Analysis of clinical cure outcome, macrophages number, cytokines levels and expression of annexin-A1 in the cutaneous infection in patients with Leishmania braziliensis.

BACKGROUND: Leishmania braziliensis, a protozoan prevalent in Brazil, is the known causative agent of cutaneous leishmaniasis (CL). The activation of M1 macrophages is a pivotal factor in the host's ability to eliminate the parasite, whereas M2 macrophages may facilitate parasite proliferation. This study analyzed the clinical outcomes of CL and the patients' immunological profiles, focusing on the prevalence of M1 and M2 macrophages, cytokine production, and annexin-A1 (ANXA1) expression in the lesion. METHODS: Data were obtained by polymerase chain reaction (PCR) and histopathological, immunofluorescence, and cytokine analyses. RESULTS: Patients with exudative and cellular reaction-type (ECR)-type lesions that healed within 90 days showed a significant increase in M1. Conversely, patients with ECR and exudative and granulomatous reaction (EGR)types, who healed within 180 days, showed an elevated number of M2. Cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α were higher in ECR lesions that resolved within 90 days (P<0.05). In contrast, IL-9 and IL-10 levels significantly increased in both ECR and EGR lesions that healed after 180 days (P<0.001). The production of IL-21, IL-23 and TGF-ß was increased in patients with ECR or EGR lesions that healed after 180 days (P<0.05). The expression of ANXA1 was higher in M2 within ECR-type lesions in patients who healed after 180 days (P<0.05). CONCLUSIONS: These findings suggest that the infectious microenvironment induced by L. braziliensis affects the differentiation of M1 and M2 macrophages, cytokine release, and ANXA1 expression, thereby influencing the healing capacity of patients. Therefore, histopathological and immunological investigations may improve the selection of CL therapy.

Multifocal Sporotrichosis Associated with Armadillo Hunting in Midwest Brazil: An In-Depth Case Study and Comprehensive Literature Analysis.

Sporotrichosis is a globally distributed subcutaneous mycosis caused by dimorphic Sporothrix species commonly found in soil, mosses, and decaying plant matter. The lymphocutaneous manifestation, historically associated with occupational activities and sapronotic transmission, has recently been observed to also occur through animal contact, particularly notable in Brazil. We describe a rare case of lymphocutaneous sporotrichosis with simultaneous pulmonary complications resulting from the scratching of a southern three-banded armadillo, Tolypeutes matacus, primarily inhabiting the arid forests of South America's central region. Speciation using multiplex quantitative polymerase chain reaction (qPCR) established the etiological agent as S. schenckii s. str., while amplified fragment length polymorphism (AFLP) analysis unveiled a novel genotype circulating in the Midwest of Brazil. The patient received treatment with itraconazole (200 mg/day) for two months, leading to substantial clinical improvement of cutaneous and pulmonary symptoms. This case highlights the critical role of animal-mediated transmission in sporotrichosis epidemiology, particularly within regions with diverse armadillo species. The unusual epidemiology and genetic characteristics of this case emphasize the need for enhanced awareness and diagnostic vigilance in atypical sporotrichosis presentations.

A Randomized, Controlled, Noninferiority, Multicenter Trial of Systemic vs Intralesional Treatment With Meglumine Antimoniate for Cutaneous Leishmaniasis in Brazil.

BACKGROUND: Meglumine antimoniate (MA) remains the main treatment for cutaneous leishmaniasis (CL). Uncontrolled studies suggest that intralesional MA (IL-MA) may be noninferior and safer than systemic MA (S-MA). METHODS: Multicenter, randomized, controlled, open-label, phase 3 clinical trial to evaluate the efficacy and toxicity of IL-MA in 3 infiltrations at 14-day intervals compared with S-MA (10-20 mg Sb5+/kg/day, 20 days) for CL, with noninferiority margin of 20%. Primary and secondary outcomes were definitive cure at day 180 and epithelialization rate at day 90 of treatment, respectively. A 2-year follow-up was performed to assess relapses and emergence of mucosal lesions. Adverse events (AEs) were monitored according to the Division of AIDS AE grading system. RESULTS: We evaluated 135 patients. The cure rates (95% confidence interval) for IL-MA and S-MA treatment were, respectively, 82.8% (70.5-91.4) and 67.8% (53.3-78.3) per protocol (PP) and 70.6% (58.3-81.0) and 59.7% (47.0-71.5) per intention to treat (ITT). The epithelialization rates of the IL-MA and S-MA treatment were, respectively, 79.3% (66.6-88 + 8) and 71.2% (57.9-82.2) PP and 69.1% (55.2-78.5) and 64.2% (50.0-74.2) ITT. AEs in the IL-MA and S-MA groups were, respectively, clinical, 45.6% and 80.6%; laboratory, 26.5% and 73.1%; and electrocardiogram, 8.8% and 25.4%. Ten participants in the S-MA group and 1 in the IL-MA group were discontinued due to severe or persistent AEs. CONCLUSIONS: IL-MA provides a similar cure rate and results in less toxicity compared with S-MA and may be used as first-line therapy for CL patients. CLINICAL TRIALS REGISTRATION: REBEC: RBR-6mk5n4.

Cutaneous leishmaniasis treatment and therapeutic outcomes in special populations: A collaborative retrospective study.

BACKGROUND: Treatment guidance for children and older adult patients affected by cutaneous leishmaniasis (CL) is unclear due to limited representation of these groups in clinical trials. METHODS: We conducted a collaborative retrospective study to describe the effectiveness and safety of antileishmanial treatments in children ≤ 10 and adults ≥ 60 years of age, treated between 2014 and 2018 in ten CL referral centers in Latin America. RESULTS: 2,037 clinical records were assessed for eligibility. Of them, the main reason for non-inclusion was lack of data on treatment follow-up and therapeutic response (182/242, 75% of children and 179/468, 38% of adults). Data on 1,325 eligible CL patients (736 children and 589 older adults) were analyzed. In both age groups, disease presentation was mild, with a median number of lesions of one (IQR: 1-2) and median lesion diameter of less than 3 cm. Less than 50% of the patients had data for two or more follow-up visits post-treatment (being only 28% in pediatric patients). Systemic antimonials were the most common monotherapy regimen in both age groups (590/736, 80.2% of children and 308/589, 52.3% of older adults) with overall cure rates of 54.6% (95% CI: 50.5-58.6%) and 68.2% (95% CI: 62.6-73.4%), respectively. Other treatments used include miltefosine, amphotericin B, intralesional antimonials, and pentamidine. Adverse reactions related to the main treatment were experienced in 11.9% (86/722) of children versus 38.4% (206/537) of older adults. Most adverse reactions were of mild intensity. CONCLUSION: Our findings support the need for greater availability and use of alternatives to systemic antimonials, particularly local therapies, and development of strategies to improve patient follow-up across the region, with special attention to pediatric populations.

Analysis of annexin-A1 in the macrophages and apoptotic cells of patients with cutaneous leishmaniasis.

INTRODUCTION: This study aimed to determine the number of macrophages and apoptotic cells and perform annexin-A1 detection in patients with leishmaniasis. METHODS: Patients with Leishmania infection were admitted to Júlio Müller University Hospital. RESULTS: The number of apoptotic cells was higher in the exudative granulomatous reaction. The exudative cellular reaction displayed higher levels of annexin-A1 detection in macrophages and apoptotic cells. The correlation between annexin-A1 detection in apoptotic cells and macrophages was observed in exudative necrotic reaction and exudative necrotic-granulomatous reaction. CONCLUSIONS: Our data demonstrate the relevance of annexin-A1 in the regulation of apoptosis and phagocytosis in leishmaniasis.

Analysis of macrophage subtypes and annexin A1 expression in lesions of patients with cutaneous leishmaniasis.

INTRODUCTION: Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis. METHODS: Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry. RESULTS: In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively). CONCLUSIONS: These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.

Analysis of macrophage subtypes and annexin A1 expression in lesions of patients with cutaneous leishmaniasis

Abstract INTRODUCTION: Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis. METHODS: Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry. RESULTS: In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively). CONCLUSIONS: These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.

Expression of annexin A1 in Leishmania-infected skin and its correlation with histopathological features.

INTRODUCTION: The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects. METHODS: Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence. RESULTS: All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions. CONCLUSIONS: Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.

Expression of annexin A1 in Leishmania-infected skin and its correlation with histopathological features

ABSTRACTINTRODUCTION:The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects.METHODS:Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence.RESULTS:All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions.CONCLUSIONS:Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.

Varicella zoster virus reactivation during or immediately following treatment of tegumentary leishmaniasis with antimony compounds.

Antimony compounds are the cornerstone treatments for tegumentary leishmaniasis. The reactivation of herpes virus is a side effect described in few reports. We conducted an observational study to describe the incidence of herpes zoster reactivation during treatment with antimony compounds. The global incidence of herpes zoster is approximately 2.5 cases per 1,000 persons per month (or 30 cases per 1,000 persons per year). The estimated incidence of herpes zoster in patients undergoing antimony therapy is higher than previously reported.

Varicella zoster virus reactivation during or immediately following treatment of tegumentary leishmaniasis with antimony compounds

Antimony compounds are the cornerstone treatments for tegumentary leishmaniasis. The reactivation of herpes virus is a side effect described in few reports. We conducted an observational study to describe the incidence of herpes zoster reactivation during treatment with antimony compounds. The global incidence of herpes zoster is approximately 2.5 cases per 1,000 persons per month (or 30 cases per 1,000 persons per year). The estimated incidence of herpes zoster in patients undergoing antimony therapy is higher than previously reported.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identification of the parasite species.

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Possible cardioprotective effect of angiotensin-converting enzyme inhibitors during treatment of American tegumentary leishmaniasis with meglumine antimoniate.

We conducted an observational retrospective study to identify factors associated with prolongation of corrected QT (QTc) interval during treatment of American tegumentary leishmaniasis with meglumine antimoniate. A group of 108 patients with normal ECG before treatment were included. Thirty-one patients (29%) developed increase of QTc interval beyond 0.44s in the second ECG performed after an average of (mean+/-S.D.) 12.6+/-4.9 days. After univariate and multivariate analysis, the age was associated with prolonged QTc interval, and the use of angiotensin-converting enzyme (ACE) inhibitors demonstrated a protective factor. These results identify elderly as a risk factor to develop prolonged QTc due to antimonial therapy (odds ratio: 1.1; 95% confidence interval: 1.01-1.12), and suggest that use of ACE inhibitors is a possible cardioprotective agent (odds ratio: 0.3; 95% confidence interval: 0.003-0.34). Further studies using prospective methodology are necessary to define the role of ACE inhibitors as prophylactic agent in high-risk patients.

Leishmania (Viannia) braziliensis is the prevalent species infecting patients with tegumentary leishmaniasis from Mato Grosso State, Brazil.

The frequency of Leishmania (Viannia) braziliensis infection among patients of Mato Grosso, Brazil was estimated by polymerase chain reaction-PCR, DNA hybridization and by isoenzyme electrophoresis. Analysis of DNA polymorphism was carried out using random amplified polymorphic DNA-PCR (RAPDPCR) with five different primers. The patients were attended from May 1997 to February 1998 at the Reference Ambulatory for American Tegumentary Leishmaniasis at Júlio Müller University Hospital of the Federal University of Mato Grosso, Brazil. In a first screening by PCR and DNA hybridization 94.1% of 68 patients, from whom parasites were isolated in culture medium, were found to be infected with species of the Le. braziliensis complex. Only four patients (5.9%) were infected with species of Le. mexicana complex. Thirty-three samples of Le. braziliensis complex and three of Le. mexicana complex were typed by isoenzyme analysis as Le. (V.) braziliensis sensu stricto and Le. (Leishmania) amazonensis, respectively. The predominant species was Le. (V.) braziliensis, although most of the patients of this study came from the northern area of Mato Grosso, which is part of the Amazonian region of Brazil, where other known species of both subgenus Viannia (Le. braziliensis complex) and Leishmania (Le. mexicana complex) are present. The results of RAPD showed higher genetic variability among the Le. (V.) braziliensis samples from Mato Grosso. The importance of these results concerning the taxonomic status of New World Leishmania, and their implications for both clinical and epidemiological data is discussed.

[Factors associated with treatment failure of cutaneous leishmaniasis with meglumine antimoniate]. / Fatores associados ao insucesso do tratamento da leishmaniose cutânea com antimoniato de meglumina.

We investigated factors associated with treatment failure in the treatment of cutaneous leishmaniasis with meglumine antimony in a reference service in Mato Grosso State. A retrospective cohort of 151 patients with cutaneous leishmaniasis was built using medical records. The incidence of therapeutic failure was 47% (IC95%=39.2%-55%). Antimoniate doses below 10 mg/kg/day (RR=1.8; IC95: 1.1-3.0), previous leishmaniasis treatment (RR=1.7; IC95: 1.3-2.4), 3 or more skin lesions (RR=1.9; IC95: 1.4-2.5), incomplete treatment (RR=1.9; IC95: 1.3-2.6) and body weight above 68 kg (RR=1.7; IC95: 1.1-2.5) were associated with therapeutic failure. After adjustment, therapeutic failure was associated with having 3 or more cutaneous lesions (OR=4.6; IC95%=1.2-17.4), reports of previous leishmaniasis treatment (OR=4.5; IC95%=1.1-17.5), body weight above 68 kg (OR=4.3; IC95=1.5-11.9) and incomplete treatment schedule (OR=12.5; IC95%=2.1-75.4), although body weight is possibly associated with treatment failure due to the limitation of the maximum daily dose. These results help to identify patients at risk of treatment failure of cutaneous leishmaniasis with antimony therapy.

Fatores associados ao insucesso do tratamento da leishmaniose cutânea com antimoniato de meglumina / Factors associated with treatmet failure of cutaneous leishmaniasis with meglumine antimoniate

Foram investigados os fatores associados ao insucesso do tratamento da leishmaniose cutânea com antimoniato de meglumina num servico de referência para leishmanioses, em Mato Grosso. Uma coorte histórica de 151 pacientes com diagnóstico de leishmaniose cutânea foi construída com informacões dos prontuários. A incidência de insucesso após o primeiro ciclo de antimonial foi 47 por cento (IC95 por cento=39,2 por cento-55 por cento). Dose de antimonial inferior a 10mg/kg/dia (RR=1,8; IC95:1,1-3,0), tratamento prévio para leishmaniose (RR=1,7; IC95:1,3-2,4), três ou mais lesões (RR=1,9; IC95:1,4-2,5), tratamento irregular (RR=1,9; IC95:1,3-2,6) e peso maior que 68kg (RR=1,7; IC95:1,1-2,5) foram associados ao insucesso terapêutico. Após ajuste, permaneceram associados ao insucesso os seguintes fatores: 3 ou mais lesões cutâneas (OR=4,6; IC95 por cento=1,2-17,4), tratamento anterior para leishmaniose tegumentar americana (OR=4,5; IC95 por cento=1,1-7,5), peso maior que 68kg (OR=4,3; IC95 por cento=1,5-11,9) e irregularidade no tratamento (OR=12,5; IC95 por cento=2,1-75,4), embora o peso possivelmente tenha sido associado ao insucesso devido à limitacão da dose máxima. Estes achados auxiliam na identificacão de pacientes com maior risco de insucesso no tratamento da leishmaniose cutânea com antimonial.

[Entomophthoramycosis (zygomycosis) caused by Conidiobolus coronatus in Mato Grosso (Brazil): case report]. / Entomoftoromicose (zigomicose) causada por Conidiobolus coronatus em Mato Grosso (Brasil): relato de caso.

Zygomycosis is a subcutaneous mycosis caused by soil fungi, such as Conidiobolus coronatus. In general, the main clinical manifestation is a chronic rhinofacial tumor. We report the first case of zygomycosis (entomophthoramycosis) caused by Conidiobolus coronatus, occurring in Mato Grosso, West Brazil.

Entomoftoromicose (zigomicose) causada por Conidiobolus coronatus em Mato Grosso (Brasil): relato de caso / Entomophthoramycosis (zygomycosis) caused by Conidiobolus coronatus in Mato Grosso (Brazil): case report

Zigomicoses são micoses subcutâneas causadas por fungos do solo, que geralmente manifestam-se como uma infiltração granulomatosa crônica da submucosa nasal, estendendo-se para o tecido subcutâneo e pele da face. Descreve-se aqui o primeiro caso de zigomicose nasofacial causada pelo Conidiobolus coronatus, ocorrendo em Mato Grosso, Brasil.