Molecular biology in tropical dermatoses

São apresentados conceitos básicos sobre célula, código genético e síntese protéica, e sobre algumas técnicas de biologia molecular, tais como PCR, PCR-RFLP, seqüenciamento de DNA, RT-PCR e immunoblotting. São fornecidos protocolos de extração de nucleotídeos e de proteínas, como salting out no sangue periférico e métodos do fenol-clorofórmio e do trizol em tecidos. Seguem-se exemplos comentados da aplicação de técnicas de biologia molecular para o diagnóstico etiológico e pesquisa em dermatoses tropicais, com ênfase na leishmaniose tegumentar americana e hanseníase.Initially, basic concepts are presented concerning the cell, genetic code and protein synthesis, and some techniques of molecular biology, such as PCR, PCR-RFLP, DNA sequencing, RT-PCR and immunoblotting. Protocols of nucleotides and of proteins extraction are supplied, such as salting out in peripheral blood allied to phenol-chloroform and trizol methods in skin samples. To proceed, commented examples of application of those techniques of molecular biology for the etiologic diagnosis and for research in tropical dermatoses, with emphasis to American tegumentary leishmaniasis and leprosy are presented.USP - FAEPA-HCFMRPCNPqFapes

Pênfigo foliáceo endêmico (Fogo selvagem): casuística da região nordeste do Estado de São Paulo, Brasil, 1973-1998

Endemic Pemphigus Foliaceus (EPF) is a bullous autoimmune skin disease whose incidence used to be high in the State of São Paulo (SP), Brazil, during the forties, but has declined thereafter. OBJECTIVES: to report a series of EPF patients from the northeastern region of SP. METHODS: a retrospective study concerning demographic and epidemiological data of patients seen from 1973 to 1998 was conducted at the University Hospital, Faculty of Medicine of Ribeirão Preto, SP. RESULTS: bullous disease was diagnosed in 340 patients, 245 with EPF (72.1%), 9.4 cases per year, 60.4% females, and 70.2% white, 7 to 82 year-old (29.4% in their teens); 46.9% lived in the rural zone. Concerning profession, housewives predominated among women (67.6%) and agricultural workers among men (40.2%). The time of disease was less than 1 year in 62.0% of cases, followed by 1 and 5 years (27%), and more than 5 years for the remaining patients (11%). 36.7% of patients were referred by the Direção Regional de Saúde (DIR) XVIII of Ribeirão Preto, with the largest number of cases being from Ribeirão Preto and Batatais: 33.3% and 23.3%, respectively; 22% from DIR XIII (Franca); 13.5% from DIR VII (Araraquara); 2.9% from DIR IX (Barretos); 4.1% from other DIRs of SP, and 20.8% from other States (16.7% from Minas Gerais). Thirteen (5.3%) patients reported occurrence of the disease in some relative, and 4 (1.6%) in neighbors. CONCLUSIONS: the present data characterize the northeastern region of the state of São Paulo as a remaining endemic focus of EPF.FUNDAMENTOS: O pênfigo foliáceo endêmico (PFE) é doença bolhosa auto-imune, em que o Estado de São Paulo (SP) destacou-se por sua alta incidência na década de 1940, a partir da qual é descrito seu declínio. OBJETIVOS: relatar casuística do PFE na região nordeste de SP. MÉTODO: levantamento de prontuários de pacientes com doenças bolhosas, Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 1973 a 1998, anotando-se aspectos demográficos e epidemiológicos. RESULTADOS: Do total de 340 pacientes com doença bolhosa, em 245 foi diagnosticado PFE (72,1%), 9,4 casos/ano. Desses, 60,4% eram mulheres e 70,2%, brancos, de 7 a 82 anos de idade (29,4% entre 10 e 19 anos); 46,9% eram de zona rural. Predominou a profissão doméstica entre as mulheres (67,6%), e lavrador entre os homens (40,2%). Em 62,0%, a duração da doença foi menor que 1 ano; em 27,0%, entre 1 e 5 anos, e, em 11,0%, maior que 5 anos. 36,7% eram procedentes da Direção Regional de Saúde (DIR) XVIII de Ribeirão Preto, sendo que os municípios de Batatais e de Ribeirão Preto apresentaram maior número de casos: 33,3% e 23,3%, respectivamente; 22,0% da DIR XIII (Franca); 13,5% da DIR VII (Araraquara); 2,9% da DIR IX (Barretos); 4,1% de outras DIRs de SP, e 20,8% de outros Estados (16,7% de MG). Treze (5,3%) pacientes relataram PFE em algum familiar, e 4 (1,6%), em vizinhos. CONCLUSÕES: Os dados confirmam a região nordeste de SP como foco endêmico remanescente de PFE

DNA Sequencing Confirms the Involvement of Leishmania (L.) Amazonensis in American Tegumentary Leishmaniasis in the State of São Paulo, Brazil

INTRODUCTION: American tegumentary leishmaniasis (ATL) represents one of the most important public health issues in the world. An increased number of autochthonous cases of ATL in the Northeastern region of São Paulo State has been documented in the last few years, leading to a desire to determine the Leishmania species implicated. METHODS: PCR followed by DNA sequencing was carried out to identify a 120bp fragment from the universal kDNA minicircle of the genus Leishmania in 61 skin or mucosal biopsies from patients with ATL. RESULTS: DNA sequencing permitted the identification of a particular 15bp fragment (5' …GTC TTT GGG GCA AGT... 3') in all samples. Analysis by the neighbor-joining method showed the occurrence of two distinct groups related to the genus Viannia (V) and Leishmania (L), each with two subgroups. Autochthonous cases with identity to a special Leishmania sequence not referenced in Genbank predominated in subgroup V.1, suggesting the possible existence of a subtype or mutation of Leishmania Viannia in this region. In the subgroup L.2, which showed identity with a known sequence of L. (L.) amazonensis, there was a balanced distribution of autochthonous and non-autochthonous cases, including the mucosal and mucocutaneus forms in four patients. The last observation may direct us to new concepts, since the mucosal compromising has commonly been attributed to L. (V.) braziliensis, even though L. (L.) amazonensis is more frequent in the Amazonian region. CONCLUSIONS: These results confirm the pattern of distribution and possible mutations of these species, as well as the change in the clinical form presentation of ATL in the São Paulo State

Clinico-immunological spectrum of American tegumentary leishmaniasis and leprosy coinfection: A case series in Southeastern Brazil

INTRODUCTION: American tegumentary leishmaniasis (ATL) and leprosy share common areas of prevalence, but reports of coinfection are scarce. METHODS: We report a series of 9 ATL-leprosy cases and discuss the association. An integrative diagram to analyze the clinico-immunological features of coinfection with both diseases. RESULTS: Nine patients with leishmaniasis (5 cutaneous, 3 mucocutaneous, 1 disseminated case) exhibited concurrent infection with distinct clinical forms of leprosy. Our diagram-based analysis evidenced a divergent clinico-immunological spectrum for each disease in 8 out of 9 cases. CONCLUSIONS: The spectrum of ATL-leprosy comorbidity suggests that the host has a specific immune response against each pathogen

Comparison between microsatellites and Ml MntH gene as targets to identify Mycobacterium leprae by PCR in leprosy

FUNDAMENTOS: PCR tem sido frequentemente utilizada no diagnóstico molecular da hanseníase. OBJETIVOS: comparar os resultados da PCR com 4 pares de primers específicos para Mycobacterium leprae, bem como os resultados da PCR à classificação operacional, segundo a OMS, de multibacilar (MB) e paucibacilar (PB) da hanseníase. MÉTODO: Vinte e oito amostras de DNA, extraído de biópsias congeladas de pele e de imprint de biópsias em papel de filtro de 23 pacientes (14 MB e 9 PB), foram utilizadas na PCR com primers que amplificam 131pb, 151pb e 168pb de regiões de microssatélites, e um fragmento de 336pb do gene Ml MntH (ML2098) do bacilo. RESULTADOS: O bacilo pôde ser detectado em 22 (78,6%) das 28 amostras. Nove (45%) das 20 amostras de biópsia e 6 (75%) das 8 amostras de imprints foram positivas para TTC. Sete (35,5%) amostras de biópsias e 5 (62,5%) imprints foram positivos para AGT, e 11 (55%) biópsias e 4 (50%) imprints foram positivos para AT. Oito (38%) amostras de biópsias e 5 (62,5%) imprints foram positivos para o gene Ml MntH. Dentre o grupo MB, os microssatélites detectaram o bacilo em 78,5% das amostras, e o gene Ml MntH, em 57,1% das amostras, independentemente do material clínico. No grupo PB, 55,5% das amostras foram positivas para os microssatélites, enquanto que 22,2% o foram para o gene Ml MntH. CONCLUSÕES: Estes resultados mostram que, tanto as regiões específicas de microssatélites quanto o gene Ml MntH, podem representar ferramentas úteis na detecção do Ml MntH por PCR em amostras de biópsias e imprint de biópsiasBACKGROUND: The Polymerase Chain Reaction (PCR) technique has been frequently used in the molecular diagnosis of leprosy. OBJECTIVES: To compare the results of PCR with four pairs of Mycobacterium leprae specific primers as well as to compare these results to multibacillary (MB) and paucibacillary (PB) leprosy according to the WHO operational classification. METHOD: 28 DNA samples, collected from the frozen skin biopsies and biopsy imprints on filter paper of 23 patients (14 MB and PB 9), were examined for PCR using primers which amplify 131, 151 and 168bp of specific microsatellite regions and a 336 fragment of the Ml MntH (ML2098) gene. RESULTS: M.leprae bacillus could be detected in 22 (78.6%) of the 28 samples. 9 (45%) of the 20 biopsy samples and 6 (75%) of the 8 imprints were positive to TTC. 7 (35.5%) skin biopsy specimens and 5 (62.5%) imprints were positive to AGT, and 11 (55%) biopsies and 4 (50%) were positive to AGT. 11 (55%) skin biopsies and 4 (50%) imprints were positive to AT. 8(38%) skin biopsies and 5 (62.5%) imprints were positive to the Ml MntH gene. In the MB group, the microsatellites detected the bacillus in 78.5% of the samples, and the Ml MntH gene in 57.1% of the samples, independent of the clinical material. In the PB group 55.5% of samples were positive to the microsatellite primers, while 22.2% were positive to the Ml MntH gene. CONCLUSIONS: These results show that both the specific regions of microsatellites, as well as the Ml MntH gene fragment can be useful tools for detecting the M. leprae DNA by PCR in frozen skin biopsy samples and filter paper biopsy imprintsCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)(FAEPA) Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo (USP) - Fundação de Apoio ao Ensino, Pesquisa e Assistênci

Glucocorticoid Sensitivity and Proinflammatory Cytokines Pattern in Pemphigus

Glucocorticoids (GC) represent the main treatment for pemphigus; however, some patients show GC resistance. GC sensitivity was evaluated in 19 pemphigus patients and 41 controls by the number of binding sites [B-max (fmol/mg protein)] and the affinity of GC receptor [Kd (nM)] to dexamethasone (DEX) as well as by the pattern of cytokine by DEX-mediated inhibition of concanavalin-A (Con-A)-stimulated PBMC proliferation. The Kd (15.7 +/- 2.8 vs.8.1 +/- 1.3) and Bmax (6.5 +/- 0.9 vs. 3.9 +/- 0.3) were higher in pemphigus than controls (p = 0.002). Considering the values above the 95th percentile of normal group as a cut-off (K-d > 24.9 nM and B-max > 8.1 fmol/mg protein), elevated K-d and B-max were observed in 9.8% and 2.4% of controls and 15.8% and 36.8% of patients (p = 0.02). PBMC proliferation was stimulated by Con-A and inhibited by DEX (p < 0.001) in both pemphigus and control groups. IL-6 and TNF alpha (pg/mL) basal production were higher in patients than controls. There was an increment of these cytokines after Con-A stimulation, and they were inhibited by DEX (p = 0.002) in controls and remained elevated in pemphigus (p < 0.02). Patients and controls showed no difference in basal and stimulated production of IL-8 and IL-10. There is an alteration on GC sensitivity in pemphigus patients and a higher production of proinflammatory cytokines. Therefore, in pemphigus patients, proinflammatory cytokines might be involved in the mechanism of GC resistance and/or in its maintenance.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [03/09857-0]CNPq [151925/2007-0]CNP

Tzanck smears: an old but useful diagnostic tool

A citologia de Tzanck consiste em método simples e confiável para o diagnóstico de dermatoses virais, parasitárias, autoimunes e tumorais. Para exemplificar o emprego do teste de Tzanck em lesões vésico-bolhosas, um caso de pênfigo vulgar é descrito, cuja citologia e imunofluorescência direta, utilizando-se esfregaço coletado de lesão oral, confirmaram o diagnóstico de forma rápida e fácil.Tzanck smears can be used for the diagnosis of cutaneous infections, autoimmune disorders and cutaneous tumors. To exemplify its use, we report a case of a female patient with pemphigus vulgaris whose diagnosis was confirmed by positive direct immunofluorescence and conventional cytology using smears obtained by scraping the cheek mucosa.(FAEPA) Fundação de Apoio ao Ensino, Pesquisa e Assistência(FAEPA) Hospital das Clínicas - FMRP-US

Sensibilidade de teste imunoenzimático anti-L. braziliensis em relação a outros testes utilizados no diagnóstico da leishmaniose tegumentar americana

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.O diagnóstico da Leishmaniose Tegumentar Americana (LTA) é feito com base nos dados clínicos e epidemiológicos e é confirmado por meio de diferentes métodos laboratoriais. Dentre estes, são utilizados, com desempenhos variáveis, a reação de imunofluorescência indireta (IFI), a reação intradérmica de Montenegro (IRM), a pesquisa de Leishmania em material de biópsia de pele ou mucosa e a reação em cadeia da polimerase (PCR). O objetivo do presente trabalho foi avaliar o desempenho de teste imunoenzimático (ELISA) como método alternativo de diagnóstico da LTA, comparando seus resultados com os obtidos por outros métodos tradicionais de diagnóstico dessa doença. Foi utilizado teste para pesquisa de IgG anti-Leishmania no soro, utilizando antígeno bruto de Leishmania braziliensis, com os seguintes resultados de sensibilidade: ELISA = 85%; PCR = 81%; IRM = 64.4%; IFI = 58,1%; presença de parasitas na biópsia = 34%. Além disso, o teste ELISA foi positivo em parcela expressiva dos pacientes que apresentavam outros testes diagnósticos negativos (foi positivo em 100% dos pacientes com PCR negativo; em 84,8% dos casos com biópsias mostrando ausência de parasitas e em 75% dos não reativos a IRM) mostrando-se útil como método alternativo de diagnóstico da LTA