Leishmaniasis is a neglected tropical disease, transmitted by the bite of infected female sandflies and affecting the poor population. Current pharmacotherapy has remained largely unchanged for decades, and there are parasitic strains resistant to these conventional treatments. Thus, there is an urgent need to identify molecular targets to guide the rational design of new drugs. Thus, cysteine protease B (CPB) emerges as a promising target due to its roles in pathogenesis, virulence, and in modulating the host immune response. Given this potential, this review presents recent advances in CPB inhibitors, examining scaffolds, their structure-activity relationships (SARs), and the structural elements that confer selectivity for the parasitic target. Aziridine analogs and dipeptidyl nitriles stand out, and the SAR studies presented here indicate that structural modifications in the P1, P2, and P3 binding subsites influence compound affinity, thereby optimizing molecular fit in the enzyme. The stereochemical configuration (S,S) of the inhibitors is also essential for potency. Regarding selectivity, the Tyr210 residue is present in CPB but not in human cathepsin L. Taken together, these structural and mechanistic findings offer new perspectives for advancing medicinal chemistry targeting CPB, in the development of safer, more selective, and more effective antileishmanial agents.

Malaria is a tropical disease caused by protozoa of the genus Plasmodium and is responsible for several deaths worldwide. Current therapies are limited by the high incidence of adverse effects and the rising prevalence of parasite drug resistance, underscoring the need for research into new chemical scaffolds that can overcome these limitations and for the identification of novel drug targets to advance antimalarial development. In this context, quinazolines show promising potential as new antimalarials. Therefore, in this study, a new series of quinazolin-4-(3H)-one analogs was synthesized and evaluated for antiplasmodial activity. As a result, 36 compounds were synthesized and characterized, of which 3d, 3e, 4a, and 4e showed promising activity in assays against P. falciparum-3D7HT-GFP (IC50 = 4.36, 2.26, 1.70, and 4.10 μM) with no cytotoxicity (CC50 = 44.56, 22.91, 26.42, and 48.15 μM) and acceptable selectivity indexes (10.22, 10.14, 15.55, and 11.75). The compounds were evaluated against Leishmania donovani and Trypanosoma congolense but did not inhibit these parasites, suggesting that this chemical scaffold is selective for plasmodial targets. Accordingly, molecular modeling proposed N-myristoyltransferase (NMT) as a potential target and identified the specific binding mode of compound 4a for P. falciparum NMT (PfNMT) relative to L. donovani NMT (LdNMT), T. congolense NMT (TcNMT), and Homo sapiens NMT (HsNMT). It was found that the presence of Leu411, Leu369, Ser337, and Phe336 in PfNMT, substituted by Met412, Val370, Val338, and Tyr337 in LdNMT, and Met445, Tyr370, Ile371, and Tyr370 in TcNMT, may be related to the predicted target selectivity and the greater affinity of 4a for PfNMT. Finally, molecular dynamics simulations suggest that 4a is most stable against PfNMT, and MM-PBSA calculations indicate a binding energy for this target (ΔGbinding = -130.873 kJ/mol). These findings corroborate the promising potential of 4a and support its proposed selectivity against PfNMT, yielding a scaffold that can be explored in subsequent optimization studies.

Concomitant immunity (CI) can be viewed as an example of coevolution between the microorganisms and their long-lived hosts. Such an ecological trade-off may be advantageous to both the microbe and the host, as it allows protozoa and helminths to maintain their genetic features while providing the host, particularly mammals, with long-standing protection against reinfection by the same microbe. In Leishmania infection, CI is the mechanism whereby parasites remain at low-level infection in the host, which develops a strong immune reaction that protects against reinfection. Mechanistically, several CD4+ T cell populations seem to be involved in such fine immune responses. While immunity against Leishmania, Plasmodium, Taenia, Schistosoma, and Echinococcus is well known, the mechanisms of CI involving these pathogens have been poorly studied. Finally, the phenomenon of CI should be carefully assessed in the design of novel vaccine preparations.

Abstract Despite advances in the treatment of head and neck cancer, squamous cell carcinoma of the oral cavity remains primarily a surgical disease with few effective systemic therapies. Oral cavity tumors harbor both innate and acquired resistance mechanisms to cytotoxic chemotherapy. Additionally, the tumor microenvironment (TME) has limited immune cell infiltration, resulting in low response rates to immune checkpoint inhibitors. Given the need for novel systemic therapies for oral cavity squamous cell carcinoma, we investigated the role of liposomal doxorubicin (Doxil®) as both a cytotoxic agent and an immunomodulatory agent. We utilized the MOC2 syngeneic murine model of oral cavity squamous cell carcinoma, an aggressive tumor model with resistance to immune checkpoint blockade. We demonstrated that Doxil has moderate activity as a single agent in vivo for C57BL/6J mice harboring MOC2 flank tumors. We next performed flow cytometric analysis to characterize the changes in immune cell populations in the TME after treatment with Doxil. We found significantly increased numbers of innate immune cells including NK cells and myeloid cells. Multiplex immunofluorescence was also used to confirm the increase in myeloid cell tumor infiltration upon Doxil treatment. Given the changes seen in the tumor immune microenvironment, we hypothesized Doxil may improve response to immune checkpoint blockade. Therefore, we treated C57BL/6J mice inoculated with MOC2 flank tumors with Doxil alone or in combination with radiation therapy (RT) and/or anti-CTLA-4 therapy. While RT or anti-CTLA-4 alone had modest anti-tumor activity, combining either RT or anti-CTLA-4 with Doxil significantly reduced tumor growth. Moreover, the triple combination therapy of Doxil, RT, and anti-CTLA-4 had an increased effect compared to the dual therapy of Doxil and anti-CTLA-4. This included several complete responses, which resulted in a significant improvement in survival. Triple combination therapy also reduced both local and distant metastatic burden compared to single agent and dual combination therapy. Next, we selectively inhibited CD8+ T cells, myeloid cells, or NK cells to determine which immune cell populations contribute to the immunomodulatory activity of Doxil. We observed that inhibition of NK cells resulted in increased tumor growth as well as decreased survival, suggesting that Doxil-mediated infiltration of NK cells into the TME contributes to response to anti-CTLA-4 therapy. Taken together, these data provide a rationale for combining liposomal doxorubicin with anti-CTLA-4 therapy for the treatment of oral cavity squamous cell carcinoma. Citation Format: Jennifer L Anderson, Fabio H Brasil Da Costa, Allison Nipper, Laxman Devkota, Rohan Bhavane, Ratna Veeramachaneni, Sofia Cortes, Neeraja Dharmaraj, Sarah Latka, Andrew Badachhape, Renuka TR Menon, Prajwal Bhandari, Ketankumar Ghaghada, Simon Young, Ananth Annapragada, Andrew Sikora. Liposomal doxorubicin improves response to immune checkpoint blockade by enhancing innate immunity in a murine model of oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A119.

Neglected tropical diseases (NTDs) constitute a group of infectious diseases that severely affect the health of impoverished populations, and the health, economies, and health systems of affected countries. Leishmaniasis and human African trypanosomiasis (HAT) are particularly notable, and malaria, despite not being neglected, is part of the “big three” (HIV, tuberculosis, and malaria) with high incidence, increasing the probability of infection by NTDs. Therefore, efforts are ongoing in the search for new drugs targeting the enzyme N-myristoyltransferase (NMT), a potential drug target that has been explored. Thus, we provide a review here that highlights the epidemiological data for these diseases and the importance of discovering new drugs against these agents. Here, the importance of NMT and its inhibitors is clear, with this study highlighting thiochromene, pyrazole, thienopyridine, oxadiazole, benzothiophene, and quinoline scaffolds, identified by computational methods followed by biological assays to validate the findings; for example, this study shows the action of the aminoacylpyrrolidine derivative 13 against Leishmania donovani NMT (IC50 of 1.6 nM) and the pyrazole analog 23 against Plasmodium vivax NMT (IC50 of 9.48 nM), providing several insights that can be used in drug design in further work. Furthermore, the selectivity and improvement in activity are related to interactions with the residues Val81, Phe90, Tyr217, Tyr326, Tyr345, and Met420 for leishmaniasis (LmNMT); Tyr211, Leu410, and Ser319 for malaria (PvNMT); and Lys25 and Lys389 for HAT (TbNMT). We hope our work provides valuable insights that research groups worldwide can use to search for innovative drugs to combat these diseases.

Background Hormone receptor (HR)+ breast cancer (BC) causes most BC-related deaths in the US.1 Standard treatment for non-metastatic disease involves surgery plus adjuvant hormonotherapy. However, approximately 50% of patients ultimately relapse and require additional lines of treatment including chemotherapy, which is unfortunately associated with limited clinical benefits and severe toxicity. In HR+ BC patients, the efficacy of immunotherapy has also been disappointing so far. Indeed, objective responses to PD-1 blockade with pembrolizumab in women with HR+ BC have been in the range of 5–10%, with no clear advantage on survival. Thus, resistance to PD-1 blockers constitutes a major obstacle towards the implementation of immunotherapy in HR+ BC patients. Methods To obtain insights into the immunological alterations accompanying disease relapse in HR+ BC exposed to PD-1 blockade, we harnessed a unique endogenous model of BC driven in immunocompetent mice by progesterone and a carcinogen. This model recapitulates key aspects of human luminal B BC, including a relatively ´cold´ microenvironment, hence limited sensitivity to PD-1 blockage.2 To overcome PD-1 resistance we treated mice with Flt3-L to stimulate the maturation of cross-presenting dendritic cells, and radiation therapy that can act as an adjuvant to inflame tumor micro-environment.3 In parallel, we tried to achieve complete control of the primary tumor by identifying ablative doses of fractionated radiation therapy (RT). Results Immunotherapy (PD-1 + Flt3L), despite slowing down the tumor growth, failed to improve the overall survival (OS) of mice elicited by RT alone in a unique mouse model of HR+ BC, potentially linked to an accrued systemic immunosuppression (T cells exhaustion, and increase of Tregs). The addition of CTLA-4, even though providing an initial response, failed to improve the tumor growth and OS, due to immunoresistance (immature macrophages, and decrease in T cells and NK cells at the systemic level). Partially ablative RT doses were able to improve the OS and will be combined with PD-1 + Flt3L in the near future. Conclusions Breaking through resistance of HR+ tumors to PD-1 blockers can direct strategies to overcome resistance in HR+ BC patients, the majority of BC patients. If successful, this can inform therapeutic approaches to enable superior therapeutic responses in patients with HR+ BC, hence significantly reducing BC-related deaths. References Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70:7–30. Buque A, Bloy N, Perez-Lanzon M, Iribarren K, Humeau J, Pol JG, Levesque S, Mondragon L, Yamazaki T, Sato A, Aranda F, Durand S, Boissonnas A, Fucikova J, Senovilla L, Enot D, Hensler M, Kremer M, Stoll G, Hu Y, Massa C, Formenti SC, Seliger B, Elemento O, Spisek R, Andre F, Zitvogel L, Delaloge S, Kroemer G, Galluzzi L. Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer. Nat Commun. 2020;11:3819. Deng L, et al. STING-Dependent Cytosolic DNA Sensing Promotes Radiation- Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors. Immunity. 2014;41:843–852

Malaria, leishmaniasis and Chagas disease are vector-borne protozoal infections with a disproportionately high impact on the most fragile societies in the world, and despite malaria-focused research gained momentum in the past two decades, both trypanosomiases and leishmaniases remain neglected tropical diseases. Affordable effective drugs remain the mainstay of tackling this burden, but toxicicty, inneficiency against later stage disease, and drug resistance issues are serious shortcomings. One strategy to overcome these hurdles is to get new therapeutics or inspiration in nature. Indeed, snake venoms have been recognized as valuable sources of biomacromolecules, like peptides and proteins, with antiprotozoal activity. This review highlights major snake venom components active against at least one of the three aforementioned diseases, which include phospholipases A2, metalloproteases, L-amino acid oxidases, lectins, and oligopeptides. The relevance of this repertoire of biomacromolecules and the bottlenecks in their clinical translation are discussed considering approaches that should increase the success rate in this arduous task. Overall, this review underlines how venom-derived biomacromolecules could lead to pioneering antiprotozoal treatments and how the drug landscape for neglected diseases may be revolutionized by a closer look at venoms. Further investigations on poorly studied venoms is needed and could add new therapeutics to the pipeline.

Introduction: The immunosuppressive tumor microenvironment (TME) in oral cancer inhibits response to conventional treatment (surgery +/- chemoradiotherapy) and check-point inhibitor (αPD1) immunotherapy. The chemotherapeutic agent doxorubicin and its PEGylated liposomal nanoparticle formulation (PLD) have been shown to target myeloid derived suppressor cells (MDSCs) in the TME. In this preclinical work, we investigated whether PLD improves response to radio-immunotherapy in a highly aggressive and immunologically ‘cold’ oral cancer model. Experimental Procedure: Studies were performed in the MOC2 syngeneic mouse model of oral cancer. Mice were randomized to one of seven treatment groups: αPD1, Radiation (XRT), αPD1+XRT, PLD, PLD+αPD1, PLD+XRT, PLD+αPD1+XRT. High-resolution nanoparticle contrast-enhanced computed tomography (nCECT) imaging was performed, and whole-body T2-weighted magnetic resonance imaging (MRI) used to monitor primary tumor response and development of metastases. Response evaluation criteria in solid tumors (RECIST) were used to evaluate treatment outcomes. Results: Tumors in all non-PLD groups were treatment resistant and grew progressively, consistent with the immunologically ‘cold’ MOC2 model. In contrast, all PLD groups showed tumor regression with 25-38% complete responders (CR) and 12-88% partial responders (PR) while all animals in non-PLD groups showed progressive disease (PD). MR imaging identified a high incidence of regional and distant metastases in the non-PLD groups while the PLD groups showed decreased regional and no distant metastases (Table 1). Presence of metastases was confirmed by nCECT imaging and histopathological analysis. Conclusion: PLD improved response to radio-immunotherapy in a highly aggressive immunologically ‘cold’ mouse model of oral cancer while simultaneously preventing disease progression and metastasis. Citation Format: Laxman Devkota, Rohan Bhavane, Andrew Badachhape, Ratna Veeramachaneni, Renuka Menon, Prajwal Bhandari, Sofia Cortes, Fabio Henrique Brasil Da Costa, Ketan Ghaghada, Simon Young, Andrew G. Sikora, Ananth V. Annapragada. PEGylated liposomal doxorubicin improves oral cancer response to radio-immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5174.

Canine leishmaniosis (CanL) caused by Leishmania infantum is an important zoonosis in southwestern European countries where this disease is endemic, and dogs, as domestic animals in close contact with humans, are the reservoir hosts for the parasite. In Portugal, CanL is of relevant veterinary concern. The previous national study revealed an overall seroprevalence of 6.3%. Since then, new prophylactic measures, such as vaccines, have been introduced in Europe. The aim of this study was to update seroprevalence for Leishmania infection and reassess risk factors in Portugal. A cross-sectional study was conducted from January–March 2021 with 1860 client-owned dogs from continental Portugal. A questionnaire and whole blood samples on filter paper were collected and a direct agglutination test was used to calculate anti-Leishmania antibody titres. True seroprevalence was 12.5% (95% CI 10.3–13.2%). Potential risk factors associated with L. infantum infection in dogs were age ≥ 2 years (aOR = 1.68, 95% CI 1.1–2.6) and residing in the interior regions of the country (aOR = 1.92, 95% CI 1.3–2.9) and non-use of repellents (aOR = 1.75, 95% CI 1.2–2.5). The key to controlling CanL and its impact on Public Health in endemic areas lies in continuous implementation of prophylactic measures, through the correct use of repellents/insecticides and vaccines and early detection and monitoring of infected dogs.

Background Surveillance of human leishmaniasis in Europe is mostly limited to country-specific information from autochthonous infections in the southern part. As at the end of 2021, no integrated analysis has been performed for cases seen across centres in different European countries. Aim To provide a broad perspective on autochthonous and imported leishmaniasis cases in endemic and non-endemic countries in Europe. Methods We retrospectively collected records from cutaneous, mucosal and visceral leishmaniasis cases diagnosed in 15 centres between 2014 and 2019. Centres were located in 11 countries: Belgium, France, Germany, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Data on country of infection, reason for travelling, infecting species, age and sex were analysed. Results We obtained diagnostic files from 1,142 cases, of which 76%, 21% and 3% had cutaneous, visceral, and mucosal disease, respectively. Of these, 68% were men, and 32% women, with the median age of 37 years (range: 0–90) at diagnosis. Visceral leishmaniasis was mainly acquired in Europe (88%; 167/190), while cutaneous leishmaniasis was primarily imported from outside Europe (77%; 575/749). Sixty-two percent of cutaneous leishmaniasis cases from outside Europe were from the Old World, and 38% from the New World. Geographic species distribution largely confirmed known epidemiology, with notable exceptions. Conclusions Our study confirms previous reports regarding geographic origin, species, and traveller subgroups importing leishmaniasis into Europe. We demonstrate the importance of pooling species typing data from many centres, even from areas where the aetiology is presumably known, to monitor changing epidemiology.

Leishmaniasis remains one of the ten Neglected Tropical Diseases with significant morbidity and mortality in humans. Current treatment of visceral leishmaniasis is difficult due to a lack of effective, non-toxic, and non-extensive medications. This study aimed to evaluate the selectivity of 12 synthetic endoperoxides (1,2,4-trioxolanes; 1,2,4,5-tetraoxanes) and uncover their biochemical effects on Leishmania parasites responsible for visceral leishmaniasis. The compounds were screened for in vitro activity against L. infantum and L. donovani and for cytotoxicity in two monocytic cell lines (J774A.1 and THP-1) using the methyl thiazol tetrazolium assay. Reactive oxygen species formation, apoptosis, and mitochondrial impairment were measured by flow cytometry. The compounds exhibited fair to moderate anti-proliferative activity against promastigotes of the 2 Leishmania species, with IC50 values ranging from 13.0 ± 1.7 µM to 793.0 ± 37.2 µM. Tetraoxanes LC132 and LC138 demonstrated good leishmanicidal activity on L. infantum amastigotes (IC50 13.2 ± 5.2 and 23.9 ± 2.7 µM) with low cytotoxicity in mammalian cells (SIs 22.1 and 118.6), indicating selectivity towards the parasite. Furthermore, LC138 was able to induce late apoptosis and dose-dependent oxidative stress without affecting mithocondria. Compounds LC132 and LC138 can be further explored as potential antileishmanial chemotypes.

Leishmaniases are among the most impacting neglected tropical diseases. In attempts to repurpose antimalarial drugs or candidates, it was found that selected 1,2,4-trioxanes, 1,2,4,5-tetraoxanes, and pyrazole-containing chemotypes demonstrated activity against Leishmania parasites. This study reports the synthesis and structure of trioxolane–pyrazole (OZ1, OZ2) and tetraoxane–pyrazole (T1, T2) hybrids obtained from the reaction of 3(5)-aminopyrazole with endoperoxide-containing building blocks. Interestingly, only the endocyclic amine of 3(5)-aminopyrazole was found to act as nucleophile for amide coupling. However, the fate of the reaction was influenced by prototropic tautomerism of the pyrazole heterocycle, yielding 3- and 5-aminopyrazole containing hybrids which were characterized by different techniques, including X-ray crystallography. The compounds were evaluated for in vitro antileishmanial activity against promastigotes of L. tropica and L. infantum, and for cytotoxicity against THP-1 cells. Selected compounds were also evaluated against intramacrophage amastigote forms of L. infantum. Trioxolane–pyrazole hybrids OZ1 and OZ2 exhibited some activity against Leishmania promastigotes, while tetraoxane–pyrazole hybrids proved inactive, most likely due to solubility issues. Eight salt forms, specifically tosylate, mesylate, and hydrochloride salts, were then prepared to improve the solubility of the corresponding peroxide hybrids and were uniformly tested. Biological evaluations in promastigotes showed that the compound OZ1•HCl was the most active against both strains of Leishmania. Such finding was corroborated by the results obtained in assessments of the L. infantum amastigote susceptibility. It is noteworthy that the salt forms of the endoperoxide–pyrazole hybrids displayed a broader spectrum of action, showing activity in both strains of Leishmania. Our preliminary biological findings encourage further optimization of peroxide–pyrazole hybrids to identify a promising antileishmanial lead.

Background Visceral leishmaniasis (VL) is re-emerging in Armenia since 1999 with 167 cases recorded until 2019. The objectives of this study were (i) to determine for the first time the genetic diversity and population structure of the causative agent of VL in Armenia; (ii) to compare these genotypes with those from most endemic regions worldwide; (iii) to monitor the diversity of vectors in Armenia; (iv) to predict the distribution of the vectors and VL in time and space by ecological niche modeling. Methodology/Principal findings Human samples from different parts of Armenia previously identified by ITS-1-RFLP as L . infantum were studied by Multilocus Microsatellite Typing (MLMT). These data were combined with previously typed L . infantum strains from the main global endemic regions for population structure analysis. Within the 23 Armenian L . infantum strains 22 different genotypes were identified. The combined analysis revealed that all strains belong to the worldwide predominating MON1-population, however most closely related to a subpopulation from Southeastern Europe, Maghreb, Middle East and Central Asia. The three observed Armenian clusters grouped within this subpopulation with strains from Greece/Turkey, and from Central Asia, respectively. Ecological niche modeling based on VL cases and collected proven vectors ( P . balcanicus , P . kandelakii) identified Yerevan and districts Lori, Tavush, Syunik, Armavir, Ararat bordering Georgia, Turkey, Iran and Azerbaijan as most suitable for the vectors and with the highest risk for VL transmission. Due to climate change the suitable habitat for VL transmission will expand in future all over Armenia. Conclusions Genetic diversity and population structure of the causative agent of VL in Armenia were addressed for the first time. Further genotyping studies should be performed with samples from infected humans, animals and sand flies from all active foci including the neighboring countries to understand transmission cycles, re-emergence, spread, and epidemiology of VL in Armenia and the entire Transcaucasus enabling epidemiological monitoring.

Leishmaniasis is a vector-borne disease among the 10 most Neglected Tropical Diseases with diverse clinical manifestations caused by protozoan parasites of the Leishmania genus. Around 80% of leishmaniasis cases are found in the Old World affecting populations mainly in low and middle-income countries. Its control relies mostly on chemotherapy which still presents many drawbacks. Natural products may offer an inexhaustible source of chemical diversity with therapeutic potential. Despite the lack of knowledge on traditional products with activity against Leishmania parasites, many reports describe the search for natural extracts and compounds with antileishmanial properties against promastigote and amastigote parasite forms. This review summarizes the research of 74 publications of the last decade (2008-2018) focused on the identification of endemic plant-derived products that are active against Old World Leishmania parasites responsible for cutaneous and visceral leishmaniasis. The present review combines data on antileishmanial activity of 423 plants species, belonging to 94 different families, including a large range of crude extracts which lead to the isolation of 86 active compounds. Most studied plants came from Asia and most promising plant families for antileishmanial activity were Asteraceae and Lamiaceae. From the chemical point of view, terpenoids were the most frequently isolated natural products. These studies suggest that natural products isolated from Old World flora are a rich source of new chemical scaffolds for future leishmaniasis treatment as well as for other Neglected Tropical Diseases warranting further investigation.

Background Poverty, lack of resources, inadequate treatments and control programmes exacerbate the impact of infectious diseases in the developing world. Leishmaniasis is a vector-borne disease that is among the ten major neglected tropical diseases. Although endemic in more than 90 countries, the ones most affected, representing over 90% of new cases, are Bangladesh, Brazil, Ethiopia, India, Kenya, Nepal, and Sudan. In Africa south of the equator, the impact of leishmaniasis is much lower. In several countries, like Angola, little is known about this infectious neglected disease. In the 1970s, a group of Portuguese researchers described three cases of cutaneous leishmaniasis in children from Huambo district and in the 1990s visceral leishmaniasis was diagnosed in an African patient. More recently a canine survey in Luanda revealed two Leishmania -infected dogs. After some suspected cases of human cutaneous leishmaniasis in Huambo region in 2017, the Angola health authorities and the Instituto de Higiene e Medicina Tropical (IHMT), Lisbon, Portugal, established a collaboration to analyse samples from some suspected cases. Methods Three paraffin-embedded human skin samples from dermatological lesions were sent to IHMT for molecular analysis. After DNA extraction, PCR was performed by using four protocols with different molecular markers. Results One PCR protocol using a nested approach was positive in two of the samples. Sequencing analysis confirmed Leishmania sp. DNA. Conclusion This was the first time that suspected human cutaneous samples were screened for leishmaniasis by molecular methods with detection of Leishmania sp. DNA. These preliminary studies highlight the need for higher awareness of health professionals for leishmaniasis clinical forms, to recognise risk factors and the epidemiological features of leishmaniasis in the Huambo province. It would be relevant to perform further epidemiological studies to confirm if this vector-borne disease could be emergent in this country.

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife ( n = 58) and symptomatically and asymptomatically infected humans ( n = 38) and dogs ( n = 15) from south‐east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences ( n = 76) displayed a 99%–100% nucleotide identity with a L. infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L. infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L. infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L. infantum control strategies.