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Clinical diagnosis of leishmaniasis is highly complex, presenting a wide range of clinical manifestations, sometimes non-specific, and thus the epidemiological study and diagnostic need specific molecular markers for each Leishmania species. Leishmania spp. posses different Fe-SOD isoforms, one of which is excreted into the external milieu and, presenting immunogenic characteristics, is a very reliable molecular marker. Superoxide dismutases (SODs) are antioxidant metal-enzymes responsible for the dismutation of superoxide ion into hydrogen peroxide and molecular oxygen, and it is considered an important virulence factor. In this manuscript we have purified the iron(Fe)-SOD excreted by Leishmania braziliensis using ion-exchange and molecular-sieve chromatography and we have studied it as an antigen in serodiagnostic analyses in ELISA and Western blot techniques, testing 213 human sera from Mexico. Indeed, L. braziliensis Fe-SODe has been purified 123.26 times with a specific activity of about 893.66 U/mg of protein. Applying the purified enzymes in serological tests we found 17.84% sera positive. We have demonstrated that the purified enzyme is more sensitive than the non-purified ones and we also demonstrated, for the first time, the presence of antibodies against L. braziliensis, not the main species in the country, in human population from Hidalgo and Nuevo Leon States.

Abstract Background The emergence of diseases of public health concern is enhanced by factors associated with global change, such as the introduction of invasive species. The Asian tiger mosquito (Aedes albopictus), considered a competent vector of different viruses and parasites, has been successfully introduced into Europe in recent decades. Molecular screening of parasites in mosquitoes (i.e. molecular xenomonitoring) is essential to understand the potential role of different native and invasive mosquito species in the local circulation of vector-borne parasites affecting both humans and wildlife. Methods The presence of avian Plasmodium parasites was molecularly tested in mosquitoes trapped in five localities with different environmental characteristics in southern Spain from May to November 2022. The species analyzed included the native Culex pipiens and Culiseta longiareolata and the invasive Ae. albopictus. Results Avian Plasmodium DNA was only found in Cx. pipiens with 31 positive out of 165 mosquito pools tested. None of the Ae. albopictus or Cs. longiareolata pools were positive for avian malaria parasites. Overall, eight Plasmodium lineages were identified, including a new lineage described here. No significant differences in parasite prevalence were found between localities or sampling sessions. Conclusions Unlike the invasive Ae. albopictus, Cx. pipiens plays a key role in the transmission of avian Plasmodium in southern Spain. However, due to the recent establishment of Ae. albopictus in the area, further research on the role of this species in the local transmission of vector-borne pathogens with different reservoirs is required. Graphical Abstract

Leishmaniasis and Chagas disease are still considered neglected illnesses due to the lack of investment in research, despite the fact that almost one million new cases are reported every year. Four 7-oxo-5-phenyl-1,2,4-triazolo[1,5-a]pyrimidine (HftpO) first-row transition complexes (Cu, Co, Ni, Zn) have been studied for the first time in vitro against five different species of Leishmania spp. (L. infantum, L. braziliensis, L. donovani, L. peruviana and L. mexicana) as well as Trypanosoma cruzi, showing higher efficacy than the reference commercial drugs. UV and luminescence properties were also evaluated. As a proof of concept, anchoring of a model high-effective-metal complex as an antiparasitic agent on silica nanoparticles was carried out for the first time, and drug-release behaviour was evaluated, assessing this new approach for drug vehiculation.

Due to the urgent need for finding effective and free of secondary effect treatments for every clinical form of Leishmaniasis, a series of synthetic xylene, pyridine and, pyrazole azamacrocycles were tested against three Leishmania species. A total of 14 compounds were tested against J774.2 macrophage cells which were models for host cells, and against promastigote and amastigote forms of each studied Leishmania parasite. Amongst these polyamines, one proved effective against L. donovani, another one for L. braziliensis and L. infantum, and another one was selective solely for L. infantum. These compounds showed leishmanicidal activity and reduced parasite infectivity and dividing ability. Action mechanism studies gave a hint that compounds were active against Leishmania due to their ability to alter parasite metabolic pathways and reduce (except Py33333) parasitic Fe-SOD activity.

Chagas disease (CD) is a tropical and potentially fatal infection caused by Trypanosoma cruzi. Although CD was limited to Latin America as a silent disease, CD has become widespread as a result of globalization. Currently, 6–8 million people are infected worldwide, and no effective treatment is available. Here, we identify new effective agents against T. cruzi. In short, 16 aryl polyamines were screened in vitro against different T. cruzi strains, and lead compounds were evaluated in vivo after oral administration in both the acute and chronic infections. The mode of action was also evaluated at the energetic level, and its high activity profile could be ascribed to a mitochondria-dependent bioenergetic collapse and redox stress by inhibition of the Fe-SOD enzyme. We present compound 15 as a potential compound that provides a step forward for the development of new agents to combat CD.

The in vitro antimicrobial potency of the bacteriocin AS-48 is well documented, but its clinical application requires investigation, as its toxicity could be different in in vitro (haemolytic and antibacterial activity in blood and cytotoxicity towards normal human cell lines) and in vivo (e.g. mice and zebrafish embryos) models. Overall, the results obtained are promising. They reveal the negligible propensity of AS-48 to cause cell death or impede cell growth at therapeutic concentrations (up to 27 μM) and support the suitability of this peptide as a potential therapeutic agent against several microbial infections, due to its selectivity and potency at low concentrations (in the range of 0.3–8.9 μM). In addition, AS-48 exhibits low haemolytic activity in whole blood and does not induce nitrite accumulation in non-stimulated RAW macrophages, indicating a lack of pro-inflammatory effects. The unexpected heightened sensitivity of zebrafish embryos to AS-48 could be due to the low differentiation state of these cells. The low cytotoxicity of AS-48, the absence of lymphocyte proliferation in vivo after skin sensitization in mice, and the lack of toxicity in a murine model support the consideration of the broad spectrum antimicrobial peptide AS-48 as a promising therapeutic agent for the control of a vast array of microbial infections, in particular, those involved in skin and soft tissue diseases. Keywords: Cytotoxicity, Antimicrobial peptides, Haemolysis, Topical delivery, Zebrafish model, Mouse model

Chagas disease caused by the protozoan parasite Trypanosoma cruzi represents a significant public health problem in Latin America, affecting around 8 million cases worldwide. Nowadays is urgent the identification of new antichagasic agents as the only therapeutic options available, Nifurtimox and Benznidazole, are in use for >40 years, and present high toxicity, limited efficacy and frequent treatment failures in the chronic phase of the disease. Recently, it has been described the antiparasitic effect of AS-48, a bacteriocin produced by Enterococcus faecalis, against Trypanosoma brucei and Leishmania spp. In this work, we have demonstrated the in vitro potential of the AS-48 bacteriocin against T. cruzi. Interesting, AS-48 was more effective against the three morphological forms of different T. cruzi strains, and displayed lower cytotoxicity than the reference drug Benznidazole. In addition, AS-48 combines the criteria established as a potential antichagasic agent, resulting in a promising therapeutic alternative. According to the action mechanism, AS-48 trypanocidal activity could be explained in a mitochondrion-dependent manner through a reactive oxygen species production and mitochondrial depolarization, causing a fast and severe bioenergetic collapse. Keywords: AS-48, Antichagasic agent, Bacteriocin, Drug discovery, Trypanosoma cruzi

Chagas disease is usually caused by tropical infection with the insect-transmitted protozoan Trypanosoma cruzi. Currently, Chagas disease is a major public health concern worldwide due to globalization, and there are no treatments neither vaccines because of the long-term nature of the disease and its complex pathology. Current treatments are limited to two obsolete drugs, benznidazole and nifurtimox, which lead to serious drawbacks. Taking into account the urgent need for strict research efforts to find new therapies, here, we describe the in vitro and in vivo trypanocidal activity of a library of selected forty-eight selenocyanate and diselenide derivatives that exhibited leishmanicidal properties. The inclusion of selenium, an essential trace element, was due to the well-known extensive pharmacological activities for selenium compounds including parasitic diseases as T. cruzi. Here we present compound 8 as a potential compound that exhibits a better profile than benznidazole both in vitro and in vivo. It shows a fast-acting behaviour that could be attributed to its mode of action: it acts in a mitochondrion-dependent manner, causing cell death by bioenergetic collapse. This finding provides a step forward for the development of a new antichagasic agent.

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), two hydroxyphthalazine derivative compounds were prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels, by 1H NMR and TEM studies. Finally, as these compounds are potentially capable of causing oxidative damage in the parasites, the study was completed, by assessing their activity as potential iron superoxide dismutase (Fe-SOD) inhibitors. High-selectivity indices observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Compound 2 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression. Compound 2 turned out to be a great inhibitor of Fe-SOD. The high antiparasitic activity and low toxicity together with the modest costs for the starting materials render this compound an appropriate molecule for the development of an affordable anti-Chagas agent.

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.

The spectrum and efficacy of available antileishmanial drugs is limited. In the present work we evaluated in vitro the antiproliferative activity of 11 compounds based on tetradentate polyamines compounds against three Leishmania species (L. braziliensis, L. donovani and L. infantum) and the possible mechanism of action. We identified six compounds (3, 5, 6, 7, 8 and 10) effective against all three Leishmania spp both on extracellular and intracellular forms. These six most active leishmanicidal compounds also prevent the infection of host cells. Nevertheless, only compound 7 is targeted against the Leishmania SOD. Meanwhile, on the glucose metabolism the tested compounds have a species-specific effect on Leishmania spp.: L. braziliensis was affected mainly by 10 and 8, L. donovani by 7, and L. infantum by 5 and 3. Finally, the cellular ultrastructure was mainly damaged by 11 in the three Leishmania spp. studied. These identified antileishmania candidates constitute a good alternative treatment and will be further studied.

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.

An excreted iron superoxide dismutase (FeSODe) of pI 3.6 with a molecular weight of 28-30 kDa was detected in the in vitro culture of Phytomonas isolated from Euphorbia characias (SODeCHA) and from Lycopersicon esculentum (SODeTOM), in Grace's medium without serum. These FeSODe excreted into the medium had immunogenic capacity: the positivity of the anti-SODeCHA serum persisted to a dilution of 1/30,000, and for the anti-SODeTOM to 1/10,000 by Western blot. In addition, cross reaction was detected between the anti-SODe serum of Phytomonas isolated from E. characias against SODeTOM, and the anti-SODe serum from L. esculentum with SODeCHA. This characteristic offers the possibility of its use to diagnose plant trypanosomatids. The validation of the test was confirmed by experimental inoculation of tomato fruits with Phytomonas isolated from L. esculentum. At 7, 10, 15, and 21 days post infection, it was possible to detect the presence of the parasites with the anti-SODe serum of Phytomonas isolated from L. esculentum at a dilution of 1/250. These serological results were confirmed by visualization of the parasites by optical microscopy. The data of this study confirm that the SOD is sufficient to identify a trypanosomatid isolated from plants as belonging to the genus Phytomonas.

There is no effective chemotherapy against diseases caused by Phytomonas sp., a plant trypanosomatid responsible for economic losses in major crops. We tested three triazolo-pyrimidine complexes [two with Pt(II), and another with Ru(III)] against promastigotes of Phytomonas sp. isolated from Euphorbia characias. The incorporation of radiolabelled precursors, ultrastructural alterations and changes in the pattern of metabolite excretion were examined. Different degrees of toxicity were found for each complex: the platinun compound showed an inhibition effect on nucleic acid synthesis, provoking alterations on the levels of mitochondria, nucleus and glycosomes. These results, together with others reported previously in our laboratory about the activity of pyrimidine derivatives, reflect the potential of these compounds as agents in the treatment of Phytomonas sp.

A protozoan flagelate has recently been isolated from Amaranthus retroflexus. This plant grows near economically important crops in southeastern Spain, which are known to be parasitized by Phytomonas spp. The present study focuses on the characterization of the energy metabolism of this new isolate. These flagellates utilize glucose efficiently as their primary energy source, although they are unable to completely degrade it. They excrete ethanol, acetate, glycine, and succinate in lower amount, as well as ammonium. The presence of glycosomes was indicated by the early enzymes of the glycolytic pathway, one enzyme of the glycerol pathway (glycerol kinase), and malate dehydrogenase. No evidence of a fully functional citric-acid cycle was found. In the absence of catalase activity, these flagellates showed significant superoxide dismutase activity located in the glycosomal and cytosolic fractions. These trypanosomes, despite being morphologically and metabolically similar to other Phytomonas isolated from the same area, showed significant differences, suggesting that they are phylogenetically different species.

Objectives. To evaluate the in vitro leishmanicidal activity of nine flavonoid derivatives from Delphinium staphisagria against L. infantum and L. braziliensis. Design and Methods. The in vitro activity of compounds 1–9 was assayed on extracellular promastigote and axenic amastigote forms and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were carried on J774.2 macrophage cells using Glucantime as the reference drug. The mechanisms of action were analysed performing metabolite excretion and transmission electronic microscope ultrastructural alteration studies. Results. Nine flavonoids showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania infantum and L. braziliensis. These compounds were nontoxic to mammalian cells and were effective at similar concentrations up to or lower than that of the reference drug (Glucantime). The results showed that 2″-acetylpetiolaroside (compound 8) was clearly the most active. Conclusion. This study has demonstrated that flavonoid derivatives are active against L. infantum and L. braziliensis.

Objectives. To evaluate the potential of iron-oxide dismutase excreted (SODeCRU) by T. cruzi as the antigen fraction in the serodiagnosis of Chagas disease and compile new epidemiological data on the seroprevalence of this disease in the suburban population of the city of Santiago de Querétaro (Mexico). Design and Methods. 258 human sera were analyzed by the techniques of ELISA and Western blot and using the homogenate and the SODeCRU. Results. A total of 31 sera were positive against ELISA/SODeCRU (12.4%), while 30 sera proved positive by WB/SODeCRU (11.6%). The comparison between the technique of ELISA and WB showed a sensitivity of 93%, and a specificity of 99%. The positive predictive value was 93% and the negative predictive value was 99%, with a Kappa (κ) value of 1. Conclusions. These preliminary data reveal the degree of infection of nonrural areas of Mexico and demonstrated that SODeCRU is an antigen useful to diagnose Chagas disease.