Further insight into the geographic distribution of Leishmania species in Peru by cytochrome b and mannose phosphate isomerase gene analyses

To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru.


Introduction
Leishmaniasis, caused by an intracellular protozoa of the genus Leishmania, is a neglected tropical disease widely distributed worldwide, especially in tropical and subtropical areas, affecting at least 12 million people in 96 countries [1]. Approximately 20 Leishmania species belonging to the subgenera Leishmania (Leishmania), Leishmania (Viannia) and recently, Leishmania (Mundinia) are known to be pathogenic to humans and cause cutaneous, mucocutaneous and visceral disorders in infected individuals [1,2]. Since infected parasite species are known to be the major determinant of clinical outcomes and may be associated with the response to treatments in leishmaniasis [1], identification of the infected parasite is important for appropriate treatment and prognosis.
Peru is one of the most highly endemic countries for cutaneous leishmaniasis (CL) [1]. Countrywide surveillance of leishmaniasis has been carried out, and the main causative parasites were identified as L. (V.) peruviana, L. (V.) braziliensis, and L. (V.) guyanensis in the Andean highlands, in the tropical rainforest, and in the northern to central rainforest areas, respectively [3][4][5]. Other than the three dominant species, distribution of L. (V.) lainsoni and L. (L.) amazonensis has been reported in lower-altitude rainforest areas [3][4][5]. In addition, a hybrid of L. (V.) braziliensis and L. (V.) peruviana was reported in the Department of Huanuco in 1995, and the current prevalence in the same areas was confirmed [6,7]. Interestingly, CL due to the hybrid Leishmania has not been reported in other areas, although the infection seemed to be the most dominant in the Department of Huanuco [7]. On the other hand, two cases infected by L. (V.) shawi were newly reported in lowland rainforest areas in the Departments of Junin and Madre de Dios in 2010 [5]; since then, however, the infection by this species has not been reported in the country. Previous epidemiological studies have been conducted mainly in Andean areas facing the Pacific Ocean and central to northern rainforest areas, probably because of accessibility [3][4][5]. To obtain further information on prevalent Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas, was performed based on the cytochrome b (cyt b) gene sequence and PCR-RFLP analysis of the mannose phosphate isomerase (mpi) gene by using FTA (Flinders Technology Associates) card-spotted samples and smear slides as DNA sources.

Sample collection
Clinical samples were collected from patients suspected of having CL at 41 sites in 28 provinces of 13 departments in Peru during 2012 and 2017 (S1 Fig). Clinical samples spotted on FTA cards (Whatman, Newton Center, MA) and Giemsa-stained smears that were used for the diagnosis of CL were utilized in this study. To prepare FTA samples, tissue samples were taken by scraping the margins of active lesions of each patient, spotted onto an FTA Classic Card and stored at room temperature. Two-mm-diameter disks were punched out from each filter paper and washed twice with an FTA Purification Reagent (Whatman) and once with Tris-EDTA buffer. The disks were air-dried and directly subjected to PCR amplification. To extract DNA from Giemsa-stained smears obtained from skin lesions (ulcers and/or nodules) on CL patients, 50 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. Detached tissue materials in the DNA extraction buffer were transferred to 1.5 ml tubes, incubated at 37˚C overnight, and heat-inactivated at 95˚C for 5 min. Each 0.5-μl portion was directly used as a template for PCR.

Phylogenetic analysis
The Leishmania cyt b gene sequences were aligned with CLUSTAL W software [9] and examined using the program MEGA (Molecular Evolutionary Genetics Analysis) version 6 [10]. A phylogenetic tree was constructed by the maximum likelihood (ML) method with the Hasegawa-Kishino-Yano (HKY) + G (Gamma distribution with 5 rate categories) model [10]. Branch support for the ML tree was calculated using the bootstrapping method with 1,000 replicates [10]. The best ML model for analysis was selected based on the lowest BIC score (Bayesian Information Criterion) in MEGA 6 [10].

Ethics statement
Clinical samples were collected by local physicians and well-trained laboratory technicians at health centers of the Ministry of Health, Peru. For routine parasitological diagnosis, scratching smear samples of skin lesions were taken from suspected leishmaniasis patients at health centers. In this study, only residual tissue materials were collected after the routine procedure to minimize the burden on patients. Signed consent was obtained from adult subjects and from children's parents or guardians prior to the diagnostic procedures at each health center of the Ministry, providing information on the process of diagnosis and Leishmania species analysis, following the guidelines of the Ethics Committee of the Ministry. 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 a specific drug, pentavalent antimony, was also offered free of charge at each health center. The study was approved by the ethics committee of the Graduate School of Veterinary Medicine, Hokkaido University (approval number: vet26-4) and Jichi Medical University (approval number: 17-080).

Results
A total of 262 clinical samples were collected from patients suspected of having CL in 28 provinces of 13 departments in Peru. Of these, 99 samples were collected on FTA cards, and 163 samples were Giemsa-stained smears. Parasite species were identified based on cyt b gene sequence analysis and phylogenetic analysis (Fig 1). Since L. (V.) braziliensis and L. (V.) peruviana were indistinguishable by cyt b gene analysis, the two species were further differentiated by PCR-RFLP analysis of the mpi gene [5,7,8]. The PCR amplification was repeated up to three times to obtain specific gene fragments. As the result, Leishmania DNAs were successfully amplified and species were identified in 83 (83.8%) of 99 FTA-spotted samples and 59 (36.2%) of 163 Giemsa-stained smear samples. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank databases under the accession numbers LC472411-LC472486 and LC472841-LC472880. The distribution of Leishmania species by department is presented in Table 1   where the hybrid parasite has been reported [6,7] (Table 1, Fig 2). Unexpectedly, the hybrid parasite was also detected in four patients from an endemic area in the Department of Cajamarca, in which distribution of L. (V.) braziliensis and L. (V.) peruviana has been reported [3][4][5] (Table 1, Fig 2). This is the first report on the distribution of a hybrid of L. (V.) braziliensis and L. (V.) peruviana outside the Department of Huanuco. Leishmania (V.) guyanensis, a relatively dominant species in Peru, was detected in rainforest areas in Departments of Amazonas, Junin, Loreto, Madre de Dios, Puno, and San Martin, corresponding with previous observations [3][4][5] (Table 1, Fig 2). Infection by L. (V.) lainsoni is much less common in Peru; however, many cases of this infection were identified in the Department of Puno where causative species of CL have not been well-studied (Table 1, Fig 2). Two cases of L. (V.) shawi infection were reported in the Departments of Madre de Dios and Junin in 2010 [5], but no infections have been recorded since then. The present study identified L. (V.) shawi in the Departments of Cusco and Puno, suggesting that this species is widely distributed in southern Amazonian areas in Peru, although it is still less common (Table 1, Fig 2). A case of L. (L.) amazonensis infection was detected in the Department of Junin, where the infection has been reported previously [3].
In this study, all patients had typical ulcerative and/or nodular cutaneous lesions, and none had mucosal or mucocutaneous involvement. The number of cutaneous lesions per patient ranged from one to four, and the diameter of lesions ranged from 0.5 to 6cm. No marked characteristic differences in cutaneous lesions were observed among the causative Leishmania species identified.

Discussion
Countrywide epidemiological studies of leishmaniasis have been conducted in Peru, and causative parasite species have been studied mainly in Andean areas and central to northern rainforest areas. To obtain further information on leishmaniasis widely endemic in Peru, CLcausing Leishmania species were investigated, especially focusing on central to southern

Department
Leishmania species  In this study, samples spotted on FTA cards and smear slides used for parasitological diagnosis were applied as DNA sources. Use of FTA cards for direct sampling from patients' lesions has several advantages such as a minimal risk of contamination, being less invasive, and ease of sample collection as reported previously [5,11,12]. When the cards were initially applied to sample collection for the epidemiological study on leishmaniasis, the detection efficacy of the parasite gene was relatively low (61.4%, 81/132 samples), probably because of a lack of information for optimal sampling procedures [5]. However, the sampling efficacy was improved in a subsequent study (75.8%, 125/165 samples) [12], and reached 83.8% (83/99 samples) in this study, indicating that the FTA card is a powerful tool for epidemiological studies if used properly. On the other hand, in Giemsa-stained smear samples, successful identification of parasite species was lower, with a PCR-positive ratio of only 36.2% (59/163 samples). A similar result was obtained in our previous study with a positive ratio of 42.7% (114/267 samples) when Giemsa-stained smears were applied as a DNA source [7]. As discussed previously, the inefficiency was probably due to the poor DNA condition in smear samples because it was lost and damaged during the routine processes of fixation by methanol, Giemsa staining, and removal of oil after parasitological diagnosis under a microscope [7].
Distribution of a hybrid of L. (V.) braziliensis and L. (V.) peruviana has been reported only in the Department of Huanuco, located in the mid-eastern region of the Peruvian Andes [3,4,7]. The present study identified CL cases due to the hybrid strain in an area of the Department of Cajamarca located in northern Peru. This is the first report of the prevalence of the hybrid strain outside the Department of Huanuco. The hybrid strain may have newly emerged in the Department of Cajamarca rather than originating from the Department of Huanuco, since the two endemic areas are more than 500 km apart and isolated by the central and eastern mountain ranges. Detailed molecular analysis such as population genetics using microsatellite markers may clarify the relationships between the hybrid strains. Further research on the prevalence of a hybrid of L. (V.) braziliensis and L. (V.) peruviana in other departments will be necessary. Importantly, the hybrid of L. (V.) braziliensis and L. (V.) peruviana was suggested to exacerbate the disease when compared to parental strains in an experimental animal model [13]. More precise clinical and epidemiological studies on individuals infected with the hybrid should be done to elucidate the relationships between the parasite and disease severity.
In this study, a high ratio of CL cases caused by L. (V.) lainsoni was identified in endemic areas of the Department of Puno, where CL-causative species are not well-studied. L. (V.) lainsoni is not a common causative species in Peru, and the infection was reported mainly in rainforest regions. The endemic areas in the Department of Puno may be unique and suitable areas to investigate the pathophysiology caused by L. (V.) lainsoni, drug efficacy against the infection, and its transmission mechanism including reservoirs and vectors.
Leishmania (V.) shawi was originally identified as a parasite of wild animals such as monkeys, sloths and procyonids in Amazonian Brazil [14]. Human infection has been recorded in Further genetic analyses such as population genetics will be of help to reveal the origin of these parasites in each endemic area reported. In addition, it will be important to elucidate their transmission cycles, including reservoirs and vectors, to better understand and control leishmaniasis in Peru.