The precipitin test in human systemic aspergillosis

Summary The sera of healthy women and of adult men either with chest disease or some other condition associated with a mycosis have been tested for precipitins against Aspergillus extracts. Although these precipitins have been demonstrated in the serum of patients suffering from systemic aspergillosis, they have also been detected in antenatal sera and in the sera of patients with no clinical evidence of aspergillosis. The significance of the presence of precipitins againstA. fumigatus is discussed.     

Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay

An immunogold assay (IGA) was developed to detect IgG and IgE antibodies to Aspergillus fumigatus. Sixteen sera from patients with allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and normal controls were studied. All sera were also evaluated for antibodies against A. fumigatus by biotin-avidin linked enzyme immunosorbent assay (BALISA) and by agar gel double diffusion method. A. fumigatus specific IgG and IgE antibodies could be detected by IGA in all the patients' sera but not in the sera of normal controls. Both IgG and IgE antibodies to A. fumigatus could be demonstrated in all the sera by BALISA and normal controls showed only low levels of these antibodies. There was a positive correlation between the degree of reactivity detected by IGA, the BALISA titer and the precipitins by agar gel diffusion. It can be concluded that IGA is a reliable, sensitive and simple method capable of detecting both IgG and IgE antibodies against A. fumigatus in patient serum.     

Occurrence and significance of precipitating antibodies against thermophilic actinomycetes in the sera of dairy herd workers,Nangali, Delhi

The study was prompted by the lack of information on the role of thermophilic actinomycetes in hypersensitivity pneumonitis in India. It reports the occurrence of precipitating antibodies against clinically important thermophilic actinomycetes in the sera of a population sample of dairy herd workers, Nangali, Delhi. Of 112 workers investigated, 28 (25%) showed precipitins againstFaenia rectivirgula, 4 (3.2%) againstSaccharomonospora viridis, 2 againstThermoactinomyces thalpophilus and one each againstT. vulgaris andT. sacchari. The results of enzyme-linked immunosorbent assay (ELISA) indicated that IgG antibody activity againstF. rectivirgula was significantly higher in the symptomatic group than in the asymptomatic group (p<0.05) of workers and the controls (p<0.01). Significant difference inF. rectivirgula IgG activity was also obtained between the precipitin-positive symptomatic group and the precipitin-positive asymptomatic group (p< 0.05). In strong contrast, the IgG antibody activity againstT. thalpophilus was found to be uniformly low. A limited aeromicrobiological sampling of the dairy farm revealedS. viridis (55.8%) to be the commonest species followed byT. vulgaris (19.2%),T. thalpophilus (18.5%),F. rectivirgula (5%) andT. sacchari (15%). On the basis of suggestive clinical and laboratory findings, farmer's lung disease was suspected in four dairy herd workers. A comprehensive clinical evaluation including pulmonary function studies on the dairy herd workers and their long-term follow-up is indicated to determine the extent of respiratory morbidity caused byF. rectivirgula, S. viridis, T. thalpophilus, T. sacchari andT. vulgaris in India.     

Micropolyspora faeni antigens of three commercial extracts

The protein composition of three commercial extracts of Micropolyspora faeni, produced in U.S.A., England and Italy has been evaluated by agarose gel electrophoresis.By crossed immunoelectrophoresis, tandem-crossed immunoelectrophoresis and by a modification of this last technique, the antigenic composition and the common antigens of the extracts have been investigated. Hyperimmune rabbit serum and a pool of five human sera with precipitins to Micropolyspora faeni have been used as source of antibody.Different quantity and quality of protein content was observed in the available batches. Different antigenic composition was also observed, not directly related to the different proteins contained therein; three antigens were definitely common to all extracts and two of them represented the major antigens of each extract.Despite the total protein content, the major and common antigens were found in similar concentrations in all three products examined. Therefore, the discrepancies observed in the precipitin reactions using the three commercial Micropolyspora faeni extracts are due to differences in the minor antigen composition of the extracts.     

Serological difference betweenErwinia herbicola strains of plant and human origin

FiftyErwinia herbicola isolates obtained from host plants were examined in an agglutination reaction with antiserum prepared againstE. ananas (E. herbicola) strain CCM 2407 antigen of plant origin and with antiserum prepared againstEnterobacter agglomerans strain CNCTC M 269 antigen of human origin. In tests with strain CCM 2407 antiserum, 56% isolates showed a positive reaction, while in tests with strain CNCTC M 269 antiserum only 14 % isolates showed a positive reaction. AmongE. herbicola isolates which showed a positive reaction with CCM 2407 antiserum 18 % showed a positive reaction with the CNCTC M 269 antiserum too. Our results confirmed the serological heterogeneity ofE. herbicola population. In spite of the difference in the origin of the two antigens used for the preparation of antisera (plant, human; Japan, Czech Republic) our results indicate that some of ourE. herbicola strains andE. agglomerans strain CNCTC M 269 are serologically identical.     

Counterimmunoelectrophoresis and opportunistic fungal infections

Sera from 35 apparently normal humans, 37 compromised human patients, 30 hedgehogs and 30 sheep, were examined for precipitating antibodies to four opportunistic fungi — Absidia corymbifera, Aspergillus fumigatus, Candida albicans and Rhizopus arrhizus — using Counterimmunoelectrophoresis (CIE).Precipitins to A. fumigatus were almost exclusively confined to specimens obtained from the compromised human group (51% of those examined) while Candida precipitating antibodies were detected in the sera of both normal (26%) and compromised (49%) humans and in 10% of the hedgehog specimens. Serum precipitins against the two phycomycetes included in the investigations were rare.Because of the complexity of most fungal antigen extracts, it appears essential that sera be tested against a number of different antigen concentrations if CIE is to be used with confidence in fungal serology.     

Effect of Peroxynitrite on dormant spores and germlings ofAspergillus fumigatus in vitro

Peroxynitrite was tested for its effects on the opportunistic pathogenic fungusAspergillus fumigatus. It did not kill any dormant or swollen (4 h in a glucose-peptone medium) conidia in concentrations up to 6.25 mmol/L and the growth of germlings (after 6 or 9 h) was only slightly inhibited by 5 mmol/L peroxynitrite. The peroxynitrite donor SIN-1 (up to 10 mmol/L, I d in buffer) did not kill any conidia but inhibited their germination and growth, depending on the medium. Ten mmol/L SIN-1 in a poor medium was fungistatic and germination was stopped for 20 h. Nine strains ofA. fumigatus showed resistance comparable to the model strain, while 6Candida albicans strains were much more susceptible to both peroxynitrite and its donor. The results indicate that peroxynitrite does not contribute substantially to the antifungal activity of phagocytes againstA. fumigatus.     

Isolation and toxigenicity of Aspergillus fumigatus from moldy silage

Thirty-nine silage samples were collected from various siloson Terceira Island in the Azores. Samples were examined for the presence of total fungi, and isolates of Aspergillus fumigatus were analyzed for their ability to produce fumitremorgens B and C, fumigaclavines B and C, and gliotoxin. Thirty-four silage samples (87%) were contaminated with fungi, and A. fumigatus was isolated from 27 samples (69%). Samples that were taken from the surface of silos had significantly higher populations of both total fungi and A. fumigatus than did samples taken from the middle of silos. Analysis of 27 A. fumigatus isolates (one representing each positive sample) showed that 59.3% produced fumitremorgen B; 33.3% produced fumitremorgen C; 29.6% produced fumigaclavine B; 7.4% produced fumigaclavine C; and 11.1% produced gliotoxin. Fifty-two percent of the isolates produced multiple toxins, and 25.9% did not produce any of these toxins. Gliotoxin and fumigaclavine C were always produced in combination with other toxins. Because of the demonstrated potential of these A. fumigatus isolates to producemycotoxins, it is important to properly construct and manage silos to prevent their contamination with A. fumigatus.This revised version was published online in October 2005 with corrections to the Cover Date.     

Counterimmunoelectrophoresis as a routine mycoserological procedure

Counterimmunoelectrophoresis (CIE) has been compared in a diagnostic laboratory with agar gel double diffusion (DD) as a routine procedure for detection of antibodies to pathogenic and allergenic fungi and actinomycetes. It was shown to be of particular value in detecting antibodies to Aspergillus fumigatus. Thus 72 of 106 sera in which precipitins were detected were positive by CIE alone. Some sera were positive only by CIE to antigens prepared from Histoplasma capsulatum, Allescheria boydii, Candida albicans and C. parapsilosis.     

Antimicrobial activity of 3-methyl-5-furfurylidene-2-thioxo-4-thiazolidones

Substituted 3-methyl-5-furfurylidene-2-thioxo-4-thiazolidones1 –17 were tested for their action againstBacillus pumilus, B. brevis andB. megaterium and againstAspergillus flavus, A. fumigatus andA. niger. Most of the compounds possessed an activity against both bacteria and fungi. These compounds were also tested for their effectiveness as virus inhibitors employingNicotiana glutinosa as assay host against tobacco mosaic virus. Most of them exhibited potential antiviral activity bothin vitro andin vivo.     

Diagnostic Performance of Various Tests and Criteria Employed in Allergic Bronchopulmonary Aspergillosis: A Latent Class Analysis

Aim

The efficiency of various investigations and diagnostic criteria used in diagnosis of allergic bronchopulmonary aspergillosis (ABPA) remain unknown, primarily because of the lack of a gold standard. Latent class analysis (LCA) can provide estimates of sensitivity and specificity in absence of gold standard. Herein, we report the performance of various investigations and criteria employed in diagnosis of ABPA.

Methods

Consecutive subjects with asthma underwent all the following investigations Aspergillus skin test, IgE levels (total and A.fumigatus specific), Aspergillus precipitins, eosinophil count, chest radiograph, and high-resolution computed tomography (HRCT) of the chest. We used LCA to estimate the performance of various diagnostic tests and criteria in identification of ABPA.

Results

There were 372 asthmatics with a mean age of 35.9 years. The prevalence of Aspergillus sensitization was 53.2%. The sensitivity and specificity of various tests were Aspergillus skin test positivity (94.7%, 79.7%); IgE levels>1000 IU/mL (97.1%, 37.7%); A.fumigatus specific IgE levels>0.35 kUA/L (100%, 69.3%); Aspergillus precipitins (42.7%, 97.1%); eosinophil count>1000 cells/µL (29.5%, 93.1%); chest radiographic opacities (36.1%, 92.5%); bronchiectasis (91.9%, 80.9%); and, high-attenuation mucus (39.7%, 100%). The most accurate criteria was the Patterson criteria using six components followed by the Agarwal criteria. However, there was substantial decline in accuracy of the Patterson criteria if components of the criteria were either increased or decreased from six.

Conclusions

A.fumigatus specific IgE levels and high-attenuation mucus were found to be the most sensitive and specific test respectively in diagnosis of ABPA. The Patterson criteria remain the best diagnostic criteria however they have good veridicality only if six criteria are used.     

Bioleaching of Heavy Metals from Mine Tailings by Aspergillus fumigatus

The bioleaching experiment was conducted for the removal of heavy metals from mine tailings. A fungal strain was isolated from the gold mine tailings and it has been identified as Aspergillus fumigatus based on its 18S rDNA analysis. Bioleaching using A. fumigatus was carried out in bioleaching step processes (one-step and two-step) at various tailings concentrations (1%, 2%, 4%, and 8% [w/v]). In the one-step bioleaching process where fungi were cultivated in the presence of the tailings, concentration of oxalic acid was the highest among the organic acids produced. On the other hand, in the two-step bioleaching process where the metabolic products of fungal growth, which have been separated from its biomass, were used, citric acid was dominant. In the one-step process, the highest As (62%), Fe (58%), Mn (100%), and Zn (54%) removals were observed at the lowest tailings concentration (1%). The removal of Pb at 1% tailings concentration in the one-step process was 56%, whereas 88% removal was achieved in the two-step process where citric acid was dominant. In general, heavy metals removal efficiency decreased with increased tailings of the concentration in both bioleaching processes. This study shows the possibility of using A. fumigatus to bioleach hazardous heavy meals from gold mine tailings.     

Prevalence of Opportunistic Thermophilic Actinomycetes in the Atmosphere of Cairo,Egypt

Thermophilic Actinomycetes were isolated by means of Andersen Sampler from four sampling sites. The total count recorded was relatively low. Four different opportunistic species were isolated in this study, Saccharomonospora viridis (syn. Thermomonospora viridis) 17%, Thermoactinomyces vulgaris (syn. T. candidus) 16.2%, Micropolyspora faeni 6.7% and Thermoactinomyces sacchari 5.2%.     

Analysis of mechanisms regulating the expression of parental alleles at the GPD locus in mule erythrocytes

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was examined by 13% starch gel electrophoresis in 74 mules (42 females and 32 males), 35 donkeys, and ten horses. The quantitative expression of the parental alleles at the Gpd locus varies greatly in female mules from the hemizygous expression of the maternal allele to that of the paternal. The data obtained indicate that the X chromosomes are randomly inactivated in female mules. No selective advantage of a cell population with a maternally (or paternally) derived X active was found in female mule erythrocytes. It is suggested that the phenotypic variability in the expression of the parental Gpd alleles is related to the random proportions established between cells having either a maternal or paternal X active in an initiator (stem) cell group giving rise to erythroid tissue. Initiator cell numbers estimated for erythroid tissue (six or seven) are close to those reported for human females and intergeneric fox hybrids. These numbers may vary depending on the duration of the time of determination and the division rate of initiator cells at determination.     

Species-specific antigens ofMycoplasma hyopneumoniae and cross-reactions with other porcine mycoplasmas

Cell proteins from the porcine mycoplasmasMycoplasma hyorhinis, M. hyopneumoniae, andM. flocculare have been analyzed by SDS-gel electrophoresis and immunoblotting. The protein profiles ofM. hyopneumoniae andM. flocculare were similar, but the protein profile ofM. hyorhinis was quite different from the others. Antisera prepared against whole cells of the above three mycoplasmas were used in immunoblotting of electrophoretically separated antigens and in enzyme-linked immunosorbent assay. One major antigen, which had a molecular weight of 73 k, was found to be common to all three mycoplasmas. Another major antigen, with a molecular weight of 41 k, was common toM. hyopneumoniae andM. flocculare and may also be present inM. hyorhinis. Several antigens of comparatively high molecular weights (108 k, 102 k, 93 k, 89 k, and 87 k) seemed to be specific forM. hyopneumoniae. Three antisera prepared by immunization of rabbits with immunoprecipitates obtained by crossed immunoelectrophoresis ofM. hyopneumoniae were also used in blotting experiments. One of these antisera was found to be directed against the 73 k antigen common to the three porcine mycoplasmas investigated. The other two antisera were directed againstM. hyopneumoniae-specific antigens with molecular weights of 74 k, 58 k, 45 k, 44 k, and 38 k.     

Experimental avian aspergilosis

One-day-old chicks were infected by aerosolized conidia ofAspergillus fumigatus orA. flavus. Plaques containing viable organisms were observed on the third day of infection. Although plaques persisted for several weeks, cultures could not be recovered from them after about ten days. Flourescent antibody methods revealed an immune response to both agents within three days of challenge. Precipitin titers againstAspergillus active protein (AAP) did not appear until the tenth day, and was not detected after seven weeks. Aspergillus polysaccharide (APS) failed to react antigenically in infected birds. AAP was separated by Sephadex chromatography into nine distinct fractions. One fraction from each species was associated with an immediate and one with a delayed type of skin response in wattles. A low molecular weight fraction which lacked skin reactivity was an effective antigen in precipitin tests. Except for a weak skin reaction toA. fumigatus APS in rabbits, skin tests with extracts and APP fractions correlated with wattle reactions in chicks.     

The microbiology of moist barley storage in unsealed silos

The microflora of moist barley grain and whole-crop barley silage stored in top-unloading, unsealed concrete-staved silos on six farms in England depended on the initial water content of the grain (23–58%), method of covering the grain, and the rate at which it was unloaded. Fungi and actinomycetes were fewest when the initial water content was more than 30%, and the grain was covered first with a layer of wilted grass, and then a plastic sheet. During unloading, the uppermost layer of grain remained in good condition provided 7.5 cm was removed daily. With an inefficient top-seal, the top grain heated and became mouldy, as it also did when unloading was slow. As the rate of unloading slowed, heating increased, and a characteristic succession of fungi and actinomycetes developed. With unloading at 7.5 cm/day or more, only yeasts, chiefly Endomycopsis chodatii Wickerham and Hansenula anomala (Hansen) H. & P. Sydow, were abundant, but at slightly slower rates of unloading Penicillium spp. also became common. Both these groups became less common as unloading was slowed further and were replaced, first by Absidia spp. and Mucor pusillus Lindt, then Aspergillus fumigatus Fres., Humicola lanuginosa (Griffon & Maublanc) Bunce, Micropolyspora faeni Cross, Maciver & Lacey, and Thermoactinomyces vulgaris Tsiklinsky as the heating increased. The number of spores (including bacterial cells) that could be removed from samples by blowing air ranged from 0.4–428 times 10⁶/g dry weight of grain. Whole-crop barley silage contained 2.9–132 times 10⁶spores/g dry weight. Similar species were isolated from whole-crop silage as from grain. Little moulding occurred deeper than 30 cm below surface of the grain. Concentrations of airborne spores were estimated periodically during two seasons. There were always more airborne spores than is usual in outdoor air. Without disturbance the silos contained 10⁶-10⁷ spores/m³ air, but when mouldy grain was unloaded concentrations increased to a maximum of 2860times10⁶ spores/m³ air; more than half of these were bacteria and actinomycetes and a quarter Aspergillus flavus Link. Potentially pathogenic fungi and actinomycetes were frequent, particularly when they also occurred in the grain or capping materials for spontaneous heating. Some probably survived in dust deposits and were resuspended during unloading. Airborne spores were frequent around the silos when grain was unloaded and rolled. Workers should wear efficient dust respirators at these times and while inside silos.     

MonitoringAspergillus fumigatus aerosols from a composting facility

The large, outdoor Islip Yard Waste Composting Facility on Long Island, New York was investigated as a source of airborne fungus spores. The Burkard-Hirst volumetric spore trap was used for the first extensive sampling of small mold spores for this application. Samplers were operated continuously from 21 August to 30 November 1992 in the facility and in a suburban community about 540 m from the facility. A control site approximately 10 000 m from the facility was also sampled to establish background levels of fungus spores. The facility site had higher average readings ofAspergillus fumigatus spores than did the community and both were higher than the control.A. fumigatus was the only fungus among 30 categories tracked that differed significantly between the facility and control sites. It was also isolated repeatedly from the compost. Higher average levels ofA. fumigatus were measured in the community when winds blew from the facility, and also during times when the compost was moved or mixed at the facility. No correlation was found between wind direction or work times andA. fumigatus conidia at the control site. The study shows that this compost facility can produce a measurable increase in the number of airborneA. fumigatus conidia both at the edge of the facility and at 540 m downwind. It also demonstrates that the Burkard spore trap can be used for monitoring small, airborne mold spores, but it is a difficult and labor intensive task.     

Re-identification of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> sensu lato based on a new concept of species delimitation

The species concept of Aspergillus fumigatus sensu stricto has recently been defined by polyphasic taxonomy. Based on the new concept of species delimitations, 146 worldwide strains of Aspergillus fumigatus sensu lato were re-identified. Of those 146 strains, 140 (95.8%) could be identified as A. fumigatus sensu stricto, 3 (2.1%) as A. lentulus, and the remaining 3 strains as A. viridinutans complex, Neosartorya udagawae, and N. cf. nishimurae. Of 98 clinical strains, only 1 from dolphin nostril was identified as A. lentulus and not A. fumigatus sensu stricto. Random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers PELF and URP1F produced nearly the same band patterns among 136 strains of A. fumigatus sensu stricto while discriminated the species from its related species. We also discussed about identification of several atypical A. fumigatus strains from clinical environments.     

Gliotoxinogenic Aspergillus fumigatus in the dairy herd environment

The potential association between hygienic conditions in the environment of lactating cows and the presence of gliotoxinogenic Aspergillus fumigatus strains was studied. Milk samples (individual cow’s milk [ICM], bulk tank milk [BTM]) from 44 dairy farms were sampled. In ICM samples, eight different species of Aspergillus were identified. A. flavus and A. fumigatus were predominant, with 37.8 % and 26.1 % relative densities, respectively. A. fumigatus strains were isolated from 61.4 % of the BTM samples, and 34 % of these strains were able to produce gliotoxin. Principal component analysis was used to associate the presence of A. fumigatus with some hygienic and sanitary practices. A significant and positive correlation was observed between dry cow therapy and forestripping. The presence of A. fumigatus gliotoxin producers in milk was associated with high somatic cells count (SCC) samples. Good hygienic and sanitary practices were associated with absence of A. fumigatus and relatively low SCCs of <250,000 cells/ml. In general, a high percentage of dairy farms were positive for A. fumigatus in BTM samples. This is the first work that indicates the positive effects of adequate hygienic and sanitary practices in dairy herds on the control of A. fumigatus and related species. By reducing the frequency of Aspergillus spp. in the dairy environment, the risk of farm handlers’ exposure and the risk of intramammary fungal infections would also be reduced.