The contribution of galactomannan detection in the diagnosis of invasive aspergillosis in bone marrow transplant recipients.

Until recently, accurate microbiological diagnosis of invasive aspergillosis (IA) was seldom established in HSCT recipients. Blood samples are rarely positive for Aspergillus species, the reliability of the cultures depends of the specimen (if taken from a normally sterile site or not) and biopsy samples require invasive procedures, rarely recommended in patients with severe thrombocytopenia. Implementing the international consensus defining the microbiological criteria for the diagnosis of Aspergillus infection, we retrospectively evaluated the role of serum galactomannan (GM) detection by EIA to diagnose IA among HSCT patients with proven invasive fungal infection (IFI) and the impact of serum storage in GM concentrations. The EIA assay allowed categorizing as "probable" 5 of the 10 cases of "possible" aspergillosis (50%). Considering a lower cut-off level for the reaction (1.0), 80% of the cases could be categorized as "probable" aspergillosis. Positive or undetermined results were detected one to 4 months before the diagnosis of IA in eight of the 11 patients (72.7%) with proven IFI. Retesting the stored samples after a second storage for four years, we could observe lower reactivity in 20% of the samples. The detection of galactomannan by the EIA test represents a major advance in the diagnosis of IA in HSCT recipients at high risk of IA. A better understanding of the kinetics of the GM in different clinical situations is necessary to maximize the benefit of the test in Aspergillus surveillance.

Highly specific and sensitive, immunoblot-detected 54 kDa antigen from Fonsecaea pedrosoi.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by a group of different dematiaceous fungi, first described by Rudolph in 1914. In Brazil there is a clear predominance of Fonsecaea pedrosoi. Sixty sera samples obtained from patients with F. pedrosoi-caused CBM were analysed. Sera obtained from 36 sporothricosis (SPT) patients, 34 cutaneous leishmaniasis (CL) patients and from 48 blood donors (HBD) were used as control. F. pedrosoi metabolic antigen was obtained from F. pedrosoi sample no. 884 (Instituto de Medicina Tropical de São Paulo Collection). IE reaction disclosed an anodic migrating arch, which was eluted and used as antigen. Both metabolic and eluate F. pedrosoi antigens were submitted to SDS-PAGE and two fractions, weighing approximately 54 and 66 kDa were identified. The 66-kDa fraction reacted against 43 of 60 CBM (71.7%) sera samples and was recognized by 10 SPT and eight CL sera (15.3%). No reactivity was observed against HBD sera. The 54-kDa fraction reacted against 58 of 60 CBM sera (96.7% sensitivity) and was not recognized by HBD, SPT nor CL sera (100% specificity). Such high sensitivity and specificity levels suggest this antigenic fraction is immunodominant and might prove a useful tool for further studies on F. pedrosoi-caused CBM.

Comparative performance of two air samplers for monitoring airborne fungal propagules

Many studies have attempted to evaluate the importance of airborne fungi in the development of invasive fungal infection, especially for immunocompromised hosts. Several kinds of instruments are available to quantitate fungal propagule levels in air. We compared the performance of the most frequently used air sampler, the Andersen sampler with six stages, with a portable one, the Reuter centrifugal sampler (RCS). A total of 84 samples were analyzed, 42 with each sampler. Twenty-eight different fungal genera were identified in samples analyzed with the Andersen instrument. In samples obtained with the RCS only seven different fungal genera were identified. The three most frequently isolated genera in samples analyzed with both devices were Penicillium, Aspergillus and Cladophialophora. In areas supplied with a high efficiency particulate air filter, fungal spore levels were usually lower when compared to areas without these filters. There was a significant correlation between total fungal propagule measurements taken with both devices on each sampling occasion (Pearson coefficient = 0.50). However, the Andersen device recovered a broader spectrum of fungi. We conclude that the RCS can be used for quantitative estimates of airborne microbiological concentrations. For qualitative studies, however, this device cannot be recommended

Comparative performance of two air samplers for monitoring airborne fungal propagules.

Many studies have attempted to evaluate the importance of airborne fungi in the development of invasive fungal infection, especially for immunocompromised hosts. Several kinds of instruments are available to quantitate fungal propagule levels in air. We compared the performance of the most frequently used air sampler, the Andersen sampler with six stages, with a portable one, the Reuter centrifugal sampler (RCS). A total of 84 samples were analyzed, 42 with each sampler. Twenty-eight different fungal genera were identified in samples analyzed with the Andersen instrument. In samples obtained with the RCS only seven different fungal genera were identified. The three most frequently isolated genera in samples analyzed with both devices were Penicillium, Aspergillus and Cladophialophora. In areas supplied with a high efficiency particulate air filter, fungal spore levels were usually lower when compared to areas without these filters. There was a significant correlation between total fungal propagule measurements taken with both devices on each sampling occasion (Pearson coefficient = 0.50). However, the Andersen device recovered a broader spectrum of fungi. We conclude that the RCS can be used for quantitative estimates of airborne microbiological concentrations. For qualitative studies, however, this device cannot be recommended.

Dermatophytosis caused by Trichophyton raubitschekii. Report of the first case in São Paulo, Brazil.

The authors report the first case of dermatophytosis caused by Trichophyton raubitschekii in a patient from the State of São Paulo with Tinea corporis lesions localized on the buttocks. Culture on Sabouraud-agar with cycloheximide permitted the isolation and identification of the fungus, and the diagnosis was confirmed by Dr. Lynne Sigler, University of Alberta, Canada. Systemic treatment with fluconazole, 150 mg/week for 4 weeks, in combination with topical treatment with isoconazole initially yielded favorable results, with recurrence of the lesions after the medication was discontinued. This is the fifth case of this dermatophytosis published in the Brazilian medical literature.

Onychomycosis caused by Scytalidium dimidiatum. Report of two cases. Review of the taxonomy of the synanamorph and anamorph forms of this coelomycete.

The authors report two cases of onychomycosis in the dystrophic form, one of them involving an HIV-positive patient, provoked by Scytalidium dimidiatum, previously called Scytalidium lignicola. The subject is reviewed from the taxonomic viewpoint, considering the anamorph Hendersonula toruloidea as a synonym of Nattrassia mangiferae, and having Scytalidium dimidiatum as the major synanamorph. According to many mycologists, Scytalidium hyalinum may be a separate species or a hyaline mutant of Scytalidium dimidiatum. Scytalidium lignicola Pesante 1957 was considered to be the type-species of the genus by ELLIS (1971)13 and later to be a "conidial state" of Hendersonula toruloidea by the same author, today known as Nattrassia mangiferae. The microorganism lives only on the roots of certain plants (mainly Platanus and Pinus). It produces pycnidia and is not considered to be a pathogen, although it is considered as a possible emerging agent capable of provoking opportunistic fungal lesions. The importance of this topic as one of the most outstanding in fungal taxonomy, so likely to be modified over time, as well as its interest in the field of dermatologic mycology, are emphasized.

In situ hybridization in paracoccidioidomycosis.

In situ hybridization (ISH) was performed using oral biopsies from patients with paracoccidioidomycosis and guinea pig testes inoculated with a culture of Paracoccidioides brasiliensis isolated from soil, employing both a 14 base-pair specific oligoprobe (ACT CCC CCG TGG TC) and its complementary sequence. When combining ISH with the Gridley stain which detects fungal cell walls, about 2-3% of the fungal cells present in the tissues were labelled. When the complementary probe was used, labelling was higher, reaching the 3% level.

Paracoccidioidomycosis in a renal transplant recipient.

Paracoccidioidomycosis is a fungal infection rarely described in immunodeficient patients. We report a severe case of pulmonary paracoccidioidomycosis in a renal transplant recipient and demonstrate deficiencies of in vitro lymphocytic transformation assays, skin hypersensitivity tests, as well as low levels of antibodies to Paracoccidioides brasiliensis.

Responses of T and B lymphocytes to a Paracoccidioides brasiliensis cell wall extract in healthy sensitized and nonsensitized subjects.

Antigen-specific cellular immunity in paracoccidioidomycosis (PCM) has been poorly studied due to lack of standard in vitro lymphocyte proliferation assays. To standardize such an assay, we studied T and B cell responses to a Paracoccidioides brasiliensis cell wall extract (PbAg) in healthy subjects sensitized to either P. brasiliensis [Pb(+)Hc(-)] or to Histoplasma capsulatum [Hc(+)Pb(-)], and in nonsensitized persons. All subjects showed, as expected, a vigorous proliferative response to a control fungal antigen obtained from Candida albicans. Lymphocytes from Pb(+)Hc(-) donors, but not from Pb(-)Hc(-) donors, reacted to PbAg by proliferating in a dose-dependent manner with a maximum reaction after 6-9 days, suggesting a secondary specific immune response. Most activated cells were CD+CD4+ lymphocytes. However, Hc(+)Pb(-) donors' cells reacted with PbAg. Cross-reactivity with H. capsulatum was not unexpected, since both fungi, but not C. albicans, share cell wall immunogenic compounds. An enzyme-linked immunosorbent assay to detect human immunoglobulins (Ig) demonstrated that B cells from Pb(+)Hc(-) donors, but not from Pb(-)Hc(-) ones, reacted with PbAg by secreting high levels of IgG and IgM in 12-day culture supernatants. This secretion was possibly mediated by PbAg-activated CD4+ cells. We believe that analysis of T and B lymphocyte responses to PbAg will be useful in the investigation of the infection-associated immune impairment seen in some PCM patients.

[Neurocryptococcosis caused by nonencapsulated Cryptococcus neoformans]. / Neurocriptococose por Cryptococcus neoformans não capsulado.

The case of a patient with meningoencephafalitis due to a nonencapsulated strain of Cryptococcus neoformans is reported; he had no risk factors for the disease or AIDS. Clinical examination showed a chronic meningoencephalitis first diagnosed as tuberculosis. In the second cerebrospinal fluid (CSF) examination after a week from admission yeasts appeared inside macrophage cells. CSF culture in Sabouraud medium disclosed nonencapsulated Cryptococcus neoformans (biochemical identification). Sample inoculation in mouse (intraperitoneal) evidenced a capsule that disappeared in several consecutive cultures. The morphology of the yeast was studied by electronic microscopy. After treatment with amphotericin B and 5-fluorocytosine the patient had a favorable evolution. The significance of capsular material is discussed.

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis

O presente trabalho avalia a sensibilidade, especificidade e eficiencia da imunodifusao dupla (ID), contraimunoeletroforese (CIE), reacao de fixacao de complemento (FC) e imunofluorescencia indireta (IFI) no diagnostico da paracoccidioidomicose. Os pacientes portadores da micose, virgens de tratamento, tiveram o diagnostico confirmado por exame micologico e/ou histopatologico. Utilizou-se como antigenos o filtrado de cultura da fase leveduriforme do Paracoccidioides brasiliensis para os testes de ID, CIE, FC e suspensao de celulas leveduriformes de "pool" de cepas do mesmo fungo para o teste de IFI. O estudo foi realizado em 4 grupos de individuos: 46 com paracoccidioidomicose ativa (sem tratamento), 22 com outras micoses profundas, 30 com outras doencas infecciosas (tuberculose e leishmaniose tegumentar) e 47 controles normais. Os valores de sensibilidade, especificidade e eficiencia foram obtidos de acordo com a metodologia utilizada por GALEN & GAMBINO (1975). Os resultados revelaram que os testes de precipitacao em gel de agar e agarose, representados pela ID e CIE foram os melhores, apresentando maior sensibilidade (91,3 por cento e 95,6 por cento, respectivamente), maxima especificidade (100 por cento) e os maiores valores de eficiencia quando comparados a FC e IFI...

In vitro action of some disinfectants on Paracoccidioides brasiliensis yeast forms

Descreveu-se a acao fungicida do hipoclorito de sodio (0,3; 1; 2,5; 5 e 10 por cento); do formaldeido (em solucao aquosa a 2,5 e 10 por cento); e alcool etilico a 70,0 por cento sobre formas leveduriformes de 2 cepas de Paraccoccidioides brasiliensis: Pb 18 e cepa Goiana, recentemente isolada. A incubacao do fungo e desinfetantes foi realizada a temperatura ambiente por periodos de 1, 2, 24, 48 e 72 horas. A viabilidade foi avaliada pelo tratamento com diacetato de fluoresceina-brometo de etideo; pela cultura em meios solidos e liquidos a 36 graus Celsius e 26 graus Celsius; transformacao de levedura em micelio a temperatura ambiente; e estudo radiometrico da atividade metabolica. Todas as concentracoes de todos os desinfetantes estudados foram capazes de inativar ambas as cepas, exceto na incubacao com formaldeido a 2 por cento por 1 hora, em que o tratamento por diacetato de fluoresceina-brometo de etideo revelou 40 por cento e 27 por cento de celulas viaveis, respectivamente, para a cepa Pb 18 e Goiana. A transformacao de levedura em micelio foi considerada um metodo rapido, com resultados semelhantes ao cultivo em meios solidos e liquidos.

Subcutaneous phaeohyphomycosis caused by Bipolaris hawaiiensis: a case report

Os autores descrevem um caso de feohifomicose com lesao verrucosa no halux esquerdo. O paciente nao apresentava sinais clinicos de deficiencia imunologica. Os exames direto e histopatologico mostraram celulas leveduriformes e poucas hifas septadas, demacioides, essenciais ao diagnostico desta micose. O cultivo em lamina permitiu identificar o hifomiceto como Biopolaris hawaiiensis.

Serologic characterization of Paracoccidioides brasiliensis E2 antigen.

1. The behavior of the specific E2 antigen of Paracoccidioides brasiliensis was studied by agarose gel counterimmunoelectrophoresis. When the gel was read immediately after the electrophoretic run no precipitation band was visible. Visualization of the complex was possible only after incubation of the gel at room temperature overnight. 2. At alkaline pH, the E2 antigen migrates in the direction of the cathode, as do the immunoglobulins. The higher sensitivity of counterimmunoelectrophoresis when compared to double immunodiffusion for the diagnosis of paracoccidioidomycosis is due to the presence of antibodies directed against antigens which migrate to the anode. 3. The use of specific antiserum to E2 antigen as reference permits the double immunodiffusion method to be a very sensitive test for the specific serodiagnosis of paracoccidioidomycosis.

In vitro action of some disinfectants on Paracoccidioides brasiliensis yeast forms.

The fungicidal action of sodium hypochlorite (0.3, 1, 2.5, 5 and 10%); formaldehyde (2, 5, and 10%); and ethyl alcohol (70%) on yeast forms of Paracoccidioides brasiliensis Pb 18 and a newly-isolated Goiana strain was described. Contact between the fungus and the disinfectants was maintained for 1, 2, 24, 48 and 72 hours at room temperature. Viability was evaluated by the fluorescein diacetate-ethidium bromide treatment, culture in solid and liquid media (36 degrees C and 26 degrees C); yeast to mycelial germination at room temperature; and radiometric study of metabolic activity. All concentrations of disinfectants were found to be effective in inactivating Pb 18 and Goiana strains, except for the 1-hour contact with 2% formaldehyde, in which fluorescein diacetate-ethidium bromide treatment was found to reveal 40 and 27% of viable cells, respectively. The yeast to mycelial germination method was considered to reveal faster and similar results as compared to culture in solid and liquid media.

Serologic characterization of Paracoccidioides brasiliensis E2 antigen

1. The behavior of the specific E2 antigen of paracoccidioides brasiliensis was studied by agarose gel counterimmonoelectrophoresis. When immunoelectrophoresis. When the gel was read immediately after the electrophoretic run no precipitation band was visible. Visualization of the complex was possible only after incubation of the gel at room temperature overnight. 2. At alkaline pH, the, the E2 antigen migrates in the direvtion of the cathodem as do the immunoglobulins. The higher sensitivity of counterimmunoelectrophoresis when compared to double immunodiffusion for the diagnosis of paracoccidioidomycosis is due to the presence of antibodies directed agaisnt antigens which migrate to the anode. 3. The use of specific antiserum to E2 antigen reference permits the double immunodiffusion method to be a very sensitive test for the specific serodiagnosis of paracoccidioidomycosis

[Cerebrospinal fluid in infection of the central nervous system by yeasts of the genus Candida: analysis of 11 cases]. / Líquido cefalorraqueano em infecção do sistema nervoso central por leveduras do gênero Candida análise de 11 casos.

This study is based on the analysis of 44 cerebrospinal fluid (CSF) samples from 11 patients with central nervous system (CNS) Candida infection. Risk factors for CNS fungal infection were present in all patients. Five had a chronic meningitis syndrome; two had acquired immunodeficiency syndrome (AIDS); two had cranial trauma followed by chronic meningities; one had intravascular disseminated coagulation syndrome and sepsis; and one had systemic candidiasis after kidney transplant. Etiological diagnosis was made in all by the CSF examination. Nine cases had positive CSF culture for Candida. Two patients presented the yeast in the direct examination, and one of them had reagent complement fixation test for Candida in three successive samples of CSF. Changes found in the CSF composition are discussed in order to evaluate the inflammatory response to CNS infection by Candida.

Líquido cefalorraqueano em infecçäo do sistema nervoso central por leveduras do gênero candida: análise de 11 casos / Cerebrospinal fluid in central nervous system by Candida infection: analyses of 11 cases

Foram estudadas 44 amostras de líqüido cefalorraqueano (LCR) de 11 pacientes em que foi possível fazer diagnóstico etiológico de comprometimento do sistema nervoso central (SNC) por leveduras do gênero Candida. Todos os pacientes apresentavam fatores de risco para a afecçäo. Cinco apresentavam quadro de meningite crónica e esclarecer; dois, síndrome de imunodeficiência adquirida; dois, traumatismo crânioencefálico seguido de meningite crônica; um, coagulaçäo intravascular disseminada seguida de spticemia; um, candidíase sistêmica pós-transplante renal. O diagnóstico etiológico foi feito pelo exame do LCR, possibilitando em 9 o isolamento da levedura por crescimento em meio de cultura. Em dois o diagnóstico foi feito pela presença das leveduras ao exame direto e, em um deles, também pela positividade da reaçäo de fixaçäo de complemento para Candida realizada em três amostras sucessivas de LCR. Discute-se a importância das alteraçöes encontradas no exame do LCR na candidíase do SNC

Immunoenzymatic absorption test for serodiagnosis of paracoccidioidomycosis.

A sensitive enzyme-linked immunosorbent assay is described for paracoccidioidomycosis serodiagnosis, with antigen represented by filtrates from Paracoccidioides brasiliensis cultures. Cross-reactivities were, however, observed with sera from patients with other mycoses such as histoplasmosis, lobomycosis, cryptococcosis, candidiasis, and sporotrichosis. These cross-reactions did not occur when we used as antigen Yarzabal E2 component prepared by affinity fractionation of the culture filtrates. Specific results could also be obtained with the whole filtrate antigen when sera were absorbed with Histoplasma capsulatum yeast and mycelial components. Besides a very high sensitivity (100% of the 69 cases of paracoccidioidomycosis tested), this assay showed no false-positive results for the 206 non-paracoccidioidomycosis sera studied, including those from patients with other mycotic diseases.

Inquerito com paracoccidioidina em uma populacao da Bahia (Brasil). / Survey with paracoccidioidin in a population of Bahia (Brazil)

No Municipio de Una, localizado ao Sul do Estado da Bahia, em area com registro frequente de casos de leishamaniose tegumentar, foram estudados 177 individuos, na faixa etaria entre tres meses e 73 anos, atraves de provas intradermicas com paracoccidioidina (antigeno peptido-polissacaridico do Paracoccidioides brasiliensis).Positividade foi obtida em dez individuos (5,6%). Somente foi considerada positiva a reacao que apresentava enduracao igual ou maior que 5 mm. Em nenhum dos casos positivos a paracoccidioidina havia evidencia clinica de lesoes blastomicoticas. Com os soros dos individuos positivos a paracoccidioidina, foram realizadas provas de imunodifusao dupla e contraimunoeletroforese, com resultados negativos para anticorpos circulantes anti-P. brasiliensis.Este dado indica que, em nenhum dos reatores a paracoccidioidina, havia processo infeccioso em atividade. O percentual de positividade obtido com a paracoccidioidina, em que pesem eventuais reacoes cruzadas com histoplasmose, sugere a ocorrencia da paracoccidioidomicose na area estudada