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Identification of fungi in tissue sections can be difficult because of limited biopsy tissue with only a few organisms present, or mycelial elements may be the only forms present, rendering common organism types indistinguishable from one another. In situ hybridization may assist in the rapid and accurate identification of such fungi. In this study, DNA probes were directed against the 5S or 18S ribosomal RNA sequences of three groups of fungi with a high degree of specificity for each. Two of the three, Aspergillus and Zygomycetes species, are usually seen in tissue purely in their hyphal forms. The third, Candida species is seen less commonly as predominantly mycelial elements. Probes were tested on 61 formalin-fixed, paraffin-embedded tissue specimens, each with culture-proven involvement by one of these organisms (Candida species, n = 21;Aspergillus species, n = 27;Zygomycetes, n = 13). Accuracy of both in situ hybridization (ISH) and morphology, based on the examination of Grocott methanamine silver (GMS)– and periodic acid-Schiff (PAS)–stained slides, was compared with culture. The results showed that morphologic examination (GMS and PAS) showed a slightly greater sensitivity in detecting the presence of fungi (98%) compared with in situ hybridization (95%). DNA probes, however, were more accurate in correctly identifying those organisms present. Although ISH specific probes showed 97% positive predictive value (PPV), examination of GMS-and PAS-stained slides had an 86% PPV when compared with culture-based identification methods. These results show that ISH, directed against ribosomal RNA, provides a rapid and accurate technique for the identification of mycelial fungal organisms in histologic tissue sections. Its primary use lies in the ability to accurately distinguish between organisms that have similar or identical morphologic features by light microscopy.