Early Cutaneous Leishmaniasis Research Articles

Study of the in vitro and in vivo antileishmanial activities of nimodipine in susceptible BALB/c mice.

Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL), however, treatment failures are frequent. Nimodipine, a calcium channel blocker is known to show promising antiprotozoal effects. Here, we investigated the antileishmanial effect of Nimodipine in both in vitro and in vivo BALB/c mice model of CL. We also compared the in vivo effect with amphotericin B and meglumine antimoniate in the experimental CL mice model. Colorimetric alamar blue assay and J774 A.1 mouse macrophage cells were used to determine the effect of nimodipine on promastigotes and amastigotes viability, respectively. Then, the in vivo activity of nimodipine was compared to that of conventional therapies in both the early and established courses of Leishmania major infection in susceptible non-healing BALB/c mice. Nimodipine was highly active against promastigotes and amastigotes of L. major with IC50 values of 49.40 and 15.03 μM, respectively. In the early model, the combination therapy with meglumine antimoniate and nimodipine showed no parasites in the spleen or footpad of animals. The footpad thickness was significantly lower in mice treated with either nimodipine (1 mg/kg or 2.5 mg/kg) or amphotericin B compared to the control group in the established lesions model. However, no complete remission was observed in the footpad lesion of any of the treatment groups (nimodipine, amphotericin B, meglumine antimoniate, and combination therapy). The effect of nimodipine was comparable to that of amphotericin B and meglumine antimoniate in early and established CL lesion models. Since nimodipine is more cost-effective than conventional therapies, our results merit further investigation in other animal models and voluntary human subjects.

“The mosquitoes that destroy your face”. Social impact of Cutaneous Leishmaniasis in South-eastern Morocco, A qualitative study

ObjectiveTo document the psychosocial burden of Cutaneous Leishmaniasis (CL) in rural communities in Southeastern Morocco.MethodBetween March and April 2015, we conducted qualitative research in communities exposed to Leishmania major or L. tropica in Errachidia and Tinghir provinces. Twenty-eight focus groups discussions (FGDs) were realized, with a stratification by gender and tradition of medicine (users of folk versus professional medicine). Data were analyzed using content analysis.ResultsThis rural population most exposed to CL in Morocco lacks access to health care in general and clearly points out there are other major public health issues that need to be resolved. Nonetheless, respondents consider the impact of CL lesions and scars as important and similar to that of burn scar tissue. Young women with CL scars in the face are stigmatized and will often be rejected for marriage in these communities. People usually try a long list of folk remedies on the active lesions, but none was felt adequate. There was a clear demand for better treatment as well as for treatment of the scars.ConclusionsThe psycho-social impact of CL due to L.major and L.tropica is substantial, especially for young single women with facial scars. These generate social and self-stigma and diminish their marriage prospects. CL is well known, but not considered as a major health priority by these poor rural communities in South-eastern Morocco where gender discrimination is still an issue and access to basic health care is as neglected as CL. Early CL diagnosis and new treatment options with better skin outcomes are urgently needed.

Early Cutaneous Leishmaniasis Patients Infected With Leishmania braziliensis Express Increased Inflammatory Responses After Antimony Therapy.

Early cutaneous leishmaniasis (ECL) is characterized by a nonulcerated papular lesion and illness duration less than 30 days. Approximately 4 weeks later, the cutaneous leishmaniasis (CL) ulcers appear. We were surprised to find that failure after antimony therapy (Sb5) is higher in ECL than CL. We hypothesize that the inflammatory response in ECL patients may increase during Sb5 therapy, which leads to treatment failure. A cohort of 44 ECL patients infected by Leishmania braziliensis was established to evaluate the response to Sb5 and to compare immunologic responses in ECL patients with CL and healthy subjects. A hierarchical clustering based on cytokine levels showed a weak positive correlation between proinflammatory cytokine levels and those patients that failed Sb5 treatment. Although Sb5 therapy decreased interferon-γ and tumor necrosis factor levels in CL patients, we were surprised to find that an increase in these cytokines was observed in ECL patients. Moreover, interleukin (IL)-10 was less able to down-modulate immune responses in ECL. The enhanced production of proinflammatory cytokines, due in part to the decreased ability of IL-10 to down-modulate immune response during therapy in ECL, promotes the development and persistence of leishmania ulcer despite antimony therapy.

Characterization of the Histopathologic Features in Patients in the Early and Late Phases of Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL), characterized by an ulcerated lesion, is the most common clinical form of human leishmaniasis. Before the ulcer develops, patients infected with Leishmania (Viannia) braziliensis present a small papule at the site of the sandfly bite, referred to as early cutaneous leishmaniasis (E-CL). Two to four weeks later the typical ulcer develops, which is considered here as late CL (L-CL). Although there is a great deal known about T-cell responses in patients with L-CL, there is little information about the in situ inflammatory response in E-CL. Histological sections of skin biopsies from 15 E-CL and 28 L-CL patients were stained by hematoxilin and eosin to measure the area infiltrated by cells, as well as tissue necrosis. Leishmania braziliensis amastigotes, CD4+, CD8+, CD20+, and CD68+ cells were identified and quantified by immunohistochemistry. The number of amastigotes in E-CL was higher than in L-CL, and the inflammation area was larger in classical ulcers than in E-CL. There was no relationship between the number of parasites and magnitude of the inflammation area, or with the lesion size. However, there was a direct correlation between the number of macrophages and the lesion size in E-CL, and between the number of macrophages and necrotic area throughout the course of the disease. These positive correlations suggest that macrophages are directly involved in the pathology of L. braziliensis–induced lesions.

Meta-transcriptome Profiling of the Human-Leishmania braziliensis Cutaneous Lesion.

Host and parasite gene expression in skin biopsies from Leishmania braziliensis-infected patients were simultaneously analyzed using high throughput RNA-sequencing. Biopsies were taken from 8 patients with early cutaneous leishmaniasis and 17 patients with late cutaneous leishmaniasis. Although parasite DNA was found in all patient lesions at the time of biopsy, the patients could be stratified into two groups: one lacking detectable parasite transcripts (PTNeg) in lesions, and another in which parasite transcripts were readily detected (PTPos). These groups exhibited substantial differences in host responses to infection. PTPos biopsies contained an unexpected increase in B lymphocyte-specific and immunoglobulin transcripts in the lesions, and an upregulation of immune inhibitory molecules. Biopsies without detectable parasite transcripts showed decreased evidence for B cell activation, but increased expression of antimicrobial genes and genes encoding skin barrier functions. The composition and abundance of L. braziliensis transcripts in PTPos lesions were surprisingly conserved among all six patients, with minimal meaningful differences between lesions from patients with early and late cutaneous leishmaniasis. The most abundant parasite transcripts expressed in lesions were distinct from transcripts expressed in vitro in human macrophage cultures infected with L. amazonensis or L. major. Therefore in vitro gene expression in macrophage monolayers may not be a strong predictor of gene expression in lesions. Some of the most highly expressed in vivo transcripts encoded amastin-like proteins, hypothetical genes, putative parasite virulence factors, as well as histones and tubulin. In summary, RNA sequencing allowed us to simultaneously analyze human and L. braziliensis transcriptomes in lesions of infected patients, and identify unexpected differences in host immune responses which correlated with active transcription of parasite genes.

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.

CD8+ T cells in situ in different clinical forms of human cutaneous leishmaniasis

Leishmania braziliensis infection induces a large spectrum of lesions that clinically manifest as nodules or papules that progress to ulcers. Although it is already known that T helper cells predominate in the lesions, cytotoxic T cells have also been reported to be present, and their role in leishmaniasis immunopathogenesis is not well known. This study investigated the amounts of CD8+ and granzyme B+ cells in different clinical forms of human cutaneous leishmaniasis (CL). Forty tissue fragments from early (E-CL) and late CL (L-CL) lesions and from disseminated leishmaniasis (DL) - papules and ulcers - were characterized. The inflamed area per fragment was calculated, and the CD8 and granzyme B expression levels in the infiltrates were quantified by counting positive cells in 15 fields. The localization of CD8 and granzyme B was graded subjectively. Inflammation was higher in L-CL and DL ulcers. CD8 expression was increased in late ulcerated lesions compared to recent lesions. The increase in CD8+ cells also correlated with the duration of the lesion. Papules had a higher frequency of granzyme B+ cells than E-CL lesions, although the frequency was similar to those for late and DL ulcers. CD8+ cells were mostly found in the papillary dermis. CD8+ T and granzyme B+ cells are present in the inflammatory infiltrates of CL and DL and may participate in the immunopathogenesis of Leishmania infection.

Comparative immune study on cutaneous leishmaniasis patients with single and multiple sores.

Ninety-five Iraqi patients with cutaneous leishmaniasis (CL) caused by Leishmania tropica at AL-Karama Hospital in Baghdad were included in this study. Sixty patients were with single sore and the remaining with multiple sores. The study also included 10 atopic patients and 30 healthy individuals as a control group. Cellular and humoral immune response at different stages of the disease activity (early and late) were evaluated by estimation of serum IFN-γ, IL-4 and total IgE antibodies using ELISA kits while, the detection of specific anti leishmanial IgE antibodies was done manually. Specific IgE antibodies were only detected in early CL (<2months) patients 68 (71.57%) while, were not detected in late CL, atopic and healthy controls 30 (100%). The results also showed a positive relationship between this antibody and the number of sores. Th-2 predominates during the early stage of the disease then shifts to Th-1 that proceed in the late stage, but both cytokines increased in CL patients in comparison to control group. The immune response of CL infection is possibly regulated by both Th-1 and Th-2. Multiple sores patients showed an increase of anti leishmanial IgE (0.120±0.014), total IgE (120.7±39.58IU/ml), IFN-γ (87.4±30.52pg/ml) and IL-4 (63.70±20.32pg/ml) levels than single sore patients with mean value of 0.108±0.14, 92.3±35.23IU/ml, 47.2±27.80pg/ml and 51.04±15.0pg/ml respectively. It can be presented also as ratio of INF-γ/IL-4=1.37 which is greater than those for single sore 0.9. These results indicated that the immune response of multiple sores patient's is higher than that with single sores.

Accelerated healing of cutaneous leishmaniasis in non-healing BALB/c mice using water soluble amphotericin B-polymethacrylic acid

Cutaneous leishmaniasis (CL) is a neglected tropical disease that causes prominent skin scaring. No water soluble, non-toxic, short course and low cost treatment exists. We developed a new water soluble amphotericin B-polymethacrylic acid (AmB-PMA) using established and scalable chemistries. AmB-PMA was stable for 9 months during storage. In vitro, it was effective against Leishmania spp. promastigotes and amastigote infected macrophages. It was also less toxic and more effective than deoxycholate-AmB, and similar to liposomal AmB. Its in vivo activity was determined in both early and established CL lesion models of Leishmania major infection in genetically susceptible non-healing BALB/c mice. Intradermal AmB-PMA at a total dose of 18 mg of AmB/kg body weight led to rapid parasite killing and lesion healing. No toxicity was seen. No parasite relapse occurred after 80 days follow-up. Histological studies confirmed rapid parasite clearance from macrophages followed by accelerated fibroblast mediated tissue repair, regeneration and cure of the infection. Quantitative mRNA studies of the CL lesions showed that accelerated healing was associated with increased Tumour Necrosis Factor-α and Interferon-γ, and reduced Interleukin-10. These results suggest that a cost-effective AmB-PMA could be used to pharmacologically treat and immuno-therapeutically accelerate the healing of CL lesions.

Development of Cutaneous Leishmaniasis after Leishmania Skin Test

Thirty-year-old female with a previous history of a cutaneous ulcer suspicious of leishmaniasis 20 years ago presented with a new complaint of a depressed papular lesion 8 × 7 mm in the right lower extremity. The lesion was of 10-day duration. Because early cutaneous leishmaniasis (CL) lesions may have a non-ulcerated appearance, a Leishmania skin test (LST) was performed on the forearm with a strong positive result (38 × 32 mm). After 8 days, the lesion in the leg, which was diagnosed as folliculitis, completely healed. However, a typical CL ulcer (26 × 24 mm) developed at the LST site. Histopathology of the new lesion did not identifiy parasites, but the findings were consistent with a diagnosis of CL. Further analysis identified amastigotes by immunohistochemical stain. Mononuclear cells harvested from the patient were stimulated with Leishmania antigen and showed high levels of production of both tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ): 2,943 pg/mL and 2,313 pg/mL, respectively. After 40 days of treatment with antimony and pentoxifylline, the ulcer resolved. The development of CL at the LST site suggests a strong Th1 immune response, and it is an in vivo documentation of the role of the host immune response in the pathology of CL. It teaches us that LST should be cautiously, if at all, used in patients with self-healing CL ulcers.

CONCOMITANT EARLY MUCOSAL AND CUTANEOUS LEISHMANIASIS IN BRAZIL

Mucosal leishmaniasis (ML) is often clinically silent until reaching a highly advanced state. In this prospective study, 6 of 220 patients with early cutaneous leishmaniasis were diagnosed with mucosal involvement by otorhinolaryngological examination (a rate similar to the reported rate of late ML). Detection of early ML may represent an important strategy in preventing severe mucosal destruction in human leishmaniasis.

Adhesion molecule expression patterns indicate activation and recruitment of CD4+ T cells from the lymph node to the peripheral blood of early cutaneous leishmaniasis patients

Adhesion molecules play a crucial role in cell migration and recruitment. Expression of adhesion molecules that preferentially address cells to inflammatory sites is a critical event in the formation and maintenance of leishmaniasis lesions. In this work, we analyzed the expression of CD11a, CD11b and CD62L, adhesion molecules involved in cell activation and circulation, in CD4+ and CD8+ T cells from peripheral blood and lymph nodes of patients with early cutaneous leishmaniasis. The percentage of expression of CD62L, CD11a and CD11b in total lymphocytes was lower in lymph nodes as compared to peripheral blood. Moreover, differences in adhesion molecule expression between blood and lymph nodes were more striking in CD4+ than CD8+ T cells. Stimulation of PBMC from leishmaniasis patients with soluble Leishmania antigens (SLA) lead to the expansion of CD4+CD62Lhigh cells, CD4+CD11b+ cells and to an increase in the intensity of expression of CD11a in CD4+, but not CD8+ T cells. Our data suggest that early activation events that occur in the lymph nodes of patients recently infected with Leishmania lead to changes in T cell adhesion molecule expression, favoring migration to the periphery and increasing the likelihood of further recruitment to lesion sites.

Down-regulation of Th1 type of response in early human American cutaneous leishmaniasis.

This study examined the T cell responses in the early phase of Leishmania braziliensis infection. Cytokine profiles, lymphoproliferative responses, and skin test results in 25 patients with early cutaneous leishmaniasis (ECL; illness duration <60 days) were compared with those in persons with late cutaneous leishmaniasis (LCL; illness duration >2 months). Absent or low lymphoproliferative responses were observed in 8 (32%) of 25 patients and an absence of interferon (IFN)-gamma production in 9 (41%) of 22 patients prior to therapy. IFN-gamma production in ECL (mean +/- SD) was lower than in LCL (293+/-346 vs. 747+/-377 pg/mL, respectively; P<.01). In contrast, interleukin (IL)-10 production in ECL (mean +/- SD) was higher than in LCL (246+/-56 vs. 50+/-41 pg/mL, respectively; P<.01). Restoration of lymphoproliferative responses and IFN-gamma production was achieved when monoclonal antibody to IL-10 or IL-12 was added to the cultures. These results show that T cell responses during early-phase infection are down-regulated by IL-10 and may facilitate parasite multiplication.