Mannan immunoassay for diagnosis of invasive mucormycosis: assay development, preclinical testing and evaluation in an animal model.

Early diagnosis of mucormycosis would be greatly facilitated by identification of a non-invasive biomarker for infection. This study evaluated whether detection of Mucorales mannan in serum or bronchoalveolar lavage fluid (BALF) can serve as such a biomarker. To date, mannan has been found in body fluids of invasive disease caused by many ascomycetous fungal pathogens, but its presence has not been evaluated for disease-producing members of the Mucorales. We utilized a monoclonal antibody (mAb) that is reactive with Mucorales mannan to construct a lateral flow immunoassay (LFA) containing labeled europium nanoparticles as the detector reagent. Analytical inclusivity studies found that the LFA detected mannans from four of the five disease-producing Mucorales fungi. No cross-reactivity was observed with purified mannans from other major causes of invasive fungal infection, i.e. Aspergillus, Candida and Fusarium. The LFA was evaluated in a pulmonary mouse model of invasive mucormycosis using serum and BALF specimens. Mannan was detectable in both specimens and increased in concentration through the course of infection. Lastly, an exploratory study for clinical proof-of-concept was performed with a limited set of sera or plasma samples collected at one or more timepoints from patients with probable or proven mucormycosis. Four of the 12 patients had positive test results at one or more times. Treatment with amphotericin B was associated with negative LFA results: among the six patients for whom a specimen was collected while they were not receiving AmB, 4 of the 6 patients had positive test results at one or more times. One patient whose blood was collected prospectively showed a high level of mannan seven days before meeting the EORTC diagnostic criteria. Taken together, the results demonstrate, for the first time, that mannan in blood samples and BALF is a biomarker for infection by the Mucorales fungi and support further study of mannan-targeted diagnostics for mucormycosis.