Pathophysiology Of Infection And Inflammation

Inflammation is a complex tissue reaction to injury that may be caused by physical, chemical, or immunological agents or even by radiation. If the injury is caused by or involves living microbes, the injury leads to infection. Whatever the cause may be, inflammation is a nonspecific response to an injury and is a protective response to the cause as well as the consequences of such an injury [1]. Inflammatory response involves a complex network of immunologic and histochemical events, such as release of chemical mediators (histamine, serotonin, bradykinin, arachidonic acid metabolites, platelet activating factor, nitric oxide, and multiple cytokines and chemokines) and vasodilation with increased vascular permeability [2]. It causes the classical symptoms of acute inflammation; rubor (redness), calor (warmth), tumor (edema), dolor (pain) and fastio laesa (impaired function). In acute infection or inflammation, infiltrating cells are predominantly polymorphonuclear cells (PMNs). In chronic infection or inflammation, persisting for weeks or months, the cellular infiltrate mainly consist of mononuclear cells, such as lymphocytes, monocytes and macrophages [3].

Infection diagnosis techniques
The presence of infection may be suggested by signs and symptoms such as fever, pain, loss of appetite, general malaise, and abnormal laboratory results. Imaging tests often are used, however, to confirm or the presence of infection and sometimes, to monitor response to the therapy [4]. Deep seated infections such as intra-abdominal abscesses, endocarditis, osteomyelitis and infections of prosthetic devices which can be difficult to detect, resulting in delayed diagnosis and treatment [5]. The ability to identify focal sites of infection in patients who do not present with localizing symptoms is a key step in delivering appropriate medical treatment. This is particularly critical in immune compromised patients, since signs and symptoms of infection may be minimized in patients with neutropenia [6]. New techniques, especially within immunology and molecular biology, are yielding new insights into the discrimination of infection and inflammation. Clinicians usually use a variety of clues for example laboratory tests, clinical and radiological tests, to aid diagnosis of infection as early as possible and make a decision. Many current laboratory tests used to guide the diagnostic process rely on factors in the inflammatory responses such as erythrocyte sedimentation rate, white-blood cell count, acute-phase proteins and cytokines, but these tests are not specific enough to discriminate between infection and inflammation [7]. Imaging techniques can be classified as either structural or functional. Structural imaging procedures are used to evaluate macroscopic morphological changes and implant loosening. Differently, functional imaging procedures aim to visualize the specific accumulation of an injected gamma-emitter radiotracer at the site of infection [8]. Structural imaging methods like X-ray, ultrasonography (US), computed tomography (CT) and magnetic resonance imaging (MRI) are based on important anatomic alterations and the possibility of a precocious diagnosis is limited. These are not the best of methods for the localization of infection at early stages [9]. These procedures detect the morphologic alterations of the tissues after significant process has taken place in the infective site leading to abscess formation [10]. In contrast, nuclear medicine as a functional imaging technique provides information on pathophysiological and pathobiochemical processes. In this respect, it differs from other current imaging procedures such as X-ray, CT and MRI, which supply information with high resolution on the morphological changes that occur in a specific disease. In addition, it permits whole-body imaging, whereas CT and MRI routinely focus on just a part of the body [11]. Nuclear medical imaging has an important role in discriminating infections from inflammation. Inflammatory processes can be visualized in their early phases, when anatomical changes are not yet apparent [5].

Nuclear medicine techniques for infection/inflammation imaging
Radionuclide emission-based nuclear medicine modality is a noninvasive technique, which is a quick, sensitive, and specific method to detect as well as locate the lesion at any anatomical site at early stage of the disease [12]. Technetium-99m (99mTc) is one of the most desirable radionuclide that is used in clinical nuclear medicine, due to the emission of gamma ray of optimal energy (140 keV), a suitable half-life (6 h), availability from 99Mo-99mTc generator systems and low cost [13]. Nuclear medicine techniques require a reliable radiopharmaceutical that can selectively concentrate in infectious foci. Various 99mTc-labeled compounds have been developed for the scintigraphic detection of infection and sterile inflammation in humans. Unfortunately, many of these radiopharmaceuticals do not discriminate between infection and sterile inflammatory process, which is often of clinical importance [14]. Both specific and non-specific radiopharmaceuticals are used to image infection. Increased blood supply, increased vascular permeability and enhanced transudation are processes that result in accumulation of tracers, and it must be emphasized that all radiopharmaceuticals accumulate, to some extent, in this non-specific way at the site of infection or inflammation [15]. In recent years, the development of radiolabeled antimicrobial agents for specific diagnosis of infection has received considerable attention, sparking a lively debate about the infection specificity of these radiopharmaceuticals [16]. Conventional nuclear medicine techniques make the use of the following radiopharmaceuticals for imaging infection and inflammation: 67Ga-citrate, 99mTc or 111In (Indium-111) labeled leukocytes, 99mTc-labeled antigranulocyte antibodies, radiolabeled chemotactic peptides, 99mTc or 111In-labeled human immunoglobulin (HIG), 99mTc-nanocolloids, radiolabeled liposomes, cytokines, streptavidin-biotin and 18F-flourodeoxyglocose (18F-FDG). Specific approaches by targeting microorganisms include radiolabeled antibiotics, antimicrobial peptides and radiolabeled enzy??matic substrates of bacterial enzymes such as thymidine kinase (TK) substrate (FIAU) [17]. Each of these radiopharmaceuticals with their uptake mechanisms for imaging infectious and inflammatory diseases will be extensively discussed as follows.

67Ga-citrate
Gallium-67 (67Ga) citrate was one of the first radiopharmaceuticals developed for infection imaging and has been used for detecting inflammatory lesions since its introducing by Lavender et al. in 1971 [18]. Gallium 67 is a gamma-emitting radionuclide with a physical half-life of 78 hours. As the citrate salt, it is useful in the diagnosis of inflammatory disease, particularly abscesses, due to its uptake by phagocytizing cells, such as neutrophils and macrophages [19]. It binds to transferrin in the blood, extravasates at the site of inflammation, and is then bound to lactoferrin. In a low-iron environment it has a high binding affinity for siderophores. There is physiological uptake in liver, spleen, bone marrow and kidney and, later, in the colon, which may hamper visualisation of infection/inflammation in or in the vicinity of these organs [20]. 67Ga has been reported to be successful as an infection imaging radiopharmaceutical in tagging abscesses in experimental animals as well as in clinical trials [18, 21]. Gallium-67 was utilized in 34 patients suffering from clinically soft tissue, bones, and some areas of the abdomen infections by Dysine et al. in 1974. They reported that it is ideal for the diagnosis of acute and/or chronic inflammation of bone secondary to open reduction of fractures, and may also be of use in the diagnosis of primary osteomyelitis. A limitation to this technique relates to the bowel uptake of 67Ga which limits diagnosis of pelvic inflammatory disease [22]. Typical indications of 67Ga scanning for identifying inflammation and infection include sarcoidosis, pneumonia, pyelonephritis, fibrosis, AIDS (acquired immune deficiency syndrome) related inflammations, osteomyelitis, and fevers of unknown origin (FUO) [23]. Gallium is accumulated in a wide range of inflammatory, infective, and neoplastic processes but is hindered by unfavorable imaging characteristics: it emits high-energy gamma rays that decrease spatial resolution. Gallium studies have a long examination time, requiring up to 7 days to image, with resultant high radiation exposure to the patient [24]. The overall accuracy of bone/gallium imaging ranges between about 60% and 80%. The less than ideal imaging characteristics of gallium and the need for two isotopes with multiple imaging sessions over several days are additional disadvantages of the procedure [25].

99mTc or 111In-labeled leukocytes
Since the 1970s, scintigraphic imaging using isolated autologous leukocytes (WBCs), labeled either with 111In or 99mTc for clinical diagnosis of infection and inflammation has been introduced and frequently used [26]. The widely used radioisotopes for labeling of leucocytes are 111In Oxine, 111In tropolonate, and Technetium-99m hexamethyl propylene amine oxime, a lipophilic chelator (99mTc HMPAO). The amount of radioactivity which can be administered with 111In is low, because of its 67 hours half life and associated radiation dose. This results in low density in images. However, 111In labeling is very stable, with binding to intracellular macromulecules and particulates and there is minimal urinary or faecal excretion. In contrast, 99mTc has a half life of 6 hour and can be administered in higher doses, resulting in improved image quality. However 99mTc labeling is less stable because the trapped form is soluble and there is excretion of 99mTc through both kidneys and intestine which limits imaging of disease in the abdomen except at early times [27]. In the clinical setting 99mTc HMPAO labeled leucocytes are as useful as those labeled with 111In. Except when faecal excretion studies are contemplated, 99mTc HMPAO should be the preferred leucocyte label, on the grounds of availability, image quality, ease of use, and radiation dosimetry [28]. Diseases in which radiolabeled leukocytes have made a significant contribution to clinical management include inflammatory bowel disease (IBD), postoperative sepsis, intraabdominal soft tissue sepsis, and acute and chronic osteomyelities. Processes that involve nutrophilic infiltration, but to which radiolabeled granulocytes have not made such a major clinical impact, include intrathoracic sepsis such as lobar pneumonia, lung abscess and bronchiectasis, acute myocardial infarction, and rhumatoid arthritis [29]. The sensitivity and specificity of radiolabeled WBCs in abdominal abscess detection reported by Datz et al. ranged from 86% to 90%, with a slightly higher sensitivity for acute infections in which of the resulting of the increased granulocytic response, was observed [30]. Vorne et al. performed a comparison study with 99mTc HMPAO leukocyte and [67Ga] citrate scan in a series of 43 patients suspected of having various benign inflammatory conditions. The results showed that the sensitivity, specificity, and accuracy of 99mTc HMPAO leukocyte scan were 92%, 100%, and 95%, respectively. The values of [67Ga] citrate showed 96% sensitivity, but the specificity and accuracy values were lower, 63% and 81 %, respectively [31]. They concluded the quality of 99mTc leukocyte image was superior to that of [67Ga] citrate and the result was available more rapidly, and the radiation dose to the patient was lower. One meta-analysis of 99mTc-labeled leukocytes imaging of potentially infected feet in 2,889 diabetic patients from 50 studies, demonstrated a sensitivity of 86% and specificity of only 84% [32]. Recently, 99mTc-HMPAO-WBC scintigraphy along with SPECT/CT acquisition of the chest in 63 patients with suspected infections associated with cardiovascular implantable electronic devices (CIEDs), demonstrated that SPECT/CT imaging with radiolabeled WBCs increases the detection rate of infection and allows accurate assessment of disease burden. 99mTc-HMPAO-WBC scintigraphy had 94% sensitivity with no false positive results which was helpful for excluding the presence of device-related infection during a febrile episode and sepsis, with a 95% negative predictive value [33]. Although direct labeling of isolated leukocytes and reinjecting them can be considered the 'gold standard' nuclear medicine technique for imaging inflammatory process, however, the preparation of this radiopharmaceutical is laborious, requires specialized equipment and could be hazardous. Isolating and labeling a patient's white blood cells takes a trained technician approximately 3 hours. In addition, the need to handle potentially contaminated blood could lead to transmission of blood borne pathogens such as human immunodeficiency virus (HIV) and hepatitis B virus (HBV) [34]. In patients exhibiting neutropenia, there is difficulty associated with obtaining a sufficient amount of WBCs to label. In addition, the functional status of the WBCs must also be taken into account. Another concern involves patients receiving chemotherapy or other immune modulating agents that can affect WBC harvesting and/or function when injected back into the patient. Therefore, patients undergoing chemotherapy, receiving glucocorticoids, or infected with HIV may be difficult or impossible to image [35]. Radiolabeled white blood cell scintigraphy, enables detection of areas of general inflammation but cannot be used to distinguish between bacterial and nonbacterial inflammatory processes [36].

99mTc or 111In-labeled human immunoglobulin (HIG)
The research groups of Rubin et al. were the first to discover the usefulness of 111In-IgG scintigraphy in a rat model with a pseudomonal infection of the thigh for imaging infectious and inflammatory foci [37]. Human polyclonal IgG radiolabeled with either 111In or 99mTc localizes at focal sites of infection/inflammation in both animal models of infection and human subjects to a sufficient degree to permit imaging with a scintillation camera. The possible mechanism of IgG concentration in the lesions, include: specific antigen recognition by individual antibody molecules, binding to Fc receptors on inflammatory cells and nonspecific processes that include increased tissue permeability. The fact that radiolabeled IgG localizes at regions of sterile inflammation as well as at sites of bacterial or fungal infection argues against specific antigen-antibody interaction [38]. Initial studies by Rubin et al. indicated that 111In-IgG may be a superior imaging agent with practical advantages over 67Ga and 111In 'WBCs scintigraphy [37, 39]. In 1988, the first clinical study using 111In-IgG, performed by Fischman et al. with 92% sensitivity and 95% specificity in a series of 84 patients with a variety of suspected lesions in the abdomen, pelvis, lungs, bones, joints, or in vascular grafts [40]. One year later Rubin et al. studied in a larger series of 128 patients with a similar variety of suspected infections and reported sensitivity and specificity of 111In-IgG 91% and 100%, respectively [41]. The preparation of 99mTc labeled IgG by a novel method using the hydrazinonicatinamide derivative (HYNIC) of lgG in comparison with 111In-IgG for imaging focal site of infection in rats was reported by Abrams et al. in 1990 [42]. The results established that 99mTc labeled via the HYNIC is equivalent to 111In-IgG for imaging focal sites of infection in experimental animals and it is the reagent of choice for inflammation imaging due to greater general availability and superior imaging properties [42]. As a comparative study, the clinical efficacy of 99mTc HYNIC- IgG and 111In-IgG was compared by Dams et al. in 37 patients suspected of infectious and inflammatory disease [43]. 99mTc HYNIC-IgG accurately depicted all infectious and inflammatory foci, including chronic osteomyelitis, spondylodiscitis and pulmonary inflammation. The in vivo characteristics of 99mTc HYNIC- IgG showed likely similar performance and in most cases can replace the 111In-labeled compound [43]. Recently, preparation and evaluation of 99mTc-HYNIC-HIG as a kit formulation for early detection of inflammation and infection foci especially in immunodeficiency patients is used by Bakir and coworkers with good results of human biodistribution and initial clinical evaluation for human infection imaging [44]. The higher level of accumulation of 99mTc IgG in normal lung is a potential disadvantage of the agent, since delayed imaging (>24 hr after injection) with 111In-IgG has been an effective means of identifying pulmonary infections. In contrast, the lower level of accumulation of 99mTc-IgG in normal muscle may prove to be an advantage for the identification of soft tissue inflammation [42]. However, with 111In-IgG no definite discrimination is possible between infectious and sterile inflammation. This was demonstrated by the positive 111In-IgG scintigraphy in patients with a hematoma, sterile arthritis, and recent fractures [45]. Poor sensitivity of radiolabeled HIG is found in the diagnosis of endocarditis and vascular lesions in general, due to long lasting high levels of circulating activity. A general limitation is the long time span between injection and final diagnosis (24-48 h) [34].

99mTc labeled granulocyte antibodies (AGAbs)
Because in vitro leukocytes labeling is a time consuming procedure requiring facilities for handling of blood and skilled personnel, the researches have been directed toward in vivo labeling of granulocytes [46]. Monoclonal antibodies specific for surface antigens on neutrophils address many of these disadvantages and may be useful infection imaging agents when labeled with a radionuclide [47]. This procedure does not require leukocyte isolation AGAb is stored as cold kits and can selectively label granulocytes with the labeling time of about 15 minutes [48]. The first clinical study of in vivo WBC labeling was reported by Locher et al. with the high sensitivity of leukocyte immunoscintigraphy using iodine- 123 labeled monoclonal antibody (MoAb) in 1986 [49]. The antigranulocyte antibodies labeled with 99mTc have been tested for infection imaging and they were determined to accurately delineate infection and inflammation. The first approach regarding in vivo labeling was the murine IgG1k monoclonal antibody BW 250/183 (also named 99mTc-besilesomab, Scintimun??) that binds to a 95 kDa epitope non-specific cross-reacting antigen (NCA-95) expressed on granulocytes, independently from maturation stage of cells (therefore, it is used for bone marrow scintigraphy also) [48]. In the study by Lind et al. the clinical value of antigranulocyte immunoscintigraphy with MoAb BW (250/183) in 34 patients with inflammatory process was evaluated with the sensitivity and specificity of 95% and 85%, respectively [50]. Since 1992, an estimated 100,000 patients have been diagnosed with radiolabeled besilesomab (Scintimun??) in Switzerland, Hungary, the Czech Republic and Sweden on the basis of local marketing approvals and in Germany (on the basis of individual prescription). In January 2010, Scintimun?? was granted a marketing authorization for all European countries by the European Medicines Agency (EMEA) for determining the location of infection in peripheral bone in adults with suspected osteomyelitis [51]. The use of Scintimun?? for detecting infection or inflammation sites has been studied in a variety rang of clinical indications such as patients with osteomyelitis [51, 52], inflammatory bowel disease (IBD) [53], fever of unknown origin [54] and endocarditis [55]. As a comparative study, the efficacy of Scintimun?? and 99mTc-HMPAO-labeled WBCs in acute and chronic peripheral osteomyelitis was compared by Ritcher et al. in clinical setting [52]. Scintimun?? had higher sensitivity than 99mTc-HMPAO-labeled WBCs (74.8 vs 59.0%) at slightly lower specificity (71.8 vs 79.5%, respectively) [52]. However, the use of intact antibodies may cause a human antimouse antibody (HAMA) response that may result in anaphylactic shock. Elimination of Fc portion of antibodies leads to decreased nonspecific binding and rapid kinetic of the fragments that in turn may result in enhanced target/background uptake ratio and avoidance of a human anti-mouse antibody (HAMA) response [56]. Sulesomab (LeukoScan??) is the Fab' fragment of the immunoglobulin (IgG) antibody directed against the glycoprotein cross-reactive antigen-90 on nutrophils. LeukoScan?? is indicated for diagnostic imaging for determining the location and extent of infection/inflammation in bone in patients with suspected osteomyelitis, including patients with diabetic foot ulcers [57]. The first study described the use of an anti-NCA-90 antibody Fab' fragment in humans, was performed by Becker et al. 1994. The reported sensitivity of immunoscintigraphy in 20 patients with suspected bone infections was 88% with rapid localization of foci, rapid and simple use, a negligible HAMA response rate, no effect on granulocyte function and accuracy comparable to WBC scanning [58]. To evaluate immunoscintigraphy for the diagnosis of osteomyelitis, the results of imaging 24 diabetic patients with 31 suspected osteomyelitic lesions using the LeukoScan were prospectively compared with results from the bone scan coupled with 67Ga by Delcort et al. They concluded that Sulesomab is more sensitive than 67Ga for the diagnosis of foot osteomyelitis in diabetic patients and is a good alternative to 67Ga as well as to 111In- WBC by avoiding the need for labeling procedure [59]. Although initial studies with 99mTc-labelled monoclonal antigranulocyte-antibody fragments were successfully demonstrated high sensitivity and specificity [58, 60] however, only 85% sensitivity and 77% specificity were reported for orthopaedic imaging in clinical practice [61]. Another monoclonal antibody, anti stage specific embryonic antigen [anti SSEA-1] called 99mTc-fanolesomab, binds avidly to surface CD15 antigens that are expressed on human neutrophils in large numbers. Initial studies confirmed the capability of 99mTc-anti-SSEA-1 in diagnosing infectious and inflammatory diseases [62, 63]. Easy preparation and imaging after 1 hour with no HAMA allergic reactions of 99mTc-anti-SSEA-1 IgM proved that it is a suitable radiolabeled compound however high liver uptake and transient mild neutropenia that has been observed after 99mTc-anti-SSEA-1 injection in several patients were the major disadvantages [64]. Fanolesomab is an anti-CD 15 monoclonal murine M class immunoglobulin (radiolabeling with 99mTc it is also known as NeutroSpec?? and LeuTech??), binds to CD15 receptors present on leukocytes accurately diagnosed osteomyelitis in the appendicular skeleton in clinical trials [65]. The role of fanolesomab in the diagnosis of osteomyelitis in diabetic patients with pedal ulcers was studied by Palestro et al. [66]. Twenty-five diabetic patients with pedal ulcer underwent 99mTc-Fanolesomab, 111In-labeled leukocyte, and 3-phase bone imaging. The sensitivity, specificity, and accuracy of fanolesomab alone were 90%, 67%, and 76%, respectively, comparable to the values obtained with labeled leukocyte imaging alone (80%, 67%, and 72%). The results suggested that the antibody alone is sufficient for diagnosing diabetic pedal osteomyelitis and significantly more specific than three-phase bone imaging [66]. LeuTech scintigraphic imaging was shown to be convenient, rapid, and sensitive for the detection of acute appendicitis in Fifty-nine patients presenting with equivocal signs and symptoms [67]. 99mTc-fanolesomab was approved for clinical use in the United States in 2004. In December 2005 it was withdrawn from the United States market as a result of post-marketing reports of serious and life-threatening cardiopulmonary events (including cardiac arrest, hypoxia, dyspnea and hypotension) following administration [25]. A diagnostic meta-analysis of anti-granulocyte scintigraphy using 99mTc-labeled monoclonal antibodies for the diagnosis of periprosthetic joint infections (PJI) demonstrated that this method had a reasonable role in the diagnosis of PJI after total joint arthroplasty with the pooled sensitivity and specificity of 83% and 79%, respectively [68]. Additionally, some radiolabeled mAbs (such as anti-E selectin and anti-CD4) demonstrated their excellent capability for the localization of inflammatory regions, but lack of their use for the therapeutic purposes, thus limiting their further development [69]. Overall, the major potential indications for the use of monoclonal antibodies (both the whole antibody and the fragment) for imaging infection are easy to use, readily available, and provide excellent imaging qualities. Because of molecular size, the Fab' fragments may be advantages in providing diagnosis earlier and probably also localizing chronic infections such as osteomyelities of the spine. Because of a lower affinity for the epitope, the fragment has a lower bone marrow uptake. This may help in differentiation of normal bone marrow and osteomyelities and septic loosening prostheses [70]. Although antigranulocyte scintigraphy useing of MoAbs or antibody fragments is a promising diagnostic tool that has been widely used during recent years for the diagnosis of infection, including osteomyelitis in several clinical settings [71], none of these compounds, however, were specific for infection only.

Radiolabeled liposomes
Liposomes are micro-particulate or colloidal carriers, usually 0.05-0.5 ??m in diameter which form spontaneously when certain lipids are hydrated in aqueous media [72]. Liposomes have been extensively used as potential delivery systems for a variety of compounds primarily due to their high degree of biocompatibility and the enormous diversity of structures and compositions [73]. In order to utilize liposomes for diagnostic purposes in nuclear medicine, they need to be labeled with gamma radiation emitting radionuclides, such as 67Ga, 111In or, most preferably, 99mTc [74]. The first use of 99mTc labeled liposomes to image infectious foci in rats with focal Staphylococcus aureus infection was reported by Morgan et al. in 1981 [75]. They supposed that the labeled liposomes retention in the infectious foci were due to engulfment by phagocyte celles present at infectious site. In clinical setting for the first time, 99mTc-labeled liposomes were used successfully in patients with deep-seated infections such as infected prosthetic joints, tubo-ovarian abscess and osteomyelitis which were visualized within 24 hours post injection [76]. In the liposomal labeling procedure developed by Goins et al. the lipophlic chelator, hexamethyl propyleneamine oxime (HMPAO) was used for labeling due to its advantages like easy to lable, haveing high labeling efficiency and avalibility in kit form, which were tested in rats with focal S. aureus infection [77]. Laverman et al. was developed another method for labeling of liposomes with 99mTc by incorporating hydrazino nicotinamide (HYNIC) conjugated to distearoylphosphatidyl ethanolamine (DSPE) in the bilayer [78]. Coating the liposomal surface with poly (ethyleneglycol) (PEG) avoids rapid recognition by the mononuclear phagocyte system and as a consequence results in prolonged residence time of the liposomes in the circulation which results in substantial liposomal localization in infected tissue. Increased capillary permeability with resulting liposomal extravasation is thought to be important for this localization, but the exact mechanism by which PEG-liposomes localize is not yet well understood [79]. Clinical evaluation of 99mTc- PEG liposomes scintigraphy in a series of 34 patients with predominantly muscloskeletal pathology were directly compared with those of 111In labeled IgG scintigraphy was shown the sensitivity and specificity of 94% and 89% respectively, for 111In labeled IgG these values were 87% and 89%, respectively [80]. Liposomes continue to be very promising carriers for delivery of drugs to inflamed regions of the body, although, to date, no clinical products have specifically taken advantage of the inflammatory targeting of liposomes [81].

99mTc-nanocolloids
Nanocolloids as carriers for radionuclides are widely used in nuclear medicine, either for diagnosis or therapy [82]. Nanocolloids labeled with 99mTc are initially employed in the study of bone marrow: the accumulation of colloids in inflammation is related to the increased vascular permeability in inflamed tissues [83]. Nanocolloids preferentially accumulate in inflammatory lesions associated with a number of condition, for example osteomyelitis, osteoitis, rheumatoid arthritis, arthrosis, joint prostheses, and wound healing, thus nanocolloid is clinically useful for detecting osteomyelitis and other bon or joint infections [84]. 99mTc-Nanocoll??, a labeled nanometer sized, albumin-based colloid, was investigated for bone marrow scintigraphy in some inflammatory conditions with promising result. The results indicated values with sensitivity ranging from 87% to 95% and specificity between 77% and 100% [85, 86]. A comparison study between 99mTc nanocolloid and 111In leukocyte scintigraphy by Streule et al. showed a specificity of 93% for both methods and sensitivity of 81% for 111In leukocyte and 87% for 99mTc nanocolloid, concluding that both methods are equivalent [85]. They concluded that 99mTc nanocolloid scintigraphy is at least equivalent to leukocyte scintigraphy regard to sensitivity, specificity and accuracy which was in agreement with earlier study and due to recognized advantages of 99mTc nanocolloid over 111In labeled leukocyte such as time, complexity and radiation dosimetry, it appears justified to recommend more extended use of 99mTc nanocolloid in diagnosing skeletal infections [85]. Another comparison study with these two agents in 44 patients with clinically suspected osteomyelitis and prosthetic joint infections demonstrated that taken together the sensitivity with 99mTc nanocolloid was 94%, the specificity 84% and the accuracy 87%, compared with 75%, 90% and 85% with 111In-labeled leukocyte [87]. Furthermore, Remedios et al. reported the sensitivity and specificity of 99mTc nanocolloid for diagnosing osteomyelitis in a series of 9 diabetic patients with neuropathic foot disease were 100% and 60% respectively [88]. However the greatest disadvantage of radiolabeled nanocolloids is their inability to image infections outside the musculoskeletal system and, as with most of the currently available radiopharmaceuticals, distinguishing infection from inflammation [89].

Avidin or streptavidin/indium-111 labeled biotin
Avidin, a 66-kD protein found in egg whites, displays a strong avidity for biotin, a 244-D vitamin found in low concentrations in tissues and in blood. Streptavidin, a 60-kD protein obtained from streptomyces avidinii is similar to avidin in most respects; however, it is not glycosylated and is reported to show less non specific binding to tissues [90]. The application of avidin and biotin for imaging applications dates back to 1986 [90]. Avidin/111In biotin scintigraphy is based on the non-specific accumulation of avidin at sites of inflammation or infection, linked to increased transcapillary leakage of macromolecules and to interstitial oedema at these sites. Due to its extremely high affinity for and low dissociation constant with biotin, sites of infection can be imaged using avidin as a pre-target, followed by radiolabeled biotin [91]. The first application of streptavidin-biotin pre-targeting approach was studied by Rusckowski et al. for infection imaging in a mouse model compared to the administration of labeled IgG and labeled streptavidin alone in the infected mouse [92]. They demonstrated that streptavidin is a suitable alternative to nonspecific IgG as an agent for imaging sites of infection in a mouse model [92]. Scintigraphy using avidin and 111In-labeled biotin has proven to be accurate in diagnosing vascular grafts infections [93, 94]. In a clinical study, Cheisa and coworkers investigated the use of 111In-labeled avidin/biotin scintigraphy in the management of 26 patients with clinically suspected prosthetic vascular graft infection [93]. The results showed that the overall sensitivity was 100%, the specificity 95%, the accuracy 96%, the positive predictive value 86% and the negative predictive value 100%. One year later Samuel et al reported the result of this approach in twenty-five patients with a total of 29 vascular grafts. Avidin/111ln-biotin scintigraphy correctly identified all infected grafts, as confirmed by culturing surgical specimens [94]. On the basis of the similar results of these two studies, they recommended that this approach is a useful non invasive diagnostic method for early diagnosis of suspected prosthetic vascular graft infection with no allergic manifestations following the administration of avidin. Good diagnostic accuracy by a two step protocol with unlabeled streptavidin and radiolabeled biotin was demonstrated in the study of 15 patients with chronic osteomyelitis [95]. The results of a validation study of the avidin/111Indium biotin approach in 54 patients with different skeletal lesions were reported by Lazerri et al. As a comparison test, 99mTc-HMPAO-labeled leucocyte scintigraphy was performed in 39/54 patients [91]. The overall sensitivity of the avidin/111In-biotin scan was 97.7% (versus 88.9% for 99mTc-HMPAO leucocyte scintigraphy). The avidin/111In-biotin approach clearly performed better than 99mTc-HMPAO leucocyte scintigraphy in patients with suspected osteomyelitis of the trunk (100% sensitivity, specificity and accuracy versus 50% sensitivity, 100% specificity and 66.7% accuracy for 99mTc-HMPAO leucocyte scintigraphy). These results demonstrated the feasibility of the avidin/111In biotin approach for imaging of sites of infection/inflammation in the clinical setting [91]. In another study Lazzeri et al. investigated a two-step streptavidin/ 111In-biotin imaging in 55 consecutive patients with suspected vertebral osteomyelitis within 2 weeks after onset of symptoms [96]. On the basis of the obtained results (94.12% sensitivity and 95.24% specificity) they concluded that streptavidin/111In-biotin scintigraphy is highly sensitive and specific for detecting vertebral osteomyelitis in the first 2 weeks after the onset of clinical symptoms, and is potentially very useful for guiding clinical decisions on instituting appropriate therapy. Although the use of this approach shows good results in term of sensibility and specificity but the use of heterologous protein might engender immunogenic reactions (anti streptavidin (HASA) response). Therefore, in 2008, Lazzeri et al. investigated the use of 111In-biotin alone for diagnosing spinal infection in 110 patients, including 71 with suspected hematogenous infection and 39 with suspected postoperative infection [97]. 111In-biotin scintigraphy had a sensitivity of 84% and a specificity of 98% in the hematogenous infection group and a sensitivity of 100% and a specificity of 84% in the postoperative infection group [97].

Radiolabeled cytokines and chemokines
Radiolabeled cytokines are a promising class of peptide radiopharmaceuticals that may have diagnostic potential in several pathological conditions as specific receptor ligands. Cytokines are proteins and glycoproteins members of a family of overlapping and interdependent molecules with important roles in the homeostatic control of the immune system and other organs, in physiology and pathology [98]. Chemokines (CC, CXC, CX3C and C family) are chemotactic cytokines that control leukocyte trafficking in physiological or inflammatory conditions, hematopoiesis, organogenesis and angiogenesis [99]. Several cytokine/cytokine receptor systems operate in infection, autoimmunity, cancer and other pathological conditions. This enhanced receptor expression provides a target of the inflammatory disease and tumours. Many cytokines show features which favour their use as radiopharmaceuticals including a relatively low molecular weight, a short half life and rapid plasma clearance, binding of high affinity to specific receptors ready availability due to recombinant technology and lack of immunogenicity [100]. Two approaches have been explored to circumvent the adverse effects of some of these chemotactic peptides: use of lower doses, and use of less toxic members within the same family of proteins (eg, substitution of IL-1 with IL-1 receptor antagonist that lacks biological activity). On the other hand, receptor targeting by a different class of agents (IL-2 and Platelet factor 4) appears to be relatively efficient and has successfully been used to image chronic inflammation [101]. However, cytokines and receptor antagonists are inflammation but not infection specific [89].

IL-1
Interleukin 1 (IL-1) is a 17 kDa proinflammatory cytokine, mainly produced by monocytes, activated macrophages and nutrophilic granulocytes [102]. Van der laken et al. (1995) initiated investigational studies with radioiodinated IL-1 which tested in different animal models of infection or sterile inflammation [103, 104]. Specific uptake of 125I'IL-1 in mice with focal S. aureus infection was shown with high target to background ratio which increases by the time with a rapid clearance from the body [103]. Unfortunately, due to the biological side effects of IL-1 (fever, headache and hypotension) even with doses (< 10 ng/kg) the clinical application of radiolabeled IL-1 in humans excluded. Therefore, radioiodinated IL-1 receptor antagonist (IL-1ra) with similar high affinity but lacks any biologic activity was tested as an imaging agent in rabbits with focal Escherichia coli infections [104]. The results showed that the IL-1ra accumulates in E. coli abscesses in rabbits as much as 123I'IL-1. Both IL-1 and IL-1ra performed better as imaging agents than the radioiodinated chemotactic peptide, formyl-methionyl-leucyl-phenylalanine [104]. Besides, 123I'IL-1ra was tested in patients with rheumatoid arthritis by Barrera et al. [105]. Inflamed joints were visualized in these patients, but it didn't behave as a specific radiopharmaceutical and the specificity of tracer binding to infiltrating leukocytes was not confirmed in autoradiographic studies. In addition the major part of the agent cleared via the hepatobiliary route indicating this agent cannot be used for visualization of infectious lesions in abdomen [105]. Therefore, IL-1 receptor binding agents were not further developed for infection imaging agent.

IL-2
Chronic inflammation is characterized by infiltration of the target tissue by lymphocytes. It was successfully targeted with radiolabeled IL-2 through specific binding to IL-2 receptors expressed on activated T lymphocytes [15]. IL-2 is a small single-chain glycoprotein (MW: 15.5 kDa) of 133 amino acids, synthetized and secreted, in vivo, by T lymphocytes following specific antigen stimulation. Radiolabeled IL-2 allows the visualization of both lymphatic infiltration and T lymphocyte activiation [106]. Signore et al. (1992) were the first to report the application of 123I-labeled IL-2 to visualize pancreatic lymphocytic infiltration in non obese diabetic mice [107]. Beyond the preliminary studies of radiolabeled IL-2 for in vitro and biodistribution study in animals [108, 109] it was applied in several diseases such as Crohn's disease [110], Celiac disease [111], type-1 diabetes (insulin-dependent diabetes) [112] and autoimmune thyroid diseases (Hashimoto thyroiditis) [113], which were characterized by a chronic lymphocytic infiltration. Scintigraphic images of radiolabeled IL-2 in 15 patients with Crohn's disease resulted that it was able to reveal the earas of bowel infiltrated which activated by T lymphocytes [114]. On the basis of these results, using radiolabeled IL-2 suggest that it might be a convenient agent with no side effects for in vivo imaging of infiltrating mononuclear cell in several autoimmune diseases.

IL-8
Among the cytokines family members, the most attractant agent which was studied extensively as infection imaging was radiolabeled IL-8. IL-8 is a member of the CXC subfamily of the chemokines, or chemotactic cytokines, which play an important role in cell recruitment during acute inflammation, is an interesting candidate for the in vivo labeling of neutrophils. IL-8 binds the CXC type I (IL-8 type A) and CXC type II (IL-8 type B) receptors expressed on neutrophils and monocytes with high affinity (0.3'4 nmol/L) [115]. Hay et al. [116] studied first, the in vivo behavior of radioiodinated IL-8 in a rat model with carrageenan-induced sterile inflammation. They concluded that radioiodinated IL-8 had a better performance in detection of acute inflammatory lesions over than 67Ga or 111In 'leucocytes in rats [116]. The first animal study in a rabbit model with E. coli soft-tissue infection using 99mTc-HYNIC-IL-8 allowed rapid visualization of the infectious foci as early as 1 h after injection by rapid accumulation of the agent in the abscess with high target-to-background ratios. The mild transient drop of leukocyte counted and the absence of leukocytosis suggested that 99mTc-HYNIC-IL-8 may be used as an imaging agent with only mild and transient side effects [117]. The first preclinical experience was studied in rabbit model of induced osteomyelitis with the use of 99mTc-IL-8 [118]. The tracer clearly revealed the osteomyelitic lesion within 4 h after injection with good image quality, and lower radiation burden. Although the absolute uptake in the osteomyelitic area was significantly lower than that obtained with 99mTc-MDP and 67Ga-citrate, the T/Bs were significantly higher for 99mTc-IL-8 because of fast background clearance and also the direct comparison with 111In-granulocytes showed superior targeting of 99mTc-IL-8 [118]. In addition inflammatory bowel disease (IBD) was imaged successfully with 99mTc-HYNIC-IL-8 in a rabbit model of acute colitis for in vivo visualization of the extent of colonic inflammation [119]. In another animal study 99mTc-labeled IL-8 was tested for its potential to image pulmonary infection in three experimental rabbit models: aspergillosis in immunocompromised rabbits, pneumococcal (Gram-positive) pneumonia, and E. coli-induced (Gram-negative) pneumonia in immunocompetent rabbits [120]. 99mTc IL-8 enabled early (within 2 h after injection) and excellent visualization of localization and extent of pulmonary infection in each of the three experimental models of pulmonary infection. They concluded that 99mTc IL-8 proved to be feasible and offers many advantages over the used radiopharmaceuticals to image pulmonary infection, 67Ga citrate and radiolabeled leukocytes [120]. The properties of 131I-IL-8 for infection imaging in humans were investigated in 11 patients [121]. 131I-IL-8 accumulated at the sites of infection within 3 h, in 8 patients with active diabetic foot infections and in 1 patient with cellulitis of the thumb. No uptake of 131I-IL-8 was seen at the sites of successfully treated osteomyelitis in 2 patients [121]. However, the physical properties of 131I, for example, limit the spatial resolution and the delineation of small structures. Bleeker-Rovers et al. studied the first clinical evaluation of 99mTc-Labeled IL-8 scintigraphy in 20 patients with suspected localized infections (such as joint prosthesis infections, osteomyelitis, liver abscess and soft-tissue infections) to evaluate the safety of 99mTc-IL-8 in humans [122]. Injection of 400 MBq of 99mTc-IL-8 was well tolerated for the detection of infections in patients as early as 4 h after injection. In the patients with non-infectious disorders, no focal accumulation of 99mTc-IL-8 was found. Rapid accumulation at the site of infection, rapid clearance from the blood pool and non target tissues, and absence of significant side effects, made 99mTc-IL-8 scintigraphy could be a promising infection imaging technique [122].

PF-4
Platelet factor-4 (PF-4) is a 29-kDa homotetrameric protein belongs to CXC chemokines released from the alpha granules of platelets during activation [123]. Moyer et al. tested radiolabeled PF-4 analog complexed with heparin (P483H) to detect focal E. coli infections in rabbits. The agent identified intramuscular abscesses in rabbits within 4 hours after injection and showed higher abscess to contralateral and abscess to blood ratios as compared to labeled WBCs [124]. As a clinical study Palestro et al. evaluated the potential of 99mTc- P483H in 30 patients for scintigraphic detection of infection and inflammation [125]. This agent revealed 86% sensitivity, 81% specificity and 83% accuracy. Due to the high pulmonary uptake in the lungs, the agent will not suite for detection of infections in chest. Additionally, excessive thyroid uptake was observed in some patients, correlated with the peptide:heparin ratio [125].

Other cytokines
Other cytokines have been labeled with 125I or 99mTc for detecting infectious and inflammatory lesions and almost more studies include in vitro binding assay or in experimental animals. For example, the potential of 111In-labeled chemoattractant leukotriene B4 (LTB4) antagonist for imaging of pulmonary aspergillosis in rabbit model [126] and 99mTc-HYNIC LTB4 in rabbits with E. coli infection [127] were reported with promising results. Furthermore, Monocyte Chemotactic Protein 1(MCP-1) in experimentally induced atherosclerosis for identification of unstable Plaques [128] and 99mTc labeled complement factor 5a (C5a) in rabbits with intramuscular E. coli infection [129] were applied to define their diagnostic values in detecting infection and inflammation.

Radiolabeled chemotactic peptides
Chemotactic peptides are naturally released by bacteria and initiate leukocyte chemotaxis by binding to high-affinity receptors on the white blood cell membrane. These receptors are present on polymorphonuclear neutrophils and monocytes [130]. In 1991 Fischman et al. were the first to study the potential of four 111In-labeled chemotactic peptide analogs of N-formyl-methionyl-leucylphenylalanine (forMLF) for imaging E. coli infection in animal model [131]. However, the short biological half-time of the peptides makes 111In a poor choice for imaging. Due to appropriate charachtrastics of 99mTc, Babich et al. [132] synthesized and evaluated four 99mTc labeled HYNIC derivatized chemotactic peptide analogs of forMLF to study their infection localizing properties in rats and rabbits. They concluded that 99mTc-labeled chemotactice peptide analogs are effective agents for the external imaging of focal sites of infection. As a comparison study [133], the potential of this radiopharmaceutical was compared with 111In-labeled leukocytes in a rabbit model of acute bacterial infection. Their results showed that imaging of E. coli infection in rabbits with 99mTc labeled chemotactic peptides was superior to 111In-labeled white blood cells, with high target-to-background ratios at the site of infection. Another study by Fischman et al. demonstrated that, as in the rabbit, 99mTc labeled HYNIC chemotactic peptides analogs induce significant transient reductions in peripheral leukocyte levels in monkeys and also, are attective agents for imaging mild focal sites of inflammation in monkeys [134]. The biodistribution and infection imaging properties of a 99mTc labeled chemotactic peptide and 111In-DTPA-IgG were compared in rabbits with E. coli infection by Babich et al. [135]. The results indicated that the sites of infection were better visualized with the radiolabeled peptide and although both radiopharmaceuticals localize at the sites of infection, the radiolabeled peptide are superior reagents for the rapid detection of focal sites of infection. However, nonspecific mechanisms contribute minimally to the localization of 99mTc-chemotactic peptide analogs at the sites of infection and coinjection of a cold peptide (moderate antagonist) resulted in significantly lower target-to background ratios, so the majority of accumulation appears to be receptor mediated [135]. Furthermore, Babich et al. demonstrated that chemotactic peptide receptor antagonists can be used for infection imaging. The reagent that is used for radiolabeling ('coligand') can markedly effect the biodistribution and infection localization of 99mTc-labeled HYNIC chemotactic peptides [136]. In 2000, the effect of five 99mTc-coligand complexes (mannitol, glucamine, glucarate, tricine and glucoheptonate) to label N-formylmethionyl-leucyl-phenylalanine-lysine (f-MLFK)-HYNIC, on infection localization in rabbit model of infection were tested by Babich et al. For infection imaging, the mannitol preparation had the most favorable combination of accumulation in infected tissue, T/B ratio and biodistribution in uninfected organs [137]. While, Van der Laken et al. indicated that they selected tricine as a co-ligand because 99mTc-tricine has minimal uptake in the infection uptake [138]. Hartwig et al. demonstrated that pancreatic uptake of the chemotactic peptide 99mTc-fMLFK-HYNIC quantitatively correlates with leukocyte infiltration in acute pancreatitis. This was the first study showing that chemotactic peptides localize at an acute intra-abdominal inflammatory site in rat model. The finding confirms that leukocyte accumulation is more important than plasma leakage in chemotactic peptide localization [130]. Although 111In and 99mTc-labeled chemotactic peptides accumulate at the sites of infection with high target-to-background ratio, receptor specificity has not been completely established and a significant amount of localization could be due to nonspecific processes, such as increased tissue permeability, blood pool or blood flow characteristics of inflammatory lesions, or characteristics of the peptides that are not related to for-MLF receptor binding [136]. However, their development as radiopharmaceuticals has been curtailed because they cause significant leucopenia at physiological concentration [89].

18F-flourodeoxyglocose (18F-FDG)
FDG is an analogue of glucose which concentrates in areas of high glycolytic activity such as rapidly dividing cells. Neutrophils and macrophages have increased FDG uptake allowing localization of infection and inflammation [139]. The first term of PET (positron emission tomography) and the first PET radiopharmaceutical 18F-Fluorodeoxyglucose (18F-FDG) were used in 1973. In vivo imaging was succeded with the introduction of radiolabeling a glucose analogue. For the imaging of biodistribution within the body, deoxyglucose was labeled with 18F successfully [140]. Clinically, 18F-FDG PET is most widely used for cancer detection and has proven its efficacy in general oncologic imaging [141]. Tumor, inflamed or infected tissues internalize FDG more quickly than normal tissues due to their over expression of glucose transporters, enhanced glucose metabolism and increased tissue permeability [142]. FDG therefore accumulates in sites of infection, although it is a nonspecific tracer that also accumulates in regions of aseptic inflammation, as well as in malignant lesions. These characteristics make FDG-PET suitable for imaging of various inflammatory and infectious diseases, mostly nonosseous infections but also osteomyelitis [143]. The clinical indication of 18F-FDG for PET scanning was approved in 2000 by FDA and after that PET obtained a larger utilization in nuclear medicine practice and oncology [140]. Advantages of 18F-FDG-PET include high tracer uptake by inflammatory cells, an image acquisition time of less than 2 h, absence of artifacts produced by metallic prosthetic components, and better resolution of PET cameras compared to classical gamma cameras [144]. A comparative biodistribution study in animal with bacterial infections showed that FDG uptake is higher in the infection site than the other tested radiotracers, including thymidine, L-methionine, Ga-67-citrate, and I-125-HAS (Human Serum Albumin) [145]. Since Tahara et al. (1989) performed the first FDG pet study for detecting human abdominal abscesses with high FDG uptake resulting, it has been confirmed that FDG-PET imaging is sensitive enough to precisely detect infections, non-infectious inflammatory diseases [146]. As a prospective study by Meller et al. FDG-PET was reported to be superior to 67Ga scintigraphy in imaging fever of unknown origin (FUO) with sensitivity and specificity of 81% and 86% respectively [147]. The value of FDG-PET imaging in patients with painful joint replacement and musculoskeletal infection have demonstrated accuracy ranging from 47% to 95% [148, 149]. In recent years, dual-time-point imaging with FDG-PET or FDG-PET/CT has been proposed as a method to differentiate between malignant and inflammatory processes in settings where such a distinction is essential for optimal patient management. This is due to the observation that standardized uptake values (SUVs) of inflammatory and non neoplastic lesions tend to remain stable or decrease, while those of the malignant lesions tend to increase over time [150]. PET/CT using 18F-FDG for the diagnosis of vascular graft infections in 39 patients with suspected vascular graft infection demonstrated the feasibility and incremental value of 18F-FDG PET/CT with a sensitivity of 93%, specificity of 91%. Increased 18F-FDG uptake provided by PET/CT enables accurate differentiation between graft and soft-tissue infection [151]. Although the use of FDG-PET may be limited because of the lack of specificity and its incapability of discriminating bacterial infection from other inflammatory disorders, it has already had a highly impact on diagnosis, staging of bacterial infection and evaluation of therapy and will definitely take a part in the diagnosis and management of inflammatory or infectious diseases [142]. However, based on the numerous reported studies it is obvious that FDG accumulates at the sites of various inflammatory and infectious processes and FDG-PET has been successfully used in the evaluation of various both non osseous soft tissue infections and osseous infection. The potential applications of FDG-PET imaging for detecting inflammatory processes include painful joint prosthesis [152], acquired immunodeficiency syndrome (AIDS)-related disorders [153], fever of unknown origin [154], diabetic foot infection [155], vasculitis [156] and vascular graft infection [157]. Unfortunately, 18F-FDG is of limited value for differentiation between infectious bacterial and nonbacterial inflammatory processes, such as autoimmune conditions, including vasculitis, fever of unknown origin, fungal, tuberculosis infections, and cancer. Nevertheless, in the right clinical perspective, 18F-FDG can be a very good alternative to conventional radiopharmaceuticals and morphological imaging methods to image various clinical infectious diseases, as reported in numerous reports from different centers [158].

Properties of an ideal infection imaging agent
The ideal radiopharmaceutical to image infection should meet the following criteria: (1) rapid delineation of foci and extent of lesion; (2) no significant physiological accumulation in the blood or organs such as liver, spleen, gastrointestinal tract, bone, bone marrow, kidneys, and target; (3) rapid washout from background and retention in target; (4) discrimination between infection and non-microbial inflammation; (5) low toxicity and absence of immune response; and (6) low cost and ease of preparation [20]. Considering these criteria for an ideal radiopharmaceutical for infection imaging, the best techniques which fit this criteria better, are following by next topic.

Targeting the microorganisms approaches
A totally different strategy is to target microorganisms directly in vivo without the need for intervening leukocytes, an approach that has been adopted in the development of new radiopharmaceuticals such as radiolabeled ciprofloxacin and antimicrobial peptides to improve both the sensitivity and specificity of infection imag??ing [158]. In this respect, a wide variety of radiolabeled antibiotics and antimicrobial peptides and newly labeled enzy??matic substrates of bacterial enzymes such as FIAU were investigated for targeting infection imaging and their abilities in discrimination between infection and non infectious inflammatory diseases were evaluated.

Radiolabeled antibiotics
Erythromycin and streptomycin, were the first antibiotics labeled with 99mTc (1992) used to image turpentine oil induced inflammatory lesions lesions in animals [159]. Following experimental studies of radiolabeling antibiotics mainly have been focused on imaging the site of induced infections in compare to induced inflammatory lesions to assess their abilities in discriminating infection from sterile inflammation. The technique of radiolabeling antibiotics has the enormous potential for diagnostic use. Radiolabeled antibiotics have none of the disadvantages of the in-vitro labeled white blood cell or antigranulocyte antibody procedures. Presumably, the radiolabeled antibiotic is incorporated and metabolized by bacteria present in the infectious focus resulting in accurate and specific localization of the infection [160]. Radiolabeled antibiotics are advantageous due to the selective toxicity and the localizing agent for infective foci. Besides, radiolabeled antibiotics may make a differential diagnosis between infection and sterile inflammation [161]. Radiolabeling of various antibiotics such as antibacterial, antifungal and anti mycobacterium tuberculosis agents for targeting diagnosis of infectious foci, were investigated up to now.

Radiolabeled antibacterial agents
Antimicrobial drugs are classified according to their mechanism of action, for example, cell wall inhibiting, cell membrane inhibiting, protein synthesis inhibiting and nucleic acid inhibiting [162]. Among the antibacterial groups, fluoroquinolones and cephalosporins, were the most antibiotics which radiolabeled for scintigraphic imaging of infection.

Fluoroquinolones
The labeled antibiotic that was most extensively evaluated and has been substantially studied in clinical trials is 99mTc-labeled ciprofloxacin (infecton) a member of fluoroquinolone group that was introduced by Solanki et al. in 1993 as a new class of radiopharmaceutical for infection imaging [163]. Ciprofloxacin which is taken up by Gram-positive and Gram-negative bacteria inhibits DNA synthesis by binding to bacterial DNA gyrase and topoisomerase IV enzymes. Ciprofloxacin penetrates into white cells, is not retained in the absence of bacterial infection [164]. The first clinical application of 99mTc-ciprofloxacin was performed by Vinjamuri et al. in 1996 and the ability of 99mTc infecton imaging in comparison with radiolabeled white blood cell imaging for evaluating of bacterial infection, were investigated [165]. 99mTc-ciprofloxacin had 84% sensitivity and 96% specificity which were superior to white blood cell results (81% and 77% sensitivity and specificity, respectively). They concluded that 99mTc-ciprofloxacin had several advantages over radiolabeled white blood cells. First, 99mTc-ciprofloxacin does not require handling of blood during preparation, thus reducing the risks of hepatitis and HIV infection. Second, 99mTc-ciprofloxacin is technically easier and less labour-intensive to prepare than radiolabeling white cells. Third, this method is independent of the patient's white-cell status, which is advantageous in leukopaenic patients. Finally, 99mTc-ciprofloxacin is not taken up by bone marrow and is thus better able to identify infection in the spine and the proximal parts of limbs [165]. Many clinical applications of 99mTc infecton imaging in patients with various infectious diseases were reported with different ranges of sensitivity and specificity including detecting chronic skeletal infection [166], suspected osteomyelitis or septic arthritis [167], postoperative spinal infection [168], bone infection [169], prosthetic joint infections [170], acute or chronic cholecystitis [171], pediatric osteomyelitis [172]. The largest worldwide multi-center study on 99mTc-ciprofloxacin involving 879 patients with suspected bacterial infection in 8 countries under the auspices of the International Atomic Energy Agency (IAEA) showed that Tc-99m labeled ciprofloxacin supplied as a two phase kit formulation is able to diagnose and localize a wide range of bacterial infections accurately with the overall sensitivity and specificity (85.4% and 81.7%, respectively) [173]. Despite such promising results, low specificities of 99mTc infecton in discrimination between bacterial infection and sterile inflammation in animal models and in clinics, were found in some cases [167, 174]. Such nonspecific accumulation of 99mTc-ciprofloxacin in aseptic sites and inability to discriminate between infection and sterile inflammation may be explained by exudation in the interstitial space, favored by an increased capillary permeability. However, the persistence of increased activity on 24-h images may indicate other, more specific mechanisms for tracer uptake [174]. Langer et al. presented an alternative approach by labeling of ciprofloxacin with 18F (half-life 109.8 min) developing a potentially infection-specific radiopharmaceutical for PET [175]. [18F] ciprofloxacin was applied in four patients with microbiologically proven bacterial soft tissue infections [176]. The results indicated that [18F] ciprofloxacin is not suited as a bacteria-specific infection imaging agent for PET. Because the radioactivity was not retained in infected tissue and appeared to wash out with a similar elimination half-life as in uninfected tissue, suggesting that the kinetics of [18F] ciprofloxacin in infected tissue are governed by increased blood flow and vascular permeability due to local infection rather than by a binding process [176]. Besides, possible explanations for these contradicting results include the presence of ciprofloxacin-resistant bacteria, biofilm, insufficient numbers of viable intra-lesional bacteria and the use of similar antibiotic therapy before imaging. It was also reported that the discrepancy in the ciprofloxacin performance was due to different kit formulations for the radio-ciprofloxacin preparation used in different studies [177]. However, the low binding affinity of 99mTc-ciprofloxacin to bacteria and the risk of emerging antibiotic-resistant microorganisms make this radiopharmaceutical unattractive for imaging bacterial infections [178]. Moreover, radiolabeling of some other members of the quinolone antibiotics such as sparfloxacin [179], enrofloxacin [180], pefeloxacin [181], lomefloxacin and ofloxacin [182], moxifloxacin [183], norfloxacin [184], gemifloxacin [185], rufloxacin [186], clinafloxacin [187], garenoxacin [188], gatifloxacin [189], levofloxacin [190], temafloxacin [191] and these radiopharmaceauticals were evaluated.

Cephalosporins
Ceftizoxime, a third generation cephalosporin, was the first cephalosporin antibiotic to be labeled with 99mTc by Barreto et al. (2000) [192]. It has a wide spectrum of activity to the betalactamases, binds to the bacterial wall, inhibits the synthesis of peptidoglycan and therefore inhibits the synthesis of bacterial wall, which drives to bacterial death. As opposed to Infecton, ceftizoxime is more rapidly depurated from the organism (half life of 1.7 hours), thus diminishing the circulating pool and favoring the specific capture by the infectious site over the nonspecific [192]. Animal experiment study in rats with E. coli induced infection showed higher uptake of 99mTc ceftizoxime than in rats with sterile induced abscess [193]. 99mTc ceftizoxime behavior in clinical study on 23 patients with different infectious and inflammatory diseases resulted 100% sensitivity, 83% specificity and 94 % accuracy [194]. A kit formulation of this radiotracer was prepared by Diniz et al. (2005) and its capability of identifying infectious foci in an experimental model of infection in rats was evaluated [9]. Another member of the third generation cephalosporin group is ceftriaxone with a broad spectrum activity against the majority of aerobic and anaerobic gram positive and gram negative pathogenic bacteria [195]. Radiolabeling of ceftriaxone with 99mTc was firstly introduced by Mostafa et al. (2010) to assess biodistribution of this agent in E. coli induced infection [196]. Fazli et al. recently performed an experimental animal study for scintigraphic imaging of 99mTc-ceftriaxone in mouse model due to staphylococcal infection [197]. In vitro binding of 99mTc-ceftriaxone complex with S. aureus (viable and heat killed bacteria) showed saturated binding of 99mTc-ceftriaxone to viable S. aureus with a maximum of 45 ?? 4.6 % in comparison with heat killed bacteria within 3 h. In the competition assay, when 100-fold excess of unlabeled ceftriaxone as a competitor was used, the inhibition of binding of 99mTc-ceftriaxone to viable S. aureus was observed about 70 % that indicates specificity of the complex for binding to the bacterial cells. The results of biodistribution study indicated that the highest target (infected muscle) to non-target (normal muscle) ratio of 99mTc-ceftriaxone was obtained at 3 h post injection which was more than three times higher than the normal muscle (3.39 ?? 0.6). The scintigraphic images completely visualized the infection foci in left thigh muscles of the mice as the area of increased radiotracer accumulation. [197]. Kaul et al. (2013) formulated 99mTc-ceftriaxone into a ready-to-use single-vial cold kit with a shelf-life of over 6 months which was used to identify active septic foci in the experimental model of S. aureus -induced infectious lesions in the rabbit and also it was examined as a preliminary clinical study in 36 patients with a clinical and radiological suspicion of an orthopedic infection [198]. This clinical evaluation of 99mTc labeled ceftriaxone showed a diagnostic accuracy of 83.3%, and a sensitivity and specificity of 85.2% and 77.8%, respectively. In comparison with uptake level of radioactivity of 99mTc'ciprofloxacin, they concluded that the interaction of 99mTc-ciprofloxacin with various physiological factors (such as ciprofloxacin structure) prior to its interaction with the bacterial chromosomes and its lesser protein binding (30%) causes reduced availability in the blood pool. Meanwhile, in the case of infection 99mTc-ceftriaxone, high protein binding results in the increased availability of circulating drug and consequently it continues to accumulate at the site of infection [198]. Among the other cephalosporin group, cefoperazone [10], cefuroxime axetil [7], cefotaxime [199], cefepime [185], cefuroxime [200] and ceftazidime [201] were investigated for radiolabeling to evaluate their ability for infection detection in animal models.

Other antibiotics
Kanamycin is an aminoglycoside antibiotic used to treat a wide variety of infections, directly labeled with 99mTc by Roohi et al. which was used for in vivo biodistribution study in rats and scintigraphic imaging in rabbit model. S. aureus infection in rabbit thigh muscle was visualized clearly 15 minute post administration and target-to-background ratios obtained from 99mTc-Kanamycin ranged 2.5 to 4:1. The results indicated that 99mTc-kanamycin localizes and detects bacterial infection sites significantly. However, its usefulness has not been studied in animals with sterile inflammatory lesions [202]. Roohi et al. in another study investigated the in vivo biodistribution manner of 99mTc labeled vancomycin, a glycopeptide antibiotic, for discriminating staphylococcal induced infection with turpentine oil sterile inflammation. The highest T/NT 1.5 was achieved at 1 hour post injection and the localization at sterile inflammation was insignificant [203]. 99mTc'rifampicin, belonged to rifamycin group was synthesized specifically for the infection localization caused by methicillin-resistant S. aureus (MRSA). Imaging of the MRSA infected rabbit confirmed the usefulness of the 99mTc'rifampicin as a precise radiotracer for MRSA infection imaging and the ratios of the infected muscle to normal muscle and inflamed muscle to normal muscle in rats were 7.34??74 and 1.20??0.85, respectively [204].

Radiolabeled antitubercular agents
Mycobacterial infections have been shown to be increasing in number worldwide, mainly due to a global increase in developing countries, the increased number of patients with HIV infection and AIDS disease worldwide, an increasing the number of elderly patients and the emergence of multidrug resistant tuberculosis [205]. Radiolabeling of isoniazid (INH) (an antitubercular drug which binds to mycolic acid in the cell walls of living mycobacteria) using 99mTc for imaging mycobacterium tuberclusis cold abscesses in rabbits was reported by Singh et al. (2003). Scintigraphic imaging of induced tuberclusis cold abscesses with rapid washout of 99mTc- isoniazid from S. aureus infected abcesses (as a control) suggested that this agent maybe useful for detection of tuberculous lesions. Overall more than 95% sensitivity and high specificity were observed in the animal study [206]. Singh et al. (2009) performed a clinical trial to establish the efficacy of 99mTc-INH in humans with sensitive as well as resistant tuberculosis was conducted in 20 patients. Bone lesions were visualized within 1 h while pulmonary lesions accumulated 99mTc-INH very slowly with time and 24 h acquisition appeared essential for the diagnostic interpretation. They concluded that the 99mTc-INH with low possibility of allergic or antigenic response and being safe for human use may be sensitive in both pulmonary and peripheral forms of the infection including resistant tuberculosis [207]. Ethambutol (EMB) is used as a first-line drug for the treatment of tuberculosis which acts by inhibiting cell wall mycolic acid synthesis. 99mTc- ethambutol was used for detection of cold abscess caused by M.tuberculosis in thigh muscle of rabbits and mice [208]. Imaging results demonstrated that this tracer accumulated and bound only to cold abscess as early as 2 hours after injection, which increased at 4 hours and persisted till 24 hours with good specificity. They concluded that the agent was a stable, reproducible, safe and cost effective imaging method for the early diagnosis of tuberculosis [208]. Singh et al. in 2010 evaluated the efficacy of 99mTc-Ethambutol to detect and locate the tubercular lesions for clinical trial in humans. Scintigrams of 99mTc-EMB in fourteen patients showed localization of this agent in pulmonary and bone tubercular lesions with no adverse reactions. Their clinical study suggested that 99mTc-EMB has high potential to qualify as a specific tuberculosis imaging radiopharmaceutical and is safe for human use for sensitive as well as resistant tubercular lesion detection and localization [12].

Radiolabeled antifungal agents
In recent years, an increase (from 19 % to 25 %) in the incidence of infections caused by opportunistic mould pathogens including Aspergillus, Candida, zygomycete, Fusarium, Scedosporium and Acremonium species has been observed, with invasive aspergillosis (IA) being the predominant infection [209]. Candida albicans is the most common fungal pathogen, and is the organism responsible for the majority of localized fungal infections in humans [210]. Fluconazole, the triazole antifungal agent in treating Candida infections, was successfully labeled with 99mTc by Lupetti et al. (2002) and used for the diagnosis of C. albicans infections in mice to compare its efficacy for detecting bacterial and sterile inflammatory lesions [211]. The results concluded that this agent only detected infections due to C. albicans, no binding to bacterial infections or accumulation at sterile inflammatory sites in animals. Besides, to compare its ability with a radiolabeled antimicrobial peptide, the superiority of 99mTc fluconazole in differentiating between fungal and bacterial infections was shown. However, they reported that it was not suitable choice to detect in case of Aspergillus fumigatus infections [211]. Siaens et al. (2004) used another approach for in vivo detection of fungal infections by using 123I-labeled chitinase, which recognizes chitin on the cell wall of fungi in mice with infected thigh muscles [212]. C. albicans as well as A. fumigatus infections could be visualized with scintigraphy by specific accumulation at site of infections with good target-to-non target ratios (with no accumulation in tissue infected with gram-positive or gram-negative bacteria, or in sterile inflammations) which remained as high as 5.1 %ID/g at 4 h after intravenous injection [212]. Furthermore, synthesis and evaluation of technetium-99m HYNIC labeled chitin-binding protein (like 99mTc-HYNIC-CBP21) as potential specific radioligand due to 99mTc favorable characteristics, for the detection of fungal infections in mice was investigated [213]. 99mTc-fluconazole proved to be an excellent tracer for C. albicans infections as it did not accumulate in bacterial infections and inflammatory processes. However this tracer poorly detected A. fumigatus infections. Furthermore, 123I-chitinase and 99mTc-HYNIC-CBP21 accumulated in both C. albicans and A. fumigatus infections in mice at later time points. Besides, radiolabeled antimicrobial peptides as antifungal imaging agent, have the ability to distinguish local C. albicans and A. fumigatus infections from sterile inflammatory processes, but not from bacterial infections [214].

Radiolabeled FIAU
The possibility of using a naturally occurring virus-encoded molecule as an imaging reporter was first explored in the early 1980s, when acyclovir was approved as an antiviral drug for the treatment of HSV (herpes simplex virus) infections. The ability of this and other nucleoside analogues to selectively inhibit HSV replication is based on their ability to undergo phosphorylation by the viral thymidine kinase (TK), but not by corresponding host enzymes [215]. Among various substrates, radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-??-D-arabinofuranosyluracil (FIAU) demonstrated high sensitivity and selectivity for the detection of HSV1-tk expression. FIAU is trapped intracellularly only in the presence of HSV1-tk [216]. Bettagotha et al. (2005) were the first developed a simple method for imaging bacterial infections in mice that relies on the phosphorylation and trapping of the thymidine kinase (TK) substrate, FIAU within bacteria [217]. They found that FIAU can also be a substrate for the TK of multiple pathogenic bacterial strains, as demonstrated through in vitro experiment and in vivo uptake of [125I] FIAU in mice with focal infections induced by five different genera of bacteria were imaged as early as 2 h after injection of radiolabeled compound [217]. This preclinical study was led to successful imaging of human musculoskeletal infections using 124I-FIAU by Diaz et al. in 2007 [218]. They employed 124I-FIAU as a PET probe to detect staphylococcal infections following joint surgery. Seven patients with proven musculoskeletal infections demonstrated positive [124I] FIAU-PET/CT signals in the sites of concern at two hours after radiopharmaceutical administration with no observation of adverse reactions [218]. Recently Pullambhatla et al. reported the use of [125I] FIAU to image murine pulmonary bacterial using SPECT-CT with specificity for bacterial infection rather than sterile inflammation. Radiolabeled FIAU showed that it can be used to estimate the number of bacteria present within lungs, with a limit of detection of 109 colony forming units per mL on the X-SPECT scanner [219]. However, not all bacteria possess the TK enzyme and as a result not all bacterial infections can be visualized using radiolabeled FIAU. The usefulness of radiolabeled FIAU in diagnosing bacterial infections in patients with genetic immunodeficiencies with Pseudomonas infections demonstrated that it would not be useful in these patients, considering the apparent lack of TK enzyme in Pseudomonas [220].

Radiolabeled antimicrobial peptides
Antimicrobial peptides (AMPs) are widely distributed throughout the animal and plant kingdoms where they play a critical role in the defence system of multicellular organisms against bacteria, fungi and viruses. Antimicrobial peptides usually contain hydrophobic and cationic amino acids, which are able to organize in an amphipatic structure [221]. The peptides are broadly classified into five major groups namely (a) peptides that form '-helical structures, (b) peptides rich in cysteine residues, (c) peptides that form '-sheet, (d) peptides rich in regular amino acids namely histatin, arginine and proline and (e) peptides composed of rare and modified amino acids. Most of these peptides are believed to act by disrupting the plasma membrane leading to the lysis of the cell [222]. Due to their cationic nature, AMPs are electrostatically attracted to negatively charge microbial surfaces such as lipopolysaccharide (LPS) in Gramnegative bacteria and teichoic and teichuronic acids in Gram-positive bacteria [223]. AMPs which are generated in various types of host cells and tissues that were exposed to pathogens display a broad microbicidal activity against bacteria, fungi, viruses, or parasites in vitro and in laboratory animals they show effective microbicidal efficacy against experimental systemic, soft-tissue, and bone infections caused by various pathogens. Besides their direct microbial killing properties, AMPs can neutralize bacterial toxins and LPS and up-regulate the host defense as chemoattractants or by other immunostimulatory effects. Besides their microbicidal activities AMPs may play an important role in anti-tumor activity bone repair, neoangiogenesis, enzymatic activity and anti-biofilm activity [224]. Because antimicrobial peptides preferentially bind to bacterial membranes, radiolabeling of these peptides would offer the medical community the novel candidates for the development of bacteria-seeking radiopharmaceuticals [178]. Various natural and synthetic antimicrobial peptides have been investigated for imaging of bacterial infections, such as synthetic peptides derived from domains on human antimicrobial peptide ubiquicidin (UBI), human lactoferrin (hLF), and natural peptide human defensin.
The first studies in this field dates backs to 1998 by Welling et al. They were evaluated the potential use of human neutrophil peptide-1 (HNP-1) which is a member of the defensins family as a tracer for rapid visualization of bacterial infections in mice [178, 225]. HNP-1 previously was studied for antibacterial therapy of experimental infections in mice in which the reduction of bacterial numbers was found to be associated with an increased influx of neutrophils into the infected area [225]. They concluded that 99mTc- HNP-1 allows rapid visualization of bacterial infections (T/NT was higher than 1.3 for 99mTc-HNP-1 within 5 min) after administration of the tracer to mice [178]. Another animal experiments compared the possibilities and limitations of 99mTc labeled synthetic peptides derived from two human antimicrobial peptides, ubiquicidin (UBI) and lactoferrin (hLF), for the scintigraphic detection of bacterial (S. aureus and Klebsiella pneumoniae )and fungal ( fluconazole-resistant C. albicans) infections in mice and rabbits [14]. 99mTc-labeled antimicrobial peptides UBI 29'41 (ubiquicidin-derived cationic human antimicrobial peptide fragment), UBI 18'35, UBI 31'38, hLF 1'11 (derived from the first 11 amino acids of human lactoferrin), and defensins accumulated significantly in tissues infected with gram-positive and gram-negative bacteria and C. albicans with significantly lower (P<0.01) accumulation of these peptides occured in sterile inflamed tissues. These data indicated that the peptides preferentially tag microorganisms at the site of infection, which is in agreement with their preferential binding to the microorganisms in vitro and in vivo. However, they preferred 99mTc-labeled UBI peptides to 99mTc-labeled hLF peptides, because of hLF accumulation in liver and deposits in intestines, therefore made them less favorable for imaging infections and also 99mTc-labeled UBI peptides can be prepared synthetically under good manufacturing conditions in large amounts, with no adverse effects were found under the conditions of experiments [14]. The possibility of monitoring the efficacy of antibacterial therapy in S. aureus infected mice using 99mTc-UBI 29-41 and scintigraphy was investigated by Nibbering et al. [226]. The results indicated this tracer could be used for monitoring the efficacy of antibacterial agents in animals infected with S. aureus suggesting that at sites of infection mainly extracellular bacteria are targeted by 99mTc-UBI 29-41. Brouwer et al. studied the effect of various routes of administration of 99mTc-hLF 1'11 (either intravenously, subcutaneously, intraperitoneally, or orally) and various doses of this agent on the bactericidal activity against Multidrug-resistant S. aureus (MRSA) and the biodistribution were evaluated and compared to that of mice injected intravenously with 99mTc-hLF 1'11 [227]. Intravenously and orally administrated 99mTc-hLF 1'11 accumulates in infected tissues and is highly effective against experimental infections with MRSA. Moreover, scintigraphy is an excellent tool to study the pharmacology of experimental compounds and to determine the uptake in infected tissues [227]. Akhtar et al. in 2004 performed an experiment using 99mTc-UBI (29-41) in a freeze-dried kit to evaluate its scintigraphic potential as a bacterial infection seeking agent in S. aureus and E. coli induced infections in rabbits [228]. A higher accumulation of 99mTc-UBI (29-41) at sites of S. aureus infected animals (T/NT ratio, 2.2 ?? 0.5) compared with that of E. coli infected animals (T/NT ratio, 1.7 ?? 0.4) at 60 min after injection was observed. They concluded that relatively low T/NT ratios were observed in E. coli induced infections may be due to a low virulence of E. coli used in experiments or may be by differences in the pathogenicity mode of the 2 microorganisms [228]. To compare direct labeling of UBI 29-41 by 99mTc with the labeling of this peptide through the intermediacy of the bifunctional chelator HYNIC or a protected diaminedithiol (N2S2), were assessed by Welling et al. (2004) and the radiochemical and biological characteristics, as well as the ability of these three tracers to detect bacterial infections in mice were evaluated [229]. The results demonstrated that purified 99mTc-HYNIC-UBI 29-41 and 99mTc-N2S2-UBI 29-41 were as effective as 99mTc-UBI 29-41 in detecting infections in mice injected intramuscularly with bacteria. However, 99mTc-N2S2-UBI 29-41 should not be advised for the imaging of abdominal infections as this tracer, in contrast to the other tracers, is cleared via the liver and intestines [229]. Similarly, the results revealed insignificant accumulation of labeled peptide in sterile inflamed thigh muscles of mice and rabbits [14, 178, 228, 229]. 99mTc-UBI 29-41 scintigraphy for detection of experimental Staphylococcus aureus prosthetic joint infections in a rabbit model showed specific early detection of acute prosthetic joint infection with well differentiating from chronic sterile prosthetic joint inflammation [230] Besides, UBI 29-24 was radiolabeled with 18F by Zijlstra et al. and in vitro experiment for pharmacokinetic analyses of 18F-UBI 29-41 reported a substantial binding to S. aureus [231]. In clinical setting, Melendez-Alafort et al. (2003) were the first to establish a 99mTc-UBI biokinetic model and evaluate its feasibility as an infection imaging agent in humans (six children between 2 and 15 years old with suspected bone infection) using 67Ga-citrate as a control [232]. Biokinetic data showed a rapid background clearance, minimal accumulation in non-target tissues and a low effective radiation absorbed dose with no adverse reactions after administration of 99mTc-UBI in humans. The sites of infection were successfully imaged at short times (1-2 h) which is an important advantage in the clinical diagnosis of infections and especially in pediatrics. Furthermore, all 99mTc-UBI positive images were in agreement with those obtained with 67Ga-citrate [232]. Another clinical study by Akhtar et al. (2005) was performed for scintigraphic imaging of 99mTc-UBI 29-41 in 18 patients with suspected bone, soft-tissue, or prosthesis infections [233]. The optimum imaging time for delineation between infectious and inflammatory process is 30 min after intravenous administration of radiotracer. The sensitivity, specificity, and overall diagnostic accuracy of 99mTc-UBI 29'41 in their study for infection localization were 100%, 80%, and 94.4%, respectively. They concluded that 99mTc-UBI 29'41 is a highly sensitive and specific agent for localizing infective foci in bone and soft tissues of humans [233]. Furthermore, the clinical use of 99mTc-UBI 29-41 antimicrobial peptide for the scintigraphic detection of mediastinitis after cardiac surgery was evaluated [234] resulting in the ability of 99mTc-UBI 29-41 to distinguish between sterile inflammation and mediastinitis with a significant positive predictive value. Qualitative analysis of the scintigrams with 99mTc-UBI 29-41 correctly identified the infection in 5/6 patients with mediastinitis. The potentiality of [99mTc/Tricine/HYNIC0] UBI 29'41 prepared from lyophilized kits was tested by Gandomkar et al. as an infection imaging agent in seven patients with suspected bone or soft tissue infections [235]. Images showed minimal accumulation in non target tissues, and the maximum absorption obtained 30 min after injection had a T/NT ratio of 2.10??0.33% which was similar to that observed in animal experiments by Welling et al. [235]. Moreover, clinical application of 99mTc-UBI 29-41 to assess diagnostic value as an infection imaging radiopharmaceutical were reported in detecting of infectious diseases such as osteomyelitis [236], fever of unknown origin (FUO) [237], Vertebral osteomyelitis [238] and suspected orthopedic implants infection [239]. Although there are limitations attributed to synthesis/isolation of antimicrobial peptides, labeling with isotopes, minimum detection limit of 103 Colony-Forming Unit (CFU) of bacteria, and inability to distinguish between bacterial and fungal infections. In addition, different bacterial types reveal different tracer accumulation (S. aureus versus E. coli). Currently no evidence regarding resistance against antimicrobial peptides has been reported. Considering the merits and demerits of radiolabeled peptides and radiolabeled antibiotics, it can currently be concluded that radiolabeled peptides are better specific infection localizing agents than radiolabeled antibiotics [240].

Conclusion
Discrimination between infectious and noninfectious inflammation continues to pose a diagnostic challenge, and the search continues for methods that can reveal infectious foci rapidly and effectively. As discussed above, various conventional radiopharmaceuticals which are basically on the uptake mechanism of targeting host inflammatory response (increased blood supply, increased vascular permeability and enhanced transudation) are not specific for infection imaging. In contrast, the use of radiopharmaceuticals for specific targeting of microorganisms responsible for infection, have been proposed. Examples of potential microorganism targeting agents include labeled antibiotics, labeled antimicrobial peptides and enzy??matic substrates of bacterial enzymes. Radiolabeled antimicrobials (such as antibiotics and antimicrobial peptides) represent a novel approach to the diagnosis of deep seated infection due to the direct targeting of the microorganisms. Antibiotics play a critical role in modern medicine in the treatment of infections caused by pathogenic microorganisms. However, the increasing antibiotic resistance in bacteria due to the misuse of antibiotics and microbial genetic mutations has become a worldwide threat to human health. In addition, some authors have shown that radiolabeled antibiotics (for example 99mTc-ciprofloxacin) cannot discriminate between infection and inflammation due to nonspecific accumulation in inflammatory sites. Another targeting approach is radiolabeled FIAU which has been proposed as an agent to image bacterial infections, based on presence of endogenous TK (thymidine kinase) substrate within bacteria, but not for TK-negative bacteria. Because fungi in general do not have TK, would not be expected to phosphorylate FIAU either. Furthermore, there are limitations regarding radionuclide availability and elaborate synthesis and purification steps. Radiolabeling of antimicrobial peptides would offer the novel candidates for the development of the radiopharmaceutical displaying a preferential binding to microorganisms to discriminate infections from sterile inflammatory lesions due to insignificant accumulation at the site of sterile inflammation and also, to monitor the efficacy of antibiotic therapy. Despite some limitations attributed to antimicrobial peptides, no evidence regarding resistance against them has been reported. Considering more advantages of antimicrobial peptides over antibiotics, we suggest that, antimicrobial peptides with wide promising properties as the infection imaging agents have the ability to be used in clinical settings in patients with suspected infections for more accurate diagnosis. However, it is necessary to continue research on seeking the ideal infection imaging agent to address infections caused by microorganisms such as viruses, fungi, parasites and intracellular pathogens. In addition, by growing the number of immunocompromised patients in the era of HIV and tuberculosis, future agents should work well in immunosuppressed and immunocompetent patients to over??come the limitations of currently available agents/techniques.

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