Detection of Leishmania infantum DNA in Pintomyia evansi and Lutzomyia longipalpis in an endemic area of Non-ulcerative cutaneous leishmaniasis from Honduras

Wilfredo Humberto Sosa-Ochoa (  wilfredo.sosa@unah.edu.hn ) Universidad Nacional Autonoma de Honduras https://orcid.org/0000-0003-1387-7488 Javier Varela Amador Hospital Militar de Honduras Yokomi N Lozano-Sardaneta Centro de Medicina Tropical, División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México Gabriela Rodriguez Segura Microbiology Research Institute, UNAH Concepcion Zúniga Health Surveillance Department, University School Hospital, Tegucigalpa Honduras Gabriela V. Araujo Laboratory of Pathology of Infectious Diseases, Medical School, São Paulo University Carmen M. Sandoval Laboratory of Pathology of Infectious Diseases, Medical School, São Paulo University Márcia D. Laurenti Laboratory of Pathology of Infectious Diseases, Medical School, São Paulo University Fredy Galvis-Ovallos Department of Epidemiology, School of Public Health, São Paulo University

Leishmaniasis is a vector-borne disease caused by parasite species of the genus Leishmania (Trypanosomatidae: Trypanosomatidae). The geographic distribution occurs mainly in the tropical and subtropical regions, and in 98 countries throughout Europe, Africa, Asia, and America [1]. More than 1,000 sand y species have been described worldwide, from which 530 species are present in the Americas [2,3] and at least 30 species are considered as Leishmania vectors [4]. In Honduras, 29 sand y species have been reported [2,[5][6][7]. Lutzomyia (Lutzomyia) longipalpis and Pintomyia (Pifanomyia) evansi are the most abundant species in the endemic area for Visceral Leishmaniasis (VL) and Non -ulcerative cutaneous leishmaniasis (NUCL) in the southern region of Honduras [6,8]. However, Lutzomyia (Lutzomyia) longipalpis is the only specie reported as vector of Leishmania (L.) infantum [9]. Nevertheless, the role of Pintomyia evansi as vector of Leishmania (L.) infantum in Honduras is unknown, although evidences about it vector competence has been reported in South America [10]. For that reason, the aim of this study was to evaluate the DNA circulation of Leishmania (L.) infantum in Lutzomyia (Lutzomyia) longipalpis and Pintomyia (Pifanomyia) evansi by on a Mesoamerican Paci c island.

Study area and sand ies collections
The study was carried out in Amapala municipality (N13 15.618, W87 37.463), Valle department, with an area of 80.7 km 2 . The municipality comprises two islands, Zacate Grande and El Tigre, located in the Gulf of Fonseca in the southern Honduras. Sand ies were sampled for ve consecutive nights in May 2018 on ve localities (Las Pelonas, Punta Honda, Tigüilotada, Islitas and Playa Grande). Samplings was carried out to 18:00 at 6:00 h, using automatic CDC miniature light traps (model 512; John W. Hock Co., Gainesville, FL, USA) in neighborhoods selected by the presence of active cases of Non-ulcerative or atypical cutaneous leishmaniosis (NUCL). The traps were installed in peri-domiciliary environment, mainly near of decomposing organic matter, or next to the latrines. The specimens were separated and processed the day after the capture.

Taxonomic identi cation of Sand y species
The phlebotomine sand ies were mounted for morphological identi cation, following the procedures outlined by Mejía et al., 2018 [6]. The sand y species were identi ed according to Young and Duncan, 1994 [5], and the genera and species classi cation is presented according Galati´s key, 2017 [11].
Genomic material extraction and polymerase chain reaction (PCR) Genomic DNA was extracted only from the bowel dissection of individual female sand ies using Chelex® 100 (Bio-Rad Lab Inc., Hercules, California, USA). As an internal control of the extraction of DNA, the cacophony IVS6 gene present in the genome of sand ies was ampli ed [12]. For the detection of genus Leishmania we use the primers Leish1: 5′-AACTTTTCTCTGGTCCTCCGGGTAG-3′ and Leish2: 5′-ACCCCCAGTTTCCCGCC-3 ′ in order to amplify a ≈120-base-pair fragment of the Leishmania kinetoplast DNA minicircle [13]. Ampli cations were performed using a commercial kit (Master Mix 2X -Promega). Each reaction was performed by adding 4 µL of target DNA and 0.6 µmol/L of each primer in a nal volume of 20 µL. The PCR reactions were done in a Applied Biosystem 2770 Thermal Cycler (ThermoFisher Scienti c/USA), under the following conditions initial denaturation cycle at 94°C for 5 minutes, followed by 35 cycles alt 94°C for 15s, 60°C for 20s and 72°C for 60s, and nal extension of 72°C for 10 min. The ampli cation products were analyzed by electrophoresis in a 1.5% agarose gel.
To characterize the Leishmania species, PCR-RFLP was performed which ampli es a speci c region of the hsp70 gene [14] . Primers used were hsp70 sense (5 'GACGGTGCCTGCCTACTTCAA 3') and hsp70 antisense (5 'CCGCCCATGCTCTGGTACATC 3'). Reaction mixture was prepared in a nal volume of 50 µL with 25 µL of Master Mix 2X (Promega), 5 µL of target DNA and 0.6 µmol/L of each primer. The PCR reactions were done under the following conditions initial denaturation at 94°C for 5 min, followed by 37 cycles at 94°C for 30 s, 61°C for 1 min and 72°C for 3 min, and a nal extension cycle at 72°C for 10 min.

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The ampli cation products were analyzed by electrophoresis in a 2% agarose gel. To perform the restriction of PCR products, the enzyme Hae III (Promega) was used, adding 5 µL of ampli ed DNA to the reaction and incubated at 37ºC for 3 h. The pro les of each species were observed using a 2% agarose gel subjected to electrophoresis as described by Montalvo [15].

Result And Discussion
A total of 222 specimens were collected in, being predominant males (66%). The number of sand ies according sex and species is presented in Table 1. Six species were identi ed through morphological characters, with predominance of Lutzomyia (Lutzomyia) longipalpis, followed by Pintomyia (Pifanomyia) evansi. Other species captured were Micropygomyia (Micropygomyia) cayennensis cayennensis, Micropygomyia (Coquillettimyia) chiapanensis, Dampfomyia (Coromyia) beltrani and Lutzomyia (Tricholateralis) gomezi. Lutzomyia (Lutzomyia) longipalpis has been previously studied in the region and incriminated as the vector of Leishmania (L.) infantum [9]. Additionally, behavioral characteristics of this species in the study area were described by Carrasco et al. [7]. Recently, Mejía et al. [6] described aspects of the feeding preference of sand ies in the Paci c Honduran area. These authors observed the predominance of Pintomyia (Pifanomyia) evansi and Lutzomyia (Lutzomyia) longipalpis, but couldn't determine the presence of Leishmania (L.) infantum in those species [6]. Similar results were described by Zeledón et al., (1984) and Raymond et al., (2010) who reported those species as the two most common species in areas endemic to NUCL in Costa Rica and Nicaragua, respectively. The PCR trial showed positive results to the Leishmania genus in 37 of 96 analyzed female specimens analyzed (Fig. 1). Nine samples showed presence of Leishmania (L.) infantum DNA, 7 from Lutzomyia (Lutzomyia) longipalpis and 2 from Pintomyia (Pifanomyia) evansi. The Leishmania (L.) infantum infection rate was 9.4% for Lutzomyia (Lutzomyia) longipalpis and 2.7% for Pintomyia (Pifanomyia) evansi. All the analyzed samples, threw an ampli ed product of 220 pb corresponding to a Lutzomyia spp. constitutive gene (cacophony), con rming the insect DNA preparation integrity and the absence of PCR inhibitors. Our results are the rst report of the presences of Leishmania (L.) infantum DNA in Pintomyia (Pifanomyia) evansi females in Central America. In Colombia, the natural infection of Leishmania (L.). infantum in endemic areas of VL associated with Pintomyia (Pifanomyia) evansi has been recorded [10,16] with an infection rate of 0.10 and 0.34%. Meanwhile in Lu. longipalpis the natural infection rates between 0.5 and 1.1%, when done with direct observation on intestine dissection [9,17] or using intestine dissection followed by PCR [18][19][20]. We reported 9.4% infection of Leishmania (L.) infantum in Lutzomyia (Lutzomyia) longipalpis, which agree with other reports [19,20]. Although the Leishmania DNA detection in sand ies not represent the ability of a specie to transmit the parasite, our results present evidence of contact between the specie Pintomyia evansi and natural host of Leishmania (L.). infantum in the study area. Considering that the vector competence of this specie was previously described (Travi et al. 1990). They could be involved with this parasite specie transmission in south Honduras.