title: Intravital excitation increases detection sensitivity for pulmonary tuberculosis by whole-body imaging with β-lactamase reporter enzyme fluorescence.
abstract: Tuberculosis is a pulmonary disease with an especially high mortality rate in immuno-compromised populations, specifically children and HIV positive individuals. The causative agent, Mycobacterium tuberculosis (Mtb), is a very slow growing and difficult organism to work with, making both diagnosis and development of effective treatments cumbersome. We utilize a fiber-optic fluorescence microendoscope integrated with a whole-body imaging system for in vivo Mtb detection. The system exploits an endogenous enzyme of Mtb (β-lactamase, or BlaC) using a BlaC-specific NIR fluorogenic substrate. In the presence of BlaC, this substrate is cleaved and becomes fluorescent. Using intravital illumination of the lung to excite this probe, sensitivity of the optical system increases over trans- and epi-illumination methods of whole-body fluorescence imaging. We demonstrate that integration of these imaging technologies with BlaC-specific fluorescent reporter probe improves the level of detection to ∼100 colony forming units, a 100× increase in sensitivity in comparison to epi-illumination and a 10× increase in sensitivity in comparison to previous work in intravital excitation of tdTomato-expressing Mtb. This lower detection threshold enables the study of early stage bacterial infections with clinical strains of Mtb and longitudinal studies of disease pathogenesis and therapeutic efficacy with multiple time points in a single animal.
authors.affiliation: Biomedical Engineering Department, 3120 TAMU, Texas A&M University, College Station, TX, 77843, United States.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Nooshabadi
authors.affiliation: Microbial Pathogenesis and Immunology Department, Texas A&M University Health Science Center, Bryan, TX, 77807, United States.
authors.forename: Hee-Jeong
authors.initials: HJ
authors.lastname: Yang
authors.affiliation: Radiology Department, Stanford University, Stanford, CA, 94304, United States.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Biomedical Engineering Department, 3120 TAMU, Texas A&M University, College Station, TX, 77843, United States.
authors.forename: Madeleine S
authors.initials: MS
authors.lastname: Durkee
authors.affiliation: Radiology Department, Stanford University, Stanford, CA, 94304, United States.
authors.forename: Hexin
authors.initials: H
authors.lastname: Xie
authors.affiliation: Radiology Department, Stanford University, Stanford, CA, 94304, United States.
authors.forename: Jianghong
authors.initials: J
authors.lastname: Rao
authors.affiliation: Microbial Pathogenesis and Immunology Department, Texas A&M University Health Science Center, Bryan, TX, 77807, United States.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Cirillo
authors.affiliation: Biomedical Engineering Department, 3120 TAMU, Texas A&M University, College Station, TX, 77843, United States.
authors.forename: Kristen C
authors.initials: KC
authors.lastname: Maitland
mesh_terms: D000818:Animals; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis; D061848:Optical Imaging; D012680:Sensitivity and Specificity; D014397:Tuberculosis, Pulmonary / Q000000981:diagnostic imaging*; D051598:Whole Body Imaging; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D001618:beta-Lactamases
keywords: fluorescence; in vivo imaging; intravital microscopy; tuberculosis; whole-animal imaging