpis, the principal vector of L. (L.) infantum (=L. (L.) chagasi), on the basis of the mitochondria ND4 gene [55]. On the other hand, an absence of geographic distribution for both ITS2 and mitochondria ND4 genes was reported in P. papatasi, the principal vector of L. (L.) major [39], and for ITS1 and ITS2 genes in both Lu. hartmanni and Lu. ayacuchensis, which are the vector species of L. (V.) panamensis and L. (L.) mexicana, respectively [52]. The genetic heterogeneity described in P. sergenti and Lu. longipalpis may be associated with morphological variation that is observed in the two species but not in Lu. hartmanni, Lu. ayacuchensis and P. papatasi [39,52,59]. More recently, a correlation between microsatellite markers and geographical origin was described in P. papatasi, suggesting multi-locus microsatellite typing to be effective for population analysis in sand flies [61]. Thus, further extensive study should disclose markers appropriate for population genetics in sand flies, resulting in the elucidation of vectorial capacity among population structures.

4. Advances in the Detection and Identification of Leishmania Species within Naturally Infected Sand Flies

Detection and identification of Leishmania species within sand flies are important for predictions of the risk and expansion of the disease in endemic and surrounding areas since the infected species is the major determinant of the clinical outcome in humans [13]. The infection of sand flies with Leishmania promastigotes has been examined by the dissection of individual sand flies under a microscope. For this purpose, specimens need to be fresh, and the dissection of tiny sand flies requires a highly skilled technique. The procedure takes a relatively long time, and additionally, a large number of specimens have to be examined to obtain informative data for each area since the infection rate of sand flies with Leishmania is generally very low (0.01–1%) even in endemic areas [62,63]. To improve on conventional methods, several PCR-based techniques have been developed [45,47,6473], and infection of Leishmania species within sand flies was identified, e.g., L. (L.) major in P. papatasi [65], L. (L.) infantum in P. major [69] and L. (L.) donovani in P. argentipes [72] in the Old World, and L. (L.) mexicana in Lu. ayacuchensis [45,47] and Lu. ovallesi [66], L. (L.) amazonensis in Lu. longipalpis [67,73], L. (L.) infantum chagasi in Lu. longipalpis [67] and Lu. almerioi [73], L. peruviana in Lu. peruensis [47], L. (V.) naiffi in Lu. tortura [48] and L. (V.) braziliensis in Lu. ovallesi [66], Lu. gomezi [66] and Lu. neivai [70] in the New World. In most studies, DNA was extracted from pooled sand fly samples or from individual sand flies by use of an expensive kit or by a complicated conventional protocol using proteinase K-containing DNA extraction buffer followed by phenol/chloroform extraction and ethanol precipitation. These methods are sensitive enough to detect Leishmania species within sand flies. However, several improvements were desirable for the analysis of a large number of sand flies with less effort and cost. In addition, it is better to analyze sand flies individually because several species co-exist in most endemic areas and the use of pooled samples may spoil important information on vector epidemiology such as the prevalent sand fly species and the relationships between Leishmania and vector species. Recently, a method of mass screening sand flies for Leishmania infection was established, and its usability for field research confirmed [46,47]. The protocol is represented in Figure 3. With this method, 96 individual samples can be analyzed at once with few processes. The method has a number of advantages: 1) purification of DNA is not required; that is, ethanol-fixed sand fly samples lysed in conventional DNA extraction buffer overnight at 37 °C without homogenization can be directly used as PCR templates, 2) the sensitivity and specificity are very high; leishmanial DNA is detectable if only one parasite exists in a sample, 3) data on individual sand flies can be obtained, 4) there is minimum risk of DNA loss and contamination among samples because of few processes, and 5) each sample can be used as a template for 100–150 PCRs. In addition, the sand fly species can be identified by molecular biological methods such as PCR-RFLP using the same template DNA if some genetic information is available on the prevalent sand fly species in the given endemic areas. Further, Leishmania species detected within sand flies can be identified by the analysis of leishmanial genes such as the mitochondrial cytochrome b gene [4548,7476]. Thus, application of the mass screening method in different areas will provide important information on risk factors, hopefully leading to the control and/or surveillance of leishmaniasis.

Figure 3.
A molecular mass screening method for the detection of Leishmania within individual sand flies and identification of both sand fly and Leishmania species.

5. Concluding Remarks

Molecular biological techniques are now becoming powerful tools for sand fly research. Therefore, more detailed information on the risk factors for leishmaniasis, such as the prevalent sand fly species as well as the seasonal variation in the infection rate and transmission risk, can be accumulated by continuous efforts using such techniques in various endemic areas in different seasons. In addition, elucidation of the relationships between Leishmania and vector species, which requires enormous effort with current methods, by use of molecular methods mentioned, will contribute to not only epidemiological research on leishmaniasis but also basic studies on parasite-vector interactions.

References

1. Desjeux P. The increase of risk factors for leishmaniasis worldwide. Trans. R. Soc. Trop. Med. Hyg. 2001;95:239–243. [PubMed]
2. Desjeux P. Leishmaniasis: current situation and new perspectives. Comp. Immunol. Microbiol. Infect. Dis. 2004;27:305–318. [PubMed]
3. Choi CM, Lerner EA. Leishmaniasis as an emerging infection. J. Invest. Dermatol. Symp. Proc. 2001;6:175–182. [PubMed]
4. Grimaldi G, Jr, Tesh RB, McMahon-Pratt D. A review of the geographic distribution and epidemiology of leishmaniasis in the New World. Am. J. Trop. Med. Hyg. 1989;41:687–725. [PubMed]
5. Marsden PD. Mucosal leishmaniasis (“espundia” Escomel, 1911) Trans. R. Soc. Trop. Med. Hyg. 1986;80:859–876. [PubMed]
6. Naiff RD, Talhari S, Barrett TV. Isolation of Leishmania guyanensis from lesions of the nasal mucosa. Mem. Inst. Oswaldo Cruz. 1988;83:529–530. [PubMed]
7. Barral A, Pedral-Sampaio D, Grimaldi G, Jr, Momen H, McMahon-Pratt D, Ribeiro de Jesus A, Almeida R, Badaro R, Barral-Netto M, Carvalho EM, Johnson WD., Jr Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. Am. J. Trop. Med. Hyg. 1991;44:536–546. [PubMed]
8. Osorio LE, Castillo CM, Ochoa MT. Mucosal leishmaniasis due to Leishmania (Viannia) panamensis in Colombia: clinical characteristics. Am. J. Trop. Med. Hyg. 1998;59:49–52. [PubMed]
9. Bryceson ADM. Leishmaniasis. In: Cook CC, editor. Manson’s Tropical Disease. 20th ed. W.B. Saunders Comp. Ltd; London, Philadelphia, Tronto, Sydney and Tokyo: 1996. pp. 1213–1245.
10. Bates PA. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int. J. Parasitol. 2007;37:1097–1106. [PMC free article] [PubMed]
11. Bates PA, Rogers ME. New insights into the developmental biology and transmission mechanisms of Leishmania. Curr. Mol. Med. 2004;4:601–609. [PubMed]
12. Kamhawi S. Phlebotomine sand flies and Leishmania parasites: friends or foes? Trends Parasitol. 2006;22:439–445. [PubMed]
13. Peters NC, Sacks DL. The impact of vector-mediated neutrophil recruitment on cutaneous leishmaniasis. Cell. Microbiol. 2009;11:1290–1296. [PMC free article] [PubMed]
14. Laskay T, van Zandbergen G, Solbach W. Neutrophil granulocytes—Trojan horses for Leishmania major and other intracellular microbes? Trends Microbiol. 2003;11:210–214. [PubMed]
15. Ribeiro-Gomes FL, Otero AC, Gomes NA, Moniz-De-Souza MC, Cysne-Finkelstein L, Arnholdt AC, Calich VL, Coutinho SG, Lopes MF, DosReis GA. Macrophage interactions with neutrophils regulate Leishmania major infection. J. Immunol. 2004;172:4454–4462. [PubMed]
16. van Zandbergen G, Klinger M, Mueller A, Dannenberg S, Gebert A, Solbach W, Laskay T. Cutting edge: neutrophil granulocyte serves as a vector for Leishmania entry into macrophages. J. Immunol. 2004;173:6521–6525. [PubMed]
17. Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, Kamhawi S, Lawyer P, Fay MP, Germain RN, Sacks D. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science. 2008;321:970–974. [PMC free article] [PubMed]
18. Jochim RC, Teixeira C. Leishmania commandeers the host inflammatory response through neutrophils. Trends Parasitol. 2009;25:145–147. [PubMed]
19. Young DG, Duncan MA.Guide to the identification and geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae), Memoirs of the American Entomological Institute, 54, Associated Publishers—American Entomological Institute, Gainsville, FL, USA, 1994
20. Munstermann LE. Phlebotomine sand flies, the Psychodidae. In: Marquardt WC, Black WC, Freier JE, Hagedorn HH, Hemingway J, Higgs S, James AA, Kondratieff B, Moore CG, editors. Biology of Disease Vectors. 2nd ed. Elsevier; San Diego, CA, USA: 2004. pp. 141–151.
21. Killick-Kendrick R. The biology and control of phlebotomine sand flies. Clin. Dermatol. 1999;17:279–289. [PubMed]
22. Sacks DL. Leishmania-sand fly interactions controlling species-specific vector competence. Cell. Microbiol. 2001;3:189–196. [PubMed]
23. Sacks DL, Modi G, Rowton E, Späth G, Epstein L, Turco SJ, Beverley SM. The role of phosphoglycans in Leishmania-sand fly interactions. Proc. Natl. Acad. Sci. USA. 2000;97:406–411. [PubMed]
24. Kamhawi S, Ramalho-Ortigao M, Pham VM, Kumar S, Lawyer PG, Turco SJ, Barillas-Mury C, Sacks DL, Valenzuela JG. A role for insect galectins in parasite survival. Cell. 2004;119:329–341. [PubMed]
25. Bauzer LG, Souza NA, Maingon RD, Peixoto AA. Lutzomyia longipalpis in Brazil: a complex or a single species? A mini-review. Mem. Inst. Oswaldo Cruz. 2007;102:1–12. [PubMed]
26. Ready PD, Smith DF, Killick-Kendrick R. DNA hybridizations on squash-blotted sandflies to identify both Phlebotomus papatasi and infecting Leishmania major. Med. Vet. Entomol. 1988;2:109–116. [PubMed]
27. Ready PD, Lainson R, Shaw JJ, Souza AA. DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera:Psychodidae) Mem. Inst. Oswaldo Cruz. 1991;86:41–49. [PubMed]
28. Ready PD, de Souza AA, Rebelo JM, Day JC, Silveira FT, Campbell-Lendrum D, Davies CR, Costa JM. Phylogenetic species and domesticity of Lutzomyia whitmani at the southeast boundary of Amazonian Brazil. Trans. R. Soc. Trop. Med. Hyg. 1998;92:159–160. [PubMed]
29. Booth DR, Ready PD, Smith DF. Retrotransposons and evolution in phlebotomines. Parassitologia. 1991;33:105–112. [PubMed]
30. Booth DR, Ready PD, Smith DF. Isolation of non-LTR retrotransposon reverse transcriptase-like sequences from phlebotomine sandflies. Insect Mol. Biol. 1994;3:89–96. [PubMed]
31. Booth DR, Ready PD, Smith DF. Evolution of multiple families of non-LTR retrotransposons in phlebotomine sandflies. Genet. Res. 1996;67:227–237. [PubMed]
32. Esseghir S, Ready PD, Killick-Kendrick R, Ben-Ismail R. Mitochondrial haplotypes and phylogeography of Phlebotomus vectors of Leishmania major. Insect Mol. Biol. 1997;6:211–225. [PubMed]
33. Aransay AM, Scoulica E, Chaniotis B, Tselentis Y. Typing of sand flies from Greece and Cyprus by DNA polymorphism of 18S rRNA gene. Insect Mol. Biol. 1999;8:179–184. [PubMed]
34. Aransay AM, Scoulica E, Tselentis Y, Ready PD. Phylogenetic relationships of phlebotomine sandflies inferred from small subunit nuclear ribosomal DNA. Insect Mol. Biol. 2000;9:157–168. [PubMed]
35. Aransay AM, Ready PD, Morillas-Marquez F. Population differentiation of Phlebotomus perniciosus in Spain following postglacial dispersal. Heredity. 2003;90:316–325. [PubMed]
36. Campbell-Lendrum DH, Brandão-Filho SP, Pinto MC, Vexenat A, Ready PD, Davies CR. Domesticity of Lutzomyia whitmani (Diptera: psychodidae) populations: field experiments indicate behavioural differences. Bull. Entomol. Res. 2000;90:41–48. [PubMed]
37. Di Muccio T, Marinucci M, Frusteri L, Maroli M, Pesson B, Gramiccia M. Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, psychodidae) by ITS2 rDNA sequences. Insect Biochem. Mol. Biol. 2000;30:387–393. [PubMed]
38. Depaquit J, Ferté H, Léger N, Lefranc F, Alves-Pires C, Hanafi H, Maroli M, Morillas-Marquez F, Rioux JA, Svobodova M. Volf, P. ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera, Psychodidae): possible consequences in their ability to transmit Leishmania tropica. Int. J. Parasitol. 2002;32:1123–1131. [PubMed]
39. Depaquit J, Lienard E, Verzeaux-Griffon A, Ferté H, Bounamous A, Gantier JC, Hanafi HA, Jacobson RL, Maroli M, Moin-Vaziri V, Müller F, Ozbel Y, Svobodova M, Volf P, Léger N. Molecular homogeneity in diverse geographical populations of Phlebotomus papatasi (Diptera, Psychodidae) inferred from ND4 mtDNA and ITS2 rDNA epidemiological consequences. Infect. Genet. Evol. 2008;8:159–170. [PubMed]
40. Testa JM, Montoya-Lerma J, Cadena H, Oviedo M, Ready PD. Molecular identification of vectors of Leishmania in Colombia: mitochondrial introgression in the Lutzomyia townsendi series. Acta Trop. 2002;84:205–218. [PubMed]
41. Torgerson DG, Lampo M, Velazquez Y, Woo PT. Genetic relationships among some species groups within the genus Lutzomyia (Diptera: Psychodidae) Am. J. Trop. Med. Hyg. 2003;69:484–493. [PubMed]
42. Beati L, Caceres AG, Lee JA, Munstermann LE. Systematic relationships among Lutzomyia sand flies (Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA sequences. Int. J. Parasitol. 2004;34:225–234. [PubMed]
43. Yahia H, Ready PD, Hamdani A, Testa JM, Guessous-Idrissi N. Regional genetic differentiation of Phlebotomus sergenti in three Moroccan foci of cutaneous leishmaniasis caused by Leishmania tropica. Parasite. 2004;11:189–199. [PubMed]
44. Pesson B, Ready JS, Benabdennbi I, Martín-Sánchez J, Esseghir S, Cadi-Soussi M, Morillas-Marquez F, Ready PD. Sandflies of the Phlebotomus perniciosus complex: mitochondrial introgression and a new sibling species of P. longicuspis in the Moroccan Rif. Med. Vet. Entomol. 2004;18:25–37. [PubMed]
45. Kato H, Uezato H, Katakura K, Calvopiña M, Marco JD, Barroso PA, Gomez EA, Mimori T, Korenaga M, Iwata H, Nonaka S, Hashiguchi Y. Detection and identification of Leishmania species within naturally infected sand flies in the Andean areas of Ecuador by a polymerase chain reaction. Am. J. Trop. Med. Hyg. 2005;72:87–93. [PubMed]
46. Kato H, Uezato H, Gomez EA, Terayama Y, Calvopiña M, Iwata H, Hashiguchi Y. Establishment of a mass screening method of sand fly vectors for Leishmania infection by molecular biological methods. Am. J. Trop. Med. Hyg. 2007;77:324–329. [PubMed]
47. Kato H, Cáceres AG, Gomez EA, Mimori T, Uezato H, Marco JD, Barroso PA, Iwata H, Hashiguchi Y. Molecular mass screening to incriminate sand fly vectors of Andean-type cutaneous leishmaniasis in Ecuador and Peru. Am. J. Trop. Med. Hyg. 2008;79:719–721. [PubMed]
48. Kato H, Gomez EA, Yamamoto Y, Calvopiña M, Guevara AG, Marco JD, Barroso PA, Iwata H, Hashiguchi Y. Natural infection of Lutzomyia tortura with Leishmania (Viannia) naiffi in an Amazonian area of Ecuador. Am. J. Trop. Med. Hyg. 2008;79:438–440. [PubMed]
49. Barón S, Martín-Sánchez J, Gállego M, Morales-Yuste M, Boussaa S, Morillas-Márquez F. Intraspecific variability (rDNA ITS and mtDNA Cyt b) of Phlebotomus sergenti in Spain and Morocco. Acta Trop. 2008;107:259–267. [PubMed]
50. Barroso PA, Marco JD, Kato H, Tarama R, Rueda P, Cajal SP, Basombrío MA, Korenaga M, Taranto NJ, Hashiguchi Y. The identification of sand fly species, from an area of Argentina with endemic leishmaniasis, by the PCR-based analysis of the gene coding for 18S ribosomal RNA. Ann. Trop. Med. Parasitol. 2007;101:247–253. [PubMed]
51. Terayama Y, Kato H, Gomez EAL, Uezato H, Calvopiña M, Iwata H, Hashiguchi Y. Molecular typing of sand fly species (Diptera, Psychodidae, Phlebotominae) from areas endemic for leishmaniasis in Ecuador by PCR-RFLP of 18S ribosomal RNA gene. J. Vet. Med. Sci. 2008;70:907–913. [PubMed]
52. Kuwahara K, Kato H, Gomez EA, Uezato H, Mimori T, Yamamoto YI, Calvopiña M, Cáceres AG, Iwata H, Hashiguchi Y. Genetic diversity of ribosomal RNA internal transcribed spacer sequences in Lutzomyia species from areas endemic for New World cutaneous leishmaniasis. Acta Trop. 2009;112:131–136. [PubMed]
53. Krzywinski J, Besansky NJ. Molecular systematics of Anopheles: from subgenera to subpopulations. Annu. Rev. Entomol. 2003;48:111–139. [PubMed]
54. Obsomer V, Defourny P, Coosemans M. The Anopheles dirus complex: spatial distribution and environmental drivers. Malar. J. 2007;6:26. [PMC free article] [PubMed]
55. Soto SI, Lehmann T, Rowton ED, Vélez BID, Porter CH. Speciation and population structure in the morphospecies Lutzomyia longipalpis (Lutz & Neiva) as derived from the mitochondrial ND4 gene. Mol. Phylogenet. Evol. 2001;18:84–93. [PubMed]
56. Bottecchia M, Oliveira SG, Bauzer LG, Souza NA, Ward RD, Garner KJ, Kyriacou CP, Peixoto AA. Genetic divergence in the cacophony IVS6 intron among five Brazilian populations of Lutzomyia longipalpis. J. Mol. Evol. 2004;58:754–761. [PubMed]
57. Bauzer LG, Souza NA, Ward RD, Kyriacou CP, Peixoto AA. The period gene and genetic differentiation between three Brazilian populations of Lutzomyia longipalpis. Insect. Mol. Biol. 2002;11:315–323. [PubMed]
58. Bauzer LG, Gesto JS, Souza NA, Ward RD, Hamilton JG, Kyriacou CP, Peixoto AA. Molecular divergence in the period gene between two putative sympatric species of the Lutzomyia longipalpis complex. Mol. Biol. Evol. 2002;19:1624–1627. [PubMed]
59. Maingon RD, Ward RD, Hamilton JG, Bauzer LG, Peixoto AA. The Lutzomyia longipalpis species complex: does population sub-structure matter to Leishmania transmission? Trends Parasitol. 2008;24:2–7. [PubMed]
60. Lins RM, Souza NA, Peixoto AA. Genetic divergence between two sympatric species of the Lutzomyia longipalpis complex in the paralytic gene, a locus associated with insecticide resistance and lovesong production. Mem. Inst. Oswaldo Cruz. 2008;103:736–740. [PubMed]
61. Hamarsheh O, Presber W, Yaghoobi-Ershadi MR, Amro A, Al-Jawabreh A, Sawalha S, Al-Lahem A, Das ML, Guernaoui S, Seridi N, Dhiman RC, Hashiguchi Y, Ghrab J, Hassan M, Schönian G. Population structure and geographical subdivision of the Leishmania major vector Phlebotomus papatasi as revealed by microsatellite variation. Med. Vet. Entomol. 2009;23:69–77. [PubMed]
62. Hashiguchi Y, Gomez EAL. A review of leishmaniasis in Ecuador. Bull. Pan Am. Hlth. Org. 1991;25:64–76. [PubMed]
63. Hashiguchi Y. Leishmaniasis. In: Otsuru M, Kamegai S, Hayashi S, editors. Progress of Medical Parasitology in Japan. Vol. 7. Megro Parasitological Museum; Tokyo, Japan: 2003. pp. 537–553.
64. Aransay AM, Scoulica E, Tselentis Y. Detection and identification of Leishmania DNA within naturally infected sand flies by seminested PCR on minicircle kinetoplast DNA. Appl. Environ. Microbiol. 2000;66:1933–1938. [PMC free article] [PubMed]
65. Parvizi P, Mauricio I, Aransay AM, Miles MA, Ready PD. First detection of Leishmania major in peridomestic Phlebotomus papatasi from Isfahan province, Iran: comparison of nested PCR of nuclear ITS ribosomal DNA and semi-nested PCR of minicircle kinetoplast DNA. Acta Trop. 2005;93:75–83. [PubMed]
66. Jorquera A, González R, Marchán-Marcano E, Oviedo M, Matos M. Multiplex-PCR for detection of natural Leishmania infection in Lutzomyia spp. captured in an endemic region for cutaneous leishmaniasis in state of Sucre, Venezuela. Mem. Inst. Oswaldo Cruz. 2005;100:45–48. [PubMed]
67. Paiva BR, Secundino NF, Nascimento JC, Pimenta PF, Galati EA, Junior HF, Malafronte RS. Detection and identification of Leishmania species in field-captured phlebotomine sandflies based on mini-exon gene PCR. Acta Trop. 2006;99:252–259. [PubMed]
68. Córdoba-Lanús E, De Grosso ML, Piñero JE, Valladares B, Salomón OD. Natural infection of Lutzomyia neivai with Leishmania spp. in northwestern Argentina. Acta Trop. 2006;98:1–5. [PubMed]
69. Azizi K, Rassi Y, Javadian E, Motazedian MH, Asgari Q, Yaghoobi-Ershadi MR. First detection of Leishmania infantum in Phlebotomus (Larroussius) major (Diptera: Psychodidae) from Iran. J. Med. Entomol. 2008;45:726–731. [PubMed]
70. Pita-Pereira D, Souza GD, Zwetsch A, Alves CR, Britto C, Rangel EF. First report of Lutzomyia (Nyssomyia) neivai (Diptera: Psychodidae: Phlebotominae) naturally infected by Leishmania (Viannia) braziliensis in a periurban area of south Brazil using a multiplex polymerase chain reaction assay. Am. J. Trop. Med. Hyg. 2009;80:593–595. [PubMed]
71. Silva EA, Andreotti R, Dias ES, Barros JC, Brazuna JC. Detection of Leishmania DNA in phlebotomines captured in Campo Grande, Mato Grosso do Sul, Brazil. Exp. Parasitol. 2008;119:343–348. [PubMed]
72. Pandey K, Pant S, Kanbara H, Shuaibu MN, Mallik AK, Pandey BD, Kaneko O, Yanagi T. Molecular detection of Leishmania parasites from whole bodies of sandflies collected in Nepal. Parasitol. Res. 2008;103:293–297. [PubMed]
73. Savani ES, Nunes VL, Galati EA, Castilho TM, Zampieri RA, Floeter-Winter LM. The finding of Lutzomyia almerioi and Lutzomyia longipalpis naturally infected by Leishmania spp. in a cutaneous and canine visceral leishmaniases focus in Serra da Bodoquena, Brazil. Vet. Parasitol. 2009;160:18–24. [PubMed]
74. Luyo-Acero G, Uezato H, Oshiro M, Kariya K, Katakura K, Gomez EAL, Hashiguchi Y, Nonaka S. Sequence variation of the cytochrome b gene of various human infecting members of the genus Leishmania and their pathology. Parasitol. 2004;128:483–491. [PubMed]
75. Marco JD, Bhutto AM, Soomro FR, Baloch JH, Barroso PA, Kato H, Uezato H, Katakura K, Korenaga M, Nonaka S, Hashiguchi Y. Multilocus enzyme electrophoresis and cytochrome b gene sequencing-based identification of Leishmania isolates from different foci of cutaneous leishmaniasis in Pakistan. Am. J. Trop. Med. Hyg. 2006;75:261–266. [PubMed]
76. Marco JD, Uezato H, Mimori T, Barroso PA, Korenaga M, Nonaka S, Basombrio MA, Taranto NJ, Hashiguchi Y. Are cytochrome b gene sequencing and polymorphism-specific polymerase chain reaction as reliable as multilocus enzyme electrophoresis for identifying Leishmania spp. from Argentina? Am. J. Trop. Med. Hyg. 2006;75:256–260. [PubMed]

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