Diagnostics for Wound Infections

Abstract

Significance: Infections can significantly delay the healing process in chronic wounds, placing an enormous economic burden on health care resources. Identification of infection biomarkers and imaging modalities to observe and quantify them has seen progress over the years.

Recent Advances: Traditionally, clinicians determine the presence of infection through visual observation of wounds and confirm their diagnosis through wound culture. Many laboratory markers, including C-reactive protein, procalcitonin, presepsin, and bacterial protease activity, have been quantified to assist diagnosis of infection. Moreover, imaging modalities like plain radiography, computed tomography, magnetic resonance imaging, ultrasound imaging, spatial frequency domain imaging, thermography, autofluorescence imaging, and biosensors have emerged for real-time wound infection diagnosis and showed their unique advantages in deeper wound infection diagnosis.

Critical Issues: While traditional diagnostic approaches provide valuable information, they are time-consuming and depend on clinicians' experiences. There is a need for noninvasive wound infection diagnostics that are highly specific, rapid, and accurate, and do not require extensive training.

Future Directions: While innovative diagnostics utilizing various imaging instrumentation are being developed, new biomarkers have been investigated as potential indicators for wound infection. Products may be developed to either qualitatively or quantitatively measure these biomarkers. This review summarizes and compares all available diagnostics for wound infection, including those currently used in clinics and still under development. This review could serve as a valuable resource for clinicians treating wound infections as well as patients and wound care providers who would like to be informed of the recent developments.

Liping Tang, PhD

Scope and Significance

Through decades of research and development, tremendous improvements have been made in the efficiency and accuracy of wound infection diagnostics. In this review, we provide a comprehensive view on different aspects of wound infection diagnostics, ranges from approaches that are actively used in the clinic to innovative studies that are undergoing laboratory development, from clinical biological assay to imaging modalities, as well as sensor-based instrumentation. The ultimate goal of the review is to provide both wound care providers and patients a better evaluation of available wound infection diagnostics.

Translational Relevance

Many new technologies have been created in recent years to diagnose infected wounds.^1,2 In addition to physiological assays, traditional imaging modalities such as radiography and magnetic resonance imaging (MRI), as well as new hybrid imaging techniques, including single-photon emission computed tomography (SPECT)/computed tomography (CT) and positron emission tomography (PET)/MRI, are being utilized as wound infection diagnostic tools.³ Innovative imaging approaches such as ultrasonography, thermography, and multispectral imaging have gained prominence to fulfill the need for fast, cost-effective, and accurate diagnosis of wound infection,⁴ although they have been developed for research use only.

Clinical Relevance

Wound infections encumber not only the patients but also the health care systems worldwide that provide care for the patients.⁵ While existing tools and techniques have provided reasonable success in wound infection management, emerging diagnostic instruments and technologies hold tremendous potential in the clinical management of wounds. There is great potential in using these techniques to provide additional information about wound infection diagnosis and direct further treatment plans. Insight into these diagnostics can improve the accuracy of clinical validation in light of the wide diversity of wound types, anatomic locations, and tissue involved.

Background

Chronic wounds affect ∼20 million individuals worldwide and annual cost for their treatment and management is estimated to be over $31 billion.^5–7 Many factors contribute to delay in the wound healing process, including chronic metabolic disease (diabetes), neurological defect, vascular insufficiency, nutritional deficiency, aging, and infection.^8,9 Among these, infection is a major factor that delays the wound healing rate and requires immediate treatment. A wound is characterized as a chronic wound when it does not progress through the normal stages of healing within 3 months or relapses.

There are three stages in the wound infection continuum, namely contamination, colonization, and infection.¹⁰ Wound contamination is the presence of nonreplicating microbes in an open wound. The presence of small numbers of microbes does not affect normal inflammatory responses and wound healing processes.¹¹ However, microbial replication causes wound colonization. The increased number and persistent presence of microbes can prolong the inflammatory phase of wound healing and lead to further tissue damage.¹² When the microbes migrate deep into the wound bed and reproduce rapidly, they can trigger either a local or systemic immune reaction with the characteristics of infection.¹³

Furthermore, some microbes can produce a complex protective glycocalyx—also called biofilm—which makes the infected wounds hard to be detected and treated.¹⁴ The presence of biofilm is a hallmark of chronic wounds and most chronic wounds possess biofilms.¹⁵ Certain microbial phenotypes enhance inflammation causing further tissue damage and protect from antimicrobial therapy as well as the immune system.^16,17 Within less than 24 h of infection,¹⁸ the invading microbes can skew early innate immune responses to allow the establishment of the biofilm phenotype.¹⁹ With further progressing, the wound infection may jump from topical wounds to systemic complications such as cellulitis, osteomyelitis, and septicemia.

To diagnose wound infection, most practitioners rely on clinical characteristics 98% of the time, followed by patient-reported symptoms (88%) and wound cultures (70%).²⁰ Clinical signs of a superficial infection include purulent drainage, abnormal granulation tissue, abnormal foul odor, increasing temperature, additional breakdown, edema, induration, and erythema.²¹ Due to the varieties of wound infections and considering the history of wound infection diagnosis, wound cultures have become the gold standard in infection diagnosis.

Traditionally, techniques including swab culture (Levine technique), needle aspiration, and tissue biopsy are used to identify pathogens in the wound bed.²² Among them, swab culture is the most frequently applied approach since it is simple, cheap, and convenient. However, this invasive approach can be time-consuming. Therefore, fast and noninvasive diagnostics that can detect a wide range of microbes regardless of wound types and locations are greatly needed.

Discussion of Findings and Relevant Literature

Visual observations

Before using any diagnostic tool, most clinicians suspect wound infections by visually inspecting the wounds for signs and symptoms of infection. Typical signs of an acute infection include pain, erythema, heat, and purulent exudate. However, this may be modified in a chronic wound setting by underlying comorbidities. In addition to these signs, an infected chronic wound may show signs of delayed healing, discolored and friable granulation tissue, serous exudate, epithelial bridging/pocketing in granulation tissue, foul odor, and wound breakdown.^12,23,24 Acute wound infection has apparent signs and symptoms since it is generally caused by a single organism, while chronic wound infection has less obvious signs due to multiple pathogens.²⁵ Anyhow, due to the wound infection continuum, it is vital that clinicians must be able to distinguish colonization, in which multiplication of organisms interferes with wound healing without invading, from bacterial infection.¹⁰ Unfortunately, the visual inspection does not provide any definite determination. Signs like changes in exudate, granulation tissue, discoloration of the wound bed, and wound bridging can be associated with colonization rather than infection. A comprehensive list (Table 1) summarizing signs and symptoms at different stages of the wound infection continuum can be a useful tool for clinicians to assess patients' wounds.²⁵ As demands on health care systems increase, expanding the role of point-of-care medical personnel (i.e., nurses) in assessing the state of wounds is essential. The Bate-Jensen wound assessment tool,²⁶ a holistic approach to record wound parameters such as size, depth, necrotic tissue type, necrotic tissue content, edge characterization, and undermining presence, can be used for wound diagnosis and captures much of the information listed above. This can help to streamline the diagnosis and move toward implementing therapy in a timelier manner.

Table 1.

Signs and symptoms within wound infection continuum

Contamination	All open wounds may contain microorganisms. They will not multiply or persist until suitable nutritive and physical conditions are available for each microbial species, or they successfully evade host's defenses. Consequently, their presence is only transient and wound healing is not delayed.
Colonization	Microbial species successfully grow and divide, but do not cause damage to the host or initiate wound infection.
Local infection	Covert (subtle) signs of local infection:
	Hypergranulation (excessive “vascular tissue”); bleeding, friable granulation; epithelial bridging and pocketing in granulation tissue; wound breakdown and enlargement; delayed wound healing beyond expectations; new or increasing pain; increasing malodor.
	overt (classic) signs of local infection:
	Erythema; local warmth; swelling; purulent discharge; delayed wound healing beyond expectations; new or increasing pain; increasing malodor.
Spreading infection	Extending in duration +/− erythema; lymphangitis; crepitus; wound breakdown/dehiscence with or without satellite lesions; malaise/lethargy or nonspecific general deterioration; loss of appetite; inflammation, swelling of lymph glands.
Systemic infection	Severe sepsis; septic shock; organ failure; death.

International Wound Infection Institute (IWII) Wound infection in clinical practice. Wounds International 2016.

Since a delayed wound healing process is an important indicator for wound infection, wound mapping tools such as wound tracing, scaled photographs, and planimetry can be used to indirectly detect wound infection by monitoring wound size changes.²⁷ If there is no noticeable improvement of the wound within 3 weeks, the wound could be infected.²⁸ It should be noted that the impediment to wound healing can be associated with many factors other than an infection. Therefore, further investigation is inevitably needed to confirm the diagnosis.

Odor sensing and pain assessment

Odor sensing and pain assessment are also widely used by the clinicians for wound infection diagnosis. Wound odor (or malodor) is often led by necrosis or extremely poor vascularization of tissues, promoting bacteria as well as fungal colonization or infection. Hence, it is clinically used as an indication for wound infection.²⁹ Deteriorating, highly exuding and fungating wounds usually possess malodor due to the fermentation of amino acids in anaerobes to malodorous organic amines.³⁰

Wound pain has been considered the most frequent sign of heralding the onset of infection.³¹ The pain mainly comes from the cellular injury and host immunological reactions caused by the growth, multiplication, and invasion of the microorganisms.³² Wound pain assessment tools can help to assess the nature and severity of pain. Thorough and frequent pain assessment are critical for early detection of infection as well as comprehension of the patient's distress and discomfort.³³ Although neither provides a definite prediction of wound infection, both odor sensing and pain assessment cannot identify microbial species and the extent of colonization, which are important parameters for the instruction of further treatment. This necessitates further clinical/laboratory assessments to acquire quantitative information within the infected wounds.

Clinical and laboratory assessments

Wound culture

Wound cultures are performed when an infection is suspected through visual observation. The culture is used to confirm the initial diagnosis, identify species of resident microorganisms, and determine effective antibiotics to treat the wound.³⁴ Current literature provides three techniques on laboratory methods for diagnosing wound infections, including deep-tissue biopsy, needle aspiration, and swab culture.³⁵

A deep-tissue biopsy is a qualitative and quantitative culture of wound tissue, which is accomplished by using an aseptic technique in obtaining a tissue sample by punch biopsy, needle biopsy, or a scalpel.³⁶ Through microscopic examinations, biopsy results are generally reported as the number of organisms per gram of tissue. Compared with other tests, deep-tissue biopsy result is considered more conclusive and accurate for the detection of microorganisms invading wound tissue, which makes the technique the gold standard for identifying wound infection.³⁷ However, the biopsy procedure itself is not only time-consuming, costly, invasive, and painful, and requires special equipment as well as special training, but also the risk for postsurgical trauma, wound disruption, and bacteremia is fairly high.³⁵ Most importantly, the feasibility of biopsy procedure varies from the anatomic location of the wound, patient's comorbidities, and/or provider's willingness. Therefore, it is usually not the primary choice for wound cultures.

Needle aspiration of wound fluid is a common technique used in puncture wounds or postsurgical wounds with suspected abscess. This method can obtain microbes below the surface of the wound by inserting a fine-gauge needle into the tissue to aspirate fluid and to quantify the concentrations of microbes.³⁸ Although needle aspiration is less invasive compared to tissue biopsy, it is still painful and the results are not always reproducible.³⁷

Swab culture is the most commonly used technique in the clinic due to its practical, noninvasive, reproducible, and inexpensive features. It has been reported that swab culture has sufficient correlation with tissue biopsy to identify causative organisms in an infected wound.^39–41 Among existing approaches, the Levine technique is considered one of the best methods to obtain a swab culture.⁴² Usually, for quantitative swab cultures, the wound fluid-stained swab is placed in 1 mL of diluent and vortexed to release microorganisms. After incubation under aerobic condition, the type and the number of bacteria are measured and reported as the number of organisms per swab.²¹ The major concern associated with swab culture is that only the surface-colonizing bacteria will be reflected instead of the pathogenic strain invading deeper tissues. Moreover, swab cultures can be unreliable in the context of biofilm infection.⁴³ A comparison of these three wound culture techniques has been listed in Table 2.

Table 2.

Comparisons among wound culture techniques

	Descriptions	Advantages	Disadvantages
Deep-tissue biopsy	Obtain tissue sample by punch/needle biopsy or a scalpel; quantitative results acquired by microscopic examinations.	Conclusive and accurate result for detecting invading microorganisms; gold standard for wound infection diagnosis.	Time-consuming, costly, invasive, painful, require special equipment and special training; high risk for postsurgical trauma, wound disruption, and bacteremia.
Needle aspiration	Obtain microbes below the surface of the wound by inserting a fine-gauge needle into tissue to aspirate fluid.	Feasible for small open wound and detecting subcutaneous microorganisms; less invasive.	Time-consuming, painful; may underestimate bacterial isolates.
Swab culture	Press sterile culture swab against the wound base to extract wound fluid; using eluent for incubation and quantification.	Practical, noninvasive, reproducible, and inexpensive; has sufficient correlation with tissue biopsy outcome.	Time-consuming; cannot detect pathogenic strain invading deeper tissues; weak in detection of biofilm infection.

Many different types of microorganisms may exist in chronic wounds. Even though different clinical approaches may be able to identify the type of microorganisms in wounds, these methods are unable to determine a causative organism or combination of organisms in complex chronic wounds. Such complexities need to be appreciated and considered in the development of wound infection diagnostics.

Laboratory markers

In addition to the wound culture techniques, laboratory markers can also be measured to aid the diagnosis of wound infection. These markers include C-reactive protein (CRP), procalcitonin (PCT), presepsin, microbial DNA, and bacterial protease activity (BPA).

In response to inflammation and infection, CRP, a peptide produced in the liver, is stimulated by cytokines, primarily interleukin-6, and employed in complement binding and phagocytosis by macrophages.⁴⁴ By using light scattering from an aggregation of CRP-specific antibody, the concentration of CRP can be measured within 15–30 min.⁴⁵ Several studies have investigated the relationship between CRP production and wound infection. For example, a recent study showed that a higher grade of diabetic foot infection possessed a significantly higher level of CRP.⁴⁶ Moreover, elevated serum CRP levels were found to correlate well with incisional surgical site infection.⁴⁷ Although elevated CRP is frequently associated with acute infection, it has been suggested that CRP measurement be considered supportive data instead of being used for direct diagnosis of infection.^45,48

PCT is a peptide hormone secreted by non-neuroendocrine parenchymal cells, can be measured by time-resolved amplified cryptate emission, and serum level of PCT can increase 500–8,000 times in patients with severe sepsis compared with healthy individuals.⁴⁹ A series of studies has reported that patients with bacterial infections have elevated PCT levels.⁵⁰ It has also been shown that PCT values were significantly higher among patients who developed postsurgical infections compared with those who did not.² However, due to the relatively low levels of PCT in infected individuals (ng/mL), a high-sensitivity PCT test may be needed to detect infection at an early phase.⁵¹

Presepsin, a soluble CD14 subtype, has been considered a new, emerging, early indicator for diagnosis and prognosis of wound infections. It is secreted by monocytes and functions to stimulate the phagocytosis of monocytes within innate immunity responses.⁵² Presepsin has been investigated as a potential biomarker for the detection of bacterial infection using presepsin ELISA kits. Subsequently, a highly sensitive and fully automated PATHFAST presepsin measurement system was developed for faster processing.⁵³ Based on the results of several clinical studies, presepsin levels in patients with systemic bacterial infections were significantly higher than those without infections.^54–56 However, this approach is limited by pathophysiological conditions,⁵⁷ burn wounds that elevate levels,⁵⁸ and by chronic kidney syndromes.⁵⁹

As an adjunct to wound cultures, culture-independent investigation of the microbial DNA applying pyrosequencing,⁶⁰ polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis,⁶¹ and quantitative real-time PCR⁶² have identified a greater range of bacteria than traditional culture techniques. However, these identification techniques are costly and they require special training as well as instruments that make them less translational into clinical application. Recently, DxWound, a PCR-based microbial DNA analysis system, has been developed to provide accurate and sensitive detection of an array of microbes, including aerobic bacteria, anaerobic bacteria, and fungi. Moreover, the procedure is easy to handle with results available within 1 day. This comprehensive testing, although not rapid, has great potential in diagnosing wound infection.⁶³

Bacterial protease is a large and diverse group of proteases produced exclusively by all microorganisms and functions mainly in degrading host tissue proteins to sustain the bacteria.⁶⁴ Bacterial proteases are often secreted into the infected wound environment and therefore can be utilized as diagnostic markers for wound infection. Moreover, the measurement of BPA can help to understand the pathological behavior of the microorganisms within the wound and provide valuable information on wound infection.⁶⁵ The most common and basic method to assess BPA is the incorporation of substrates of bacterial proteases, such as collagen, gelatin, and casein, into microbiological agar and a zone of clearance in the agar will appear where extracellular bacterial protease presents.⁶⁶ However, this technique is time-consuming and lacks specificity. A point-of-care swab-based test, WOUNDCHEK™ Bacterial Status, has been developed by WOUNDCHEK Laboratories to help clinicians detect BPA within 15 min.⁶⁷ The product was recently approved by the U.S. Food and Drug Administration for clinical use in the diagnosis and prognosis of nonhealing wounds. While this device is designed to detect one enzyme produced by an organism that might not be associated with a wound infection, this device has not been approved for infection diagnosis in the United States. It must be noted that some laboratory markers like CRP and PCT are nonspecific as they can be elevated by conditions unrelated to infection. A table that summarizes and compares all the available laboratory markers for wound infection diagnosis is listed for better comprehension (Table 3).

Table 3.

Laboratory markers for wound infection diagnosis

	Description	Pros and Cons	Sensitivity	Specificity
CRP	In response to inflammation and infection, CRP is stimulated by cytokines, primarily interleukin-6. By using light scattering from aggregation of CRP-specific antibody, concentration of CRP can be measured.	✓ Simple; Fast; Noninvasive.	High	Low
		× Short half-life; Nonspecific; Cannot determine microbial species.
PCT	PCT is a peptide hormone secreted by non-neuroendocrine parenchymal cells and can be measured by time-resolved amplified cryptate emission. Patients with bacterial infections have elevated PCT levels.	✓ Fast; Noninvasive.	High	Low
		× Costly; Nonspecific; require high-sensitivity facilities; cannot determine microbial species.
Presepsin	Presepsin is a soluble CD14 fragment released into blood when monocytes are activated during an infection. It can be quantified by either an assay kit or PATHFAST presepsin measurement system.	✓ Fast; Noninvasive; high sensitivity; prognostic.	High	Low
		× Altered by other pathophysiological conditions; Nonspecific; cannot determine microbial species.
Microbial DNA	Culture-independent investigation of the microbial DNA can identify a greater range of bacteria species than traditional culture techniques. It can also be detected by DxWound, a PCR-based microbial DNA analysis system.	✓ Accurate; sensitive; noninvasive; can determine microbial species.	High	High
		× Costly; require special training and instruments.
BPA	Bacterial proteases are often secreted into the infected wound environment to degrade host tissue proteins for bacterial sustenance.	✓ Fast; noninvasive; prognostic.	Medium	Low
		× Nonspecific; cannot determine microbial species.

BPA, bacterial protease activity; CRP, C-reactive protein; PCR, polymerase chain reaction; PCT, procalcitonin.

Imaging modalities and instrumentations

Traditional imaging modalities

In the clinic, to confirm the diagnosis from visual observation/blood test and examine the existence of systemic/deep tissue infection, several current imaging modalities are frequently applied to aid in the diagnosis of wound infections. These imaging modalities include plain radiography (X-ray), CT, MRI, ultrasound imaging, PET, a SPECT, and combined imaging modalities, such as PET/CT and SPECT/CT. These imaging modalities are not used as primary diagnostic tools for wound infection due to the fact that they require specific instruments, imaging facility, are costly, and cannot quickly identify infection status in complex chronic wounds.

The initial examination of patients with soft-tissue infections typically begins with plain radiography. This approach is widely used due to its simple operation, low cost, and wide availability.⁶⁸ However, it should be noted that plain radiography is nonspecific and cannot distinguish between infection and other conditions, such as trauma, venous insufficiency, systemic causes of subcutaneous edema, and deep venous thrombosis.⁶⁹

CT plays an important role in the diagnosis of soft-tissue infection and intra-abdominal abscesses due to its wide availability, fast scanning speed, high spatial resolution, and multiplanar reformatting capabilities.⁷⁰

MRI has been used for the diagnosis of soft-tissue infection due to its high spatial and contrast resolution. MRI provides anatomic and pathophysiologic information about the extent of infection within both soft tissue and the underlying bone.⁷¹ However, it is difficult to distinguish foreign bodies from adjacent structures, such as scar tissue, calcifications, and tendons in superficial wounds.³

Ultrasound imaging stands out among other imaging modalities because it is portable, cost-effective, and readily, available and does not provide exposure to radiation.⁷² As air can interfere with ultrasound imaging, when utilized for intra-abdominal infection diagnosis, loops of intestines with intraluminal air can obscure its detection capabilities.⁷² Operator skill is also a major factor in distinguishing an abscess from other causes of skin swelling.⁷³

PET relies on developing a three-dimensional image from the accumulation of specific radioisotopes and resulting emission of gamma photons by positron annihilations to construct a three-dimensional image of the area of isotope accumulation. One of the drawbacks of this approach is that the equipment and radioisotope tracers used to perform these scans are expensive.

Innovative imaging modalities

Several innovative imaging approaches are still being developed in laboratories. These imaging approaches include spatial frequency domain imaging (SFDI), thermography, and fluorescence imaging. These fast and noninvasive imaging technologies have a great potential to revolutionize wound infection diagnosis.

SFDI is a new noncontact imaging approach that can quantify volume fraction of tissue chromophores, such as oxyhemoglobin, deoxyhemoglobin, and water.⁷⁴ It can study the optical properties of wound tissue by separating and quantifying absorbed and scattered incoherent monochromatic light.⁷⁵ The technique has been applied to identify infection in burn wound through quantifying changes in absorption and reduced scattering that correlate with bacterial infection.⁷⁶ Although SFDI technology is a new entrant in the field of cutaneous wound research, it is promising for diagnosis of burn wound infection and has great potential in assessing infections in diabetic wounds as well as pressure ulcers.^77,78

Thermography is a technique wherein an infrared camera is applied to measure infrared radiation emitted from the wound tissue.⁷⁹ As a marker of inflammation, the elevated temperature has been shown in multiple studies to diagnose infection around wound site.⁸⁰ The recent emergence of smartphone-based thermography has opened doors for the development of simple, portable, and inexpensive techniques to detect and monitor wound healing.^81,82 Although the technique has limited accuracy and specificity,⁸³ it can offer complementary information on wound infection and has the potential to be utilized remotely from the clinic.

Luminescence imaging, an emerging imaging modality that captures visible photons emitted by Cherenkov radiation,⁸⁴ has recently been used to detect infection in skin wounds.⁸⁵ A portable luminescence imaging device for detecting both inflammatory responses and infection in superficial wounds has been developed and tested using a full-thickness cutaneous wound model in pigs.⁸⁵ Since reactive oxygen species (ROS) levels can increase by an order of magnitude in an infected wound,⁸⁶ this imager can display 2D ROS activity distribution in real-time through visualizing ROS-associated luminescence. Moreover, by analyzing ROS intensity and distribution within infected wounds on a pig model, this approach has been developed to distinguish infected wounds from uninfected ones. With unique properties like being simple, noninvasive, real time, and portable, the imager has shown great potential in being utilized in clinics as a wound infection diagnostic tool.

Autofluorescence imaging is becoming an innovative approach for the diagnosis of wound infection due to the light-absorbing properties of endogenously produced bacterial porphyrins. Bacterial porphyrins are not only important for the metabolism of molecular oxygen and diatomic gases but also involved in gene regulation.⁸⁷ Many clinically relevant bacterial species, such as Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli, Enterococcus spp., Proteus spp., Klebsiella pneumonia, and Enterobacter spp., have been detected using this approach without application of a contrast agent.^88,89 A handheld portable autofluorescence imaging device has been developed and tested to detect bacterial infection around diabetic foot ulcers in real time.^90,91 Moreover, a product, MolecuLight i:X™, has been commercialized based on the prototype. However, the device cannot detect infection and all causative microorganisms since not all of them possess porphyrins. Finally, the wound must be free of blood and blood clots because the presence of blood can block fluorescence signal.⁹² The device may be used to detect microbial “hot spots” in wound, therefore helping to assist with wound debridement and other treatment decisions at the point of care.^93,94

Detection of biofilms

Biofilms on wounds may be suspected when wounds do not progress for multiple weeks.⁹⁵ They show no evident signs until they are large and produce a viscous exopolymeric substance.⁹⁵ Diagnosis of biofilms on wounds is difficult since there are no point-of-care tools that can detect biofilms. While standard clinical microbiology culture method can be used to identify infectious microbes, especially on superficial wounds, it cannot be used to identify the main pathogen in the biofilm located in deep tissue.⁹⁶ Microscopic techniques, especially bright-field microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), are some of the most reliable diagnostic methods for biofilms on wounds.^97–100 By visualization of debridement samples from diabetic foot wounds, a study has shown that bright-field microcopy, CLSM, and environmental SEM were able to visualize the microcolonies associated with the biofilm phenotype and biofilm structure.⁹⁹ Molecular techniques such as 16S rRNA PCR, Bacterial tag-encoded FLX amplicon pyrosequencing, full ribosomal amplification, cloning and Sanger sequencing, partial ribosomal amplification and pyrosequencing, and peptide nucleic acid fluorescence in situ hybridization have been explored to detect microbes in biofilm (please see review Wu et al.⁹⁶). More recently, investigations have probed the presence of monomicrobes or multimicrobes in biofilms using combined SEM and molecular techniques like peptide nucleic acid fluorescent in situ hybridization with CLSM.¹⁰¹ With increasing evidence on the important role of biofilm in wound healing, there is tremendous need for reliable biofilm detection techniques that can serve at the point of care and serve as an adjunct tool for wound care specialists. Coincidentally, a recent study has shown that the creation of a fast diagnosis tool for biofilm might be possible. This study shows that the presence of biofilms in wounds can be identified by using a wound blotting technique with prewet nitrocellulose membrane and then stained with a polysaccharide staining dye, ruthenium red or alcian blue.¹⁰²

Microwave-microfluidic device

A small and inexpensive microwave-microfluidic biosensor was recently developed for rapid, contactless, and noninvasive quantification of E. coli within medium solutions to increase the efficacy of clinical wound infection assessment.¹⁰³ By measuring the variation of resonant amplitude and frequency responses of the microwave system, different concentrations of bacteria can be detected in solutions with different pH values. The minimum prepared optical transparency of bacteria was tested at an OD₆₀₀ value of 0.003, which indicates the high sensitivity of this device. Moreover, the growth of bacteria can be monitored over time and that reveals the potential to use the device for rapid and real-time monitoring of bacterial infection in the wound. A table that summarizes and compares all the available imaging modalities and instrumentations for wound infection diagnosis is listed for better comprehension (Table 4).

Table 4.

A summary of imaging modalities and instrumentations for wound infection diagnosis

	Application in Wound Infection Diagnosis	Pros and Cons	Sensitivity	Specificity
Plain radiography	Initial examination of soft-tissue infections.	✓ Simple operation, low cost, wide availability; radioactive; reveals inflammatory changes	Low	Low
		× Can distinguish swelling due to infection from fractures; nonspecific findings
		× Misled by other conditions.
Computed tomography	Diagnosis of soft-tissue infection and intra-abdominal abscesses; evaluation of deeper structures and the extent of surrounding inflammations; identification of small infected collections.	✓ Wide availability; fast scanning speed; high spatial resolution; multiplanar reformatting capabilities; high penetration depth.	High	High
		×. Radioactive, sometimes requires contrast agent.
Magnetic resonance imaging	Diagnosis of soft-tissue infection; Can provide anatomic and pathophysiologic information about the extent of infection within both soft tissue and the underlying bone.	✓ High spatial/contrast resolution; nonradioactive	High	High
		×. Costly; low availability; requires special training/facility; hard to distinguish foreign bodies from adjacent structures within superficial wounds.
Ultrasound imaging	Diagnosis of skin and soft tissue infections. Can evaluate suspected radiolucent foreign bodies.	✓ Fast; Accurate; cost-effective; portable; available in many clinics; no ionizing radiation.	Medium	Medium
		×. Interference from air; low penetration depth; relies on operator's skill.
PET	Diagnose and predict remission of antibiotic treatment for diabetic foot infections by developing 3D images from accumulation of radioisotopes.	✓ High-resolution 3D imaging; high penetration depth.	High	High
		× Costly; Short tracer half-life; requires special training/facility; Possible misdiagnosis resulting from sterile inflammation.
SFDI	Identify burn wound infection by quantifying volume fraction of tissue chromophores.	✓ Noncontact; distinguishes infected and noninfected burn wounds.	High	High
		×. Limited scanning area and wound types.
Thermography	Use an infrared camera to measure infrared radiation emitted from the wound tissue. Smart phone-based thermography has been developed for diabetic foot ulcer detection and wound healing prediction.	✓ Simple; portable; cost-effective; real-time imaging; noninvasive; remote diagnosis.	Low	Medium
		×. Limited accuracy and specificity.
Luminescence imaging	Portable imager distinguishes infected wounds from uninfected ones in a pig model based on intensity and distribution of visible photons emitted y Chernow radiation.	✓ Simple; portable; cost-effective; real-time imaging; noninvasive; remote diagnosis.	Medium	Medium
		×. Limited specificity; requires more research
Autofluorescence imaging	A handheld portable device diagnoses bacterial infection in diabetic foot ulcers in real time by detecting autofluorescence due to the light absorbing properties of endogenously produced bacterial porphyrins.	✓ Simple; portable; cost-effective; real-time imaging; noninvasive; sensitive; remote diagnosis.	High	Low
		×. Low specificity; Cannot determine microbial species.
Microwave-microfluidic biosensor	A microwave-microfluidic biosensor for quantification of Escherichia coli within medium solutions to increase the efficacy of clinical wound infection assessment; the growth of bacteria can be monitored over time.	✓ Small; cost-effective; rapid; contactless; real-time measurement; noninvasive; sensitive.	High	N/A
		×. Limited detectable bacterial species

PET, positron emission tomography; SFDI, spatial frequency domain imaging.

Summary

There is an exponentially growing population with wounds and associated co-morbidities that increase the risk of wound infections. Consequently, there is an urgent need to develop rapid, inexpensive, noninvasive, accurate, simple, and specific techniques to assist wound care providers in the diagnosis and monitoring of wound infections. Current techniques used in clinical practice, including visual observation for clinical signs and symptoms, clinical/laboratory assessments, and imaging modalities/instrumentations, are speculative, expensive, or sometimes off target. The recent development of wound infection diagnostic products, such as PCR kits DxWound and handheld portable bacteria imager MolecuLight i:X, may be considered small, but significant steps before we reach our ultimate goal of developing inexpensive, noninvasive, accurate, simple, and specific wound infection diagnostic devices.

Take-Home Messages

• Wound infection is one of the main factors for delayed wound healing.

• The wound infection continuum has three stages, including contamination, colonization, and infection.

• Current wound infection diagnostics like the visual observation of signs and symptoms of wound infection cannot identify these stages.

• Wound culture is costly and time-consuming, and lacks accuracy.

• Multiple laboratory markers have been used for wound infection diagnosis, such as CRP, PCT, presepsin, Microbial DNA, and BPA.

• Other than traditional imaging modalities like MRI, CT, ultrasound, PET, and SPECT/CT, new imaging modalities, including SFDI, thermography, luminescence imaging, and autofluorescence imaging, have been used for wound infection diagnosis. Summary and comparison of all the imaging modalities have been listed.

• Microscopic and molecular techniques have been used for the detection of biofilms on wounds.

• Several new testing kits and devices have been developed and investigated for wound infection diagnosis as well.

Abbreviations and Acronyms

BPA

bacterial protease activity

CLSM

confocal laser scanning microscopy

CRP

C-reactive protein

CT

computed tomography

ELISA

enzyme-linked immunosorbent assay

MRI

magnetic resonance imaging

PCR

polymerase chain reaction

PCT

procalcitonin

PET

positron emission topography

ROS

reactive oxygen species

SEM

scanning electron microscopy

SFDI

spatial frequency domain imaging

SPECT

single-photon emission computed tomography

Author Disclosure and Ghostwriting

Tang has a potential research conflict of interest due to a financial interest with Progenitec, Inc. A management plan has been created to preserve objectivity in research, in accordance with UTA policy. P.R. has a potential research conflict of interest due to employment as a full-time staff scientist at Smith & Nephew, plc. and his graduate studies are funded through an employee tuition reimbursement program. A management plan has been created to preserve objectivity in research in accordance with UTA policy. No competing financial interests exist for the other authors. The content of this article was expressly written by the author(s) listed. No ghostwriters were used to write this article.

About the Authors

Shuxin Li, PhD, is a bioengineering doctoral student at the University of Texas at Arlington. His research focuses on fabricating probes and probe-loaded gauze for wound monitoring and healing. Paul Renick is a quantitative biology doctoral student at the University of Texas at Arlington. His research focuses on the development of bacterial specific PET probes and the development of activated antimicrobial peptides. He is also employed as a Staff Scientist at Smith & Nephew plc. and has worked in the pharmaceutical industry in the discovery and development of novel antibacterial and biologic therapies. Jon Senkowsky, MD, is a vascular surgeon with over 30 years of clinical experience in wound management. He provides care for patients with chronic wounds daily using many techniques and products for the treatment of chronic, nonhealing wounds in patients with vascular disease, venous stasis, and diabetes. Ashwin Nair, PhD, is a scientist and project leader at Progenitec, Inc., with expertise in foreign body reactions to materials and tissue regeneration techniques. Liping Tang, PhD, is a bioengineering professor at the University of Texas at Arlington. His expertise covers a broad area of biocompatibility, biomaterials, tissue engineering, inflammation imaging, infection, and stem cell therapies.