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The most severe clinical form of American tegumentary leishmaniasis (ATL) due to Leishmania braziliensis is mucosal leishmaniasis (ML), characterized by destructive lesions in the facial mucosa. We performed a retrospective cohort study of 109 ATL patients from Rio de Janeiro State, Brazil, where ATL is caused by L. braziliensis, to evaluate the influence of intestinal parasite coinfections in the clinical course of ATL. Parasitological stool examination (PSE) was performed with samples from all patients by the sedimentation, Kato-Katz and Baermann-Moraes methods. The diagnosis of ATL was made from lesion biopsies by direct observation of amastigotes in Giemsa-stained imprints, isolation of Leishmania promastigotes or histopathological examination. All patients were treated with meglumine antimoniate. Patients with positive PSE had a frequency of mucosal lesions significantly higher than those with negative PSE (p<0.005). The same was observed for infections with helminths in general (p<0.05), with nematodes (p<0.05) and with Ascaris lumbricoides (p<0.05), but not for protozoan infections. Patients with intestinal parasites had poor response to therapy (therapeutic failure or relapse) significantly more frequently than the patients with negative stool examination (p<0.005). A similar difference (p<0.005) was observed between patients with positive and negative results for intestinal helminths, but not for intestinal protozoa. Patients with positive PSE took significantly longer to heal than those with negative PSE (p<0.005). A similar difference was observed for intestinal helminth infections (p<0.005), but not for protozoan infections. Our results indicate a deleterious influence of intestinal helminth infections in the clinical course of ATL and evidence for the first time an association between ML and these coinfections, particularly with nematodes and A. lumbricoides., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We evaluated the effectiveness and safety of intralesional meglumine antimoniate (MA) in 24 not submitted to previous treatment patients with cutaneous leishmaniasis (CL) and with contraindication to systemic therapy. Each treatment consisted of one to four intralesional applications of MA at 15-day intervals. Patients' age ranged from 3 to 90 years; fourteen were females. Intralesional treatment in the absence of any relevant toxicity was successful in 20 (83.3%) patients. Three patients required additional treatment with amphotericin B and one required systemic MA. None of the patients developed mucosal lesions when followed up to 60 months. Intralesional MA is an effective and less toxic alternative treatment of patients with CL and contraindication to systemic therapy.

We have described a case of a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, two opposite polar forms of these spectral diseases. In the present follow-up study, we investigated the effect of the addition of Mycobacterium leprae antigens on interferon-gamma (IFN-γ) production in Leishmania antigen-stimulated cultures of peripheral blood mononuclear cells (PBMC) from this patient. For this purpose, PBMC cultures were stimulated with crude L. braziliensis and/or M. leprae whole-cell antigen extracts or with concanavalin A. In some experiments, neutralizing anti-human interleukin (IL)-10 antibodies were added to the cultures. IFN-γ and IL-10 levels in culture supernatants were measured by ELISA. During active leprosy, M. leprae antigens induced 72.3% suppression of the IFN-γ response to L. braziliensis antigen, and this suppression was abolished by IL-10 neutralization. Interestingly, the suppressive effect of M. leprae antigen was lost after the cure of leprosy and the disappearance of this effect was accompanied by exacerbation of mucosal leishmaniasis. Considered together, these results provide evidence that the concomitant lepromatous leprosy induced an IL-10-mediated regulatory response that controlled the immunopathology of mucosal leishmaniasis, demonstrating that, in the context of this coinfection, the specific immune response to one pathogen can influence the immune response to the other pathogen and the clinical course of the disease caused by it. Our findings may contribute to a better understanding of the Leishmania/M. leprae coinfection and of the immunopathogenesis of mucosal leishmaniasis.

Acroangiodermatitis, often known as pseudo-Kaposi sarcoma, is an uncommon angioproliferative entity related to chronic venous insufficiency, arteriovenous fistulae, paralysed limbs, amputation stumps, vascular syndromes and conditions associated with thrombosis. It presents most frequently as purple macules, papules or plaques in the dorsal aspects of the feet, especially the toes, and the malleoli. We report a case of acroangiodermatitis in the plantar aspect of the foot, misdiagnosed for two years, in which haematoxylin-eosin hystopathological stain and immunolabeling with CD34 histochemistry examination were decisive for diagnosis. Patient had chronic venous insufficiency. The lesion responded well to the treatment with a combination of leg elevation and compression.

Skin inflammation plays an important role during the healing of American tegumentary leishmaniasis (ATL), the distribution of cells in active lesions may vary according to disease outcome and parasite antigens in ATL scars have already been shown. We evaluated by immunohistochemistry, 18 patients with 1- or 3-year-old scars and the corresponding active lesions and compared them with healthy skin. Small cell clusters in scars organized as in the active lesions spreaded over the fibrotic tissue were detected, as well as close to vessels and cutaneous glands, despite a reduction in the inflammatory process. Analysis of 1-year-old scar tissue showed reduction of NOS2, E-selectin, Ki67, Bcl-2 and Fas expression. However, similar percentages of lymphocytes and macrophages were detected when compared to active lesions. Only 3-year-old scars showed reduction of CD3(+), CD4(+) and CD8(+)T cells, in addition to reduced expression of NOS2, E-selectin, Ki67 and BCl-2. These results suggest that the pattern of cellularity of the inflammatory reaction observed in active lesions changes slowly even after clinical healing. Analysis of 3-year-old scars showed reduction of the inflammatory reaction as demonstrated by decrease in inflammatory cells and in the expression of cell-activity markers, suggesting that the host-parasite balance was only established after that period.

The present study investigated the diagnostic value of polymerase chain reaction (PCR) performed in parallel to conventional methods at an American tegumentary leishmaniasis (ATL) referral centre for diagnosis. Accuracy parameters for PCR were calculated using 130 patients with confirmed ATL (ATL group), 15 patients established with other diseases and 23 patients with a lesion suggestive of ATL, but without parasitological confirmation (NDEF group). PCR showed 92.3% sensitivity, 93.3% specificity, a 99.2% positive predictive value and a 13.84 positive likelihood ratio. In the NDEF group, PCR confirmed ATL in 13 of the 23 patients, seven of whom responded to leishmaniasis treatment and six who presented spontaneous healing of the lesion. PCR should be included in the routine diagnostic procedures for ATL, especially for cases found to be negative by conventional methods.

It is known that the same antigen can induce different immune responses, depending upon the way that it is presented to the immune system. The objective of this study was to compare cytokine responses of peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients and subjects immunized with a first-generation candidate vaccine composed of killed Leishmania amazonensis promastigotes to a whole-cell promastigote antigen extract (La) and to the recombinant protein LACK (Leishmania analogue receptor for activated C kinase), both from L. amazonensis. Thirty-two patients, 35 vaccinees and 13 healthy subjects without exposure to Leishmania, were studied. Cytokine production was assessed by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay. The interferon (IFN)-gamma levels stimulated by La were significantly higher and the levels of interleukin (IL)-10 significantly lower than those stimulated by LACK in the patient group, while LACK induced a significantly higher IFN-gamma production and a significantly lower IL-10 production compared with those induced by La in the vaccinated group. LACK also induced a significantly higher frequency of IFN-gamma-producing cells than did La in the vaccinated group. The contrast in the cytokine responses stimulated by LACK and La in PBMC cultures from vaccinated subjects versus patients indicates that the human immune response to crude and defined Leishmania antigens as a consequence of immunization differs from that induced by natural infection.

Diffuse cutaneous leishmaniasis (DCL) is characterised by multiple and progressive cutaneous lesions, resistance to chemotherapy and Leishmania-specific T-cell anergy. We report the first autochthonous DCL case and the first human infection with Leishmania amazonensis in Rio de Janeiro State, Brazil, where only L. braziliensis is considered to be the causative agent of cutaneous leishmaniasis. Leishmania amazonensis was identified by multilocus enzyme electrophoresis and PCR-RFLP. Our case was diagnosed as DCL according to clinical, parasitological, histopathological and immunological criteria. These observations indicate that L. amazonensis is increasing its geographical distribution in Brazil, accounting for unusual clinical presentations in new transmission areas.

The first line drugs for the treatment of leishmaniasis are antimonial derivatives. Poor clinical response may be credited to factors linked to the host, the drug, or the parasite. We determined the sensitivity of Leishmania sp. promastigotes and amastigotes by counting parasites exposed to increasing concentrations of meglumine antimoniate (Glucantime). Leishmania braziliensis promastigotes were significantly more sensitive than those belonging to other species. The sensitivity of L. braziliensis isolates from patients with unfavorable clinical outcome, such as therapeutic failure or relapse, was significantly lower than those from patients who had clinical cure. Poor clinical response to therapy (therapeutic failure or relapse) was also associated with inadequate antimonial therapy. We also found a significant and positive correlation between promastigotes and intracellular amastigotes with regard to their in vitro susceptibilities to meglumine antimoniate. Our data provide evidence for an association between the sensitivity of promastigotes to antimonials in vitro and clinical response to therapy in American tegumentary leishmaniasis. The high sensitivity of the local L. braziliensis to meglumine antimoniate in vitro provides an explanation for the good clinical response of cutaneous leishmaniasis in the municipality of Rio de Janeiro, Brazil, even when low-dose regimens are employed.

Whole-cell and soluble extracts of Leishmania promastigotes have both been used as skin test antigens and have also been tested as vaccine candidates. However, the differences in antigenicity between soluble and particulate Leishmania fractions are not known. We evaluated in vitro responses of PBMC from 30 American tegumentary leishmaniasis (ATL) patients and seven noninfected donors to different antigen preparations from Leishmania promastigotes, namely Leishmania amazonensis and L. braziliensis whole-cell extracts, as well as soluble and particulate fractions of L. amazonensis. All Leishmania antigen preparations stimulated significantly higher proliferation and interferon (IFN)-gamma production (but not interleukin (IL)-10 production) in PBMC from the leishmaniasis patients than in cells from the control subjects. The L. braziliensis whole-cell extract stimulated significantly higher cell proliferation and IFN-gamma production than the L. amazonensis whole-cell extract in the group of patients but not in the control group. This result can be explained by the fact that the patients were infected with L. braziliensis. Again in the group of patients, the PBMC proliferative responses as well as the levels of IFN-gamma and IL-10 stimulated by L. amazonensis whole-cell extract were significantly greater than those elicited by the L. amazonensis soluble fraction but were not significantly different from those elicited by the L. amazonensis particulate fraction. We found a higher antigenicity of the particulate fraction as compared to the soluble fraction, what suggests that the antigens present in the particulate fraction account for most of the antigenicity of whole-cell Leishmania promastigote antigen extracts.

Background: Tegumentary leishmaniasis and leprosy display similar spectra of disease phenotypes, which are dependent on cell-mediated immunity to specific antigens. Diffuse cutaneous leishmaniasis and lepromatous leprosy represent the anergic end of the spectrum, whereas mucocutaneous leishmaniasis and tuberculoid leprosy are associated with marked antigen-specific cellular immune response., Methods: We characterized and compared the cell-mediated response to Leishmania and Mycobacterium leprae antigens in a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, which are at opposite ends of the immunopathological spectra of these diseases. This was done by performance of skin tests and by assessment of the cell proliferation and cytokine production of peripheral blood mononuclear cells (PBMCs)., Results: Strong skin-test reactions and PBMC proliferation were observed in response to Leishmania antigens but not to M. leprae antigens. The stimulation of PBMCs with Leishmania and M. leprae antigens induced comparable levels of tumor necrosis factor- alpha , interleukin-5, and interleukin-10. However, the interferon- gamma response to Leishmania antigens was remarkably high, and that to M. leprae antigens was almost nil., Conclusions: We found that concomitant leprosy and tegumentary leishmaniasis can produce opposite polar forms associated, respectively, with absent or exaggerated cell-mediated immune responses to each pathogen. This suggests that independent mechanisms influence the clinical outcome of each infection. Moreover, interferon- gamma appears to play a major role in the clinical expression of these intracellular infections.

This study reviews a series of cutaneous leishmaniasis cases diagnosed and treated in outpatient units in the municipality of Rio de Janeiro, where the intermittent schedule of antimonial therapy was replaced by the continuous regimen. Both schedules were based on daily intramuscular injections of pentavalent antimonial. Forty-nine subjects received the intermittent regimen, consisting of three ten-day series alternated with ten-day rest intervals whereas seventy-one patients received the continuous regimen during 20 consecutive days. The study groups had similar composition regarding age, sex and clinical condition. The cure rate was significantly higher in the group receiving the intermittent schedule than in the group receiving continuous therapy (89.8% vs 63.3%). Moreover, loss to follow-up was significantly more frequent in the group receiving continuous therapy (19.7% vs 4.1% in the intermittent therapy). Under field conditions, the intermittent regimen provided higher effectiveness and adherence than the continuous schedule.

T-cell immune responses in patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) were studied during the active disease, at the end of therapy, and 1 to 17 years posttherapy (long-term follow-up). Lymphocyte proliferative responses, phenotypic characterization of CD4(+) and CD8(+) Leishmania-reactive T cells, and cytokine production were assayed. Patients with active ML and CL showed higher proportions of CD4(+) than CD8(+) T cells. In CL, the healing process was associated with a decrease of CD4(+) and an increase of CD8(+), leading to similar CD4(+) and CD8(+) proportions. This pattern was only seen in ML after long-term therapy. Long-term follow-up of patients with CL showed a positive CD4(+)/CD8(+) ratio as observed during the active disease, although the percentages of these T cell subsets were significantly lower. Patients with CL did not show significant differences between gamma interferon (IFN-gamma) and interleukin-5 (IL-5) production during the period of study. Patients with active ML presented higher IFN-gamma and IL-5 levels compared to patients with active CL. IL-4 was only detected during active disease. Patients long term after cure from ML showed increasing production of IFN-gamma, significant decrease of IL-5, and no IL-4 production. Two apparently beneficial immunological parameters were detected in tegumentary leishmaniasis: (i) decreasing proportions of CD4(+) Leishmania-reactive T cells in the absence of IL-4 production associated with cure of CL and ML and (ii) decreasing levels of IL-5 long after cure, better detected in patients with ML. The observed T-cell responses maintained for a long period in healed patients could be relevant for immunoprotection against reinfection and used as a parameter for determining the prognosis of patients and selecting future vaccine preparations.

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.