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.

Comparative analysis of the severity and progression of cutaneous leishmaniasis caused by Leishmania tropica in untreated and glucantime-treated patients.

Millions of people worldwide are affected by cutaneous leishmaniasis (CL), a disease that has a significant impact on morbidity and mortality. Understanding the immune responses responsible for tissue damage or the process of lesion healing plays a pivotal role in shaping optimal treatment strategies. In this study, we investigated immunological phenotypes for three groups: glucantime treated (n = 30) and untreated (n = 30) CL patients infected with Leishmania tropica (L. tropica), and healthy controls (n = 20). T-lymphocytes (CD4+ and CD8+), and B lymphocytes (CD14+ and CD19+) were isolated using antibody-conjugated microbeads and magnetic field isolation to achieve high purity. A higher significant difference was observed between T-lymphocytes (CD4+ and CD8+), and B-lymphocytes (CD14+ and CD19+) cells in CL-infected groups before and after treatment (p < 0.0001). When compared, there was also a significant difference among T-lymphocytes (CD4+ and CD8+), B lymphocytes (CD14+ and CD19+) p < 0.0001, p < 0.0005, and p < 0.0007, respectively between CL-infected individuals (before and after treatment) to controls. Our findings suggest that an increased proportion of these cells seen in treated patients may mediate healing, while it is also possible that they may contribute to tissue injury. Understanding the immune system and lesion size of CL can help develop immunotherapies and comprehend the evolution of this parasitic disease.

A Broad Spectrum Antiparasitic Activity of Organotin (IV) Derivatives and Its Untargeted Proteomic Profiling Using Leishmania donovani.

Metals have been used in medicine since ancient times for the treatment of different ailments with various elements such as iron, gold and arsenic. Metal complexes have also been reported to show antibiotic and antiparasitic activity. In this context, we tested the antiparasitic potential of 10 organotin (IV) derivatives from 4-(4-methoxyphenylamino)-4 oxobutanoic acid (MS26) against seven eukaryotic pathogens of medical importance: Leishmania donovani, Trypanosoma cruzi, Trypanosoma brucei, Entamoeba histolytica, Giardia lamblia, Naegleria fowleri and Schistosoma mansoni. Among the compounds with and without antiparasitic activity, compound MS26Et3 stood out with a 50% effective concentration (EC50) of 0.21 and 0.19 µM against promastigotes and intracellular amastigotes of L. donovani, respectively, 0.24 µM against intracellular amastigotes of T. cruzi, 0.09 µM against T. brucei, 1.4 µM against N. fowleri and impaired adult S. mansoni viability at 1.25 µM. In terms of host/pathogen selectivity, MS26Et3 demonstrated relatively mild cytotoxicity toward host cells with a 50% viability concentration of 4.87 µM against B10R cells (mouse monocyte cell line), 2.79 µM against C2C12 cells (mouse myoblast cell line) and 1.24 µM against HEK923 cells (human embryonic kidney cell line). The selectivity index supports this molecule as a therapeutic starting point for a broad spectrum antiparasitic alternative. Proteomic analysis of host cells infected with L. donovani after exposure to MS26Et3 showed a reduced expression of Rab7, which may affect the fusion of the endosome with the lysosome, and, consequently, impairing the differentiation of L. donovani to the amastigote form. Future studies to investigate the molecular target(s) and mechanism of action of MS26Et3 will support its chemical optimization.

Biosynthesis of anti-leishmanial natural products in callus cultures of Artemisia scoparia.

Clinically, available synthetic chemotherapeutics in the treatment for leishmaniasis are associated with serious complications, such as toxicity and emergence of resistance. Natural products from plants can provide better remedies against the Leishmania parasite and can possibly minimize the associated side effects. In this study, various extracts of the callus cultures of Artimisia scoparia established in response to different plant growth regulators (PGRs) were evaluated for their anti-leishmanial effects against Leishmania tropica promastigotes, followed by an investigation of the possible mechanism of action through reactive apoptosis assay using fluorescent microscopy. Amongst the different callus extracts, higher anti-leishmanial activity (IC50:19.13 µg/mL) was observed in the callus raised in-vitro in the presence of 6-Benzylaminopurine (BA) plus 2,4-Dichlorophenoxyacetic Acid (2,4-D) at the concentration of 1.5 mg/L, each. Further, the results of apoptosis assay showed a large number of early-stage apoptotic (EA) and late-stage apoptotic (LA) cells in the Leishmania under the effect of callus extract grown in-vitro at BA plus 2,4-D. For the determination of the potent natural products in the callus extracts responsible for the anti-leishmanial activity, extracts were subjected to Gas chromatography-mass spectrometry (GC-MS) for the metabolite analysis. Nonetheless, higher levels of the metabolites, such as nerolidol (22%), pelletierine (18%), aspidin (15%) and ascaridole (11%) were detected in the callus grown in vitro at BA plus 2,4-D (1.5 mg/L, each). This protocol determines a novel method of production of anti-leishmanial natural products through callus cultures of A. scoparia, a medicinal plant.

Natural compounds from plants controlling leishmanial growth via DNA damage and inhibiting trypanothione reductase and trypanothione synthetase: an in vitro and in silico approach.

In the present study, four different natural compounds including quercetin, gallic acid, rutin, and lupeol were studied for their anti-leishmanial potentials with anticipated mechanism of action through in vitro and in silico approaches. Results showed that rutin was exceedingly active (IC50; 91.2 µg/ml) against the promastigote form of Leishmania tropica compared to quercetin (IC50; 182.3 µg/ml), gallic acid (IC50; 198.00 µg/ml) and lupeol (IC50; 200.77 µg/ml). Similarly, rutin was highly active against the amastigote form as well, followed by quercetin, gallic acid and lupeol with IC50 values of 101.3 µg/ml, 137.4 µg/ml, 277.2 µg/ml, and 298.9 µg/ml, respectively. These compounds were found to be nontoxic to human blood erythrocytes even at the highest concentration (1000 µg/ml) tested. Rutin and lupeol showed promising DNA degradation/fragmentation activity against the DNA of treated promastigotes which increased with the increase in concentration of the compounds. The in silico investigation revealed that these ligands have high affinity with the important catalytic residues of trypanothione reductase (Try-R) where, rutin showed the lowest docking score (i.e., - 6.191) followed by lupeol (- 5.799), gallic acid and quercetin. In case of ligands' interaction with trypanothione synthetase (Try-S), rutin again showed highest interaction with docking score of - 6.601 followed by quercetin (- 4.996), lupeol and gallic acid. The ADMET prediction of these compounds showed that all the parameters were within the acceptable range as defined for human use while molecular dynamics simulation supported the good interaction of quercetin and rutin against both enzymes. These findings suggest that the studied compounds may control leishmanial growth via DNA damage and inhibiting Try-R and Try-S, the two unique but critical enzymes for leishmania growth.