Erika Janotka

Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI-TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI-TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI-TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI-TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.

Detection of microbial antigens in metal working fluids

In recent years there have been several reports of hypersensitivity pneumonitis (HP) or an HP-like illness occurring among machinists working with water-based metal working fluids (MWF). Microbial contamination of the MWF is common and microbial agents have been suspected to be causal agents for the HP-like illness, but no specific etiologic agent has been identified to date. In particular, gram negative bacteria and biocide resistant mycobacterial species may colonize the MWF, and may stimulate an inflammatory response if inhaled. Because direct culture techniques provide data only about viable organisms present at the time the sample was collected, we have been evaluating techniques to detect microbial substances (antigens) that may be present and persist in the MWF. We have found that the endotoxin of gram negative bacteria can be detected in MWF using the limulus amoebocyte lysate (LAL) assay, and may be present in high concentrations. In addition, MWF samples have been analyzed by Western Blot techniques using polyclonal antibodies to mycobacteria to demonstrate the presence of mycobacterial antigens in these samples. The use of non-culture-based techniques for the assessment of microbial contamination of MWF may help to determine the role of microorganisms in the etiology of HP associated with MWF exposure.

Monoclonal Antibodies to Hyphal Exoantigens Derived from the Opportunistic Pathogen Aspergillus terreus

ABSTRACT Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.

Post-hire asthma among insect-rearing workers.

Objective: To evaluate the incidence of post-hire asthma (PHA) among insect-rearing workers, defined as asthma, the symptoms of which appeared after hire at the current workplace. Methods: We surveyed the health of workers at three insect-rearing facilities and an associated office facility. We calculated the incidence and estimated hazard ratios for PHA. Results: Post-hire asthma incidence in 157 insect-rearing workers was 16.2 per 1000 person-years compared with 9.2 per 1,000 person-years in 70 office workers. Workers with predominant exposure to Lepidoptera had an incidence of 26.9 per 1000 person-years and a hazard ratio of 5.5 (95% confidence interval: 1.6 to 23.9) adjusted for sex, race, and parental asthma. In contrast, the presence of specific immunoglobulin E to Lepidoptera antigens was not associated with PHA. Conclusion: Insect-rearing workers had a high incidence of PHA, primarily accounted for by workplace exposure to Lepidoptera.

Production and Characterization of IgM Monoclonal Antibodies Against Hyphal Antigens of Stachybotrys Species

Stachybotrys is a hydrophilic fungal genus that is well known for its ability to colonize water-damaged building materials in indoor environments. Personal exposure to Stachybotrys chartarum allergens, mycotoxins, cytolytic peptides, and other immunostimulatory macromolecules has been proposed to exacerbate respiratory morbidity. To date, advances in Stachybotrys detection have focused on the identification of unique biomarkers that can be detected in human serum; however, the availability of immunodiagnostic reagents to Stachybotrys species have been limited. In this study, we report the initial characterization of monoclonal antibodies (MAbs) against a semi-purified cytolytic S. chlorohalonata preparation (cScp) derived from hyphae. BALB/c mice were immunized with cScp and hybridomas were screened against the cScp using an antigen-mediated indirect ELISA. Eight immunoglobulin M MAbs were produced and four were specifically identified in the capture ELISA to react with the cScp. Cross-reactivity of the MAbs was tested against crude hyphal extracts derived from 15 Stachybotrys isolates representing nine Stachybotrys species as well as 39 other environmentally abundant fungi using a capture ELISA. MAb reactivity to spore and hyphal antigens was also tested by a capture ELISA and by fluorescent halogen immunoassay (fHIA). ELISA analysis demonstrated that all MAbs strongly reacted with extracts of S. chartarum but not with extracts of 39 other fungi. However, four MAbs showed cross-reactivity to the phylogenetically related genus Memnoniella. fHIA analysis confirmed that greatest MAb reactivity was ultrastructurally localized in hyphae and phialides. The results of this study further demonstrate the feasibility of specific MAb-based immunoassays for the detection of S. chartarum.