In situ hybridization in cutaneous deep fungal infections: a valuable diagnostic adjunct to fungal morphology and tissue cultures

Abstract

Dimorphic fungal infections (histoplasmosis, blastomycosis, coccidioidomycosis, and cryptococcosis) can occur in immunocompromised and healthy individuals. Cutaneous involvement is often secondary and may be the presenting sign of systemic disease. These ominous infections are frequently clinically indistinct, and patient prognosis is influenced by a timely diagnosis and treatment. Morphologic differentiation between these organisms is not definitive, and tissue cultures represent the diagnostic gold standard in current day practice. However, tissue cultures are rarely obtained and merely represent an afterthought in seemingly unsuspecting cases. Furthermore, when performed, they may take several days or weeks for completion. In situ hybridization (ISH) utilizing oligonucleotide probes directed against fungal ribosomal RNA is a rapid and accurate assay for the identification of dimorphic fungi in paraffin-embedded tissue sections. We present five patients in whom ISH both prospectively and retrospectively confirmed the presence of a cutaneous infection (histoplasmosis, blastomycosis, coccidioidomycosis, and cryptococcosis). In all of the skin sections analyzed, dimorphic fungi were morphologically apparent but not diagnostically discernible. In summary, ISH is a valuable tool in the prompt diagnosis of cutaneous deep fungal infections.

Challenges and Pitfalls of Morphologic Identification of Fungal Infections in Histologic and Cytologic Specimens

“…Special stains (Gomori-methenamine silver or periodic acid-Schiff with diastase) were used in 86% (32/37) of the correctly identified subgroup and in 70% (7/10) of the incorrectly identified subgroup (P = .34), but neither subgroup incorporated immunohistochemical studies or molecularbased techniques. 1,[17][18][19][20] For accurate fungal identification by subgroup, 13 surgical pathologists were involved in the 37 cases in the correctly identified subgroup, and 9 surgical pathologists were involved in the 10 cases in the incorrectly identified subgroup, indicating that errors were evenly distributed among pathologists (Table 1). In addition, no apparent trend was identified when analyzing overall operator accuracy in fungal identification.…”

“…Although newer immunohistochemical and/or molecular methods have demonstrated improved identification and classification of yeast and hyphal organisms compared with conventional histologic (H&E, special stains) methods, 1,17-20 many of these technologies are not commercially available or are not in widespread use and may also require technical and interpretative proficiency. Of these newer techniques, in situ hybridization is the most promising 1,17,19,20 and may prove useful in providing a rapid, accurate preliminary diagnosis for slow-growing fungal cultures or for cases in which the morphologically visible organism may actually not grow. Familiarity of likely fungal organisms by body site location and immune status can also be advantageous in establishing a differential diagnosis ❚Table 4❚.…”

In Situ Hybridization for rRNA Sequences in Anatomic Pathology Specimens, Applications for Fungal Pathogen Detection

“…31-42 ISH for fungi using DNA oligonucleotide probes show high degrees of specificity (often >90%) and various levels of sensitivity depending on the fungal agent being studied (ranging from 50% to 95%), can be performed in most instances in 1 to 3 hours, and have been used in a variety of settings including evaluation of invasive and noninvasive fungal rhinosinusitis, deep cutaneous fungal infections, pulmonary fungal infections, and for the evaluation of fatal culture negative disseminated fungal infections 26,[34][35][36]40 (Fig. 14-18,31-42 Subsequently, a variety of fungal pathogens have been detected in tissues by ISH with either biotin or digoxigenin probes including yeast (B. dermatitidis, C. immitis, Cryptococcus neoformans, H. capsulatum, and S. schenckii) and filamentous fungi (Mucorales, Pseudallsecheria bodyii, Fusarium sp, Candida sp.…”

Articular Involvement in Disseminated Histoplasmosis in a Kidney Transplant Patient Taking Azathioprine: Figure 1.

“…Diagnosis is based on clinical suspicion with detection of histoplasma antigen in urine or serum or successful demonstration of H. capsulatum in cultures or histologic specimens by in situ hybridization or PCR 8,9 . Traditionally, successful treatment has been accomplished with amphotericin B 10 in patients with fulminant disease or associated sepsis but triazole antifungals are noted to be as effective.…”