Standardization and characterization of antigens for the diagnosis of aspergillosis

The aim of this study was to develop and characterize antigens for the diagnosis of aspergillosis. Nine strains of Aspergillus species Aspergillus fumigatus , Aspergillus flavus , and Aspergillus niger were grown in Sabouraud and Smith broth to produce exoantigens. The antigens were tested by immunodiffusion against sera from patients with aspergillosis and other systemic mycoses. The protein fraction of the antigens was detected by SDS-PAGE; Western blot and representative bands were assessed by mass spectrometry coupled to a nano Acquity UltraPerformance LC and analyzed by the Mascot search engine. Concurrently, all sera were tested with Platelia Aspergillus EIA. The most reactive antigens to sera from patients infected by A. fumigatus were produced by A. fumigatus MG2 Sabouraud and pooled A.?fumigatus Sabouraud samples, both with a sensitivity of 93% and specificity of 100% and 97%, respectively. Aspergillus niger and A. flavus antigens were reactive against A. niger and A. flavus sera, each one with a sensitivity and specificity of 100%. Two proteins, probably responsible for antigenic activity, β-glucosidase in A. fumigatus and α-amylase in A. niger were attained. The commercial kit had a specificity of 22%, sensitivity of 100%, positive predictive value of 48%, and negative predictive value of 100%. The antigens produced showed high sensitivity and specificity and can be exploited for diagnostics of aspergilloma.     

Circulating antibodies against thermophilic actinomycetes in farmers and mushroom workers

Sera from individuals exposed to moldy hay, mushroom compost and unexposed individuals, were studied for antibodies against thermophilic actinomycetes and Aspergillus fumigatus. Antigens extracted from Thermoactinomyces vulgaris strains isolated from China were used in the study along with antigens from standard strains to screen the sera of individuals exposed to farms and unexposed controls from both China and the United States. It was found that very little antibody response to A. fumigatus and F. rectivirgula was demonstrated in the sera of both individuals exposed to farm environments and unexposed controls. On the other hand, antibodies to Thermoactinomyces vulgaris and T. candidus strains were seen in the sera of both exposed and unexposed individuals from China, indicating that these organisms are prevalent in all the environments of China. The results also indicate that antigenicity of the strains vary considerably in their reactivity with sera.     

Specificity of antigens from pathogenic Aspergillus species. II. Studies with line immunoelectrophoresis

Somatic (mycelial) and metabolic (culture filtrate) antigens of Aspergillus flavus, A. fumigatus, A. nidulans, A. niger and A. terreus were compared by line immunoelectrophoresis with sera from patients with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma, or from immunized animals. Number of lines observed when tested with human sera were similar for somatic and metabolic preparations of A. fumigatus, but up to 33 lines were present when both types of antigens were tested simultaneously. Cross-reactions between heterologous antigens and sera from patients with aspergilloma or ABPA were uncommon. In contrast, cross-reactions were common when standard antisera prepared in animals against heterologous species of Aspergillus were tested against A. fumigatus antigens. Lines of identity between homologous antigens and those from A. fumigatus were observed in 5 of 9 lines obtained with A. flavus, 4 of 16 lines of A. nidulans, 4 of 9 lines of A. niger and 4 of 8 lines of A. terreus.     

Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Variation of a 470 000 daltons antigen complex and catalase antigen in clinical isolates of Aspergillus fumigatus

Antigens in ruptured mycelium of 18 Aspergillus strains including 14 clinical isolates of A. fumigatus were studied by immunoelectrophoresis. One antigenic component of molecular weight 470 000 previously characterized by hydrophobic interaction chromatography and gel filtration and a second component with catalase activity were detected in all A. fumigatus isolates but in varying quantities. The 470 000 antigen complex cross-reacted with antigens in A. flavus and A. nidulans but not in A. niger or A. terreus. A. fumigatus catalase antigen cross-reacted with catalase in A. flavus, A. nidulans and A. terreus, but not in A. niger. One A. fumigatus isolate produced two catalase antigens showing a reaction of partial identity. A. flavus also produced two catalase antigens, one of which was species-specific.     

A strategy for identifying serodiagnostically relevant antigens of Leishmania or other pathogens in genetic libraries.

Different individuals, when infected with the same parasite, rarely produce antibodies against the same set of antigens. Indeed, unless a particular antigen happens to be recognized by antibodies in all individuals, the use of a single antigen in the serodiagnosis of parasitic diseases leads, invariably, to false-negative results. A straightforward method for pin-pointing, in genetic libraries, the precise antigens that would increase serodiagnostic assay sensitivities is presented. The method is based on the utilization of sera that produced false-negative results against previously available antigens. Employing this false-negative serum-selection methodology for the identification of new Leishmania infantum recombinant antigens (rAgs), the sensitivity of a dipstick assay for anti-Leishmania antibodies in a panel of sera from patients with visceral leishmaniasis was increased from 83.3% to 98.1%, without affecting its specificity, by the inclusion of a fifth and a sixth L. infantum rAg.     

Characterization of Aspergillus fumigatus protein disulfide isomerase family gene.

Aspergillus fumigatus is an opportunistic fungus which causes pulmonary complications in humans and animals. The clinical spectrum observed with A. fumigatus is attributed to the multifunctional nature of its antigens. Lack of understanding on the molecular processes and complexity of the fungus have spurred interest in the identification and characterization of its antigens/allergens with biological activities and virulence functions. For identification of some of these antigens/allergens, a cDNA library of A. fumigatus was screened with antibodies of allergic bronchopulmonary aspergillosis (ABPA) patients. One of the reactive clones was sequenced and observed to have an open reading frame of 1095 nucleotides corresponding to a polypeptide of 364 amino acids. The nucleotide and deduced amino acid sequence showed significant homology with the protein disulfide isomerase (PDI) superfamily. The expressed recombinant fusion protein exhibited specific IgG and IgE binding with antibodies present in ABPA patients' sera. The recombinant protein in vitro catalyzed folding of scrambled RNase. The probable epitopic regions of the deduced amino acid sequence were mapped by algorithmic analysis. This is the first report of isolation of a gene encoding a member of the PDI family from A. fumigatus. The PDI superfamily of proteins may play an important role in the protein folding mechanisms of A. fumigatus antigens/allergens for their interaction with the host.     

Immunoreactivity of antigen extracts of Aspergillus fumigatus isolated from different sources

Allergic bronchopulmonary aspergillosis (ABPA) is the result of hypersensitivity to Aspergillus antigens in patients with long-standing atopic asthma. In the present study mycelial and culture filtrate antigens from Aspergillus fumigatus cultures isolated from diverse sources were tested against sera of 10 ABPA patients and 10 control individuals by an ELISA methodology. The results indicate higher antibody reactivity against both antigens in the sera of ABPA patients, while culture filtrate antigens also gave non-specific reactivity with control sera. Mycelial extracts, in general, were useful in the diagnosis of ABPA.     

Expression of fibroblast growth factor receptor-1 in rat heart H9c2 myoblasts increases cell proliferation

Aspergillus fumigatus is a highly pathogenic fungus causing a wide spectrum of diseases in immunocompromised as well as immunocompetent hosts. The present work was undertaken to evaluate the cytotoxic nature of fractionated antigens of A. fumigatus against the mammalian cell lines (J774, RAW, CHO and L929). An enriched protein antigenic fraction of A. fumigatus was subjected to con A Sepharose and phenyl Sepharose chromatography. Antigenic fractions, ConAub (conA unbound) and PSC III (fraction III of phenyl Sepharose column) containing low mw antigens showed higher cytotoxicity as compared to other antigenic fractions. PSC III was further purified on HPLC resulting in an 18 kDa homogeneous protein. The purified protein showed high ELISA absorbance values for specific IgG and IgE antibodies in sera of ABPA patients. Monoclonal antibody raised against Asp fl, a major allergen/antigen of A. fumigatus recognised the purified 18 kDa by ELISA and western blot. The 18 kDa allergen/antigen or Asp fl showed similar toxicity towards all the four cell lines (macrophage and fibroblast) with an IC50 of 75 ng/ml or 4.16 nM. Reduction in toxicity of 18 kDa at low temperatures and potentiation in presence of ammonium chloride and monensin indicates mechanism of internalisation of 18 kDa in eukaryotic cells is similar to -sarcin. The present work shows that the 18 kDa allergen/antigen (Asp fl) is a major cytotoxin secreted by A. fumigatus which may play multiple roles in the pathogenesis of Aspergillosis through allergenicity, antigenicity and cytotoxicity. (Mol Cell Biochem 167: 89-97, 1997)     

Antibodies to the rhamnose determinants of the polysaccharide of streptococci group A in the sera of rheumatism patients and donors]

In the sera of patients with recurrent rheumocarditis, and especially in cases of primary rheumatism, the level of antibodies to group A streptococcal polysaccharide (A-PS) has been found, according to the results of the enzyme immunoassay, to be considerably higher than in the sera of healthy donors. The level of antibodies to rhamnose determinants (RD) of A-PS has been determined by the inhibition of the immunoenzyme reaction with A-PS under the influence of a variant of group A streptococcus and rhamnose disaccharides with the bonds alpha 1-2 and alpha 1-3. In patients with recurrent rheumocarditis the level of antibodies to A-PS has been shown to be considerably higher than in healthy donors having these antibodies. In acute primary rheumatism a high level of antibodies to A-PS has been detected only in a few cases, and at the same time the prevalence of antibodies to the specific RD of A-PS, bound with beta-N-acetylglucosamine, is observed. In the sera of patients with recurrent rheumocarditis and donors having a high content of antibodies to the rhamnose site of A-PS antibodies, seemingly active against at least two RD, have been detected. In acute primary rheumatism an insignificant amount of antibodies to the rhamnose site of A-PS may probably cause the autoimmune process accompanying rheumatism. This suggestion is substantiated by the previously established capacity of these antibodies for inducing the suppression of cytotoxic cell reactions to microbial antigens.     

Dot-immunobinding assay in the detection of IgG antibodies against farmer's lung antigens

Circulating antibodies against Faenia rectivirgula, Thermoactinomyces candidus, T. vulgaris and Aspergillus fumigatus were studied in the sera of 14 clinically proven farmer's lung patients and 10 normal controls using three immunological methods. These methods were agar gel double diffusion (DD), biotin-avidin-linked immunosorbent assay (BALISA) and dot-immunobinding assay (DIBA). Agar gel diffusion, the least sensitive of the three methods, failed to detect antibodies in some of the patients, while BALISA detected antibodies even in the normal controls. However, the sensitivity of dot-immunobinding assay was in between DD and BALISA while the specificity was comparable to DD to all the antibodies except against A. fumigatus antigens. Dot-immunobinding assay gave faster results than DD and the blots can be stored as record for longer periods of time without fading.     

Antigenic profile of some typical and septate phialide-strains of Aspergillus fumigatus

Antigens from typical and septate phialide-strains of Aspergillus fumigatus, A. fumigatus var. ellipticus, A. fischeri, A. flavus and A. niger were analysed by fused rocket and two-dimensional immunoelectrophoresis. Depsite strain-to-strain variations, isolates of the two morphological forms of A. fumigatus showed a close similarity in their antigenic profile; the percentage of qualitative sharing ranged between 80% and 100%. The number of antigens shared by the isolates of A. fumigatus var. ellipticus and A. fischeri as compared with a typical A. fumigatus isolate was relatively high. By contrast isolates of A. flavus and A. niger, which are taxonomically not so closely related to A. fumigatus, revealed a qualitative sharing of only 16% and 8%, respectively. The number of common antigenically active components identified in culture filtrate antigens from the typical and septate phialide-strains of A. fumigatus was around 29. Two-dimensional electrophoresis with tandem and intermediate gel proved further that the two types of A. fumigatus isolates are antigenically homologous. Present findings also support the view that the antigenically active components of this pathogen are predominantly protein-glycoprotein in nature.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia lipopolisacharydes and Yop proteins by ELISA

The antibodies against the somatic antigens of Y. enterocolitica O3, O8, O9, O5,27,Y. pseudotuberculosis I, and released proteins Yop were detected using the ELISA in 1634 serum samples and 84 synovial fluids collected from 1290 persons suspected for yersiniosis, as well as 200 serum samples from healthy individuals (blood donors). The presence of antibody in diagnostically significant titres for somatic antigens of Yersinia were detected by ELISA in 20.5% and 50.6%, antibodies for released proteins Yop in 11.5% and 28.4% respectively of blood donors and patients suspected for yersiniosis. The antibody against the O3 antigen of Y. enterocolitica was the most frequently detected antibody while the most infrequent was the antibody for the antigen from the 08 serologic group. The results of the study showed that the humoral response picture to Yersinia antigens in the course of yersiniosis in humans is dependent on the age and sex of the patient, duration of the infection, and clinical manifestations. Most frequently the elevated antibody levels were detected among patients with erythema nodosum and patients with gastrointestinal symptoms. The frequency of occurrence of antibodies for most antigens of Yersinia, together with age increased reaching its peak, on the average, among individuals aged 21 - 40 years. Analysis of individual cases showed that by the end of the first week of infection, elevated levels of antibodies for somatic antigens of Yersinia are evident. On the other hand, antibodies for released proteins Yop as a matter of rule appear in the second week from the onset of clinical symptoms. Within this early phase of infection immunoglobulins of the A and M classes dominate reaching their highest level in the second to third week of the infection. In majority of the individuals studied antibodies of the IgG class reached their highest level much later in relation to those of the IgA and IgM classes. Significant differences were found in IgA antibody detection among individuals with clinical manifestations of stomachaches and arthritis. Nevertheless, among individuals with clinical symptoms of stomachaches, these immunoglobulins as a matter of principle disappear with a period of 2-3 months from the onset of clinical symptoms. In individuals with arthritis however the aforementioned immunoglobulins maintained at considerable levels even after a year. In joint-fluid samples obtained from patients with arthritis antibodies for Yersinia antigens were detected in similar levels just as obtained simultaneously serum from those individuals.     

Aspergillosis: Mechanisms of pathogenicity implicated and approach to laboratory diagnosis.

Aspergillosis comprises a wide range of clinical conditions, of which the most serious is invasive aspergillosis, which particularly affects immunodeficient individuals. In the present work we present a review centred on three main aspects of this disease: 1) Possibility of differentiation of strains of Aspergillus fumigatus by phenotyphic and genotypic methods, 2) Mechanisms of pathogenicity of this species and especially the relationships of elastase activity, and 3) Laboratory diagnosis of aspergillosis, principaly in terms of determination of circulating antigens and DNA sequences in the blood and urine of patients.     

Production and standardization of Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus antigens to be used in immunodiagnosis. Comparison between immunodiffusion and immunoelectrophoresis

Soluble antigens (Ag) from Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus were prepared and standardized by double immunodiffusion (DID) and immunoelectroosmophoresis (IEOP). No difference in sensitivity was observed between the two techniques; 100% of standard patient sera were positive with P. brasiliensis and A. fumigatus Ag and 83.3% were positive with H. capsulatum Ag. The specificity of the tests was verified testing 96 sera from patients with paracoccidioidomycosis, histoplasmosis, systemic candidiasis, sporotrichosis, tuberculosis, lung cancer, visceral or cutaneous leishmaniasis and 18 sera from healthy individuals. All the three antigens were 100% specific with the DID (using the identification pattern indicated by the confluence of test serum with standard serum precipitin lines as a positive criterium). However in the IEOP, the specificity varied with each Ag. Positive reactions with P. brasiliensis Ag were observed in 16.7% of histoplasmosis sera and in 10% of cutaneous leishmaniasis sera. On the other hand 31.8% of paracoccidioidomycosis and 10% of cutaneous leishmaniasis sera reacted with H. capsulatum Ag. The high sensitivity and specificity of the DID test, its easy reproducibility and low cost, led us to consider it highly appropriate as a routine procedure for the screening of patients with respiratory infections.     

Immunogens of interest for the diagnosis of Campylobacter jejuni infections

In order to identify the C. jejuni immunogens of interest for the diagnosis of Campylobacter infections, we analyzed the humoral response of 153 patients by using complement fixation (CF) and western blot assays. A first group of 79 sera was from C. jejuni infected patients suffering from enteritis (n=16), Guillain-Barré syndrome (GBS) (n=40) and arthritis (n=23). A second group of 49 sera was from healthy blood donors and a third group consisted of 25 sera from children under 4 years old. Using the CF test, 88.6% of the C. jejuni infected patients were seropositive versus 28.5% of the healthy blood donors and none of the children. The Western blot assay allowed detection of antibodies directed against seven selected antigens ranging from 14 to 67 kDa. Three of these antigens with a molecular size of 29, 37 and 43 kDa were detected by 86.0%, 84.8% and 91.1% of the C. jejuni infected patients, respectively. These three antigens seem to be good candidates for the development of assays suitable for direct and indirect diagnosis of Campylobacter infections.     

我国部分地区自然发酵豆酱中的曲霉菌及其有性型

Twenty species of Aspergillus and their teleomorphs were isolated from naturally fermented soybean pastes collected in 42 regions of China. Of which A. tubingensis, A. flavus, A. niger, A. ochraceus, A. candidus and A. fumigatus were predominant. The high frequency of A. flavus from the samples indicates that the traditional making method of soybean paste might possibly be harmful to food safety. Some Aspergillus species or strains isolated from the traditional food samples may be hopeful of using in pure leaven making and industrial production of soybean paste with special flavor.     

我国部分地区自然发酵豆酱中的曲霉菌及其有性型

从我国42个地区的自然发酵酱醅和酱醪样品中分离得到曲霉Aspergillus及其有性型真菌20种,其中优势曲霉菌种类为A.tubingensis、A.flavus、A.niger、A.ochraceus、A.candidus和A.fumigatus.调查研究发现,从酱醅和酱醪样品中分离出黄曲霉的频率较高,表明自然发酵豆酱存在一定的安全隐患.研究中分离获得的曲霉菌株有望用于纯种制曲或特殊风味豆酱的工业化生产.     

Effect of Antifungal Compounds on Aspergillosis in Hatching Chick Embryos

Aspergillosis was induced experimentally in hatching chick embryos by dipping them in water containing spores of Aspergillus fumigatus or A. flavus. The addition to the dip water of antifungal compounds prevented the development of this syndrome. Of the compounds studied, amphotericin B was most effective, followed by pimaricin and nystatin. Sorbic acid was without effect.     

Immunoblotting reactivity of human sera from various sources against purified epstein-barr virus

The immunoblotting technique was used to analyse polypeptides of purified Epstein-Barr virus reacting with antibodies present in sera from clinically healthy individuals, from patients with infectious mononucleosis (IM) or AIDS, and from renal transplant recipients with molecular sizes in the range of 40–290 kDa were detected.The 47- and 160-kDa nucleocapsid polypeptides, as well as the 72-, 74-, 140-, 220- and 290-kDa membrane polypeptides were the major viral proteins detected in the sera. Sera from clinically healthy individuals contained antibodies directed against all EBV membrane and nucleocapsid antigens. Sera from renal transplant recipients, from patients with IM and from patients with AIDS failed to react with certain nucleocapsid and membrane antigens; in particular, sera from AIDS patients and renal transplant recipients did not react with the 220-kDa polypeptide, one of the major membrane antigens, while sera from subjects with IM and from healthy individuals did.A high proportion of sera from patients with IM (38% vs 5% of clinically healthy individuals and 0–5% of the AIDS patients and renal transplant recipients) reacted with a 42-kDa polypeptide, suggesting its possible role in acute EBV infection.