Genetic diversity and epidemiological insights into cutaneous leishmaniasis in Pakistan: a comprehensive study on clinical manifestations and molecular characterization of Leishmania species.

Cutaneous leishmaniasis (CL) stands out as a significant vector-borne endemic in Pakistan. Despite the rising incidence of CL, the genetic diversity of Leishmania species in the country's endemic regions remains insufficiently explored. This study aims to uncover the genetic diversity and molecular characteristics of Leishmania species in CL-endemic areas of Baluchistan, Khyber Pakhtunkhwa (KPK), and Punjab in Pakistan. Clinical samples from 300 CL patients were put to microscopic examination, real-time ITS-1 PCR, and sequencing. Predominantly affecting males between 16 to 30 years of age, with lesions primarily on hands and faces, the majority presented with nodular and plaque types. Microscopic analysis revealed a positivity rate of 67.8%, while real-time PCR identified 60.98% positive cases, mainly L. tropica, followed by L. infantum and L. major. Leishmania major (p = 0.009) showed substantially greater variation in nucleotide sequences than L. tropica (p = 0.07) and L. infantum (p = 0.03). Nucleotide diversity analysis indicated higher diversity in L. major and L. infantum compared to L. tropica. This study enhances our understanding of CL epidemiology in Pakistan, stressing the crucial role of molecular techniques in accurate species identification. The foundational data provided here emphasizes the necessity for future research to investigate deeper into genetic diversity and its implications for CL control at both individual and community levels.

Leishmania spp. genetic factors associated with cutaneous leishmaniasis antimony pentavalent drug resistance: a systematic review.

BACKGROUND: Leishmaniasis is a neglected zoonosis caused by parasites of Leishmania spp. The main drug used to treat cutaneous leishmaniasis (CL) is the antimoniate of meglumine. This drug, which has strong adverse and toxic effects, is usually administered intravenously, further complicating the difficult treatment. Factors such as Leishmania gene expression and genomic mutations appear to play a role in the development of drug resistance. OBJECTIVES: This systematic review summarises the results of the literature evaluating parasite genetic markers possibly associated with resistance to pentavalent antimony in CL. METHODS: This study followed PRISMA guidelines and included articles from PubMed, SciELO, and LILACS databases. Inclusion criteria were studies that (i) investigated mutations in the genome and/or changes in gene expression of Leishmania associated with treatment resistance; (ii) used antimony drugs in the therapy of CL; (iii) used naturally resistant strains isolated from patients. The Joanna Briggs Institute Critical Appraisal Checklist was used to assess article quality and risk of bias. FINDINGS: A total of 23 articles were selected, of which 18 investigated gene expression and nine genomic mutations. Of these 23 articles, four examined gene expression and genomic mutations in the same samples. Regarding gene expression, genes from the ABC transporter protein family, AQP1, MRPA, TDR1 and TRYR were most frequently associated with drug resistance. In one of the articles in which mutations were investigated, a mutation was found in HSP70 (T579A) and in three articles mutations were found in AQP1 (A516C, G562A and G700A). A limitation of this review is that in most of the included studies, parasites were isolated from cultured lesion samples and drug resistance was assessed using in vitro drug susceptibility testing. These approaches may not be ideal for accurate genetic evaluation and detection of treatment failure. MAIN CONCLUSIONS: The development of further studies to evaluate the genetic resistance factors of Leishmania spp. is necessary to elucidate the mechanisms of the parasite and improve patient treatment and infection control.

Molecular Diagnosis of Human Cutaneous Leishmaniasis and Identification of the Causative Leishmania Species in Iran: A Narrative Review

Cutaneous leishmaniasis (CL) is a common form of leishmaniasis in underdeveloped countries. Although CL tends to be self-limiting, it can cause significant scars and may progress to more severe manifestations. Additionally, Leishmania species vary in susceptibility to the available treatments. The selection of treatment and clinical outcome of CL depend on the accurate determination of the Leishmania species. This mini-review aims to provide an overview of the molecular diagnosis techniques such as PCR-based assays, nucleic acid sequence-based amplification, and loop-mediated isothermal amplification utilized in the identification of Leishmania species in Iran.

Diagnostic Accuracy of Polymerase Chain Reaction Against Giemsa Staining on Tissue Biopsy for Cutaneous Leishmaniasis.

OBJECTIVE: To evaluate the diagnostic accuracy of a commercial real-time polymerase chain reaction (PCR) kit targeting 18S rRNA against Giemsa-stained tissue slides in patients clinically suspected of cutaneous leishmaniasis (CL). STUDY DESIGN: Cross-sectional analytical study. Place and Duration of the Study: Department of Microbiology, Armed Forces Institute of Pathology / National University of Medical Sciences, Rawalpindi, Pakistan, from July to December 2022. METHODOLOGY: Samples of skin tissue in 98 patients suspected of CL were evaluated. These samples were subjected to Giemsa-staining for microscopy and real-time PCR. Sensitivity, specificity, and accuracy of the PCR were calculated keeping Giemsa-stained tissue slide microscopy as gold standard. RESULTS: Out of the 98 tissue samples, 37 were found positive for leishmaniasis on PCR while 13 were found Leishmania positive on microscopy of Giemsa-stained slides. The sensitivity, specificity, and accuracy of the PCR for the detection of Leishmania species were 100%, 71.8%, and 91.8%, respectively with 100% negative predictive value. CONCLUSION: This study demonstrates that the commercial PCR is a reliable diagnostic test for the diagnosis of CL. The ease, rapidity, and reliability of the PCR make it a dependable tool in diagnostic repertoire of CL. KEY WORDS: Giemsa stain, Leishmania spp., Polymerase chain reaction, Viasure.

Abundance and Leishmania infection patterns of the sand fly Psathyromyia cratifer in Southern Mexico.

BACKGROUND: Localized cutaneous leishmaniasis (LCL) is a serious public health problem in Southern Mexico. Six species of Phlebotominae (Diptera: Psychodidae) have been found to be infected with Leishmania (Leishmania) mexicana, the causative agent of LCL in the region. However, little is known about the biology and potential participation of Psathyromyia cratifer in the Leishmania transmission cycle in Mexico, and the Americas. The present study provides evidence of temporal infection caused by Leishmania in Psathyromyia cratifer as well as data on its population dynamics in a LCL endemic area during the well-known transmission cycle of Leishmania in Southern Mexico. METHODOLOGY/PRINCIPAL FINDINGS: Individual specimens of Psathyromyia cratifer were collected in four sites over the course of five months (from November 2020 through March 2021) using animal-baited, human-baited, and light traps. The temporal activity pattern (month + hour) of Psathyromyia cratifer was assessed along with its relationship with environmental variables. Moreover, Leishmania DNA and blood meals were analyzed and detected in female sand flies. This evidenced an infection rate ranging from 8% to 83%, and the record of Homo sapiens and Ototylomys phyllotis as blood hosts of this sand fly species. High abundances of these sand flies in human-baited traps were recorded which revealed the marked anthropophilic behavior of Psathyromyia cratifer. As regards the transmission dynamics of the parasite within the region, it was observed that the potential highest epidemiological risk for Leishmania transmission by Psathyromyia cratifer occurred during the months of January and March. CONCLUSION: This is the first contribution ever made to both the population dynamic and the temporal Leishmania prevalence patterns in Psathyromyia cratifer. The resulting findings suggest that this sand fly specimen is the sixth potential vector of L. (L.) mexicana in Southern Mexico. Nonetheless, various biology, behavior, and ecology strands are yet to be addressed. The latter, to determine the role it plays in the transmission dynamics of the parasite within the region, and other areas of the country.

Antigenic reactivity of Leishmania (Viannia) lainsoni axenic amastigote proved to be a suitable alternative for optimizing Montenegro skin test.

BACKGROUND: Laboratory diagnosis of American cutaneous leishmaniasis (ACL) requires a tool amenable to the epidemiological status of ACL in Brazil. Montenegro skin test (MST), an efficient immunological tool used for laboratory diagnosis of ACL, induces delayed-type hypersensitivity (DTH) response to the promastigote antigens of Leishmania; however, human immune responses against infection are modulated by the amastigote of the parasite. Leishmania (V.) lainsoni induces strong cellular immunity in humans; therefore, the antigenic reactivity of its axenic amastigote (AMA antigen) to MST was evaluated for the laboratory diagnosis of ACL. METHODS: Among 70 individuals examined, 60 had a laboratory-confirmed diagnosis of ACL; 53 had localized cutaneous leishmaniasis (LCL), and 7 had mucosal leishmaniasis (ML). Patients were treated at the Evandro Chagas Institute's leishmaniasis clinic, Pará State, Brazil. Ten healthy individuals with no history of ACL (control group) were also examined. Leishmania (V.) braziliensis promastigote antigen (PRO) was used to compare the reactivity with that of AMA antigen. Paired Student's t-test, kappa agreement, and Spearman test were used to evaluate the reactivity of AMA and PRO. RESULTS: The mean reactivity of AMA in ACL patients was 19.4 mm ± 13.3, which was higher (P < 0.001) than that of PRO: 12.1 mm ± 8.1. MST reactivity according to the clinical forms revealed that AMA reactivity in LCL and ML, 18.8 mm ± 13.3 and 24.3 mm ± 13.7, was higher (P < 0.001) than that of PRO, 11.8 mm ± 8.2 and 14.6 mm ± 8.4, respectively. CONCLUSION: AMA reactivity was higher than that of PRO, indicating that AMA is a promising alternative for optimizing MST in the laboratory diagnosis of ACL.

Comparative analysis of the microbiota of sand fly vectors of Leishmania major and L. tropica in a mixed focus of cutaneous leishmaniasis in southeast Tunisia; ecotype shapes the bacterial community structure.

Phlebotomine sand flies are vectors of the protozoan parasite Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the impact of host species and environment on the gut microbiome. To address this issue, a comparative analysis of the microbiota of sand fly vector populations of Leishmania major and L. tropica in a mixed focus of cutaneous leishmaniasis in Tunisia was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize and compare the overall bacterial and fungal composition of field-collected sand flies: Phlebotomus papatasi, Ph. perniciosus, Ph. riouxi, and Ph. sergenti. Thirty-eight bacterial genera belonging to five phyla were identified in 117 female specimens. The similarities and differences between the microbiome data from different samples collected from three collections were determined using principal coordinate analysis (PCoA). Substantial variations in the bacterial composition were found between geographically distinct populations of the same sand fly species, but not between different species at the same location, suggesting that the microbiota content was structured according to environmental factors rather than host species. These findings suggest that host phylogeny may play a minor role in determining the insect gut microbiota, and its potential to affect the transmission of the Leishmania parasite appear to be very low. These results highlight the need for further studies to decode sand fly Leishmania-microbiota interactions, as even the same bacterial species, such as Enterococcus faecalis, can exert completely opposite effects when confronted with different pathogens within various host insects and vice versa.

NLRP1-dependent activation of Gasdermin D in neutrophils controls cutaneous leishmaniasis.

Intracellular pathogens that replicate in host myeloid cells have devised ways to inhibit the cell's killing machinery. Pyroptosis is one of the host strategies used to reduce the pathogen replicating niche and thereby control its expansion. The intracellular Leishmania parasites can survive and use neutrophils as a silent entry niche, favoring subsequent parasite dissemination into the host. Here, we show that Leishmania mexicana induces NLRP1- and caspase-1-dependent Gasdermin D (GSDMD)-mediated pyroptosis in neutrophils, a process critical to control the parasite-induced pathology. In the absence of GSDMD, we observe an increased number of infected dermal neutrophils two days post-infection. Using adoptive neutrophil transfer in neutropenic mice, we show that pyroptosis contributes to the regulation of the neutrophil niche early after infection. The critical role of neutrophil pyroptosis and its positive influence on the regulation of the disease outcome was further demonstrated following infection of mice with neutrophil-specific deletion of GSDMD. Thus, our study establishes neutrophil pyroptosis as a critical regulator of leishmaniasis pathology.

Leishmania major-induced alteration of host cellular and systemic copper homeostasis drives the fate of infection.

Copper plays a key role in host-pathogen interaction. We find that during Leishmania major infection, the parasite-harboring macrophage regulates its copper homeostasis pathway in a way to facilitate copper-mediated neutralization of the pathogen. Copper-ATPase ATP7A transports copper to amastigote-harboring phagolysosomes to induce stress on parasites. Leishmania in order to evade the copper stress, utilizes a variety of manipulative measures to lower the host-induced copper stress. It induces deglycosylation and degradation of host-ATP7A and downregulation of copper importer, CTR1 by cysteine oxidation. Additionally, Leishmania induces CTR1 endocytosis that arrests copper uptake. In mouse model of infection, we report an increase in systemic bioavailable copper in infected animals. Heart acts as the major organ for diverting its copper reserves to systemic circulation to fight-off infection by downregulating its CTR1. Our study explores reciprocal mechanism of manipulation of host copper homeostasis pathway by macrophage and Leishmania to gain respective advantages in host-pathogen interaction.

Autochthonous Leishmaniasis Caused by Leishmania tropica, Identified by Using Whole-Genome Sequencing, Sri Lanka.

Cutaneous leishmaniasis is atypical in Sri Lanka because Leishmania donovani, which typically causes visceral disease, is the causative agent. The origins of recently described hybrids between L. donovani and other Leishmania spp. usually responsible for cutaneous leishmaniasis remain unknown. Other endemic dermotropic Leishmania spp. have not been reported in Sri Lanka. Genome analysis of 27 clinical isolates from Sri Lanka and 32 Old World Leishmania spp. strains found 8 patient isolates clustered with L. tropica and 19 with L. donovani. The L. tropica isolates from Sri Lanka shared markers with strain LtK26 reported decades ago in India, indicating they were not products of recent interspecies hybridization. Because L. tropica was isolated from patients with leishmaniasis in Sri Lanka, our findings indicate L. donovani is not the only cause of cutaneous leishmaniasis in Sri Lanka and potentially explains a haplotype that led to interspecies dermotropic L. donovani hybrids.

Emerging Leishmania donovani Lineages Associated with Cutaneous Leishmaniasis, Himachal Pradesh, India, 2023.

The clinical manifestation of leishmaniasis has historically been determined by the Leishmania species involved. However, recent emergence of novel Leishmania lineages has caused atypical pathologies. We isolated and characterized 2 new Leishmania donovani parasites causing cutaneous leishmaniasis in Himachal Pradesh, India.

Assessment of the antileishmanial activity of diallyl sulfide combined with meglumine antimoniate on Leishmania major: Molecular docking, in vitro, and animal model.

Currently, no safe vaccine against leishmaniasis is available. So far, different control strategies against numerous reservoir hosts and biological vectors have not been environment-friendly and feasible. Hence, employing medicinal components and conventional drugs could be a promising approach to developing novel therapeutic alternatives. This study aimed to explore diallyl sulfide (DAS), a dynamic constituent of garlic, alone and in a mixture with meglumine antimoniate (MAT as standard drug) using in vitro and animal model experiments against Leishmania major stages. The binding affinity of DAS and four major defense elements of the immune system (iNOS, IFN-É£, IL-12, and TNF-α) was used to predict the predominant binding mode for molecular docking configurations. Herein, we conducted a broad range of experiments to monitor and assess DAS and MAT potential treatment outcomes. DAS, combined with MAT, displayed no cytotoxicity and employed a powerful anti-leishmanial activity, notably against the clinical stage. The function mechanism involved immunomodulation through the induction of Th1 cytokine phenotypes, triggering a high apoptotic profile, reactive oxygen species (ROS) production, and antioxidant enzymes. This combination significantly decreased cutaneous lesion diameter and parasite load in BALB/c mice. The histopathological findings performed the infiltration of inflammatory cells associated with T-lymphocytes, particularly CD4+ phenotypes, as determined by biochemical markers in alleviating the amastigote stage and improving the pathological changes in L. major infected BALB/c mice. Therefore, DAS and MAT deserve further advanced therapeutic development and should be considered as possible candidates for treating volunteer cases with cutaneous leishmaniasis in designing an upcoming clinical trial.

The Immune Memory Response of In Vitro-Polarised Th1, Th2, and Th17 Cells in the Face of Ovalbumin-Transgenic Leishmania major in a Mouse Model.

Th1 and Th2 cytokines determine the outcome of Leishmania major infection and immune protection depends mainly on memory T cells induced during vaccination. This largely hinges on the nature and type of memory T cells produced. In this study, transgenic Leishmania major strains expressing membrane-associated ovalbumin (mOVA) and soluble ovalbumin (sOVA) were used as a model to study whether fully differentiated Th1/Th2 and Th17 cells can recall immune memory and tolerate pathogen manipulation. Naïve OT-II T cells were polarised in vitro into Th1/Th2 cells, and these cells were transferred adoptively into recipient mice. Following the transferral of the memory cells, the recipient mice were challenged with OVA transgenic Leishmania major and a wild-type parasite was used a control. The in vitro-polarised T helper cells continued to produce the same cytokine signatures after being challenged by both forms of OVA-expressing Leishmania major parasites in vivo. This suggests that antigen-experienced cells remain the same or unaltered in the face of OVA-transgenic Leishmania major. Such ability of these antigen-experienced cells to remain resilient to manipulation by the parasite signifies that vaccines might be able to produce immune memory responses and defend against parasitic immune manipulation in order to protect the host from infection.

The Role of Melatonin in the Inflammatory Process in Patients with Hyperglycemia and Leishmania Infection.

Type 2 diabetes mellitus is a metabolic disorder that causes chronic high blood sugar levels, and diabetic patients are more susceptible to infections. American cutaneous leishmaniasis is an infectious disease caused by a parasite that affects the skin and mucous membranes, leading to one or multiple ulcerative lesions. Chronic inflammation and functional changes in various organs and systems, including the immune system, are the primary causes of both diseases. Melatonin, an essential immunomodulatory, antioxidant, and neuroprotective agent, can benefit many immunological processes and infectious diseases, including leishmaniasis. Although, limited reports are available on diabetic patients with leishmaniasis. The literature suggests that melatonin may play a promising role in inflammatory disorders. This study was designed to assess melatonin levels and inflammatory mediators in diabetic patients affected by leishmaniasis. Blood samples from 25 individuals were analyzed and divided into four groups: a control group (without any diseases), a Leishmania-positive group, patients with type 2 diabetes mellitus, and patients with a combination of both diseases. This study measured the serum levels of melatonin through ELISA, while IL-4 and TNF-α were measured using flow cytometry, and C-reactive protein was measured through turbidimetry. This study found that patients with leishmaniasis significantly increased TNF-α and decreased melatonin levels. However, the group of diabetic patients with leishmaniasis showed higher melatonin levels than the control group. These observations suggest that TNF-α may influence melatonin production in patients with American cutaneous leishmaniasis, potentially contributing to the inflammatory characteristics of both diseases.

Semen Cannabis and Oleum Hyperici: Antileishmanial activity against Leishmania tropica promastigotes and intracellular amastigotes.

The exploration of alternative agents and novel drug candidates for the effective treatment of cutaneous leishmaniasis has garnered significant attention, driven by the high cost, toxic effects, and the emergence of drug resistance associated with current therapeutic options. Plant extracts derived from Semen Cannabis, the seeds of the Cannabis sativa L. (hemp) plant, and Oleum Hyperici, the oily macerate of Hypericum perforatum L. (St. John's Wort) plant, were prepared by using solvents of varying polarity (n-hexane, chloroform, ethanol, and 60% aqueous ethanol). The primary objective of this study was to research in vitro and ex vivo antileishmanial efficacy of Semen Cannabis and Oleum Hyperici plant extracts against Leishmania tropica promastigotes and intracellular amastigotes. The efficacy of plant extracts against promastigotes were assessed using the cell counting by hemocytometer and the CellTiter-Glo assay. Additionally, their impact on infected THP-1 macrophages and the quantity of intracelluler amastigotes were investigated. Cytotoxicity was evaluated in THP-1 macrophages. Among the tested plant extracts, chloroform extract of Oleum Hyperici demonstrated significant antileishmanial activity against promastigotes (SI: 12.6) and intracellular amastigotes (SI: 16.8) of L. tropica without inducing cytotoxic effects and hold promise for further investigation as potential antileishmanial agents.

Microemulsions strongly promoted the activity of α-bisabolol against different Leishmania species and its skin permeation.

This study aimed to develop microemulsions (MEs) containing α-bisabolol for the topical treatment of cutaneous leishmaniasis (CL). Initially, pseudoternary phase diagrams were developed using α-bisabolol as the oil phase, Eumulgin® CO 40 as the surfactant, Polymol® HE as the co-surfactant, and distilled water as the aqueous phase. Two transparent liquid systems (TLS) containing 5% of α-bisabolol were selected and characterized (F5E25 and F5EP25). Next, skin permeation and retention assays were performed using Franz cells. The interaction of the formulation with the stratum corneum (SC) was evaluated using the FTIR technique. The cytotoxicity was evaluated in murine peritoneal macrophages. Finally, the antileishmanial activity of microemulsions was determined in promastigotes and amastigotes of L. amazonensis (strain MHOM/BR/77/LTB 0016). As a result, the selected formulations showed isotropy, nanometric size (below 25 nm), Newtonian behavior and pH ranging from 6.5 to 6.9. The MEs achieved a 2.5-fold increase in the flux and skin-permeated amount of α-bisabolol. ATR-FTIR results showed that microemulsions promoted fluidization and extraction of lipids and proteins of the stratum corneum, increasing the diffusion coefficient and partition coefficient of the drug in the skin. Additionally, F5E25 and F5EP25 showed higher activity against promastigotes (IC50 13.27 and 18.29, respectively) compared to unencapsulated α-bisabolol (IC50 53.8). Furthermore, F5E25 and F5EP25 also showed antileishmanial activity against intracellular amastigotes of L. amazonensis, with IC50 50 times lower than free α-bisabolol and high selectivity index (up to 15). Therefore, the systems obtained are favorable to topical administration, with significant antileishmanial activity against L. amazonensis promastigotes and amastigotes, being a promising system for future in vivo trials.

First detection of Leishmania major in dogs living in an endemic area of zoonotic cutaneous leishmaniasis in Tunisia.

BACKGROUND: Dogs are considered the main domestic animals that may be a reservoir for Leishmania infantum, the agent of zoonotic visceral leishmaniasis (ZVL) in several countries of the world. The dog may host other Leishmania species, but its epidemiological role in the maintenance and spreading of these parasites is not completely elucidated. Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major, affects thousands of people every year and is particularly diffused in many countries of North Africa and Middle East Asia. In ZCL endemic countries, few reports of L. major-positive dogs have been reported, probably because most human cases occur in poor rural areas where the social role of the dog and its medical management is not well considered. The aim of the present study is to better understand the possible involvement of domestic dogs in the epidemiology of ZCL. METHODS: Our research focused on a well-established endemic focus of ZCL, in the area of Echrarda, Kairouan Governorate, central Tunisia. A total of 51 dogs with no or mild clinical signs of vector borne diseases were selected in small villages where human cases of ZCL are yearly present. All dogs were sampled for the Leishmania spp. diagnosis, by using the following procedures: blood sample for serology and buffy coat quantitative polymerase chain reaction (qPCR), popliteal fine needle aspiration, and cutaneous biopsy punch for lymph node and skin qPCR. RESULTS: The results demonstrated a high percentage (21.6%) of dogs positive at least at one or more test; the most sensitive technique was the lymph node qPCR that detected 8/11 positive dogs. Nine, out of the eleven positive dogs, resulted as infected by Leishmania infantum; ITS1-PCR-sequencing allowed Leishmania major identification in the remaining two cases, both from the popliteal lymph node samples, which can suggest a possible visceral spread of a cutaneous Leishmania species in the dog. Interestingly, one of the two L. major-positive dogs was living in the same house where 6-year-old children showed cutaneous lesions referred to as ZCL. CONCLUSIONS: To our knowledge, this is the first report of L. major-positive dogs in Tunisia, the  epidemiological role of which remains under investigation.

Unveiling the Enigmatic nature of six neglected Amazonian Leishmania (Viannia) species using the hamster model: Virulence, Histopathology and prospection of LRV1.

American tegumentary leishmaniasis (ATL) is highly endemic in the Amazon basin and occurs in all South American countries, except Chile and Uruguay. Most Brazilian ATL cases are due to Leishmania (Viannia) braziliensis, however other neglected Amazonian species are being increasingly reported. They belong to the subgenus L. (Viannia) and information on suitable models to understand immunopathology are scarce. Here, we explored the use of the golden hamster Mesocricetus auratus and its macrophages as a model for L. (Viannia) species. We also studied the interaction of parasite glycoconjugates (LPGs and GIPLs) in murine macrophages. The following strains were used: L. (V.) braziliensis (MHOM/BR/2001/BA788), L. (V.) guyanensis (MHOM/BR/85/M9945), L. (V.) shawi (MHOM/BR/96/M15789), L. (V.) lindenbergi (MHOM/BR/98/M15733) and L. (V.) naiffi (MDAS/BR/79/M5533). In vivo infections were initiated by injecting parasites into the footpad and were followed up at 20- and 40-days PI. Parasites were mixed with salivary gland extract (SGE) from wild-captured Nyssomyia neivai prior to in vivo infections. Animals were euthanized for histopathological evaluation of the footpads, spleen, and liver. The parasite burden was evaluated in the skin and draining lymph nodes. In vitro infections used resident peritoneal macrophages and THP-1 monocytes infected with all species using a MOI (1:10). For biochemical studies, glycoconjugates (LPGs and GIPLs) were extracted, purified, and biochemically characterized using fluorophore-assisted carbohydrate electrophoresis (FACE). They were functionally evaluated after incubation with macrophages from C57BL/6 mice and knockouts (TLR2-/- and TLR4-/-) for nitric oxide (NO) and cytokine/chemokine production. All species, except L. (V.) guyanensis, failed to generate evident macroscopic lesions 40 days PI. The L. (V.) guyanensis lesions were swollen but did not ulcerate and microscopically were characterized by an intense inflammatory exudate. Despite the fact the other species did not produce visible skin lesions there was no or mild pro-inflammatory infiltration at the inoculation site and parasites survived in the hamster skin/lymph nodes and even visceralized. Although none of the species caused severe disease in the hamster, they differentially infected peritoneal macrophages in vitro. LPGs and GIPLs were able to differentially trigger NO and cytokine production via TLR2/TLR4 and TLR4, respectively. The presence of a sidechain in L. (V.) lainsoni LPG (type II) may be responsible for its higher proinflammatory activity. After Principal Component analyses using all phenotypic features, the clustering of L. (V.) lainsoni was separated from all the other L. (Viannia) species. We conclude that M. auratus was a suitable in vivo model for at least four dermotropic L. (Viannia) species. However, in vitro studies using peritoneal cells are a suitable alternative for understanding interactions of the six L. (Viannia) species used here. LRV1 presence was found in L. (V.) guyanensis and L. (V.) shawi with no apparent correlation with virulence in vitro and in vivo. Finally, parasite glycoconjugates were able to functionally trigger various innate immune responses in murine macrophages via TLRs consistent with their inflammatory profile in vivo.

Development and accuracy evaluation of a new loop-mediated isothermal amplification assay targeting the HSP70 gene for the diagnosis of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a global public health problem caused by species on the genus Leishmania and is the most prevalent clinical form of leishmaniasis. The aim of this study was to develop a new LAMP assay for Leishmania sp. based on HSP70 gene and evaluate it clinically for molecular diagnosis of CL. The study was carried out in the following stages: i) design of primers based on HSP70 gene of Leishmania sp.; ii) evaluation of detection limit and analytical specificity; iii) estimation of the accuracy of LAMP-Leish/HSP70 assay for diagnosing CL. A total of 100 skin biopsy samples from patients, comprising 60 CL cases and 40 non-cases, were analyzed in this study. One LAMP assay using HSP70 gene as molecular target were standardized, and the observed detection limit was 100fg of L. braziliensis purified DNA. The LAMP-Leish/HSP70 assay was specific for Leishmania spp. The LAMP-Leish/HSP70 assay showed an accuracy of 92%, and positivity rates were not affected by lesion onset time or parasite load. This novel LAMP assay targeting the HSP70 gene of Leishmania sp. has the potential to be a useful tool to integrate into routine diagnosis for suspected cases of CL.