Detection Of Bartonella Henselae Dna In Clinical Samples Including Peripheral Blood Of Immune Competent And Immune Compromised Patients By Three Nested Amplifications [detecção De Dna De Bartonella Henselae Em Amostras Clínicas, Incluindo Sangue Periférico, De Pacientes Imunocompetentes E Imunodeprimidos Por Meio De Três Amplificações Duplas]

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. 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Usefulness of matrix-assisted laser desorption ionisation–time-of-flight mass spectrometry for identifying clinical Trichosporon isolates

AbstractTrichosporon spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for therapeutic management, but conventional identification based on biochemical traits is hindered by the lack of updates to the species databases provided by the different commercial systems. In this study, 93 strains, or isolates, belonging to 16 Trichosporon species were subjected to both molecular identification using IGS1 gene sequencing and matrix-assisted laser desorption ionisation–time-of-flight (MALDI–TOF) analysis. Our results confirmed the limits of biochemical systems for identifying Trichosporon species, because only 27 (36%) of the isolates were correctly identified using them. Different protein extraction procedures were evaluated, revealing that incubation for 30 min with 70% formic acid yields the spectra with the highest scores. Among the six different reference spectra databases that were tested, a specific one composed of 18 reference strains plus seven clinical isolates allowed the correct identification of 67 of the 68 clinical isolates (98.5%). Although until recently it has been less widely applied to the basidiomycetous fungi, MALDI–TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level

Obtenção de exoantígenos de Histoplasma capsulatum em meio de neopeptona, glicose, tiamina e asparagina (NGTA) Histoplasma capsulatum exocellular antigens. Obtention in neopeptone, glucose, thiamine and asparagine medium (NGTA)

O presente trabalho teve como objetivo a produção de exoantígenos H e M das amostras 58, B-679, A-811 e O187 de Histoplasma capsulatum, utilizando o meio NGTA (neopeptona, glicose, tiamina e asparagina) em períodos de cultivo de 1, 2 e 3 meses, a 36ºC, sob agitação constante (50 v.p.m.). Os antígenos brutos foram avaliados contra anti-soro e antígeno de Histoplasma capsulatum de referência (Center for Disease Control), 4 soros de pacientes portadores de paracoccidioidomicose, 7 de histoplasmose e soro hiperimune anti-H. capsulatum produzido em coelhos, através da reação de imunodifusão dupla. Verificou-se que, com exceção de B-679 com 1 mês de crescimento, todos os demais exoantígenos apresentaram as frações H e M de precipitação. Os exoantígenos obtidos de A-811 apresentaram só a banda H. Excetuando-se os exoantígenos 58 e B-679 com 1 mês de crescimento, todos os demais exoantígenos reagiram contra soros de pacientes com histoplasmose. Em relação aos soros de pacientes com paracoccidioidomicose, somente os exoantígenos 58 e O187 não apresentaram reação cruzada. Todos os exoantígenos reagiram frente ao soro hiperimune de coelho anti-H. capsulatum. Para obtenção de exoantígenos de H. capsulatum, sugerimos que as amostras sejam cultivadas sob as condições anteriormente descritas, adotando-se o período de 3 meses de crescimento, utilizando-se exoantígenos de referência como controles da reação.<br>The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1,2 and 3 months, at 36ºC and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta - USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoc-cidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them

Gallium-67 imaging in a patient with paracoccidioidomycosis: a case report Pesquisa de corpo inteiro com gálio-67 em uma paciente com paracoccidioidomicose: relato de caso

A 26 year-old female was admitted with abdominal pain, fever and weight loss. The clinical and laboratory investigations led to the diagnosis of paracoccidioidomycosis. Gallium-67 whole body images correlated well with the clinical course of the disease and with the patient's prognosis.<br>Paciente do sexo feminino de 26 anos foi internada com dor abdominal, febre e emagrecimento. A investigação clínico-laboratorial estabeleceu o diagnóstico de paracoccidioidomicose. Os achados cintilográficos com citrato de gálio-67 correlacionaram-se com o curso clínico da paciente

Paracoccidioides cerebriformis Moore, 1935. MYCOLOGIC AND IMMUNOCHEMICAL STUDY

The present study concern on mycologic and immunochemical data obtained from two samples of a fungus considered as belonging to the species Paracoccidioides cerebriformis described by Moore in 1935, and maintained since then on Sabouraud’s agar in the mycology collection of the Instituto de Medicina Tropical de São Paulo. After 60 years, the samples exhibited the same characteristics described by Moore (1935). However, experimental lesions did not resulted in guinea-pigs inoculated intratesticularly. The dominant antigen in Paracoccidioides brasiliensis, 43 kDa glicoprotein (gp43), could not be demonstrated by SDS PAGE and Western blotting. Immunoelectrophoresis did not demonstrated the E arch of cathodic migration using a policlonal anti gp43 serum. According to these findings, it is concluded that the fungus described by Moore (1935) as P. cerebriformis does not belong to the genus Paracoccidioides. Paracoccidioidomycosis should therefore be considered as resulting from infection by a single species, Paracoccidioides brasiliensis (Splendore, 1912) as asserted by Almeida (1930). Further studies, through molecular biology methods, could identify the mentioned fungus<br>Paracoccidioides cerebriformis Moore, 1935. Estudo micológico e imunoquímico O presente estudo trata dos resultados obtidos, do ponto de vista micológico e imunoquímico, de duas amostras de Paracoccidioides consideradas como pertencentes à espécie cerebriformis, criada por MOORE in 1935 e mantidas desde aquela época, através de repiques em ágar-Sabouraud, na Micoteca do Instituto de Medicina Tropical de São Paulo. Após cerca de 60 anos, tais amostras conservavam as mesmas características descritas por MOORE (1935). Não foram registradas lesões experimentais em cobaios inoculados por via intratesticular, não se demonstrando, também, pelas técnicas de SDS PAGE e Western blotting, o antígeno dominante do Paracoccidioides brasiliensis, representado pela glicoproteína de 43 kDa (gp43). A imunoeletroforese também não revelou o arco E de migração catódica, utilizando-se um soro policlonal anti-gp43. Em vista desses resultados, concluímos que Paracoccidioides cerebriformis não pertence àquele gênero, devendo-se considerar a paracoccidioidomicose como infecção fúngica causada por uma única espécie de Paracoccidioides - Paracoccidioides brasiliensis (SPLENDORE, 1912) Almeida, 1930. No futuro, através de métodos de biologia molecular, talvez possamos identificar este fung