First Human Cases of Leishmania (Viannia) lainsoni Infection and a Search for the Vector Sand Flies in Ecuador

An epidemiological study of leishmaniasis was performed in Amazonian areas of Ecuador since little information on the prevalent Leishmania and sand fly species responsible for the transmission is available. Of 33 clinical specimens from patients with cutaneous leishmaniasis (CL), causative parasites were identified in 25 samples based on cytochrome b gene analysis. As reported previously, Leishmania (Viannia) guyanensis and L. (V.) braziliensis were among the causative agents identified. In addition, L. (V.) lainsoni, for which infection is reported in Brazil, Bolivia, Peru, Suriname, and French Guiana, was identified in patients with CL from geographically separate areas in the Ecuadorian Amazon, corroborating the notion that L. (V.) lainsoni is widely distributed in South America. Sand flies were surveyed around the area where a patient with L. (V.) lainsoni was suspected to have been infected. However, natural infection of sand flies by L. (V.) lainsoni was not detected. Further extensive vector searches are necessary to define the transmission cycle of L. (V.) lainsoni in Ecuador.


Introduction
Leishmaniases are caused by infection with protozoan parasites of the genus Leishmania transmitted by bites of female sand flies [1,2]. The genus Leishmania is further divided into two subgenera, Leishmania (Leishmania) and Leishmania (Viannia), originally distinguished by their development in the digestive tract of sand fly vectors and later confirmed by phylogenetic studies. This group of diseases is distributed worldwide, especially in tropical and subtropical areas, affecting at least 12 million people in 98 countries [2]. Approximately 20 Leishmania species are known to be pathogenic to humans, and the infecting species is the major determinant of clinical outcome [2]. Therefore, identification of the parasite species in endemic areas is important for both appropriate treatment and prognosis.
In Ecuador, leishmaniasis is a major public health concern and is reported in 21 of 24 provinces of the country, in Pacific coast subtropical, Amazonian, and Andean highland areas [3].  [4,5,7,8,9,10,11]. On the other hand, in Amazonian areas, although L. (V.) guyanensis, L. (V.) braziliensis, and recently, L. (V.) naiffi have been identified as causative agents for CL and MCL [4,5,6,12,13,14], epidemiological studies on leishmaniasis have been very limited and consequently little information is available regarding prevalent parasite species as well as epidemiological situation.
Molecular biological methods are widely used for identification of Leishmania species using DNA extracted from clinical material of patients' lesion, and is a powerful tool for epidemiological studies of leishmaniasis [15,16,17,18]. DNA extracted from Giemsa-stained smears obtained from patients' skin ulcers, which are used routinely for microscopic diagnosis in the laboratory to detect parasites in the lesion, have been used also as templates for detection and identification of Leishmania DNA although the sensitivity is low because of the limitation of the DNA source [19,20,21,22]. Recently, to facilitate sample collection and DNA extraction processes, a Flinders Technology Associates (FTA) card (Whatman), a filter paper that readily lyses the spotted materials and fixes nucleic acids, was used for direct sampling of patients' material in an epidemiological study of leishmaniasis, and its usability was reported [18,23]. In the present study, using smear slides and FTA card-spotted samples as DNA sources, an epidemiological survey of leishmaniasis was performed in Amazonian areas where little information on the endemic Leishmania species and vector sand flies is available.

Sample collection
Clinical samples were collected from patients suspected of having CL who visited health centers in Cascales, Lago Agrio (Province of Sucumbíos), Coca, La Joya de los Sachas, and Nuevo Rocafuerte (Province of Orellana) for the diagnosis and treatment of leishmaniasis (S1 Fig). Tissue samples were taken by scraping the margins of active lesions on patients, spotting them onto FTA Classic Cards (Whatman, Newton Center, MA) and storing them at room temperature. Two-mm-diameter disks containing the sample spot were punched out from each filter paper and washed three times with FTA Purification Reagent (Whatman) and once with Tris-EDTA buffer. The disks were air-dried and directly subjected to PCR amplification. For the extraction of DNA from Giemsa-stained smears taken from patients' skin ulcers that were used for diagnosis of CL, 30 μl of DNA extraction buffer [150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 10 mM EDTA and 0.1% sodium dodecyl sulfate (SDS)] containing 100 μg/ml of proteinase K were spotted on each smear, and the tissue sample was collected into 1.5 ml microtube. The sample was incubated at 37°C overnight, heated at 95°C for 5 min, and then 0.5 μl of each sample was directly used as a template for PCR amplification.

Sand fly collection
Sand flies were captured with mouth aspirator on protected human bait, CDC light traps, and the modified Shannon light traps [24] between 18:30 and 21:00 for 14 nights on February 2015 at mountain and forest areas around patient houses in Sucumbíos Province where a patient was suspected to have acquired an infection. Female sand flies were dissected and identified to species level based mainly on the morphology of their spermathecae [25]. Sand flies were examined under light microscopy for natural flagellate infections and positive samples were fixed individually in absolute ethanol. Ethanol-fixed specimens were individually lysed in 50 μl of DNA extraction buffer with proteinase K, and 0.5 μl of the extract was directly used as PCR templates [6,26,27,28].

Phylogenetic analysis
The Leishmania cyt b gene sequences were aligned with CLUSTAL W software [30] and examined using the program MEGA (Molecular Evolutionary Genetics Analysis) version 6 [31]. Phylogenetic trees were constructed by the neighbor-joining method with the distance algorithms available in the MEGA package [30].

Ethics statement
The collection was performed by local physicians and well-trained laboratory technicians with the approval of the research ethics committee of the Graduate School of Veterinary Medicine, Hokkaido University (license number: vet26-4). Informed consent was obtained from the adult subjects and from the children's parents or guardians, prior to collection of diagnostic materials at each health center of the Ecuadorian Ministry of Health (Provinces of Sucumbíos and Orellana). Signed consent was obtained after the explanation of the process of diagnosis and Leishmania species analysis at the time of routine diagnosis carried out at rural health centers, following the guidelines of the Ethics Committee of the Ministry of Health, Ecuador. The subjects studied were volunteers in routine diagnosing/screening and treatment programs promoted by the Ministry. All routine laboratory examinations were carried out free of charge, and treatment with specific drug (Glucantime) was also offered free of charge at each health center of the Ministry.

Results
Identification of Leishmania species from patient specimens Of 33 clinical samples, nine samples from patients in Sucumbíos Province and 24 from patients in Orellana Province were obtained (Table 1). Of these, 6 and 14 samples from Sucumbíos and Orellana patients, respectively, were Giemsa-stained smears used for routine diagnosis of CL, and others (3 and 10 samples, respectively) which were specimens spotted on FTA cards (Table 1). Patients had one to four skin lesions on their face, arms or/and legs with a diameter of 1 to 3 cm, typical of those observed on patients with CL in the area. The leishmanial cyt b gene was successfully amplified from two smears and three FTA card samples from Sucumbíos, and seven smear and nine FTA card samples from Orellana (Table 1)

Sand fly collection and dissection
A total of 1,104 female sand flies were captured and dissected. Of these, 732 were captured on protected human bait, and 372 flies with light traps (CDC light trap and the modified Shannon   Table 2). Four species, Lu. yuilli yuilli (71.7%), Lu. tortura (12.3%), Lu. davisi (7.3%), and Lu. napoensis (5.0%) accounted for 96.3% of the sand flies (Table 2). Among these, Lu. napoensis was captured only by light traps. In addition to these four species, Lu. sherlocki, Lu. trapidoi, Lu. gomezi, Lu marinkelli, Lu dysponeta, Lu camposi, Lu. robusta, Lu hirusta hirusta, Lu micropyga, and five unidentified species were captured (Table 2) [32,33], indicating that the flagellate infections in Lu. yuilli yuilli were Endotrypanum. On the other hand, the sequences of the parasite from Lu. davisi had only 81.2-82.3% homology with those of Leishmania and Endotrypanum species, indicating that the flagellate is neither Leishmania nor Endotrypanum. These results were supported by a phylogenetic analysis showing that nine flagellates from Lu. yuilli yuilli were located in the clade of Endotrypanum species, while the one from Lu. davisi was distant from Leishmania and Endotrypanum species (Fig 2). We suspect that the flagellates observed in Lu. davisi may belong to the genus Trypanosoma since some sand fly species are reported to transmit performed by the neighbor-joining method together with sequences from 12 Leishmania species. The scale bar represents 0.02% divergence. Bootstrap values are shown above or below branches.
doi:10.1371/journal.pntd.0004728.g001 Table 2. Identification of sand fly species and detection of flagellates within individual sand flies by microscopic examinations.

Discussion
An epidemiological study of leishmaniasis was conducted in Amazonian areas of Ecuador where very little information is available on prevalent parasite species or sand fly species associated with their transmission. In addition to L. Leishmaniasis is widely distributed in Pacific coast subtropical climate areas, Andean highland areas, and Amazonian tropical areas in Ecuador [4,5]. To date, most clinical and parasitological studies of leishmaniasis have been reported from Pacific coast and Andean highland areas [4,5,7,8,9,10,11], and little information is available on endemic parasite and sand fly species in Amazonian areas, mainly because of difficulty in gaining access [4,5,6,12,13,14]. In this study, Giemsa-stained smears used for routine diagnosis of CL were utilized as a DNA source in addition to FTA card-collected samples. The detection ratio was lower in DNA samples from smear slides when compared to FTA card collection (2/6 vs. 3/3 in Sucumbíos and 7/ 14 vs. 9/10 in Orellana), reflecting the amount of DNA recoverable and the condition of the smear slides. The thin smear samples were methanol-fixed, Giemsa-stained, and then examined under oil immersion. Thus, part of specimens and DNA may be lost and damaged when the immersion oil was wiped from the slide. Nevertheless, stored smear slides, which will not be used for any further purpose than diagnosis by microscopic examination, can be useful for identification of Leishmania species in endemic areas where sample collection for epidemiological study is difficult.
In this study, L. (V.) guyanensis and L. (V.) braziliensis were identified as causative agents in Amazonian areas as reported previously [4,5,6,12,13,14]. In addition, L. (V.) lainsoni is implicated as a causative agent of CL in Ecuador for the first time. Leishmania (V.) lainsoni was originally identified from patients in Brazilian Amazon [36]. It causes CL with characteristic lesions similar to those caused by other Leishmania (Viannia) species: small ulcers or small self-limiting nodules [37]. The parasite was subsequently identified from patients in Sub-Andean and Amazonian areas of Peru [18,38,39,40], in subtropical climate areas and Sub Andean areas of Bolivia [41,42], Suriname [43], and French Giana [44]. Two L. (V.) lainsoniinfected patients were found during this study. One was from the northern part of Ecuadorian Amazon (Sucumbios) near the border with Colombia, and the other was from the eastern frontier with Peru (Orellana), more than 200 km away. These findings suggest that L. (V.) laisoni is widely distributed in South America.
Natural infections with L. (V.) lainsoni in sand flies have been detected in Lu. ubiquitalis in Brazil [45], Lu. nuneztovari anglesi in Bolivia [42], and Lu. auraensis in Peru [46]. Microscopic examinations of sand flies collected in this study revealed no natural infection with L. (V.) lainsoni, and none of the above three Lutzomyia species were collected. Of the three dominant human-biting species in our collections, infection with Endotrypanum species, flagellate parasites of sloths, which are non-pathogenic to humans [32,33], were detected in the most dominant species, Lu. yuilli yuilli, as reported in another Amazonian area of Ecuador [6]. Although natural infection was not detected in this study, the next dominant species, Lu. tortura, was already incriminated as a vector species of L. (V.) naiffi in Ecuadorian Amazon [6,13]. Furthermore, Lu. davisi was reported to transmit L. (V.) naiffi in Brazil [47] and L. (V.) braziliensis in Peru [46]. In this study, natural infection of Lu. davisi by flagellates was observed. However, the parasite was neither Leishmania nor Endotrypanum. To identify the vector of L. (V.) lainsoni, further research is necessary to understand the natural transmission cycle of this parasite in Ecuador.
Our finding of the first human cases of CL caused by L. (V.) lainsoni infection in two separate areas of Ecuador suggests that this parasite is widely distributed in South America. Extensive countrywide surveillance is necessary to gain a proper understand of the status of L. (V.) lainsoni, as well as the sand flies responsible for its transmission in Ecuador.