A Guide to Diagnostic Techniques for DFU Soft Tissue Infection and Osteomyelitis

The International Diabetes Federation (IDF) recently reported approximately 537 million people globally have diabetes mellitus.¹ In 2021, over 6.7 million deaths globally were attributed to diabetes.¹ There are a variety of diabetes-associated complications, but the diabetic foot ulcer (DFU) is associated with a very high morbidity and chronicity.² The open wound of a DFU requires continuous wound care and is at continuous risk of infection and additional trauma.² All of these complications contribute to a remarkable and unsustainable economic burden. The IDF reported the United States spent more than $415 billion in 2021 on diabetes-related health care alone.¹

DFUs are a common occurrence in patients with diabetes and more than half of all DFUs will become infected.³ The soft tissue infection can rapidly spread within the tissues and can infect the bone, resulting in osteomyelitis.³ Diabetic foot osteomyelitis (DFO) can progress quickly and leads to amputation in more than 80% of patients.^3-5 Unfortunately, there is a high mortality risk associated with diabetes-related amputation.⁶ Regardless of the amputation etiology, the 5-year survival rate is a dismal 40%.⁶ Quick and accurate diagnosis and management of soft tissue infection and osteomyelitis in DFUs continues to be a key principle in improving healing outcomes and avoiding amputations.⁷

Diagnosis and Specimen Collection for Soft Tissue Infection

Early recognition of the area of involved tissue can facilitate appropriate management and prevent progression of the infection. The International Working Group on the Diabetic Foot (IWGDF) Updated 2019 Guidelines suggest the diagnosis of diabetic foot soft tissue infection should be based on local or systemic signs and symptoms of inflammation.⁷ Examples of these findings include the presence of purulent discharge, erythema, pain, tenderness, warmth, induration, and/or the presence of malodor.

However, local inflammatory findings may be less prominent or absent in some diabetic foot infections. For example, neuropathic patients may exhibit a reduction in, or absence of, pain and tenderness, whereas erythema may be absent in those with vascular disease.⁸ In patients exhibiting symptoms for systemic illness or infection, also consider blood cultures during diagnosis.⁹ The IWGDF guidelines also encourage assessing the severity of the soft tissue infection to document the presentation objectively using the Infectious Diseases Society of America (IDSA)/IWGDF classification.^7,10

Physicians commonly use 3 techniques to obtain a wound culture in a wound suspected of being infected: tissue biopsy, needle aspiration, and swab culture.⁹ See Table 1 for a brief comparison of techniques.

Tissue biopsy. For infected DFUs without suspicion of DFO, take a tissue specimen from the ulcer by biopsy or curettage and send the specimen for microbial culturing.⁷ In order to avoid contamination, cleanse and debride the ulcer prior to the tissue biopsy or curettage.^7,8 Once the area to be sampled is clean, take the tissue biopsy using a punch biopsy or by scraping the base of the ulcer or infected tissue with a scalpel or curette (see Figure 1A and 1B). The quality of the sample impacts the quality of the results on which antibiotic treatment will be based. Soft tissue collection via biopsy can cause patient discomfort and minor bleeding but provides better guidance for antibiotic selection by culturing and Gram staining.⁷ Early microbial culturing and guided treatment of soft tissue infection may help prevent contiguous progression to DFO. Soft tissue infections that have deep tissue specimens that culture a single causative pathogen may eliminate the need for a bone biopsy.⁷

Needle aspiration. In wounds where there is little loss of skin such as puncture wounds or postsurgical wounds, needle aspiration of wound fluid is a good alternative for tissue biopsy.⁹ This method is also useful for collecting wound fluid to evaluate for suspected abscess.⁹ As the name implies, a fine-gauge needle can aspirate wound fluid for microbial cultures.⁹

Swab culture. In 1976, Levine and colleagues characterized the quantitative swab culture technique for quantifying bacterial burden in burn patients, commonly referred to as the Levine technique.^9,11 While at the time the Levine technique was useful in the acute burn wound, the chronic diabetic foot ulcer wound is etiologically and structurally very different. Although a swab can easily collect a tissue or fluid specimen superficially using a swab, several systematic reviews and studies have now shown cultures from swabs or semi-quantitative swabs are not as sensitive and specific as tissue biopsies, especially in chronic wounds.⁷ The superficial nature of a wound lends itself to more contaminates and microbial flora than the microbiome deeper within the tissues. Swab cultures are known to give false-positive results, especially in the situation of inadequately cleaning and preparing the wound bed, and typically obtains only a culture of the surface bacteria.⁹ The use of superficial swabs has a low concordance with tissue biopsies and could adversely skew antibiotic selection.^7,12 Use of swab cultures in DFUs is not recommended.

Diagnosis and Specimen Collection for Osteomyelitis

Both the IWGDF and the IDSA state that for a definitive diagnosis of osteomyelitis, one should use a bone specimen to culture the DFO microbial flora and examine this histologically.^7,10,13 There are three main techniques in evaluating the presence of osteomyelitis: probe to bone, percutaneous bone biopsy, and per-wound bone biopsy.^7,12 See Table 1 for a brief comparison of techniques.

Probe to bone (PTB). Diagnosing osteomyelitis is often difficult for a variety of reasons. The IWGDF has several recommendations to rule in or rule out osteomyelitis in the diabetic foot.⁷ One recommendation for suspected osteomyelitis is a combination approach of a probe to bone (PTB) test, standard X-rays, and erythrocyte sedimentation rate (ESR) test for the initial study for diagnosis. If the X-rays and lab results are compatible with osteomyelitis, then no further imaging is needed. If the diagnosis is not certain, then consider advanced imaging. Note that one could use a C-reactive protein and/or procalcitonin test instead of ESR at initial assessment and test results may vary between facilities.  

The PTB test is a clinically useful technique that is inexpensive and easy to learn.¹⁴ After ulcer debridement, use a sterile, blunt, metal or wood probe to gently palpate the depths of the ulcer to determine if palpable bone is present (see Figure 2).¹⁴ A positive PTB would be when the assessor palpates a hard, possibly gritty structure (ie, bone).¹⁴ A positive PTB test is suggestive of osteomyelitis, whereas a negative PTB can rule out DFO in a low-risk patient.⁷ The PTB test has a high sensitivity (0.87) and specificity (0.83) for detecting DFO as demonstrated in a systematic literature review.¹⁵ The interrater reliability is moderate, and the test reliability can be affected by both the ulcer location and the clinician’s experience.⁷ The early detection of osteomyelitis using PTB allows the clinician to then proceed with procuring a specimen for microbial and histological investigation of the DFU.

Percutaneous bone biopsy (PBB). One can use PBB to collect an adequate bone specimen or this can be perioperative if in the operating room.⁷ Both methods require aseptic technique in order to avoid contamination, and both are considered safe for the patient with diabetes.⁷ While the specimen accurately determines the pathogens and the antibiotic susceptibility, the procedure can be expensive and time-consuming, rendering this method impractical for all patients.⁷ Whether it is the impracticality of the procedure, the expense, or the perception that PBB results in few positive cultures, clinicians do not perform PBBs as frequently as would be expected given the bone biopsy is considered a requirement for the “gold standard” diagnosis.¹⁶

DFO is definitive when the PBB culture is positive and the specimen histology shows pathological deterioration.⁷ PBB microbial cultures can reveal not only the causative pathogen, but also the antimicrobial sensitivity. Unfortunately, the histopathology has a low inter-rater reliability. Likewise, the concordance between the histology and cultures is also low.⁷ Studies have shown soft tissue cultures near the bone do not accurately detect DFO pathogens in the bone with the concordance rate for soft tissue cultures being less than 50%.⁷ In at-risk patients who may be more susceptible to antibiotic resistant pathogens, or where the causative pathogen is difficult to determine, a PBB can provide important clinical and treatment information.⁷

Per-wound bone biopsy. It is not recommended to use per-wound bone biopsy, in which bone is removed via the ulcer.^4,7 Bone specimens taken from an abscess or ulcer site are more likely to be contaminated and unlikely to reveal the true causative pathogen upon culturing. In a study comparing bone biopsies taken per-wound versus percutaneous biopsies, the results showed only 42% of the microbial results were identical between the two biopsy methods.¹⁷ Given that microbial cultures help guide the antibiotic regimen, per-wound bone biopsy does not lend itself to quality specimen sampling and accurate identification, this technique is not recommended.

A Closer Look at Microbial Aspects

Multidisciplinary teams facilitate successful wound healing outcomes. This collaboration is especially true for the clinical and microbial departments that work and communicate effectively with one another for the most accurate results. Clinical microbiology departments may have limitations on what services they can perform in-house, and many cannot perform newer techniques.⁷ Understanding what services your clinical microbiology department can perform may impact your clinical care practice and may require an outside contract, especially in smaller hospitals and rural areas. It can be helpful for the lab staff to provide clinical information about the patient and the specimen such as wound etiology, infection characteristics, specimen site, and recent antimicrobial usage.⁷

The microbiology laboratory can provide detailed information on the best way to collect and transport specimens depending on the tissue type. Most specimens are time sensitive, and the culture quality degrades the longer it takes the specimen to be processed, which impacts the accuracy of the results.¹⁸ Likewise, using the wrong transport medium may inadvertently skew the microbial growth and cultures. The microbiology laboratory should provide the clinician with both preliminary and final organism identifications, detailed reports about the organisms cultured, and the antibiotic susceptibility results.⁷ The microbial report should contain enough detailed pathogen information either to confirm or alter the antibiotic therapeutic course.

Use of conventional microbiology culture methods remains the technique of choice for identifying causative pathogens and their antibiotic sensitivity profiles regardless of specimen type.⁷ A new molecular technique such as 16S RNA genetic profiling is exciting in that it can provide information on anaerobic and aerobic organisms, but it cannot distinguish between living and dead organisms and antibiotic sensitivity profiles cannot be performed.^4,7,19 Genetic sequencing techniques provide an interesting look into the DFU and DFO microbiomes with many more organism identifications than conventional techniques. However, the additional information does not necessarily help determine the prime pathogens to direct therapy towards at this time.⁷

In addition to clinical labs not being able to perform the molecular techniques, the increase in expense and time required often renders this technique to the research arena. As the newer techniques improve and standardize, the impact on clinical decision pathways may also improve in the future.

Key Conclusions

In order to salvage limbs and improve healing outcomes, each clinician needs to focus on quickly and accurately diagnosing soft tissue infection and DFO and focus on utilizing pathogen directed antimicrobial treatment. Appropriately using tools and techniques and knowing when and how to take quality tissue or bone specimens for accurate microbial culture should improve patient outcomes.

Dr. Tickner is the Director of Podiatric Wound Care Education at Saint Vincent Hospital and Medical Director of Saint Vincent Hospital/Restorix Wound Healing Center in Worcester, MA.

Dr. Bolling is a second-year resident at Saint Vincent Hospital in Worcester, MA.

Dr. Ghannoum is a first-year resident at Saint Vincent Hospital in Worcester, MA.

References
1.    International Diabetes Federation. IDF Diabetes Atlas, 10th ed. Available at: https://www.diabetesatlas.org. Accessed September 30, 2022.
2.    Amin N, Doupis J. Diabetic foot disease: From the evaluation of the “foot at risk” to the novel diabetic ulcer treatment modalities. World J Diabetes. 2016;7(7):153–164.
3.    Armstrong DG, Boulton AJM, Bus SA. Diabetic foot ulcers and their recurrence. N Engl J Med. 2017;376(24):2367–2375.
4.    Lavery LA, Ryan EC, Ahn J, et al. The infected diabetic foot: re-evaluating the Infectious Diseases Society of America diabetic foot infection classification. Clin Infect Dis. 2020;70(8):1573–1579.
5.    Lindbloom BJ, James ER, McGarvey WC. Osteomyelitis of the foot and ankle: diagnosis, epidemiology, and treatment. Foot Ankle Clin. 2014;19(3):569–588.
6.    Huang YY, Lin CW, Yang HM, et al. Survival and associated risk factors in patients with diabetes and amputations caused by infectious foot gangrene. J Foot Ankle Research. 2018;11:1.
7.    Lipsky BA, Senneville E, Abbas ZG, et al. Guidelines on the diagnosis and treatment of foot infection in persons with diabetes (IWGDF 2019 update). Diabetes Metab Res Rev. 2020;36(S1),e3280.
8.    Bader MS. Diabetic foot infection. Am Fam Physician. 2008;78(1):71-79.
9.    Spear M. Best technique for obtaining wound cultures. Plast Surg Nurs. 2012;32(1):34-36.
10.    Lavery LA, Armstrong DG, Murdoch DP, et al. Validation of the Infectious Diseases Society of America’s diabetic foot infection classification system. Clin Infect Dis. 2007;44(4):562–65.
11.    Levine NS, Lindberg RB, Mason AD, et al. The quantitative swab culture and smear: A quick, simple method for determining the number of viable aerobic bacteria on open wounds. J Trauma. 1976;16(2):89–94.
12.    Mutluoglu M, Uzun G, Turhan V, et al. How reliable are cultures of specimens from superficial swabs compared with those of deep tissue in patients with diabetic foot ulcers? J Diabetes Complicat. 2012;26(3):225–229.
13.    Lavery LA, Crisologo PA, La Fontaine J, et al. Are we misdiagnosing diabetic foot osteomyelitis? Is the gold standard gold? J Foot Ankle Surg. 2019;58(4):713–716.
14.    Grayson ML, Gibbons GW, Balogh K, et al. Probing to bone in infected pedal ulcers. A clinical sign of underlying osteomyelitis in diabetic patients. JAMA. 1995; 273(9):721–3.
15.    Lam K, van Asten SA, Nguyen T, et al. Diagnostic accuracy of probe to bone to detect osteomyelitis in the diabetic foot: a systematic review. Clin Infect Dis. 2016;63(7):944–948.
16.    Schechter MC, Ali MK, Risk BB, et al. Percutaneous bone biopsy for diabetic foot osteomyelitis: A systematic review and meta-analysis. Open Forum Infect Dis. 2020;7(10):ofaa393.
17.    Couturier A, Chabaud A, Desbiez F, et al. Comparison of microbiological results obtained from per-wound bone biopsies versus transcutaneous bone biopsies in diabetic foot osteomyelitis: A prospective cohort study. Eur J Clin Microbiol Infect Dis. 2019;38(7):1287–1291.
18.    Wilson ML. General principles of specimen collection and transport. Clin Infect Dis. 1996;22(5):766–77.
19.    Travis J, Malone M, Hu H, et al. The microbiome of diabetic foot ulcers: a comparison of swab and tissue biopsy wound sampling techniques using 16S rRNA gene sequencing. BMC Microbiol. 2020;20(1):163.