Leishmaniasis is a disease caused by obligate intracellular protozoan and flagellated parasites of the genus Leishmania, belonging to the family Trypanosomatidae, order Kinetoplastidia. The parasite is transmitted to humans via the bite of infected female sandflies of the genus Phlebotomus and Lutzomyia [1]. Leishmaniasis is endemic in nearly 100 countries [2]. They are geographically divided into two groups: "New World" leishmaniasis - typically found in America - and the "Old World" leishmaniasis, which is endemic in several tropical countries of Asia and Africa, and also in countries of the Mediterranean region [3, 4]. However, the spread of endemic areas owing mostly to the change in sandfly habitats and human mobility are of recent concerns [5].

The main clinical forms of leishmaniasis are cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leishmaniasis (VL) and its dermal complication called post-Kala-azar dermal leishmaniasis (PKDL) [1]. CL is most commonly caused by L. major, L. tropica, L. aethiopica, L. amazonensis, L. mexicana, L. braziliensis, L. panamensis and L. guyanensis, depending on the geographical spread of the species in endemic regions [6, 7]. Common pathological consequences include various skin manifestations such as ulcerations covered by crusts, raised margins, and erythematous papules, plaques and nodules. Mucocutaneous leishmaniasis (MCL) can be caused by L. braziliensis, L. panamensis and L. guyanensis and is developed often after CL [8]. Visceral leishmaniasis (VL) or "Kala-azar" caused by the L. donovani complex is the most fatal form of leishmaniasis if left untreated and is highly endemic in the Indian subcontinent and in East Africa [3]. Parasite infection in viscerotropic organs can lead to splenomegaly, pancytopenia, hepatomegaly, fever, weight loss, and mucosal bleeding. PKDL is an atypical and chronic dermatosis that often develops as sequelae of VL - mostly after treatment. PKDL manifestation is in the form of painless macular or papulo-nodular lesions or a mix of both that harbour parasites. Another less common type is the diffuse cutaneous leishmaniasis (DCL) caused by L. amazonensis and L. aethiopica [1], which features typical nodular or plaque-shaped solitary lesions.

The diagnostic protocol for leishmaniasis involves provisional diagnosis, which is based on clinical presentation and symptoms, usually followed by a confirmatory test, which is usually either culture, microscopy or polymerase chain reaction (PCR). Cases of leishmaniasis may pose a diagnostic dilemma as the signs and symptoms are often similar to those of other infectious and/or genetic diseases, particularly in imported cases and for species of Leishmania, which are non-endemic at a particular region. For instance, pathological manifestations of VL often mimic symptoms commonly resulting from bacterial and other protozoan parasites (e.g., tuberculosis, toxoplasmosis), viral infections, malignancies (e.g., leukaemia, lymphoma), liver cirrhosis and autoimmune disorders such as Systemic Lupus Erythematosus and autoimmune hepatitis [9], [10], [11], [12]. Differential diagnosis of PKDL usually includes leprosy, vitiligo and miliaria rubra [13] while clinical appearance of CL and MCL often resembles bacterial (lupus vulgaris) and fungal (sporotrichosis) infections, autoimmune disorders (Localized Lupus Erythematosus), various cancers (oral carcinoma, T-cell lymphoma, squamous cell carcinoma) and tumors, as discussed elsewhere [14].

In such cases, parasite detection using culture and/or microscopy could be limiting as for sensitivity [15], whereas PCR methods are mostly targeted for only one species or a particular form of leishmaniasis. Moreover, antibody detection tests such as rk39 rapid diagnostic test largely cannot differentiate between active infection and previous episodes [16, 17], while anti-leishmanial IgE, IgM and IgG have shown promises for detecting recent infection [18], [19], [20]. On the other hand, intradermal test (also called leishmanin skin test), which is possible for CL or MCL is about 95% sensitive and 96-100% specific, however it cannot diagnose VL. Of note, leishmanin antigen is no longer produced under good manufacturing practice conditions anywhere in the world. Consequently, the use of skin tests has declined in favor of serological and molecular tests [21].

PCR is known to be more sensitive, and to be a specific and robust test for detection of active infection [22], [23], [24], [25], however, it is limited to laboratories with relatively complex technical equipment [26]. A low cost and easy to establish simple molecular diagnostic method such as recombinase polymerase amplification (RPA) would leverage the setup at the often resource-poor settings for the detection of Leishmania spp in endemic countries. RPA runs significantly faster (15-20 minutes) and at a constant temperature of about 37-42°C to produce results that demonstrated sensitivity and specificity comparable to PCR based methods. The unique feature of the RPA amplification lies in the formation of recombinase enzyme-primer complexes that upon homology in double-stranded DNA template, performs strand-invasion to insert the primer sequence. In this way, the strand-displacing polymerase can ultimately bind to the primer and synthesize the desired amplicon [27]. For real-time detection, an exo-probe is used and the emitted fluorescence signal can be measured. The lyophilized pellets of the enzyme mix are relatively stable in ambient temperature requiring no cold-chain maintenance, which facilitates its operation in mobile settings in a portable suitcase [28].

Several RPA assays have been developed to date that are either specific for one or a few species of Leishmania. We have previously developed RPA assay targeting the kDNA, which was found to be very sensitive and specific for the detection of L. donovani that typically cause visceral- and post-Kala-azar dermal leishmaniasis, and cross-reactive to L. major, L. aethiopica and L. infantum but not the other infectious species [29]. Furthermore, this qualitative RPA assay was found to have absolute agreement with an established real-time qPCR assay in terms of sensitivity and specificity, and so had the quantitative RPA assay that used the same target [30]. The aim of the present study was to develop a pan-Leishmania RPA assay for the detection of all Leishmania pathotypes. Two RPA assays targeting the 18S ribosomal RNA gene- (18S rRNA-RPA assay) and the cathepsin L-like cysteine proteinase B (Cpb) gene (Cpb-RPA assay) were developed. Their ability to detect common pathogenic species was tested, and the better performer between the assays was selected for clinical validation.