Search Results (101)

An automated cartridge-based Vcheck M Canine Vector 8 Panel for qualitative detection of Leishmania spp. DNA in canine lymph node aspirates, an off-label specimen type, using laboratory qPCR as the comparator, was evaluated. Fifty-seven residual lymph node aspirate suspensions from dogs investigated for suspected canine leishmaniosis (CanL) were tested. Reference qPCR detected L. infantum DNA in 29 samples. Vcheck M was positive in 22/29 qPCR-positive samples and negative in 28/28 qPCR-negative samples, corresponding to positive percent agreement/sensitivity of 75.9% (95% CI, 56.5–89.7) and negative percent agreement/specificity of 100.0% (95% CI, 87.7–100.0). Agreement was substantial (Cohen’s kappa, 0.76), and discordance was asymmetric (McNemar p = 0.016). Vcheck-negative/qPCR-positive results were mainly observed at low qPCR parasite loads: 6/7 discordant samples contained ≤30 parasites/mL, whereas all samples with ≥500 parasites/mL were Vcheck positive. Among Vcheck-positive clinical samples, Vcheck Ct correlated inversely with log10 qPCR parasite load (Spearman rho = −0.77; p < 0.001). In a single-run dilution series, the lowest instrument-positive L. infantum standard was 10³ parasites/mL. Purified L. major, L. braziliensis, and L. tropica DNA were also detected. Vcheck M showed high specificity as a rapid rule-in test for Leishmania spp. detection in canine lymph node aspirates. However, negative results should not exclude infection in symptomatic or strongly suspected dogs and should be confirmed by qPCR, particularly when low parasite burden is plausible or when the result is critical for diagnostic or therapeutic decision-making. Full article

Background: Leishmaniasis remains a major public health problem in Peru, and the Apurímac, Ene and Mantaro River Valley (VRAEM) is an endemic area where, despite its epidemiological relevance, information on incidence and circulating Leishmania species remains limited. Methods: We estimated district-level cumulative incidence of leishmaniasis in the 60 districts of the VRAEM during 2015–2024, using national surveillance data and census population figures. In addition, molecular species typing was performed on 112 Giemsa-stained tissue smears obtained in health facilities located in Ayacucho districts belonging to the VRAEM region. Results: A total of 3589 cases were reported, yielding an estimated annual incidence rate of 80.01 cases per 100,000 inhabitants, which is 4.3-fold higher than the national average. Marked spatial heterogeneity was observed, with districts such as Pangoa, Río Tambo, Llochegua, and Pichari reporting estimated annual incidence rates above 130 cases per 100,000 inhabitants. Furthermore, of 55 samples processed by High-Resolution Melting Analysis, Leishmania braziliensis was found in 54.6% of samples, followed by Leishmania guyanensis in 21.8%. Conclusions: The VRAEM constitutes a high-transmission endemic focus of tegumentary leishmaniasis with marked inter-district heterogeneity and circulation of multiple Leishmania species, with Leishmania braziliensis as the predominant species. These findings support the need for targeted surveillance and species-informed clinical management strategies. Full article

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America. Its diagnosis is often delayed due to the limited availability of accessible and rapid molecular tools in endemic settings. In this study, we developed and conducted a preliminary evaluation of a recombinase polymerase amplification assay coupled with a lateral flow readout (RPA-LFA) for genus-level detection of Paracoccidioides spp. using reference strains and DNA extracted from clinical specimens. The assay targets the ITS1–5.8S–ITS2 ribosomal region using dual-labeled primers, which enable visual detection on LFA strips. Analytical specificity, analytical sensitivity, and clinical concordance were evaluated using reference strains, non-target microorganisms, and an exploratory set of clinical samples. No cross-reactivity was observed with Coccidioides posadasii, Emmonsia crescens, Histoplasma capsulatum, or Leishmania braziliensis. The preliminary analytical limit of detection (LOD) was 100 copies/µL. Visual results were obtained within 35 min, including 20 min of amplification at 39 °C and 15 min for LFA readout. In the clinical sample set analyzed, the assay showed complete concordance with direct microscopy. The RPA-LFA approach addresses several operational limitations of conventional molecular methods by combining isothermal amplification, a short turnaround time, visual interpretation, and low equipment requirements. This work provides a proof of concept for a point-of-care-oriented molecular approach for Paracoccidioides detection in clinical specimens. Further validation in larger and more diverse clinical cohorts is required to establish its diagnostic performance and potential implementation in endemic settings. Full article

Background/Objective: Leishmaniasis is a neglected tropical disease caused by species of the genus Leishmania, with a broad clinical spectrum that can overlap with other infectious and non-infectious conditions. Accurate species identification is critical for appropriate treatment and prognosis; however, parasitological methods are limited by suboptimal sensitivity, specificity, and inability to reliably differentiate species. This study aimed to develop and validate a real-time PCR assay based on melting-curve analysis (Leish-qPCR) for the detection of Leishmania spp. and the differentiation of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. Methods and Results: Genus-specific primers were designed based on the kDNA (kinetoplast DNA) minicircle consensus sequences of Leishmania species, while species-specific primers targeted the internal transcribed spacer 2 (ITS2) consensus regions of the ribosomal RNA locus of L. (L.) amazonensis and L. (V.) braziliensis. Analytical performance was evaluated in silico and in vitro using a panel of protozoa, fungi, and bacteria, exhibiting 100% specificity with no cross-amplification. The limit of detection was one copy per reaction for all targets using positive controls. Clinical validation was performed using skin biopsy specimens from patients with granulomatous lesions. The optimized Leish-qPCR assay, performed in separate reaction tubes within the same run, demonstrated reliable analytical specificity and sensitivity, with distinct and reproducible melting temperature (Tm) peaks across plasmid controls, parasite DNA, and clinical samples. Comparative analysis with histopathological examination demonstrated moderate agreement between the methods, supporting the applicability of the assay for sensitive detection and species-level discrimination of Leishmania spp. in clinical samples. Conclusions: The Leish-qPCR assay presented high sensitivity, specificity, and diagnostic accuracy, representing a promising tool for routine diagnosis of leishmaniasis and for the differentiation of L. (L.) amazonensis and L. (V.) braziliensis in clinical samples. Full article

Background/Objectives: Many parasitic diseases remain without an effective treatment and cause many deaths worldwide. Leishmaniasis is a complex disease that belongs to the category of Neglected Tropical Diseases, as its treatment relies on outdated drugs that also lead to resistance and negative side-effects. To address this problem, two new chemical families have been tested in vitro against three of the most common parasites from the genus Leishmania. Methods: One family is formed by the polyamine tris(2-aminoethyl)amine functionalised either in one or its three primary amines with different aryl group, and the other is a group of azamacrocyclic cyclophanes containing either one or two aromatic spacers. Results: From the first family, only one compound showed activity against Leishmania donovani, and from the second family, three compounds were selective, two of them for Leishmania braziliensis and a different one against L. donovani, another parasite of the studied genus. Conclusions: The anti-Leishmania activity seems to be related to the compounds’ ability to inhibit the iron superoxide dismutase activity and to alter the parasite metabolism by inhibiting glucose intake in L. braziliensis or by accelerating it in L. donovani and by attacking the parasite defences against ROS, both effects triggering a mitochondrial membrane depolarization that enhances damage, leading to cell death. Full article

Leishmaniasis remains an important neglected tropical disease, and current treatments are limited by toxicity, resistance, and low bioavailability. In this study, novel guanidine derivatives were evaluated through an integrated approach, combining in silico physicochemical profiling with in vitro biological assays using Leishmania (Viannia) braziliensis, the etiological agent of American Tegumentary Leishmaniasis (ATL). Most compounds exhibited favorable drug-like properties, though variations in lipophilicity and solubility influenced biological performance. Among the tested molecules, FURL-G5 emerged as the most promising candidate, showing potent activity against promastigote forms and low cytotoxicity in murine macrophages, resulting in high selectivity indices (SI > 10), comparable to those of LQOF-G1, a compound with previously established leishmanicidal effects. These compounds were also tested on intracellular amastigotes, drastically reducing the infection rate of macrophages. The integration of an in silico approach and biological validation enabled rational compound prioritization and supports the early-stage development of these scaffolds. Overall, this study reinforces the potential of guanidine-based compounds as leads for innovative ATL drug discovery and demonstrates the value of multidisciplinary strategies for identifying selective and safe therapeutic candidates. Full article

Background/Objective: American tegumentary leishmaniasis is a neglected tropical disease with limited therapeutic options characterized by high toxicity and poor tolerability. Drug repurpose offers a pragmatic strategy to accelerate the development of safer treatments. This study evaluated the antileishmanial activity of three clinically approved acetylcholinesterase (AChE) inhibitors—donepezil hydrochloride (DH), rivastigmine tartrate (RT), and galantamine hydrobromide (GH), tested individually and in combination with amphotericin B (AmpB) against Leishmania species relevant to tegumentary leishmaniasis. Methods: Antileishmanial activity was assessed against Leishmania (Leishmania) amazonensis promastigotes and intracellular amastigotes and Leishmania (Viannia) braziliensis promastigotes and axenic amastigotes. Cytotoxicity was evaluated in mammalian cell lines. The synergy with AmpB was analyzed at different proportions. Mechanistic studies included morphological analysis using light and scanning electron microscopy, flow cytometry, AChE activity assays, choline supplementation experiments, and membrane fluidity measurements. Results: All three AChE inhibitors demonstrated antileishmanial activity with selectivity indices > 1. DH emerged as the most promising candidate (IC₅₀ = 16.82 μM against promastigotes; SI = 10.25), with superior potency compared to other repurposed drugs. Strong synergistic interactions with AmpB were observed for all inhibitors (χΣFIC ≤ 0.17), with DH-AmpB displaying the most robust synergy (χΣFIC = 0.09), reducing the IC ₅₀ of AmpB by nearly 90-fold. DH induced distinct morphological alterations and acted through non-cholinergic mechanisms. The DH-AmpB combination retained maximal efficacy against L. (V.) braziliensis, with enhanced activity against clinically relevant amastigotes. Conclusions: Repurposed AChE inhibitors, particularly donepezil hydrochloride, are highly promising therapeutic candidates for tegumentary leishmaniasis. The robust synergistic effect with amphotericin B, together with their favorable safety profiles and non-antimicrobial mechanisms, positions these drugs as viable partners in dose-sparing combination regimens that could improve treatment adherence and reduce toxicity in endemic areas. Full article

There is increasing evidence on the occurrence of Crithidia spp. in patients presenting either cutaneous or visceral leishmaniasis, solely or associated with Leishmania. We analyzed growth, morphology, and temperature tolerance of two C. fasciculata strains, the reference strain COLPROT048 and patient isolate COLPROT606. We also evaluated their co-cultivation with L. braziliensis, macrophage infectivity, and infections in hamsters, BALB/c mice, and sandflies. In culture, both Crithidia strains survived at 32 °C for 96 h, showing major morphological alterations and decreased mitochondrial membrane potential, with ΔΨm reducing to 52% in COLPROT606. At 34 °C, the patient isolate showed an 80% reduction in cell number. Mixed cultivation of Crithidia-Leishmania led to recovery of only Crithidia. In macrophages, C. fasciculata alone was virtually eliminated, and in co-infection only Leishmania was detected. No Crithidia lesion or RNA were found in infected mice or hamsters, while L. braziliensis reached 1145–1625 parasites/mg of tissue. In sandflies, C. fasciculata successfully established infection for up to 7 days, both alone and in coinfections. Amphotericin B IC₅₀ values at 72 h were 4- to 5-fold higher in C. fasciculata strains compared to L. braziliensis. Our results indicate that both C. fasciculata strains are unable to reproduce the pathogenic effect in vitro and in vivo models. Full article

Leishmaniasis is difficult to control due to clinical and vector diversity associated with the complex life cycle of Leishmania parasites, which are transmitted by sandflies. This study investigated the presence of Leishmania DNA in sandfly vectors, their blood meal sources, and their distribution in relation to environmental and climatic variables in four municipalities in Piauí state, Brazil. Between 2020 and 2022, sandflies were collected, morphologically identified, and analyzed for the presence of parasite DNA and blood meal sources (PCR, sequencing). Climate data were correlated with the density of collected insects. Among the 10,245 specimens collected, Lutzomyia longipalpis (54.87%) and Nyssomyia whitmani (30.41%) were the most abundant in the collection areas. Leishmania braziliensis DNA was detected in Lu. longipalpis, while L. braziliensis and Leishmania infantum DNAs were recovered from Ny. whitmani. Homo sapiens was the main blood meal source (~73%). Vector density was associated with humidity, temperature, and precipitation in Teresina and Pedro II, with significant results for Ny. whitmani. In conclusion, Lu. longipalpis, widely adapted to anthropized environments, can act as a potential vector of the etiological agent of cutaneous leishmaniasis in Teresina and Oeiras. In Pedro II, the detection of L. infantum DNA in Ny. whitmani suggests a possible role of this species in the transmission cycle of visceral leishmaniasis, reinforcing the complex ecoepidemiology of Leishmania spp. in Piauí. Full article

Cutaneous leishmaniasis (CL) is a vector-borne zoonotic disease affecting the skin and mucous membranes of animals and humans. While CL is commonly diagnosed and studied in humans in Panama, limited information exists on its occurrence in domestic animals and their potential role as reservoirs. In this study, samples from twelve domestic animals (ten dogs and two horses) with suspected CL lesions were collected between 2021 and 2025 in endemic regions of Panama and evaluated using multiple diagnostic methods. Leishmania infection was confirmed in six of them (50%): five dogs and one horse. Three dogs were infected with Leishmania (Viannia) braziliensis, representing the first molecularly confirmed cases of this species in dogs from Panama and Central America. Two dogs tested positive for Leishmania (Leishmania) infantum, though epidemiological evidence suggests these were imported cases. Notably, Leishmania (Mundinia) martiniquensis was identified in a horse, marking the first report of this species in equines in Central America. These findings indicate a broader diversity of Leishmania species circulating in domestic animals than previously recognized and highlight their potential role in sustaining transmission cycles. The study underscores the need for enhanced surveillance of animal reservoirs to better understand the epidemiology and public health risks of CL in Panama. Full article

Human cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis is a complex parasitic disease marked by dynamic host–parasite interactions and immunomodulation. Extracellular vesicles (EV) derived from immune cells have emerged as key mediators of intercellular communication and potential biomarkers in infectious diseases. In this study, we combined a modified lymphocyte proliferation assay with nano-flow cytometry to quantify and phenotype EV released by CD4⁺, CD8⁺, and CD14⁺ cells in PBMC cultures from CL patients at different clinical stages: before treatment (PBT), during treatment (PDT), and post-treatment (PET) with antimonial. Healthy individuals (HI) were included as physiological controls. Upon stimulation with L. (V.) braziliensis antigens, we observed a distinct modulation of EV subsets. In the PBT group, CD4⁺ and CD14⁺ EV were significantly reduced, while CD8⁺ EV remained elevated. During PDT and PET, EV concentrations were restored across all subsets. These findings suggest that L. (V.) braziliensis selectively modulates the release of immune cell–derived EV, possibly as an immune evasion mechanism. The restoration of EV release following antimonial therapy highlights their potential as sensitive biomarkers for disease activity and treatment monitoring. This study offers novel insights into the immunoregulatory roles of EV in CL and underscores their relevance in host–parasite interactions. Full article

Background/Objectives: Leishmaniasis remains a neglected tropical disease, with nearly one million new cases annually and limited investment in research. Current treatments, primarily based on pentavalent antimonials, are associated with severe side effects and increasing resistance. This study aims to develop a novel therapeutic strategy using a nanomaterial functionalized with sialic acid (SA) and colchicine (COL) to selectively target Leishmania braziliensis parasites. Methods: A nanostructured system was engineered by functionalizing its surface with SA and COL. SA was chosen to mimic host cell surfaces, enhancing parasite attraction, while COL was selected for its known leishmanicidal properties. The nanomaterial was designed to concentrate extracellular parasites on its surface via SA-mediated interactions, thereby increasing local COL efficacy. Results: The functionalized nanomaterial demonstrated a dual mechanism: SA facilitated the selective accumulation of Leishmania braziliensis parasites on the nanostructure surface, while COL exerted a cytotoxic effect. This synergistic interaction resulted in enhanced parasite mortality in vitro, suggesting improved selectivity and potency compared to conventional treatments. Conclusions: The proposed nanomaterial offers a promising alternative for leishmaniasis treatment by combining targeted parasite attraction with localized drug delivery. This strategy may reduce systemic toxicity and improve therapeutic outcomes. Full article

Zoonotic diseases present a growing public health challenge, particularly in sub-Saharan Africa (SSA) due to close interactions between humans and animals and poor diagnostic capacity. This pilot study investigated human exposure to zoonotic pathogens in Zambia among 47 suspected COVID-19 patients from whom nasopharyngeal samples were collected between November 2020 and February 2021 at two major COVID-19 referral centers in Lusaka. Using metagenomic next-generation sequencing (mNGS), the study identified a diverse range of pathogens, including bacterial, fungal, viral, and parasitic species. The prevalence of zoonotic pathogens was 57.4%. Noteworthy zoonoses included Bacillus anthracis, Sporothrix schenckii, Listeria monocytogenes, Yersinia pestis, Streptococcus suis, Vibrio parahaemolyticus, Brucella melitensis, Rickettsia prowazekii, Shewanella algae, Rickettsia japonica, Coxiella burnetii, Leptospira borgpetersenii, Erysipelothrix rhusiopathiae, Brucella abortus, Bartonella quintana, Banna virus, Vibrio alginolyticus, Bartonella clarridgeiae, Rickettsia canadensis, Leishmania braziliensis, Trypanosoma brucei, Pasteurella multocida, and Arcobacter butzleri. Despite moderate diversity in the microbial community, no significant demographic or health-related factors, including age, gender, or comorbidities such as HIV, were found to be statistically associated with zoonotic pathogen infection. The findings provide valuable data on the presence of zoonotic pathogens in humans in Zambia and highlight the need for more comprehensive research into zoonotic diseases in both clinical and non-clinical settings. Full article

American cutaneous leishmaniasis (ACL), caused by Leishmania (Leishmania) amazonensis and L. (Viannia) braziliensis, presents a wide spectrum of clinical and immunopathological manifestations, ranging from localized cutaneous leishmaniasis (LCL) to severe forms like anergic diffuse cutaneous (ADCL) and mucocutaneous leishmaniasis (MCL). Despite evidence of the immune response’s complexity, the role of inflammasomes in disease severity and parasite persistence remains unclear. We investigated the transcriptomic and immunopathological profiles of inflammasome components in patient lesions across the clinical spectrum. Genes such as NLRP3, AIM2, NLRP12, NLRC4, CASP1, CASP5, GSDMD, and IL1B and all evaluated proteins, showed higher expression in ACL compared to healthy controls. Distinct inflammasome activation patterns were observed: MCL, the hyperreactive form, showed elevated NLRP3, AIM2, and IL-1β, indicating an intensified inflammatory environment. ADCL, the hyporeactive form, displayed increased NLRP12 and NLRC4 expression with reduced GSDMD. Localized forms showed transitional profiles, highlighting ACL’s multifactorial pathogenesis. These findings advance our understanding of inflammasome mechanisms in ACL, identifying potential therapeutic targets to modulate inflammation and improve management. Full article

Background/Objectives: In Brazil, Leishmania braziliensis is the main etiological agent of cutaneous leishmaniasis and represents an important public health problem. The actual pharmacotherapy of leishmaniasis has several disadvantages, making the development of new therapeutic options essential. The present study aimed to carry out the bioprospecting and selection of products of Amburana cearensis, including extracts and active principles with a leishmanicidal effect and to evaluate its possible mechanism of action. Methods: A dry extract of A. cearensis (DEAC) was characterized by HPLC, with the following active markers: coumarin (CM), amburoside A (AMR), and vanillic acid (VA). The leishmanicidal effect of DEAC was assessed, and the in vitro inhibitory action of the phenolic fraction, including CM, AMR, and VA, on promastigote and amastigote forms were determined. Results: CM showed the best reductions (maximal inhibition: 57%) of the promastigote form of L. braziliensis, followed by the plant extract (40% inhibition) and other test drugs (maximal reduction: 29%). The treatment of macrophages infected by L. brasiliensis with CM (10 μg/mL) reduced the intracellular parasite load (amastigote form, maximal reduction: 50%), increased the production of nitric oxide, TNF-α, IL-12, and IL-10, and decreased the production of IL-4. These effects were not related to cytotoxicity (MTT test). Glucantime (4 mg/mL, standard drug) reduced the amastigote form by 65%. Conclusions: CM showed promising leishmanicidal activity against both forms of L. brasiliensis, and this effect seems to be associated, at least in part, to its immunomodulatory action by tilting the Th1/Th2 imbalance in favor of Th1. Full article