Breath-Based Diagnosis of Invasive Mucormycosis (IM)
Authors: Koshy Seena , Ismail Nour , Astudillo Carmen Leon , Haeger Christina Mallarino , Aloum Obadah , Acharige Mahesh Thalavitiya , Farmakiotis Dimitrios , Baden Lindsey R , Marty Francisco M , Kontoyiannis Dimitrios P , Fredenburgh Laxura , Koo Sophia 
Published: October 2017

Abstract

Background
Timely diagnosis of IM remains a major challenge in clinical mycology. Because of the lack of specific diagnostic methods for IM and the frequently fulminant nature of this infection, IM-associated mortality remains high.

Methods
We examined breath volatile metabolite profiles in a neutropenic murine model of IM, using the 3 Mucorales species that most commonly cause human IM - Rhizopus arrhizus var. arrhizus, R. arrhizus var. delemar, and R. microsporus - and for comparison, Aspergillus fumigatus. We infected female balb/c mice (N = 4 per group) treated with cyclophosphamide and cortisone followed by intranasal administration of 106 conidia of each species. 3 days post-infection, we collected breath samples from each mouse via tracheostomy using a flexiVent murine ventilator, examining breath volatile metabolites using thermal desorption gas chromatography/tandem mass spectrometry (GC-MS/MS). We also sampled breath prospectively from five patients eventually diagnosed with proven IM caused by R. microsporus, analyzing breath volatile metabolites using thermal desorption GC-MS/MS.

Results
Each Mucorales species produced a consistent profile of breath sesquiterpene secondary metabolite VOCs in our murine models, which distinguished these species from each other and from murine invasive aspergillosis (Figure A). These fungi shifted their secondary metabolism significantly in vivo, compared with their previously characterized in vitro metabolism. We found overlapping VOC sesquiterpene metabolites between breath samples from the murine model of R. microsporus infection and 5 of 5 patients with R. microsporus IM, with additional sesquiterpene secondary metabolites detected in patient breath, compared with the murine IM model (Figure B). In one patient with serial breath samples, these sesquiterpenes declined in abundance and disappeared with antifungal therapy, in parallel with clinical improvement (Figure C).

Conclusion
The three Mucorales species that cause most human IM have distinct breath sesquiterpene profiles that can be used to identify these infections in vivo noninvasively. These profiles distinguish these infections from each other and from aspergillosis, and may be useful in monitoring clinical response to treatment.

Disclosures
F. M. Marty, Astellas Pharma US: Consultant and Grant Investigator, Consulting fee and Grant recipient; Chimerix: Consultant and Grant Investigator, Consulting fee and Grant recipient; Fate Therapeutics: Scientific Advisor, Consulting fee; Gilead Sciences: Consultant and Grant Investigator, Consulting fee and Grant recipient; LFB: Consultant, Consulting fee; Merck: Consultant, Grant Investigator and Scientific Advisor, Consulting fee and Grant recipient; Roche Molecular Systems: Consultant, Consulting fee; Shire: Consultant and Grant Investigator, Consulting fee and Grant recipient; D. P. Kontoyiannis, Pfizer: Research Contractor, Research support and Speaker honorarium; Astellas: Research Contractor, Research support and Speaker honorarium; Merck: Honorarium, Speaker honorarium; Cidara: Honorarium, Speaker honorarium; Amplyx: Honorarium, Speaker honorarium; F2G: Honorarium, Speaker honorarium