RESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (S1, PS2, and PS3) and by the new species, P. lutzii. Considering that genetic differences in the Paracoccidioides genus could elicit distinct immune responses by the host, current research investigated serum IgG levels to antigens from P. brasiliensis B339 (S1), P. brasiliensis LDR3 (PS2), and atypical strain LDR2 (P. lutzii), in patients with chronic PCM from the northern and west regions of Paraná, Brazil (n = 35). Cell-free antigen (CFA) and high molecular mass fraction (hMM) were produced from each strain. Samples were analyzed by ELISA and immunodiffusion (ID). ELISA positivity using CFA: B339-100 %, LDR3-83 %, and LDR2-74 %. Response to CFA from B339 was more intense (p < 0.05), while there was no difference between LDR3-LDR2. IgG anti-hMM was higher for antigens from B339 or LDR3, when compared with LDR2 (p < 0.05). There was a positive correlation for each strain between CFA-hMM and for hMM between B339-LDR3 and LDR3-LDR2. ID positivity with CFA: B339-63 %, LDR3-66 %, and LDR2-60 %. We conclude that the intensity of reaction of the patients' sera varies with the strain used; hMM influences tests that use CFA, independently of strain; using ID, positive rates were very similar, but there was a large number of false negative results; ELISA tests using antigens from P. brasiliensis S1 were able to detect a larger number of patients than PS2 and P. lutzii (which had a considerable number of false negative results), and therefore, its use may be more appropriate in this region of Brazil.
Assuntos
Anticorpos Antifúngicos/sangue , Antígenos de Fungos , Imunoglobulina G/sangue , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/microbiologia , Antígenos de Fungos/imunologia , Brasil , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Paracoccidioidomicose/diagnóstico , Sensibilidade e Especificidade , Soro/imunologiaRESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb). The cyclosporin A (CsA) is an immunosuppressant drug that inhibits calcineurin and has been described as a potential antifungal drug. The present study investigated the effect of CsA on the immune response, fungal load/antigenemia in experimental murine PCM. It was used four groups of BALB/c mice: (a) infected with 1 x 105 Pb18 yeast cells (Pb), (b) infected and treated with CsA every other day 10 mg/kg of CsA (s.c.) during 30 days (Pb/CsA), (c) treated with CsA (CsA) and (d) no infected/treated (PBS). The immune response was evaluated by lymphocyte proliferation, DTH assays to exoAgs, ELISA for IgG anti-gp43 (specific immune responses) and cytokine serum levels (IFN-γ, TNF-α, IL-4 and IL-10). Fungal load was determined by lung colony-forming units (CFU) counts, lung and liver histopathology analysis and antigenemia determined by inhibition-ELISA. As expected, CsA was able to inhibit the specific cellular and humoral immune response (P < 0.05), with decrease in serum IFN-γ, TNF-α and IL-4 levels (P < 0.05). Cyclosporin A treatment also resulted in significantly decreased lung Pb CFU (P < 0.05) as well as a lower number of yeasts in the lung and liver (P < 0.05) by histopathology. In concordance, the decreased antigenemia was observed in Pb/CsA group (P < 0.05). In conclusion, even with immunosuppressive action, treatment with CsA results in decreased lung fungal load/antigenemia in experimental PCM in BALB/c mice. Further study is required to determine whether this represents less severe disease or protection by CsA.
Assuntos
Ciclosporina/uso terapêutico , Pulmão/microbiologia , Paracoccidioides , Paracoccidioidomicose/tratamento farmacológico , Animais , Anticorpos Antifúngicos/sangue , Antígenos de Fungos/sangue , Antígenos de Fungos/imunologia , Contagem de Colônia Microbiana , Ciclosporina/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/sangue , Proteínas Fúngicas/imunologia , Glicoproteínas/sangue , Glicoproteínas/imunologia , Hipersensibilidade Tardia/imunologia , Imunoglobulina G/sangue , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-4/sangue , Fígado/microbiologia , Fígado/patologia , Pulmão/patologia , Ativação Linfocitária , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/patologia , Fator de Necrose Tumoral alfa/sangueRESUMO
Histoplasma capsulatum var. capsulatum is a thermally dimorphic fungus that causes histoplasmosis. Fungal hemagglutination activity and cases of reactive hemophagocytic syndrome (RHS) have been reported in the disseminated form of disease. In the present study, soluble components of H. capsulatum var. capsulatum have been investigated for hemagglutinin activity and the capacity to induce hemophagocytosis in the mouse system. To analyze hemagglutinating activity, mouse red blood cells (RBC) (1% v/v in PBS) were incubated (37 degrees C, 1 h) with cell-free antigen (CFAg) from H. capsulatum var. capsulatum (isolate IMT/HC128) (RBC-CFAg) or previously heated CFAg (56 degrees C, 30 min) (RBC-hCFAg) or as control with PBS (RBC-PBS). Hemophagocytosis was analyzed by incubating BALB/c mouse peritoneal phagocytic cells (5 x 10(6) cells) with syngeneic RBC, sensitized or not with CFAg. In addition, mouse polyclonal antibodies were raised against syngeneic RBC-CFAg (anti-RBC-CFAg) and used to analyze CFAg chromatographic fractions (Sephadex G75/120) by immunoenzymatic assay (ELISA). Hemagglutinin activity was observed with RBC-CFAg, but not with RBC-hCFAg or RBC. Also, hemophagocytosis was observed with RBC-CFAg, but not with RBC. The anti-RBC-CFAg antibodies reacted with CFAg fractions corresponding to a molecular mass (MM) higher than 150 kDa. In conclusion, the yeast form of H. capsulatum var. capsulatum releases thermolabile soluble components with hemagglutinin activity and it has been demonstrated for the first time that soluble components of the same fungus induce syngeneic hemophagocytosis in the in vitro mouse system. Also, indirect analysis with antibodies suggests that high-MM components (>150 kDa) are responsible for the interaction with RBC.
Assuntos
Proteínas Fúngicas/imunologia , Proteínas Fúngicas/isolamento & purificação , Hemaglutinação , Histoplasma/química , Fagocitose , Animais , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/química , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peso MolecularRESUMO
The fungus Paracoccidioides brasiliensis is the pathogen of paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. The loop-mediated isothermal amplification method (LAMP) was used in this study to detect the presence of P. brasiliensis in sputa samples from patients with chronic PCM, suspected PCM, and a negative control. The target P. brasiliensis gp43 gene was amplified in less than 4 hr in 11 of 18 sputa samples tested. The LAMP method had the advantage of speed and simplicity compared with the classic diagnostic methods such as the histopathological test or biological material culture and did not require sophisticated technical apparatus. It would be an important aid in cases where immediate treatment would mean patient survival, especially in immune-suppressed patients.
Assuntos
Antígenos de Fungos/genética , Proteínas Fúngicas/genética , Glicoproteínas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/diagnóstico , Escarro/microbiologia , Adulto , Idoso , Erros de Diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Paracoccidioides/genética , Sensibilidade e EspecificidadeRESUMO
Paracoccidioidomycosis, a systemic mycosis, is rarely diagnosed in its initial phase and can remain latent for up to 40 years. Although PCR is sensitive for the identification of Paracoccidioides brasiliensis (Pb) in different samples, no study using paraffin-embedded human tissue has been published. The size of the amplicon, the fixation method and the time of the storage may affect the reaction. Recently the more sensitive Primer-Extension-Preamplification (PEP)-Nested-PCR has been used for amplification of small samples. Our aims were to detect Pb in paraffin embedded biopsies using (PEP)-Nested-PCR and to correlate the data with histopathological parameters. Analyses were carried out in 107 biopsies from tegument, lymph node, lung and tongue. The fungal DNA was detected in 29.9% of the biopsies by (PEP)-nested-PCR against 5% of Nested-PCR. The positivity correlated with numbers of fungi and fungal viable cells, and there was no correlation with the granuloma pattern.
Assuntos
Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/patologia , Reação em Cadeia da Polimerase , Biópsia , DNA Bacteriano/análise , Humanos , Paracoccidioides/genéticaRESUMO
In this study, Swiss mice were experimentally infected with Paracoccidoides brasiliensis (Pb18) and we investigated the levels of gp43 in urine and plasma, anti-gp43 and IgG-gp43 immune complexes in plasma. These levels were correlated with the histopathological findings. Blood and urine samples were collected from mice at 7, 28, 56 and 84 days after intravenous inoculation of 10(5) yeast cells, and analysed by ELISA. The results showed increased levels of soluble gp43 in the plasma in all periods, and anti-gp43 IgG and immune complexes after day 28. High gp43 levels were detected in the urine, except for day 28, coincident with the presence of compact granulomas in lungs. All the infected mice showed fungal cells in the lungs, with initial granulomatous lesions at day 7, dissemination of lesions to other organs at day 56, and granulomas lacking the surrounding mononuclear cells infiltration, especially at days 56 and 84. Our results suggest that gp43 diffuses passively into the urine, and the determination of gp43 levels in urine samples may be a non-invasive alternative method for diagnosis and follow up of PCM. Further studies are needed to determine if the cellular immune response correlate with decreased urine gp43 levels.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Antígenos de Fungos/urina , Proteínas Fúngicas/imunologia , Glicoproteínas/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Animais , Antígenos de Fungos/sangue , Antígenos de Fungos/imunologia , Proteínas Fúngicas/sangue , Glicoproteínas/sangue , Imunoglobulina G/análise , Masculino , Camundongos , Paracoccidioides/genética , Paracoccidioidomicose/sangue , Paracoccidioidomicose/patologiaRESUMO
Cryptococcus neoformans is an important fungal pathogen in immunocompromised hosts. Capsulation, urease and melanin synthesis activity of the fungus are well known virulence factors. Although artificial melanin-deficient mutants of Cr. neoformans have been investigated, the clinical mutant is rare. We found a Cr. neoformans isolate in the cerebrospinal fluid of an AIDS patient which produced a light tan colony on a caffeic acid cornmeal agar (CACA) plate. The mycological feature of the isolate was as follows; normal capsulation, defective inositol assimilation ability, serotype A; urease-positive; mating type alfa; haploid; extremely slow growth in RPMI 1640 medium, Sabouraud dextrose broth, brain heart infusion broth and yeast nitrogen base; lower production of melanin with L-DOPA substrate; and low virulence to ddY mice. We also investigated the partial DNA sequence of CNLAC1 gene between the 3085th to 3623rd base. There were many substitutions, 3 insertions and 3 deletions in the isolate compared with GenBank accession number L22866. The result indicated some functional disorder in the gene. Although the CACA plate is an excellent selective medium for Cr. neoformans, other identification methods should also be used.
Assuntos
Síndrome da Imunodeficiência Adquirida/microbiologia , Cryptococcus neoformans/isolamento & purificação , Adulto , Cryptococcus neoformans/genética , Feminino , Humanos , Hospedeiro Imunocomprometido , Técnicas MicrobiológicasRESUMO
Twelve isolates of Paracoccidioides brasiliensis generated cerebriform colonies at room temperature on potato glucose agar slants (PDA). These isolates contained abundant chlamydospores and yeast-like cells and are a subset of the 65 isolates obtained from nine-banded armadillos (Dasypus novemcinctus). They grew as a yeast form with typical multiple buddings at 37 degrees C on brain heart infusion agar supplemented with 1% glucose. After replating on PDA and culturing at room temperature for 2 months, the mutants appeared as cottonous colonies, which indicated that the morphological characteristics were unstable.
Assuntos
Tatus/microbiologia , Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/veterinária , Animais , Meios de Cultura , Micologia/métodos , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/microbiologia , TemperaturaRESUMO
Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. An increase in PCM has been reported in recent years and the disease is now recognized as one of the imported fungal infections in Japan. To date, more than 15 cases of PCM have been reported in our country, and five of them were diagnosed by clinical and histopathological findings without mycological study. We applied 2 nested polymerase chain reaction (PCR) amplification methods for detecting P. brasiliensis genes from paraffin-embedded tissue specimens. Successfully amplified were: a 473 base pairs fragment of gp43 gene of P. brasiliensis (located from 741st to 1,213rd base), and a 418 base pairs fragment of 5.8S ribosomal RNA gene of P. brasilienisis which included internal transcribed spacers (ITS) 1 and 2 (located from 131st at ITS1 to 195th at ITS2) in paraffin-embedded murine tissues infected with P. brasiliensis yeast cells. The authenticity of the PCR products was confirmed by nucleotide sequence analysis. These results indicate that the two nested PCR methods may be useful for diagnosis of PCM.
Assuntos
Antígenos de Fungos , Proteínas Fúngicas , Genes Fúngicos , Glicoproteínas/genética , Oligossacarídeos/genética , Paracoccidioides/genética , Paracoccidioidomicose/diagnóstico , RNA Fúngico/análise , RNA Ribossômico 5,8S/análise , Animais , Glicoproteínas/análise , Humanos , Camundongos , Oligossacarídeos/análise , Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/microbiologia , Inclusão em Parafina , Reação em Cadeia da PolimeraseRESUMO
Internal transcribed spacer (ITS) genes including the 5.8S ribosomal (r)RNA of Paracoccidioides brasiliensis were amplified and the DNA sequences were determined. Based on a comparison of the sequence information, a new polymerase chain reaction (PCR) primer pair was designed for specific amplification of DNA for P. brasiliensis. This primer pair amplified a 418-bp DNA sequence and was 100% successful in identifying 29 strains of P. brasiliensis (including the reference strains) isolated from the regions of Brazil, Costa Rica, Japan, Argentina or from different sources. The results of specificity tests of these primers to compare the fungus with those of Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei are also reported.