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Immunohistologic identification of Aspergillus spp. and other hyaline fungi by using polyclonal fluorescent
L Kaufman1, P G Standard, M Jalbert
1Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
Abstract:
Isolation and identification of pathogenic Aspergillus and Fusarium spp. from clinical materials provide the most accurate means for establishing a diagnosis of infections by these molds. Such efforts, however, are not always successful. Histologic diagnosis also has its limitations. In vivo the hyphae of Aspergillus and Fusarium spp. are very similar and their in situ manifestations are not pathognomonic. To improve the histologic diagnosis of infections by Aspergillus and Fusarium species, we developed polyclonal fluorescent-antibody reagents to Aspergillus fumigatus and Fusarium solani and evaluated their diagnostic utilities. Our studies revealed that A. fumigatus and F. solani share epitopes not only with one another but also with other Aspergillus and Fusarium spp. as well as with Paecilomyces lilacinus and Pseudallescheria boydii. Adsorption of the A. fumigatus conjugate with cells of Fusarium proliferatum and F. solani and F. solani antiserum with cells of Aspergillus flavus resulted in reagents that distinguished Aspergillus spp. from Fusarium spp. but that still cross-stained P. lilacinus and P. boydii. Adjunctive use of a specific P. boydii conjugate enabled the identification of Aspergillus spp., Fusarium spp., P. lilacinus, and P. boydii in formalin-fixed tissue sections from 19 humans with culture-proven cases of mycotic infection.
Insights
Developing fluorescent-antibody reagents improves the diagnosis of mold infections. These antibodies help identify Aspergillus and Fusarium species in clinical samples, overcoming limitations of traditional methods for accurate mold infection diagnosis.
Area of Science:
- Medical Mycology
- Immunohistochemistry
- Diagnostic Microbiology
Background:
- Accurate diagnosis of mold infections, particularly those caused by Aspergillus and Fusarium species, is crucial for effective treatment.
- Traditional diagnostic methods like culture and histology have limitations, including potential failure and difficulty in differentiating morphologically similar fungi.
- In vivo, the hyphae of Aspergillus and Fusarium species appear similar, complicating definitive histologic diagnosis.
Purpose of the Study:
- To develop and evaluate polyclonal fluorescent-antibody reagents for improved histologic diagnosis of Aspergillus and Fusarium infections.
- To enhance the specificity of antibody reagents to accurately differentiate between Aspergillus, Fusarium, and other potentially cross-reacting fungal species.
- To assess the utility of these reagents in identifying multiple fungal pathogens in formalin-fixed tissue sections.
Main Methods:
- Development of polyclonal fluorescent-antibody reagents targeting Aspergillus fumigatus and Fusarium solani.
- Evaluation of antibody cross-reactivity with various Aspergillus, Fusarium, Paecilomyces lilacinus, and Pseudallescheria boydii species.
- Adsorption techniques were employed to enhance reagent specificity, distinguishing Aspergillus from Fusarium.
- Adjunctive use of a specific Pseudallescheria boydii conjugate was implemented for comprehensive identification.
Main Results:
- Initial reagents showed cross-reactivity between Aspergillus and Fusarium species, as well as with Paecilomyces lilacinus and Pseudallescheria boydii.
- Adsorption of conjugates significantly improved differentiation between Aspergillus and Fusarium species.
- Cross-staining with Paecilomyces lilacinus and Pseudallescheria boydii persisted after adsorption.
- The combined use of specific conjugates successfully identified Aspergillus spp., Fusarium spp., Paecilomyces lilacinus, and Pseudallescheria boydii in 19 clinical cases.
Conclusions:
- Polyclonal fluorescent-antibody reagents, when properly adsorbed, can improve the histologic differentiation of Aspergillus and Fusarium species.
- Adjunctive use of specific conjugates is essential for accurate identification of multiple fungal pathogens, including Paecilomyces lilacinus and Pseudallescheria boydii.
- This immunofluorescence approach offers a valuable tool for diagnosing complex mycotic infections in formalin-fixed tissues.