A microscopy-free approach for detection of fungal elements in tissue using mannan immunoassay

ABSTRACT KOH microscopy is a routine method to determine the presence of fungal elements in infected tissue. Although widely used, the procedure requires trained personnel and the infrastructure needed for microscopy. An alternative approach is the extraction of fungal antigens and detection by immunoassay. In this study, we used oxidative release with sodium hypochlorite to extract mannan from fungal cells. Quenching of free sodium hypochlorite was done using an excess of bovine serum albumin or sodium thiosulfate. Quenching reagents were incorporated into sample pads of lateral flow immunoassays (LFIAs), followed by detection of released mannan using mannan-specific monoclonal antibodies coupled to colloidal gold on the LFIA platform. Cells of Candida spp. or Aspergillus spp. were used to evaluate the extraction/quenching steps. The results showed that sodium thiosulfate was particularly well suited for hands-free, in-strip neutralization. The analytical sensitivity (minimum limit of detection, LOD) of the Candida LFIA was 67 ng/mL of formaldehyde-inactivated, whole Candida albicans cells. The LOD of the Aspergillus LFIA was 250 ng/mL of formaldehyde-inactivated, whole Aspergillus fumigatus cells. Proof-of-concept for oxidative release of mannan followed by detection of antigen by LFIA was provided using an ex vivo porcine corneal model of fungal keratitis. The results demonstrated that the test is a rapid two-step, specimen-to-result protocol suitable for point-of-care use. Such a diagnostic would be particularly valuable in settings that lack access to the trained personnel or infrastructure needed for KOH microscopy. IMPORTANCE KOH microscopy is widely used to detect fungal infection, but it requires trained personnel and microscopy infrastructure. An alternative approach is extraction of antigens from fungal cells within infected tissue and detection by lateral flow immunoassay. In this study, we developed a <20 min, specimen-to-result protocol suitable for point-of-care use that combines (i) oxidative release via sodium hypochlorite to rapidly extract fungal mannan from specimens, (ii) on-strip (hands-free) quenching of sodium hypochlorite with sodium thiosulfate or bovine serum albumin, and (iii) visual detection of positive signal via novel, mannan-specific monoclonal antibodies coupled to colloidal gold. Analytical performance of the Candida and Aspergillus tests was evaluated with cultured fungal cells. Clinical proof-of-concept for each assay was provided using an ex vivo porcine corneal model of fungal keratitis. This diagnostic strategy would be particularly valuable in settings that lack access to trained personnel or the infrastructure needed for KOH microscopy.