Jariyapan et al. Parasites & Vectors (2018) 11:351 https://doi.org/10.1186/s13071-018-2908-3 RESEARCH Open Access Leishmania (Mundinia) orientalis n. sp. (Trypanosomatidae), a parasite from Thailand responsible for localised cutaneous leishmaniasis Narissara Jariyapan1*, Teerada Daroontum2, Krit Jaiwong3, Wetpisit Chanmol1, Nuchpicha Intakhan1, Sriwatapron Sor-suwan4, Padet Siriyasatien4, Pradya Somboon1, Michelle D. Bates5 and Paul A. Bates5* Abstract Background: Leishmaniasis is an emerging disease in Thailand with an unknown incidence or prevalence. Although the number of properly characterized and clinically confirmed cases is about 20, it is suspected that this low number masks a potentially high prevalence, with clinical disease typically manifesting itself against an immunocompromised background, but with a substantial number of subclinical or cured cases of infection. To date leishmaniasis in Thailand has been mainly ascribed to two taxa within the recently erected subgenus Mundinia Shaw, Camargo & Teixeira, 2016, Leishmania (Mundinia) martiniquensis Desbois, Pratlong & Dedet, 2014 and a species that has not been formally described prior to this study. Results: A case of simple cutaneous leishmaniasis was diagnosed in a patient from Nan Province, Thailand. Molecular analysis of parasites derived from a biopsy sample revealed this to be a new species of Leishmania Ross, 1908, which has been named as Leishmania (Mundinia) orientalis Bates & Jariyapan n. sp. A formal description is provided, and this new taxon supercedes some isolates from the invalid taxon “Leishmania siamensis”. A summary of all known cases of leishmaniasis with a corrected species identification is provided. Conclusions: Three species of parasites are now known to cause leishmaniasis is Thailand, L. martiniquensis and L. orientalis n. sp. in the subgenus Mundinia, which contains the type-species Leishmania enriettii Muniz & Medina, 1948, and a single case of Leishmania infantum Nicolle, 1908. This study now enables epidemiological and other investigations into the biology of these unusual parasites to be conducted. It is recommended that the use of the taxonomically invalid name “L. siamensis” should be discontinued. Keywords: Leishmania orientalis, Mundinia, Thailand, Cutaneous leishmaniasis Background study has been performed to date and there is little Leishmaniasis is an emerging disease in Thailand. The experience in diagnosis of leishmaniasis in Thailand. first confirmed autochthonous case was reported in To date a total of 22 parasitologically confirmed cases 1999 [1], but since then there have been further cases have been reported, and of these 19 have been identi- described that undoubtedly reflect a much higher fied using various molecular methods. There has been underlying prevalence of undiagnosed cases and asymp- one case of visceral leishmaniasis due to Leishmania tomatic infections, given that no active case detection infantum Nicolle, 1908 infection [2], but no subsequent reports. * Correspondence: [email protected]; [email protected] The most frequent parasite found so far is L. martini- 1 Department of Parasitology, Faculty of Medicine, Chiang Mai University, quensis Desbois, Pratlong & Dedet, 2014, accounting Chiang Mai, Thailand 5Division of Biomedical and Life Sciences, Faculty of Health and Medicine, for 15 of the identified cases. This parasite is named Lancaster University, Lancaster, UK after the Caribbean island of Martinique, where it was Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jariyapan et al. Parasites & Vectors (2018) 11:351 Page 2 of 9 first isolated [3, 4]. In some of the early Thailand re- Results portsthisisreferredtoas“L. siamensis”;however,there Case report are two problems with this usage. The first problem is The patient was a 57-year-old woman who lives in Chiang that “L. siamensis” has never been formally described, Klang District, Nan Province, northern Thailand. She is a sothenameistaxonomicallyinvalid(anomen nudum), gardener and has never been abroad, only travelling to without any type specimen for reference, and therefore Phitsanulok and Phijit, provinces near Nan in Thailand. technically should not have been used in any publica- The patient presented in May 2014 at Chiang Klang Hos- tions. The second problem is subsequent evidence that pital with a single skin nodule on her left cheek (1.0 × 1.5 the group of isolates that have previously been called cm), and also with crusting at the left angle of the mouth “L. siamensis” has been found not to be monophyletic, (Fig. 1a). No skin nodules in other sites of the body were and includes two taxa [5, 6]. One of these includes the observed. Two pieces of formalin-fixed skin biopsy from aforementioned parasites that appear identical to L. the cheek nodule (0.6 × 0.5 × 0.4 cm3 and0.5×0.5×0.2 martiniquensis and,sincethisisavalidspecies,this cm3) were sent to the Department of Pathology, Faculty of name takes precedence for these particular isolates. Medicine, Chiang Mai University for histopathological in- However, in the second taxon there are two isolates of vestigation. Histopathological analyses revealed epidermal “L. siamensis” that are very similar to each other but ulceration with a heavy, chronic inflammation of the der- different from L. martiniquensis based on molecular mis (Fig. 2a) and numerous intracellular small round or analysis, the PCM2 Trang strain from a patient in oval-shaped bodies, with the appearance of amastigotes southern Thailand [7], which is phylogenetically (1–2 μminwidthand2–4 μminlength)ofLeishmania distinct [6, 8], and a recent isolate from central spp. (Fig. 2b). A week later, a fresh skin biopsy from the Thailand [9]. These two isolates were both from nodule (0.4 × 0.5 × 0.3 cm3) was collected and sent to patients that were HIV-infected and presented with Department of Parasitology, Faculty of Medicine, Chiang disseminated cutaneous leishmaniasis, the former also Mai University for confirmation of diagnosis by parasite with visceral disease. culture and species identification. The skin biopsy sample Here we describe a case of simple cutaneous leish- was cultured in Schneider’s insect medium supplemented maniasis from northern Thailand that was caused by a with 20% foetal bovine serum (FBS) and 50 International parasite apparently very similar to those responsible for Units penicillin/ml, 50 μg/ml streptomycin at 25°C. Motile these two previous cases, and we formally name this as promastigotes were first observed on day 3 of the culture. Leishmania orientalis Bates & Jariyapan n. sp., thereby Therefore, the patient was confirmed as diagnosed with establishing a taxonomically valid name for these para- cutaneous leishmaniasis. She was treated with oral sites, and simultaneously eliminating the need to use amphotericin B at 1 mg/kg/day for 1 day and fluconazole the unavailable name “L. siamensis”. We place this new at 200 mg/day for 45 days. The skin lesion had disap- species in the recently erected Leishmania subgenus peared completely by six months after treatment (Fig. 1b). Mundinia Shaw, Camargo & Teixeira, 2016 [10]and Pre-treatment laboratory investigation showed only mild provide an updated identification of all known previous anaemia with a haemoglobin concentration of 10.9 g/dl, isolates from Thailand into L. infantum, L. martiniquen- white blood count of 7700 cells/mm3, and platelet count sis and L. orientalis n. sp. of 483,000/mm3. There was no hepatosplenomegaly or Fig. 1 Clinical presentation of cutaneous leishmaniasis. a A nodule on the cheek and a crusted sore in the angle of the lips of the patient before treatment, both arrowed. b The same view of the patient’s skin after treatment Jariyapan et al. Parasites & Vectors (2018) 11:351 Page 3 of 9 Fig. 2 Histopathology of skin biopsy from a nodule on the left cheek (Giemsa stain). a Low power magnification photomicrograph showing pseudo-epitheliomatous hyperplasia of the epidermis (arrows) and heavy chronic inflammation of dermis (starbursts). b High power magnification photomicrograph showing numerous Leishmania amastigotes within the cytoplasm of macrophages and in the extracellular matrix (arrows). Scale-bars: a, 200 μm; b,10μm palpable lymph nodes. Liver function was not investi- [11]. Although free-swimming individual promastigotes gated, renal function was within normal limits and were readily observed, rosettes and large aggregates of HIV serology was negative. The patient declined a promastigotes were prevalent in culture (Additional file 1: bone marrow biopsy for evaluation of visceral leish- Video S1). maniasis. She did not report any other underlying dis- ease, routine drug use, or any other symptoms, and Molecular analysis in general was in a good state of health. Four different sequences were analysed by polymerase chain reaction (PCR) and deoxyribonucleic acid Parasite characterization (DNA) sequencing: the ribosomal