The clinical and epidemiological significance of Leishmania DNA in extralesional sites is obscured by uncertainty of whether the DNA derives from viable parasites. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients. The feasibility of discriminating parasite viability by PCR of Leishmania 7SLRNA was evaluated in relation with luciferase activity of luc transfected intracellular amastigotes in dose-response assays of Glucantime cytotoxicity. Monocytes, tonsil swabs, aspirates of normal skin and lesions of 28 cutaneous and 2 mucocutaneous leishmaniasis patients were screened by kDNA amplification/Southern blot. Positive samples were analyzed by quantitative PCR of Leishmania 7SLRNA genes and transcripts. 7SLRNA amplification coincided with luciferase activity, confirming discrimination of parasite viability. Of 22 patients presenting kDNA in extralesional samples, Leishmania 7SLRNA genes or transcripts were detected in one or more kDNA positive samples in 100% and 73% of patients, respectively. Gene and transcript copy number amplified from extralesional tissues were comparable to lesions. 7SLRNA transcripts were detected in 13/19 (68%) monocyte samples, 5/12 (42%) tonsil swabs, 4/11 (36%) normal skin aspirates, and 22/25 (88%) lesions; genes were quantifiable in 15/19 (79%) monocyte samples, 12/13 (92%) tonsil swabs, 8/11 (73%) normal skin aspirates. Viable parasites are present in extralesional sites, including blood monocytes, tonsils and normal skin of dermal leishmaniasis patients. Leishmania 7SLRNA is an informative target for clinical and epidemiologic investigations of human leishmaniasis. Understanding of the dynamics and distribution of Leishmania in the human host is fundamental to the targeting of control measures and their evaluation. Amplification of parasite gene sequences in clinical samples from cutaneous leishmaniasis patients has provided evidence of Leishmania in blood, other tissues and sites distinct from the lesion and of persistence of infection after clinical resolution of disease. However, there is uncertainty about the interpretation of the presence of Leishmania DNA as indicative of viable parasites. Because RNA is short-lived and labile, its presence provides an indicator of viability. We amplified Leishmania 7SLRNA, a molecule involved in intracellular protein translocation, to establish viability and estimate parasite load in blood monocytes, tonsil swab samples, and tissue fluid from healthy skin of patients with dermal leishmaniasis. Results showed that during active dermal leishmaniasis, viable Leishmania are present in blood monocytes, tonsils and normal skin in quantities similar to that in lesions, demonstrating widespread dissemination of infection and subclinical involvement of tissues beyond the lesion site. Leishmania 7SLRNA will be useful in deciphering the role of human infection in transmission.

Cysteine, a sulfur-containing amino acid, plays an important role in a variety of cellular functions such as protein biosynthesis, methylation, and polyamine and glutathione syntheses. In trypanosomatids, glutathione is conjugated with spermidine to form the specific antioxidant thiol trypanothione (T[SH]2) that plays a central role in maintaining intracellular redox homeostasis and providing defence against oxidative stress.

Leishmaniasis is a parasitic disease treatable and curable, however, the chemotherapeutic agents for their treatment are limited. In South American countries, pentavalent antimonials are still the first line of treatment for cutaneous leishmaniasis with an efficacy of about 75%, but the toxicity of the drug causes serious side effects and remains as the main obstacle for treatment. New knowledge aimed to improve drug delivery into the intracellular environment is essential, especially for drugs currently used in the clinic, to develop new anti-Leishmania formulations. In the present study, we analyzed the scientific literature to highlight the progress achieved in the last decade regarding the use of nanotechnology for improving the current leishmaniasis treatments. Results allowed us to conclude that the encapsulated Glucantime liposomal formulation can be improved by means of nanoparticle functionalization processes, resulting in new drug delivery systems that can be potentially proposed as alternative therapies for leishmaniasis treatment.

Chagas disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Current therapeutic management is limited to treatment with nitroheterocyclic drugs, such as nifurtimox (NFX) and benznidazole (BZ). Thus, the identification of affordable and readily available drugs to treat resistant parasites is urgently required worldwide. To analyse the effects of BZ on human macrophage gene expression, a quantitative PCR (qPCR) array analysis was performed using drug transporter and oxidative stress pathway genes to compare the gene expression profiles of human differentiated THP-1 macrophage (THP-1 MΦ) cells infected or not with benznidazole-sensitive (CL Brener) and naturally benznidazole-resistant (Colombiana) T. cruzi parasites followed by treatment with BZ. The gene expression analysis indicated that the expression levels of 62 genes were either up- or downregulated at least 3-fold in the host upon infection with CL Brener and BZ treatment, of which 46 were upregulated and 16 were downregulated. Moreover, the expression level of 32 genes was altered in THP-1 MФ cells infected with Colombiana and treated with BZ, of which 29 were upregulated and 3 were downregulated. Our results revealed that depending on the specific condition, human THP-1 MΦ cells infected with T. cruzi strains with sensitive or resistant phenotypes and treated with BZ expressed high mRNA levels of AQP1, AQP9 and ABCB1 (MDR1) compared to those of the control cells. Our findings suggest that the proteins encoded by AQP1, AQP9 and ABCB1 may be implicated in benznidazole detoxification. Therefore, studies on gene expression are required to better understand the host response to pathogens and drug treatment integrated with functional and metabolic data to identify potentially novel targets for the treatment of this important and neglected tropical disease.

Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania -infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (Sb ^V ), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S -transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of Sb ^V . By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1 , GSS , and ABCB5 resulted in an increased leishmanicidal effect of Sb ^V exposure in vitro . Our results suggest that human MDMs infected with L. braziliensis and treated with Sb ^V express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime.

Background: Chagas disease is a potentially fatal disease caused by the parasite Trypanosoma cruzi. There is growing scientific interest in finding new and better therapeutic alternatives for this disease’s treatment. Methods: A total of 81 terpene compounds with potential trypanocidal activity were screened and found to have potential T. cruzi cysteine synthase (TcCS) inhibition using molecular docking, molecular dynamics, ADME and PAIN property analyses and in vitro susceptibility assays. Results: Molecular docking analyses revealed energy ranges from −10.5 to −4.9 kcal/mol in the 81 tested compounds, where pentacyclic triterpenes were the best. Six compounds were selected to assess the stability of the TcCS–ligand complexes, of which lupeol acetate (ACLUPE) and α-amyrin (AMIR) exhibited the highest stability during 200 ns of molecular dynamics analysis. Such stability was primarily due to their hydrophobic interactions with the amino acids located in the enzyme’s active site. In addition, ACLUPE and AMIR exhibited lipophilic characteristics, low intestinal absorption and no structural interferences or toxicity. Finally, selective index for ACLUPE was >5.94, with moderate potency in the trypomastigote stage (EC₅₀ = 15.82 ± 3.7 μg/mL). AMIR’s selective index was >9.36 and it was moderately potent in the amastigote stage (IC₅₀ = 9.08 ± 23.85 μg/mL). Conclusions: The present study proposes a rational approach for exploring lupeol acetate and α-amyrin terpene compounds to design new drugs candidates for Chagas disease.