Radiolabeled analogs of FIAU have been used to image bacterial infection in experimental models and in human subjects previously [18
]. Due to the presence of sequence homology within the consensus catalytic domain of TK in several different bacterial strains, most bacteria and mycoplasma are able to metabolize FIAU. However, the consensus sequence does not exist in mammalian TKs, which explains the poor capability of mammalian cells to metabolize FIAU. We undertook the current study with two goals in mind: 1) to validate the method, i.e., determine whether or not [125
I]FIAU was specific for infection, or could also image sterile inflammation; and, 2) to estimate the sensitivity of detection of this radiopharmaceutical-based technique and define whether it could be used effectively in longitudinal murine infection models to test new antibiotics. We demonstrate using SPECT-CT that [125
I]FIAU indeed does not show specific uptake in sterile inflammation, corroborating earlier experiments that employed this tracer in ex vivo
studies performed in rats [30
]. In contrast to earlier studies in which [125
I] FIAU was used to image localized bacterial infection [18
] or caseous lesions within the lungs caused by M. tuberculosis
], the method of bacterial infection by the intranasal route employed in this study resulted in infections that assume a more diffuse pattern within the lungs. Although in the present study we used [125
I] FIAU, using positron-emitting analogs of FIAU combined with positron emission tomography (PET) would enable higher sensitivity (5 – 50 fold) [31
], which would enable imaging of fewer bacteria. Despite the diffuse pattern of the infection and the use of a low-energy radionuclide not optimized for imaging ([125
I]FIAU), we showed that we were able to detect and image lung infection following inoculation with TK-positive E. coli
. The level of detection that can be visualized in a SPECT scan corresponded to a bacterial concentration in the lungs of 109
CFU/mL. In spite of the fact that the %ID per mL within the lungs is significantly higher than the controls at a much lower bacterial load, we define the level of detection when a distinct signal can be visualized over the lungs, and this corresponds to 109
CFU/mL. Although the number of bacteria present on the image to allow visual detection is several orders of magnitude higher than for fluorescent probes [32
] and bioluminescence (~ 106
CFU/lung), bioluminescence requires genetic manipulation of the cells (introduction of a luciferase) [35
], administration of large amounts of luciferin (on the order of 100 mg/kg), and is not readily amenable to quantification due to the attenuation of signal through the tissues. Furthermore, in contrast to bioluminescence, SPECT or PET imaging using radiolabeled versions of FIAU is directly translatable to human applications, and therefore of critical importance for establishing the link between antibiotics investigated in preclinical models and in patients.
We also applied FIAU imaging methodology to monitor a course of routinely used antibiotics. We were able to show decreased lung signal in infected animals over time with treatment that achieved significance in the case of doxycycline and approached significance for ampicillin. Imaging eliminates the need for time-consuming and invasive methods that require collection and culture of specimens that are currently in use. Moreover, since this method is noninvasive, fewer animals can be imaged repeatedly, serving as their own controls, enabling a more statistically robust study. FIAU at high concentrations (> 32 μg/mL) is known to inhibit the growth of bacteria that have endogenous TK [18
]. In our current experiments, the standard dose of [125
I]FIAU per animal of 2 – 5 mCi (74 – 185 MBq) is equivalent to 0.34 – 0.85 μg of FIAU per animal, far below the pharmacologic dose.
Radiolabeled FIAU satisfies most criteria for functioning as an effective radiopharmaceutical for imaging infection. Radiolabeled FIAU demonstrates rapid accumulation at specific sites of infection – even if those infections are diffuse, such as within the lungs, rather than being focal, such as with an abscess – with low uptake in uninfected regions. It is nontoxic, safe and easy to prepare. Above all, it demonstrates the ability to differentiate between infection and sterile inflammation, and compares favorably in this regard with [18F]fluorodeoxyglucose and other agents being considered for imaging infection. We have also shown that it can be used, in principle, to follow antibiotic treatment in a preclinical model of infection, although the sensitivity of detection is lower than for optically-based techniques.