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Background. Cytokines and growth factors involved in the tissue inflammatory process influence the outcome of Leishmania infection. Insulin-like growth factor (IGF-I) constitutively present in the skin may participate in the inflammatory process and parasite-host interaction. Previous work has shown that preincubation of Leishmania (Leishmania) amazonensis with recombinant IGF-I induces accelerated lesion development. However, in human cutaneous leishmaniasis (CL) pathogenesis, it is more relevant to the persistent inflammatory process than progressive parasite proliferation. In this context, we aimed to investigate whether IGF-I was present in the CL lesions and if this factor may influence the lesions’ development acting on parasite growth and/or on the inflammatory/healing process. Methodology. Fifty-one CL patients’ skin lesion samples from endemic area of L. (Viannia) braziliensis infection were submitted to histopathological analysis and searched for Leishmania and IGF-I expression by immunohistochemistry. Results. In human CL lesions, IGF-I was observed preferentially in the late lesion (more than 90 days), and the percentage of positive area for IGF-I was positively correlated with duration of illness ( r = 0.42 , P < 0.05 ). IGF-I was highly expressed in the inflammatory infiltrate of CL lesions from patients evolving with good response to therapy ( 2.8 % ± 2.1 % ; median = 2.1 % ; n = 18 ) than poor responders ( 1.3 % ± 1.1 % ; median: 1.05%; n = 6 ; P < 0.05 ). Conclusions. It is the first time that IGF-I was detected in lesions of infectious cutaneous disease, specifically in American tegumentary leishmaniasis. IGF-I was related to chronicity and good response to treatment. We may relate this finding to the efficient anti-inflammatory response and the known action of IGF-I in wound repair. The present data highlight the importance of searching nonspecific factors besides adaptive immune elements in the study of leishmaniasis’ pathogenesis.

Infection of humans with Leishmania braziliensis typically results in localized cutaneous leishmaniasis (LCL). Rarely, after months or years of apparent clinical cure, some patients develop the destructive mucosal leishmaniasis (ML). ML results from L. braziliensis dissemination, probably via phagocytic cells. As the preferred cells for Leishmania spp. colonization, macrophages are critical to infection control, and may contribute to parasite dissemination. However, the host factors that determine this outcome are unknown. Matrix metalloproteinase 9 (MMP-9) is known to be important for immune cell migration, macrophage recruitment, and effective granuloma formation. Moreover, MMP-9 has been involved in Mycobacterium tuberculosis dissemination. Here, we demonstrate that in vitro infection of human macrophages with L. braziliensis increased the secretion and activation of MMP-9. We also demonstrate that macrophages from healthy cured individuals with previous history of ML had increased MMP-9 activity compared to LCL cured individuals. These findings may represent a fundamental difference in host innate immunity that could contribute to the clinical leishmaniasis presentation.

The diagnosis of visceral leishmaniasis (VL) generally requires the use of invasive tests for the collection of infected tissue (aspirates of bone marrow, spleen, liver or lymph nodes). This difficulty has led to the search for safer and less painful techniques to confirm the occurrence of the disease in children. Polymerase chain reaction (PCR) is a method that is advantageous in that it allows the use of peripheral blood samples for diagnosis. This paper reports the utilisation of PCR on peripheral blood samples to diagnose VL in 45 children in Mato Grosso do Sul, Brazil. This technique is compared with methods carried out using tissue collected by invasive procedures, including direct microscopy, culture and detection of Leishmania DNA by PCR in bone marrow aspirates. The results show that PCR of peripheral blood provides great sensitivity (95.6%) that is similar to that from the PCR of bone marrow aspirates (91.1%) and higher than that achieved with microscopy (80%) or culture (26.7%) methods. PCR of peripheral blood proved to be a suitable tool for the diagnosis of VL in children because it is highly sensitive and safe, with tissue collection being less invasive than in traditional tests.

Introducción. La reacción a la prueba cutánea de Montenegro (a leishmanina) y la lesión de leishmaniasis comparten características fundamentales de la reacción de hipersensibilidad de tipo retardado. Objetivos. Determinar si la respuesta cutánea a leishmanina se aproxima y podría modelar la respuesta inflamatoria e inmune temprana a la infección por Leishmania. Materiales y métodos. Este estudio comparó la respuesta inflamatoria de biopsias de lesiones agudas (tiempo de evolución £1 mes) y lesiones crónicas (tiempo de evolución ³6 meses) con su respectiva reacción a la prueba cutánea de Montenegro, y con la reacción a la prueba de Montenegro de individuos infectados asintomáticos. Resultados. La proporción de linfocitos T CD4+ y CD8+, células fagocíticas mononucleares y granulocitos fue similar entre lesiones agudas y sus reacciones a la prueba cutánea a leishmanina. En contraste, los linfocitos CD4+ (32,6%) representaron una proporción significativamente mas baja, y los linfocitos B (20%) y macrófagos (27%) una proporción significativamente más alta del infiltrado celular en lesiones crónicas que en sus correspondientes reacciones a la prueba cutánea de Montenegro (CD4+: 43,7%, linfocitos B: 0.9%; macrófagos: 17,5%). Se encontró una asociación positiva entre linfocitos T CD8+ y macrófagos ( P=0,038) en la reacción a la prueba cutánea de Montenegro de los individuos con infección asintomática, mientras que los linfocitos T CD8+ y CD4+ estuvieron asociados positivamente en las reacciones a la prueba cutánea de Montenegro de pacientes crónicos ( P=0,002). Adicionalmente, la proporción de linfocitos B en lesiones crónicas (20%) fue más alta que en lesiones agudas (5,3%) ( P=0,002). Conclusión. La reacción cutánea de Montenegro permitió diferenciar la respuesta celular inmune entre infección asintomática y enfermedad crónica, y simula la respuesta temprana a la infección.

The utility of 2 polymerase chain reaction (PCR)-based assays amplifying genus or Viannia subgenus Leishmania minicircle kDNA for the diagnostics of ML was assessed. The Viannia subgenus product was yielded after PCR from isolates of L. (Viannia) braziliensis, L. (Viannia) colombiensis, and L. (Viannia) guyanensis, whereas no product was obtained with the non-Viannia-pertaining species: L. (Leishmania) amazonensis, L. (Leishmania) donovani, and L. (Leishmania) chagasi. With both assays, 11 of 13 (86.4%) patients with confirmed ML could be identified, whereas only 2 (16.7%) of these patients were positive by microscopy. All amplified genus-specific products gave a positive signal by hybridization with a Leishmania (Viannia) subgenus-specific radioactive probe. The Viannia subgenus-specific kDNA PCR represents a sensitive and specific tool for the diagnosis of ML, remarkably improving the sensitivity of parasitological methods and offering an alternative for the radioactive-dependent assays for subgenus characterization.

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Eosinophilic panniculitis may be induced by several triggering factors and this syndrome is described here following the intramuscular injection of antimonial compounds for treatment of American muco-cutaneous leishmaniasis. Three patients, treated with intramuscular injections of pentavalent antimony (Glucantime®) were studied through clinical examination, histopathology and immunologic methods. The three patients developed deep infiltrated plaques at the site of antimony injection. Histopathology disclosed a prominent inflammatory infiltrate of the subcutaneous fat with numerous eosinophils. Immunologic studies could not detect immunoglobulins or complement fractions in the lesions. The final diagnosis was eosinophilic panniculitis occurring as a side-effect of antimonial therapy. The pathogenesis was not clear and it is suggested that the disease could be induced by a physical factor such as pressure or an immunological reaction to antimony.

The inflammasome is a multiprotein signalling platform involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated gene and protein expression of the inflammasome molecules AIM2 and NLRP3 in active lesions from patients with L. (V.) braziliensis-associated tegumentary leishmaniasis (TL) and correlated these findings with the clinical presentations and responses to therapy. Real-time PCR assays showed a significantly higher AIM2 gene expression in mucosal leishmaniasis (ML) compared with that in cutaneous leishmaniasis (CL). Additionally, AIM2 mRNA expression was significantly higher in lesions from poor responders than in lesions from good responders. In situ protein quantification analyses revealed greater AIM2 expression in ML lesions than in CL lesions. The percentage of AIM2-producing cells was higher in poor responders than in good responders. Although not quite significant, IL-1β+ cells were slightly more prominent in poor responders than in good responders. Similar results were observed when patients were evaluated according to clinical form. GP63 immunostaining was identified in all samples, but no significant variation between mucosal and cutaneous lesions was observed. GP63 could be associated with reduced NLRP3 inflammasome expression in CL and ML patients. Taken together, these data demonstrate that AIM2 is an important component of the inflammasome in TL patients and is directly associated with the severity of lesions.

Response to treatment with antimonial drugs varies considerably depending on the parasite strain involved, immune status of the patient and clinical form of the disease. Therapeutic regimens with this first line drug have been frequently modified both, in dose and duration of therapy. A regimen of 20 mg/kg/day of pentavalent antimony (Sb5+) during four weeks without an upper limit on the daily dose is currently recommended for mucosal disease ("espundia"). Side-effects with this dose are more marked in elderly patients, more commonly affected by this form of leishmaniasis. According to our experience, leishmaniasis in Rio de Janeiro responds well to antimony and, in cutaneous disease, high cure rates are obtained with 5 mg/kg/day of Sb5+ during 30 to 45-days. In this study a high rate of cure (91.4%) employing this dose was achieved in 36 patients with mild disease in this same geographic region. Side-effects were reduced and no antimony refractoriness was noted with subsequent use of larger dose in patients that failed to respond to initial schedule. A resposta de pacientes com leishmaniose ao tratamento antimonial pode variar em função de fatores como a cepa do parasito envolvido, estado imunológico do paciente e a forma clínica da doença. Os esquemas terapêuticos com antimônio pentavalente (Sb5+) têm sido frequentemente modificados quanto à dose e duração da terapia. Os efeitos colaterais observados com o uso de 20 mg/kg/dia de Sb5+ durante quatro semanas (dose recomendada para o tratamento da forma mucosa) são mais acentuados em pacientes idosos, os mais afetados pela forma mucosa (espundia) da doença. Nossa experiência demonstra que a forma cutânea ocorrendo no Rio de Janeiro responde muito bem ao tratamento com a dose de 5 mg/kg/dia de Sb5+ durante 30 ou 45 dias. Neste estudo foi obtida elevada eficácia (91,4%) com o mesmo regime terapêutico em 36 pacientes com espundia (que evoluem, contudo, com lesão crônica menos destrutiva do que o observado em outras áreas endêmicas) procedentes desta região. Os efeitos colaterais foram reduzidos, não havendo também qualquer resistência ao emprego de doses maiores nos casos que não responderam ao tratamento inicial.

Purpose To describe American cutaneous leishmaniasis of the eyelids and highlight the main clinical and diagnostic features of lesions, which are rare in this location. Design Retrospective, noncomparative case series Methods Leishmanin skin test, touch preparations, histopathologic analysis, and culture in appropriate media were used for clinical confirmation and parasitologic diagnosis. Positive cultures were identified by the iso-enzymes technique. All patients were treated with pentavalent antimony applied intramuscularly. Results Leishmanin skin test was positive in all five patients. Touch preparations, histopathologic analysis, and culture were performed in four patients. Touch preparations were positive (presence of Leishman's bodies) in two patients; histopathologic analysis showed a granulomatous infiltrate in four patients and parasite was present in two patients; culture was positive in three patients, and in two the parasite was identified as Leishmania ( Viannia ) braziliensis . Therapy was effective for all patients. Conclusions Cutaneous leishmaniasis of the eyelids is uncommon in the Americas. The disease may present diagnostic difficulties when appearing in nonendemic areas. The clues for diagnosis are the clinical aspect of lesions, the epidemiologic data, and a positive Leishmanin skin test. Demonstration of parasite is not always possible. Pentavalent antimonial compounds are the therapy of choice. Formerly, transmission of leishmaniasis occurred only when humans penetrated forested areas and became an incidental host. Now, eyelid lesions are part of the changing pattern in the transmission of the disease. With the increase in ecotourism, these lesions may begin to be seen in air travelers returning to other parts of the world.

In Brazil, the most common etiological agent of American tegumentary leishmaniasis is Leishmania ( Viannia ) braziliensis . In general, diagnostic techniques envisage the visualization of the parasite, but that technique has a low sensitivity. The main purpose of the present work was to evaluate the PCR as a routine tool for the diagnosis of leishmaniasis. Biopsy specimens from cutaneous or mucosal lesions were taken from 230 individuals from areas where Leishmania is endemic: 216 patients who had a clinical picture suggestive ofl leishmaniasis and 14 individuals with cutaneous lesions due to other causes. Each specimen was processed for histopathologic examination, culture, touch preparation, and DNA isolation. Oligonucleotides that amplify the conserved region of the minicircle molecules of Leishmania were used in a hot-start PCR. While at least one conventional technique was positive for Leishmania for 62% (134 of 216) of the patients, PCR coupled to hybridization was positive for 94% (203 of 216) of the patients. The 14 patients whose clinical picture was not suggestive of leishmaniasis had negative results by all techniques. The impact of the PCR was striking in mucosal disease. While the disease in only 17% (4 of 24) of the patients could be diagnosed by conventional techniques, PCR was positive for 71% (17 of 24) of the patients. Hybridization showed that all cases of disease were caused by parasites belonging to the Viannia subgenus. Altogether, the results indicate that PCR is a valuable tool for the diagnosis of leishmaniasis on a routine basis and is likely to provide valuable epidemiological information about the disease in countries where it is endemic.

Background Leishmaniasis recidiva cutis (LRC) is rare in New World leishmaniasis. Only seven cases have been reported so far. Materials and methods Four cases are reported here. Parasite diagnosis was performed by classical methods of touch preparations, histopathologic sections, and cultures. In addition, the detection of parasite DNA by the polymerase chain reaction (PCR) was performed in all cases. Results Parasites were detected by at least one of the classical methods in all primary lesions; however, only the PCR was positive in the recidivant lesions. Discussion LRC cases most likely represent a reactivation of an initial infection, probably due to the persistence of parasites in scarred tissue. Although lupoid leishmaniasis (LL) has been used as a synonym of LRC, a clear difference between LRC and LL can be defined as LL is the initial clinical presentation while LRC is a recurrent lesion. Conclusions The results indicate that it is not appropriate to use these two denominations as synonyms. The designation of LRC should be maintained in order to define recidives occurring at the border of an old scar of cutaneous leishmaniasis, avoiding the confusion with the lupoid form of the disease.

Leishmaniasis is an endemic disease in developing countries. The efficacy of therapy is usually evaluated through clinical parameters. To define the parasitologic cure, 20 patients were biopsied before and 1 month to 8 years after treatment. Paraffin-embedded tissue was used for DNA isolation. All patients had a positive polymerase chain reaction (PCR) result before therapy, except 1, for whom no histopathologic material was available. The causative agent was identified as belonging to the Leishmania (Viannia) subgenus by hybridization. Despite clinical healing and absence of reactivation or development of mucosal lesions, PCR was positive in scars of 16 patients (80%). The results suggest that parasites persist in the skin for many years despite treatment. Depending on specific pathogenetic features of the parasite and the immune status of the host, this phenomenon might result in mucosal lesions. Alternatively, it could have a role in the maintenance of immunologic memory in patients living in areas in which leishmaniasis is endemic.

A partir de relato de diagnóstico de leishmaniose visceral em cinco cães dos municípios de Santa Maria, Itaara e Júlio de Castilhos, no centro do Estado do Rio Grande do Sul, foi realizado levantamento sorológico através de teste de imunofluorescência indireta em sangue obtido em papel de filtro de 40 cães de Santa Maria, 20 de Itaara e 11 de Júlio de Castilhos, em geral relacionados aos cinco cães considerados como positivos. Além destes, foi também examinado o sangue de 44 cães vadios de Júlio de Castilhos, 68 de Cachoeira do Sul e 20 de Caçapava do Sul. Soros fracamente reagentes com títulos < 1:40 foram avaliados em ensaios de Ensaio Imuno-enzimático. Amostras de fígado, baço, pulmão e linfonodos de um sexto cão de Santa Maria, considerado como positivo à necropsia, foram examinadas através de histopatologia e PCR. Todos os resultados dos 204 cães foram negativos, indicando que, apesar de não ser impossível a ocorrência de leishmaniose visceral na região, por vários fatores aqui discutidos, ela deve ser pelo menos extremamente rara, sendo necessários estudos mais detalhados do material dos cinco cães tidos como positivos.

Immunological, parasitological, and molecular techniques were applied to blood samples of dogs to diagnose Leishmania infections. In 1997, 644 domestic dogs were studied. Peripheral blood samples were collected for serological diagnosis and detection of Leishmania parasite by polymerase chain reaction (PCR). The indirect immunofluorescence test was positive in 139 (21.6%) of 644 dogs examined. The PCR was performed in 70 blood samples and 3 bone marrow aspirates. A 120-bp fragment specific for Leishmania was present in PCR hybridization analysis of all seropositive samples in the molecular assays. The PCR hybridization test, which used a minicircle of Leishmania chagasi as a probe, was negative in 20 seronegative dogs. These results suggest that a combined PCR-Southern hybridization technique is a highly sensitive approach to diagnose leishmaniasis in dogs, which are a zoonotic reservoir of leishmaniasis for humans.

Summary Cutaneous lesions caused by Leishmania braziliensis infection occasionally heal spontaneously, but with antimonials therapy heal rapidly in approximately 3 weeks. However, about 15% of the cases require several courses of therapy. Matrix metalloproteinase-2 (MMP-2) and MMP-9 are gelatinases that have been implicated in other chronic cutaneous diseases and skin re-epithelialization. These enzymes are controlled by their natural inhibitors [tissue inhibitors of metalloproteinase (TIMPs)] and by some cytokines. Uncontrolled gelatinase activity may result in intense tissue degradation and, consequently, poorly healing wounds. The present study correlates gelatinase activity to therapeutic failure of cutaneous leishmaniasis (CL) lesions. Our results demonstrate an association between gelatinase activity and increased numbers of cells making interferon (IFN)-γ, interleukin (IL)-10 and transforming growth factor (TGF)-β in lesions from poor responders. Conversely, high levels of MMP-2 mRNA and enhanced MMP-2 : TIMP-2 ratios were associated with a satisfactory response to antimonials treatment. Additionally, high gelatinolytic activity was found in the wound beds, necrotic areas in the dermis and within some granulomatous infiltrates. These results indicate the importance of gelatinase activity in the skin lesions caused by CL. Thus, we hypothesize that the immune response profile may be responsible for the gelatinase activity pattern and may ultimately influence the persistence or cure of CL lesions.

Background American tegumentary leishmaniasis (ATL) caused by Leishmania (Viannia) braziliensis is endemic in Rio de Janeiro State (RJ), where the disease shows epidemiologic and clinical characteristics distinct from those of ATL in other Brazilian regions. Paraty is the second most important endemic area in RJ; however, reports on leishmaniasis in this region refer to the occurrence of the disease without describing its characteristics. Methods The clinical features of 71 cases of ATL reported between 1991 and 1997 in Paraty are presented. Thirty patients were re-evaluated 10 years later. Results Males and females were affected in similar proportions, and the disease was more prevalent in patients aged between 10 and 49 years (63.4%). Cutaneous leishmaniasis was the most prevalent clinical form observed. Unique lesions were present in 69% of cases, 91.6% of which displayed an ulcerated aspect. Although mucosal leishmaniasis was not observed, severe clinical manifestations, such as disseminated cutaneous lesions caused by L. braziliensis, were diagnosed in two patients. These patients presented skin lesions with different clinical aspects spread throughout the body, as well as low cellular immune responses. Montenegro skin test (92% positivity) and serology (8% IgM and 56% IgG anti-Leishmania positive results) were the most utilized tests for supporting the diagnosis of leishmaniasis. Parasites, detected in 27 of the 33 cases analyzed, were characterized as L. braziliensis. Conclusion ATL in Paraty shares the clinical and laboratory characteristics reported for ATL in other regions of RJ, probably because of the similar epidemiologic context related to the Atlantic rainforest region.

Epidermal changes from 32 cutaneous and 3 mucosal American cutaneous leishmaniasis (ACL) active lesions were studied for HLA-DR, -DQ and -DP expression, Langerhans cells and lymphocyte infiltration. In addition to a DR and DQ positivity at the surface of the cells of the inflammatory infiltrate, a strong reaction for DR antigens was detected on keratinocytes. Hyperplasia of Langerhans cells was present in all cutaneous lesions and epidermis was infiltrated by T lymphocytes. When healed lesions of 14 of these subjects were re-biopsied 1 to 12 months after the end of pentavalent antimonial therapy, MHC class II antigens could no longer be seen on keratinocytes. Our data represent evidence for the reversibility of the abnormal HLA-DR expression by keratinocytes in ACL after Glucantime therapy or spontaneous scar formation, demonstrating that this expression is restricted to the period of active lesions. The present findings can be regarded as an indirect evidence that keratinocytes may be involved in the immunopathology of ACL.

SUMMARY A limiting dilution analysis (LDA) was utilized to estimate the frequency of L. braziliensis braziliensis reactive T cells (Lbb-T cells) in peripheral blood and in the lesions of patients with mild localized cutaneous leishmaniasis (LCL) or with severe mucosal leishmaniasis (MCL). The frequencies of Lbb-T cells in peripheral blood varied from 1:107 300 to 1:3587 and were not significantly different in MCL and LCL patients. However, a significant difference was encountered (P

The aim of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) in the detection of Leishmania DNA in archived Giemsa-stained bone marrow slides for diagnosis of visceral leishmaniasis (VL), and to compare PCR with conventional diagnostic techniques, like direct microscopy and parasite culture. Specimens of archived Giemsa-stained bone marrow slides from 91 patients with VL and from 79 controls with other diseases or conditions were studied. PCR showed the highest sensitivity (92.3%) and had good specificity (97.5%). Direct examination detected 79.1% and culture 59% of positive samples. In addition, PCR was able to detect VL in 16 of 19 patients (84.2%) with negative microscopy. PCR in Giemsa-stained bone marrow slides is a suitable tool for confirming diagnosis in patients with VL and may be useful in the diagnosis of difficult cases. Slide smears are easily stored, do not require special storage conditions such as low temperatures, and can be easily mailed to centers where PCR is available, making it an excellent option for diagnosis in the field.

BackgroundInterferon-gamma is a key cytokine in the protective responses against intracellular pathogens. A single nucleotide polymorphism (SNP) located in the first intron of the human IFN-γ gene can putatively influence the secretion of cytokine with an impact on infection outcome as demonstrated for tuberculosis and other complex diseases. Our aim was to investigate the putative association of IFNG+874T/A SNP with American tegumentary leishmaniasis (ATL) and also the influence of this SNP in the secretion of IFN-γin vitro.MethodsBrazilian ATL patients (78 cutaneous, CL, and 58 mucosal leishmaniasis, ML) and 609 healthy volunteers were evaluated. The genotype of +874 region in the IFN-γ gene was carried out by Amplification Refractory Mutational System (ARMS-PCR).Leishmania-induced IFN-γ production on peripheral blood mononuclear cell (PBMC) culture supernatants was assessed by ELISA.ResultsThere are no differences between +874T/A SNP frequency in cases and controls or in ML versus CL patients. Cutaneous leishmaniasis cases exhibiting AA genotype produced lower levels of IFN-γ than TA/TT genotypes. In mucosal cases, high and low IFN-γ producers were clearly demonstrated but no differences in the cytokine production was observed among the IFNG +874T or A carriers.ConclusionOur results suggest that +874T/A polymorphism was not associated with either susceptibility or severity to leishmaniasis. Despite this, IFNG +874T/A SNP could be involved in the pathogenesis of leishmaniasis by influencing the amount of cytokine released by CL patients, although it could not prevent disease development. On the other hand, it is possible that in ML cases, other potential polymorphic regulatory genes such as TNF-α and IL-10 are also involved thus interfering with IFN-γ secretion.

One of the potential dangers of American tegumentary leishmaniasis (ATL) caused by Leishmania (Viannia) braziliensis is the development of mucosal lesions. Haematogenous dissemination of the parasite is the most likely mechanism to explain this occurrence, but most attempts to isolate the parasite from blood have so far been unsuccessful. The presence of Leishmania in peripheral blood was therefore evaluated by PCR using DNA samples isolated from patients presenting active cutaneous or mucosal disease, and from individuals cured by antimonial treatment as well as individuals without a past history of leishmaniasis but with a positive Montenegro skin test, all living in L. (V.) braziliensis-endemic areas. Leishmania DNA was found not only in those patients presenting active cutaneous (24.8%) or mucosal (35%) lesions, but also in samples isolated from healed individuals (27.3%) as well as in asymptomatic skin-test-positive residents of endemic areas (37.5%). Overall, PCR showed the presence of parasite DNA in the blood of 26.2% of the 225 examined samples. These data suggest that persistence of parasites within the host may last for many years and, rather than being a risk factor, might be important in maintaining the protective response in those living in endemic areas.

Seventeen patients proceeding from the municipality of Rio de Janeiro, Brazil presenting with the cutaneous ulcerative form of American leishmaniasis were treated with one ampoule of pentavalent antimony daily for 30 days. With this regimen the individuals doses varies greatly: from 3.8 mg/kg of body weight to 22.3 mg/kg. After five years, patients receiving either a smaller dose or a bigger one, showed the same therapeutic result: cutaneous scars and no mucosal lesions.

The present studies on infections withLeishmania(Viannia)braziliensisin rhesus macaques were made to characterize the evolution of different parasite strains and the immune responses they elicited in this experimental host. A standardized inoculum of promastigotes was injected intradermally either above the eyelid or on the forearm of each monkey. Sixteen infected monkeys developed longstanding infections which lasted until the end of the observation period (33 months). The time required for lesion development was very variable, not only for the isolates showing molecular differences but also for individual animals in groups infected with the same parasite strain. The inocula produced lesions of variable severity, ranging from localized cutaneous leishmaniasis (CL) with a tendency to spontaneous healing to non-healing disease. One infected animal developed persistent metastatic skin and mucosal lesions. Anti-Leishmaniaantibodies and parasite-specific T-cell responses were induced by the experimental infections. As the granulomatous inflammatory response found at the lesions inL. (V.)braziliensis-infectedM. mulattawas similar to that in patients with CL, this primate model could be useful for studying the pathophysiology and immunoregulatory events associated with disease evolution, as well as for the evaluation of new drugs or candidate vaccines.

A long-term evaluation of human American tegumentary leishmaniasis patients was conducted to detect immunological and/or parasitological indicators associated with cure or protection against leishmaniasis. Cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) patients from endemic areas of Leishmania braziliensis infection in Brazil were studied during the active disease, at the end of therapy, and up to 10 years after the end of therapy. For immunological studies, lymphocyte proliferative responses, phenotypic characterization of CD4+ and CD8+ T cells reactive to L. braziliensis and cytokine production in vitro were assayed. In CL, with its tendency for healing lesions, at or shortly after the completion of therapy the ratio of CD4+ to CD8+ T cells was approximately one and production of interferon γ (IFNγ) remainded roughly constant. In ML, these apparently beneficial CD4 + CD8 + ratios and cytokine patterns appeared later. The long-term memory T cell responses were associated with preferential induction of CD4+ subpopulations and IFNγ production that probably led to protection against relapses or reinfection. Deoxyribonucleic acid (DNA) was isolated from peripheral blood and oligonucleotides that amplify the conserved region of the minicircle molecules of Leishmania were used in a ‘hot-start’ polymerase chain reaction (PCR). Leishmania DNA was found in about one-quarter of the patients with active disease as well as in individuals who had received chemotherapy. The PCR was also positive in one-third of the individuals with a positive skin test but no past or present history of leishmaniasis. The well-modulated T cell response leading to long-term protection observed in CL patients could result from a favourable host genetic background and/or a particular parasite genotype, leading to a beneficial T cell immune response even in the presence of parasite antigens. The possibility of parasite persistence after clinical cure suggests that the immune response can control, but not fully eliminate, the infection. It could prevent the parasite from causing disease, maintaining a leishmanial antigen-specific response and hampering reinfection.

The efficacy of an antimony regimen at the dose of 20 mg/kg/day for a 3-4-week period is well established in the treatment of American cutaneous leishmaniasis. Several drug side effects, however, have been described and the search for more suitable regimens is advisable. In the present paper, the effect of a low dose (5 mg/kg/day for 30 days) of antimony was evaluated in 159 individuals from endemic regions of Rio de Janeiro State, Brazil, an area of Leishmania (V.) braziliensis transmission. Patients presented typical cutaneous lesions and parasites were demonstrated in all cases. One hundred forty-three patients were available for evaluation and of these, 120 (84%) were cured by the end of therapy. Twenty-three patients (16%) were considered treatment failures. Side effects were observed in only six patients (4%). Extensive follow-up (up to 10 years) disclosed no relapses or mucosal lesions. The results show that a low dose of antimony is less toxic, more appropriate, especially in children and elderly people, and has the same final result as that obtained with larger doses.

Background The drug of choice for leishmaniasis is pentavalent antimony and different regimens are under continuous evaluation. The ideal therapy should be simple, effective, and with no or minor side-effects, in this paper we have studied the efficacy of intralesionally applied antimony in New World cutaneous leishmaniasis. Methods Seventy-four patients from Rio de Janeiro state, Brazil, and presenting with single ulcerative cutaneous lesions mainly located on the trunk or extremities were enrolled in the study. The drug employed was N-methyl glucamine (425 mg of Sbv in each 5 ml ampoule). Each lesion was infiltrated with the drug at the four cardinal points in order to achieve complete blanching. Results Of the 74 patients, 59 (80%) were healed after a 12–week interval. Extensive follow-up (up to 10 years) disclosed no relapses or the development of mucosal lesions. Conclusions The aim of therapy in New World cutaneous leishmaniasis is the healing of the cutaneous lesion and the prevention of late mucosal damage. Both conditions were achieved with the treatment employed with no side-effects and a considerable decrease in costs. In addition, the method is easy to apply in the field.

The host response to infection appears to be regulated by specific patterns of local cytokine production. In the mouse, resistance to many pathogens including Leishmania is associated with a THl cytokine profile, IL-2 and IFN-,y; whereas susceptibility to infection is associated with production of TH2 cytokines, IL4, IL-5, and IL-10. To determine the cytokine patterns of the local immune response to Leishmania infection in humans, we used the polymerase chain reaction to compare cytokine mRNAs in biopsy specimens of American cutaneous leishmaniasis. In localized cutaneous leishmaniasis and the Montenegro delayed-type hypersensitivity reaction, type 1 cytokine mRNAs such as IL-2, IFN-y, and lymphotoxin were relatively predominant. In the chronic and destructive mucocutaneous form of leishmaniasis, there was a mixture of type 1 and type 2 cytokines, with a striking abundance ofIL4mRNA in lesions. These results suggest that clinical course ofinfection with Leishmania braziliensis in man is associated with specific local patterns of cytokine production. (J. Clin. Invest. 1993.

The majority of T cells bear the T-cell receptor (TCR) alpha beta complex which recognizes foreign antigen peptides only in the context of self major histocompatibility complex (MHC) molecules. Such T cells function in a variety of effector roles and secrete cytokines that mediate the activation and differentiation of other cells in the immune system. Recently, a small subpopulation T cells was found to bear a distinct TCR composed of gamma and delta subunits. In man, TCR gamma delta+ cells are distributed as approximately 5 per cent of the CD3+ cells in all organized lymphoid organs as well as in the skin- and gut-associated lymphoid tissues. Although a limited number of germ-line genes encode the TCR gamma and delta subunits, extensive junctional variation particularly in the delta gene, results in unprecedented diversity for this receptor. The nature of the specificity and immunological functions of these T cells remains enigmatic. We report here that in contrast to the normal low frequency of gamma delta-bearing cells in lymphoid tissues, peripheral blood, or normal skin, the frequency is increased five to eightfold in particular granulomatous reactions of leprosy. TCR gamma delta+ lymphocyte lines from these leprosy skin lesions proliferate in vitro specifically to mycobacterial antigens. This reactivity to foreign antigens appears to require presentation in the context of self-molecules. Moreover, culture supernatants from activated gamma delta T lymphocytes induce adhesion and aggregation of bone-marrow monocytes in the presence of granulocyte monocyte-colony stimulating factor (CSF), suggesting that products of gamma delta-bearing T cells may play a role in the immune response, possibly by stimulating granuloma formation.

From July 1984 to September 1986, 105 cases of American cutaneous leishmaniasis were studied in a locality closely situated to an urbanized area of the city of Rio de Janeiro, Brazil. Settement in this area was established at least 20 years ago but the first cases were noted six months prior to the beginning of this study. Cases were almost exlusively cutaneous and ulcerated, with one to six months of evolution. Montenegro's skin tests were positive in all cases and anti-Leishmania antibodies were detected by indirect immunofluorescence test in 74.3% of the patients. Parasites were demonstrated in 69.5% of cases. Domestic animals were easily found infected; 32% of the examined dogs and 30.8% of the examined equines were positive to the presence of Leishmania in cutaneous ulcerated lesions. Parasite isolates from human, dog andequines were immunologically characterized and identified as L. b. braziliensis. 73,0% of the sandfly population were Lutzomyia intermedia mainly caught on human baits and on domestic animals. Our observations suggest that this is an area of recent established L. b. braziliensis infection and that transmission probably occurs indoors or outdoors close to the houses. De julho de 1984 a setembro de 1986, 105 casos de leishmaniose cutânea foram estudados numa localidade situada na imediata vizinhança da área urbanizada da região metropolitana da cidade do Rio de Janeiro. A ocupação do sítio deu-se aproximadamente há 20 anos, mas os primeiros casos foram registrados somente seis meses antes do início de nosso estudo. Os casos eram quase que exclusivamente cutâneos, da forma clínica ulcerada com um e seis meses de evolução. O teste de Montenegro foi positivo em todos os casos e anticorpos antileishmania foram detectados por imunofluorescência indireta em 74,3% dos pacientes. A demonstração do parasito foi obtida em 69,5%. Animais domésticos infectados foram facilmente encontrados: 32% dos cães examinados e 30,8% dos equinos mostravam presença de leishmânia em lesões ulceradas. Parasitos isolados, tanto de casos humanos como de cães e equinos, foram imulogicamente caracterizados e identificados com L. b. braziliensis. Da população de flebotomíneos encontrados 73% eram de Lutzomyia intermedia capturados principalmente com iscas humanas e de animais domésticos. Nossas observações sugerem que esta é uma área de estabelecimento recente da infecção por L. b. braziliensis e que a transmissão ocorre provavelmente tanto no peridomicílio como no interior das habitações.

Four mongrel dogs were intradermically inoculated with 3 x 10**6 Leishmania braziliensis braziliensis promastigotes. Three out of the four animals developed cutaneous lesions respectively 4, 7, and 8 months after. The fourth dog did not develop lesion at the inoculation site, but a mucosal ulcer was seen 16 months after the inoculum. Clinical, histopathological, and serological findings were similar to what is found in natural canine infection as well as in the human disease. These results suggest that dogs may be an useful model for L. b. braziliensis infection.Quatro cães foram inoculados por via intredérmica com 3 x 10**6 promastigotos de Leishmania brasiliensis braziliensis. Três deles desenvolveram lesões no sítio do inóculo, mas uma úlcera mucosa foi detectada 16 meses após o inóculo. Os achados foram similares áqueles verificados na infecção canina natural, bem como na doença humana. Estes resultados sugerem que o cão pode representar um modelo experimental para a infecção causada pela L. b. brasiliensis.