Geographic Distribution of Leishmania Species in Ecuador Based on the Cytochrome B Gene Sequence Analysis

A countrywide epidemiological study was performed to elucidate the current geographic distribution of causative species of cutaneous leishmaniasis (CL) in Ecuador by using FTA card-spotted samples and smear slides as DNA sources. Putative Leishmania in 165 samples collected from patients with CL in 16 provinces of Ecuador were examined at the species level based on the cytochrome b gene sequence analysis. Of these, 125 samples were successfully identified as Leishmania (Viannia) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, and L. (Leishmania) mexicana. Two dominant species, L. (V.) guyanensis and L. (V.) braziliensis, were widely distributed in Pacific coast subtropical and Amazonian tropical areas, respectively. Recently reported L. (V.) naiffi and L. (V.) lainsoni were identified in Amazonian areas, and L. (L.) mexicana was identified in an Andean highland area. Importantly, the present study demonstrated that cases of L. (V.) braziliensis infection are increasing in Pacific coast areas.


Introduction
Leishmaniasis is caused by protozoan parasites of the genus Leishmania, which is further divided into two subgenera, Leishmania (Leishmania) and Leishmania (Viannia). The disease is widely distributed around the world, especially in tropical and subtropical areas, affecting at least 12 million people in 98 countries [1]. Approximately 20 Leishmania species are known to be pathogenic to humans, and the infecting species is the major determinant of clinical outcome [1]. 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 reported in 21 of 24 provinces of the country, in Pacific coast subtropical areas, Amazonian tropical areas and Andean highland areas [2][3][4]. During 2010 and 2014, 6,608 cases were registered in the Ministry of Public Health, Ecuador, ranging yearly from 899 to 1,629 (average 1,321.6), and in 2014, 262 (22.1%) of the 1,183 cases were derived from Pichincha province, followed by Santo Domingo de los Tsáchilas (148 cases, 12.5%), Esmeraldas (136 cases, 11.5%), Orellana (94 cases, 7.9%), Sucumbios (88 cases, 7.4%), and Morona Santiago (87 cases, 7.4%) provinces (Departamento de Epi lainsoni have been identified as causative agents for CL and MCL [5][6][7][8][9]. Currently, molecular biological methods are widely used for identification of Leishmania species using DNA extracted from clinical samples of patients' lesions, and they have become a powerful tool for epidemiological studies of leishmaniasis [10][11][12]. DNA extracted from Giemsa-stained smears obtained from patients' skin lesions, which have been used for the microscopic diagnosis to detect parasites in the lesions, has also been used as a template for detection and identification of Leishmania, although the sensitivity is not so high because of limitations of the DNA source [13][14][15][16][17]. Recently, to facilitate sample collection and DNA extraction processes, an FTA card, a filter paper that readily lyses spotted materials and fixes nucleic acids, was used for direct sampling of patients' samples in an epidemiological study of leishmaniasis, and its usability for field epidemiology was reported [18][19][20]. In the present study, a countrywide epidemiological survey was performed to elucidate the current geographic distribution of causative species of CL in Ecuador, by using FTA card-spotted samples and smear slides as DNA sources.  Fig). Tissue samples were taken by scraping the margins of active lesions of a patient, spotted onto an FTA Classic Card (Whatman, Newton Center, MA) and stored at room temperature. Two-mm-diameter disks were punched out from each filter paper and washed three times with an 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 obtained from skin lesions (ulcers and/or nodules) on CL patients, 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 mixed well, and detached tissue materials in the DNA extraction buffer were transferred to 1.5 ml tubes. The sample was incubated at 37°C overnight, and heated at 95°C for 5 min. Each 0.5-μl portion was directly used as a template for PCR.

Identification of Leishmania species
Leishmania species were identified by cytochrome b (cyt b) gene sequence analysis [18,19]. PCR amplification with a pair of specific primers, L.cyt-AS (5'-GCGGAGAGRARGAAAAGG C-3') and L.cyt-AR (5'-CCACTCATAAATATACTATA-3'), was performed with 30 cycles of denaturation (95°C, 1 min), annealing (55°C, 1 min) and polymerization (72°C, 1 min) using Ampdirect Plus reagent (Shimadzu Biotech, Tsukuba, Japan). Each 0.5-μl portion of the PCR product was reamplified with L.cyt-S (5'-GGTGTAGGTTTTAGTYTAGG-3') and L.cyt-R (5'-CTACAATAAACAAATCATAATATRCAATT-3'). The products were cloned into the pGEM-T Easy Vector System (Promega, Madison, WI) and sequences were determined by the dideoxy chain termination method using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The parasite species were identified based on the homology with cyt b gene sequences from Leishmania reference strains. The result was confirmed by a phylogenetic analysis and a distant matrix using the program MEGA (Molecular Evolutionary Genetics Analysis) version 5.2.

Phylogenetic analysis
The Leishmania cyt b gene sequences were aligned with CLUSTAL W software [21] and examined using the program MEGA version 5.2 [22]. Phylogenetic trees were constructed by the maximum likelihood (ML) method with Kimura 2 parameter [22]. Branch support for ML tree was calculated using the bootstrapping method with 1,000 replicates in MEGA 5.2 [22].

Ethics statement
Sample 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. Signed consent was obtained after explanation of the process of diagnosis and Leishmania species analysis during 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 diagnosis/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
A total of 165 samples (162 FTA cards and three smear samples) were collected from patients living in endemic areas of Ecuador who were suspected of having cutaneous leishmaniasis. The samples were subjected to PCR targeting the leishmanial cyt b gene, and the amplification was repeated, not more than twice, to obtain gene fragments of the parasites from samples negative in the first PCR. Leishmanial cyt b gene fragments were obtained from 125 (123 FTA cards and two smear samples) of 165 patients' samples (75.8%). Parasites were identified to species on the basis of cyt b gene sequence analysis (S2 Fig) [18,19]. The nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers LC055618-LC055621 and LC153160-LC153277. The distribution of Leishmania species by province in Ecuador is presented in Table 1 and  Fig 1). Leishmania (V.) braziliensis was identified in northern and central provinces (Esmeraldas, Manabi, Pichincha, and Santo Domingo de los Tsáchilas) (Table 1, Fig 1). In Andean highland areas, a CL case from the province of Chimborazo was examined and infecting parasites identified as L. (L.) mexicana. In Amazonian tropical provinces (Orellana, Pastaza, Sucumbíos, Zamora-Chinchipe), L. (V.) braziliensis was identified as the dominant species as reported previously [5,8,9]. In addition, other spe- reported previously in Ecuador, the latter two rather recently [7,8] (Table 1, Fig 1). Cases of L. (V.) naiffi infection were identified in the previously reported area (Shangrila) in Orellana province, and L. (V.) lainsoni infections were identified in Sucumbíos province. In the southern Amazonian province (Zamora-Chinchipe), only L. (V.) braziliensis was identified.
In the present study, all patients had typical ulcerative and/or nodular cutaneous lesions; none had mucosal or mucocutaneous lesions. The number of cutaneous lesions per patient ranged from one to six, and the diameter of lesions ranged from 0.5 to 5cm. The one Leishmania (L.) mexicana infection in an Andean area caused a typical small ulcerative lesion (0.5cm), the so-called "Andean-type CL" [4]. No marked characteristic differences in cutaneous lesions among causative Leishmania species were observed.  [3][4][5]. Of these, wide distribution of L. (V.) panamensis was identified by multilocus enzyme electrophoresis (MLEE) in Pacific coast areas [3,5], whereas L. (L.) amazonensis, for which samples were not examined in this study, was identified from only a few areas [3,5]. Leishmania (V.) panamensis is very closely-related to L. (V.) guyanensis, and a previous study questioned the distinctness of the two species by MLEE and genetic analyses of Leishmania isolates in Ecuador [23]. Previous studies reported that L. (V.) panamensis and L. (V.) guyanensis were separated in distinct clades by phylogenetic analysis targeting the cyt b gene [24,25]; however, multiple genetic analyses of Ecuadorian isolates identified as L. (V.) panamensis or L. (V.) guyanensis by MLEE revealed discordant results among targeted genes, which is in agreement with a previous enzymatic and genetic analyses of the two species [23]. Therefore, it is speculated that L. (V.) guyanensis identified in this study includes Leishmania species previously identified as L. (V.) guyanensis and L. (V.) panamensis by MLEE. Since the present classification of Leishmania species has been defined by MLEE and genetic analyses of Leishmania suggested that the number of species could be very large, reclassification of Leishmania species including L. (V.) panamensis and L. (V.) guyanensis may be needed using extensive multiple genetic analyses [26,27]. Leishmania (L.) major-like has been reported in Andean areas as a minor species causing CL [4]; however, this infection has not been detected recently. The present study examined only one sample from an Andean area, and the infection parasite was identified as L. (L.) mexicana, the major causative species in Andean highland areas [3][4][5]. The characteristic cyt b gene sequence, which composes a separate clade from other L. (L.) mexicana strains including reference strains by a phylogenetic analysis [18], was confirmed. In Amazonian areas, four species, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi, and L. (V.) lainsoni, were identified, of which distributions of the latter two species have been reported recently [7,8]. Distribution of L. (V.) lainsoni, the most recently reported species in Ecuador [7,8], was recorded in several areas in the Sucumbíos and Orellana provinces. On the other hand, cases of L. (V.) naiffi were identified only in a military training camp at Shangrila, Orellana province, as reported previously [7]. The vector species of L. (V.) naiffi has been identified as Lutzomyia (Lu.) tortura in the same area [7]. Although natural infection of Lu. tortura by L. (V.) naiffi has also been detected in Arajuno, Pastaza province [6], no human cases of infection with L. (V.) naiffi have been reported in this area. It may be interesting to compare its transmission cycle, including reservoir animals and the vector's host preferences in the two areas to understand different occurrences of CL caused by L. (V.) naiffi.

Discussion
The present study revealed wide distribution of L. (V.) guyanensis and L. (V.) braziliensis in Pacific coast and Amazonian areas. Wide distribution of the two species has been reported in other South American countries [28], reflecting the broad vector and reservoir ranges of these Leishmania species. One of the most important findings of this study is that cases of L. (V.) braziliensis infection seem to be increasing in Pacific coast areas of Ecuador when compared to past studies [3,5]. Distribution of L. (V.) braziliensis and its sand fly vectors may be expanding in these areas. Alternatively, parasite isolation, which is required for MLEE, may be inefficient in L. (V.) braziliensis when compared to other species, resulting in fewer identifications of this species in past studies. The procedures for isolation of L. (V.) braziliensis parasites from patient's lesions and its maintenance/culture in vitro are very difficult compared to other Leishmania species because of the extremely limited presence of amastigotes in the lesions and/or maladaptation of the species to an artificial culture medium. Genetic analysis of directly sampled materials as conducted in this study can overcome this issue. Since infection by L. (V.) braziliensis is associated with destructive mucocutaneous lesions [1], continuous surveillance will be needed. At present, MCL has been reported rarely in the Pacific coast areas in Ecuador. Several factors such as patients' genetic background and/or pathogenicity of parasite strains may be associated with the formation of mucocutaneous lesions.
The present countrywide surveillance revealed the current geographic distribution of causative species of CL in Ecuador. The less-invasive and easy-to-use FTA card will be a useful tool for further continuous monitoring of prevalent Leishmania species. Together with prevalent parasite species, vector and reservoir research will be needed since this information is limited in Ecuador despite its importance for control of leishmaniasis.