Usefulness of Gram Stains for Identification of Morphologically Similar Fungi in Tissue Sections and Cytologic Preparations.
Ashley A Kelly, Raul E Villanueva, Thomas E Davis, Alison S Burris, Stephen D Allen. Indiana University, Indianapolis
Background: Fungi cause significant morbidity and mortality. Microscopic features are useful in differentiating fungi, but several commonly encountered species share similar appearances making definitive identification difficult. Atypical morphology of some fungi can also lead to errors in diagnosis. Gram's stain, when used in combination with other special stains, can aid in making diagnoses. However, differences in the results of tissue Gram stain methods can lead to ambiguity or cause diagnostic errors. The primary object of this study was to evaluate the usefulness of Gram stains as a means of differentiating between commonly encountered fungi in both paraffin sections of tissue and in cytologic preparations.
Design: Paraffin-embedded tissue specimens from infections caused by species of Candida, Histoplasma, Blastomyces, Cryptococcus, Aspergillus, and Zygomycetes were studied. In Part one, we compared the following four different tissue Gram stain procedures: an instrument procedure (IP, Artisan™, Dako), Brown-Hopps (BH), Brown and Brenn (B&B), and a modified Hucker and Conn (H&C) manual Gram stain (Sigma-Aldrich®). Included in Part two of the study were cytologic specimens from the last 2.5 years that were all analyzed using Diff-Quik, Giemsa, GMS, calcofluor white and Gram stains. Identities of fungi were confirmed by microbiologic cultures or DNA probes.
Results: The modified H&C and BH procedures showed consistently reliable staining of Candida (14/14). The B&B (13/14) and IP (12/14) procedures were less reliable. Cryptococcus also showed consistent positive staining (5/5) with the modified H&C. Histoplasma (0/13) and Blastomyces (0/6) consistently failed to stain with the modified H&C method, but less consistently with the other Gram stain methods. In cytologic preparations, all Candida species (20/20) were stained dark blue. Again, Histoplasma (0/10) and Blastomyces (0/3) did not retain the crystal violet stain. Candida pseudohyphae stained stongly blue with the Gram stain (10/10); Aspergillus and Zygomycetes (0/8) stained irregularly or not at all.
Conclusions: Gram's stain aids in differentiating both yeast-like and filamentous fungi. Species of Candida and Cryptococcus yeasts as well as Candida pseudohyphae stain intensely dark blue (positive) with the Gram stain while other common pathogenic yeasts and filamentous fungal hyphae do not.
Wednesday, March 2, 2011 1:00 PM
Poster Session VI # 225, Wednesday Afternoon