Rapid oxidative release of fungal mannan for detection by immunoassay.

Detection of fungal cells in infected tissue by procedures such as potassium hydroxide (KOH) microscopy and histopathology are well-established methods in medical mycology. However, microscopy requires skilled personnel, specialized equipment, and may take considerable time to a result. An alternative approach is immunoassay for detection of fungal mannans in tissue as a biomarker for the presence of fungal cells. However, mannan is a component of the fungal cell wall, and detection of mannan would require a facile means for mannan extraction prior to detection by immunoassay. In this study, we evaluated a broad spectrum of extraction reagents using Trichophyton rubrum mycelia and Saccharomyces cerevisiae Mnn2 blastoconidia as model fungi. Oxidative release by treatment with dilute bleach proved to be a novel and highly effective procedure. Complete extraction occurred in as little as 2-4 min. Detergents, chaotropes, and acid were ineffective. Strong base released mannan but was less efficient than oxidative release and required the use of highly corrosive reagents. Oxidative release of cell wall mannans from fungal mycelia and blastoconidia may be an effective first step in immunodetection of fungi in tissues from infected humans, animals, or plants that could be done at or near the diagnostic point of need.

Mannans are components of the fungal cell wall that play a role in disease production and are potential biomarkers for the diagnosis of infection. Oxidative release of mannans from intact cell walls is a novel method for mannan extraction that is rapid, uses relatively mild reagents, and yields soluble mannans that are readily detected by immunoassay.

Conservation of Mannan Synthesis in Fungi of the Zygomycota and Ascomycota Reveals a Broad Diagnostic Target.

Ascomycetes and zygomycetes account for the majority of (i) fungi responsible for cutaneous, subcutaneous, and invasive human fungal infections, (ii) plant fungal pathogens, (iii) fungi that threaten global biodiversity, (iv) fungal agents of agricultural spoilage, and (v) fungi in water-damaged buildings. Rapid recognition of fungal infection (or contamination) enables early treatment (or remediation). A bioinformatics search found homologues of Saccharomyces cerevisiae Mnn9p present in members of the Zygomycota and Ascomycota phyla and absent in members of the Chytridiomycota and Basidiomycota. Mnn9p is a component of the yeast mannan polymerization complex and is necessary for α-1,6 mannan production. A monoclonal antibody (2DA6) was produced that was reactive with purified mannans of Mucor, Rhizopus, Aspergillus, Fusarium, and Candida species. Experimentation using a 2DA6 antigen capture enzyme-linked immunosorbent assay (ELISA) and extracts of fungi from the four phyla found agreement between the presence or absence of Mnn9p homologues and production or lack of production of mannan reactive with 2DA6. Studies of cell extracts from yeast mannan mutants identified α-1,6 mannan as the epitope recognized by 2DA6. To translate this finding into a point-of-use diagnostic, a 2DA6 lateral flow immunoassay was constructed that detected mannan in (i) extracts of dermatophytes and fungi that produce trauma-related infection and (ii) tissue from plants infected with Grosmannia clavigera or Sclerotium cepivorum These studies (i) revealed that the conservation of α-1,6-linked mannan in fungi of the Zygomycota and Ascomycota can be exploited as a broad diagnostic target and (ii) have provided a means to detect that target in an immunoassay platform that is well suited for clinic or field use.IMPORTANCE A key question asked when faced with an infection, an infestation, or environmental damage is whether it is a fungus. Identification of fungi as the cause of the problem can lead to remediation or treatment. Zygomycetes and ascomycetes account for the vast majority of fungal causes of human, animal, and plant disease, large-scale biodiversity loss, agricultural spoilage, and contamination of water-damaged buildings. These studies revealed the conservation of a common cell wall structural component of zygomycetes and ascomycetes to be a diagnostic target applicable to multiple pathogenic fungi and have leveraged that insight for practical use. Monoclonal antibodies reactive with this pan-fungal structure were produced and used to construct immunoassays (including ELISA and lateral flow assay) for detection of a broad range of pathogenic fungi.