Mucocutaneous lymph node syndrome or Kawasaki syndrome is very severe disease. The most common symptom includes a high fever which is probably due to inflammation of blood vessels so-called vasculitis. The characteristic statement is that the etiology of disease remains unknown and the origin is not clarified. Several months old babies to preschool children are the most often affected group of patients. It is relatively rare, modern and mystery disease in contemporary and industrial developed society. The first appearance of this disorder is in 20th century. The disorder was first described by well-known Tokyo origin pediatrician Tomisaku Kawasaki in Japan, who studied this disease very thoroughly. This rare disease is considered autoimmune in origin triggering by an infectious agent especially in those who are genetically predisposed. Specific and typical symptoms of Kawasaki disease include long term fever, conjunctivitis, erythema,lymphadenopathy, mucosal changes as red swollen lips and strawberry tongue and multiple rashes. Related cardiovascular complications should be pointed out especially coronary or other major arteries aneurysms and their ruptures, pericardial effusion, heart inflammations, coronary thrombosis, pericardial exudates, arrhythmias, or mitral valve disease. There is no specific test for identification of Kawasaki disease despite the contemporary technological and diagnostic options. So, the easiest way to establish diagnosis is to recognize typical symptoms, blood/urine/spinal fluid testing and then performing X-ray, electrocardiogram and echocardiogram. This disease has very low mortality, the short-term prognosis is excellent and relapse of symptoms is rare. In total, 337 children were diagnosed with Kawasaki disease and admitted to hospital during the evaluation period (2007-2015) in the Czech Republic. This is significantly lower incidence comparing to the other countries in the world. Diploma thesis "Kawasaki syndrome in contemporary society from a nurse´s point of view" was designed as theoretical with a supplement of short case study. The goal of presence of this case study is to better comprehend presented topic. The scope of problem is described from theoretical point of view in partial chapters of this diploma thesis. All the information mentioned in this thesis quotes verified sources, publications written by the Czech and foreign specialists in this particular field. The goal of the thesis was to describe, based on available literature, the issue of Kawasaki disease in children focused on specifics of nursing. All the information was searched in bibliographical issued writings, in databases, or on the Internet. Based on the goal determined in advance, scientific methods such as explanation, analysis, synthesis and demonstration of data were chosen for composing this diploma thesis. The output of theoretical work is to present complex view on the issue of Kawasaki syndrome particularly for non-medical professionals. It came out that the profession of nurse has its own irreplaceable place in the context of Kawasaki disease. As well as competences of nurse are essential. These competences are provided for improving quality of life of children with this unfamiliar and life-threatening disease.

Mon projet de thèse a consisté en l’identification et la caractérisation moléculaire et immunologique de patients présentant une susceptibilité accrue aux infections fongiques par Candida sp. dans le syndrome Mendélien de candidose cutanéo-muqueuse chronique (CCMC).La CCMC est caractérisée par des infections persistantes ou récurrentes de la peau, des ongles et des muqueuses par les champignons Candida, principalement C. albicans. Elle est fréquemment associée à d’autres infections opportunistes dans certaines immunodéficiences primaires ou acquises, ou bien elle peut être associée à un tableau auto-immun. La CCMC peut finalement être isolée (CCMCi) sans autre tableau clinique sévère: la plupart des cas rapportés sont sporadiques, mais il existe également des cas familiaux avec une hérédité mendélienne autosomique principalement dominante (AD) ou plus rarement récessive (AR).Basés sur les données de la littérature, qui démontrent un rôle majeur de l’immunité dépendante des IL-17s dans la résistance aux infections mucocutanées vis-à-vis de C. albicans et nos résultats récents, qui démontrent un défaut de cette immunité dans certaines immunodéficiences primaires associées à une CCMC [les syndromes AD-HIES et AR APS-1, ainsi que chez les patients déficients en CARD9, nous avons émis l’hypothèse que parmi les patients atteints de CCMCi, certains pourraient présenter un défaut génétique affectant spécifiquement l’immunité IL-17-dépendante. Au début de ma thèse, j’ai participé à l’identification des deux premières étiologies génétiques de la CCMCi : le défaut autosomique récessif (AR) complet en IL-17RA et autosomique dominant (AD) en IL-17F. Plus récemment, j’ai identifié la troisième et la plus fréquente étiologie génétique de la CCMC par l’identification de mutations gain de fonction dans le gène STAT1 suite à une approche explorant l’ensemble du génome (séquençage de l’ensemble des exons). Ces mutations engendrent une « hyper-réponse » aux interférons de type I et II et à l’IL-27 qui inhibent la différentiation des lymphocytes T sécréteurs d’IL-17, impliqués dans l’immunité mucocutanée vis-à-vis de C. albicans chez l’homme.En conclusion, nous avons identifié, en 2011, des trois premières étiologies génétiques de la CCMCi, avec les défauts AR en IL-17RA, AD en IL-17F et des mutations gain-de-fonction de STAT1, toutes associées à un défaut de l’immunité dépendante de l’IL-17. Des mutations gain-de-fonction de STAT1 représentent à ce jour la cause génétique la plus fréquente de la CCMCi avec au total 94 patients rapportés dans la littérature depuis 2011. Nous avons ainsi démontré que la CCMCi est une immunodéficience primaire, associée à un défaut de l’immunité réalisée par les IL-17s. Ces travaux ont des implications majeures dans le domaine immunologique avec la description et la caractérisation des mécanismes biologiques impliqués dans l’immunité protectrice spécifique de C. albicans et une meilleure compréhension des mécanismes physiopathologiques associés à une susceptibilité accrue aux infections fongiques, dans des conditions naturelles d’infection ; et dans le domaine médical, avec la possibilité de diagnostics moléculaires, un conseil génétique en cas de diagnostic positif, une meilleure prise en charge des patients. / My project consists in the molecular and immunological identification and characterization of patients with increased susceptibility to fungal infections with Candida sp. suffering from the Mendelian syndrome of chronic mucocutaneous candidiasis (CMC).CMC is characterized by persistent or recurrent infections of the skin, nails and mucosae by Candida fungi, especially C. albicans. CMC is frequently associated with other opportunistic infections in some acquired or primary immunodeficiencies, or can be associated with autoimmune disorders. Finally, CMC may be present as an isolated form (chronic mucocutaneous candidiasis disease or CMCD) without any other severe infectious or autoimmune clinical manifestation: most reported cases are sporadic, but there are also familial cases with autosomal dominant (AD) or recessive (AR) Mendelian inheritance.Based the literature, which demonstrated a major role of IL-17 cytokines in mucocutaneous immunity with C. albicans, and our recent results, which show an impairment of IL-17 immunity in some primary immunodeficiencies associated with CMC (AD-HIES syndrome, AR APS-1, and CARD9-deficient patients), we hypothesized that among CMCD patients, some might have a genetic defect affecting specifically the IL-17-dependent immunity.At the beginning of my PhD, I participated in the identification of the first two genetic etiologies of CMCD: complete AR IL-17RA and partial AD IL-17F deficiencies. More recently, I identified the third and most common genetic etiology of CMCD by identifying gain of function mutations in the STAT1 gene following an approach exploring the whole genome (sequencing of all exons). These mutations are responsible for a "hyper-response" to type I and II interferons and IL-27, which inhibit the differentiation of IL-17-producing T cells. Impaired IL-17 immunity results in reduced mucocutaneous defenses against C. albicans in humans. In conclusion, we have identified in 2011, the first three genetic etiologies of CMCD with AR IL-17RA and AD IL-17F deficiencies and gain-of-function STAT1 mutations, all associated with an impaired IL-17-dependent immunity. Gain-of-function STAT1 mutations represent the most frequent genetic cause of CMCD with a total of 94 patients reported in the literature since 2011. We have shown that CMCD is a primary immunodeficiency associated with inborn errors of IL-17 immunity. This work has important implications in the field of immunology with the description and characterization of the biological mechanisms involved in protective immunity specific to C. albicans and a better understanding of the pathophysiological mechanisms associated with increased susceptibility to fungal infections in natural conditions of infection, and in the medical field, with the possibility of molecular diagnostics, genetic counseling for a positive diagnosis, and a better follow-up of the patients.

A pele e as mucosas constituem as primeiras barreiras na defesa contra infecções e os macrófagos são componentes essenciais do sistema imune inato, importante neste aspecto. O envolvimento destas células pode ser verificado em grande percentual das imunodeficiências primárias. Desta forma, a avaliação da função fagocitária é de extrema relevância para o reconhecimento dos distúrbios imunológicos que acometem a pele. O objetivo do presente estudo foi avaliar a metodologia laboratorial para a detecção de defeitos funcionais dos fagócitos. Para isto foram estabelecidos os seguintes testes laboratoriais: Nitro Blue Tetrazolium (NBT), Dihidrorodamina (DHR), quimiotaxia, fagocitose e a aderência de S. aureus e C. albicans por citometria de fluxo (CF), além de morte intracelular de S. aureus e C. albicans (CF). Para verificar a integridade do sistema complemento realizou-se ensaios hemolíticos para as vias clássica e alternativa (CH50 e AP50). A metodologia proposta foi aplicada em indivíduos normais para a padronização dos testes. O burst oxidativo avaliado pelo teste da dihidrorodamina (DHR) foi aplicado em 101 indivíduos saudáveis e em paralelo, 50 indivíduos sadios para o teste do NBT. Os mesmos testes foram realizados em pacientes com Candidíase mucocutânea crônica (CMC) (n=9 ), Candidíase persistente (n=5), Suspeita de distúrbios de fagócitos (SDF) (n=14), Doença Granulomatosa Crônica (DGC)(n= 7) e portadores de DGC (n=5). A quimiotaxia foi padronizada em 34 controles para neutrófilos estimulados com Lipopolissacarídeo de E.Coli (LPS) e 5 com fungo Candida albicans. A técnica de fagocitose e aderência de patógenos foi padronizada com os mesmos estímulos (n=7 para fungos/n=5 para bactéria). Após a padronização, o ensaio foi aplicado em pacientes com candidíase persistente (n=5 para bactéria e n=5 para fungo) e em pacientes com CMC (n= 3 para bactéria e n=4 para fungo). Os ensaios de fagocitose e morte intracelular (capacidade bactericida e fungicida) foram padronizados em 18 indivíduos sadios para bactérias e os ensaios de morte intracelular para S. aureus foi aplicado em pacientes com CMC (n=5), com CP (n=6), com SDF (n =9) e com DGC (n=2), para os ensaios de fagocitose com morte intracelular para fungos foram utilizados 22 indivíduos saudáveis e após a padronização do ensaio foram aplicados em pacientes com CMC (n=8), pacientes com CP ( n= 7), pacientes com DGC (n=2) e indivíduos com SDF (n= 13) O ensaio de DHR foi padronizado e estabelecido em 80% de intensidade de fluorescência para células estimuladas com PMA e 15% de intensidade de fluorescência para células sem estímulo. Nos resultados do DHR encontrou-se diferença significativa no grupo de DGC (n=7)(P= 0,0001), no grupo de portadores (n=5)(P=0,0005) e no grupo de SDF (n=14)(P= 0,0053). O ensaio do DHR foi repetido após 24 horas da coleta (n=7), não se verificando alteração da resposta. A quimiotaxia mostrou diferença significativa entre C (n=4) vs SDF (n=3)(P=0,0001) e pacientes com CMC apresentaram redução da capacidade quimiotática para bactérias (n=3)e fungos (n= 4) com soro autólogo (P= 0,0246 e P=0,0109, respectivamente). Na fagocitose e aderência de bactérias inativadas ,os grupos de CMC, CP E SDF não mostraram diferenças significativas com bactérias não opsonizadas ou opsonizadas com soro AB e apresentaram menor índice de fagocitose (C x CMC)(P=0,0357) quando foram opsonizadas com soro autólogo. Na fagocitose e aderência de fungos inativados, controles e grupos de pacientes apresentaram resposta semelhante com fagocitose preservada. Os ensaios de morte intracelular para bactérias não opsonizadas houve menor expressão de fagocitose no grupo de C x SDF (P=0,0044). Na capacidade bactericida verificou-se diferença significativa entre os grupos CxCMC (P=0,0403). A opsonização das bactérias com soro AB foi significativamente diferente entre os grupos CxCP (P=0,0129) e CxSDF (P=0,0048) e com capacidade bactericida diferente entre grupos CxCP (P=0,0258) e CxSDF (P=0,0205). Na avaliação da fagocitose de bactérias opsonizadas com soro autólogo foi verificada diferença significativa entre os grupos CxCP (P=0,0013) e CxSDF (P=0,0048). Não houve diferença na capacidade bactericida dos grupos de pacientes com o controle. Os ensaios de fagocitose e morte intracelular para fungos sem opsonização não mostrou diferença estatisticamente significativa. A morte intracelular mostrou-se diferente para o grupo CxCMC (P=0,0155) e quando opsonizado com soro AB houve diferença CxCP (P=0,0369). A fagocitose com opsonização por soro autólogo significativa no grupo CxSDF (P=0,0001) e um paciente de CMC com sua fagocitose comprometida quando comparado com o controle do dia. A morte intracelular foi diferente nos grupos CxCMC (P=0,0018) e CxCP (p=0,0203). Não houve diferença estatisticamente significativa à avaliação do complemento. O ensaio do DHR mostrou ser sensível e preciso para o diagnóstico de DGC e portadores de DGC, porém pode detectar outras alterações de fagócitos. O ensaio de aderência e fagocitose mostraram-se variáveis dificultando a padronização de valores de normalidade e exclusão de defeitos. Ensaios de fagocitose com morte intracelular mostraram-se como a melhor forma de detectar distúrbios de fagócitos além do diagnóstico de DGC. A aplicação de controles do dia mostrou-se necessária e importante para a detecção de defeitos funcionais. O presente trabalho mostrou que a avaliação de distúrbios de fagócitos por morte intracelular por citometria de fluxo pode ser aplicado em outras situações clínicas com comprometimento imunológico / Skin and mucosa are part of the first barriers in the defense against infections, and the macrophages are essential components of the innate immune system, important when related to this aspect. The involvement of these cells can be seen in a large percentage of the primary immunodeficiencies. Therefore, the assessment of the phagocitary function is extremely important for the recognition of immunological disorders which affect the skin. The present study focus on the evaluation of the laboratorial methodology for the detection of functional defects of phagocytes. For this the following laboratorial tests were established: Nitro Blue Tetrazolium (NBT), chemotaxis, phagocytosis and adherence of S. aureus and C. albicans through flow cytometry (FC), besides the intracellular death of S. aureus and C. albicans (FC). To assess the integrity of the complement system hemolytic assays were performed for the classic and alternative pathways (CH50 and AP50). The proposed methodology was applied to normal individuals for the standardization of the assays. The oxidative burst evaluated through the dihydrorodamine essay (DHR) was applied to 101 healthy individuals and in parallel, 50 healthy individuals for the NBT assay. The same assays were performed on patients with Chronic mucocutaneous candidiasis (CMC)(n=9), persistent candidiasis (n=5), Phagocytes disorders suspicious (PDS) (n=14), Chronicle granulomatous disease (CGD)(n=7) and CGD carriers (n=5). Chemotaxis was standardized using 34 controls for neutrophils stimulated by lipopolisacharydes from e. coli (LPS) and 5 by C. albicans. Phagocytosis and adherence of pathogens were standardized using the same stimuli (n=7 for fungi and n=5 for bacteria). Following the standardization, the assay was applied to patients with persistent candidiasis (n=5 for fungi and n=5 for bacteria) and on patients with CMC (n=4 for fungi and n=3 for bacteria). Phagocytosis and intracellular death assays (bactericidal and fungicidal capacity) were standardized using 18 healthy individuals for bacteria and the intracellular death assays for S. aureus were applied on patients suffering from CMC (n=5), from PC (n=6), from PDS (n=9) and from CGD (n=2), for the phagocytosis with fungi intracellular death assays 22 healthy individuals were used, and following the standardization the assay was applied to patients suffering from CMC (n=8), from PC (n=7), from CGD (n=2) and PDS individuals (n=13). The DHR assay was standardized and established according to fluorescence intensity 80% for cells stimulated by PMA and fluorescence intensity 15% for cells without stimuli. In the DHR results a significant difference in the CGD group (n=7)(P= 0,0001), in the carriers group (n=5)(P=0,0005) and in the PDS group (n=14)(P= 0,0053) was found. The DHR assay was performed once again 24 hours after the sample collection (n=7) and no changes in the response were seen. Chemotaxis showed a significant difference between C (n=4) vs PDS (n=3)(P=0,0001) and patients suffering from CMC showed decreased ability in the chemotaxis of bacteria (n=3) and fungi (n=4) with autologous serum (P= 0,0246 e P=0,0109, respectively). In the phagocytosis and adherence of inactivated bacteria, the CMC, PC and PDS groups showed no significant differences with non-opsonizated bacteria or opsonizated with AB serum and presented a lower phagocytosis level (C x CMC)(P=0,0357) when they were opsonizated by autologous serum. In the phagocytosis and adherence of inactivated fungi, controls and patient groups presented a similar response with preserved phagocytosis. In the intracellular death assays for non-opsonizated bacteria there was a lower phagocytosis expression in the C x SDF group (P=0,0044). In the bactericidal ability a significant difference between the groups C x CMC was seen (P=0,0403). The opsonization of bacteria with AB serum showed a significant difference among the groups C x CP (P=0,0129) and C x SDF (P=0,0048) and with different bactericidal ability among the groups C x CP (P=0,0258) and C x SDF (P=0,0205). In the evaluation of the phagocytosis of bacteria opsonizated by autologous serum a significant difference among the groups C x CP (P=0,0013) and C x SDF (P=0,0048) was seen. There was no difference between the bactericidal ability of the patients group and control group. The phagocytosis and intracellular assays for fungi without opsonization presented no significant statistical difference. Intracellular death was different for the C x CMC group (P=0,0155) and when opsonizated by AB serum difference was shown C x CP (P=0,0369). The phagocytosis with opsonization by autologous serum presented significant difference in the C x SDF group (P=0,0001) and in a CMC patient with compromised phagocytosis when compared with the daily control. Intracellular death was different in the C x CMC (P=0,0018) and C x CP (p=0,0203) groups. There was no significant statistical difference according to the complement evaluation. The DHR assay was seen as very sensitive and precise for the diagnosis of CGD, however it can detect other phagocyte alterations. The phagocytosis and adherence assay varied a lot making the standardization of normal values and defects exclusion very difficult. Phagocytosis with intracellular death assays showed the best performance to detect phagocytes disorders besides CGD diagnosis. The use of daily controls was seen as very necessary and important to detect functional disorders. This study demonstrated that phagocytes disorder evaluation through intracellular death using flow cytometry can be applied to other clinical situations which are immunologically compromised

Em áreas endêmicas para leishmaniose visceral, a AIDS aumenta o risco de aparecimento desta doença de cem a mil vezes. Conhecem-se muitos estudos sobre leishmaniose visceral (LV) em HIV/AIDS no Mediterrâneo, mas, pouco sobre leishmaniose tegumentar (LT) em HIV/AIDS. No Brasil não se constatou, até o momento, um aumento de casos de co-infecção HIV-Leishmania, mas, isto pode ser conseqüente ao subdiagnóstico por várias causas incluindo o não conhecimento da apresentação clínica nesses casos. Pela prevalência maior da LT em relação à LV no Brasil, é de se supor que ocorram com freqüência casos de co-infecção com a forma tegumentar de leishmaniose no nosso meio. Como no Brasil não dispomos de estudos de descrição sistemática da apresentação clínica nos casos de co-infecção, um parâmetro importante visando o diagnóstico, realizou-se um estudo transversal e descritivo, durante um período de dois anos a partir de fevereiro de 2002, em dois centros de tratamento de doenças infecciosas em São Paulo. Partimos de uma população alvo de 113 pacientes com sorologia positiva para HIV, procedentes de área endêmica para leishmanioses, 69 com algum sintoma sugestivo de leishmaniose visceral ou tegumentar e 44 assintomáticos. Em dez pacientes foi diagnosticada leishmaniose por encontro de parasitos ou antígeno de Leishmania em tecidos, maioria (8/10) por busca ativa, sendo cinco casos de LV e cinco de LT. Baseados nesses casos, observamos que, o contato com área endêmica de leishmaniose mesmo por um período curto como duas semanas deva ser valorizado, principalmente se foi recente. Houve dificuldade no encontro de casos porque a leishmaniose não é considerado como diagnóstico diferencial de infecções intercorrentes nos casos de HIV/AIDS no nosso meio, houve presença concomitante de outras doenças oportunistas mais prevalentes e manifestações clínicas atípicas ou semelhantes a infecções oportunistas. xvi Os casos de LV apresentaram manifestações clássicas, enquanto que os casos de LT apresentaram lesões diversas, desde úlcera única até lesões múltiplas e polimórficas (placas infiltrativas, pápulas, nódulos, máculas), com tendência à disseminação atingindo mucosa e pele, incluindo área genital (em quatro dos cinco pacientes com LT). Em dois pacientes observaram-se aparecimento de lesões (num caso) e piora (noutro caso), após início da terapia antirretroviral altamente efetiva (HAART), com características sugestivas de síndrome inflamatória de recuperação imune. Quanto à classificação de HIV segundo CDC, oito pacientes eram C3 e dois B3 e todos apresentavam contagem de linfócitos T CD4+ menor que 200 células/?l. A sorologia para leishmaniose foi positiva em todos os casos de LV mas, positiva em três dos cinco casos de LT. O teste de Montenegro foi negativo nos dois casos de LV onde o teste foi realizado e positivo em todos os três pacientes de LT submetidos ao teste. No tratamento da leishmaniose, freqüentemente os pacientes apresentaram efeitos adversos aos medicamentos e obervou-se mortalidade alta (40%) e freqüência alta de recidiva nos pacientes sobreviventes (100% dos LT e 66% dos LV) / In endemic areas of visceral leishmaniasis (VL), AIDS increases the risk for VL in 100 to 1000 times. There are many studies on VL in HIV/AIDS patients from the Mediterranean basin but data on tegumentary leishmaniasis (TL) are scarce. In Brazil, no increase in the number of cases of HIV/Leishmania co-infection was reported so far, but it may be due to subdiagnosis owing to various reasons including ignorance on clinical features of the co-infected patients.. Since the prevalence of TL is greater than VL in Brazil, we presume that cutaneous or mucosal leishmaniasis would be frequent in cases of coinfection in our country. Since there are no studies on systematic description of clinical manifestations of HIV/Leishmania co-infection in Brazil, an important basis for diagnosis, study was done for a period of two years from February 2002 aiming description of clinical manifestations in a transversal study in two infectious disease reference centers in Sao Paulo city, Brazil. The target population was constituted by 113 HIV positive patients that reported some kind of contact with endemic area for leishmaniasis, 69 being with some symptoms suggestive of visceral or tegumentary leishmaniasis, and 44 asymptomatic. In ten patients diagnosis of leishmaniasis was established by the detection of amatigotes or Leishmania antigens in tissue samples, five presented VL and five TL. Eight of 10 were found by active search. Based in these cases, we noted that contact with endemic area, even for short time like two weeks has to be considered in the diagnosis, mainly if it occurred recently. The search for these cases were difficult because leishmaniasis is not considered within differential diagnosis ofopportunistic infections in HIV positive patients among physicians, and in addition more prevalent opportunistic infections were concomitantly present and the clinical features were atypical for leishmaniasis or similar to other opportunistic infections. Clinical manifestation of VL was classic, while that of TL was quite variable, ranging from single to multiple ulcers, with polimorphic xviii presentations (papules, nodules, infiltrative plaques, macules) that tended to disseminate to the other areas of skin and mucosa, including genital area (in four from five TL patients). In two patients appearance of lesions (in one case) and worsening of tegumentary manifestations (in another case) after introduction of highly active anti-retroviral therapy (HAART) were observed that were suggestive of the so called immune reconstitution inflammatory syndrome. Concerning CDC classification of HIV, eight patients were C3 and two B3, and all presented CD4+ T cell count lower than 200/?l. All the VL patients had anti-Leishmania antibody test positive, however in TL three of five cases of was positive. Leishmanin skin (Montenegro) test was negative in two cases of VL submitted to this test, and positive in all three TL patients evalued. Frequent toxicity and adverse events with treatments were observed, as well as high mortality (40%), and high frequency of relapses among survivors (100% of TL and 66% of VL cases)

Em áreas endêmicas para leishmaniose visceral, a AIDS aumenta o risco de aparecimento desta doença de cem a mil vezes. Conhecem-se muitos estudos sobre leishmaniose visceral (LV) em HIV/AIDS no Mediterrâneo, mas, pouco sobre leishmaniose tegumentar (LT) em HIV/AIDS. No Brasil não se constatou, até o momento, um aumento de casos de co-infecção HIV-Leishmania, mas, isto pode ser conseqüente ao subdiagnóstico por várias causas incluindo o não conhecimento da apresentação clínica nesses casos. Pela prevalência maior da LT em relação à LV no Brasil, é de se supor que ocorram com freqüência casos de co-infecção com a forma tegumentar de leishmaniose no nosso meio. Como no Brasil não dispomos de estudos de descrição sistemática da apresentação clínica nos casos de co-infecção, um parâmetro importante visando o diagnóstico, realizou-se um estudo transversal e descritivo, durante um período de dois anos a partir de fevereiro de 2002, em dois centros de tratamento de doenças infecciosas em São Paulo. Partimos de uma população alvo de 113 pacientes com sorologia positiva para HIV, procedentes de área endêmica para leishmanioses, 69 com algum sintoma sugestivo de leishmaniose visceral ou tegumentar e 44 assintomáticos. Em dez pacientes foi diagnosticada leishmaniose por encontro de parasitos ou antígeno de Leishmania em tecidos, maioria (8/10) por busca ativa, sendo cinco casos de LV e cinco de LT. Baseados nesses casos, observamos que, o contato com área endêmica de leishmaniose mesmo por um período curto como duas semanas deva ser valorizado, principalmente se foi recente. Houve dificuldade no encontro de casos porque a leishmaniose não é considerado como diagnóstico diferencial de infecções intercorrentes nos casos de HIV/AIDS no nosso meio, houve presença concomitante de outras doenças oportunistas mais prevalentes e manifestações clínicas atípicas ou semelhantes a infecções oportunistas. xvi Os casos de LV apresentaram manifestações clássicas, enquanto que os casos de LT apresentaram lesões diversas, desde úlcera única até lesões múltiplas e polimórficas (placas infiltrativas, pápulas, nódulos, máculas), com tendência à disseminação atingindo mucosa e pele, incluindo área genital (em quatro dos cinco pacientes com LT). Em dois pacientes observaram-se aparecimento de lesões (num caso) e piora (noutro caso), após início da terapia antirretroviral altamente efetiva (HAART), com características sugestivas de síndrome inflamatória de recuperação imune. Quanto à classificação de HIV segundo CDC, oito pacientes eram C3 e dois B3 e todos apresentavam contagem de linfócitos T CD4+ menor que 200 células/?l. A sorologia para leishmaniose foi positiva em todos os casos de LV mas, positiva em três dos cinco casos de LT. O teste de Montenegro foi negativo nos dois casos de LV onde o teste foi realizado e positivo em todos os três pacientes de LT submetidos ao teste. No tratamento da leishmaniose, freqüentemente os pacientes apresentaram efeitos adversos aos medicamentos e obervou-se mortalidade alta (40%) e freqüência alta de recidiva nos pacientes sobreviventes (100% dos LT e 66% dos LV) / In endemic areas of visceral leishmaniasis (VL), AIDS increases the risk for VL in 100 to 1000 times. There are many studies on VL in HIV/AIDS patients from the Mediterranean basin but data on tegumentary leishmaniasis (TL) are scarce. In Brazil, no increase in the number of cases of HIV/Leishmania co-infection was reported so far, but it may be due to subdiagnosis owing to various reasons including ignorance on clinical features of the co-infected patients.. Since the prevalence of TL is greater than VL in Brazil, we presume that cutaneous or mucosal leishmaniasis would be frequent in cases of coinfection in our country. Since there are no studies on systematic description of clinical manifestations of HIV/Leishmania co-infection in Brazil, an important basis for diagnosis, study was done for a period of two years from February 2002 aiming description of clinical manifestations in a transversal study in two infectious disease reference centers in Sao Paulo city, Brazil. The target population was constituted by 113 HIV positive patients that reported some kind of contact with endemic area for leishmaniasis, 69 being with some symptoms suggestive of visceral or tegumentary leishmaniasis, and 44 asymptomatic. In ten patients diagnosis of leishmaniasis was established by the detection of amatigotes or Leishmania antigens in tissue samples, five presented VL and five TL. Eight of 10 were found by active search. Based in these cases, we noted that contact with endemic area, even for short time like two weeks has to be considered in the diagnosis, mainly if it occurred recently. The search for these cases were difficult because leishmaniasis is not considered within differential diagnosis ofopportunistic infections in HIV positive patients among physicians, and in addition more prevalent opportunistic infections were concomitantly present and the clinical features were atypical for leishmaniasis or similar to other opportunistic infections. Clinical manifestation of VL was classic, while that of TL was quite variable, ranging from single to multiple ulcers, with polimorphic xviii presentations (papules, nodules, infiltrative plaques, macules) that tended to disseminate to the other areas of skin and mucosa, including genital area (in four from five TL patients). In two patients appearance of lesions (in one case) and worsening of tegumentary manifestations (in another case) after introduction of highly active anti-retroviral therapy (HAART) were observed that were suggestive of the so called immune reconstitution inflammatory syndrome. Concerning CDC classification of HIV, eight patients were C3 and two B3, and all presented CD4+ T cell count lower than 200/?l. All the VL patients had anti-Leishmania antibody test positive, however in TL three of five cases of was positive. Leishmanin skin (Montenegro) test was negative in two cases of VL submitted to this test, and positive in all three TL patients evalued. Frequent toxicity and adverse events with treatments were observed, as well as high mortality (40%), and high frequency of relapses among survivors (100% of TL and 66% of VL cases)

A pele e as mucosas constituem as primeiras barreiras na defesa contra infecções e os macrófagos são componentes essenciais do sistema imune inato, importante neste aspecto. O envolvimento destas células pode ser verificado em grande percentual das imunodeficiências primárias. Desta forma, a avaliação da função fagocitária é de extrema relevância para o reconhecimento dos distúrbios imunológicos que acometem a pele. O objetivo do presente estudo foi avaliar a metodologia laboratorial para a detecção de defeitos funcionais dos fagócitos. Para isto foram estabelecidos os seguintes testes laboratoriais: Nitro Blue Tetrazolium (NBT), Dihidrorodamina (DHR), quimiotaxia, fagocitose e a aderência de S. aureus e C. albicans por citometria de fluxo (CF), além de morte intracelular de S. aureus e C. albicans (CF). Para verificar a integridade do sistema complemento realizou-se ensaios hemolíticos para as vias clássica e alternativa (CH50 e AP50). A metodologia proposta foi aplicada em indivíduos normais para a padronização dos testes. O burst oxidativo avaliado pelo teste da dihidrorodamina (DHR) foi aplicado em 101 indivíduos saudáveis e em paralelo, 50 indivíduos sadios para o teste do NBT. Os mesmos testes foram realizados em pacientes com Candidíase mucocutânea crônica (CMC) (n=9 ), Candidíase persistente (n=5), Suspeita de distúrbios de fagócitos (SDF) (n=14), Doença Granulomatosa Crônica (DGC)(n= 7) e portadores de DGC (n=5). A quimiotaxia foi padronizada em 34 controles para neutrófilos estimulados com Lipopolissacarídeo de E.Coli (LPS) e 5 com fungo Candida albicans. A técnica de fagocitose e aderência de patógenos foi padronizada com os mesmos estímulos (n=7 para fungos/n=5 para bactéria). Após a padronização, o ensaio foi aplicado em pacientes com candidíase persistente (n=5 para bactéria e n=5 para fungo) e em pacientes com CMC (n= 3 para bactéria e n=4 para fungo). Os ensaios de fagocitose e morte intracelular (capacidade bactericida e fungicida) foram padronizados em 18 indivíduos sadios para bactérias e os ensaios de morte intracelular para S. aureus foi aplicado em pacientes com CMC (n=5), com CP (n=6), com SDF (n =9) e com DGC (n=2), para os ensaios de fagocitose com morte intracelular para fungos foram utilizados 22 indivíduos saudáveis e após a padronização do ensaio foram aplicados em pacientes com CMC (n=8), pacientes com CP ( n= 7), pacientes com DGC (n=2) e indivíduos com SDF (n= 13) O ensaio de DHR foi padronizado e estabelecido em 80% de intensidade de fluorescência para células estimuladas com PMA e 15% de intensidade de fluorescência para células sem estímulo. Nos resultados do DHR encontrou-se diferença significativa no grupo de DGC (n=7)(P= 0,0001), no grupo de portadores (n=5)(P=0,0005) e no grupo de SDF (n=14)(P= 0,0053). O ensaio do DHR foi repetido após 24 horas da coleta (n=7), não se verificando alteração da resposta. A quimiotaxia mostrou diferença significativa entre C (n=4) vs SDF (n=3)(P=0,0001) e pacientes com CMC apresentaram redução da capacidade quimiotática para bactérias (n=3)e fungos (n= 4) com soro autólogo (P= 0,0246 e P=0,0109, respectivamente). Na fagocitose e aderência de bactérias inativadas ,os grupos de CMC, CP E SDF não mostraram diferenças significativas com bactérias não opsonizadas ou opsonizadas com soro AB e apresentaram menor índice de fagocitose (C x CMC)(P=0,0357) quando foram opsonizadas com soro autólogo. Na fagocitose e aderência de fungos inativados, controles e grupos de pacientes apresentaram resposta semelhante com fagocitose preservada. Os ensaios de morte intracelular para bactérias não opsonizadas houve menor expressão de fagocitose no grupo de C x SDF (P=0,0044). Na capacidade bactericida verificou-se diferença significativa entre os grupos CxCMC (P=0,0403). A opsonização das bactérias com soro AB foi significativamente diferente entre os grupos CxCP (P=0,0129) e CxSDF (P=0,0048) e com capacidade bactericida diferente entre grupos CxCP (P=0,0258) e CxSDF (P=0,0205). Na avaliação da fagocitose de bactérias opsonizadas com soro autólogo foi verificada diferença significativa entre os grupos CxCP (P=0,0013) e CxSDF (P=0,0048). Não houve diferença na capacidade bactericida dos grupos de pacientes com o controle. Os ensaios de fagocitose e morte intracelular para fungos sem opsonização não mostrou diferença estatisticamente significativa. A morte intracelular mostrou-se diferente para o grupo CxCMC (P=0,0155) e quando opsonizado com soro AB houve diferença CxCP (P=0,0369). A fagocitose com opsonização por soro autólogo significativa no grupo CxSDF (P=0,0001) e um paciente de CMC com sua fagocitose comprometida quando comparado com o controle do dia. A morte intracelular foi diferente nos grupos CxCMC (P=0,0018) e CxCP (p=0,0203). Não houve diferença estatisticamente significativa à avaliação do complemento. O ensaio do DHR mostrou ser sensível e preciso para o diagnóstico de DGC e portadores de DGC, porém pode detectar outras alterações de fagócitos. O ensaio de aderência e fagocitose mostraram-se variáveis dificultando a padronização de valores de normalidade e exclusão de defeitos. Ensaios de fagocitose com morte intracelular mostraram-se como a melhor forma de detectar distúrbios de fagócitos além do diagnóstico de DGC. A aplicação de controles do dia mostrou-se necessária e importante para a detecção de defeitos funcionais. O presente trabalho mostrou que a avaliação de distúrbios de fagócitos por morte intracelular por citometria de fluxo pode ser aplicado em outras situações clínicas com comprometimento imunológico / Skin and mucosa are part of the first barriers in the defense against infections, and the macrophages are essential components of the innate immune system, important when related to this aspect. The involvement of these cells can be seen in a large percentage of the primary immunodeficiencies. Therefore, the assessment of the phagocitary function is extremely important for the recognition of immunological disorders which affect the skin. The present study focus on the evaluation of the laboratorial methodology for the detection of functional defects of phagocytes. For this the following laboratorial tests were established: Nitro Blue Tetrazolium (NBT), chemotaxis, phagocytosis and adherence of S. aureus and C. albicans through flow cytometry (FC), besides the intracellular death of S. aureus and C. albicans (FC). To assess the integrity of the complement system hemolytic assays were performed for the classic and alternative pathways (CH50 and AP50). The proposed methodology was applied to normal individuals for the standardization of the assays. The oxidative burst evaluated through the dihydrorodamine essay (DHR) was applied to 101 healthy individuals and in parallel, 50 healthy individuals for the NBT assay. The same assays were performed on patients with Chronic mucocutaneous candidiasis (CMC)(n=9), persistent candidiasis (n=5), Phagocytes disorders suspicious (PDS) (n=14), Chronicle granulomatous disease (CGD)(n=7) and CGD carriers (n=5). Chemotaxis was standardized using 34 controls for neutrophils stimulated by lipopolisacharydes from e. coli (LPS) and 5 by C. albicans. Phagocytosis and adherence of pathogens were standardized using the same stimuli (n=7 for fungi and n=5 for bacteria). Following the standardization, the assay was applied to patients with persistent candidiasis (n=5 for fungi and n=5 for bacteria) and on patients with CMC (n=4 for fungi and n=3 for bacteria). Phagocytosis and intracellular death assays (bactericidal and fungicidal capacity) were standardized using 18 healthy individuals for bacteria and the intracellular death assays for S. aureus were applied on patients suffering from CMC (n=5), from PC (n=6), from PDS (n=9) and from CGD (n=2), for the phagocytosis with fungi intracellular death assays 22 healthy individuals were used, and following the standardization the assay was applied to patients suffering from CMC (n=8), from PC (n=7), from CGD (n=2) and PDS individuals (n=13). The DHR assay was standardized and established according to fluorescence intensity 80% for cells stimulated by PMA and fluorescence intensity 15% for cells without stimuli. In the DHR results a significant difference in the CGD group (n=7)(P= 0,0001), in the carriers group (n=5)(P=0,0005) and in the PDS group (n=14)(P= 0,0053) was found. The DHR assay was performed once again 24 hours after the sample collection (n=7) and no changes in the response were seen. Chemotaxis showed a significant difference between C (n=4) vs PDS (n=3)(P=0,0001) and patients suffering from CMC showed decreased ability in the chemotaxis of bacteria (n=3) and fungi (n=4) with autologous serum (P= 0,0246 e P=0,0109, respectively). In the phagocytosis and adherence of inactivated bacteria, the CMC, PC and PDS groups showed no significant differences with non-opsonizated bacteria or opsonizated with AB serum and presented a lower phagocytosis level (C x CMC)(P=0,0357) when they were opsonizated by autologous serum. In the phagocytosis and adherence of inactivated fungi, controls and patient groups presented a similar response with preserved phagocytosis. In the intracellular death assays for non-opsonizated bacteria there was a lower phagocytosis expression in the C x SDF group (P=0,0044). In the bactericidal ability a significant difference between the groups C x CMC was seen (P=0,0403). The opsonization of bacteria with AB serum showed a significant difference among the groups C x CP (P=0,0129) and C x SDF (P=0,0048) and with different bactericidal ability among the groups C x CP (P=0,0258) and C x SDF (P=0,0205). In the evaluation of the phagocytosis of bacteria opsonizated by autologous serum a significant difference among the groups C x CP (P=0,0013) and C x SDF (P=0,0048) was seen. There was no difference between the bactericidal ability of the patients group and control group. The phagocytosis and intracellular assays for fungi without opsonization presented no significant statistical difference. Intracellular death was different for the C x CMC group (P=0,0155) and when opsonizated by AB serum difference was shown C x CP (P=0,0369). The phagocytosis with opsonization by autologous serum presented significant difference in the C x SDF group (P=0,0001) and in a CMC patient with compromised phagocytosis when compared with the daily control. Intracellular death was different in the C x CMC (P=0,0018) and C x CP (p=0,0203) groups. There was no significant statistical difference according to the complement evaluation. The DHR assay was seen as very sensitive and precise for the diagnosis of CGD, however it can detect other phagocyte alterations. The phagocytosis and adherence assay varied a lot making the standardization of normal values and defects exclusion very difficult. Phagocytosis with intracellular death assays showed the best performance to detect phagocytes disorders besides CGD diagnosis. The use of daily controls was seen as very necessary and important to detect functional disorders. This study demonstrated that phagocytes disorder evaluation through intracellular death using flow cytometry can be applied to other clinical situations which are immunologically compromised