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Background: Leishmaniasis is a neglected zoonosis caused by parasites of Leishmania spp. The main drug used to treat cutaneous leishmaniasis (CL) is the antimoniate of meglumine. This drug, which has strong adverse and toxic effects, is usually administered intravenously, further complicating the difficult treatment. Factors such as Leishmania gene expression and genomic mutations appear to play a role in the development of drug resistance., Objectives: This systematic review summarises the results of the literature evaluating parasite genetic markers possibly associated with resistance to pentavalent antimony in CL., Methods: This study followed PRISMA guidelines and included articles from PubMed, SciELO, and LILACS databases. Inclusion criteria were studies that (i) investigated mutations in the genome and/or changes in gene expression of Leishmania associated with treatment resistance; (ii) used antimony drugs in the therapy of CL; (iii) used naturally resistant strains isolated from patients. The Joanna Briggs Institute Critical Appraisal Checklist was used to assess article quality and risk of bias., Findings: A total of 23 articles were selected, of which 18 investigated gene expression and nine genomic mutations. Of these 23 articles, four examined gene expression and genomic mutations in the same samples. Regarding gene expression, genes from the ABC transporter protein family, AQP1, MRPA, TDR1 and TRYR were most frequently associated with drug resistance. In one of the articles in which mutations were investigated, a mutation was found in HSP70 (T579A) and in three articles mutations were found in AQP1 (A516C, G562A and G700A). A limitation of this review is that in most of the included studies, parasites were isolated from cultured lesion samples and drug resistance was assessed using in vitro drug susceptibility testing. These approaches may not be ideal for accurate genetic evaluation and detection of treatment failure., Main Conclusions: The development of further studies to evaluate the genetic resistance factors of Leishmania spp. is necessary to elucidate the mechanisms of the parasite and improve patient treatment and infection control.

The clinical manifestation of leishmaniasis has historically been determined by the Leishmania species involved. However, recent emergence of novel Leishmania lineages has caused atypical pathologies. We isolated and characterized 2 new Leishmania donovani parasites causing cutaneous leishmaniasis in Himachal Pradesh, India.

Cutaneous leishmaniasis is atypical in Sri Lanka because Leishmania donovani, which typically causes visceral disease, is the causative agent. The origins of recently described hybrids between L. donovani and other Leishmania spp. usually responsible for cutaneous leishmaniasis remain unknown. Other endemic dermotropic Leishmania spp. have not been reported in Sri Lanka. Genome analysis of 27 clinical isolates from Sri Lanka and 32 Old World Leishmania spp. strains found 8 patient isolates clustered with L. tropica and 19 with L. donovani. The L. tropica isolates from Sri Lanka shared markers with strain LtK26 reported decades ago in India, indicating they were not products of recent interspecies hybridization. Because L. tropica was isolated from patients with leishmaniasis in Sri Lanka, our findings indicate L. donovani is not the only cause of cutaneous leishmaniasis in Sri Lanka and potentially explains a haplotype that led to interspecies dermotropic L. donovani hybrids.

Background: Cutaneous Leishmaniasis (CL) remains a significant public health concern, particularly in the tropical and subtropical regions. Present treatment options for CL such as Fluconazole (FLZ) face limitations, including low solubility and bioavailability. This study aimed to address these challenges by investigating the use of nano-emulsions (NEs) to enhance the efficacy of FLZ against Leishmania major(L.major)., Materials and Methods: FLZ-NEs were formulated with oleic acid, Tween-20, and ethanol using low-energy emulsification at various surfactant/co-surfactant ratios. Subsequently, a comprehensive analysis was conducted to assess the physicochemical characteristics of the samples. This analysis encompassed stability, zeta potential, pH, viscosity, refractive index, and droplet size. We then studied the anti-parasitic properties of these optimized FLZ-NEs both in vitro and in vivo., Results: The selected nano-emulsion (NE) formulation (2 % oleic acid, 20 % Tween 20, 10 % ethyl alcohol) showcased desirable properties like small droplet size (10.51 ± 0.24 nm), low dispersity (0.19 ± 0.03), and zeta potential value (- 0.41 ± 0.17 mV), key for stability and targeted drug delivery. This optimal formulation translated into remarkable efficacy. In vitro, FLZ-NEs demonstrated a threefold increase in their ability to combat promastigotes and a remarkable thirtyfold increase in their ability to combat amastigotes. Additionally, they demonstrated a ninefold advantage in their ability to specifically target parasites within infected macrophages, thereby attacking the infection site. These promising in vitro results translated into improved outcomes in vivo. Compared to other chemicals studied, FLZ-NE-treated mice showed decreased disease severity, weight growth, and quicker ulcer healing. It was further supported by histopathological research, which showed reduced tissue damage linked to Leishmania infection., Conclusion: These findings show the potential of nanotechnology-based drug delivery in improving anti-leishmanial treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest. All authors contributed equally to this work.

Cutaneous Leishmaniasis, an infectious disease is globally the most prevalent form of leishmaniasis accounting for approximately 1 million cases every year as per world health organization. Infected individuals develop skin lesion which has been reported to be infiltrated by immune cells and parasite with high sodium accumulation creating hypertonic environment. In our work, we tried to mimic the hypertonic environment in virtual environment to study dynamicity of SHP-1 and NFAT5 along with their interactions through molecular dynamics simulation. We validated the SHP-1 and NFAT5 dynamics in infection and HSD conditions to study the impact of hypertonicity derived NFAT5 mediated response to L.major infection. We also evaluated our therapeutic peptides for their binding to SHP-1 and to form stable complex. Membrane stability with the peptides was analyzed to understand their ability to sustain mammalian membrane. We identified PepA to be a potential candidate to interact with SHP-1. Inhibition of SHP-1 through PepA to discern IL-10 and IL-12 reciprocity may be assessed in future and furnish us with a potential therapeutic molecule. HSD mice exhibited high pro-inflammatory response to L.major infection which resulted in reduced lesion size. Contrary to observations in HSD mice, infection model exhibited low pro-inflammatory response and increased lesion size with high parasite load. Thus, increase in NFAT5 expression and reduced SHP-1 expression may result in disease resolving effect which can be further studied through incorporation of synthetic circuit using PepA to modulate IL-10 and IL-12 reciprocity., Competing Interests: Declaration of competing interest The authors potentially declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Post Kala-azar dermal leishmaniasis (PKDL) arises as a significant dermal sequel following Visceral leishmaniasis (VL) caused by protozoan parasite Leishmania donovani (LD). PKDL acts as a significant constrain for VL elimination serving as a crucial reservoir for LD. PKDL patients exhibit depigmented macular and papular lesions on their skin, which results in social discrimination due to loss of natural skin color. Inflammatory reactions, prevalent in both VL and PKDL, potentially lead to tissue damage in areas harboring the parasite. Disruption of the immune-inflammasomal network not only facilitates LD persistence but also leads to the skin hypopigmentation seen in PKDL, impacting social well-being. Activation of inflammasomal markers like STAT1, NLRP1, NLRP3, AIM2, CASP11, and NLRP12 have been identified as a common host-defense mechanism across various Leishmania infections. Conversely, Leishmania modulates inflammasome activation to sustain its presence within the host. Nevertheless, in specific instances of Leishmania infection, inflammasome activation can worsen disease pathology by promoting parasite proliferation and persistence. This study encompasses recent transcriptomic analyses conducted between 2016 and 2023 on human and murine subjects afflicted with VL/PKDL, elucidating significant alterations in inflammasomal markers in both conditions. It offers a comprehensive understanding how these markers contribute in disease progression, drawing upon available literature for logical analysis. Furthermore, our analysis identifies validated miRNA network that could potentially disrupt this crucial immune-inflammasomal network, thereby offering a plausible explanation on how secreted LD-factors could enable membrane-bound LD, isolated from the host cytoplasm, to modulate cytoplasmic inflammasomal markers. Insights from this study could guide the development of host-directed therapeutics to impede transmission and address hypopigmentation, thereby mitigating the social stigma associated with PKDL., Competing Interests: Declaration of competing interest The authors declare that the systematic review was conducted without any conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Many areas of Iran are endemic regarding Cutaneous leishmaniasis (CL) as a parasitic disease transmitted by a female sand fly vector's bite. The present study investigated the distribution of sand flies in Harand and Egieh in Isfahan province, Central Iran. Overall, 408 out of 1260 collected sand flies were identified morphologically, among which 353 and 55 were isolated from Harand and Egieh, respectively. Also, 66.4% and 33.6% of the sand flies were female and male, respectively. The most prevalent sand fly species were Phlebotomus papatasi (52%), followed by Ph . caucasicus (40.4%), Sergentomyia sintoni (4.9%), and Ph. kazeruni (0.7%). Among 180 molecularly-analyzed sand flies, 14 (7.77%) were found infected with L . major , with 9 out of 103 (8.73%) Ph . papatasi and 5 out of 75 (6.66%) Ph. caucasicus .

Currently, no safe vaccine against leishmaniasis is available. So far, different control strategies against numerous reservoir hosts and biological vectors have not been environment-friendly and feasible. Hence, employing medicinal components and conventional drugs could be a promising approach to developing novel therapeutic alternatives. This study aimed to explore diallyl sulfide (DAS), a dynamic constituent of garlic, alone and in a mixture with meglumine antimoniate (MAT as standard drug) using in vitro and animal model experiments against Leishmania major stages. The binding affinity of DAS and four major defense elements of the immune system (iNOS, IFN-ɣ, IL-12, and TNF-α) was used to predict the predominant binding mode for molecular docking configurations. Herein, we conducted a broad range of experiments to monitor and assess DAS and MAT potential treatment outcomes. DAS, combined with MAT, displayed no cytotoxicity and employed a powerful anti-leishmanial activity, notably against the clinical stage. The function mechanism involved immunomodulation through the induction of Th1 cytokine phenotypes, triggering a high apoptotic profile, reactive oxygen species (ROS) production, and antioxidant enzymes. This combination significantly decreased cutaneous lesion diameter and parasite load in BALB/c mice. The histopathological findings performed the infiltration of inflammatory cells associated with T-lymphocytes, particularly CD4+ phenotypes, as determined by biochemical markers in alleviating the amastigote stage and improving the pathological changes in L. major infected BALB/c mice. Therefore, DAS and MAT deserve further advanced therapeutic development and should be considered as possible candidates for treating volunteer cases with cutaneous leishmaniasis in designing an upcoming clinical trial., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zarrinkar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study investigated nine provinces in northern Morocco and collected 275 skin scraping, 22 bone marrow aspirates, and 89 fine needle aspirations from suspected cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) patients and potentially infected dogs. Molecular analysis using ITS1 RFLP PCR and RT-PCR revealed a higher prevalence of L. infantum (66.18 %; χ 2  = 28.804; df = 1; P-value = 8.01e-08) than L. tropica in skin scraping, with L. infantum being the sole causative agent for both VL and canine leishmaniasis. L. infantum was predominantly found in most provinces, while L. tropica was relatively more dominant in Taza Province. Discriminant Analysis of Principal Components (DAPC) revealed distinct clustering between L. tropica and the other three species. However, no small subset of SNPs could clearly differentiate between Infantum_CL, Infantum_VL, and CanL, as they likely share a significant genetic background. The high rate of L. infantum could be attributed to the abundance of sand fly species transmitting VL. In Taza Province, Phlebotomus sergenti, responsible for anthroponotic CL, is the most abundant species. DNA sequencing demonstrated sequence heterogeneity in L. infantum (variants 1-9) and L. tropica (variants 1-7). Phylogenetic analysis showed a distinct separation between L. tropica and L. infantum strains, with an overlap among L. infantum strains isolated from cutaneous, visceral, and canine cases, and dogs serving as the central population for L. infantum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Cutaneous leishmaniasis (CL) is a global public health problem caused by species on the genus Leishmania and is the most prevalent clinical form of leishmaniasis. The aim of this study was to develop a new LAMP assay for Leishmania sp. based on HSP70 gene and evaluate it clinically for molecular diagnosis of CL. The study was carried out in the following stages: i) design of primers based on HSP70 gene of Leishmania sp.; ii) evaluation of detection limit and analytical specificity; iii) estimation of the accuracy of LAMP-Leish/HSP70 assay for diagnosing CL. A total of 100 skin biopsy samples from patients, comprising 60 CL cases and 40 non-cases, were analyzed in this study. One LAMP assay using HSP70 gene as molecular target were standardized, and the observed detection limit was 100fg of L. braziliensis purified DNA. The LAMP-Leish/HSP70 assay was specific for Leishmania spp. The LAMP-Leish/HSP70 assay showed an accuracy of 92%, and positivity rates were not affected by lesion onset time or parasite load. This novel LAMP assay targeting the HSP70 gene of Leishmania sp. has the potential to be a useful tool to integrate into routine diagnosis for suspected cases of CL., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Soares et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Th1 and Th2 cytokines determine the outcome of Leishmania major infection and immune protection depends mainly on memory T cells induced during vaccination. This largely hinges on the nature and type of memory T cells produced. In this study, transgenic Leishmania major strains expressing membrane-associated ovalbumin (mOVA) and soluble ovalbumin (sOVA) were used as a model to study whether fully differentiated Th1/Th2 and Th17 cells can recall immune memory and tolerate pathogen manipulation. Naïve OT-II T cells were polarised in vitro into Th1/Th2 cells, and these cells were transferred adoptively into recipient mice. Following the transferral of the memory cells, the recipient mice were challenged with OVA transgenic Leishmania major and a wild-type parasite was used a control. The in vitro-polarised T helper cells continued to produce the same cytokine signatures after being challenged by both forms of OVA-expressing Leishmania major parasites in vivo. This suggests that antigen-experienced cells remain the same or unaltered in the face of OVA-transgenic Leishmania major. Such ability of these antigen-experienced cells to remain resilient to manipulation by the parasite signifies that vaccines might be able to produce immune memory responses and defend against parasitic immune manipulation in order to protect the host from infection.

American tegumentary leishmaniasis (ATL) is highly endemic in the Amazon basin and occurs in all South American countries, except Chile and Uruguay. Most Brazilian ATL cases are due to Leishmania (Viannia) braziliensis, however other neglected Amazonian species are being increasingly reported. They belong to the subgenus L. (Viannia) and information on suitable models to understand immunopathology are scarce. Here, we explored the use of the golden hamster Mesocricetus auratus and its macrophages as a model for L. (Viannia) species. We also studied the interaction of parasite glycoconjugates (LPGs and GIPLs) in murine macrophages. The following strains were used: L. (V.) braziliensis (MHOM/BR/2001/BA788), L. (V.) guyanensis (MHOM/BR/85/M9945), L. (V.) shawi (MHOM/BR/96/M15789), L. (V.) lindenbergi (MHOM/BR/98/M15733) and L. (V.) naiffi (MDAS/BR/79/M5533). In vivo infections were initiated by injecting parasites into the footpad and were followed up at 20- and 40-days PI. Parasites were mixed with salivary gland extract (SGE) from wild-captured Nyssomyia neivai prior to in vivo infections. Animals were euthanized for histopathological evaluation of the footpads, spleen, and liver. The parasite burden was evaluated in the skin and draining lymph nodes. In vitro infections used resident peritoneal macrophages and THP-1 monocytes infected with all species using a MOI (1:10). For biochemical studies, glycoconjugates (LPGs and GIPLs) were extracted, purified, and biochemically characterized using fluorophore-assisted carbohydrate electrophoresis (FACE). They were functionally evaluated after incubation with macrophages from C57BL/6 mice and knockouts (TLR2-/- and TLR4-/-) for nitric oxide (NO) and cytokine/chemokine production. All species, except L. (V.) guyanensis, failed to generate evident macroscopic lesions 40 days PI. The L. (V.) guyanensis lesions were swollen but did not ulcerate and microscopically were characterized by an intense inflammatory exudate. Despite the fact the other species did not produce visible skin lesions there was no or mild pro-inflammatory infiltration at the inoculation site and parasites survived in the hamster skin/lymph nodes and even visceralized. Although none of the species caused severe disease in the hamster, they differentially infected peritoneal macrophages in vitro. LPGs and GIPLs were able to differentially trigger NO and cytokine production via TLR2/TLR4 and TLR4, respectively. The presence of a sidechain in L. (V.) lainsoni LPG (type II) may be responsible for its higher proinflammatory activity. After Principal Component analyses using all phenotypic features, the clustering of L. (V.) lainsoni was separated from all the other L. (Viannia) species. We conclude that M. auratus was a suitable in vivo model for at least four dermotropic L. (Viannia) species. However, in vitro studies using peritoneal cells are a suitable alternative for understanding interactions of the six L. (Viannia) species used here. LRV1 presence was found in L. (V.) guyanensis and L. (V.) shawi with no apparent correlation with virulence in vitro and in vivo. Finally, parasite glycoconjugates were able to functionally trigger various innate immune responses in murine macrophages via TLRs consistent with their inflammatory profile in vivo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Soares et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Dogs are considered the main domestic animals that may be a reservoir for Leishmania infantum, the agent of zoonotic visceral leishmaniasis (ZVL) in several countries of the world. The dog may host other Leishmania species, but its epidemiological role in the maintenance and spreading of these parasites is not completely elucidated. Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major, affects thousands of people every year and is particularly diffused in many countries of North Africa and Middle East Asia. In ZCL endemic countries, few reports of L. major-positive dogs have been reported, probably because most human cases occur in poor rural areas where the social role of the dog and its medical management is not well considered. The aim of the present study is to better understand the possible involvement of domestic dogs in the epidemiology of ZCL., Methods: Our research focused on a well-established endemic focus of ZCL, in the area of Echrarda, Kairouan Governorate, central Tunisia. A total of 51 dogs with no or mild clinical signs of vector borne diseases were selected in small villages where human cases of ZCL are yearly present. All dogs were sampled for the Leishmania spp. diagnosis, by using the following procedures: blood sample for serology and buffy coat quantitative polymerase chain reaction (qPCR), popliteal fine needle aspiration, and cutaneous biopsy punch for lymph node and skin qPCR., Results: The results demonstrated a high percentage (21.6%) of dogs positive at least at one or more test; the most sensitive technique was the lymph node qPCR that detected 8/11 positive dogs. Nine, out of the eleven positive dogs, resulted as infected by Leishmania infantum; ITS1-PCR-sequencing allowed Leishmania major identification in the remaining two cases, both from the popliteal lymph node samples, which can suggest a possible visceral spread of a cutaneous Leishmania species in the dog. Interestingly, one of the two L. major-positive dogs was living in the same house where 6-year-old children showed cutaneous lesions referred to as ZCL., Conclusions: To our knowledge, this is the first report of L. major-positive dogs in Tunisia, the  epidemiological role of which remains under investigation., (© 2024. The Author(s).)

Type 2 diabetes mellitus is a metabolic disorder that causes chronic high blood sugar levels, and diabetic patients are more susceptible to infections. American cutaneous leishmaniasis is an infectious disease caused by a parasite that affects the skin and mucous membranes, leading to one or multiple ulcerative lesions. Chronic inflammation and functional changes in various organs and systems, including the immune system, are the primary causes of both diseases. Melatonin, an essential immunomodulatory, antioxidant, and neuroprotective agent, can benefit many immunological processes and infectious diseases, including leishmaniasis. Although, limited reports are available on diabetic patients with leishmaniasis. The literature suggests that melatonin may play a promising role in inflammatory disorders. This study was designed to assess melatonin levels and inflammatory mediators in diabetic patients affected by leishmaniasis. Blood samples from 25 individuals were analyzed and divided into four groups: a control group (without any diseases), a Leishmania-positive group, patients with type 2 diabetes mellitus, and patients with a combination of both diseases. This study measured the serum levels of melatonin through ELISA, while IL-4 and TNF-α were measured using flow cytometry, and C-reactive protein was measured through turbidimetry. This study found that patients with leishmaniasis significantly increased TNF-α and decreased melatonin levels. However, the group of diabetic patients with leishmaniasis showed higher melatonin levels than the control group. These observations suggest that TNF-α may influence melatonin production in patients with American cutaneous leishmaniasis, potentially contributing to the inflammatory characteristics of both diseases.

Leishmania major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC 50 values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC 50 values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We report an immunocompromised patient with a complex cutaneous leishmaniasis (CL) who suffered from singular bone involvement of the little left toe due to Leishmania (L.) infantum infection. The diagnosis was confirmed by positive polymerase chain reaction (PCR) testing in skin and bone tissue. The patient was successfully treated with miltefosine. In immunocompromised patients with CL, extracutaneous manifestations should always be ruled out. This is the first case report describing osseous involvement in CL due to Leishmania infantum., (© 2024. The Author(s).)

Purpose: Leishmaniasis, caused by the parasite of the genus Leishmania, is a neglected tropical disease which is endemic in more than 60 countries. In South-East Asia, Brazil, and East Africa, it mainly occurs as kala-azar (visceral leishmaniasis, VL), and subsequently as post kala-azar dermal leishmaniasis (PKDL) in a smaller portion of cases. As stated per WHO roadmap, accessibility to accurate diagnostic methods is an essential step to achieve elimination. This study aimed to test the accuracy of a portable minoo device, a small battery-driven, multi-use fluorimeter operating with isothermal technology for molecular diagnosis of VL and PKDL., Methods: Fluorescence data measured by the device within 20 min are reported back to the mobile application (or app) via Bluetooth and onward via the internet to a backend. This allows anonymous analysis and storage of the test data. The test result is immediately returned to the app displaying it to the user., Results: The limit of detection was 11.2 genome copies (95% CI) as determined by screening a tenfold dilution range of whole Leishmania donovani genomes using isothermal recombinase polymerase amplification (RPA). Pathogens considered for differential diagnosis were tested and no cross-reactivity was observed. For its diagnostic performance, DNA extracted from 170 VL and PKDL cases, comprising peripheral blood samples (VL, n = 96) and skin biopsies (PKDL, n = 74) from India (n = 108) and Bangladesh (n = 62), was screened. Clinical sensitivity and specificity were 88% and 91%, respectively., Conclusion: Minoo devices can offer a convenient, cheaper alternative to other molecular diagnostics. Its easy handling makes it ideal for use in low-resource settings to identify parasite burden., (© 2024. The Author(s).)

In this present study, carried out between November 2020 and July 2023 at Londrina's University Hospital, patients with active lesions of cutaneous leishmaniasis (CL) were analyzed regarding pain perception and anatomopathological aspects of the ulcers. Pain was assessed using a numerical rating scale (NRS) to compare five patients diagnosed with CL with four control patients diagnosed with vascular skin ulcers. Histopathological evaluations were used to investigate the nociceptor neuron-Leishmania interface. Patients with CL ulcers reported less pain compared to patients with vascular ulcers (2.60 ± 2.30 and 7.25 ± 0.95, respectively, p = 0.0072). Histopathology evidenced Leishmania spp. amastigote forms nearby sensory nerve fibers in profound dermis. Schwann cells marker (S100 protein) was detected, and caspase-3 activation was not evidenced in the in the nerve fibers of CL patients' samples, suggesting absence of apoptotic activity in nerve endings. Additionally, samples taken from the active edge of the lesion were negative for bacilli acid-alcohol resistant (BAAR), which excludes concomitant leprosy, in which painless lesions are also observed. Thus, the present data unveil for the first time anatomopathological and microbiological details of painless ulcers in CL patients, which has important clinical implications for a better understanding on the intriguing painless clinical characteristic of CL., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Cutaneous leishmaniasis (CL) is understudied in sub-Saharan Africa. The epidemiology of CL is determined by the species involved in its transmission. Our objectives were to systematically review available data on the species of Leishmania, along with vectors and reservoirs involved in the occurrence of human cases of CL in sub-Saharan Africa, and to discuss implications for case management and future research., Methods: We systematically searched PubMed, Scopus, Cochrane and African Index Medicus. There was no restriction on language or date of publication. The review was conducted according to PRISMA guidelines and was registered on PROSPERO (CRD42022384157)., Results: In total, 188 published studies and 37 reports from the grey literature were included. An upward trend was observed, with 45.7% of studies published after 2010. East Africa (55.1%) represented a much greater number of publications than West Africa (33.3%). In East Africa, the identification of reservoirs for Leishmania tropica remains unclear. This species also represents a therapeutic challenge, as it is often resistant to meglumine antimoniate. In Sudan, the presence of hybrids between Leishmania donovani and strictly cutaneous species could lead to important epidemiological changes. In Ghana, the emergence of CL in the recent past could involve rare species belonging to the Leishmania subgenus Mundinia. The area of transmission of Leishmania major could expand beyond the Sahelian zone, with scattered reports in forested areas. While the L. major-Phlebotomus duboscqi-rodent complex may not be the only cycle in the dry areas of West Africa, the role of dogs as a potential reservoir for Leishmania species with cutaneous tropism in this subregion should be clarified. Meglumine antimoniate was the most frequently reported treatment, but physical methods and systemic agents such as ketoconazole and metronidazole were also used empirically to treat L. major infections., Conclusions: Though the number of studies on the topic has increased recently, there is an important need for intersectional research to further decipher the Leishmania species involved in human cases of CL as well as the corresponding vectors and reservoirs, and environmental factors that impact transmission dynamics. The development of molecular biology in sub-Saharan Africa could help in leveraging diagnostic and research capacities and improving the management of human cases through personalized treatment strategies., (© 2024. The Author(s).)

Cutaneous leishmaniasis (CL) is often considered a 'great imitator' and is the most common form of leishmaniasis. The Leishmania species responsible for CL varies among countries, as these species exhibit specific distribution patterns. The increased mobility of people across countries has resulted in the imported incidences of leishmaniasis caused by non-endemic species of Leishmania. During 2023, we confirmed three CL cases caused by L. major from Kerala, India, and upon detailed investigation, these were identified to be imported from the Middle East and Kazakhstan regions. This is the first report of CL caused by L. major from Kerala. The lesion morphology, detection of anti-rK 39 antibody and Leishmania parasite DNA from the blood samples were the unique observations of these cases. Kerala, being an emerging endemic zone of visceral leishmaniasis (VL) and CL, the imported incidences of leishmaniasis by non-endemic species can pose a significant threat, potentially initiating new transmission cycles of leishmaniasis caused by non-endemic species., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Exosomes produced by the protozoan parasite Leishmania (LeishEXO) are well-established drivers of virulence, though mechanisms underlying their exacerbation of experimental leishmaniasis remain elusive. Expression of Annexin A1 (ANXA1), a protein implicated in exosome-mediated pathologies and viral internalization, has been shown to correlate with cutaneous leishmaniasis severity. Given ANXA1's regulation of myeloid cells - the canonical hosts for Leishmania - we studied the potential role of ANXA1 and its receptors FPR1/2 in exerting LeishEXO's effects., Methods: Murine and in vitro ANXA1 -/- models were used to study the generation of protective T H 1 responses during experimental L. major infection with and without LeishEXO. Recruitment of inflammatory cells was assessed using a peritoneal cell recruitment assay and immunophenotyping, and production of inflammatory mediators was measured using a cytokine and chemokine array. Treatment of experimental models with FPR2 antagonist WRW4 and FPR1/2 agonist WKYMVm was used to delineate the role of the FPR/ANXA1 axis in LeishEXO-mediated hyperpathogenesis., Results: We established that ANXA1 deficiency prohibits LeishEXO-mediated pathogenesis and myeloid cell infection, with minimal alterations to adaptive and innate immune phenotypes. FPR2 blockade with WRW4 similarly inhibited leishmanial hyperpathogenesis, while direct activation of FPRs with WKYMVm enhanced infection and recapitulated the LeishEXO-mediated phenotype. This research describes LeishEXO's utilization of the ANXA1/FPR axis to facilitate parasitic internalization and pathogenesis, which may be leveraged in the development of therapeutics for leishmaniasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 da Silva Lira Filho, Lafleur, Alvarez, Piccirillo and Olivier.)

Background: In Brazil, Leishmania (Leishmania) infantum is a widely distributed protozoan parasite. The human leishmaniasis caused by this species is often associated with visceral form. Tegumentary leishmaniasis (TL) cases due to L. (L.) infantum in the country are considered rare but may be underestimated. Although probably uncommon, these cases represent a new challenge to the prevention and control of leishmaniasis., Objectives: Here, we describe two distinct cases of TL with atypical clinical presentations caused by L. (L.) infantum., Methods and Findings: Parasites were isolated from cutaneous lesions of the two patients and typed as L. (L.) infantum after sequencing of the ribosomal DNA internal transcribed spacer. The dermotropic L. (L.) infantum isolates were compared in terms of growth culture patterns, metacyclogenesis and in vitro infectivity in macrophages., Main Conclusions: This study addresses the emergence of L. (L.) infantum as a causative agent of cutaneous disease in a visceral leishmaniasis hotspot located in northeast Brazil. The data presented provides novel information about the presence of dermotropic L. (L.) infantum in the country and demonstrates the infectivity potential of theses isolates.

Introduction: Post-kala-azar dermal leishmaniasis (PKDL) arises as a dermal complication following a visceral leishmaniasis (VL) infection. Current treatment options for PKDL are unsatisfactory, and there is a knowledge gap regarding the distribution of antileishmanial compounds within human skin. The present study investigated the skin distribution of miltefosine in PKDL patients, with the aim to improve the understanding of the pharmacokinetics at the skin target site in PKDL., Methods: Fifty-two PKDL patients underwent treatment with liposomal amphotericin B (20 mg/kg) plus miltefosine (allometric dosing) for 21 days. Plasma concentrations of miltefosine were measured on study days 8, 15, 22 and 30, while a punch skin biopsy was taken on day 22. A physiologically based pharmacokinetic (PBPK) model was developed to evaluate the distribution of miltefosine into the skin., Results: Following the allometric weight-based dosing regimen, median miltefosine concentrations on day 22 were 43.73 µg/g (IQR: 21.94-60.65 µg/g) in skin and 33.29 µg/mL (IQR: 25.9-42.58 µg/mL) in plasma. The median individual concentration ratio of skin to plasma was 1.19 (IQR: 0.79-1.9). In 87% (45/52) of patients, skin exposure was above the suggested EC90 PK target of 10.6 mg/L associated with in vitro susceptibility. Simulations indicated that the residence time of miltefosine in the skin would be more than 2-fold longer than in plasma, estimated by a mean residence time of 604 versus 266 hours, respectively., Conclusion: This study provides the first accurate measurements of miltefosine penetration into the skin, demonstrating substantial exposure and prolonged retention of miltefosine within the skin. These findings support the use of miltefosine in cutaneous manifestations of leishmaniasis. In combination with parasitological and clinical data, these results are critical for the future optimization of combination therapies with miltefosine in the treatment of PKDL., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Combination therapy at appropriately suitable doses presents a promising alternative to monotherapeutic drugs. In this study, Cinnamomum verum and Syzygium aromaticum essential oils and their major compounds have exhibited substantial leishmaniacidal potential against both promastigote and amastigote forms of Leishmania (L.) major . However, they displayed high cytotoxicity against Raw264.7 macrophage cells. Interestingly, when combined with each other or with amphotericin B, they demonstrated a synergistic effect (FIC<0.5) with low cytotoxicity. These combinations are able to modulate the production of nitric oxide (NO) by macrophages. Notably, the combination of S. aromaticum Essential oil with amphotericin B stimulates macrophage cells by increasing NO production to eliminate leishmanial parasites. Furthermore, investigation of the molecular mechanism of action of these synergistic combinations reveals potent inhibition of the sterol pathway through the inhibition of the CYP51 gene expression. The findings suggest that combination therapy may offer significant therapeutic benefits in both food and pharmaceutical fields.

The obligate intracellular parasite Leishmania binds several receptors to trigger uptake by phagocytic cells, ultimately resulting in visceral or cutaneous leishmaniasis. A series of signaling pathways in host cells, which are critical for establishment and persistence of infection, are activated during Leishmania internalization. Thus, preventing Leishmania uptake by phagocytes could be a novel therapeutic strategy for leishmaniasis. However, the host cellular machinery mediating promastigote and amastigote uptake is not well understood. Here, using small molecule inhibitors of Mitogen-activated protein/Extracellular signal regulated kinases (MAPK/ERK), we demonstrate that ERK1/2 mediates Leishmania amazonensis uptake and (to a lesser extent) phagocytosis of beads by macrophages. We find that inhibiting host MEK1/2 or ERK1/2 leads to inefficient amastigote uptake. Moreover, using inhibitors and primary macrophages lacking spleen tyrosine kinase (SYK) or Abl family kinases, we show that SYK and Abl family kinases mediate Raf, MEK, and ERK1/2 activity and are necessary for uptake. Finally, we demonstrate that trametinib, a MEK1/2 inhibitor used to treat cancer, reduces disease severity and parasite burden in Leishmania-infected mice, even if it is started after lesions develop. Our results show that maximal Leishmania infection requires MAPK/ERK and highlight potential for MAPK/ERK-mediated signaling pathways to be novel therapeutic targets for leishmaniasis., Competing Interests: Declaration of competing interest The authors have no competing financial interests., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The visceral and atypical cutaneous leishmaniasis (VL and CL) caused by Leishmania donovani is an emerging infectious disease in the Western Ghats, Kerala, India. In this study, L. donovani specific kinetoplast minicircle DNA (k-DNA) sequence analysis was conducted to ascertain the genetic variability among the L. donovani isolates from the Western Ghats. Out of 23 CL and 5 VL suspected patient samples, 18 CL and 3 VL tested positive for k-DNA diagnostic PCR. Subsequently, 17 CL and 3 VL samples were found positive for L. donovani specific k-DNA PCR. Although the genetic diversity among the VL and CL isolates was low, there was clear variation from the parasites reported from other countries. The parasites characterized from the current study were more related to those reported from East Africa and India., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cutaneous leishmaniasis (CL), a neglected tropical disease, is a major public health concern in Yemen, with Leishmania tropica identified as the main causative agent. This study aims to investigate the occurrence and distribution of Leishmania parasites in domestic and wild animals in CL endemic areas in the western highlands of Yemen. A cross-sectional study was conducted in the Utmah District of western Yemen. Blood and skin scraping specimens were collected from 122 domestic and wild animals and tested for the Leishmania DNA using internal transcribed spacer 1 (ITS1) nested polymerase chain reaction. Phylogenetic analyses were performed on 20 L. tropica sequences obtained from animals in this study and 34 sequences from human isolates (collected concurrently from the same study area) retrieved from the GenBank. Overall, L. tropica was detected in 16.4% (20/122) of the examined animals, including 11 goats, two dogs, two bulls, one cow, one donkey, one rabbit, one rat and one bat. None of the examined cats and sheep was positive. The animal sequences were segregated into four different L. tropica haplotypes, with the majority of the animal (15/20) and human (32/34) sequences composed of one dominant haplotype/genotype. These findings represent the first confirmed evidence of natural L. tropica infections in different kinds of domestic and wild animals in western Yemen, suggesting these animals potentially have a role in the transmission of CL in Yemen. Therefore, a One Health approach is required for the effective prevention and control of this devastating disease among endemic populations., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Gold miners working illegally in mines live in poor health conditions related to their strenuous work and precarious housing. Therefore, they are at higher risk for infectious diseases. American tegumentary leishmaniasis (ATL) appears to be of great concern to the population living in the Guiana Shield region. Our aim was to describe their demographic characteristics, the clinical features of cutaneous leishmaniasis (CL), and the frequency of Leishmania infection in people working in illegal gold mines in French Guiana. A cross-sectional study was carried out from October to December 2019 in Oiapoque city, Amapá, Brazil. Indeed, many gold miners working in French Guiana are originally from Brazil, and from Oiapoque in particular. A total of 105 participants from 31 different mining sites in French Guiana were recruited. Suspected Leishmania infection was confirmed by the following: detection of kDNA in blood or the lesion site; detection of specific antibodies; or detection of IFN-γ release after blood incubation with leishmanial antigens (IGRA-Leish). Nine active CL cases, 38 healed ATL (hATL) and 58 cases with no history of ATL (noATL), were identified. Only half of the treated hATL (50.0%; n = 14) reported having been assisted by a health care unit and the others treated themselves. PCR-kDNA for Leishmania was positive in the blood of 100% of CL cases. Curiously, blood PCR-kDNA was positive in 13% of hATL patients and in 15.5% of noATL patients. The IGRA-Leish was positive in 60.5% of hATL and in 37.9% of noATL. In addition to scars suggestive of CL, 71% of hATL had laboratory evidence of Leishmania infection. Restriction fragment polymorphism (RFLP) of the hsp70 gene identified a sympatric circulation of L. (V.) guyanensis (n = 4), L. (V.) braziliensis (n = 1), L. (L.) amazonensis (n = 2), L. (V.) shawi (n = 1) and L. (V.) naiffi/shawi (n = 1). Taking the laboratory techniques and the clinical evaluations together, 76% (n = 80) of the 105 participants had evidence of Leishmania infection. These results suggests that illegal gold miners working in French Guiana are at high risk for infection with different species of Leishmania, but their illegal condition and remoteness make it difficult for them to access health services., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mosquera Atehortua et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cutaneous leishmaniasis caused by different species of Leishmania is transmitted by Phlebotominae sandflies. This disease remains a public health concern in Iran. Therefore, the present study aimed to examine Leishmania infection in sandflies and reservoir rodents in six rural regions of Nahavand, located in western Iran. From May to October 2022, sandflies and rodents were collected and identified at the species level. Additionally, rodents' skin lesions and earlobe specimens were collected separately for microscopic and molecular examination. All specimens were tested for Leishmania DNA by PCRs targeting the parasite's ITS-2 and 18S rRNA gene and positive were Sanger sequenced. A total of 3396 sandflies belonging to seven subgenera and 11 species, i.e., Phlebotomus papatasi (42.7%), P. major (20.6%), P. mascitti (0.3%), P. neglectus (0.2%), P. alexandri (0.2%), P. turanicus (0.3%), Sergentomyia murgabiensis (18.1%), S. dentata (10.5%), S. theodori (5.8%), S. antennata (1.1%), and S. pawlowski (0.1%) were identified. Based on the species population, 29 pools of sandflies were examined for the presence of Leishmania DNA using conventional PCR (cPCR), and individual DNAs were tested when positive. Leishmania major DNA was detected in two P. papatasi and Leishmania sp. in one P. major individual sandfly. This is the first report of Leishmania infection in sandflies from Hamadan province. The captured rodents (n = 61) belonged to four families and seven species, i.e., Arvicola amphibius (37.7%), Mus musculus (29.5%), Microtus socialis (13.1%), Apodemus sylvaticus (11.5%), Talpa davidiana (4.9%), Apodemus witherbyi (1.6%), and Rattus norvegicus (1.6%). Microscopic and molecular examinations of the rodent lesions and earlobes scored negative results. The presence of Leishmania in the Phlebotominae sandflies in Nahavand indicates a potential threat to humans and animals in the region. Regular monitoring and examination of the sandflies' population and timely diagnosis and treatment of new patients are strongly recommended., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Pathogenesis of cutaneous leishmaniases involves parasite growth, persistent inflammation, and likely participation of lipoproteins (LP). The cholesteryl ester transfer protein (CETP), involved in LP remodeling, has been shown to participate in the inflammatory response and the evolution of infectious conditions., Methods: We evaluated the impact of the presence of CETP on infection by Leishmania (L.) amazonensis in an experimental model of cutaneous leishmaniasis using C57BL6/J mice transgenic for human CETP (CETP), having as control their littermates that do not express the protein, wild-type (WT) mice. The progression of the lesion after infection in the footpad was monitored for 12 weeks. Two groups of animals were formed to collect the plantar pad in the 4th and 12th week post-infection., Results: The lesion increased from the 3rd week onwards, in both groups, with a gradual decrease from the 10th week onwards in the CETP group compared to the WT group, showing a reduction in parasitism and an improvement in the healing process, a reduction in CD68+ cells, and an increase in CD163+ and CD206, characterizing a population of M2 macrophages. A reduction in ARG1+ cells and an increase in INOS+ cells were observed. During infection, the LP profile showed an increase in triglycerides in the VLDL fraction in the CETP group at 12 weeks. Gene expression revealed a decrease in the CD36 receptor in the CETP group at 12 weeks, correlating with healing and parasite reduction. In vitro , macrophages derived from bone marrow cells from CETP mice showed lower parasite load at 48 h and, a reduction in arginase activity at 4 h accompanied by increased NO production at 4 and 24 h compared to WT macrophages, corroborating the in vivo findings., Discussion: The data indicate that the presence of CETP plays an important role in resolving Leishmania (L.) amazonensis infection, reducing parasitism, and modulating the inflammatory response in controlling infection and tissue repair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Batista-Dantas, Ozaki, Santana, Nunes, Uscata, Siess-Portugal, Reis, Yamashiro-Kanashiro, Tafuri, Duarte-Neto, Sotto, Goto and Cazita.)

Background: In Southeast Asia, treatment is recommended for all patients with post-kala-azar dermal leishmaniasis (PKDL). Adherence to the first-line regimen, twelve weeks of miltefosine (MF), is low and ocular toxicity has been observed with this exposure period. We assessed the safety and efficacy of two shorter-course treatments: liposomal amphotericin B (LAmB) alone and combined with MF., Methodology/principal Findings: An open-label, phase II, randomized, parallel-arm, non-comparative trial was conducted in patients with parasitologically confirmed PKDL, 6 to ≤60 years. Patients were assigned to 20 mg/kg LAmB (total dose, in five injections over 15 days) alone or combined with allometric MF (3 weeks). The primary endpoint was definitive cure at 12 months, defined as complete resolution of papular and nodular lesions and >80% re-pigmentation of macular lesions. Definitive cure at 24 months was a secondary efficacy endpoint. 118/126 patients completed the trial. Definitive cure at 12 months was observed in 29% (18/63) patients receiving LAmB and 30% (19/63) receiving LAmB/MF (mITT), increasing to 58% and 66%, respectively, at 24 months. Most lesions had resolved/improved at 12 and 24 months for patients receiving LAmB (90%, 83%) and LAmB/MF (85%, 88%) by qualitative assessment. One death, unrelated to study drugs, was reported; no study drug-related serious adverse events were observed. The most frequent adverse drug reactions were MF-related vomiting and nausea, and LAmB-related hypokalaemia and infusion reactions. Most adverse events were mild; no ocular adverse events occurred., Conclusions/significance: Both regimens are suitably safe and efficacious alternatives to long-course MF for PKDL in South Asia., Trial Registration: CTRI/2017/04/008421., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: MdB declares consultancy fees from DNDi. The remaining authors report no conflicts of interest., (Copyright: © 2024 Sundar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Zoonotic cutaneous leishmaniasis (ZCL) is a neglected tropical disease caused by Leishmania (L.) major. This zoonosis is characterized by a broad-spectrum clinical polymorphism and may be underestimated and poorly treated since it is a simulator of various dermatoses. The aim of our study was to analyze the clinical polymorphism of patients with ZCL. A total of 142 patients with confirmed CL based on the microscopic examination of skin lesion biopsies were included in this study. Molecular typing of Leishmania species revealed that all patients were infected with L. major. In total, 14 clinical forms were observed. Six were typical and eight were atypical. The typical ZCL forms are grouped as follows: papular (26.76%), ulcero-crusted (26.05%), ulcerated (13.38%), impetiginous (9.86%), nodular (9.15%), and papulo-nodular (5.63%) lesions. In atypical ZCL forms, we described erythematous (2.81%), erysipeloid (1.4%), sporotrichoid, (1.4%), keratotic (0.7%) lupoid (0.7%), lichenoid (0.7%), psoriasiform (0.7%), and zosteriform (0.7%) lesions. Here, the lichenoid and the keratotic forms caused by L. major were reported for the first time in Tunisia. These findings will help physicians to be aware of the unusual lesions of ZCL that could be confused with other dermatological diseases. For this reason, it will be necessary to improve the diagnosis of CL especially in endemic areas. Such large clinical polymorphism caused by L. major may be the result of a complex association between the vector microbiota, the parasite, and the host immune state, and further studies should be carried out in order to reveal the mechanisms involved in clinical polymorphism of ZCL., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Valencia-Portillo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Post-kala-azar dermal leishmaniasis (PKDL) patients are a key source of Leishmania donovani parasites, hindering the goal of eliminating visceral leishmaniasis (VL). Monitoring treatment response and parasite susceptibility is essential due to increasing drug resistance. We assessed the drug susceptibility of PKDL isolates ( n = 18) from pre-miltefosine (MIL) era (1997-2004) with isolates ( n = 16) from the post-miltefosine era (2010-2019) and post-miltefosine treatment relapse isolates ( n = 5) towards miltefosine and amphotericin B (AmB) at promastigote stage and towards sodium antimony gluconate (SAG) at amastigote stage. PKDL isolates were examined for mutation in gene-encoding AQP1 transporter, C26882T mutation on chromosome 24, and miltefosine-transporter (MT). PKDL isolates from the post-miltefosine era were significantly more susceptible to SAG than SAG-resistant isolates from the pre-miltefosine era ( P = 0.0002). There was no significant difference in the susceptibility of parasites to miltefosine between pre- and post-miltefosine era isolates. The susceptibility of PKDL isolates towards AmB remained unchanged between the pre- and post-miltefosine era. However, the post-miltefosine era isolates had a higher IC 50 value towards AmB compared with PKDL relapse isolates. We did not find any association between AQP1 gene sequence variation and susceptibility to SAG, or between miltefosine susceptibility and single nucleotide polymorphisms (SNPs in the MT gene. This study demonstrates that recent isolates of Leishmania have resumed susceptibility to antimonials in vitro . The study also offers significant insights into the intrinsic drug susceptibility of Leishmania parasites over the past two decades, covering the period before the introduction of miltefosine and after its extensive use., Importance: Post-kala-azar dermal leishmaniasis (PKDL) patients, a key source of Leishmania donovani parasites, hinder eliminating visceral-leishmaniasis. Assessment of the susceptibility of PKDL isolates to antimony, miltefosine (MIL), and amphotericin-B indicated that recent isolates remain susceptible to antimony, enabling its use with other drugs for treating PKDL., Competing Interests: The authors declare no conflict of interest.

Cutaneous leishmaniasis caused by Leishmania parasites exhibits a wide range of clinical manifestations. Although parasites influence disease severity, cytolytic CD8+ T cell responses mediate disease. Although these responses originate in the lymph node, we found that expression of the cytolytic effector molecule granzyme B was restricted to lesional CD8+ T cells in Leishmania-infected mice, suggesting that local cues within inflamed skin induced cytolytic function. Expression of Blimp-1 (Prdm1), a transcription factor necessary for cytolytic CD8+ T cell differentiation, was driven by hypoxia within the inflamed skin. Hypoxia was further enhanced by the recruitment of neutrophils that consumed oxygen to produce ROS and ultimately increased the hypoxic state and granzyme B expression in CD8+ T cells. Importantly, lesions from patients with cutaneous leishmaniasis exhibited hypoxia transcription signatures that correlated with the presence of neutrophils. Thus, targeting hypoxia-driven signals that support local differentiation of cytolytic CD8+ T cells may improve the prognosis for patients with cutaneous leishmaniasis, as well as for other inflammatory skin diseases in which cytolytic CD8+ T cells contribute to pathogenesis.

Background: Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis., Methods and Results: 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR., Conclusion: The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

In the context of neglected diseases, tegumentary leishmaniasis (TL) presents an emerging and re-emerging character in the national territory and in the world. The treatment of TL has limitations, such as intravenous administration route, high toxicity, and high treatment costs. Thus, several researchers work on new therapeutic strategies to improve the effectiveness of the treatment of leishmaniasis. In this light, the present study used a topical formulation, containing 8-hydroquinoline (8-HQN), for the treatment of Balb/c mice infected with L. amazonensis. After the treatment, the mean diameter of the lesion was measured, as well as the parasite load in organs and immunological parameters associated with the treatment. The results showed that the animals treated with 8-HQN 5%, when compared to controls, showed a reduction in the mean diameter of the lesion and in the parasite load. The animals treated with the ointment showed a type 1 cellular immune response profile associated with the production of cytokines such as INF-γ and TNF-α. In addition, the treatment did not demonstrate toxicity to mice. Therefore, the topical formulation containing 8-HQN 5% is a promising candidate in the topical treatment and could be considered, in the future, as an alternative for the treatment of TL., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In visceral and mucocutaneous leishmaniasis, humoral immune response can reflect disease severity and parasite burden. Cutaneous leishmaniasis (CL) in Sri Lanka is caused by a usually visceralizing parasite, Leishmania donovani. We assessed the parasite burden (relative quantity-RQ) in 190 CL patients using quantitative real-time PCR (qPCR-with primers designed for this study) and smear microscopy, then correlated it with clinical parameters and IgG response. RQ of parasite DNA was determined with human-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The qPCR sensitivity was tested with serially diluted DNA from cultured L. donovani parasites. Smears were assigned a score based on number of parasites per high power field. Data from previous studies were used for comparison and correlation; nested Internal Transcribed Spacer 1 (ITS1) PCR as reference standard (RS) and IgG antibody titers to the Leishmania rKRp42 antigen as the immune response. The qPCR amplified and quantified 86.8% of the samples while demonstrating a fair and significant agreement with ITS1-PCR and microscopy. Parasite burden by qPCR and microscopy were highly correlated (r = 0.76; p = 0.01) but showed no correlation with the IgG response (r = 0.056; p = 0.48). Corresponding mean RQs of IgG titers grouped by percentiles, showed no significant difference (p = 0.93). Mean RQ was higher in early lesions (p = 0.04), decreased with lesion size (p = 0.12) and slightly higher among papules, nodules and wet ulcers (p = 0.72). Our study established qPCR's efficacy in quantifying parasite burden in Sri Lankan CL lesions but no significant correlation was observed between the parasite burden and host IgG response to the Leishmania rKRP42 antigen., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC 50 ) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC 50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Cutaneous leishmaniasis (CL) is a vector-borne disease characterized by skin lesions that can evolve into high-magnitude ulcerated lesions. Thus, this study aimed to develop an innovative nanoemulsion (NE) with clove oil, Poloxamer® 407, and multiple drugs, such as amphotericin B (AmB) and paromomycin (PM), for use in the topical treatment of CL., Methods: Droplet size, morphology, drug content, stability, in vitro release profile, in vitro cytotoxicity on RAW 264.7 macrophages, and antileishmanial activity using axenic amastigotes of Leishmania amazonensis were assessed for NEs., Results: After optimizing the formulation parameters, such as the concentration of clove oil and drugs, using an experimental design, it was possible to obtain a NE with an average droplet size of 40 nm and a polydispersion index of 0.3, and these parameters were maintained throughout the 365 days. Furthermore, the NE showed stability of AmB and PM content for 180 days under refrigeration (4 °C), presented a pH compatible with the skin, and released modified AmB and PM. NE showed the same toxicity as free AmB and higher toxicity than free PM against RAW 264.7 macrophages. The same activity as free AmB, and higher activity than free PM against amastigotes L. amazonensis., Conclusion: It is possible to develop a NE for the treatment of CL; however, complementary studies regarding the antileishmanial activity of NE should be carried out., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The infectious inoculum of a sand fly, apart from its metacyclic promastigotes, is composed of factors derived from both the parasite and the vector. Vector-derived factors, including salivary proteins and the gut microbiota, are essential for the establishment and enhancement of infection. However, the type and the number of bacteria egested during salivation is unclear. In the present study, sand flies of Phlebotomus papatasi were gathered from three locations in hyperendemic focus of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan Province, Iran. By using the forced salivation assay and targeting the 16S rRNA barcode gene, egested bacteria were characterized in 99 (44%) out of 224 sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. Ten top genera of Spiroplasma, Ralstonia, Acinetobacter, Reyranella, Undibacterium, Bryobacter, Corynebacterium, Cutibacterium, Psychrobacter, and Wolbachia constituted >80% of the saliva microbiome. Phylogenetic analysis displayed the presence of only one bacterial species for the Spiroplasma, Ralstonia, Reyranella, Bryobacter and Wolbachia, two distinct species for Cutibacterium, three for Undibacterium and Psychrobacter, 16 for Acinetobacter, and 27 for Corynebacterium, in the saliva. The abundance of microbes in P. papatasi saliva was determined by incorporating the data on the read counts and the copy number of 16S rRNA gene, about 9,000 bacterial cells, per sand fly. Both microbiological and metagenomic data indicate that bacteria are constant companions of Leishmania, from the intestine of the vector to the vertebrate host. This is the first forced salivation experiment in a sand fly, addressing key questions on infectious bite and competent vectors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Maleki-Ravasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L. amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L. amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ferreira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity. CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5, Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Amorim Sacramento et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 μg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fernández et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cutaneous leishmaniasis is the most prevalent form of leishmaniasis worldwide. Although various anti-leishmanial regimens have been considered, due to the lack of efficacy or occurrence of adverse reactions, design and development of novel topical delivery systems would be essential. This study aimed to prepare artemether (ART)-loaded niosomes and evaluate their anti-leishmanial effects against Leishmania major. ART-loaded niosomes were prepared through the thin-film hydration technique and characterized in terms of particle size, zeta potential, morphology, differential scanning calorimetry, drug loading, and drug release. Furthermore, anti-leishmanial effect of the preparation was assessed in vitro and in vivo. The prepared ART-loaded niosomes were spherical with an average diameter of about 100 and 300 nm with high encapsulation efficiencies of > 99%. The results of in vitro cytotoxicity revealed that ART-loaded niosomes had significantly higher anti-leishmanial activity, lower general toxicity, and higher selectivity index (SI). Half-maximal inhibitory concentration (IC50) values of ART, ART-loaded niosomes, and liposomal amphotericin B were 39.09, 15.12, and 20 µg/mL, respectively. Also, according to the in vivo study results, ART-loaded niosomes with an average size of 300 nm showed the highest anti-leishmanial effects in animal studies. ART-loaded niosomes would be promising topical drug delivery system for the management of cutaneous leishmaniasis., (© 2024. The Author(s).)

Despite efforts, available alternatives for the treatment of leishmaniasis are still scarce. In this work we tested a class of 15 quinolinylhydrazone analogues and presented data that support the use of the most active compound in cutaneous leishmaniasis caused by Leishmania amazonensis. In general, the compounds showed activity at low concentrations for both parasitic forms (5.33-37.04 μM to promastigotes, and 14.31-61.98 μM to amastigotes). In addition, the best compound (MHZ15) is highly selective for the parasite. Biochemical studies indicate that the treatment of promastigotes with MHZ15 leads the loss of mitochondrial potential and increase in ROS levels as the primary effects, which triggers accumulation of lipid droplets, loss of plasma membrane integrity and apoptosis hallmarks, including DNA fragmentation and phosphatidylserine exposure. These effects were similar in the intracellular form of the parasite. However, in this parasitic form there is no change in plasma membrane integrity in the observed treatment time, which can be attributed to metabolic differences and the resilience of the amastigote. Also, ultrastructural changes such as vacuolization suggesting autophagy were observed. The in vivo effectiveness of MHZ15 in the experimental model of cutaneous leishmaniasis was carried out in mice of the BALB/c strain infected with L. amazonensis. The treatment by intralesional route showed that MHZ15 acted with great efficiency with significantly reduction in the parasite load in the injured paws and draining lymph nodes, without clinical signs of distress or compromise of animal welfare. In vivo toxicity was also evaluated and null alterations in the levels of hepatic enzymes aspartate aminotransferase, and alanine aminotransferase was observed. The data presented herein demonstrates that MHZ15 exhibits a range of favorable characteristics conducive to the development of an antileishmanial agent., (© 2024 John Wiley & Sons Ltd.)

Background: Cutaneous leishmaniasis (LC) is an infectious vector-borne disease caused by parasites belonging to the genus Leishmania. Metallic nanoparticles (MNPs) have been investigated as alternatives for the treatment of LC owing to their small size and high surface area. Here, we aimed to evaluate the effect of MNPs in the treatment of LC through experimental, in vitro and in vivo investigations., Methods: The databases used were MEDLINE/ PubMed, Scopus, Web of Science, Embase, and Science Direct. Manual searches of the reference lists of the included studies and grey literature were also performed. English language and experimental in vitro and in vivo studies using different Leishmania species, both related to MNP treatment, were included. This study was registered in PROSPERO (CRD42021248245)., Results: A total of 93 articles were included. Silver nanoparticles are the most studied MNPs, and L. tropica is the most studied species. Among the mechanisms of action of MNPs in vitro, we highlight the production of reactive oxygen species, direct contact of MNPs with the biomolecules of the parasite, and release of metal ions., Conclusion: MNPs may be considered a promising alternative for the treatment of LC, but further studies are needed to define their efficacy and safety., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)