Received Date : 17-Dec-2014

Revised Date : 04-Feb-2015

Accepted Article
Accepted Date : 10-Mar-2015
Article type : Invited Review

Challenges in diagnosing infection in the diabetic foot

A. W. J. M. Glaudemans1, I. Uçkay2,3 and B. A. Lipsky2,4

1
Department of Nuclear Medicine and Molecular Imaging, University of Groningen, University

Medical Center Groningen, Groningen, The Netherlands, 2Service of Infectious Diseases and
3
Orthopaedic Surgery Service, Geneva University Hospitals and Faculty of Medicine, Geneva,

Switzerland, and 4Division of Medical Sciences, University of Oxford, Oxford, UK

Accepted

Correspondence to: Benjamin A. Lipsky. E-mail: dblipsky@hotmail.com

Abstract

Diagnosing the presence of infection in the foot of a patient with diabetes can sometimes be a

difficult task. Because open wounds are always colonized with microorganisms, most agree that

infection should be diagnosed by the presence of systemic or local signs of inflammation.

Determining whether or not infection is present in bone can be especially difficult. Diagnosis

begins with a history and physical examination in which both classic and 'secondary' findings

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 suggesting invasion of microorganisms or a host response are sought. Serological tests may be
Accepted Article
helpful, especially measurement of the erythrocyte sedimentation rate in osteomyelitis, but all

(including bone biomarkers and procalcitonin) are relatively non-specific. Cultures of properly

obtained soft tissue and bone specimens can diagnose and define the causative pathogens in

diabetic foot infections. Newer molecular microbial techniques, which may not only identify

more organisms but also virulence factors and antibiotic resistance, look very promising.

Imaging tests generally begin with plain X-rays; when these are inconclusive or when more

detail of bone or soft tissue abnormalities is required, more advanced studies are needed. Among

these, magnetic resonance imaging is generally superior to standard radionuclide studies, but

newer hybrid imaging techniques (single-photon emission computed tomography/computed

tomography, positron emission tomography/computed tomography and positron emission

tomography/magnetic resonance imaging) look to be useful techniques, and new

radiopharmaceuticals are on the horizon. In some cases, ultrasonography, photographic and

thermographic methods may also be diagnostically useful. Improved methods developed and

tested over the past decade have clearly increased our accuracy in diagnosing diabetic foot

infections.

Introduction

As the incidence of diabetes mellitus increases worldwide and people with this disease live

longer, the number of patients developing a diabetic foot complication is growing dramatically.

The most common foot problem is an ulceration, which is most often related to the consequences

of prolonged peripheral neuropathy, often in association with peripheral arterial disease. While

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 all foot wounds require treatment, those that are infected are at the highest and most urgent risk
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of dire consequences, including lower extremity amputation and occasionally infection-related

death [1]. They are also the only foot wounds that require antimicrobial therapy. Thus,

diagnosing infection is key to proper treatment of diabetic foot wounds.

How, then, should we define infection of the diabetic foot? Many clinicians might respond, 'I

know it when I see it', but infection appears to be mostly in the eye of the beholder. Infections

are generally defined in one of two ways: 1) the laboratory isolation of pathogenic

microorganisms from a normally sterile site (e.g. blood, cerebral spinal fluid, sterilely collected

deep tissue) or 2) a constellation of clinical signs and symptoms compatible with an infectious

syndrome. The classic clinical manifestations of infection, dating from antiquity, are: erythema

(rubor), warmth (calor), swelling (tumor), pain or tenderness (dolor). There are two major

problems with diagnosing diabetic foot infections. First, all open wounds are colonized with

microorganisms, making culture results diagnostically non-definitive. Second, the presence of

peripheral neuropathy and vascular disease can either diminish or mimic inflammatory findings,

both in the soft tissue and underlying bones, reducing their usefulness. At presentation for

medical care, about half of these wounds are clinically infected [2,3].

Clinical presentation and probe-to-bone testing

Clinical evaluation (see Table 1) begins with the patient’s history. Patients with diabetic foot

infections will typically have a history of a current or recent (although occasionally forgotten)

wound that caused a break in the protective skin envelope. These may be caused by mechanical,

chemical or thermal trauma, but are most often attributable to pressure on a neuropathic

(deformed and insensate) foot. On physical examination, infections are more likely to be present

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 in wounds that are chronic (present for >2 weeks), large (>2 cm2) or deep (>3mm). Other than
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cases of erysipelas/cellulitis or surgical site infections, infection in the diabetic foot is an

epiphenomenon of underlying problems related to various concomitant comorbidities. Most

important among these are peripheral neuropathy (affecting sensory, motor and autonomic

nerves) and peripheral arterial disease. These disorders can either lead to a diminution of the

expected inflammatory response or be the cause of these signs and symptoms [4]. The great

majority of patients with a diabetic foot infection have peripheral neuropathy affecting the feet.

In a sizeable percentage of them, Charcot neuro-osteoarthropathy may be present. In the acute

stages this can manifest as a red, warm, painful foot, mimicking soft tissue infection, while in the

chronic phase it can lead to bony abnormalities that may suggest osteomyelitis [5]. Similarly,

peripheral arterial disease, which is present in the majority of patients with diabetic foot

infections, may impair manifestations of erythema, warmth or induration, or cause pain

(claudication) or dependent rubor [6]; thus, seeking cardinal or 'textbook' signs of inflammation,

such as warmth, redness, pain/tenderness, swelling, or loss of function alone may not be

sufficient to diagnose infection. Clinicians must often seek other potential manifestations of

infection, such as fever, shivering, or purulent secretions, and ask about any spreading

inflammation over the preceding hours or days [7,8]. Because these findings are often lacking in

diabetic foot infection, some advocate defining infection of a wound by the presence of

'secondary' findings, such as foul odour, serous exudate, undermining of the wound rim,

discoloured or friable granulation tissue [9].

Based on the available evidence, the 2012 guidelines on diabetic foot infections produced by

both the Infectious Diseases Society of America and the International Working Group on the

Diabetic Foot advocate defining infection as the presence of at least two of the classic findings

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 of inflammation or purulence [10]. Because of the problems discussed above, in situations where
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clinicians are uncertain about whether or not infection is present, some advocate empirical

treatment with antibiotic agents for 2 or 3 days to see if clinical signs or symptoms improve. We

do not condone this approach, as it is likely to lead to overtreatment of uninfected wounds based

on a misperceived belief that the response is to antibiotic therapy. Because diabetic foot wounds

respond to standard wound care, such as cleansing, debridement, appropriate dressings, pressure

offloading and improved glycaemic control, improvement may not actually be related to

antibiotic-induced killing of infecting pathogens. Our recommended approach in such a situation

is to optimize wound care and carefully observe the patient; should more clear evidence of

infection appear, cultures and antibiotic treatment are then appropriate.

Diagnosing osteomyelitis of the diabetic foot is particularly problematic. Patients with a history

of foot wounds, or with deep wounds (especially over bony prominences) are more likely to

develop infection of the underlying bone [2]. In almost all cases, diabetic foot osteomyelitis

occurs in a patient who has a current or recent soft tissue wound through which contiguous

infection leads to bone involvement. Notably, bone infection can sometimes occur under what

appears to be a clinically uninfected ulcer [11]. The presence of a 'sausage toe', a red, swollen,

warm digit, is typical of diabetic foot osteomyelitis. The only virtually pathognomonic clinical

sign of osteomyelitis, however, is the presence of fragments of bone in the wound or dressing, or

fragments found during debridement. In contrast to long bones, osteomyelitis of the diabetic toe

often lacks a sequestrum or sinus tract that can been easily distinguished from an overlying ulcer

[12].

The probe-to-bone test can be helpful in diagnosing diabetic foot osteomyelitis, but only if it is

performed and interpreted correctly. The clinician should probe after debriding the wound, using

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 a blunt metal (not wood or plastic) probe; a characteristic feel of a hard, gritty surface constitutes
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a positive test [1]. The test is, however, not pathognomonic for bone infection. Indeed, based on

several reports, the sensitivity ranges from ~60 to 87%, the specificity from 85 to 91%, and the

positive predictive value from 87 to 90%. The negative predictive value is only 56–62% [13–15].

Thus, a key issue in interpreting the test is the pretest probability of osteomyelitis in the patient

population being studied. Where the clinical or imaging features make the likelihood of

osteomyelitis high, a positive test may be sufficient for diagnosing probable osteomyelitis. By

contrast, where the suspicion of bone infection is low, a negative test is helpful in ruling out the

diagnosis. The test requires some experience to gain mastery and the interobserver concordance

is relatively low, with a κ index of ~50% [16]. On its own, the performance characteristics of the

probe-to-bone test are similar to those of other less regarded variables, such as an ulcer area >2

cm2 or an erythrocyte sedimentation rate of >70 mm/h [17].

Serum inflammatory markers

As with other local infections, serum inflammatory markers are frequently not elevated in

diabetic foot infections, especially in chronic cases. As the available literature reports conflicting

performance characteristic results, recommendations on the relative usefulness of the several

available tests vary. Some have found higher baseline levels of C-reactive protein, erythrocyte

sedimentation rate and white blood cell levels in osteomyelitis cases than in those of soft-tissue

infections [18]. While serological tests cannot be used alone, and the recommended thresholds

for differentiating these two vary, most consider an erythrocyte sedimentation rate of >60–70 to

be perhaps the most suggestive of osteomyelitis [19]. The serum procalcitonin level is one of the

newer tests promulgated for diagnosing infections. Only a few studies have reported results in

patients with skin and soft tissue infections; these have found that procalcitonin correlates with

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 disease classification (being more often elevated in complicated than uncomplicated infections),
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clinical course of infection, and other laboratory markers of inflammation [20]. Results in

patients with diabetic foot infections, especially osteomyelitis, have generally been disappointing

[21–23]. Levels are higher in infected than in uninfected diabetic foot ulcers, but the

performance characteristics are not as good as the erythrocyte sedimentation rate or C-reactive

protein levels [24]. Our review of the available studies suggests that, in the absence of systemic

manifestations of localized infection, procalcitonin does not help to distinguish acute infection

from acute ischaemia or other non-infectious conditions, or to differentiate soft-tissue infection

from osteomyelitis.

Investigators have sought other serum markers that may help diagnose infection, especially

osteomyelitis. One such marker is bone sialoprotein because Staphylococcus aureus isolates

from patients with osteomyelitis express bone sialoprotein-binding protein that binds the

corresponding bone matrix protein. In one pilot study of patients with a diabetic foot ulcer,

serological assays for bone sialoprotein-binding protein discriminated cases of osteomyelitis

from those with just soft tissue infections [25]. Another innovative approach could be the

measurement of bone turnover markers, which might indirectly help with the diagnosis and

monitoring of patients with osteomyelitis. Bone alkaline phosphatase and serum amino-terminal

telopeptides are two such markers; however, in a recent study analysing their performance in 54

patients with diabetes, neither marker was useful in detecting osteomyelitis, either at baseline or

follow-up, nor did they help predict outcome [26]. One new idea is to investigate the value of

measuring local cytokine titres in patients with osteomyelitis. A preliminary retrospective study

of immunohistochemical staining of bone biopsy specimens of patients with diabetic foot

osteomyeltis showed that stains for interleukin-6 were intensively positive in cases with acute

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 infection while stains for tumour necrosis factor-α were positive in chronic or reparative states
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[27]. Further studies are needed to see if these markers may prove useful as, for example,

interleukin-6 expression is also high in the acute Charcot foot [28]

Histopathology

In some cases osteomyelitis can only be diagnosed by examining a specimen of bone, obtained

either at the time of surgery or by percutaneous biopsy. Most believe the criterion standard for

diagnosing bone infection is obtaining positive results on both culture and histopathological

examination of bone. This is because bone cultures can be falsely negative if a patient is taking

antibiotics and falsely positive because of contamination of the specimen, while histopathology

can be falsely negative if the infected area is missed or inaccurate when read by an inexperienced

pathologist. Unfortunately, cultures of soft tissue, even deep aspiration near the infected bone, do

not provide sufficiently accurate results compared with bone specimens. Even needle bone

puncture appears to provide significantly fewer positive results (58%) compared with

transcutaneous bone biopsy (97%) [29]. A recent study has shown that when bone cultures are

negative, subsequent occurrence of osteomyelitis within 2 years' follow-up is infrequent [30].

Bone specimens from patients with long-standing diabetes may be found to have myelofibrosis,

osteonecrosis and osteoporosis at affected sites, but are usually normal or unremarkable when the

bone is not infected [31]. Thus, the presence in bone of inflammatory cells (particularly

polymorphonuclear leukocytes, but also mononuclear cells) and the presence of necrosis indicate

bone infection, especially if microorganisms are visible microscopically and there is no other

reason for chronic inflammation. The criteria for histopathological diagnosis of osteomyelitis,

however, have not been validated or standardized. The findings of a recent study showed a

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 remarkable lack of agreement among pathologists who independently reviewed 39 consecutive
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diabetic foot bone biopsy specimens blinded to the patients’ clinical characteristics [32]. In only

one-third of cases was there complete agreement on the presence or absence of osteomyelitis and

in 41% of cases there was a clinically important disagreement between at least two of the

pathologists. This distressing result may have been at least partly related to the absence of an

agreed-upon classification scheme for diabetic foot osteomyelitis. In light of this finding and

based on extensive experience, Cecilia-Matilla et al. [33] proposed a well defined scheme of four

histopathological types of diabetic foot osteomyelitis according to the cell groups present and the

histopathological changes in the bone samples: acute osteomyelitis; chronic osteomyelitis; acute

chronic osteomyelitis; and fibrosis stage (an unresolved final process of bone infection with

fibrotic, avascular tissue). Using this scheme they showed much less intra-observer variability

between two pathologists [34]. One member of this group developed a clinical classification

scheme for types of osteomyelitis: without ischaemia or soft tissue involvement (class 1); with

ischaemia but without soft tissue involvement (class 2); with soft tissue involvement (class 3);

and with ischaemia and soft tissue involvement (class 4). Applying these criteria in a study of 48

patients showed that the classes were associated with a statistically significant trend among the

four types toward increased severity, amputation rates and mortality [35]. Other groups will need

to test these classification schemes and it remains to be seen if they gain wider acceptance.

Culture

Although detecting microorganisms within a diabetic foot wound (see Table 2) does not

currently define the presence of infection, it is a necessary step for selecting the optimum

therapeutic approach. For over 150 years, we have used methods developed by Pasteur and

others to detect and classify pathogens. These methods have been useful, but are limited by

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 several problems. Firstly, we only grow the organisms we know how to look for, and these easy-
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to-grow species may actually be 'laboratory weeds' and we may be missing true pathogens that

standard techniques fail to identify. Secondly, cultivating microorganisms and determining their

sensitivity patterns usually takes at least 2–3 days, even with the newer more rapid techniques.

During this waiting period we are forced to treat the patient with an empiric antibiotic regimen,

which has been shown to be inappropriate in almost a quarter of cases [36]. With the growing

pandemic of antibiotic resistance pathogens this problem is likely to worsen, and it has major

clinical and financial consequences [37]. Thirdly, with over a third of diabetic foot infections

being polymicrobial, we cannot currently determine which of the microorganisms are truly

playing a pathogenic role and which are merely colonizers. Fourthly, standard culture methods

lead to false-negative results in patients who are already being treated with antimicrobial agents,

a common clinical situation. Finally, we have learned that bacteria in wounds are commonly

found in biofilms, making them more difficult to culture (as well as to treat) [38]. So, how do we

address these problems?

To start, while we still depend on the microbiology laboratory, clinicians need to properly collect

and quickly send them optimum specimens. Although taking a swab of a wound is easy and

inexpensive, it clearly provides a suboptimum specimen, giving culture results that are both less

sensitive and less specific than with a tissue specimen [39]. This is clearly a situation where

'garbage in' (a poorly obtained wound specimen, especially when it takes hours to get to the

laboratory) leads to 'garbage out' (an unhelpful laboratory report such as 'mixed cutaneous flora'

or 'no S. aureus found'). Quantitative microbiology, championed by some over the past 50 years,

is not the answer, both because it has not been shown to prove a wound is infected and because

non-research clinical laboratories do not provide this complex and expensive service.

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 The future of microbiology certainly appears to be the newer molecular technologies that are
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currently making their way into many diagnostic laboratories. These techniques will allow rapid

identification (probably in less than an hour) of all of the microorganisms in a wound.

Furthermore, they will rapidly report on the presence of genes that code for pathogenicity and for

resistance to commonly used antibiotic agents. If this sounds like a distant dream, or an episode

of 'Crime Scene Investigation,' it is not [40]. Newer molecular tools allow detection of the >500

species of microorganisms that constitute the microbiota on various colonized surfaces, including

the skin. Remarkable progress in sequencing bacterial genomes and the development of new

molecular approaches has facilitated an understanding of the steps in the evolution of the

complex flora of skin microbiota as well as the development of wound infection.

We now recognize that bacteria within a diabetic foot wound are often in biofilms, i.e.

composites of aggregated cells encased in an extracellular matrix of hydrated polymers and

debris, which impair wound healing and protect the enmeshed bacteria from host immune

responses and antimicrobials. This has led to the concept of microorganisms in a wound

behaving as 'functionally equivalent pathogroups', in which species that usually behave in a non-

pathogenic manner on their own may co-aggregate to act synergistically to cause a chronic

infection. Molecular methods have uniformly shown that most diabetic foot ulcers host many

more bacterial species in greater numbers than previously appreciated. Available data suggest

that diabetic foot infections more often arise from the presence of specific combinations of

pathogens than a simple increase in the microbial load of any one opportunistic microbe.

Furthermore, compared with the superficial flora, those from the deep tissue are more complex

and diverse, and especially rich in anaerobic species. Investigations in the diabetic foot have

shown that the enrichment of the number of S. aureus organisms may be a precursor to

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 developing clinically apparent infection in a diabetic foot ulcer. Microbiota studies have also
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shown that certain wound flora are associated with several specific clinical characteristics of the

diabetic foot, including ulcer depth (a surrogate for wound severity) and duration (which may be

a surrogate for delayed wound healing) [41].

Studies examining the role of S. aureus, the predominant pathogen in diabetic foot infection,

have highlighted the co-existence of several populations, and that a combination of five specific

genes may help distinguish colonized from infected wounds and predict ulcer outcome, which

contributes to more appropriate use of antibiotics [42]. Studies have also shown that using

oligonucleotide arrays to determine the type of clonal complex of S. aureus isolates by DNA

arrays is a promising technique for distinguishing uninfected from infected wounds, predicting

ulcer outcome and thereby contributing to more appropriate use of antibiotics [43]. Metagenomic

approaches have vastly increased our knowledge on the genomes, activity and functionality of

the complex ecosystem residing within the diabetic foot ulcer.

Imaging techniques

The role of imaging in managing the infected diabetic foot (see Table 3) is expanding and now

often plays a key role in both diagnosis and successful treatment. The aims of all existing

imaging techniques include helping to either exclude an infection or to 'confirm' the diagnosis, to

evaluate the extent of an existing infection, and to differentiate among bone infection

(osteomyelitis), soft tissue infection and neuro-osteoarthropathy (Charcot foot). Unfortunately,

there is no single imaging technique that can routinely and accurately provide all of this

information. Key concerns are that some imaging tests are insensitive when used for an early

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 diagnosis of the disease, while others are non-specific, as they cannot easily differentiate
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between bony changes related to neuro-osteoarthropathy vs. infection.

Radiological imaging techniques

Plain X-ray

Plain radiography of the foot, taken in at least two different projections, should virtually always

be the initial imaging test [44,45]. It is inexpensive, widely available, and can detect major bone

structural changes as well as tissue gas and foreign bodies. Characteristic changes in the early

phase of osteomyelitis are focal lucency, loss of trabecular pattern and cortical destruction; later

abnormalities include periosteal reaction, sclerosis and new bone formation [45]. Overall, the

sensitivity (~ 60%) and the specificity (~ 80%) of plain radiography in diagnosing osteomyelitis

in the infected diabetic foot are relatively low [5]. This relates to three major limitations: 1) bony

changes are only visible when there is demineralization of >30–50% of the bone, which usually

takes at least 2–4 weeks; 2) radiography is suboptimal for detecting soft tissue infection,

although some non-specific signs (induration, obliteration of peri-articular fat planes) may be

seen; and, 3) differentiating infection from neuro-osteoarthropathy, which may sometimes co-

exist, is difficult. It is possible to overcome some of these limitations by performing serial

radiographs (e.g. every 2 weeks), which may be useful in detecting changes that are

characteristic of osteomyelitis over time [10]. For many patients in whom the likelihood of

osteomyelitis is either very high or low, the results of plain radiographs may be sufficient to

confirm the clinical suspicion.

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 Magnetic resonance imaging
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When plain radiography fails to provide a clear answer about the involvement of bone in a

diabetic foot, advanced imaging is usually needed. Magnetic resonance imaging (MRI) is widely

considered the best available radiological imaging technique currently available to detect the

presence and extent of bone and soft tissue involvement. It is useful, when available and not

contraindicated, to identify the extent of the involved soft tissue and bone, provide information

on vascular perfusion (by different MRI perfusion sequences, such as arterial spin labelling and

dynamic susceptibility contrast imaging) and may help guide surgical options [46,47]. When

osteomyelitis is present MRI of affected bone shows a decreased bone marrow signal on T1-

weighted sequences, and increased signal intensity on T2-weighted images. A finding of

increased T2 signal and normal T1 signal represents oedema suggestive of soft tissue infection

[47]. Administering i.v. gadolinium aids evaluation of soft tissue involvement and may help

demonstrate abscesses, synovitis, deep tissue necrosis and sinus tracts [47,48] and to differentiate

cellulitis (which enhances with gadolinium) from non-infectious oedema (with no enhancement)

[49]; however, as gadolinium is relatively contraindicated in patients with renal impairment,

which is found in many patients with diabetic foot infection, its use to enhance MRI is limited in

these patients. For the diagnosis of diabetic foot osteomyelitis MRI has an overall sensitivity of

~90% and a specificity ~80% [45].

The major limitation of MRI is that it cannot always reliably differentiate between infection and

neuro-osteoarthropathy. Findings supporting neuro-osteoarthropathy are the presence of intra-

articular bodies or subchondral cysts and involvement of multiple joints, while findings

suggesting osteomyelitis are diffuse signal enhancement in an entire bone, replacement of fat

adjacent to abnormal bone and the presence of a concurrent skin ulcer or a sinus tract [50].

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 Differentiating these two conditions is especially problematic when an infectious process is
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superimposed on a Charcot foot, when a patient has had a recent surgical intervention in the foot,

or when osteosynthesis material is present at the site of interest. Other potential limitations of

MRI are its limited availability in many locations, high costs, the need for a skilled specialist

radiologist and its inability to image patients with various types of implanted devices (e.g.

orthopaedic metalwork, pacemaker, surgical clips) or who have claustrophobia.

Ultrasonography/computed tomography

Diagnostic ultrasonography and computed tomography (CT) each play a limited role in the

evaluation of diabetic foot disorders and are not recommended in most published diabetic foot

guidelines [46,49,51,52]. If other and better techniques are not available, ultrasonography may

be used to detect the presence of soft tissue fluid collections, joint effusion and foreign bodies

[49] and to guide peri-articular aspirations or soft tissue biopsies [53]. CT is more accurate than

plain radiography for evaluation of cortical erosions, focal areas of lucency, bone sequestra [45]

and soft tissue gas. Compared with MRI, however, the soft tissue contrast is lower and it is more

difficult to demarcate between healthy and infected tissue.

Nuclear medicine imaging techniques

Nuclear medicine techniques detect in vivo pathophysiological changes, sometimes even before

anatomical changes are observable. The role of nuclear medicine techniques for imaging

infectious diseases has been enhanced by new insights into methods to acquire and interpret

standard imaging techniques, recent developments in integrated camera systems that combine

physiological and anatomical data and the availability of more specific tracers.

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 Bone scintigraphy
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Three-phase bone scintigraphy was among the first, and remains the most widely used, nuclear

imaging procedure for the diagnosis of musculoskeletal infections. This technique has a high

sensitivity (i.e. when all three phases are negative infection is highly unlikely) but unfortunately

a low specificity (i.e. many false-positive results). Any cause of increased bone formation (e.g.

recent surgery, fractures, malignancy, metabolic bone disease, prosthetic loosening) may cause

increased uptake of diphosphonates in the late (third) phase. Furthermore, in several of these

conditions there may also be increased blood flow (first phase) or blood pool (second phase).

Meta-analyses of the use of three-phase bone scintigraphy for detection of diabetic foot infection

using only planar imaging, or combined with single-photon emission computed tomography

(SPECT), estimate a sensitivity of ~ 90% but a specificity of ~50% [45,48,54]. Although recent

development in camera systems (SPECT/CT) may lead to better diagnostic accuracy, the many

causes of high diphosphonate uptake make this technique a secondary imaging technique for

diagnosing diabetic foot infection. Its main usefulness is that a negative scan largely rules out an

infection, although scintigraphy may be falsely negative in patients with lower extremity

ischaemia

Labelled white blood cells

The use of radiolabelled white blood cells (WBC) is still considered the best nuclear imaging

technique for musculoskeletal infections. Labelling with 99mTechnetium is preferred to 111Indium

as it has better radiation characteristics, requires a lower radiation dose, has higher image

resolution and lower costs. This technique is quite specific for detecting leukocytic infiltration,

its results are not affected by recent or current antibiotic treatment [55,56] and it can accurately

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 detect both acute and chronic (even low grade) infections. The disadvantages of WBC
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scintigraphy include the fact that its preparation is laborious, it must be performed under sterile

conditions, it takes a trained technician ~3 h to do the labelling and it exposes the patient to a

relatively high dose of radiation. In addition, the test’s requirement for handling potentially

infectious blood puts both technicians and patients at risk.

The role of radiolabelled WBCs in diabetic foot infections has been extensively investigated,

with reported sensitivities of 72–100% and specificities of 67–100% [48,54,57,58]. The poorer

results are generally reported from studies using only planar imaging with poor spatial resolution

and no bony landmarks to help differentiate soft tissue infection from osteomyelitis. The reasons

for the variability in reported diagnostic accuracies include variations in labelling procedures,

acquisition protocols and interpretation criteria [59,60].

Recently, data from two studies led to a proposal for a new flow chart for the correct acquisition

and interpretation of WBC scintigraphy including dual-time point imaging (3–4 h and 20–24 h

after administration) with time decay-corrected acquisition [55,61]. Over time, an increase in

WBC uptake strongly supports an infection, whereas a decrease in uptake makes infection highly

unlikely. In doubtful or equivocal cases, performing a semi-quantitative analysis using the

contralateral side as reference may be useful. Following this flowchart results in high diagnostic

accuracy; its implementation in new guidelines developed by the Infection and Inflammation

Committee of the European Association of Nuclear Medicine should lead to better and more

comparable study results at different centres.

In the past decades, some suggested that the combining of bone scans with WBC scintigraphy, or

bone scan/WBC scintigraphy and bone marrow imaging, might improve diagnostic accuracy.

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 Unfortunately, studies found that using these techniques together did not substantially improve
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the results [59,62]. We believe that when nuclear medicine specialists use the correct protocol for

WBC imaging and interpretation there is no need for additional bone and/or bone marrow

imaging.

Single-photon emission computed tomography/computed tomography

Using new integrated camera systems that combine physiological and anatomical data has now

become standard procedure. SPECT/CT has several advantages over standard or dual isotope

scans: it can provide excellent cortical spatial resolution, is less expensive than dual scintigraphy,

delivers a lower radiation dose, and can be used with several different isotope agents. In WBC

scintigraphy adding SPECT/CT to the early images (3–4 h) leads to better localization of the site

and the extent of the infection, and better differentiation between soft tissue infection and

osteomyelitis [61], thereby achieving better diagnostic accuracy [63]. Using a composite severity

index (a standardized hybrid image-based scoring system) appears to add prognostic value for

diagnosing diabetic foot osteomyelitis to 99mTc-SPECT/CT [64]. Another additional potential use

of SPECT/CT is helping to determine when diabetic foot osteomyelitis has resolved. This is a

key issue for knowing when to discontinue antibiotic therapy and whether or not surgical

treatment may be needed, yet it is probably even more difficult than diagnosing infection. One

study with 29 patients with diabetic foot osteomyelitis found that a negative WBC SPECT/CT at

the end of antibiotic therapy had a 100% negative predictive value (and 71.5% positive

predictive value) for detecting relapse of infection [65].

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 18
F-fluorodeoxyglucose positron emission tomography
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The newer technology of positron emission tomography (PET) with 18F-fluorodeoxyglucose

(FDG) has several theoretical advantages over standard scintigraphy: it avoids the need for blood

manipulation (like WBC scintigraphy); acquisition time is shorter; image resolution is higher;

and physiological FDG background uptake is low. The major limitation of this technique is that,

in addition to infection, FDG accumulates in malignancies and inflammation, because cells

involved in all three of these processes metabolize glucose as a source of energy [66]. Increased

glucose metabolism of leucocytes, macrophages, monocytes, lymphocytes and giant cells occurs

in infectious and inflammatory diseases, but uptake is also seen in regenerating and traumatic

processes.

The FDG-PET method may be helpful in the diagnosis of diabetic foot infection, but its role in

the evaluation of diabetic foot infection has yet to be clarified [67]. Diagnostic accuracies have

varied from 54 to 94% [68–71]. A recent systematic review and meta-analysis found nine studies

comprising 299 patients evaluated for diabetic foot problems. The quantitative analysis of four

selected studies provided the following results on a per patient-based analysis: sensitivity 74%;

specificity 91%; positive likelihood ratio 5.56; negative likelihood ratio 0.37; and diagnostic

odds ratio 17 [72]. What is now needed is a consensus about which criteria should be used to

declare the results of a scan as positive or negative and how best to differentiate between

infectious and non-infectious entities. For now, when FDG-PET imaging shows no increased

uptake infection is unlikely, but when FDG uptake is increased, it is challenging to differentiate

between the various causes: infection, inflammation, neuro-osteoarthropathy, recent surgery,

fractures, osteophytes, enthesopathy and degenerative changes. Currently, PET camera systems

are combined with CT to allow precise anatomical localization of the FDG uptake (Fig. 1). This

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 facilitates differentiation between osteomyelitis and soft tissue infection, but does not solve the
Accepted Article
problem of differentiating among infection, inflammation and osteoarthropathy. Notably,

elevated blood glucose levels negatively influence the accuracy of FDG-PET scans; obviously,

this is a common finding in patients with suspicion of an infected diabetic foot.

Other nuclear tracers

67
Gallium (67Ga)-citrate. 67Ga-citrate was previously used extensively for imaging infections but

its suboptimum intrinsic characteristics (poor spatial resolution, non-specific binding) and the

development of better tracers have resulted in it rarely being used currently. In a recent study of
67
Ga-SPECT/CT with 55 patients with suspected diabetic foot osteomyelitis it had a negative

predictive value of 100%, but a positive predictive value of only 50% [73].

Antigranulocyte antibodies. Considerable efforts have been devoted to developing in vivo

methods for WBC labelling that could overcome the limitations of in vitro-labelled WBCs.

Although production of antigranulocyte monoclonal antibodies (e.g. Scintimun®, LeukoScan®)

has been promising, the results have not been found to be better than in vitro 99mTc-labelled

WBCs.

Labelled WBCs for PET imaging. WBCs have also been labelled in vitro with FDG in an attempt

to develop a more specific PET tracer. Only a few published studies are available on this tracer

(none of which included patients with an infected diabetic foot) and results have varied [59,74].

Unfortunately, this technique delivers high amounts of radioactivity and, because of the short

half-life of 18F (110 min), it is technically not feasible to perform imaging > 4–5 h after injection.

Which imaging technique is the first choice?

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 Both the Infectious Disease Society of America and by the International Working Group on the
Accepted Article
Diabetic Foot have included recommendations in their guidelines for imaging for diabetic foot

infections. Recently, a promising combined diagnostic flow chart was proposed by a committee

of expert clinicians, radiologists and nuclear medicine specialists [56]. Plain radiography is

recommended in all patients who present with a potential diabetic foot infection, to look for bony

as well as soft tissue abnormalities [1]. If both the clinical presentation and X-rays are most

compatible with osteomyelitis, no further diagnostic evaluation is needed. For patients in whom

either the diagnosis or the optimum surgical approach is unclear, additional imaging with MRI is

recommended [1]. Nuclear medicine imaging techniques should be considered in cases in which

clinical suspicion and MRI/radiographic findings are incongruent or inconclusive, or when MRI

is contraindicated or not available. There is no general consensus within the nuclear medicine

community on which nuclear technique should be the procedure of choice, but most would

recommend either labelled WBCs with SPECT/CT or FDG-PET/CT. Large-scale studies,

preferably comparing these techniques with MRI, are needed to determine the most appropriate

imaging tool and to analyse the cost-effectiveness of all available imaging techniques.

Future perspectives in imaging

Positron emission tomography/magnetic resonance imaging

Simultaneous imaging with the recently developed combined PET/MRI camera system has the

potential to become the premier technique for assessing the infected diabetic foot. It combines

acquisition and quantification of functional data at the molecular level with superior soft tissue

resolution and anatomical detail. Advantages include providing an absolute match between the

tissue information of both techniques under the same physiological conditions (PET/CT is

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 performed sequentially, not simultaneously), better localization of the PET signal within soft
Accepted Article
tissues, no radiation exposure with MRI, and a 'one-stop-shopping' approach for the patient by

allowing the acquisition of diagnostic quality imaging results of nuclear medicine and

radiological techniques in one visit. This hybrid PET/MRI imaging could optimally differentiate

among soft tissue infection, osteomyelitis, inflammation and neuropathic osteoarthropathy [75].

Possible new positron emission tomography radiopharmaceuticals

In most clinical situations, and particularly in the low grade infected diabetic foot, imaging at

late time points (e.g. 24 h after injection) is necessary because of the slow leukocyte

accumulation in infected sites as compared with bone marrow. Regrettably, this delay between

injection and imaging is not possible using 18F as radiolabel, but fortunately, 64Copper, with a

half-life of 12.7 h, appears to be a more suitable radionuclide for labelling of white blood cells;

the first attempts to in vitro labelling of WBCs with 64Copper have been successful [76], and we

are anxiously waiting for the first clinical results.

68
Ga-citrate PET/CT imaging is another emerging technology. The imaging characteristics of the

PET tracer 68Ga are superior to those of 67Ga because it provides higher spatial resolution and

because of the quantification potential of PET. 68Ga-citrate has other advantages, including rapid

blood clearance, quick diffusion and the fact that it can be produced in any hospital by generator

without the need of a cyclotron on site; however, to date there has been only one study in 31

patients with suspected osteomyelitis (showing a diagnostic accuracy of 90%) and there is no

experience in patients with infected diabetic foot [77].

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 Tailored radiopharmaceuticals
Accepted Article
Advances in molecular techniques and translational medicine have taken nuclear medicine to the

threshold of a new diagnostic era. Personalized medicine, based on individual characteristics of

the patient and the pathogen, is now under investigation in large oncological trials and has the

potential to provide optimum treatment in infectious diseases. Nuclear medicine techniques

provide the opportunity to characterize pathophysiological processes histologically, highlight the

cell type(s) involved, detect the presence of a potential target, quantify the pathogenic bacteria

and biologically active molecules (e.g. cytokines and chemokines) and detect the presence of

apoptopic and autoreactive cells [78]. It may also allow evidence-based biological therapy by

assessing which molecule will localize in an infected area, then using the same unlabelled

molecule therapeutically [78].

Photographic foot imaging and infrared thermography

Patients at risk of foot ulcers should be screened regularly by an appropriately trained healthcare

professional. In some situations, however, this rather time-consuming, relatively intrusive and

costly procedure may not be logistically possible. In those situations, using telemedicine

diagnostic support in the home environment may allow the required foot assessment. Recently,

investigators developed a photographic foot imaging device to use for home monitoring for the

early diagnosis of foot ulcers and pre-ulcerative lesions in patients with diabetes [79]. The device

provided high-quality digital photographs of the plantar foot surface that could be remotely

assessed by a foot specialist.

As infections tend to cause inflammation and increased blood flow, increased skin temperature is

another important sign of possible foot infection. Home monitoring of foot temperatures by

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 infrared thermometry has been shown to be effective in patients with diabetes [80]. In fact,
Accepted Article
infrared thermal cameras may be useful to either detect infections or to predict which patients are

at risk of future foot complications [81], including infections [82]. In a recent study of 38

patients with a diabetic foot complication assessed with photographic and temperature sensing

devices, diagnosis of infection from photographs was specific (>85%) but not very sensitive

(<60%), while thermography was sensitive (>90%) but not very specific (<25%). Diagnosis

based on the combination of both techniques was both sensitive (> 60%) and specific (>79%)

with good intra-observer agreement [83]. These techniques are promising for the home

monitoring of high-risk patients with diabetes to facilitate early diagnosis of signs of infection

[83].

Conclusions

In the past decade we have made great strides in diagnosing infection in the diabetic foot.

Clinical examination (history, physical, probe-to-bone) remains the first and most important

diagnostic approach. Laboratory tests, especially the erythrocyte sedimentation rate, but

disappointingly not procalcitonin, provide some help, especially with diagnosing and following

osteomyelitis, but we need better tests. The coming availability of molecular microbiology in

clinical laboratories will almost certainly help to not only more rapidly identify causative

pathogens, but also to provide information on their potential virulence. Advanced imaging

techniques, particularly hybrid imaging possibilities (SPECT/CT and PET/MRI) have made

these tests more useful in both diagnosing infection and helping to direct therapy. Our clinical

forbears would be jealous of our diagnostic armamentarium, but our students seem poised to

benefit from the next generation of the new techniques that are now emerging.

This article is protected by copyright. All rights reserved.

 References
Accepted Article
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FIGURE 1 Example of 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/

computed tomography (CT) imaging in a patient with diabetes with suspicion of an infected

foot. (a) sagittal PET view. (b) Sagittal PET/CT fusion. (c) Transversal PET view. (d)

Transversal PET/CT fusion. Note increased FDG uptake most compatible with osteomyelitis

involving the plantar aspect of the fifth metatarsus and infection in the adjacent soft tissues

(Figure provided courtesy of Dr Zohar Keidar, Ramban Health Care Campus, Haifa, Israel).

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 Table 1. Summary of potentially useful clinical findings in diagnosing diabetic foot infection
Accepted Article
A. History

1. Long duration (> 4 weeks) of foot wound

2. Previous infection at the same or a nearby site

3. Presence of new pain in the wound (especially in a previously insensate foot)

4. Presence of immunosuppressive condition (beyond that related to diabetes)

B. Physical examination

1. Large wound (>2 cm2 )

2. Deep wound (>3 mm)

3. Classic signs of inflammation (tenderness, pain, redness, warmth, induration)

4. Secondary signs of infection (foul odour, friable or discoloured granulation tissue, rim

undermining, purulent or non-purulent secretions)

5. Signs of a systemic inflammatory response (fever, tachycardia, tachypnoea,

hypotension)

Table 2 Comparison of methods for identifying causative pathogens in diabetic foot infections

Type of Major advantages Major disadvantages When to order Other comments

processing

Standard Widely available Only identifies known If only method available Can be supplemented by

culture pathogens Gram-stained smear

Relatively Unusual organisms

Results depend on type of

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 inexpensive specimen collected and unlikely
Accepted Article rules regarding reporting

Molecular More sensitive Not widely available yet Unusual organisms more May also provide

diagnostic likely information on the

Identifies wider More expensive
methods presence of genes coding
range of organisms Severe infection
Uncertainty about how to for virulence factors and

More rapid interpret results Patient on antibiotic antibiotic resistance

therapy

Rapid identification

important

Table 3 Types of imaging tests for diabetic foot infections

Imaging Major Major Relative Sensitivity/ When to order Other comments

test advantages disadvantages costs ( ) specificity

Plain X- Relatively Bone changes 100–200 Sensitivity Should usually be Serial repeat

rays inexpensive only visible ~60% the first imaging radiographs could

when test ordered overcome several

Widely Specificity
demineralization limitations by
available ~80%
> 30–50%; detecting changes

Can detect over time

Suboptimal for
major bone
detecting soft
structural
tissue infection,
changes,
differentiating
tissue gas
infection from
and foreign

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 bodies osteoarthropathy
Accepted Article
MRI Able to Cannot always 500–800 Sensitivity In patients who Extensively studied

identify differentiate ~90% require additional and widely

extent of infection from imaging when soft considered the best

Specificity
involved soft osteoarthropathy tissue available imaging
~80%
tissue and involvement is technique
Often limited
bone suspected or
availability
diagnosis remains
Provides
Requires skilled uncertain after
information
radiologist preliminary
on vascular
evaluations
perfusion Cannot image

patients who
May help
have various
guide
types of
surgical
implanted
options
devices or who

No radiation have

exposure claustrophobia

CT Good Soft tissue 300–400 Sensitivity Only when other Not recommended

imaging of contrast lower ~80% better advanced by diabetic foot

soft tissue than with MRI imaging infection guidelines

Specificity
fluid col- techniques are not
Difficult to ~70%
lection, joint

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 effusion, demarcate available
Accepted Article foreign healthy from

bodies, bone infected tissue

cortex

To guide

aspirations

or biopsies

Bone scan Widely Low specificity 300–400 Sensitivity To rule out Although a positive

available because of ~90% infection when bone scan is as

+
increased uptake suspicion is low; likely to be a false-
SPECT/CT Long Specificity
in any cause of otherwise only a as a true-positive, a
experience ~50%
increased bone secondary negative scan

SPECT/CT formation technique largely rules out an

increases infection

diagnostic

accuracy

WBC scan Specific for Requires 600–800 Sensitivity 72– Consider when Flow-chart for

+ leukocytic laborious 100% degree of clinical correct acquisition

SPECT/CT infiltration preparation suspicion and and interpretation

Specificity 67–
under sterile MRI/radiographic results in higher
Results not 100%
conditions findings are diagnostic accuracy
affected by
incongruent or
antibiotic Associated with Leads to better
when MRI is
treatment risk to patient localization of site
contraindicated or
and technicians and extent of
Accurately not available *
from handling infection
detects both
potentially
acute and

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 chronic infectious blood
Accepted Article infections

FDG- Short Increased 800– Sensitivity 29– Consider when Need consensus

PET/CT acquisition uptake in 1200 100% degree of clinical criteria on when to

time inflammation suspicion and declare a scan

Specificity 67–
and malignancy, MRI/radiographic positive or negative
High image 93%
as well as findings are and how to
resolution
infection incongruent or differentiate

Avoids need when MRI is between infection,

No consensus
for blood contraindicated or inflammation and
criteria for
manipulation not available * osteoarthropathy
interpretation
Low

physiological

background

uptake

PET/MRI Absolute Expensive 1000– Not available Unknown Has the potential to

simultaneous 1500 become the premier

Limited
match advanced imaging
availability
between technique—ideal

tissue Long one-stop shopping

information acquisition time approach

of both
Limited
techniques
published

Better experience

localization

of PET

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 signal
Accepted Article
No radiation

exposure

with MRI

MRI, magnetic resonance imaging; CT, computed tomography; SPECT, single-photon emission computed

tomography; FDG-PET, 18F-fluorodeoxyglucose-positron emission tomography. WBC: white blood cells

*No consensus within the nuclear medicine community on which nuclear technique is the procedure of choice.

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 A B
Accepted Article

C D

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