Bacterial Swabs and the Chronic Wound: When, How, and What Do They Mean

T he ability to diagnose infection in chronic wounds should be a well-honed clinical skill grounded in knowledge of human physiology, immunology, and microbiology. Instead, it has become a topic mired in controversy. Although it has been suggested that much of this controversy arises from lack of evidence, more than 5,000 "wound infection" articles were published in peer-reviewed journals over the past 12 years. Confusion may be based not so much on lack of evidence but rather on lack of balanced interpretation of evidence. Poor clinical performance in diagnosing chronic wound infection may partly account for the fact that soft tissue infection is the leading indication for outpatient parenteral antibiotic therapy and the third most common indication for oral antibiotic use.1,2 An exhaustive study of chronic wound prevalence in Sweden has revealed that 60% of patients either were actively receiving antibiotics or had been treated with antibiotics in the previous 6 months.3 Another example of clinical perplexity is the current North American clinical practice guideline on management of pressure ulcers.4 Although this guideline recommends obtaining quantitative bacterial cultures rather than swab cultures, quantitative culture techniques are rarely utilized in the management of most chronic wounds.5 Culture methodology in particular has been prone to controversy. The technique of using a swab for microbiological sampling now has been practiced for more than 100 years. However, the swab remains much maligned, with critics questioning many aspects of this technique: What type of swab should be used? Should the swab be dry or premoistened? Does superficial swabbing of the wound bed reflect deeper tissue cultures? Does the swab miss fastidious organisms? How should the swab be rolled across the wound surface? Is it important to quantitate bacterial growth in chronic wounds? When Should a Chronic Wound Be Cultured? Determining when is far more important than determining how to culture a wound. As in much of medical practice, the timing of clinical action is sometimes more important than the action itself. A wound should be cultured after wound infection has been clinically diagnosed. Clinical diagnosis of infection is essential before culturing because 100% of wounds are contaminated at the time of wounding (inoculated with organisms which subsequently do not actively divide).6 Soon after the inoculation event, 100% of wounds become colonized (where some micro-organisms replicate within a wound but do not harm the host). Wound infection by definition implies that replicating micro-organisms within a wound are having a detrimental effect on the host. In a healthy host, the local signs of wound infection are seldom subtle and include swelling, induration, erythema, increased temperature, pain, increased exudate, foul odor, and eventual progressive wound breakdown. If allowed to progress, local infection becomes systemic. Systemic signs of sepsis include fever, rigors, chills, hypotension, multi-organ failure, and death. Unfortunately, these florid complications of chronic wound infection culminating in septicemic death continue to occur.7 Infection in chronic wounds may be quite subtle.8 Covert infections often are described as being out of bacterial balance when the pathogens in question have overcome the host immune response. Such wound infections are characterized by progressive wound breakdown or by a wound that appears to stall after previously showing signs of healing progress. In such wounds, ruling out other causes for wound failure (eg, lack of compliance with pressure downloading, inadequate perfusion, or inappropriate wound care) is important. Specific clinical attributes of these wounds include atrophy of previously exuberant granulation tissue, often with discoloration of the granulation tissue to a pale grey or deep red hue, with increased friability and bleeding. These wounds often have excessive exudate. This exudate may be more watery or serous than purulent due to lysis of neutrophils by bacterial toxins. Wounds that are not in bacterial balance also tend to have significant undermining and foul odor. Alternatively, some wounds with marked signs of inflammation may not be infected (eg, Pyoderma gangrenosum or cutaneous vasculitis). Because the signs of local and even systemic infection can be subtle or misleading, bacterial quantitation has been proposed as a potential technique to diagnose wound infection for 40 years.9 Quantitative biopsy has been promulgated as the gold standard in the diagnosis of wound infection.10 This has evolved into the excessive and indiscriminate tendency to culture wounds under the false hope that this will identify underlying infection. Wound cultures will almost always be positive,6 further promoting this false belief. However, quantitative biopsy is merely a technique and no microbiological technique can supplant clinical assessment skills. Wounds should be cultured only when infection is suspected so patients rather than culture results are treated. How Should a Chronic Wound Be Cultured? The clinician needs to determine whether quantitative or qualitative microbiologic sampling techniques (ie, quantitative tissue biopsy versus simple qualitative swab technique) should be employed. Quantitative microbiology clearly has merits, particularly as a research tool. A strong association exists between the number of organisms in a wound and the ability of that wound to achieve subsequent healing, with an overall trend toward impaired wound healing once bacterial growth attains a quantity of 106 colony forming units/g of tissue or greater. Kucan at el11 demonstrated this in a trial comparing topical silver sulfadiazine to povidone iodine or saline dressings in patients with active pressure ulceration. Use of silver sulfadiazine was associated with superior healing compared to the other two agents, and healing was associated with attaining bacterial counts of less than 106 colony forming units/mL of wound fluid. Similar findings have been reported using quantitative biopsy techniques in studies involving delayed closure of surgical wounds,9,12,13 skin graft survival in experimental wound infections,14 and rate of healing in chronic wounds.15,16 Positive findings that correlate bacterial quantity with successful wound healing must be considered with a balanced perspective. First, at least 20% of heavily colonized wounds with greater than 105 colony forming units/g of tissue will still heal.17 Further, beta-hemolytic Streptococci can interfere with wound healing when present in a wound at any quantity, an observation first made in 1918 by a group which included Sir Alexander Fleming.18 Other organisms detrimental to the host when isolated in wounds in any quantity include Mycobacterium tuberculosis, Treponema pallidum, Corynebacteria diphtheriae, Bacillus anthracis, Francisella species, and Brucella species, not to mention numerous other bacterial, viral, fungal, and protozoan pathogens. Alternatively, normal skin flora has been shown to enhance wound healing despite proliferation to very high quantities within wound fluid.19 Other pitfalls associated with quantitative biopsy include the fact that this technique is traumatic, expensive, and time consuming. Woolfrey et al20 showed that quantitative biopsy also may have poor sensitivity and reliability. Their work indicated a 25% chance of missing an organism using the biopsy technique, likely due to uneven distribution of organisms within the wound bed and to techniques used to cleanse the specimen, which may reduce microbial recovery. Quantitative results can vary by 2 logs in 27% of paired isolates, suggesting suboptimal reliability. The simple premise that quantitation of bacteria will help identify wound infection does not take into account the fact that most infections in chronic wounds are polymicrobial and that a significant and dynamic interaction occurs between multiple bacterial populations within these wounds. Selkon21 has demonstrated that obligate anaerobes - frequent pathogens in chronic wounds - significantly inhibit the intracellular killing of aerobic bacterial pathogens by polymorphonuclear leukocytes. Further, aerobic bacteria promote anaerobic growth by inducing tissue hypoxia and reduced redox potential and by producing necessary nutrients for anaerobic growth such as vitamin K and succinate.22 Thus, qualitative aspects of wound microbiology are at least as important as quantitative aspects. Quantitative aspects themselves are essentially irrelevant if a wound is healing in a progressive fashion. Finally, growing evidence suggests that the qualitative microbiology obtained by a swab may adequately approximate qualitative findings obtained from tissue biopsy. Wheat et al23 showed that qualitative results obtained with swabs are similar to tissue biopsy results. Although the authors noted a fairly high rate of false positive and false negative results using the swab technique, they observed that most of the false positives were commensal organisms commonly isolated in the setting of wound colonization and usually not directly targeted for antimicrobial therapy. Further, organisms that appeared to be missed by performing a swab technique were not considered crucial - for example, missing one of three anaerobic organisms isolated would not alter antibiotic therapy. The researchers determined that 92% of antibiotic therapy would be adequate for diabetic foot infection based on qualitative swab data alone. Similarly, Sapico et al24 demonstrated a 75% concordance between swab and biopsy microbiology in samples obtained from chronic pressure ulcers. Quantitative biopsy in this study did not accurately predict the success of subsequent surgical flap closure. Ehrenkranz et al25 showed that an irrigation-aspiration technique using a saline irrigant held significant concordance with qualitative microbiology obtained by biopsy. It is important to note that all the studies that demonstrated adequate correlation between swab and biopsy results utilized specific wound bed preparation. The wound beds in these studies generally were cleansed with saline and superficially debrided, so the culture was more likely to represent the microbiology in the deep wound compartment. While the superficial wound compartment may be more likely to contain colonizing organisms rather than pathogens, the presence of Staphylococcus aureus in a superficial swab has been shown to correlate with the presence of that pathogen in the deeper tissues as well.26 Chronic wounds undergo a predictable alteration in microbial flora over time.8 Early on, the wound is colonized by cutaneous flora as described above, but within the first 1 to 4 weeks, these are soon accompanied by pathogenic Gram-positive aerobic cocci, particularly beta-hemolytic Streptococci and S. aureus, that often infect wounds together in a synergistic fashion. After approximately 4 weeks, chronic wounds are more likely to become colonized by Gram-negative facultative anaerobic bacteria, particularly coliforms, and thereafter by anaerobic bacteria and Pseudomonas species. Thus, infection in an early chronic wound is predictably a suppurative, Gram-positive, monomicrobial process. Infections in older wounds are polymicrobial mixtures of aerobic and anaerobic pathogens usually associated with tissue necrosis, undermining, and deep structure involvement. How Should a Swab Be Performed? Under most conditions of routine clinical practice, wounds should not be cultured without evidence of infection; if infection is subsequently diagnosed, a properly obtained wound swab would be sufficient for most clinical purposes. How to properly obtain a swab from a chronic wound is the subject of much discussion. Because fatty acids contained in cotton swabs can inhibit bacterial growth for some fastidious organisms, some clinicians have recommended that an alginate or rayon-tipped swab be used.27 However, the organisms that routinely cause wound infection are hearty, ubiquitous, and seldom require these special measures. Further, although pre-moistening a swab improves yields when sampling a dried surface,27 swabbing a desiccated wound is seldom indicated. The most important aspect of obtaining a swab culture is preparation of the wound bed. The wound bed should be cleansed with saline and superficially debrided to obtain access to the deep compartment of the wound. The area then can be sampled by simply rolling the tip of the swab on its side for one full rotation over the area in question. Although clinicians recommended that an ulcer be cultured by simultaneously rotating and zigzagging a swab across the wound to cover as much surface area as possible,28 this technique has never been validated and is as likely to increase the yield of nonsignificant superficial colonizers as it is to yield true pathogens. Probably, sampling the part of the wound with the most dramatic signs of infection is the best approach. Once obtained, the swab immediately should be placed in transport medium. The holding media contained within transport containers are designed to maintain viability of fastidious organisms while preventing overgrowth of heartier pathogens. Standard aerobic transport media such as Stuart's and Amies are considered inadequate for isolation of anaerobic flora.8 However, anaerobes can be successfully isolated from these transport media as long as they undergo planting within approximately 2 hours from the time the specimen is obtained. Generally, prereduced anaerobically sterilized media are ideal for isolating both aerobes and anaerobes in mixed culture. These specimens are clinically useful only if the location of the wound and the identity of the patient are adequately documented on the transport specimen. Although much emphasis has been placed on how to properly obtain a swab, if a loculated collection of pus or a discrete abscess is noted, the fluid from the abscess cavity should be aspirated into a syringe using a needle. The needle can be discarded (using forceps) into a sharps container and a plastic nipple placed on the syringe. This would suffice as a transportation device for the aspirated fluid and serve as an ideal specimen for aerobic and anaerobic culture. During the process of debridement, obtaining deep curettings for culture is appropriate because curettings correlate even more closely to biopsy results than swab samples.29 In the case of patients with systemic features of sepsis, obtaining blood cultures at the time that local wound cultures are obtained is important. Using Swab Culturing Techniques in Chronic Wound Management Cultures need not be obtained from patients with a diagnosis of covert infection (ie, a wound out of bacterial balance). When a wound stops healing or shows subtle signs of deterioration, an appropriately obtained deep culture from the wound bed would be helpful. However, covert infections usually do not involve deep tissue invasion. If no signs of cellulitis along the wound margins are evident, using superficial debridement and a topical antimicrobial regimen often places these wounds back into bacterial balance. In a pressure ulcer exhibiting marked undermining with pale edematous granulation tissue with increased serous exudate but no surrounding cellulitis (see Figure 1), the US Department of Health and Human Services clinical practice guideline for management of pressure ulceration would recommend debridement and topical antimicrobial and not culture.4 In fact, using a swab under conditions of covert infection probably would not alter therapy, because debridement and topical antimicrobials are usually successful. Due to concerns regarding sensitivity and reliability of quantitative biopsy, as well as the many drawbacks mentioned, that technique would not alter clinical management of this ulcer. The ulcer healed completely with debridement and use of topical silver sulfadiazine (see Figure 2). A wound surrounded by a zone of cellulitis indicates that the wound already has progressed beyond the covert stage of infection. In this case, systemic antimicrobial therapy would be required to supplement local wound care techniques such as wound cleansing and debridement. A wound infection like this can be adequately assessed using a properly obtained swab. The Stage IV pressure ulcer shown in Figure 3 represents a florid infection in a wound with necrotic tissue, significant undermining, and palpable bone at the base of this ulcer. A large zone of cellulitis surrounds this wound and is associated with copious and malodorous exudate. This patient had systemic signs of wound sepsis. Quantitative biopsy would not be indicated because the diagnosis of wound infection is obvious, and qualitative microbiology using a deep swab technique has been shown to provide satisfactory microbiologic results to direct appropriate therapy. A patient with this presentation also should have blood cultures drawn; in this case, subsequent cultures grew S. aureus in blood and S. aureus, group B Streptococcus, Escherischia coli, and Bacteroides fragilis from the wound. The patient responded well to systemic parenteral antibiotic therapy and wound debridement. Conclusion Diagnosing infection is a clinical skill and not a microbiologic technique. It depends not only on the quantity of organisms involved but also the virulence of microbial species and the synergistic interplay between these species. The most important indicator of infection involves the host response (both local and systemic), recognizing that this response will vary directly depending upon the underlying physiology of the host. Patients with impaired defense mechanisms (eg, tissue ischemia, diabetes, or alcoholism) should be scrutinized closely, as they are more likely to have occult, covert infections where the only manifestation of that infection may be wound healing failure or subtle wound deterioration. Because quantitative microbiology focuses on only one aspect of wound infection, it has not been embraced in common practice despite its recommendation in clinical practice guidelines. Under most conditions of routine clinical practice, properly obtaining a swab after thorough cleansing and debridement of the wound bed should provide sufficient information to direct appropriate antimicrobial therapy. The choice of microbiologic sampling technique is dependent on the individual clinical presentation. Patients with systemic signs of sepsis should undergo blood cultures, localized abscesses should be aspirated, and local wound infections should be assessed with either deep swab or curettage specimens. Quantitative techniques should be utilized primarily for research purposes.

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