Forms Of American Tegumentary Leishmaniasis Research Articles

MicroRNAs regulating macrophages infected with Leishmania L. (V.) Braziliensis isolated from different clinical forms of American tegumentary leishmaniasis.

Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania. There are still no vaccines, and therapeutic options are limited, indicating the constant need to understand the fine mechanisms of its pathophysiology. An approach that has been explored in leishmaniasis is the participation of microRNAs (miRNAs), a class of small non-coding RNAs that act, in most cases, to repress gene expression. miRNAs play a role in the complex and plastic interaction between the host and pathogens, either as part of the host's immune response to neutralize infection or as a molecular strategy employed by the pathogen to modulate host pathways to its own benefit. Monocyte-derived macrophages from healthy subjects were infected with isolates of three clinical forms of L. braziliensis: cutaneous (CL), mucosal (ML), and disseminated (DL) leishmaniasis. We compared the expression of miRNAs that take part in the TLR/NFkB pathways. Correlations with parasite load as well as immune parameters were analyzed. miRNAs -103a-3p, -21-3p, 125a-3p -155-5p, -146a-5p, -132- 5p, and -147a were differentially expressed in the metastatic ML and DL forms, and there was a direct correlation between miRNAs -103a-3p, -21-3p, -155-5p, -146a-5p, -132-5p, and -9-3p and parasite load with ML and DL isolates. We also found a correlation between the expression of miR-21-3p and miR-146a-5p with the antiapoptotic gene BCL2 and the increase of viable cells, whereas miR-147a was indirectly correlated with CXCL-9 levels. The expression of miRNAs is strongly correlated with the parasite load and the inflammatory response, suggesting the participation of these molecules in the pathogenesis of the different clinical forms of L. braziliensis.

Anti-Leishmania IgG is a marker of disseminated leishmaniasis caused by Leishmania braziliensis

BackgroundIn this study, we determined the accuracy of anti-Leishmania IgG and IgG subclasses to distinguish clinical forms of American tegumentary leishmaniasis (ATL) and and determined the relationship between antibodies levels with cytokine production and severity of ATL. MethodsParticipants were 40 patients with cutaneous leishmaniasis (CL), 20 patients with mucosal leishmaniasis (ML), 20 patients with disseminated leishmaniasis (DL), and 20 individuals with subclinical Leishmania braziliensis infection (SC). Diagnosis was performed by DNA of L. braziliensis or IFN-γ production in SC. IgG and subclasses of IgG to soluble Leishmania antigen and cytokine levels in supernatants of mononuclear cells were detected by ELISA. ResultsIgG was detected in 95%, 95%, and 100% of patients with CL, ML, and DL, respectively. Higher levels of anti-Leishmania IgG and IgG2 were seen in DL compared to CL, ML, and SC. ROC analysis confirmed the ability of IgG to distinguish DL from the other clinical forms. A direct correlation was observed between IgG titers and levels of IFN-γ and CXCL10 in CL and DL, and IgG2 antibodies were correlated with the number of lesions in DL. ConclusionsHigh anti-Leishmania IgG and IgG2 levels are characteristic of DL, and while IgG was correlated with pro-inflammatory cytokines, IgG2 was direct correlated with the number of lesions.

Quantitative analysis of proteins secreted by Leishmania (Viannia) braziliensis strains associated to distinct clinical manifestations of American Tegumentary Leishmaniasis

The role of Leishmania braziliensis in the development of different clinical forms of American Tegumentary Leishmaniasis (ATL) is unclear, but it has been suggested that molecules secreted/released by parasites could modulate the clinical outcome. Here, we analyzed the infection rate and cytokine profile of macrophages pretreated with the secretome of two L. braziliensis strains associated with polar clinical forms of ATL: one associated with localized self-healing cutaneous leishmaniasis (LCL) and other associated with the disseminated form (DL). Besides, we use an iTRAQ-based quantitative proteomics approach to compare the abundance of proteins secreted by those strains. In vitro infection demonstrated that pretreatment with secretome resulted in higher number of infected macrophages, as well as higher number of amastigotes per cell. Additionally, macrophages pretreated with LCL secretome exhibited a proinflammatory profile, whereas those pretreated with the DL one did not. These findings suggest that secretomes made macrophages more susceptible to infection and that molecules secreted by each strain modulate, differentially, the macrophages' cytokine profile. Indeed, proteomics analysis showed that the DL secretome is rich in molecules involved in macrophage deactivation, while is poor in proteins that activate proinflammatory pathways. Together, our results reveal new molecules that may contribute to the infection, persistence and dissemination of the parasite. SignificanceLeishmania braziliensis is associated to localized self-healing cutaneous lesions (LCL), disseminated leishmaniasis (DL), and mucocutaneous lesions (MCL). To understand the role of the parasite in those distinct clinical manifestations we evaluated infection rates and cytokine profiles of macrophages pre-treated with secretomes of two L. braziliensis strains associated with DL and LCL, and quantitatively compared these secretomes. The infection index of macrophages pretreated with the DL secretome was significantly higher than that exhibited by non-treated cells. Interestingly, whereas the LCL secretome stimulated a proinflammatory setting, favoring an effector cell response that would explain the proper resolution of the disease caused by this strain, the DL strain was not able to elicit such response or has mechanisms to prevent this activation. Indeed, DL secretome is rich in peptidases that may deactivate cell pathways crucial for parasite elimination, while is poor in proteins that could activate proinflammatory pathways, favoring parasite infection and persistence.

American tegumentary leishmaniasis in Brazil: a critical review of the current therapeutic approach with systemic meglumine antimoniate and short-term possibilities for an alternative treatment.

Meglumine antimoniate (MA; Glucantime®), the 80-year-old first-line systemic treatment for all forms of American tegumentary leishmaniasis (ATL) caused by Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis and Leishmania (Leishmania) amazonensis, is highly toxic, presents adverse side-effects and may not attain clinical and parasitological cure. This critical review examines the necessity for intramuscular/intravenous administration of MA, the alternatives to this approach, and the possibilities of developing affordable, accessible and non-toxic drugs or new delivery methods. PubMed searches were performed using the terms 'cutaneous leishmaniasis' or 'American tegumentary leishmaniasis' in combination with 'meglumine antimoniate' or 'N-methyl glucamine' or 'drug repositioning' or 'nanotechnology'. Searches covered a period of 20years of peer reviewed journals and technical bulletins. We explored the mode of action, pharmacokinetics, toxicity and efficacy of MA, evaluated the progress of ATL therapy in Brazil, and examined the potential of drug repositioning and nanotechnology in accelerating the introduction and/or optimisation of an alternative treatment. The evidence suggests that ATL therapy will continue to rely on systemic MA in the foreseeable future even though an intralesional subcutaneous route has evolved over the last 10years. The chances of developing a novel drug for ATL or a new mode of delivery of MA are low. While MA nanocarriers afford a promising approach, this technology is still in its infancy. A more immediate solution would be the production of a bioequivalent of miltefosine, an efficacious oral agent no longer protected by patent. Development of a contemporary treatment requires governmental commitment in bringing together private and public sectors.

Disseminated leishmaniasis: clinical, pathogenic, and therapeutic aspects

Disseminated leishmaniasis is a severe and emerging form of American tegumentary leishmaniasis. Disseminated leishmaniasis is defined by the presence of more than 10 polymorphic cutaneous lesions, distributed over more than two noncontiguous parts of the body. Nasal mucosal involvement is observed in almost half of cases. Disseminated leishmaniasis patients present with a decreased production of Th1 cytokines in the peripheral blood due to the attraction of leishmania-activated T cells to the multiple cutaneous lesions. Disseminated leishmaniasis development is poorly understood and is related to a complex network involving environmental, host immune response, and parasite factors, in which L. braziliensis polymorphism plays an important role. Disseminated leishmaniasis is a challenging disease to cure, presenting a high failure rate of 75% to pentavalent antimony therapy. Despite its importance and severity, this form of American tegumentary leishmaniasis has been poorly studied and documented, deserving greater attention from professionals working in endemic areas.

Leishmania (Viannia) guyanensis in tegumentary leishmaniasis.

Leishmania (Viannia) guyanensis is a causal agent of American tegumentary leishmaniasis (ATL). This protozoan has been poorly investigated; however, it can cause different clinical forms of ATL, ranging from a single cutaneous lesion to severe lesions that can lead to destruction of the nasopharyngeal mucosa. L. (V.) guyanensis and the disease caused by this species can present unique aspects revealing the need to better characterize this parasite species to improve our knowledge of the immunopathological mechanisms and treatment options for ATL. The mechanisms by which some patients develop a more severe form of ATL remain unclear. It is known that the host immune profile and parasite factors may influence the clinical manifestations of the disease. Besides intrinsic parasite factors, Leishmaniavirus RNA 1 (LRV1) infecting L. guyanensis can contribute to ATL immunopathogenesis. In this review, general aspects of L. guyanensis infection in humans and mouse models are presented.

In Vitro Immunomodulatory Activity of a Transition-State Analog Inhibitor of Human Purine Nucleoside Phosphorylase in Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) is the most common clinical form of American tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis. CL is associated with a strong Th1 immune response. This exacerbated inflammatory response is correlated with severity of disease and delays the healing time of the ulcer. The fourth-generation immucillin derivative (DI4G), a potent inhibitor of purine nucleoside phosphorylase, has been proposed as a promising agent in the treatment of diseases associated with T cell activation. Herein, we evaluated the in vitro immunomodulatory activity of DI4G in cells of patients presenting with CL. Peripheral blood mononuclear cells (PBMC) from CL patients were stimulated with soluble leishmania antigen (SLA), in the presence or absence of DI4G, and IFN-γ, TNF, CXCL9, and CXCL10 levels were determined by ELISA. Lymphocyte proliferation in the presence or absence of DI4G was also evaluated, using flow cytometry. DI4G was able to decrease (p < 0.05) IFN-γ production but did not change the TNF, CXCL9, and CXCL10 levels. DI4G decreased (p < 0.05) the lymphoproliferative response mediated by CD8+ T cells, but not that by CD4+ T cells. DI4G is able to attenuate the exaggerated immune response in CL, exhibiting immunomodulatory activity in IFN-γ production and in CD8+ T cell proliferation.

Effectiveness of an immunohistochemical protocol for Leishmania detection in different clinical forms of American tegumentary leishmaniasis

American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa=0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis.

Insulin-like growth factor-I serum levels and their biological effects on Leishmania isolates from different clinical forms of American tegumentary leishmaniasis.

sBackgroundAmerican tegumentary leishmaniasis (ATL) in Brazil is mostly caused by Leishmania (Viannia) braziliensis, with known forms of the disease being cutaneous (CL), mucosal (ML) and disseminated (DL) leishmaniasis. The development of the lesion in ATL is related both to the persistence of the Leishmania in the skin and to the parasite-triggered immune and inflammatory responses that ensue lesions. In this context one factor with expected role in the pathogenesis is insulin-like growth factor (IGF)-I with known effects on parasite growth and healing and inflammatory processes. In the present study, we addressed the effect of IGF-I on intracellular amastigote isolates from CL, ML and DL patients within human macrophage and we evaluated the IGF-I and IGF-binding protein-3 (IGFBP3) serum levels in patients presenting different clinical forms and controls from the endemic area.MethodsWe evaluated biological variability in the responses of intracellular amastigotes of Leishmania isolates derived from CL, ML, and DL patients from an area for ATL in response to IGF-I. Intracellular amastigote growth was evaluated using the human macrophage cell line THP-1. Arginase activity in infected cells was evaluated quantifying the generated urea concentration. Serum samples from patients and controls were assayed using chemiluminescent immunometric assay to determine IGF-I and IGFBP3 levels.ResultsWe observed an increase in intracellular parasitism upon IGF-I stimulus in 62.5 % of isolates from CL, in 85.7 % from ML and only 42.8 % from DL cases. In DL, the basal arginase activity was lower than that of CL. We then evaluated the IGF-I and IGFBP3 serum levels in patients, and we observed significantly lower levels in ML and DL than in CL and control samples.ConclusionsThe data suggest that IGF-I is modulated distinctly in different clinical forms of tegumentary leishmaniasis. IGF-I seemingly exerts effect on parasite growth likely contributing to its persistence in the skin in earlier phase. In addition the decreased IGF-I serum levels may affect the modulation of inflammation and lesion healing in chronic phase. In view of potential role of IGF-I in the pathogenesis of ATL we can speculate on therapeutic procedures taking into account the local IGF-I level.

In vitro characterization of Leishmania (Viannia) braziliensis isolates from patients with different responses to Glucantime® treatment from Northwest Paraná, Brazil

Leishmaniasis is a group of diseases that presents various clinical manifestations. Many studies have shown that the parasite plays an important role in the clinical manifestations and prognosis of this disease. The cutaneous and mucosal forms of American tegumentary leishmaniasis (ATL) are associated with Leishmania (Viannia) braziliensis, which exhibits intraspecific genetic polymorphisms and various clinical manifestations. The present study focused on four different L. braziliensis strains that were isolated from patients with distinct Glucantime® treatment responses. The isolates were described based on their molecular, biological, and infective characteristics. Growth patterns in culture medium and different grow phases were analyzed, MID-Logarithimic (Mid-LOG), Logarithimic (LOG) and Stationary (STAT) phases. Complement resistance was evaluated using guinea pig serum. Infection to murine peritoneal macrophages, cytokine and nitric oxide were analyzed. Ultrastructural features were determined by transmission electron microscopy, and molecular characteristics were determined based on random amplified polymorphic DNA (RAPD). All of the L. braziliensis isolates showed typical growth and similar complement sensitivity patterns. Markedly lower infectivity indexes were observed for all strains in the LOG phase, with different cytokine profiles. The ultrastructure analysis revealed distinct differences between the MID-LOG, LOG, and STAT phases. The RAPD results showed a divergence between the isolates of the L. braziliensis. The in vitro characterization of L. braziliensis isolates from humans with different treatment responses using various parameters enabled us to observe differences among the isolates. Molecular and in vivo characterizations are currently under study to improve understanding of the parasite-host interaction that can imply in the clinical manifestation differences.

Disseminated Leishmaniasis by Leishmania viannia Subgenus: A Series of 18 Cases in Southeastern Brazil.

Background. Disseminated leishmaniasis (DL) is an emerging clinical form of American tegumentary leishmaniasis (ATL) that occurs mainly in Northeastern Brazil. This study describes 18 cases where DL has not yet been reported.Methods. Disseminated leishmaniasis cases were extracted from ATL recorded cases between 1987 and March 2015. Disseminated leishmaniasis identification was based on ≥10 mixed-type lesions, located in ≥2 body parts.Results. Eighteen (5.4%) patients were identified as DL. Polymerase chain reaction followed by enzymatic digestion confirmed Leishmania viannia subgenus in 17 patients; amastigotes forms were identified in another one.Conclusions. Considering that DL diagnosis and management is challenging, clinicians must be aware of this emerging clinical form of the disease.

Intestinal helminth coinfection is associated with mucosal lesions and poor response to therapy in American tegumentary leishmaniasis

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.

Association between an Emerging Disseminated form of Leishmaniasis and Leishmania (Viannia) braziliensis Strain Polymorphisms

Leishmania (Viannia) braziliensis causes three main types of American tegumentary leishmaniasis (ATL), localized cutaneous leishmaniasis (CL), mucosal leishmaniasis (ML), and disseminated leishmaniasis (DL). All forms are observed among individuals of Corte de Pedra, Brazil. We previously used random amplified markers to identify a multiclonal population among L. (V.) braziliensis isolates from ATL patients, defining parasite clades associated with different clinical syndromes. Herein we compared sequences of random amplified markers to identify genotypes of L. (V.) braziliensis recovered from lesions of CL, ML, and DL patients. Six polymorphic genomic loci were sequenced from 35 parasite isolates. Single-nucleotide polymorphisms (SNPs) and insertions-deletions (indels) at each locus allowed us to segregate the L. (V.) braziliensis population according to haplotypes. Several SNPs, indels, and haplotypes were significantly associated with an increased risk of DL. Molecular genotyping may provide markers to identify L. (V.) braziliensis strains likely to cause this emerging, hard-to-treat form of ATL.

Transcriptome Patterns from Primary Cutaneous Leishmania braziliensis Infections Associate with Eventual Development of Mucosal Disease in Humans

IntroductionLocalized Cutaneous Leishmaniasis (LCL) and Mucosal Leishmaniasis (ML) are two extreme clinical forms of American Tegumentary Leishmaniasis that usually begin as solitary primary cutaneous lesions. Host and parasite factors that influence the progression of LCL to ML are not completely understood. In this manuscript, we compare the gene expression profiles of primary cutaneous lesions from patients who eventually developed ML to those that did not.MethodsUsing RNA-seq, we analyzed both the human and Leishmania transcriptomes in primary cutaneous lesions.ResultsLimited number of reads mapping to Leishmania transcripts were obtained. For human transcripts, compared to ML patients, lesions from LCL patients displayed a general multi-polarization of the adaptive immune response and showed up-regulation of genes involved in chemoattraction of innate immune cells and in antigen presentation. We also identified a potential transcriptional signature in the primary lesions that may predict long-term disease outcome.ConclusionsWe were able to simultaneously sequence both human and Leishmania mRNA transcripts in primary cutaneous leishmaniasis lesions. Our results suggest an intrinsic difference in the immune capacity of LCL and ML patients. The findings correlate the complete cure of L. braziliensis infection with a controlled inflammatory response and a balanced activation of innate and adaptive immunity.

Morita-Baylis-Hillman adduct showsin vitroactivity againstLeishmania (Viannia) braziliensisassociated with a reduction in IL-6 and IL-10 but independent of nitric oxide

Current treatments for different clinical forms of leishmaniasis are unsatisfactory, highly toxic and associated with increasing failure rates resulting from the emergence of resistant parasites. Leishmania (Viannia) braziliensis is the main aetiological agent of different clinical forms of American tegumentary leishmaniasis, including the mucosal form for which treatment has high failure rates. The aim of this work was to investigate the activity of the Morita-Baylis-Hillman adduct, methyl 2-{2-[hydroxy(2-nitrophenyl)methyl])acryloyloxy} benzoate in vitro against isolates of L. (V.) braziliensis obtained from patients with different clinical manifestations of tegumentary leishmaniasis: localized cutaneous leishmaniasis, mucosal leishmaniasis and disseminated cutaneous leishmaniasis. The adduct effectively inhibited the growth of promastigotes of the different isolates of L. (V.) braziliensis (IC(50) ≤ 7·77 μg/ml), as well as reduced the infection rate of macrophages infected with these parasites (EC(50) ≤ 1·37 μg/ml). It is remarkable to state that the adduct was more effective against intracellular amastigotes (P ≤ 0·0045). The anti-amastigote activity correlated with an immunomodulatory effect, since the adduct was able to decrease the production of IL-6 and IL-10 by the infected macrophages. However, its effect was independent of nitric oxide production. This work demonstrates the anti-leishmanial activity of methyl 2-{2-[hydroxy(2-nitrophenyl)methyl])acryloyloxy} benzoate and suggests its potential in the treatment of human infections caused by L. (V.) braziliensis.

Disseminated Leishmaniasis: Clinical, immunological, and therapeutic aspects

AbstractDisseminated leishmaniasis (DL) is a distinct, severe, and emerging form of American tegumentary leishmaniasis. DL has been characterized by more than 10 multiple and pleomorphic cutaneous lesions, distributed in more than two noncontiguous parts of the body. Mucosal involvement is detected in up to 44% of cases. The development of the DL involves a complex and poorly understood network involving parasite, host immune response, and the environment, where L. braziliensis polymorphism plays a major role. A decrease in the type 1 immune response in the peripheral blood of DL patients appears to be caused by the attraction of Leishmania‐activated T cells to the multiple cutaneous lesions. DL is a difficult to cure disease and failure to pentavalent antimony therapy is observed in at least 60% of cases. Drug Dev Res 72:437–441, 2011. © 2011 Wiley‐Liss, Inc.

SP5-33 Systematic review of the adverse effects of cutaneous leishmaniasis treatment in the new world

IntroductionPentavalent antimonials are first-line drugs for the treatment of the cutaneous form of American tegumentary leishmaniasis. Second-line drugs include amphotericin B and pentamidine. Although these drugs have been used for...

Insulin-like growth factor-I induced and constitutive arginase activity differs among isolates of Leishmania derived from patients with diverse clinical forms of Leishmania braziliensis infection

Arginase activity has been related to leishmaniasis development, thus we studied the constitutive and insulin-like growth factor (IGF) I-induced arginase activity of Leishmania (Viannia) braziliensis isolates from patients with different clinical forms of American tegumentary leishmaniasis (ATL). Isolates from mucosal leishmaniasis presented higher basal levels of arginase activity than isolates from other clinical forms of ATL. Isolates from disseminated leishmaniasis that present mucosal lesion in some cases reached the arginase activity similar to that of isolates from mucosal leishmaniasis upon IGF-I stimulation. Differences in arginase activity may influence disease outcomes such as evolution to mucosal lesion in patients with L. (V.) braziliensis infection.

Foxp3 expression in lesions of the different clinical forms of American tegumentary leishmaniasis

As the diversity in clinical presentation of American tegumentary leishmaniasis (ATL) is determined mainly by the immune response of host, our aim was to evaluate the in situ expression of Foxp3 [marker of regulatory T (Treg) cell] in lesions of the different clinical forms of ATL. Foxp3(+) cells were observed in 39.5% (32/81) of the samples and the number of positive cells was low in all the clinical forms. Even presenting a significantly lower number of CD4(+) T cells, diffuse cutaneous leishmaniasis (DCL) showed a higher expression of Foxp3 when compared with localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). In LCL and MCL, the number of Foxp3(+) cells correlated positively with the number of apoptotic cells (active caspase-3(+) cells). A positive correlation was also observed between the expression of active caspase-3 and FasL in these clinical forms. Our data suggest that increased number of Treg cells may be associated to the hyporesponsiveness observed in DCL and also indicate that the apoptosis may be a possible mechanism of action of Foxp3(+) Treg cell in LCL and MCL. However, further studies are required to better understand the mechanism of action of Treg cell.

Accuracy of an ELISA and indirect immunofluorescence for the laboratory diagnosis of American tegumentary leishmaniasis

We compared the accuracy of ELISA and indirect immunofluorescence (IIF) using Leishmania braziliensis and L. major-like antigens and antigens from the Bio-Manguinhos kit for serological diagnosis of American tegumentary leishmaniasis (ATL). Cut-off values were defined by the area under the receiver-operating characteristic curve. For ELISA, statistical analyses revealed better accuracy [95.7% sensitivity, 100% specificity, 100% positive predictive value (PPV), 97.5% negative predictive value (NPV)] and reliability [intraclass correlation coefficient (ICC): 0.940] for L. braziliensis antigen compared with L. major-like antigen (78.7% sensitivity, 82.8% specificity, 73.3% PPV, 86.6% NPV, ICC: 0.833). ELISA optical density values obtained for both antigens were higher in mucosal forms of ATL. For IIF, sensitivity and specificity were 81.5 and 86.2%, respectively, for the L. braziliensis antigen, compared with 95.4 and 77.7% for the L. major-like antigen and 75.4 and 89.2% for the Bio-Manguinhos kit. No difference in the specificity of the IIF test was observed between antigens, whereas sensitivity differed between the L. braziliensis and L. major-like antigens and the Bio-Manguinhos kit. Parallel ELISA and IIF testing increased sensitivity, irrespective of the antigen employed, and serial testing increased overall specificity. These results support the recommendation that ELISA employing L. braziliensis antigen be used as a diagnostic tool for suspected cases of ATL in L. braziliensis-endemic areas.