Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

The absence of eosinophils is associated with early metastatic lesions in Leishmania amazonensis-infected mice

BACKGROUND Eosinophils are granulocytes that rapidly increase frequency in the bloodstream during helminthic infections and allergic responses. They are found in tissue infected by Leishmania during early disease, but their role during infection is not entirely understood. OBJECTIVES We aim to compare the disease due to Leishmania amazonensis in BALB/c and Δdbl-GATA1 mice, which lack eosinophils. METHODS BALB/c and Δdbl-GATA1 mice infected with L. amazonensis were observed for several weeks. The parasite load and dissemination pattern were assessed. FINDINGS The Δdbl-GATA1 mice developed an anticipated dissemination of L. amazonensis and a worsening disease. No differences were found in the lesion development or the parasite load in the footpad among Δdbl-GATA1 mice and BALB/c eight weeks after infection. However, nine weeks after infection, massive growth of metastatic lesions appeared in several parts of the skin in Δdbl-GATA1 mice, weeks earlier than BALB/c. We observed increased parasites in the bloodstream, probably an essential dissemination route. Thirteen weeks after infection, metastatic lesions were found in all Δdbl-GATA1 mice. MAIN CONCLUSION These results suggest a protective role of eosinophils in delaying the disease caused by L. amazonensis, although several limitations of this mice strain must be considered.

Immune Response to LinB13, a Lutzomyia Intermedia Salivary Protein Correlates With Disease Severity in Tegumentary Leishmaniasis.

BACKGROUND: We have previously shown that seropositivity to rLinB-13, a salivary protein from Lutzomyia intermedia, predicted sand fly exposure and was associated with increased risk of developing cutaneous leishmaniasis (CL). METHODS: Here, we investigated the cellular immune response to saliva from Lu. intermedia, using rLinB-13 as a surrogate antigen in naturally exposed individuals presenting positive serology to LinB-13. We also investigated the response to rLinB-13 in leishmaniasis patients, displaying active ulcers and positive PCR for Leishmania braziliensis. RESULTS: Peripheral blood mononuclear cells (PBMCs) stimulated in vitro with rLinB-13 secreted elevated levels of interleukin-10 (IL-10), IL-4, IL-1ß, IL-1α, IL-6, and chemokines (CCL3, CCL4, CCL5, and CXCL5). CL and disseminated leishmaniasis (DL) patients displayed a significantly higher immunoglobulin G (IgG) response to rLinB-13 compared with healthy subjects, and anti-rLinB-13 IgG was positively correlated with the number of lesions in DL patients. Positive serology to rLinB-13 was also associated with chemotherapy failure. PBMCs from DL patients stimulated with rLINB-13 secreted significantly higher levels of IL-10 and IL-1ß compared with CL individuals. CONCLUSIONS: In this study, we observed an association between humoral and cellular immune response to the sand fly salivary protein rLinB-13 and disease severity in tegumentary leishmaniasis. This study brings evidence that immunity to rLinB-13 influences disease outcome in L. braziliensis infection and results indicate that positive serology to rLinB-13 IgG can be used as a marker of DL, an emerging and severe form of disease caused by L. braziliensis.

The Influence of Infection by Different Leishmania (Viannia) braziliensis Isolates on the Pathogenesis of Disseminated Leishmaniasis.

Disseminated Leishmaniasis (DL) is an emerging and severe form of Leishmania (Viannia) braziliensis infection defined by the presence of 10 and up to more than 1,000 skin lesions. The mechanisms underlying parasite dissemination remain unknown. Genotypic differences among species of L. braziliensis have been associated with different clinical forms of disease. The present work compared the function of monocytes obtained from patients with cutaneous leishmaniasis (CL) and DL in response to infection with L. braziliensis isolates of both these two clinical forms of disease. Mononuclear cells obtained from DL and CL patients were infected with different L. braziliensis isolates, and numbers of infected cells, parasite load, respiratory burst, TLR2 and TLR4 expression and cytokine production were evaluated. DL isolates infected more monocytes, induced greater respiratory burst, and more cytokine production compared to isolates from CL patients regardless of the origin of monocytes (DL or CL). However, greater parasite multiplication and higher TLR2 and TLR4 expression were seen in monocytes from DL patients compared to CL following infection with DL isolates. Our results indicate the participation of both parasite genotype and host factors in the pathogenesis of DL.

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

BACKGROUND: In 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. METHODS: Participants 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. RESULTS: IgG 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. CONCLUSIONS: High 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.

Leishmania braziliensis isolated from disseminated leishmaniasis patients downmodulate neutrophil function.

AIMS: The polymorphism observed in Leishmania braziliensis is associated with different clinical forms of leishmaniasis. Neutrophils (PMNs) participate in the pathogenesis of leishmania infection, and here, we evaluate neutrophil function after infection with isolates of L. braziliensis from cutaneous leishmaniasis (CL) or disseminated leishmaniasis (DL) patients. METHODS AND RESULTS: Neutrophils from 30 healthy subjects (HS) were infected with isolates of L. (V.) braziliensis obtained from three CL and three DL patients. They were infected at the ratio of 3:1 parasites per neutrophil, and leishmania uptake was evaluated by microscopy. The neutrophil activation markers and oxidative burst by expression of dihidrorhodamine (DHR) were evaluated by flow cytometry and cytokine production by ELISA. The frequency of infected cells and the number of amastigotes were higher in neutrophils infected with CL isolates compared to DL isolates (P < 0.05). The DHR and CD66b expression after infection with DL isolate was lower than with CL isolates. There was no difference regarding chemokine production. CONCLUSION: The L. (V.) braziliensis isolates of DL induced lower respiratory burst and neutrophils activation markers compared with CL isolates which may contribute to parasite survival and dissemination in DL patients.

Cutaneous Manifestations of Human and Murine Leishmaniasis.

The leishmaniases are diseases caused by pathogenic protozoan parasites of the genus Leishmania. Infections are initiated when a sand fly vector inoculates Leishmania parasites into the skin of a mammalian host. Leishmania causes a spectrum of inflammatory cutaneous disease manifestations. The type of cutaneous pathology is determined in part by the infecting Leishmania species, but also by a combination of inflammatory and anti-inflammatory host immune response factors resulting in different clinical outcomes. This review discusses the distinct cutaneous syndromes described in humans, and current knowledge of the inflammatory responses associated with divergent cutaneous pathologic responses to different Leishmania species. The contribution of key hematopoietic cells in experimental cutaneous leishmaniasis in mouse models are also reviewed and compared with those observed during human infection. We hypothesize that local skin events influence the ensuing adaptive immune response to Leishmania spp. infections, and that the balance between inflammatory and regulatory factors induced by infection are critical for determining cutaneous pathology and outcome of infection.

Disseminated Cutaneous Leishmaniasis in Colombia: Report of 27 Cases.

Disseminated leishmaniasis (DL) is a poorly described disease that is frequently misdiagnosed as other clinical manifestations of cutaneous leishmaniasis (CL) such as diffuse CL or post-kala-azar dermal leishmaniasis. Twenty-seven cases of DL diagnosed between 1997 and 2015 are described. A higher prevalence was observed in men (mean age 32 years). The number of lesions per patient ranged from 12 to 294, distributed mainly in the upper extremities, face and trunk. The lesions were mostly plaques or nodules. Seven patients had nasal mucous damage, 74% of the patients were of mixed race, 92% lived in northwestern Colombia, and Leishmania (Viannia) panamensis was identified as the causative agent in 58% of cases. Eighteen patients recovered with pentavalent antimonial. The importance of distinguishing DL from those other clinical presentations is based on the fact that disseminated, diffuse and post-kala-azar CL are very different in etiology, clinical manifestations and response to treatment and prognosis.

Treatment of Disseminated Leishmaniasis With Liposomal Amphotericin B.

BACKGROUND: Disseminated leishmaniasis (DL) is a severe and emerging form of American tegumentary leishmaniasis, associated primarily with infection by Leishmania brasiliensis. DL is defined by the presence of ≥10 mixed-type lesions such as inflammatory papules and ulcers, located in ≥2 body parts. Most patients have hundreds of lesions all over the body, and mucosal involvement is detected in up to 44% of cases. DL is a difficult to cure disease and pentavalent antimony (Sb(v)) is used as standard treatment, its highest dosage being 20 mg/kg/day, for 30 days. However, less than 25% of DL cases will be cured after standard therapy, and the majority of cases will require more than one course of Sb(v) for a cure. In this context, new therapies are needed that offer a higher cure rate and a better safety profile, with convenience in drug administration. METHODS: We have evaluated liposomal amphotericin B in 20 patients with DL in an open clinical trial. The total dose ranged from 17 to 37 mg/kg, used in 7 to 14 days of treatment. RESULTS: Cure rate at 3 months after therapy was 70%. One relapse was documented 4 months after treatment, producing a final cure rate of 65%. Although liposomal amphotericin B was considered well tolerated, mild adverse events were documented in 75% of the patients. CONCLUSIONS: Liposomal amphotericin B is an effective therapy for DL, with a higher final cure rate of 75% observed when used in a total dose above 30 mg/kg. CLINICAL TRIALS REGISTRATION: NCT02025491.

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.

Inflammation in disseminated lesions: an analysis of CD4+, CD20+, CD68+, CD31+ and vW+ cells in non-ulcerated lesions of disseminated leishmaniasis

Disseminated leishmaniasis (DL) differs from other clinical forms of the disease due to the presence of many non-ulcerated lesions (papules and nodules) in non-contiguous areas of the body. We describe the histopathology of DL non-ulcerated lesions and the presence of CD4-, CD20-, CD68-, CD31- and von Willebrand factor (vW)-positive cells in the inflamed area. We analysed eighteen biopsies from non-ulcerated lesions and quantified the inflamed areas and the expression of CD4, CD20, CD68, CD31 and vW using Image-Pro software (Media Cybernetics). Diffuse lymphoplasmacytic perivascular infiltrates were found in dermal skin. Inflammation was observed in 3-73% of the total biopsy area and showed a significant linear correlation with the number of vW+ vessels. The most common cells were CD68+ macrophages, CD20+ B-cells and CD4+ T-cells. A significant linear correlation between CD4+ and CD20+ cells and the size of the inflamed area was also found. Our findings show chronic inflammation in all DL non-ulcerated lesions predominantly formed by macrophages, plasmacytes and T and B-cells. As the inflamed area expanded, the number of granulomas and extent of the vascular framework increased. Thus, we demonstrate that vessels may have an important role in the clinical evolution of DL lesions.

Disseminated Leishmaniasis Caused by Leishmania tropica in a Puppy from Karaj, Central Iran.

A 5-month old puppy with muco-cutaneous lesions in the chin, around lips and eyes was examined physically and microscopically for leishmaniasis. Muco-cutaneous lesions containing a large number of amastigotes of Leishmania spp. were observed. Amastigotes were also detected in liver and spleen of the puppy. The animal was positive with Dipstick rK39 kit and high level of anti-Leishmania antibodies was detected by direct agglutination test (DAT). DNA, Using PCR-RFLP technique extracted from cultured Leishmania promastigotes and L. tropica was identified. This is the first report of concurrent mucosal and visceral involvement of L. tropica in a puppy from Iran.

Estudo clínico-molecular na leishmaniose mucocutânea: diagnóstico e rastreamento de subpopulações de leishmania (Viannia) braziliensis nos níveis inter e intrapacientes / Clinical and molecular study in mucocutaneous leishmaniasis: Diagnoses and screening of subpopulation of leishmania (Viannia) braziliensis in the inter and intrapatient levels

O presente estudo teve como principal objetivo avaliar a diversidade genética de Leishmania (Viannia) braziliensis nos níveis inter e intrapacientes, diretamente em lesões cutâneas e mucosas de indivíduos com leishmanioses mucocutânea (LMC), disseminada (LD) e mucosa (LM), incluindo indivíduos coinfectados pelo vírus da imunodeficiência humana (HIV). Um total de 61 amostras procedentes de 38 pacientes foi analisado pelas técnicas da reação em cadeia da polimerase (PCR), da reação em cadeia da polimerase com primer único em condições de baixa estringência (LSSP-PCR) e da análise fenética, tendo como alvo molecular a região variável do minicírculo do DNA do cinetoplasto (kDNA). Neste estudo, predominaram indivíduos do sexo masculino e com acometimento mucoso nasal. A presença de DNA do parasita foi evidenciada pela banda diagnóstica de 750 pb, em todas as amostras analisadas, possibilitando o diagnóstico específico. Na investigação do perfil genotípico de subpopulações de L. (V.) braziliensis, através da LSSP-PCR, foi revelado o polimorfismo genético intrafragmento traduzido como uma assinatura do kDNA do parasito para cada amostra. Assinaturas de kDNAs similares em amostras de paciente coletadas ao mesmo tempo (mucosa oral e nasal), e a divergência nos perfis genéticos em amostras coletadas em tempos diferentes na mesma localização (mucosa nasal) sugerem a clonalidade do inóculo inicial, como consequência da estrutura populacional clonal de Leishmania. No estudo da variabilidade genética de L. (V.) braziliensis nos níveis inter e intrapacientes foram evidenciadas similaridades genotípicas entre as amostras de lesões cutânea e mucosa intrapacientes. As análises fenética e estatística possibilitaram afirmar que a diversidade genética no nível intrapacientes é menor do que a observada entre os pacientes...

The present study has as its main objective to evaluate the genetic diversity of Leishmania(Viannia) braziliensis in the inter and intrapatient levels, directly from cutaneous and mucosallesions of individuals with mucocutaneous (MCL), disseminated (DL) and mucosal (ML)leishmaniasis, including individuals with the human immunodeficiency virus (HIV) infection.A total of 61 samples recovered from 38 patients was analyzed by the techniques ofpolymerase chain reaction (PCR), low-stringency single-specific-primer PCR (LSSP-PCR)and phenetic analysis, directed to the variable region of the kinetoplast DNA (kDNA)minicircles. In this study, male individuals with nasal mucosa involvement predominated. Thepresence of the parasite’s DNA was revealed by the diagnosis band of 750 bp, in all analyzedsamples, making the specific diagnosis possible. In the investigation of the genotypic profileof the subpopulations of L. (V.) braziliensis, through LSSP-PCR, it was revealed theintrafragment genetic polymorphism translated as a kDNA signature for each sample. SimilarkDNAs signatures in patient’s samples collected simultaneously (oral and nasal mucosa), andthe divergence in the genetic profiles in samples collected at different times on the samelocation (nasal mucosa) suggest the clonality of the initial inoculum, as a consequence of theclonal population structure of Leishmania. In the study of the genetic variability of L. (V.)braziliensis in the inter and intrapatient levels, genotypic similarities were observed amongthe cutaneous and mucosal lesions intrapatients...