Cutaneous leishmaniases (CL) constitute a wide range of cutaneous vector-borne diseases caused by diverse species of Leishmania genus parasites. This group represents one of the most neglected infectious diseases. In Old World, more than 1 million CL cases are reported each year, of which 80% occur in the Middle East and North Africa (MENA) region. Moreover, the MENA is at increased risk of emergence and epidemics due to the changes in epidemiological trends related to environmental or climate changes, migrations, or conflicts (reviewed by Tabbabi, 2019). Indeed, CL in MENA, caused by four species (L. major, L. tropica, L. infantum, and L. donovani), have complex epidemiology due to species diversity and co-endemicity and even co-sympatry, their different levels of prevalence, microfocal distribution, and many unknowns in transmission cycles.

Zoonotic cutaneous leishmaniasis (ZCL) due to L. major is endemic in all countries in the MENA region (reviewed by Tabbabi, 2019). It has diverse clinical manifestations and is known to be epidemic to hyperendemic in these countries while the transmission of other species encountered is less frequent to sporadic, with exceptions of Syria or Sudan where L. tropica and L. donovani are highly endemic respectively, and Morocco where L. tropica is increasingly prevalent (El Idrissi Saik et al., 2022). Furthermore, while several Sub-Saharan African countries face gaps in identifying the etiology of Leishmania species responsible for CL, others, such as Burkina Faso, Nigeria, Niger, and Cameroon, have reported L. major as the sole species causing CL in these regions (reviewed by Blaizot et al., 2024), a statement that may be revised by the increasing use of DNA assays for species detection and identification.

While CL is not fatal, it is highly morbid with disfiguring and/or lifelong scars leading to social stigma and psychological distress (Chahed et al., 2016; Elfaki et al., 2024). Furthermore, CL was recently called “the great imitator” (Gurel et al., 2020) as it could mimic a wide range of skin diseases, which makes it difficult to diagnose on the sole base of clinical manifestation. CL diagnosis is mainly done by microscopic direct examination (DE) of smears of Giemsa-stained lesion that requires experienced microscopists, time-consuming, and laborious when parasite loads are low. DE is 100% specific but is poorly sensitive (43%) (Mouttaki et al., 2014) and does not identify Leishmania species given the similar morphology of their amastigote forms. Only molecular DNA assays accurately identify the species. Etiologic diagnosis of CL is crucial as it is known that treatment protocols depend on the species (Arevalo et al., 2007; Mosimann et al., 2013); efficiency of CL treatment is also highly dependent on Leishmania species (Madusanka et al., 2022).

Several PCR-based methods have been developed for the specific and sensitive detection of Leishmania parasites responsible for CL (Table 1). These include conventional PCR assays (Rostami et al., 2020), real-time PCR, PCR-RFLP, and PCR sequencing, among others. While these methods can detect Leishmania parasites, species identification often requires complementary techniques, such as RFLP (Schönian et al., 2003; Bel Hadj Ali et al., 2021) or sequencing (Stevenson et al., 2010), to enable species identification. Real-time PCR is among the most highly sensitive methods for detecting Leishmania DNA (Stevenson et al., 2010; Nath-Chowdhury et al., 2016). However, the requirement for specialized equipment, such as real-time PCR machines, can limit its accessibility in resource-limited settings.

In low-resource or remote settings where there is a lack of equipped laboratories, molecular diagnostics with a point-of-care (POC) format would guarantee an accurate diagnosis and equitable and better access of patients to health care wherever they are. Actually, healthcare challenges in such settings mainly relate to infrastructure and budget (Maphumulo and Bhengu, 2019). This emphasizes the importance of point-of-care molecular diagnostics for prevention, timely patient management, and disease control.

Over the last decade, the POC format has been given a special attention, and a broad spectrum of technologies, reagents, test kits, and equipment were developed (Kozel and Burnham-Marusich, 2017). With the advent of PCR as the gold standard technology in molecular diagnostics for pathogen detection, a range of DNA amplification technologies have been developed to expand their use at the POC, thus allowing to address the need for POC CL testing, requiring minimal equipment while being fast in delivering DNA products. The most commonly used are the loop-mediated isothermal amplification (LAMP) (Erber et al., 2022; Soares et al., 2024; Taye et al., 2024) and the recombinase-based amplification [recombinase polymerase amplification (RPA) or recombinase-aided amplification (RAA)] (Bharadwaj et al., 2021; Khan et al., 2021; Travi et al., 2021; Mondal et al., 2016). RPA/RAA is an isothermal DNA amplification technology with a simple experimental design, by contrast to LAMP that requires four to six primer pairs, to amplify one DNA target. Indeed, RPA operates at low temperatures (37°C–42°C) and requires only two primers or two primers and a probe depending on the used chemistry (Piepenburg et al., 2006). RAA is another designation for this technology (Xue et al., 2020). Using RPA or RAA (according to the kit supplier) to develop molecular diagnostics potentially overcomes current limitations of cost, complexity, and resources while ensuring rapidity, efficiency, and accuracy of the results (Ghosh et al., 2021).

In this study, we aimed to develop a simple, rapid sensitive, and cost-effective L. major-specific DNA-based assay. The test based on an isothermal recombinase-based amplification coupled to lateral flow immuno-assay detection would contribute to diagnosing L. major-infected patients or their screening in remote areas.

Cutaneous leishmaniases represent a wide range of cutaneous diseases characterized by their diverse clinical manifestations. These diseases are not fatal, but they are highly morbid and have a negative psychological impact on patients due to the lesions, the disfiguring scars, and subsequent stigma (Chahed et al., 2016; Pires et al., 2019).

CL are poverty-related infections that occur mainly in low-income and middle-income countries (Moya-Salazar et al., 2021) where health centers lack essential equipment for their diagnosis. Routinely Leishmania parasites from CL lesions are detected by direct microscopy examination, a laborious technique that lacks sensitivity. In addition, this technique is not able to identify Leishmania species, an important step that should be taken into consideration to guide CL treatment and patient management (Arevalo et al., 2007; Mosimann et al., 2013). This highlights the need for POC CL diagnostics to ensure healthcare equity for patients wherever they are.

The World Health Organization’s (WHO) roadmap to 2030, in terms of Research and Development on diagnostics needs to cover all NTDs, pointed to inadequate existing tools. Concurrently, in this study, we aimed to enhance access to CL diagnosis in endemic areas by developing an isothermal amplification coupled with lateral flow detection of L. major-infecting parasites. This test intends to improve CL diagnosis in low-resource settings where infections due to L. major are predominant like, for instance, in North African countries, or highly endemic like in other countries in Africa, the Middle East, and Asia. According to WHO, an ideal POC test should be affordable, sensitive, specific, user-friendly, and equipment-free and delivered to patients (ASSURED) (Kosack et al., 2017). In this study, we provided a proof of concept in a laboratory environment that RPA/LF assays are suitable for the detection and identification of L. major in clinical samples. The method has several inherent advantages; it is simple, rapidly processed, and needs low-cost basic laboratory equipment. The time to result delivery is 1 h, and it includes amplification, detection, and identification of L. major parasites. The RPA ready-to-use lyophilized reagents provided in a single tube make the reactions simple and rapid to perform without any expertise needed. In addition, the design of the assay is simpler than LAMP, an isothermal amplification technology that requires four to six primer pairs. It is also known that RPA tolerates PCR inhibitors (Kersting et al., 2014; Krõlov et al., 2014; Rosser et al., 2015; Tan et al., 2022), so it could be coupled to minimally processed and simplified methods for DNA extraction (Saldarriaga et al., 2016; Moore and Jaykus, 2017; Munawar et al., 2020). All these advantages are favoring factors for the widespread adoption of the RPA/LF platform to support POC diagnosis of cutaneous leishmaniases.

RPA technology was used for the detection of diverse pathogens including bacteria, viruses, and parasites (Kersting et al., 2014; Krõlov et al., 2014; Rosser et al., 2015; Munawar et al., 2020). During the last decade, many studies reporting RPA assays for the detection of Leishmania parasites were published (Mondal et al., 2016; Saldarriaga et al., 2016; Gunaratna et al., 2018; Cossio et al., 2021; Ghosh et al., 2021; Khan et al., 2021; Mesa et al., 2021). These assays use protocols with different end-point readouts including LF (Saldarriaga et al., 2016) or real-time portable tubes scanner (Mondal et al., 2016) to detect, namely, L. donovani (Mondal et al., 2016) and L. viannia parasites (Cossio et al., 2021; Travi et al., 2021; Saldarriaga et al., 2016). A recent study evaluated a mobile laboratory suitcase that includes an RPA method for the detection of L. donovani parasites in VL and PKDL cases in a multi-country phase 2 study and demonstrated the accuracy of this test for VL and PKDL diagnosis (Ghosh et al., 2021). However, investigations using RPA technology for CL diagnosis were lacking. Thus, we intended to develop a CL RPA test that could be used as a screening assay to complement other DNA methods in countries/regions where L. major is highly prevalent. To our knowledge, this is the first report describing an RPA test that specifically detects L. major parasites. It is known that L. major infections occur mostly in rural arid areas of North Africa and the Middle East where it frequently causes epidemics (Gradoni, 2017). L. major is also a widespread species across Africa and Asia. All these transmission areas typically lack healthcare infrastructure; point-of-care diagnostics that could be deployed as portable solutions, relying on basic equipment, is therefore needed for timely patient management and early alert on emergence. However, it is clear that a cost-effectiveness analysis of the RPA/LF assays is requested as it is another crucial factor for diagnostics scalability and implementation in resource-limited settings (Aerts et al., 2019).

In this study, we report on the development of an L. major RPA/LF assay in which we encountered two major challenges. The first one was the design of primers/probes that selectively detect L. major strains. RPA is known to tolerate mismatches in priming sites (Boyle et al., 2013; Daher et al., 2015). However, Boyle et al. (2013) were unable to amplify one HIV variant using an RPA reaction because of the presence of six nucleotide changes and one insertion in the primer/probe binding sites (Boyle et al., 2013). Thus, our strategy has relied on selecting an extensive number of mismatches between L. major and other Leishmania DNAs, flanking our target fragment, to maximize the chances to amplify selectively L. major DNA. In fact, the finally retained assay, m22-RPA, targeted a non-coding fragment in L. major, originally identified as an L. infantum-specific coding gene. In L. major, the selected primers/probe sites include five mismatches in the forward primer, nine in the reverse one, and nine in the probe when compared to L. infantum or L. tropica DNA sequences. These mismatches are specific to L. major strains and are present in all sequenced L. major DNA fragments amplified covering strains from North Africa, Asia, and the Middle East and in the sequences extracted from the database of two Middle Eastern (Friedlin and LV39) and one African strains (SD75). This confirms the relevance of the primers and probe set for a consistent reactivity within the species. In addition, in the cases of L. aethiopica and L. donovani, the priming site for the reverse primer was absent, while for the other species, the priming sites exhibited significant divergence from the L. major sequence, with less than 80% similarity. The UPGMA tree is consistent with these results, further highlighting the taxonomic potential of the selected m22 target. It was further confirmed experimentally with the panel of Leishmania species and strains tested. In addition, our results corroborated that efficient RPA reaction leading to species-specific amplification may depend on the number and positions of mismatches (Daher et al., 2015). The type of nucleotide involved in the mismatch was also shown to significantly impact RPA reaction efficiency, with primers’ terminal cytosine–thymine and guanine–adenine mismatches having the most detrimental effects (Higgins et al., 2022).

The second challenge was the formation of primers and probe/primer dimers during the RPA reaction. Indeed, the length of the primers/probes together with the low reaction temperature contribute to primer dimers noise that could appear in the LF-negative tests and lead to confusing results. Some studies opted for a post-RPA heating step (Liu et al., 2017) or used self-avoiding molecular recognition systems (SAMRS) oligonucleotides (Sharma et al., 2014) to overcome this noise issue. In our case, we have aimed at delivering simple assays performing at a single and low temperature. Therefore, to resolve this problem, we have designed and tested many primers to produce reliable assays and clear negative results in the LF device with the negative samples and the control samples without template DNA. We also carefully selected the oligonucleotides after checking their in silico properties (secondary structure, GC content, and repeats) but also their specificity, by performing a blast analysis with all DNA sequences available in NCBI and TritrypDB databases. It was reported that the non-specific background DNA could interfere with RPA reactions and diminish its efficiency depending on the primers/probes used and their level of interaction with the background DNA (Rohrman and Richards-Kortum, 2015). In this study, we used CL samples during the whole process of development in order to verify potential cross-reactivity of the tests under development with the human DNA and other pathogens that could be present in cutaneous lesions. Then, we extended the study by evaluating our assays on a larger number of DNAs extracted from cutaneous samples from different geographic origins in countries from MENA, including Tunisia, Morocco, and Lebanon. We used harmonized protocols for sampling. Tunisian, and Moroccan teams succeeded in using lesions scrapping for the detection and identification of parasites. However, the Lebanese team performed DNA extraction on swabs taken on the scrapped lesions. As standard techniques for RPA assay validation, we used the combination of direct examination and ITS1 PCR for the detection of Leishmania parasites, and ITS1 PCR-HaeIII RFLP for parasites identification. Our L. major RPA-/LF-specific test was able to detect 100% of L. major infections within CL patients, independently of the parasite load estimated by microscopy on the slides. Importantly, the test performed well in all cases where the parasite load was low, highlighting the relevance of the assay to support diagnosis as a complement or alternative to microscopy. Among the negative samples, seven showed positive results. These samples belong to patients with CL-like clinical features.

Four of them (PER340295, PER340337, PER570110, and PER570107) showed positive results with the generic Lei70 PCR. These samples are considered true positive. The three remaining samples (PER340294, PER570106, and PER570104) were negative with the generic Lei70 PCR. Two of these patients (PER570106 and PER570104) exhibited a faint β-globin amplification band, suggesting poor DNA quality or the presence of PCR inhibitors in the extracted DNA. Unlike PCR, however, it is well documented that a wide range of inhibitors in the reaction can be tolerated, making it a more robust method for challenging samples (Kersting et al., 2014; Krõlov et al., 2014; Rosser et al., 2015; Tan et al., 2022). For patient PER340294, the parasite load in the lesion sampled may be very low and therefore not detectable by the standard molecular tests used nor by microscopic direct examination. Two hypotheses could be stated in this case. The first one is that these samples are false positives, and then, our test is not 100% specific (Sp = 88%). The second hypothesis is that these samples are true positive, and then, our test is more sensitive than the used reference diagnosis tests.

Given the ongoing climatic change impacting the complex epidemiology of Leishmaniases, particularly in Africa and the Middle East, there is a growing risk of the emergence of new Leishmania species in areas where L. major is prevalent, which may lead to co-sympatry, mixed infections, and generation of interspecific hybrids. Expansion of L. major into other areas so far free of leishmaniases could also occur. Accurate diagnosis of leishmaniases, L. major cases in particular, is therefore increasingly important. In instances where the developed test is negative for L. major, additional tests should be considered to confirm whether the sample is truly negative or is infected with another Leishmania species. It was shown that using multiple diagnostics tests achieves a greater specificity than any single test (Gass, 2020). In addition, establishing an algorithm to guide diagnosis should be envisaged as it may depend on the tested population, disease prevalence, and specimens (Hurt et al., 2017). Developing a simple and rapid molecular test to screen L. major CL infections in regions where this species is predominant is in line with such a purpose.

Expanding the RPA-LF platform to encompass CL cases due to other Leishmania species of relevance is in our perspectives, permitting us to detect and identify all CL cases including those that are caused by mixed infections or by natural interspecific hybrids such as the ones recently reported in Ethiopia (Hadermann et al., 2023). As a Leishmania infantum RPA/LF assay has already been developed and validated for the diagnosis of visceral leishmaniasis (Castellanos-Gonzalez et al., 2015), it would be worthwhile to explore its potential application for cutaneous leishmaniasis caused by this species, and the closely related L. donovani. Furthermore, as a future direction, the development of L. tropica and L. aethiopica species-specific RPA/LF assays could address a critical diagnostics gap, particularly in the African and Middle Eastern region, where L. major, L. tropica, L. aethiopica, and L. infantum/L. donovani are the most prevalent species causing cutaneous leishmaniases. Expanding the RPA/LF platform to cover these key species would greatly enhance diagnostics capacity in these endemic areas. Moreover, incorporating into the assays an internal control for human DNA compatible with an LF detection will enhance the reliability of the test by verifying its validity and confirming negative results.

Additional developments to our current RPA/LF assay and future platform should be considered, bringing them closer to the point of care by shortening further the timeline of the workflow, reducing the costs, and automating the readouts. Indeed, the current DNA extraction step presents a challenge for implementing the test in resource-limited settings and its scalability. Therefore, the integration of an upstream simple DNA extraction and a less costly method within the workflow, as described by Mondal et al. (2016), will enhance the feasibility and usability of the test at the point of care. Moreover, developing and using a lateral flow smartphone reader application could significantly enhance test interpretation and minimize observer bias. This technology is particularly valuable for resolving ambiguities associated with faint bands and overcoming challenges like the one we encountered during our evaluation of the analytical limit of detection of assay.

We have developed a platform of an L. major-specific and sensitive RPA/LF assays. Through this proof-of-concept study, we have provided evidence of the efficacy of our RPA-LF assays for the detection and identification of L. major species in CL lesions. Our tests could be expanded/used to complement other recombinase-based amplifications to detect and identify Leishmania parasites in a point-of-need context.