Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing.

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multi-locus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.

Canine visceral leishmaniasis: Seroprevalence and georeferencing in the state of Santa Catarina, Brazil.

Canine visceral leishmaniasis (CVL) has long been considered an endemic disease in the northern and northeastern regions of Brazil, while the southern region remains non-endemic. However, in recent years, several cases of CVL have been reported in southern states. The objective of this work was to determine the seroprevalence of CVL in dogs in the state of Santa Catarina, Brazil, through immunochromatographic tests (DPP®) and ELISA (Enzyme-Linked Immunosorbent Assay) and its correlation with environmental characteristics through georeferencing. Blood samples from dogs (n = 1227) were collected in six mesoregions of the state and evaluated by the rapid test (DPP®). Positive samples were sent to Lacen (Central Public Health Laboratory) in Santa Catarina to be tested using ELISA. Information obtained from the epidemiological questionnaire was subjected to statistical analysis (Chi-square and Student's t-test; P < 0.05) to verify the correlation between serology and the analyzed variables. The locations (GPS) of the samples were used for georeferencing and creating heatmaps (Kernel Method). Four animals that died from CVL were necropsied and organ samples were collected for molecular analysis (PCR), immunohistochemistry, and histopathology (HE). Of the 1227 dogs analyzed, 22 (1.8%) were reactive in the DPP® and of these, 7 (0.6%) were also positive in the ELISA. A correlation (P < 0.01) was observed between positive serology and region, environment, access to the street, and clinical signs. The positive cases were concentrated in the eastern region of the state, in low-altitude areas with average rainfall and higher average temperatures, and in more populated areas close to forest fragments. PCR, HE, and immunohistochemistry, along with serology, have proven to be efficient for characterizing positive cases.

Differential structure and immunomodulatory functions of lipophosphoglycan between Leishmania spp.

Leishmaniasis is a collective term for several tropical, neglected diseases caused by protozoans of the species Leishmania, 20 of which causing disease in humans ranging from localised self-healing lesions to chronic manifestations which affect the skin or inner organs. Although millions of infections are accounted for annually, treatment options are scarce and limited to medication associated with heavy side-effects and increasing antibiotic resistance. Case studies point towards immunotherapy as effective alternative treatment relying on immunomodulatory properties of e.g., the Bacillus Calmette-Guérin vaccine. Leishmania parasites are also known to modulate the immune system, yet the underlying macromolecules and surface molecules remain widely under characterised. With this short review, we aim to provide a complete summary of the existing literature describing one of the most expressed surface molecule on Leishmania spp, lipophosphoglycan (LPG), which shows great variability between different lifecycle stages and different Leishmania spp. Complete characterisation of LPG may aid to improve treatment and aid the development of vaccination strategies, and open new avenues to exploit the immunomodulatory properties of LPG in unrelated conditions.

Finding Priority Areas in the Evaluation of Strategies for the Prevention of Leishmaniasis in an Endemic Municipality of Brazil.

Visceral leishmaniasis is a zoonotic disease that affects humans and dogs. The infection is endemic in the municipality of Araçatuba, São Paulo, Brazil. Given the role of dogs in the epidemiology of leishmaniasis, strategies to enhance surveillance and reduce transmission are focused on dogs. In this study, we retrospectively analyzed records of canine visceral leishmaniasis from 2013 to 2022. According to this database, the prevalence of dogs testing positive for leishmaniasis fluctuated, with an average of 65.04% (6590/10,133). Cases were clustered in 10 statistically significant areas. Environmental analyses identified a significant geographical association between animals testing positive and higher vegetation density rates compared with animals testing negative. The period from sample collection to diagnosis and euthanasia, as recommended by the Brazilian Ministry, correlated with disease prevalence and decreased over time. These findings serve to implement different action plans against leishmaniasis for each geographic region and to understand the impact and efforts of strategies in an endemic area.

Leishmania spp. amastigotes surrounding sensory nerve fibers in human painless skin ulcers: Evidence of pathogen-neuron proximity and absence of neuronal apoptosis.

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.

Extracellular Vesicles: Translational Agenda Questions for Three Protozoan Parasites.

The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll on the disease landscape of the human population in sub-Saharan Africa and Latin America. Control measures have helped reduce the burden of their respective diseases-malaria, leishmaniasis and Chagas disease-in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub-Saharan Africa and Latin America.

Promising aryl selenoate derivatives as antileishmanial agents and their effects on gene expression.

Leishmaniasis remains one of the main public health problems worldwide, with special incidence in the poorest populations. Selenium and its derivatives can be potent therapeutic options against protozoan parasites. In this work, 17 aryl selenoates were synthesized and screened against three species of Leishmania (Leishmania major, Leishmania amazonensis, and Leishmania infantum). Initial screening in promastigotes showed L. infantum species was more sensitive to selenoderivatives than the others. The lead Se-(2-selenocyanatoethyl) thiophene-2-carboselenoate (16) showed a half-maximal effective concentration of 3.07 µM and a selectivity index > 32.57 against L. infantum promastigotes. It was also the most effective of all 17 compounds, decreasing the infection ratio by 90% in L. infantum-infected macrophages with amastigotes at 10 µM. This aryl selenoate did not produce a hemolytic effect on human red blood cells at the studied doses (10-100 µM). Furthermore, the gene expression of infected murine macrophages related to cell death, the cell cycle, and the selenoprotein synthesis pathway in amastigotes was altered, while no changes were observed in their murine homologs, supporting the specificity of Compound 16 against the parasite. Therefore, this work reveals the possible benefits of selenoate derivatives for the treatment of leishmaniasis.

GSK-3 kinase a putative therapeutic target in trypanosomatid parasites.

Trypanosomatids are an important group of parasites that predominate in tropical and subtropical areas of the planet, which cause diseases that are classified as forgotten and neglected by the world health organization. In this group of parasites, we find Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma brucei rhodesiense and Leishmania spp, for which there is no vaccine available, and its control has focused mainly on pharmacological treatment. Due to the poverty situation where these diseases are found and the biological complexity of these parasites, there are multiple variables to control, including the diversity of species, the complexity of their life cycles, drug resistance, cytotoxicity, the limited use in pregnant women, the high costs of treatment and the little-known pharmacological mechanisms of action, among others. It is therefore necessary to find new strategies and approaches for the treatment of these parasitic diseases. Among these new approaches is the rational search for new targets based on the allosteric inhibition of protein kinases, which have been little studied in trypanosomatids. Among these kinases, we find Glycogen Synthase Kinase-3 (GSK-3), a kinase of great pharmacological interest, which is under intense basic and clinical research by pharmaceutical companies for the treatment of cancer. This kinase, highly studied in the PI3K/AKT/mTOR pathway signaling in humans, has an orthologous gene in these parasites (GSK-3 s), which has proven to be essential for them in response to different challenges; Therefore, it is notable to increase research in this kinase in order to achieve a broad structural and functional characterization in the different species of trypanosomatids.

Salvadoran Celastraceae Species as a Source of Antikinetoplastid Quinonemethide Triterpenoids.

Chagas disease and leishmaniasis are among the most widespread neglected tropical diseases, and their current therapies have limited efficacy and several toxic side effects. The present study reports the chemical and antikinetoplastid profiles of extracts from five Salvadoran Celastraceae species against the Trypanosoma cruzi epimastigotes stage and Leishmania amazonensis and Leishmania donovani promastigote forms. The phytochemical profile evinced the presence of flavonoids, tannins, sterols, and triterpenes as the main components in all plant species, whereas quinonemethide triterpenoids (QMTs) were restricted to the root bark of the studied species. Antikinetoplastid evaluation highlights the root bark extracts from Zinowewia integerrima, Maytenus segoviarum, and Quetzalia ilicina as the most promising ones, exhibiting higher potency against T. cruzi (IC50 0.71-1.58 µg/mL) and L. amazonensis (IC50 0.38-2.05 µg/mL) than the reference drugs, benznidazole (IC50 1.81 µg/mL) and miltefosine (IC50 2.64 µg/mL), respectively. This potent activity was connected with an excellent selectivity index on the murine macrophage J774A.1 cell line. These findings reinforce the potential of QMTs as antikinetoplastid agents for the development of innovative phytopharmaceuticals and the plant species under study as a source of these promising lead compounds.

Characterization of Novel Trypanosoma cruzi-Specific Antigen with Potential Use in the Diagnosis of Chagas Disease.

Chagas disease is caused by the parasite Trypanosoma cruzi. In humans, it evolves into a chronic disease, eventually resulting in cardiac, digestive, and/or neurological disorders. In the present study, we characterized a novel T. cruzi antigen named Tc323 (TcCLB.504087.20), recognized by a single-chain monoclonal antibody (scFv 6B6) isolated from the B cells of patients with cardiomyopathy related to chronic Chagas disease. Tc323, a ~323 kDa protein, is an uncharacterized protein showing putative quinoprotein alcohol dehydrogenase-like domains. A computational molecular docking study revealed that the scFv 6B6 binds to an internal domain of Tc323. Immunofluorescence microscopy and Western Blot showed that Tc323 is expressed in the main developmental forms of T. cruzi, localized intracellularly and exhibiting a membrane-associated pattern. According to phylogenetic analysis, Tc323 is highly conserved throughout evolution in all the lineages of T. cruzi so far identified, but it is absent in Leishmania spp. and Trypanosoma brucei. Most interestingly, only plasma samples from patients infected with T. cruzi and those with mixed infection with Leishmania spp. reacted against Tc323. Collectively, our findings demonstrate that Tc323 is a promising candidate for the differential serodiagnosis of chronic Chagas disease in areas where T. cruzi and Leishmania spp. infections coexist.

GSK-3 kinase a putative therapeutic target in trypanosomatid parasites

ABSTRACT Trypanosomatids are an important group of parasites that predominate in tropical and subtropical areas of the planet, which cause diseases that are classified as forgotten and neglected by the world health organization. In this group of parasites, we find Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma brucei rhodesiense and Leishmania spp, for which there is no vaccine available, and its control has focused mainly on pharmacological treatment. Due to the poverty situation where these diseases are found and the biological complexity of these parasites, there are multiple variables to control, including the diversity of species, the complexity of their life cycles, drug resistance, cytotoxicity, the limited use in pregnant women, the high costs of treatment and the little-known pharmacological mechanisms of action, among others. It is therefore necessary to find new strategies and approaches for the treatment of these parasitic diseases. Among these new approaches is the rational search for new targets based on the allosteric inhibition of protein kinases, which have been little studied in trypanosomatids. Among these kinases, we find Glycogen Synthase Kinase-3 (GSK-3), a kinase of great pharmacological interest, which is under intense basic and clinical research by pharmaceutical companies for the treatment of cancer. This kinase, highly studied in the PI3K/AKT/mTOR pathway signaling in humans, has an orthologous gene in these parasites (GSK-3 s), which has proven to be essential for them in response to different challenges; Therefore, it is notable to increase research in this kinase in order to achieve a broad structural and functional characterization in the different species of trypanosomatids.

Advances in the Development of Carbonic Anhydrase Inhibitors as New Antiprotozoal Agents.

BACKGROUND: Parasitic diseases are a public health problem despite the existence of drugs for their treatment. These treatments have variable efficacy and, in some cases, serious adverse effects. There has been interest in the enzyme carbonic anhydrase (CA) in the last two decades since it is essential in the life cycle of various parasites due to its important participation in processes such as pyrimidine synthesis, HCO3- transport across cell membranes, and the maintenance of intracellular pH and ion transport (Na+, K+, and H+), among others. OBJECTIVE: In this review, CA was analyzed as a pharmacological target in etiological agents of malaria, American trypanosomiasis, leishmaniasis, amoebiasis, and trichomoniasis. The CA inhibitors´ design, binding mode, and structure-activity relationship are also discussed. CONCLUSION: According to this review, advances in discovering compounds with potent inhibitory activity suggest that CA is a candidate for developing new antiprotozoal agents.

Assessment of the diagnostic performance of serological tests in areas where Leishmania infantum and Leishmania tarentolae occur in sympatry.

BACKGROUND: Visceral leishmaniosis caused by infection with the zoonotic protozoan Leishmania infantum is a life-threatening disease affecting dogs and humans. The sympatric occurrence of L. infantum and Leishmania tarentolae in an area of southern Italy endemic for canine leishmaniosis, where dogs are also exposed to the latter species, suggests the persistence of herpetophilic L. tarentolae in a non-permissive host, therefore raising questions about the performance of serological diagnostic tests routinely employed. METHODS: The diagnostic performance of serological tests such as the immunofluorescence antibody test (IFAT), two commercial immunoenzymatic assays (i.e. NovaTec VetLine Leishmania ELISA® and rK39 ICT®) and an in-house enzyme-linked immunosorbent assay (ELISA) was evaluated in healthy dogs seropositive to L. infantum, whereas the only IFAT available was used to detect antibodies to L. tarentolae. RESULTS: With the IFAT, out of a total of 104 dogs tested, 15 were seronegative for L. infantum of which three were L. tarentolae seropositive' and 89 were L. infantum seropositive. Of the latter 89 dogs, representing the highest proportion of seropositive animals (85.6%) detected by IFAT' 66 were also seropositive for L. tarentolae. Cohen's kappa (κ) agreement coefficient between the IFAT results and those of all the other tests was very low, and the IFAT results were significantly different from those of all the other serological tests as calculated by Cochran's Q-test. Analysis using the Bayesian latent class (Bayes-LCA) showed that the in-house ELISA and IFAT contributed the most towards identifying infected and non-infected dogs, respectively. The IFAT test showed low positive predictive value (59.5%), but high negative predictive value (100%). CONCLUSIONS: These results demonstrate that the IFAT for L. infantum, although highly sensitive, may not be considered a useful diagnostic test due to its low specificity. Therefore, an accurate serological tool with high specificity is mandatory for avoiding cross-reaction in epidemiological contexts where the two species of Leishmania occur in sympatry.

Zoonotic Microparasites in Invasive Black Rats (Rattus rattus) from Small Islands in Central Italy.

Invasive species have a detrimental impact on native populations, particularly in island ecosystems, and they pose a potential zoonotic and wildlife threat. Black rats (Rattus rattus) are invasive species that disrupt native flora and fauna on islands and serve as potential competent reservoirs for various pathogens and parasites. Microparasites screening was conducted in rat populations from small islands in central Italy (the Pontine Islands and Pianosa) with the aim of assessing the role of rats in maintaining infections, particularly in cases where key reservoir hosts were scarce or absent. We focused on microparasites of zoonotic and veterinary relevance. A total of 53 rats was kill-trapped and target tissues were analysed with molecular techniques. We observed the absence or very low prevalence of Anaplasma spp., while Babesia was found in rats from all locations, marking the first recorded instance of Babesia divergens in wild rats. Data from Pianosa strongly suggest the presence of an autochthonous Leishmania infantum cycle in the Tuscan archipelago islands. Neospora caninum was absent from all islands, even in areas where dogs, the main reservoirs, were present. Toxoplasma gondii was only recorded on the Pontine Islands, where genotyping is needed to shed light on infection dynamics. This study confirms that invasive species, such as rats, may be responsible for maintaining an increased parasitological threat to fauna and human communities in certain ecosystems.

The application of MD simulation to lead identification, vaccine design, and structural studies in combat against leishmaniasis - A review.

Drug discovery, vaccine design, and protein interaction studies are rapidly moving toward the routine use of molecular dynamics simulations (MDS) and related methods. As a result of MDS, it is possible to gain insights into the dynamics and function of identified drug targets, antibody-antigen interactions, potential vaccine candidates, intrinsically disordered proteins, and essential proteins. The MDS appears to be used in all possible ways in combating diseases such as cancer, however, it has not been well documented as to how effectively it is applied to infectious diseases such as Leishmaniasis. As a result, this systematic review aims to survey the application of MDS in combating leishmaniasis. We have systematically collected articles that illustrate the implementation of MDS in drug discovery, vaccine development, and structural studies related to Leishmaniasis. Of all the articles reviewed, we identified that only a limited number of studies focused on the development of vaccines against Leishmaniasis through MDS. Also, the PCA and FEL studies were not carried out in most of the studies. These two were globally accepted utilities to understand the conformational changes and hence it is recommended that this analysis should be taken up in similar approaches in the future.

Intravenous Liposomal Amphotericin B Efficacy and Safety for Cutaneous and Mucosal Leishmaniasis: A Systematic Review and Meta-analysis.

Background: Tegumentary leishmaniasis is often subject to limited funding, underpowered studies, and a paucity of high-quality interventional studies. Intravenous liposomal amphotericin B (L-AmB) has been increasingly used to treat cutaneous and mucosal leishmaniasis (CL and ML, respectively) despite the lack of well-conducted interventional studies. We conducted a systematic review to consolidate the descriptive evidence on the efficacy and safety of L-AmB in treating CL and ML. Methods: Several online databases and the reference lists of included studies were searched to extract data from 132 studies comprising both case reports and case series. The population, intervention, comparison, outcome, and study design strategy and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were used. Results: Of 132 studies included, 92 were case reports and 40 were case series. Of the 92 cases, 65 (82.3%) were considered cured after receiving L-AmB as part of their treatment regimen. Twenty-one of the 92 (22.8%) cases reported adverse reactions to L-AmB. A pooled cure rate of 87.0% (95% CI, 79.0%-92.0%) was reported for the 38 case series that reported on treatment efficacy; 40.7% of the cases were associated with an adverse reaction. Conclusions: Observational data on cure rates using L-AmB suggest efficacy between 80% and 90%, similar to rates reported for other antileishmanial drugs. The highest efficacy rates were observed when a single cycle of L-AmB was administered to patients with mild-moderate CL and ML. The limitations of this study include the heterogeneity observed among the included studies and the increased likelihood of publication bias associated with the inclusion of case reports and case series. This systematic review further illustrates the need for high-quality comparative trials of intravenous L-AmB for the treatment of tegumentary leishmaniasis.

Effects of Synthetic Ligustrazine-Based Chalcone Derivatives on Trypanosoma brucei brucei and Leishmania spp. Promastigotes.

Current medication therapy for leishmaniasis and trypanosomiasis remains a major challenge due to its limited efficacy, significant adverse effects, and inaccessibility. Consequently, locating affordable and effective medications is a pressing concern. Because of their easy-to-understand structure and high functionalization potential, chalcones are promising candidates for use as bioactive agents. Thirteen synthetic ligustrazine-containing chalcones were evaluated for their ability to inhibit the growth of leishmaniasis and trypanosomiasis in etiologic agents. The tetramethylpyrazine (TMP) analogue ligustrazine was chosen as the central moiety for the synthesis of these chalcone compounds. The most effective compound (EC50 = 2.59 µM) was the chalcone derivative 2c, which featured a pyrazin-2-yl amino on the ketone ring and a methyl substitution. Multiple actions were observed for certain derivatives, including 1c, 2a-c, 4b, and 5b, against all strains tested. Eflornithine served as a positive control, and three ligustrazine-based chalcone derivatives, including 1c, 2c, and 4b, had a higher relative potency. Compounds 1c and 2c are particularly efficacious; even more potent than the positive control, they are therefore promising candidates for the treatment of trypanosomiasis and leishmaniasis.

Degron Pathways and Leishmaniasis: Debating Potential Roles of Leishmania spp. Proteases Activity on Guiding Hosts Immune Response and Their Relevance to the Development of Vaccines.

Degrons are short peptide sequences that signalize target sites for protein degradation by proteases. Herein, we bring forth the discussion on degrons present in proteins related to the immune system of Mus musculus that are potential targets for cysteine and serine proteases of Leishmania spp. and their possible roles on host immune regulation by parasites. The Merops database was used to identify protease substrates and proteases sequence motifs, while MAST/MEME Suite was applied to find degron motifs in murine cytokines (IFN-y, IL-4, IL-5, IL-13, IL-17) and transcription factors (NF-kappaB, STAT-1, AP-1, CREB, and BACH2). STRING tool was used to construct an interaction network for the immune factors and SWISS-MODEL server to generate three-dimensional models of proteins. In silico assays confirm the occurrence of degrons in the selected immune response factors. Further analyses were conducted only in those with resolved three-dimensional structures. The predicted interaction network of degron-containing M. musculus proteins shows the possibility that the specific activity of parasite proteases could interfere with the trend of Th1/Th2 immune responses. Data suggest that degrons may play a role in the immune responses in leishmaniases as targets for parasite proteases activity, directing the degradation of specific immune-related factors.