Invasive Trichosporon infection in solid organ transplant patients: a report of two cases identified using IGS1 ribosomal DNA sequencing and a review of the literature.

Trichosporon species are rare etiologic agents of invasive fungal infection in solid organ transplant (SOT) recipients. We report 2 well-documented cases of Trichosporon inkin invasive infection in SOT patients. We also conducted a detailed literature review of Trichosporon species infections in this susceptible population. We gathered a total of 13 cases of Trichosporon species infections. Any type of organ transplantation can be complicated by Trichosporon infection. Bloodstream infections and disseminated infections were the most common clinical presentations. Liver recipients with bloodstream or disseminated infections had poor prognoses. Although the most common species was formerly called Trichosporon beigelii, this species name should no longer be used because of the changes in the taxonomy of this genus resulting from the advent of molecular approaches, which were also used to identify the strains isolated from our patients. Antifungal susceptibility testing highlights the possibility of multidrug resistance. Indeed, Trichosporon has to be considered in cases of breakthrough infection or treatment failure under echinocandins or amphotericin therapy. Voriconazole seems to be the best treatment option.

Usefulness of matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry for identifying clinical Trichosporon isolates.

Trichosporon 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.

Dual candidemia detected by nested polymerase chain reaction in two critically ill children.

The use of improved microbiological procedures associated with molecular techniques has increased the identification of Candida bloodstream infections, even if the isolation of more than one species by culture methods remains uncommon. We report the cases of two children presenting with severe gastrointestinal disorders and other risk factors that contribute to Candida infections. In the first patient, C. albicans DNA was initially detected by a nested-amplification and C. tropicalis was found later during hospitalization, while blood cultures were persistently negative. In the second child, there was amplification of C. albicans and C. glabrata DNA in the same samples, but blood cultures yielded only C. albicans. Both patients received antifungal therapy but had unfavorable outcomes. These two cases illustrate that PCR was more successful than culture methods in detecting Candida in the bloodstream of high risk children, and was also able to detect the presence of more than one species in the same patient that might impact therapy when the fungi are resistant to azole compounds.

Paracoccidioidomycosis: an epidemiologic survey in a pediatric population from the Brazilian Amazon using skin tests.

Since Paracoccidioides brasiliensis and Histoplasma capsulatum are known to be present in similar environments, there have been many epidemiologic investigations regarding the prevalences of these two organisms. However, cross-reactivity can occur in paracoccidioidin and histoplasmin skin tests, and this usually results in the overestimation of the prevalence of P. brasiliensis. The prevalence of infection with P. brasiliensis was evaluated in a cross-sectional study of 298 asymptomatic school children in the Brazilian Amazon region (Mato Grosso State). In this investigation, the reactivity of children to two different P. brasiliensis antigen preparations, paracoccidioidin and a purified 43-kD glycoprotein (gp43), was compared with or without the co-administration of histoplasmin. In the group of individuals receiving paracoccidioidin who had a positive histoplasmin skin test result, the prevalence of exposure to P. brasiliensis was 44% (16 of 36). This reactivity to P. brasiliensis was significantly higher than that observed in other groups, which ranged from 4% to 6% (P < 5 x 10(-4) for each). Overall prevalence was 4.6% (95% confidence interval = 2.5-7.7%). These data suggest that gp43 provides a better estimate of exposure to P. brasiliensis when the co-administration of histoplasmin is desired.

Randomized trial with itraconazole, ketoconazole and sulfadiazine in paracoccidioidomycosis.

Forty-two patients with active paracoccidioidomycosis were randomized to receive itraconazole (50-100 mg d(-1)), ketoconazole (200-400 mg d(-1)) or sulfadiazine (100-150 mg kg d(-1) up to 6 g d(-1)) for 4-6 months, followed by slow release sulfa until negativity of serological tests. All 14 patients in itraconazole and sulfadiazine groups and 13 in the ketoconazole group showed an adequate clinical response to the chemotherapy. One patient in the latter group showed treatment failure according to clinical and mycological criteria. The test of the hypothesis that the drugs reduced antibody levels up to ten months of treatment showed a p value equal to 0.0001 for itraconazole, 0.017 for ketoconazole and 0.0012 for sulfadiazine; this reduction was similar for the three groups. In this first randomized study for the treatment of paracoccidioidomycosis we could not show superiority of any one regimen over the others in the clinical and serological responses of patients with the moderately severe form of the disease.

Anti-idiotypic antibodies in patients with different clinical forms of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Patients with PCM show a wide spectrum of clinical and pathological manifestations depending on both host and pathogen factors. Two clinical forms of the disease are recognized: the acute or juvenile form and the chronic or adult form. The major antigenic component of the parasite is a glycoprotein of 43 kDa (gp43). All patient sera present antibodies against gp43 (anti-gp43) and, as demonstrated before by our group, spontaneous anti-idiotypic (anti-Id) antibodies (Ab2) can be detected in patient sera with high titers of anti-gp43. Since it has been postulated that anti-Id antibodies may have a modulating function, we decided to purify and characterize anti-Id antibodies in this system. The possible correlation of Ab2 titers with different clinical forms of disease was also verified. Results showed that purified human anti-Id antibodies (human Ab2) recognized specifically the idiotype of some murine monoclonal anti-gp43 (17c and 3e) but not others (40.d7, 27a, and 8a). Spontaneous anti-Id antibodies were found in all clinical forms of disease. The majority of patients (88%, n = 8) with the acute form of PCM had high titers of Ab2. However, among patients with the multifocal chronic form of the disease, only 29% (n = 14) had high titers of Ab2; 70% (n = 10) of patients with the unifocal chronic form had low titers of Ab2. A correlation between Ab2 titers and anti-gp43 titers was observed before and during antimycotic treatment. Our results suggest that titers of anti-Id antibodies correlate with the severity of PCM in humans.

Differential antibody isotype expression to the major Paracoccidioides brasiliensis antigen in juvenile and adult form paracoccidioidomycosis.

We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms. Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients'sera. Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients. IgG1 subclass levels, however, were comparable between the two clinical forms. Patients with the juvenile form had higher (P <. 001) IgG4, but lower (P =.03) IgG2 levels than patients with the adult form. The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients. In contrast, high levels of the IgG2 isotype, regulated by interferon-gamma, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients. IgG3 was either absent or detected at low levels. These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-1 or Th-2) dominant cytokine profile. A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed. Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form.

Paracoccidioides brasiliensis. A mycologic and immunochemical study of two strains.

The authors conducted a mycologic, immunochemical and molecular biology study on two strains of Paracoccidioides brasiliensis, one of them, called IBIA, isolated from soil in the municipality of IBIA (Minas Gerais) by Silva-Vergara et al. (1996, 1998), and the other, BAT, cultivated from a human case of paracoccidioidomycosis in Ribeirão Preto (São Paulo/Brazil) by Freitas Da Silva (1996). Both strains showed cotton-like (M) and yeast-like (Y) forms and were pathogenic for testicularly inoculated guinea pigs, producing granulomatous and/or suppurative orchitis. Immunochemically was demonstrated the presence of gp43 by double immunodiffusion, immunoelectrophoresis and immunoblotting.

IgG, IgM and IgA antibody response for the diagnosis and follow-up of paracoccidioidomycosis: comparison of counterimmunoelectrophoresis and complement fixation.

IgG, IgM and IgA antibodies to GP43 (glycoprotein fraction of Paracoccidioides brasiliensis) were measured by ELISA in 63 samples from 23 patients with paracoccidioidomycosis before and twice after chemotherapy was started. Antibodies against P. brasiliensis were detected by indirect immunofluorescence (IF) (IgG, IgM and IgA isotypes), counterimmunoelectrophoresis (CIE) and complement fixation. Two control groups composed of 19 healthy individuals and 12 patients with other diseases (six with histoplasmosis, three with tuberculosis and three with other mycoses). The highest efficiency percentages were found with IgG and IgA-ELISA (100%), IgG-IF (96.2%), CIE (94.4%) and the lowest with CF (75.9%). Highest positive and negative predictive values (100%) were observed for IgG and IgA ELISA. IgG and IgM-ELISA antibodies are more often found in patients with acute than chronic disease (P = 0.01). Four to six months after treatment follow-up showed decreased levels of IgG and IgM-ELISA for acute cases and decreased titres of CIE for chronic cases in relation to pretreatment levels. This study suggests that IgG-ELISA anti-GP43 represents a good marker to monitor clinical response to therapy.

Coccidioidomycosis in Brazil. A case report.

UNLABELLED: Coccidioidomycosis is an endemic infection with a relatively limited geographic distribution: Mexico, Guatemala, Honduras, Colombia, Venezuela, Bolivia, Paraguay, Argentina and the southwest of the United States. In these countries, the endemic area is restricted to the semiarid desert like regions which are similar to the northeast of Brazil. CASE REPORT: The patient is a 32 year-old male, born in the state of Bahia (Northeast of Brazil) and has been living in São Paulo (Southeast) for 6 years. He was admitted at Hospital das Clínicas, at the Department of Pneumology in October 1996, with a 6 month history of progressive and productive cough, fever, malaise, chills, loss of weight, weakness and arthralgia in the small joints. Chest x-rays and computerized tomography disclosed an interstitial reticulonodular infiltrate with a cavity in the right upper lobe. The standard potassium hydroxide preparation of sputum and broncoalveolar lavage demonstrated the characteristic thickened wall spherules in various stages of development. Sabouraud dextrose agar, at 25 degrees C and 30 degrees C showed growth of white and cottony aerial miceleium. The microscopic morphology disclosed branched hyphae characterized by thick walled, barrel shaped arthroconidia alternated with empty cells. The sorological studies with positive double immunodiffusion test, and also positive complement fixation test in 1/128 dilution confirmed the diagnosis. The patient has been treated with ketoconazole and presents a favorable clinical and radiological evolution.

Antigen-specific immunosuppression in paracoccidioidomycosis.

To characterize the immune dysfunction associated with paracoccidioidomycosis, we studied the in vitro lymphocyte reactivity to phytohemagglutinin (PHA), pokeweed mitogen (PWM), a Candida albicans antigen (CMA), and a Paracoccidioides brasiliensis antigen (PbAg) in 32 patients with the acute and the chronic form of the disease before or during the initial phase of treatment and after clinical cure. We also studied, as controls, 30 healthy individuals, 15 of them immune to P. brasiliensis. Results showed a strong hyporesponsiveness to the PbAg while responses to mitogens and CMA were comparable with those of controls. Patients with the acute form of the disease (usually more severe) had more marked PbAg hyporesponsiveness than those with the chronic form. After patients' clinical cure, PbAg proliferative responses were similar to controls and greater than those seen before pretreatment. Changes in other parameters were also seen in the treated patients; skin test anergy to paracoccidioidin, high levels of anti-P. brasiliensis antibodies, leukocytosis, and eosinophilia. These changes were usually more intense in patients with the acute form of the disease. The post-treatment CD4+, CD8+, and total lymphocyte counts were similar to those of controls. Correlation between these parameters and the lymphoproliferative responses to the various stimuli was only found with PbAg: PbAg responses correlated inversely with eosinophil and anti P. brasiliensis antibody levels. Overall, our results demonstrate an antigen-specific-cellular immunity defect, which is reversible with treatment and possibly related to a T helper cell-2 pattern of immune response during active disease.

Lack of reactivity of paracoccidioidomycosis sera in the double immunodiffusion test with the gp43 antigen: report of two cases.

Sera from two patients with chronic active paracoccidioidomycosis yielded negative double immunodiffusion results with a culture filtrate antigen from Paracoccidioides brasiliensis routinely used in our laboratory. Complement fixation tests were positive for both sera using a polysaccharide-rich antigen. This study reports the results of a more extensive serological investigation of these two sera. Both a somatic antigen and a saline extract from the fungus yielded positive results in the double immunodiffusion. However, the immunodominant 43 kDa glycoprotein antigen showed negative results, although it was recognized by both sera in the Western blot assay. The value of the double immunodiffusion as a single serological test in paracoccidioidomycosis diagnosis is discussed.

Paracoccidioides brasiliensis. A mycologic and immunochemical study of a sample isolated from an armadillo (Dasipus novencinctus).

A sample of P. brasiliensis isolated from the spleen and the liver of an armadillo (Dasipus novencinctus) has been analysed under a mycological and immunochemical viewpoint. The armadillo was captured in an area of Tucuruí (State of Pará, Brazil), the animal being already established as an enzootic reservoir of P. brasiliensis at that region of the country. This sample maintained in the fungal collection of the Tropical Medicine Institute of São Paulo (Brazil) numbered 135, has got all the characteristics of P. brasiliensis, with a strong antigenic power and low virulence for guinea-pigs and Wistar rats. The specific exoantigen of P. brasiliensis--the glycoprotein with a molecular weight of 43 kDa--was easily demonstrated with double immunodiffusion, immunoelectrophoresis, SDS-PAGE and immunobloting techniques.

[Paracoccidioidomycosis evidencing spinal cord involvement treated with success by fluconazole]. / Paracoccidioidomicose evidenciando comprometimento medular tratada com sucesso por fluconazol.

The involvement of central nervous system in paracoccidiodomycosis has rarely been described, with an incidence rate varying from 9.99% to 27.27%. There are two basic forms of clinical presentation: meningeal and tumor-like (abscesses, granulomas, nodules, and cysts). The Paracoccidioides brasiliensis is preferentially described in cerebral hemispheres, cerebellum, medulla oblonga and meninges, and exceptionally in the spinal cord. The authors present a case of paracoccidioidomycosis which diagnosis was achieved by microscopic examination of material from oral lesions and specific serology. The patient presented clinical signs of spinal cord involvement confirmed by lesions found in magnetic resonance imaging. They emphasize the inedit therapeutic response to a new antifungal agent (fluconazole) used for the first time in this kind of clinical manifestation, and the excellent prognosis when diagnosis is promptly made.

[Paracoccidioides brasiliensis, a new sample isolated from feces of a penguin (Pygoscelis adeliae)]. / Paracoccidioides brasiliensis, nova amostra isolada de fezes de um pinguim (Pygoscelis adeliae).

The Authors show the results obtained through the study of a Paracoccidioides strain isolated from a penguin in the Uruguaian Antartide by GEZUELE et al. (1989). From the fecal mater it was isolated a fungus which was recently considered as a new species of the genus Paracoccidioides--P. antarcticus. However, the mycological and immunochemical studies including the demonstration of the 43 kDa glycoprotein by immunodiffusion test, SDS-PAGE and immunoelectrophoresis disclosed that such strain is similar to P. brasiliensis. Other studies, based on molecular taxonomy, including karyotyping, are the only tools to confirm the possibility of such strain to be a variant of P. brasiliensis. The Authors report the epidemiological significance of that finding and suggest a review in the knowledge of the ecological "niche" of P. brasiliensis.

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...

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

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e., a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO. The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

[Obtention of Histoplasma capsulatum exoantigens in neopeptone, glucose, thiamine and asparagine (NGTA) media]. / Obtenção de exoantígenos de Histoplasma capsulatum em meio de neopeptona, glicose, tiamina e asparagina (NGTA).

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 36 degrees C 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 paracoccidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.