Application of an in situ hybridization method for fungal diagnosis in formalin-fixed, paraffin-embedded allergic fungal rhinosinusitis tissue

Abstract

Allergic fungal rhinosinusitis (AFRS) is a chronic inflammatory disease of the sinuses that can involve serious late complications; thus, prompt diagnosis is essential to determine the appropriate treatment. The most important diagnostic element of AFRS is the detection of noninvasive fungi within eosinophilic mucin. However, the rarity of fungal hyphae in ARFS makes it difficult to specifically identify them using histochemical staining alone. In this study, we designed a new in situ fungal detection probe for the diagnosis of fungi in formalin-fixed, paraffin-embedded AFRS tissues. Tissue sections from 49 patients with confirmed (n = 40) or suspected (n = 9) AFRS were selected for testing. A newly designed broad-spectrum probe for in situ hybridization (ISH) was compared with an anti-Aspergillus antibody in immunohistochemistry (IHC) and staining with hematoxylin and eosin and periodic acid-Schiff (PAS) to detect fungi. Hematoxylin and eosin staining had a lower detection rate (30/40 samples) than the other three methods. PAS staining led to two false-positive results in the AFRS-confirmed group and two false-negative results in the AFRS-suspected group. ISH and IHC exhibited high concordance (ĸ = 0.716); however, there was a high degree of nonspecific immunoreactivity to the anti-Aspergillus polyclonal antibody in some samples. The fungal detection rate of ISH was 95 % (38/40), with no background or nonspecific reactivity. Our novel broad-spectrum ISH probe provides more specific identification of fungi than PAS and IHC staining, exhibits no background reactivity, and may represent an essential upgrade to the in situ diagnosis of AFRS.