Immunohistochemical diagnosis of fusariosis with monoclonal antibodies

Objectives: Fusariosis is an emerging fungal infection, especially in neutropenic patients. Proper identification of Fusarium spp. is important because the choice of antifungal treatment of fusariosis differs from that of aspergillosis, candidosis or scedosporidiosis (pseudalleceriosis). Cultural isolation attempts from fusarium lesions often fail, and because the tissue forms of Fusarium spp. are histologically indistinguishable from fungal elements of aspergillosis, true hyphae containing candidosis and scedosporidiosis (pseudalleceriosis), alternative diagnostic techniques are often necessary for establishing an accurate diagnosis. Although molecular techniques (e.g. in situ hybridization and PCR) have been explored for diagnostic use, the development of specific monoclonal antibodies (Mabs) for immunohistochemical identification of Fusarium spp. will extend the availability of diagnostic options for fusariosis. A panel of newly developed Mabs for immunohistochemical diagnosis of fusariosis was screened for specificity on experimentally infected laboratory animal tissue and on skin tissue biopsies from two neutropenic patients with Fusarium sepsis. Methods: Somatic antigens were made from F. solani (CBS 166-87). Following growth in Sabouraud-broth (Oxoid) mycelium was harvested and disintegrated in an X-press (AB Biox, Sweden) using a precolled (-30 oC) cylinder operated at a maximal force of 200 MPa. Following centrifugation at 20,000g for 1h, the supernatant was sterile filtrated and constituted the somatic antigen. The production of murine Mabs was carried out as previously described for other fungi (Jensen et al., 1993, APMIS, 101, 505-516). The Mabs were isotyped and tested for reactivity in ELISA and immunoblotting (IB) with the homologous antigen. Reactive Mabs were applied on tissue sections containing homologous (fusariosis) and heterologous (aspergillosis, candidosis, and scedosporidiosis) fungal elements. Tissue reactive Mabs were then tested on skin biopsies from two patients with fusariosis sepsis with dissemination to the skin In the patients, a diagnosis of fusariosis-sepsis had been established by isolation of F. solani and F. proliferatum, respectively. Results: Four myeloma clones producing Mabs of isotype IgM, which reacted similarly with major IB bands of 51 and 63 kDa, revealed an intense staining of fungal elements of fusariosis in both experimentally infected laboratory animals and the two patients. The Mabs showed no reactivity when tested on tissues containing heterologous fungi. Conclusion: Because prompt diagnosis and initiation of treatment is essential to maximize the chance of survival for any patient with fusariosis, the present Mabs are expected to add to a quick and specific diagnosis of this infection in the future.