Biology and Life Sciences

Abstract: Cerebral Malaria (CM) is a life-threatening disease caused by Plasmodium falciparum. There are currently no reliable tools to predict the progression of cerebral syndromes in humans. Cerebral malaria results in mortality for approximately 1–2% of affected patients, with diagnosis generally established following the onset of neurological manifestations—such as coma, paralysis, or seizures—which may also be indicative of alternative medical conditions. More research is warranted to detect early biomolecular fingerprints of CM in biofluids. In this study, we have investigated the disease's early molecular markers in mice's urine. Our analysis has identified a significant decrease in methylmalonate in urine for mice with CM, a finding that not only provides a potential early detection tool for CM but also opens new avenues for research and intervention strategies.

Abstract: Clinostomum spp. are digenetic trematodes characterized by high genetic diversity and complex life cycles involving three hosts: snails, fish, and birds. These parasites signifi-cantly impact freshwater ecosystems, particularly when infecting fish as the second in-termediate host. Despite their ecological importance, no studies have examined the morpho-molecular characteristics of Clinostomum spp. from freshwater fish species in Oman, limiting our understanding of their life cycles and potential threats to the local aquatic environment. The present study investigated Clinostomum spp. infecting killifish (Aphaniops sp.) from four locations in the Muscat region of the Sultanate of Oman (Al Amirat, Al Khoud, Darsait, and Al Bahayes) using integrated morphological and mo-lecular approaches. Metacercariae were morphologically characterized through organ measurements and molecularly identified using mitochondrial cytochrome c oxidase subunit I (COI) sequences. Morphometric analysis revealed that Clinostomum parasites from Omani freshwater bodies were consistently smaller than the established species C. complanatum and C. marginatum reported from other regions. DNA sequence analysis indicated that the Omani specimens were closely related to, but distinct from, Turkish C. complanatum, suggesting they may represent a potentially novel species or subspecies.

Abstract: Background: A common zoonotic parasite protozoan with significant effects on veterinary and public health is Toxoplasma gondii. It is believed that one-third of the world's population has come into contact with this parasite. Pregnant women in Sudan have been found to have higher seroprevalence rates, and exposure has been linked to serious gestational problems such miscarriage, preterm delivery, or stunted fetal growth. In Shendi, River Nile State, Sudan, this study sought to ascertain the seroprevalence of anti-T. gondii antibodies and examine epidemiological risk factors among expectant mothers. Methods: A cross-sectional hospital and clinic-based study was carried out on 300 pregnant women in Shendi. Serum samples were collected and tested for T. gondii IgG and IgM antibodies with a commercial quick immunochromatographic test (ICT) cassette. A standardized questionnaire was used to collect sociodemographic, obstetrical, and dietary behavioral information. Results: The overall seroprevalence of T. gondii IgG antibodies in 300 individuals was 46.7% (n=140), indicating previous immunological exposure; no IgM antibodies were found, indicating the lack of active, acute infections during the sample period. T. gondii seropositivity was found to be significantly associated with raw or undercooked meat consumption (P=0.000), first-trimester gestational age (P=0.000), a maternal history of miscarriage (P=0.000), and prior deliveries of children with congenital abnormalities. There was no statistically significant link between age groups (P=0.989) or parity (P=0.636). Conclusion: The findings show a significant seroprevalence of latent maternal toxoplasmosis exposure in the Shendi region. The strong associations between dietary habits and unfavorable obstetric outcomes highlight the importance of early regular antenatal screening, enhanced food safety education, and preventative prenatal efforts to reduce vertical transmission risk.

Abstract: Urinary schistosomiasis, caused by Schistosoma haematobium, is endemic in many parts of sub-Saharan Africa. The standard microscopic diagnosis is time-consuming and requires expertise to differentiate eggs from artifacts. A study evaluated the performance of a novel staining reagent called Parasimax multicolor^® for the rapid detection of Schistosoma haematobium eggs in urine samples from two endemic countries. Samples positive for Schistosoma haematobium were identified by comparing staining techniques, including Parasimax multicolor staining, wet mount preparation and auramide staining. The results showed that Parasimax staining was faster (30 seconds) and more effective for observing Schistosoma haematobium eggs and miracidia, as well as cellular elements such as red blood cells, even when observed with lower magnification lens. These results show that staining with Parasimax reagent is a potential emergency method for detecting eggs and miracidia of Schistosoma haematobium.

Abstract: The anisakid nematode Sulcascaris (S.) sulcata is one of the most frequently reported pathogenic parasite of gastrointestinal tract of sea turtles, with wide diffusion in warm marine ecosystems of Pacific, Atlantic, and Mediterranean basin. Sea turtles, including the Mediterranean loggerhead turtle (Caretta caretta), act as definitive hosts by ingesting infected bivalve and gastropod molluscs with fourth- larval stage. Infected sea turtles typically develop ulcerative gastritis and chronic mucosal inflammation. We molecularly characterized 19 anisakid nematodes collected from the gastric tract of stranded C. caretta (n = 19) along the Adriatic and Ionian coasts and examined at local Zooprophylactic Institutes. Stomach samples from 5 severely infested turtles were also submitted to histological analysis. Species identification was performed through two conventional PCRs targeting MT-CO1 and MT-CO2; sequencing of 35 amplicons confirmed their identity as S. sulcata species revealing high genetic homogeneity. Phylogenetic analyses showed no clustering by geographic origin or host. The pathological examination revealed mucocatarrhal gastritis with ulcerative lesions, while histological analysis showed chronic gastritis characterized by lymphocytic infiltration. These findings refine current knowledge on S. sulcata distribution and host–parasite dynamics, with implications for marine turtle health and intermediatehost ecology.

Abstract: Bumped kinase inhibitors exhibit proven safety and promising efficacy against apicomplexan parasites in both in vitro and in vivo studies. The 5-aminopyrazole-4-carboxamide compound BKI-1708 effectively inhibited the vertical transmission of Toxoplasma gondii and significantly reduced the cerebral parasite loads in experimentally infected pregnant mice. In vitro experiments revealed that exposure of T. gondii tachyzoites to BKI-1708 induces the formation of atypical intracellular multinucleated complexes called “baryzoites.” These forms exhibit increased expression of bradyzoite-stage proteins while still displaying classical tachyzoite markers, and can withstand prolonged drug pressure. Differential affinity chromatography of T. gondii extracts identified numerous BKI-1708-binding proteins involved in invasion/egress, redox homeostasis, and RNA processing. To understand the transcriptional implications of BKI-1708 treatment on T. gondii tachyzoites and human foreskin fibroblast host cells, T. gondii-infected host cells, either treated with BKI-1708 or untreated, were subjected to dual RNA-seq analysis. BKI-1708 induced a significant transcriptional remodeling in the parasite, with an enrichment in pathways related to translation, RNA metabolism, and stress responses. In contrast, only limited transcriptional perturbations were observed in the host cells. These findings suggest that BKI-1708 modulates the transcriptome in a predominantly parasite-specific manner, disrupting essential biological processes in T. gondii while largely preserving host cell function.

Abstract: Trichostrongylid nematodes are globally distributed gastrointestinal parasites of major veterinary and, in some cases, medical importance. Among them, Haemonchus contortus is especially important as a highly pathogenic haematophagous species. Reliable diagnosis of trichostrongylids often requires an integrative approach because specimen damage, cryptic diversity, dimorphic species complexes, and possible hybridization may compromise identification based on morphology alone. In the Philippines, and particularly in Mindanao, previous work has been restricted largely to coproscopy, with no published studies on adult trichostrongylids using DNA-based methods or scanning electron microscopy (SEM). Here, we present an integrative characterization of H. contortus and Trichostrongylus axei recovered from a goat in Mindanao, Philippines, in 2023. Identification was based on light microscopy, SEM, morphometry with descriptive statistics, and sequence data from the CoxI and ITS regions. Phylogenetic analyses and a haplotype network for H. contortus indicated that the Philippine material belongs to a widely distributed lineage complex showing weak geographic structuring. In broadly distributed nematodes of small ruminants, the geographic origin of individual genotypes may therefore be difficult to infer from locality alone, as global host movements can facilitate admixture among parasite lineages. This study provides the first molecular record of trichostrongylid nematodes from the Philippines and the first SEM-based documentation of H. contortus in the country. These findings confirm the presence of economically important gastrointestinal nematodes in Philippine small ruminants and underscore the value of integrative diagnosis for accurate species delimitation. Broader sampling is needed to clarify parasite diversity, distribution, and genetic variation across the country.

Abstract: Lipid-rich and vesicle-like structures are recurrent features of Leishmania spp., yet their morphological diversity and stage-specific organization remain incompletely defined. Here, we provide a descriptive ultrastructural and fluorescence-based characterization of lipid-associated compartments in Leishmania (L.) amazonensis promastigotes and intracellular amastigotes using transmission electron microscopy, scanning electron microscopy, BODIPY® staining, and Nanoparticle Tracking Analysis (NTA). Promastigotes displayed electron-dense lipid bodies adjacent to the Golgi complex and abundant vesicle-like profiles within the flagellar pocket, whereas intracellular amastigotes exhibited rounded lipid-rich inclusions within parasitophorous vacuoles. NTA revealed stage-dependent differences in particle abundance and size distribution, interpreted strictly at the descriptive level in accordance with MISEV2023 guidelines. These findings establish a morphological baseline for lipid-associated structures across parasite stages. When contextualized with previously reported low-level genetic polymorphisms in Amazonian Leishmania populations, the observed structural heterogeneity suggests that microdiversity may contribute to variation in vacuolar lipid organization. This framework provides a foundation for future studies integrating ultrastructure, lipidomics, and parasite genomics.

Abstract: Sand fly midgut microbiota plays a critical role in shaping Leishmania development and vector competence, yet functional evidence from natural vector populations remains limited. In this study, sand flies were collected between 2020 and 2022 in Cukurova region, Türkiye to characterize the gut bacterial composition of Phlebotomus tobbi and evaluate the anti-leishmanial potential of cultivable isolates. A total of 1,739 sand flies were captured (878 females, 861 males), of which Ph. tobbi was the predominant species (n = 1,312). 16S rRNA amplicon sequencing (V4–V6) showed that the gut microbiota was dominated by Proteobacteria, with Erwinia aphidicola/persicina representing the most abundant species across all analyzed groups. Fourteen cultivable bacterial species were identified by MALDI-TOF MS, including Serratia liquefaciens, Pantoea agglomerans, and Micrococcus luteus. Functional XTT assays against Leishmania infantum promastigotes demonstrated variable inhibitory activity among isolates. The strongest leishmanicidal effects were observed with S. liquefaciens (32.3%) and M. luteus (28.8%). Morphological examination confirmed promastigote rounding and cell death in isolates showing >25% activity. These findings define the gut bacterial landscape of Ph. tobbi in an endemic region and identify bacterial taxa with in vitro anti-leishmanial activity, highlighting their potential for future microbiota-based or paratransgenic control strategies.

Abstract: Chagas disease, caused by Trypanosoma cruzi, remains a major public health problem in Latin America and an emerging global health concern due to population mobility. Alt-hough benznidazole and nifurtimox remain the only approved antiparasitic drugs, their limited efficacy in chronic infection, prolonged treatment regimens, frequent adverse ef-fects, and variable activity across parasite strains highlight the need for new therapeutic strategies. In addition, the pathogenesis of chronic Chagas disease is driven not only by parasite persistence but also by immune-mediated tissue damage, particularly in chronic Chagas cardiomyopathy. In this review, we examine emerging therapeutic approaches that extend beyond conventional trypanocidal chemotherapy, with emphasis on plant-derived extracts, essential oils, antimicrobial peptides, and cell-based immuno-modulatory strategies. Plant compounds and essential oils have shown antiparasitic ac-tivity through mechanisms including oxidative stress induction, membrane disruption, interference with sterol biosynthesis, and mitochondrial dysfunction, while some extracts also modulate host immune responses. Antimicrobial peptides display dual potential by directly damaging parasite membranes and organelles or by reshaping infec-tion-associated inflammatory responses. In parallel, cell-based therapies such as mesen-chymal stromal cells, tolerogenic dendritic cells, and bone marrow-derived cells have demonstrated promising cardioprotective and immunoregulatory effects in experimental chronic Chagas disease. Collectively, these approaches support a multitarget therapeutic framework in which parasite-directed and host-directed interventions may complement each other. Further mechanistic studies, standardization, and translational validation will be essential to advance these candidates toward clinically useful therapies for Chagas disease.

Abstract: Toxoplasmosis is a prevalent zoonotic disease worldwide, affecting approximately one-third of the global human population. Primary infection with Toxoplasma gondii during pregnancy can induce miscarriage or congenital infection, leading to irreversible damage to the foetus. Moreover, reactivation of T. gondii infection in immunosuppressed individuals can result in fatal outcomes. No vaccine exists to prevent human disease caused by this parasite. Thus, a vaccine that could induce complete and lasting protection against human toxoplasmosis is an unmet need. In this work, a subunit vaccine, consisting of T. gondii membrane proteins (TGMP) from the T. gondii Me49 strain plus CpG-oligodeoxynucleotide adjuvant (CpG), was tested using BALB/cByJ mice. Intranasal immunisation with TGMP plus CpG (TGMP+CpG) raised TGMP-specific serum IgG and intestinal IgA antibody levels, and parasite-specific IFN-γ-producing CD4+ and CD8+ memory T cells. Dense granule proteins (GRA) 2 and 7, surface antigen (SAG)-related sequences 25, 29B, and 34A, microneme protein (MIC) 10, toxofilin, nascent polypeptide-associated complex (NAC) domain-containing protein and NAC subunit beta were identified as immunogenic proteins in the TGMP. Mice immunised with TGMP+CpG were challenged with T. gondii tachyzoites and showed a significant reduction in the parasitic burden in the peritoneal exudate, spleen, and lungs, compared to mice sham-immunised with CpG alone. Altogether, these results indicate that mucosal immunisation with TGMP plus CpG adjuvant is worth exploring as a vaccination approach to prevent toxoplasmosis.

Abstract: The gastrointestinal and pulmonary systems are severely affected in both homozygous and heterozygous cystic fibrosis (CF) patients. Cystic fibrosis (CF) is a genetic disorder affecting epithelial ion transport and multiple organ systems. While bacterial and fungal comorbidities are well studied, parasitic infections remain underexplored. This narrative review addresses an unexplored area, inspired by the common gastrointestinal symptoms observed in both CF and certain intestinal parasites. It aims to explore potential links between CF and intestinal parasitic diseases, based on clinical case reports, literature reviews, and research articles. In this study, we used various search engines, including PubMed, ScienceDirect, Elsevier, Wiley, ResearchGate, and Google Scholar. This was constructed by employing keywords such as Cystic fibrosis, CFTR, cystic fibrosis associated with pathogens, intestinal inflammation, bowel disease, intestinal protozoa, and helminthic infections. Once similar symptoms and molecular/immune mechanisms were detected, our keywords extended to include specific parasites associated with CF and/or CFTR, as well as chronic lung inflammation, pancreatitis, or diabetes. This review also examines immunomodulation in CF, with or without parasitic infection, and the influence of CF on predisposition to parasitic infection, diagnosis, or treatment. In a nutshell, we found only a few case reports on CF-protozoan infection comorbidities in non-Middle Eastern countries, and even those are dated two decades ago. This indicates the underestimation of the possibility of CF-comorbidity, which might be life-threatening. Hence, CF diagnosis in patients with intestinal parasitic infections, family pedigree reviews before treatment, and in vivo/ex vivo research studies are recommended.

Abstract: Background/Objectives: Protozoan parasites of the genus Leishmania are causative agents of a group of devastating human diseases, known as leishmaniasis. These microorganisms possess very unusual mechanisms of gene expression that are poorly understood. This study was aimed at analyzing the tRNA repertoire encoded in the Leishmania infantum genome, a species responsible for the most severe form of disease, visceral leishmaniasis. tRNAs are adaptor molecules aimed at decoding mRNAs into proteins. Results: A total of 92 tRNA genes, dispersed on 38 loci were identified; often located in regions where unidirectional gene arrays converge. Putative intronic sequences were inferred for three tRNA genes, and, remarkably, 9 tRNAs were identified within the protein-coding sequences of annotated genes. According to structural predictions, the L. infantum tRNA repertoire covers 49 of the 61 possible anticodons, but because of the well-documented wobble phenomenon, these are sufficient to decode all codons in the 8532 protein-coding genes currently annotated in its genome. As illustrated in this study, codon usage is a well-conserved trait among different Leishmania species but differs substantially regarding the codon usage of its human host. Finally, we analyzed tRNA adaptation index (tAI) parameters, codon usage metrics, and relative protein expression levels. Conclusions: Apart from providing the tRNA gene repertoire and its genome distribution, we have shown the existence of a statistically significant, positive correlation between the tAI scores and protein expression levels in L. infantum promastigotes.

Abstract:

Bumped kinase inhibitor 1708 (BKI-1708), previously demonstrated to target apicomplexan kinases and CDPK1 and MAPKL1, exhibits remarkable activity against Toxoplasma gondii infection both in vitro and in vivo. Notably, BKI-1708 does not affect the viability of mammalian cells. Upon exposure to BKI-1708 exposure, T. gondii tachyzoites form large multinucleated complexes named baryzoites and remain trapped in within host cells. In this study, additional potential molecular targets for BKI-1708 were identified in soluble extracts of T. gondii ME49 tachyzoites and human foreskin fibroblasts (HFF) using differential affinity chromatography coupled to mass spectrometry (DAC-MS). Beyond kinases, secondary interactions in T. gondii involved the binding of proteins associated with cell division, cytoskeleton, vesicular trafficking, secretory organelles, and transcriptional and translational regulators. In non-infected HFFs, BKI-1708 interactors included cytoskeletal regulators along with multiple RNA/DNA-binding proteins. Upon infection, this profile shifted, with cytoskeletal components no longer detected, while nucleic acid–binding proteins remained present, consistent with infection-induced chromatin and transcriptional remodeling. This multi-target interference could contribute to the impaired cytokinesis and the formation of multinucleated baryzoites, aligning with the concept that antiprotozoal drugs exert efficacy through coordinated perturbation of multiple cellular processes rather than a single dominant target.

Abstract:

Toxoplasma gondii, the causative agent of toxoplamosis, a disease widely distributed, is an intracellular parasite that invades host cells of different tissues using specialized endocytic activity. Recent studies suggest that tunneling nanotubes (TNTs), thin cell surface projections, may participate in the parasite-host cell interaction process. We report results on the involvement of host cells TNTs in the adhesion and internalization of T. gondii tachyzoites to epithelial LLC-MK² cells. Microscopy analysis showed that incubating cells in 0.45 M sucrose induces reversible assembly of TNTs without affecting cell viability. The presence of extended TNTs correlated with increase on parasite adhesion and reduction of parasite entry, suggesting a structural or signaling role in mediating adhesion. TNTs assembled following sucrose incubation contain both actin and tubulin components. These results highlight the functional relevance of TNTs in T. gondii host cell interaction, especially in parasite adhesion, opening new perspectives for understanding T. gondii-host cell interaction.

Abstract: A wide range of Leishmania species, transmitted by phlebotomine, cause a group of diseases known as leishmaniasis, which present diverse clinical manifestations. Leishmaniasis has a high impact on the most vulnerable communities, primarily affecting people suffering from malnutrition or living in poor housing conditions. Due to leishmaniasis are strongly associated with poverty the access to treatment is often limited. Besides, high drug toxicity and therapeutic failure, related to the emergence of drug resistance, remain major challenges associated with currently available treatments. Therefore, there is a need for developing new therapeutic approaches safer and more effective. Drug combinations and drug repurposing are two strategies currently used in the development of new treatments targeting multiple diseases. The combination of drugs with different mechanisms of action can minimize the emergence of resistance and allow drug dosage reduction, increasing the likelihood of successful drug repurposing. The research evaluated the leishmanicidal effects of combining antitumoral agents (paclitaxel and docetaxel) with standard drugs (miltefosine and paromomycin). Results demonstrated synergistic effects at higher doses. Furthermore, the antitumoral compounds boosted the host immune response by promoting macrophage polarization toward the M1 phenotype, which is essential for parasite control. These findings highlight a promising therapeutic approach that could improve treatment efficacy and reduce resistance.

Abstract: Malaria, caused by Plasmodium parasites, remains a global health crisis, necessitating novel therapeutic strategies targeting host-parasite interactions. During liver stage in-fection, parasites exploit host vesicular trafficking machinery, particularly SNARE pro-teins that mediate membrane fusion. Using a CRISPR/Cas9 knockout system in HeLa cells combined with advanced microscopy of Plasmodium berghei-infected HeLa cells, we identified specific endolysosomal SNAREs VAMP7, VAMP8, Vti1B, and Stx7 to be re-cruited to the parasitophorous vacuole membrane (PVM) with distinct temporal profiles. This demonstrates the parasite’s precise manipulation of host endolysosomal trafficking pathways. VAMP7 and Vti1B localized to the PVM within 30 minutes post-infection suggesting potential roles during invasion, while VAMP8 and Stx7 appeared later toward 24 hpi, coinciding with increased nutrient acquisition. Single gene deletions showed minimal impact, but combinatorial knockouts revealed critical redundancy. VAMP7-VAMP8 as well as VAMP7–Vti1B double KO significantly reduced parasite in-fection and growth, with Vti1B playing a dominant role. Triple KO phenotypes mirrored VAMP7-Vti1B disruption, underscoring Vti1B’s dominant role. SNARE depletion also impaired lysosome-PVM association and LAMP1 positive vesicle recruitment. Our findings indicate Plasmodium hijacks a coordinated host SNARE network to fuse lysosomes with the PVM for nutrient uptake. Targeting Vti1B-containing complexes disrupts this pathway without host cell toxicity, offering a promising host-directed antimalarial ap-proach.

Abstract:

Between 1993 and 2024, 274 hard ticks (Ixodidae) were collected from 138 underground localities in Croatia. This study represents the most extensive survey of hard tick fauna in Croatian underground habitats to date. The collected hard ticks were classified into three genera and seven species. Ixodes was the most abundant genus, with five species, while Haemaphysalis, and Hyalomma were each represented by one species. The highest number of hard tick species was collected in underground habitats in Dalmatia, followed by north-western Croatia and Slavonia. Ixodes vespertilionis Koch, 1844 was the most abundant species in the collected sample, comprising 81.02%, and was recorded in all studied regions. Only I. vespertilionis was recorded throughout the year, while I. hexagonus was found in nine months, I. frontalis in four months, and the other species in shorter periods. The largest number of I. vespertilionis specimens was collected during the spring months (33.17%), while the fewest were collected in winter (16.58%). The discovery of Ixodes lividus Koch, 1844 in north-western Croatia represents a new country record, increasing the number of hard tick species currently known in Croatia to 28.

Abstract: Toxoplasmosis is a cosmopolitan zoonosis that particularly threatens pregnant women and immunocompromised individuals. Among people living with HIV, Toxoplasma gondii may invade the central nervous system, producing neuropathological effects associated with mental and psychiatric disorders. We assessed the seroprevalence of anti–T. gondii IgG in HIV-infected and HIV-uninfected residents of Iquitos, Peru, and evaluated an in-house ELISA based on total lysed antigen (TLA) against a commercial kit. In this observational cross-sectional study, 151 participants were enrolled: 92 HIV-positive and 59 HIV-negative. Overall IgG seroprevalence was 88.08% (133/151), reaching 91.30% (84/92) in the HIV-positive group and 83.05% (49/59) in the HIV-negative group. Seroprevalence was highest among adults aged 36–60 years (89.33%; 67/75) and among males (89.77%; 79/88) versus females (85.71%; 54/63). Mean optical density values were greater in HIV-positive participants (1.37±0.393; 95% CI 1.28–1.45) than in HIV-negative participants (0.565±0.200; 95% CI 0.510–0.620), and were higher in men overall (1.18±0.502; 95% CI 1.07–1.30). These findings indicate very high exposure to T. gondii in Iquitos, with higher IgG prevalence and antibody levels in HIV-positive individuals. The marked OD difference suggests stronger IgG responses among HIV-positive participants, highlighting potential risk for toxoplasmic encephalitis. Serological screening and preventive counseling may be warranted in high-burden Amazonian communities.

Abstract: Lipid-rich structures are frequently observed in Leishmania spp., yet their morphological diversity and stage-specific distribution remain insufficiently described. Here, we performed an ultrastructural and fluorescence-based characterization of Leishmania (L.) amazonensis promastigotes and intracellular amastigotes using transmission electron microscopy (TEM), scanning electron microscopy (SEM), BODIPY® lipid staining, and Nanoparticle Tracking Analysis (NTA). Promastigotes exhibited vesicle-like and membrane-derived compartments concentrated in the flagellar pocket, as well as electron-dense lipid bodies adjacent to the Golgi complex. Intracellular amastigotes displayed rounded morphology within parasitophorous vacuoles and contained distinct lipid-rich inclusions. BODIPY staining confirmed neutral lipid pools in promastigotes. NTA detected submicron particles in culture supernatants, with amastigotes showing higher particle abundance (3.1–5.9 × 10⁸ particles/mL) and smaller mean sizes (132–171 nm) compared with promastigotes. These observations provide a descriptive account of lipid-rich and vesicle-like structures in L. amazonensis and offer morphological context for future investigations into membrane dynamics, lipid metabolism, and parasite–host interactions. No inference of vesicle identity, biogenesis, or function is made, in accordance with MISEV2023 recommendations.