Head-to-Head Comparison of 68Ga-Citrate and 18F-FDG PET/CT for Detection of Infectious Foci in Patients with Staphylococcus aureus Bacteraemia

Purpose This study evaluated the potential of 68Ga-citrate positron emission tomography/computed tomography (PET/CT) for the detection of infectious foci in patients with Staphylococcus aureus bacteraemia by comparing it with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) PET/CT. Methods Four patients admitted to hospital due to S. aureus bacteraemia underwent both 18F-FDG and 68Ga-citrate whole-body PET/CT scans to detect infectious foci. Results The time from hospital admission and the initiation of antibiotic treatment to the first PET/CT was 4–10 days. The time interval between 18F-FDG and 68Ga-citrate PET/CT was 1–4 days. Three patients had vertebral osteomyelitis (spondylodiscitis) and one had osteomyelitis in the toe; these were detected by both 18F-FDG (maximum standardised uptake value [SUVmax] 6.0 ± 1.0) and 68Ga-citrate (SUVmax  6.8 ± 3.5, P = 0.61). Three patients had soft tissue infectious foci, with more intense 18F-FDG uptake (SUVmax  6.5 ± 2.5) than 68Ga-citrate uptake (SUVmax  3.9 ± 1.2, P = 0.0033). Conclusions Our small cohort of patients with S. aureus bacteraemia revealed that 68Ga-citrate PET/CT is comparable to 18F-FDG PET/CT for detection of osteomyelitis, whereas 18F-FDG resulted in a higher signal for the detection of soft tissue infectious foci.


Introduction
Positron emission tomography (PET) with the radiolabelled glucose analogue 2-[ 18 F]fluoro-2-deoxy--glucose ( 18 F-FDG) is a sensitive and widely used method to detect inflammation and infection according to the high glucose uptake of activated inflammatory cells. It has an important role in the diagnosis of fever of unknown origin when conventional imaging has failed [1]. Staphylococcus aureus bacteraemia is a life-threatening condition, and detection and eradication of deep infectious foci are crucial for successful treatment [2]. In previous studies, 18 F-FDG PET/CT has proven to be a sensitive method for the detection of infectious foci in patients with gram-positive bacteraemia [2,3]. 68 Ga-citrate has also been shown to be a sensitive and specific tracer for the detection of infectious lesions [4,5], although only a few human studies using 68 Ga-citrate PET/CT exist. The biological mechanism of 68 Ga-citrate accumulation in infectious foci is not fully understood. Once injected the Ga-citrate complex is quickly dissociated into 2 Contrast Media & Molecular Imaging Ga 3+ and citrate 3− within the blood. Then, 99% of the gallium ions are attached to transferrin [6,7], which accumulates in inflammatory lesions. In addition, it is assumed that some 68 Ga may attach to bacterial siderophores, lactoferrin inside neutrophils, and free lactoferrin at the site of infection [8]. According to previous studies, 68 Ga-citrate PET/CT appears to be a sensitive tool for the detection of bone infections [4,9], although until now it has not been studied in patients with S. aureus bacteraemia.
The purpose of this study was to evaluate the potential of 68 Ga-citrate PET/CT for the detection of infectious foci in patients with S. aureus bacteraemia by comparing it with 18 F-FDG in a head-to-head setting.  68 Ga was prepurified through a cationic exchanger (Strata X-C, Phenomenex Inc., Torrance, CA) by eluting with HCl/acetone-solution (800 l). Acetone was then evaporated by heating at 110 ∘ C for 240 s and after cooling of 68 GaCl 3 , the sterile isotonic sodium citrate solution (4 ml) was added to the reaction vial followed by 240 s reaction time. The product was transferred to the end product vial through a nonpyrogenic 0.22 m sterile filter and diluted with saline (9 mg/ml, 6 ml). The radiochemical purity of the 68 Ga-citrate was evaluated by instant thin layer chromatography-silica-gel technique using methanol/acetic acid (9 : 1) as a mobile phase. pH of the product was tested with indicator strips (pH range 2.0-9.0) and sterile filter integrity was assessed by a bubble point test.

Materials and Methods
Whole-body 18 F-FDG and 68 Ga-citrate PET/CT (Discovery VCT, GE Medical Systems) were performed in all patients within 1-4 days. All patients fasted before the 18 F-FDG scan. The injected radioactivity doses of 18 F-FDG and 68 Ga-citrate were 292 ± 68 MBq (range: 227-387 MBq) and 196 ± 37 MBq (range: 158-245 MBq), respectively. PET scanning started at 58 ± 7 min (range: 52-67 min) after 18 F-FDG injection and 81 ± 23 min (range: 48-100 min) after 68 Ga-citrate injection. The whole-body PET acquisition (3 min/bed position) was performed following a low dose CT for anatomical reference and attenuation correction. PET images were reconstructed using a 3D maximum-likelihood reconstruction with an ordered-subsets expectation maximization algorithm (VUE Point, GE Healthcare). Visual analysis of the images was performed by an experienced nuclear medicine specialist (J. K.), with the results being reevaluated by the research team for consensus. A positive finding was defined as an abnormal accumulation of 18 F-FDG or 68 Ga-citrate indicating infectious foci. 18 F-FDG and 68 Ga-citrate uptake in the volumes of interest were quantified and expressed as maximum standardised uptake values (SUV max ) by normalising the tissue radioactivity concentration for the injected radioactivity dose and the patient's weight. The blood background radioactivity concentration was determined from the left ventricle cavity as SUV mean , and the target-to-background ratio (TBR) was calculated as SUV max,infection /SUV mean,blood .

Statistical Analysis.
Results are expressed as mean ± SD and range. A paired -test was used to compare 18 F-FDG and 68 Ga-citrate uptake. A value of <0.05 was considered statistically significant.
Patient characteristics are presented in Table 1. All patients had a condition predisposing them to infection. In addition, Patient #3 had a cardiac pacemaker. The time interval from hospital admission and initiation of antibiotic treatment to the first PET/CT was 4-10 days, with the second PET/CT scan performed within another 1-4 days. The order of the 18 F-FDG and 68 Ga-citrate scans depended on the availability of tracers, with both being performed first in two cases. The mean C-reactive protein (CRP) levels were 89 ± 55 mg/l on the day of 18 F-FDG PET/CT and 124±118 mg/l on the day of 68 Ga-citrate PET/CT ( = 0.56). The blood background ( = 4) SUV mean was 1.4 ± 0.05 for 18 F-FDG and 2.9 ± 0.88 for 68 Ga-citrate ( = 0.043).
In addition to infectious foci, all of the patients also had other metabolic findings. Three patients (Patients #2, #3, and #4) demonstrated strong uptake of 68 Ga-citrate in the larger arteries, coincidental to visible atherosclerosis on CT. In the ascending aorta ( = 4), the SUV max of 68 Ga-citrate was 4.7 ± 1.9 (range: 2.0-6.6), whereas the SUV max of 18 F-FDG   (Figure 2(a)), which were not detected on 18 F-FDG PET/CT. In the absence of clinical symptoms, the enlarged parotid gland was not further studied by MRI or ultrasound, so the observed 68 Ga-citrate uptake remained unexplained. Patient #2 also demonstrated 68 Ga-citrate uptake in the inferior vena cava, but not 18 F-FDG uptake, with thrombosis being confirmed by ultrasonography. Patient #3 had a clear focal accumulation of both 68 Ga-citrate and 18 F-FDG in the descending colon, which was subsequently confirmed as a tubular adenoma in a colonoscopy with biopsies. The same patient also demonstrated 18 F-FDG but not 68 Ga-citrate accumulation in the caecum without specific findings on colonoscopy or biopsy. Patient #4 had reactive lymph nodes in the neck, which were indicated as being metabolically active by both imaging methods.

Discussion
We believe that this is the first study to compare 68 Ga-citrate and detection of osteomyelitis. However, the soft tissue infectious foci were clearly better visualized with 18 F-FDG than 68 Gacitrate.
In general, two different tracers must be compared with caution and taking into account their potentially different properties, such as uptake mechanisms and flow/diffusion dependency. In animal models, both 18 F-FDG and 68 Gachloride have shown increased accumulation in S. aureus osteomyelitis, whereas, in healing bones without infection, only 18 F-FDG accumulation was observed [9]. Our results are in line with the animal studies, in that accumulation of these tracers in patients with osteomyelitis does not differ in the acute phase of S. aureus infection. Further studies are warranted to clarify whether 68 Ga-citrate PET/CT can confirm the healing of osteomyelitis and whether it is superior to 18 F-FDG for the differentiation of infection from sterile bone inflammation. We found a difference between 18 F-FDG and 68 Ga-citrate PET/CT in the detection of soft tissue infection. Contrary to the clear findings of multiple small infectious foci without abscess formation in 18 F-FDG PET/CT, such lesions were only slightly visible or not visible at all on 68 Ga-citrate PET/CT (Patient #1). The difference in SUV max between 18 F-FDG and 68 Ga-citrate was also statistically significant. These findings were not controlled by other imaging modalities, but some were also detectable in clinical status (e.g., multiple infectious foci in Patient #1's arm). In this study we used 196 ± 37 MBq for 68 Ga-citrate PET and started imaging after 81±23 minutes. The observed blood background SUV mean 2.9 + 0.88 may be a strong contributing factor for not visualizing soft tissue infections with 68 Ga-citrate. It can be hypothesized that lowering of injected radioactivity dose to 100 MBq would reduce blood pool radioactivity and cause less background noise. In order to confirm the effect of a lower 68 Ga-citrate dose, further studies are warranted. A recent study comparing 18 F-FDG, 68 Ga-citrate, and other tracers in a pig-model of hematogenously disseminated S. aureus infection reported slightly different results [10]. These findings may be due to the higher blood background radioactivity of 68 Ga-citrate ( Table 2). The slight differences in the production of 68 Gacitrate might reflect different results, too.
Owing to its high sensitivity, specificity, and accuracy in the detection of osteomyelitis, MRI is the recommended imaging modality when spondylodiscitis is suspected [11]. MRI provides higher spatial resolution of the spinal cord than PET/CT, which is important if an operation is considered. However, sometimes MRI cannot be obtained because of patient-related reasons (e.g., implanted cardiac devices, claustrophobia). In such cases, 18 F-FDG PET/CT is the recommended imaging modality [11]. Traditionally, MRI is targeted to the part of the body where the patient has signs or symptoms; however, a previous study [12] showed that around one-third of infectious foci in S. aureus bacteraemia are asymptomatic. Thus, these silent infectious foci may pass unnoticed with traditional imaging, while PET/CT scanning provides information on the whole body, and as demonstrated in our cases, multiple infectious foci can be detected simultaneously.
In addition to infectious foci, both 18 F-FDG and 68 Gacitrate PET/CT also revealed other clinically significant findings. In Patient #3, PET/CT findings in the colon led to colonoscopy and the finding of a tubular adenoma. Patient #2 demonstrated an uptake of 68 Ga-citrate (but not 18 F-FDG) in the inferior vena cava where thrombosis was later confirmed by ultrasound. This can be related to pooling tracer just proximal to the narrowing caused by the thrombus. More comprehensive studies are needed to explain the differences in the findings of 68 Ga-citrate and 18 F-FDG, for example, whether they are ascribed due to infection versus sterile inflammation.
The accumulation of 68 Ga-citrate in atherosclerotic arteries warrants further studies to determine its importance. Previously, 68 Ga-chloride uptake has been demonstrated in atherosclerotic lesions in mice [13]. Instead, in the papers presented by Nanni and coworkers [4] as well as Vorster and coworkers [14], the relatively high vascular radioactivity was regarded as a normal biodistribution of 68 Ga-citrate. The identification of atherosclerosis in 3 patients by 68 Ga-citrate may be coincidental. One advantage of 68 Ga-citrate over 18 F-FDG is that patients are not required to fast before the scan. However, for detection of metastatic endovascular infection the accumulation of 68 Ga-citrate in atherosclerotic arteries can be regarded as a limitation of 68 Ga-citrate PET/CT in patients with S. aureus bacteraemia.
In the current study, the time intervals between 18 F-FDG and 68 Ga-citrate scans were short (1-4 days), and CRP remained at the same levels over these intervals. Neither surgical procedures nor changes to the antimicrobial treatment were made between the two scans. We thus consider that the infectious status of the patients did not differ markedly between 18 F-FDG and 68 Ga-citrate studies. However, time interval from commencement of antibiotic treatment to the first PET/CT was 4-10 days and we are not able to exclude the possibility that due to this delay some of the infectious lesions were not detected. We also determined the TBRs, which revealed that the blood radioactivity concentration of 68 Ga-citrate was higher than that of 18 F-FDG. Thus, in many foci, the TBRs of 68 Ga-citrate PET/CT were lower than in 18 F-FDG PET/CT. In general, the number of patients is small, which can be regarded as a limitation of this study.

Conclusion
68 Ga-citrate and 18 F-FDG are comparable PET tracers for the imaging of osteomyelitis in patients with S. aureus bacteraemia. Further studies are warranted to clarify whether 68 Ga-citrate PET/CT can detect osteomyelitis caused by other pathogens and whether it can assess the healing of infectious osteomyelitis. For the detection of soft tissue infectious foci, 18 F-FDG PET/CT shows higher intensity than 68 Ga-citrate PET/CT but the effect of lower 68 Ga-citrate dose should be verified by further studies.

Conflicts of Interest
The authors declare that they have no conflicts of interest.