Natural resistance to meglumine antimoniate is associated with treatment failure in cutaneous leishmaniasis caused by Leishmania (Viannia) panamensis.

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 µg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations.

Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.

Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

BACKGROUND: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). METHODOLOGY/PRINCIPAL FINDINGS: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. CONCLUSIONS: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

Piperine, quercetin, and curcumin identified as promising natural products for topical treatment of cutaneous leishmaniasis.

Leishmania braziliensis (L. braziliensis) causes cutaneous leishmaniasis (CL) in the New World. The costs and the side effects of current treatments render imperative the development of new therapies that are affordable and easy to administer. Topical treatment would be the ideal option for the treatment of CL. This underscores the urgent need for affordable and effective treatments, with natural compounds being explored as potential solutions. The alkaloid piperine (PIP), the polyphenol curcumin (CUR), and the flavonoid quercetin (QUE), known for their diverse biological properties, are promising candidates to address these parasitic diseases. Initially, the in vitro cytotoxicity activity of the compounds was evaluated using U-937 cells, followed by the assessment of the leishmanicidal activity of these compounds against amastigotes of L. braziliensis. Subsequently, a golden hamster model with stationary-phase L. braziliensis promastigote infections was employed. Once the ulcer appeared, hamsters were treated with QUE, PIP, or CUR formulations and compared to the control group treated with meglumine antimoniate administered intralesionally. We observed that the three organic compounds showed high in vitro leishmanicidal activity with effective concentrations of less than 50 mM, with PIP having the highest activity at a concentration of 8 mM. None of the compounds showed cytotoxic activity for U937 macrophages with values between 500 and 700 mM. In vivo, topical treatment with QUE daily for 15 days produced cured in 100% of hamsters while the effectiveness of CUR and PIP was 83% and 67%, respectively. No failures were observed with QUE. Collectively, our data suggest that topical formulations mainly for QUE but also for CUR and PIP could be a promising topical treatment for CL. Not only the ease of obtaining or synthesizing the organic compounds evaluated in this work but also their commercial availability eliminates one of the most important barriers or bottlenecks in drug development, thus facilitating the roadmap for the development of a topical drug for the management of CL caused by L. braziliensis.

Development of a topical treatment for tegumentary leishmaniasis using 8-hydroxyquinoline.

In the context of neglected diseases, tegumentary leishmaniasis (TL) presents an emerging and re-emerging character in the national territory and in the world. The treatment of TL has limitations, such as intravenous administration route, high toxicity, and high treatment costs. Thus, several researchers work on new therapeutic strategies to improve the effectiveness of the treatment of leishmaniasis. In this light, the present study used a topical formulation, containing 8-hydroquinoline (8-HQN), for the treatment of Balb/c mice infected with L. amazonensis. After the treatment, the mean diameter of the lesion was measured, as well as the parasite load in organs and immunological parameters associated with the treatment. The results showed that the animals treated with 8-HQN 5%, when compared to controls, showed a reduction in the mean diameter of the lesion and in the parasite load. The animals treated with the ointment showed a type 1 cellular immune response profile associated with the production of cytokines such as INF-γ and TNF-α. In addition, the treatment did not demonstrate toxicity to mice. Therefore, the topical formulation containing 8-HQN 5% is a promising candidate in the topical treatment and could be considered, in the future, as an alternative for the treatment of TL.

FVB/NJ strain as a mouse model for cutaneous leishmaniasis by Leishmania (L.) amazonensis.

BACKGROUND: Leishmaniases encompass a spectrum of neglected diseases caused by parasites of the genus Leishmania, grouped in two forms: tegumentary and visceral leishmaniasis. OBJECTIVES: In this study, we propose Friend Virus B NIH Jackson (FVB/NJ) mouse strain as a new experimental model of infection with Leishmania (Leishmania) amazonensis, the second most prevalent agent of tegumentary leishmaniasis in Brazil. METHODS AND FINDINGS: We performed in vitro infections of FVB/NJ macrophages and compared them with BALB/c macrophages, showing that BALB/c cells have higher infection percentages and a higher number of amastigotes/cell. Phagocytosis assays indicated that BALB/c and FVB/NJ macrophages have similar capacity to uptake parasites after 5 min incubations. We also investigated promastigotes' resistance to sera from FVB/NJ and BALB/c and observed no difference between the two sera, even though FVB/NJ has a deficiency in complement components. Finally, we subcutaneously infected FVB/NJ and BALB/c mice with 2 × 106 parasites expressing luciferase. Analysis of lesion development for 12 weeks showed that FVB/NJ and BALB/c mice have similar lesion profiles and parasite burdens. MAIN CONCLUSIONS: This work characterises for the first time the FVB/NJ mouse as a new model for tegumentary leishmaniasis caused by Leishmania (L.) amazonensis.

Monitoring the Long-Term Effectiveness of Miltefosine in Indian Post-Kala-Azar Dermal Leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL), the dermal sequel to visceral leishmaniasis (VL), is characterized by hypopigmented macules (macular) and/or papules and nodules (polymorphic). Post-kala-azar dermal leishmaniasis plays a significant role in disease transmission, emphasizing the need for monitoring chemotherapeutic effectiveness. Accordingly, this study aimed to quantify the parasite burden in PKDL patients after treatment with miltefosine by a quantitative polymerase chain reaction (qPCR). A Leishmania kinetoplastid gene-targeted qPCR was undertaken using DNA from skin biopsy specimens of patients with PKDL at three time points, i.e., at disease presentation (week 0, n = 157, group 1), upon completion of treatment (week 12, n = 39, group 2), and at any time point 6 months after completion of treatment (week ≥36, n = 54, group 3). A cycle threshold (Ct) <30 was considered the cutoff for positivity, and load was quantified as the number of parasites/µg genomic DNA (gDNA); cure was considered when samples had a Ct >30. The parasite load at disease presentation (group 1) was 10,769 (1,339-80,441)/µg gDNA (median [interquartile range]). In groups 2 and 3, qPCR results were negative in 35/39 cases (89.7%) and 48/54 cases (88.8%), respectively. In the 10/93 (10.8%) qPCR-positive cases, the parasite burdens in groups 2 and 3 were 2,420 (1,205-5,661)/µg gDNA and 22,195 (5,524-100,106)/µg gDNA, respectively. Serial monitoring was undertaken in 45 randomly selected cases that had completed treatment; all cases in groups 2 or 3 had a Ct >30, indicating cure. Overall, qPCR confirmed an 89.2% cure (as 83/93 cases showed parasite clearance), and the persistent qPCR positivity was attributed to nonadherence to treatment or unresponsiveness to miltefosine and remains to be investigated.

Leishmania braziliensis enhances monocyte responses to promote anti-tumor activity.

Innate immune cells can undergo long-term functional reprogramming after certain infections, a process called trained immunity (TI). Here, we focus on antigens of Leishmania braziliensis, which induced anti-tumor effects via trained immunity in human monocytes. We reveal that monocytes exposed to promastigote antigens of L. braziliensis develop an enhanced response to subsequent exposure to Toll-like receptor (TLR)2 or TLR4 ligands. Mechanistically, the induction of TI in monocytes by L. braziliensis is mediated by multiple pattern recognition receptors, changes in metabolism, and increased deposition of H3K4me3 at the promoter regions of immune genes. The administration of L. braziliensis exerts potent anti-tumor capabilities by delaying tumor growth and prolonging survival of mice with non-Hodgkin lymphoma. Our work reveals mechanisms of TI induced by L. braziliensis in vitro and identifies its potential for cancer immunotherapy.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Nanoemulsions containing amphotericin b and paromomycin for the treatment of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a vector-borne disease characterized by skin lesions that can evolve into high-magnitude ulcerated lesions. Thus, this study aimed to develop an innovative nanoemulsion (NE) with clove oil, Poloxamer® 407, and multiple drugs, such as amphotericin B (AmB) and paromomycin (PM), for use in the topical treatment of CL. METHODS: Droplet size, morphology, drug content, stability, in vitro release profile, in vitro cytotoxicity on RAW 264.7 macrophages, and antileishmanial activity using axenic amastigotes of Leishmania amazonensis were assessed for NEs. RESULTS: After optimizing the formulation parameters, such as the concentration of clove oil and drugs, using an experimental design, it was possible to obtain a NE with an average droplet size of 40 nm and a polydispersion index of 0.3, and these parameters were maintained throughout the 365 days. Furthermore, the NE showed stability of AmB and PM content for 180 days under refrigeration (4 °C), presented a pH compatible with the skin, and released modified AmB and PM. NE showed the same toxicity as free AmB and higher toxicity than free PM against RAW 264.7 macrophages. The same activity as free AmB, and higher activity than free PM against amastigotes L. amazonensis. CONCLUSION: It is possible to develop a NE for the treatment of CL; however, complementary studies regarding the antileishmanial activity of NE should be carried out.

Status of B-Lymphocyte Subsets and Their Homing Markers in Patients With Post-Kala-Azar Dermal Leishmaniasis.

In visceral leishmaniasis, the Type II helper T cell predominance results in B cell modulation and enhancement of anti-leishmanial IgG. However, information regarding its dermal sequel, post-kala-azar dermal leishmaniasis (PKDL), remains limited. Accordingly, this study aimed to elucidate the B cell-mediated antibody-dependent/independent immune profiles of PKDL patients. In the peripheral blood of PKDL patients, immunophenotyping of B cell subsets was performed by flow cytometry and by immunohistochemistry at lesional sites. The functionality of B cells was assessed in terms of skin IgG by immunofluorescence, while the circulating levels of B cell chemoattractants (CCL20, CXCL13, CCL17, CCL22, CCL19, CCL27, CXCL9, CXCL10 and CXCL11) were evaluated by a multiplex assay. In patients with PKDL as compared with healthy controls, there was a significant decrease in pan CD19+ B cells. However, within the CD19+ B cell population, there was a significantly raised proportion of switched memory B cells (CD19+IgD-CD27+) and plasma cells (CD19+IgD-CD38+CD27+). This was corroborated at lesional sites where a higher expression of CD20+ B cells and CD138+ plasma cells was evident; they were Ki67 negative and demonstrated a raised IgG. The circulating levels of B cell chemoattractants were raised and correlated positively with lesional CD20+ B cells. The increased levels of B cell homing markers possibly accounted for their enhanced presence at the lesional sites. There was a high proportion of plasma cells, which accounted for the increased presence of IgG that possibly facilitated parasite persistence and disease progression.

Topical application of ozonated sunflower oil accelerates the healing of lesions of cutaneous leishmaniasis in mice under meglumine antimoniate treatment.

Besides being scarce, the drugs available for treating cutaneous leishmaniasis have many adverse effects. Ozone is an option to enhance the standard treatment due to the wound-healing activity reported in the literature. In this study, we evaluated the efficiency of ozonated sunflower oil as an adjuvant in treating cutaneous lesions caused by Leishmania amazonensis. BALB/c mice were infected with L. amazonensis, and after the lesions appeared, they were treated in four different schedules using the drug treatment with meglumine antimoniate (Glucantime®), with or without ozonated oil. After thirty days of treatment, the lesions' thickness and their parasitic burden, blood leukocytes, production of NO and cytokines from peritoneal macrophages and lymph node cells were analyzed. The group treated with ozonated oil plus meglumine antimoniate showed the best performance, improving the lesion significantly. The parasitic burden showed that ozonated oil enhanced the leishmanicidal activity of the treatment, eliminating the parasites in the lesion. Besides, a decrease in the TNF levels from peritoneal macrophages and blood leukocytes demonstrated an immunomodulatory action of ozone in the ozonated oil-treated animals compared to the untreated group. Thus, ozonated sunflower oil therapy has been shown as an adjuvant in treating Leishmania lesions since this treatment enhanced the leishmanicidal and wound healing effects of meglumine antimoniate.

N6-methyltubercidin gives sterile cure in a cutaneous Leishmania amazonensis mouse model.

Leishmania is a trypanosomatid parasite that causes skin lesions in its cutaneous form. Current therapies rely on old and expensive drugs, against which the parasites have acquired considerable resistance. Trypanosomatids are unable to synthesize purines relying on salvaging from the host, and nucleoside analogues have emerged as attractive antiparasitic drug candidates. 4-Methyl-7-ß-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (CL5564), an analogue of tubercidin in which the amine has been replaced by a methyl group, demonstrates activity against Trypanosoma cruzi and Leishmania infantum. Herein, we investigated its in vitro and in vivo activity against L. amazonensis. CL5564 was 6.5-fold (P = 0.0002) more potent than milteforan™ (ML) against intracellular forms in peritoneal mouse macrophages, and highly selective, while combination with ML gave an additive effect. These results stimulated us to study the activity of CL5564 in mouse model of cutaneous Leishmania infection. BALB/c female and male mice infected by L. amazonensis treated with CL5564 (10 mg kg−1, intralesional route for five days) presented a >93% reduction of paw lesion size likely ML given orally at 40 mg kg−1, while the combination (10 + 40 mg kg−1 of CL5564 and ML, respectively) caused >96% reduction. The qPCR confirmed the suppression of parasite load, but only the combination approach reached 66% of parasitological cure. These results support additional studies with nucleoside derivatives.

Leishmaniasis in otorhinolaryngology: an emerging disease and its relationship with anti-tumor necrosis factor-alpha drugs.

PURPOSE: To investigate the clinical manifestations, management and outcomes of Leishmania lesions in the ear-nose-throat (ENT) region, and its relationship with tumor necrosis factor (TNF)-α blocking drugs. METHODS: Single-center retrospective observational study. Patients diagnosed with cutaneous and mucosal leishmaniasis in the otorhinolaryngologic area at a tertiary referral center over a period of 8 years. RESULTS: Three cases of Leishmania lesions in the ear and two in the nose were encountered at our institution. All patients were under treatment with TNF-α blocking drugs. Diagnosis was challenging, and it was important to have a clinical suspicion in order to use accurate detection techniques. All patients received systemic treatment and achieved a complete resolution of the lesions. CONCLUSIONS: With the increasing use of biologic treatments like TNF-α blockers, this type of infection will be increasingly frequent in endemic areas and also worldwide. It is important to include leishmaniasis in the differential diagnosis of inflammatory/infectious lesions in the ENT region.

Single-cell ATAC sequencing identifies sleepy macrophages during reciprocity of cytokines in L. major infection.

The hallmark characteristic of macrophages lies in their inherent plasticity, allowing them to adapt to dynamic microenvironments. Leishmania strategically modulates the phenotypic plasticity of macrophages, creating a favorable environment for intracellular survival and persistent infection through regulatory cytokine such as interleukin (IL)-10. Nevertheless, these effector cells can counteract infection by modulating crucial cytokines like IL-12 and key components involved in its production. Using sophisticated tool of single-cell assay for transposase accessible chromatin (ATAC) sequencing, we systematically examined the regulatory axis of IL-10 and IL-12 in a time-dependent manner during Leishmania major infection in macrophages Our analysis revealed the cellular heterogeneity post-infection with the regulators of IL-10 and IL-12, unveiling a reciprocal relationship between these cytokines. Notably, our significant findings highlighted the presence of sleepy macrophages and their pivotal role in mediating reciprocity between IL-10 and IL-12. To summarize, the roles of cytokine expression, transcription factors, cell cycle, and epigenetics of host cell machinery were vital in identification of sleepy macrophages, which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine expression and recruitment of immune cells.IMPORTANCELeishmaniasis is an endemic affecting 99 countries and territories globally, as outlined in the 2022 World Health Organization report. The disease's severity is compounded by compromised host immune systems, emphasizing the pivotal role of the interplay between parasite and host immune factors in disease regulation. In instances of cutaneous leishmaniasis induced by L. major, macrophages function as sentinel cells. Our findings indicate that the plasticity and phenotype of macrophages can be modulated to express a cytokine profile involving IL-10 and IL-12, mediated by the regulation of transcription factors and their target genes post-L. major infection in macrophages. Employing sophisticated methodologies such as single-cell ATAC sequencing and computational genomics, we have identified a distinctive subset of macrophages termed "sleepy macrophages." These macrophages exhibit downregulated housekeeping genes while expressing a unique set of variable features. This data set constitutes a valuable resource for comprehending the intricate host-parasite interplay during L. major infection.

Self-reporting of hearing loss and tinnitus in the diagnosis of ototoxicity by meglumine antimoniate in patients treated for American tegumentary Leishmaniasis.

INTRODUCTION: American Tegumentary Leishmaniasis (ATL) treatment is based on pentavalent antimonials (Sb5+), but these drugs have been associated to several adverse effects. Hearing loss and tinnitus during treatment with meglumine antimoniate (MA) have already been reported. This study aimed to describe the usefulness of self-reporting of hearing loss and tinnitus in diagnosing MA-induced ototoxicity. METHODS: A prospective longitudinal study was conducted with 102 patients with parasitological diagnosis of ATL, treated with different MA schemes. The presence of clinical auditory toxicity was defined as the emergence or worsening of self-reporting hearing loss and/or tinnitus during monitoring. Measures of sensitivity, specificity, and the positive and negative predictive value of the patient's self-reporting of hearing loss and tinnitus in relation to the result of the audiometric test (considered the gold standard) were calculated. RESULTS: The age of the evaluated patients ranged from 15 to 81 years, with a median of 41 years, and most were male (73.5%). Seventy-five patients (73.5%) had cutaneous leishmaniasis and 27 (26.5%) mucosal leishmaniasis. Eighty-six patients (84.3%) received intramuscular (IM) treatment and 16 (15.7%) were treated with intralesional MA. During treatment, 18 (17,6%) had tinnitus and 7 (6,9%) had complaint of hearing loss. 53 (52%) patients had cochlear toxicity confirmed by tone threshold audiometry and high frequency audiometry, from which 60% received a dose of 20 mg Sb5+/kg/day (p = 0.015) and 96.2% were treated with IM MA (p = 0.001). Tinnitus has greater specificity and positive predictive value than hearing loss, with a low number of false positives, but with a high false negative value. CONCLUSION: Although the large number of false negatives suggests that self-report of hearing loss or tinnitus cannot be considered a good screening test for referring the patient to an audiometry, the low number of false positives suggests the need to value the patient's complaint for referral. Otherwise, this study reinforces the importance of audiological monitoring during treatment with MA, especially in those patients with self-reporting of hearing loss or tinnitus when treated with 20 mg Sb5+/kg/day via IM.

Short communication: The miR-155a-5p is correlated with increased ROS and impaired apoptosis in macrophages infected by Leishmania braziliensis.

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, is a disease characterized by well-limited ulcerated lesions with raised borders in exposed parts of the body. miRNAs are recognized for their role in the complex and plastic interaction between host and pathogens, either as part of the host's strategy to neutralize infection or as a molecular mechanism employed by the pathogen to modulate host inflammatory pathways to remain undetected. The mir155 targets a broad range of inflammatory mediators, following toll-like receptors (TLRs) signaling. In this work, we evaluated the effects of the expression of miR155a-5p in human macrophages infected with L. braziliensis. Our results show that miR155a-5p is inversely correlated with early apoptosis and conversely, seems to influence an increment in the oxidative burst in these cells. Altogether, we spotted a functional role of the miR155a-5p in CL pathogenesis, raising the hypothesis that an increased miR-155 expression by TLR ligands influences cellular mechanisms settled to promote both killing and control of parasite density after infection.

In vitro growth of Leishmania parasites from biopsy samples of suspected cutaneous and visceral leishmaniasis cases in Sri Lanka: An observational study.

Sri Lanka reports a large focus of Leishmania donovani caused cutaneous leishmaniasis (CL). Subsequent emergence of visceral leishmaniasis (VL) was also reported recently. Expansion of the on-going disease outbreak and many complexities indicate urgent need to enhance early case detection methods. In vitro cultivation (IVC) of parasites causing visceral leishmaniasis (VL) is important for disease confirmation and to obtain sufficient quantities of parasites required in many scientific studies. IVC is carried out as a useful second line investigation for direct microscopy negative patients with CL in this setting. Along with the emergence of VL, current study was carried out to evaluate in vitro growth of local VL parasites and to identify their differences associated with in vitro growth characteristics. Routine parasitological diagnostic methods, i.e., light microscopy (LM), polymerase chain reaction (PCR) were used for confirmation of suspected cases. Lesion samples from 125 suspected CL cases and bone marrow or splenic aspirations from 125 suspected VL patients were used to inoculate IVCs. Media M199 (about 70 µl) supplemented with 15-20% of heat inactivated fetal bovine serum was used for initial culturing procedures in capillaries. Capillary cultures were monitored daily. Total of 44 different compositions/conditions were used for evaluating in vitro growth of VL causing parasite. Daily records on parasite counts, morphological appearance (size, shape, and wriggly movements) were maintained. In vitro transformation of Leishmania promastigotes to amastigotes and outcome of the attempts on recovery of live Leishmania from culture stabilates was also compared between CL and VL parasites. Proportion of cultures showing a transformation of promastigotes were 40/45 (88.9%) and 4/10 (40.0%) for CL and VL respectively. In the transformed cultures, parasites showing typical shape, size and movement patterns were less in VL (1/4, 25.0%) compared to CL (28/40, 70.0%). CL cultures showed a growth up to mass culturing level with mean duration of two weeks while it was about five weeks for VL cultures. Proportion of cultures that reached a parasite density of 1 × 106 cells/ml (proceeded to mass cultures) was significantly low in VL (4/10, 40%) as compared to CL (28/40, 70.0%). None of media compositions/conditions were successful for mass culturing of VL parasites while all of them were shown to be useful for growing CL strains. Also in vitro transformation to amastigote form and recovering of culture stabilates were not successful compared to CL. There were clear differences between in vitro growth of Leishmania parasites causing local CL and VL. Further studies are recommended for optimization of in vitro culturing of VL parasite which will be invaluable to enhance case detection in future.