The global incidence of invasive fungal infections (IFIs) has increased over the past few decades, mainly in immunocompromised patients, and is associated with high mortality and morbidity.
Aspergillus fumigatus
is one of the most common and deadliest IFI pathogens. Major hurdles to treating fungal infections remain the lack of rapid and definitive diagnosis, including the frequent need for invasive procedures to provide microbiological confirmation, and the lack of specificity of structural imaging methods. To develop an
Aspergillus
-specific positron emission tomography (PET) imaging agent, we focused on fungal-specific sugar metabolism. We radiolabeled cellobiose, a disaccharide known to be metabolized by
Aspergillus
species, and synthesized 2-deoxy-2-[
18
F]fluorocellobiose ([
18
F]FCB) by enzymatic conversion of 2-deoxy-2-[
18
F]fluoroglucose ([
18
F]FDG) with a radiochemical yield of 60 to 70%, a radiochemical purity of >98%, and 1.5 hours of synthesis time. Two hours after [
18
F]FCB injection in
A. fumigatus
pneumonia as well as
A. fumigatus
, bacterial, and sterile inflammation myositis mouse models, retained radioactivity was only seen in foci with live
A. fumigatus
infection. In vitro testing confirmed production of β-glucosidase enzyme by
A. fumigatus
and not by bacteria, resulting in hydrolysis of [
18
F]FCB into glucose and [
18
F]FDG, the latter being retained by the live fungus. The parent molecule was otherwise promptly excreted through the kidneys, resulting in low background radioactivity and high target-to-nontarget ratios at
A. fumigatus
infectious sites. We conclude that [
18
F]FCB is a promising and clinically translatable
Aspergillus
-specific PET tracer.