# Presentation: 396286

## Microbiologic Diagnosis of Diabetic Foot Infections

- Albert T. Sheldon, Jr. Ph.D.
- Microbiology Team Leader
- Division of Anti-infective Drug Products

## Guidance for Industry- Foot Infections in Patients with Diabetic mellitus

- Microbiological Considerations
- All patients should have pre-therapy cultures.
- Gram stain and cultures should be obtained from acceptable sources using acceptable methods.
- We prefer cultures obtained  by leading edge needle aspiration, soft tissue and joint aspiration, bone biopsy and/or surgical debridement.
- Microorganisms isolated should be assessed as true pathogens, colonizers, or contaminants.
- Only microorganisms designated as true pathogens should be considered in determining microbiological evaluability of enrolled subject.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Factors that Influence Infection Rates

- Risk of Wound Infection varies according to the following equation:*
- 	Dose of bacterial contamination x Virulence
- 			Resistance of the Host
- *Altemeire, W.A., & W.R. Culbertson (1965) Surgical Infection *In *Moyer, C. et .al. (eds.):
- Surgery, Principles and Practices, 3rd ed. Philadelphia, Lippincott Co.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Host Factors that Influence Infection Rates

- Diversity and abundance of microorganisms present in the wound are also influenced by host factors:
- Wound type, depth, location, and quality
- Presence of nonviable exogenous contamination
- Peripheral blood insufficiency
- Immune competence of the host

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Manual of Clinical Microbiology*

- “The use of specimens for bacteriological analysis requires that specific clinical  material be collected, stabilized, and transported according to exacting specifications to insure valid results.”
- *Murray, P. R., E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken. 2003. Manual of Clinical Microbiology, 8th Edition. ASM Press, Washington D.C.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Clinical Microbiology

- Implicit in this definition are two issues that are of interest to the discussion of decubitus foot infections.
- The methods used to collect the clinical sample and
- the validity of the results to assess the involvement of an organism in the etiology of the disease.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## 1. Methods used in Collection of Microbiological Wound Samples

- Deep Tissue Techniques
- Biopsy and/or surgically debrided tissue*+
- Leading edge Needle aspirate*+
- Joint fluid or synovium+
- Bone specimen+
- Blood+
- Surface Sampling Techniques
- Swab*
- Curettage*
- Dermabrasion
- Velvet pad surface imprint
- *Published methods used in decubitus ulcer sampling.
- + Method recommended in Agency guidance document are deep tissue techniques.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Comparison of Sampling MethodsSapico F.L., et. Al. (1984) Rev Infect Dis. 6:S171-S176

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Comparison of Sampling MethodsSapico F.L., et. Al. (1984) Rev Infect Dis. 6:S171-S176

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Relationship Between Biopsy & Swab:  Burn Wounds  Thomson, P.D., (1994) Amer J Surgery 167:7S-10S

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Relationship Between Quantitative Culture & Swab Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865 

- Study purpose:
- To determine the relationship between the quantity of bacteria and infection in complex extremity wounds, and
- To compare the predictive value for wound infection of quantitative cultures versus other factors considered to have predictive value for wound infection.
- I will focus on the latter purpose of the study.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Relationship Between Quantitative Culture & Swab (cont) Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865

- Evaluated 50 patients with complex wounds-defined as a soft-tissue defect that required flap for closure.
- Quantitative cultures (biopsy) compared to
- Clinical parameters (factors considered to have predictive value in wound infection; wound position, mechanism of injury, fracture type)
- Laboratory test (Swab culture)
- 28 patients had quantitative cultures obtained after debridement and high pressure wash prior to flap closure. 16 patients had swab cultures. 2-5 samples obtained per wound.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Criteria for Positive/Negative Test, Infection, and PrevalenceBreidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865

| Test | Positive test criteria | Infection | Prevalence | Negative test criteria | Infection | Prevalence |
| --- | --- | --- | --- | --- | --- | --- |
| Quantitative | >104 cfu/g | 8/9 | 89% | <104 cfu/g | 1/19 | 5% |
| Swab | Positive | 5/13 | 38% | Negative | 1/3 | 33% |
| No culture | --- | --- | --- | --- | 3/6 | 50% |
| Wound position | Lower extremity | 7/13 | 54% | Upper extremity | 11/37 | 30% |
| Mech. of injury | Farm or lawn mower | 4/11 | 36% | Other than F or LM  | 14/39 | 36% |
| Fracture type | Type IIIB or IIIC fracture | 10/28 | 36% | Type I, II, IIIA, or none | 8/22 | 36% |

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Criteria for Positive/Negative Test, Infection, and PrevalenceBreidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865

| Test | Positive test criteria | Infection | Prevalence | Negative test criteria | Infection | Prevalence |
| --- | --- | --- | --- | --- | --- | --- |
| Quantitative | >104 cfu/g | 8/9 | 89% | <104 cfu/g | 1/19 | 5% |
| Swab | Positive | 5/13 | 38% | Negative | 1/3 | 33% |
| No culture | --- | --- | --- | --- | 3/6 | 50% |
| Wound position | Lower extremity | 7/13 | 54% | Upper extremity | 11/37 | 30% |
| Mech. of injury | Farm or lawn mower | 4/11 | 36% | Other than F or LM  | 14/39 | 36% |
| Fracture type | Type IIIB or IIIC fracture | 10/28 | 36% | Type I, II, IIIA, or none | 8/22 | 36% |

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Predictive values, Sensitivity, & Specificity of Tests Studied

| Test | PPV | NPV | 100-NPV | Sensitivity | Specificity |
| --- | --- | --- | --- | --- | --- |
| Quantitative culture | 89% (56-98) | 95% (75-99) | 5%
 | 89%
 | 95% |
| Swab culture | 38% (18-64)
 | 67% (21-94) | 36%
 | 83%
 | 20% |
| Wound position | 54% (29-77) | 70% (54-83) | 33%
 | 81%
 | 19% |
| Mechanism of injury | 36%(15-65)  | 64% (48-77)
 | 30%
 | 22%
 | 78% |
| Grade III fracture
 | 36% (21-54) | 64% (43-80)
 | 36%
 | 56%
 | 44% |
| Note: Values following each % represent range of 95% confidence interval; PPV= positive predictive value= posttest probability of a positive result (infection); NPV=negative predictive value; 
100-NPV=posttest probability of a negative result (no infection). |  |  |  |  |  |

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Predictive values, Sensitivity, & Specificity of Tests Studied

| Test | PPV | NPV | 100-NPV | Sensitivity | Specificity |
| --- | --- | --- | --- | --- | --- |
| Quantitative culture | 89% (56-98) | 95% (75-99) | 5%
 | 89%
 | 95% |
| Swab culture | 38% (18-64) | 67% (21-94) | 36% | 83% | 20% |
| Wound position | 54% (29-77) | 70% (54-83) | 33%
 | 81%
 | 19% |
| Mechanism of injury | 36%(15-65)  | 64% (48-77)
 | 30%
 | 22%
 | 78% |
| Grade III fracture
 | 36% (21-54) | 64% (43-80)
 | 36%
 | 56%
 | 44% |
| Note: Values following each % represent range of 95% confidence limits. PPV= positive predictive value= posttest probability of a positive result (infection); NPV=negative predictive value; 
100-NPV=posttest probability of a negative result (no infection). |  |  |  |  |  |

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## 2. Interpretation of Microbiologic Diabetic foot infection Samples (or Qualitative Microbiology)

- Diabetic foot ulcers are polymicrobic.*
*S. aureus* is the predominant aerobic species followed by *S. epidermidis*, *Streptococcus* spp., *P. aeruginosa*, *Enterococcus* spp., and coliform bacteria.
- 	The predominant anaerobic species are *Peptostreptococcus* spp., *Bacteroides* spp., and *Prevotella* spp.
- Application of good microbiological techniques will allow isolation of anaerobes from up to 95% of decubitus ulcers.  However, such studies are not usually performed due to the labor intensive nature of anaerobic microbiological studies.
- *Bowler, P.G., et. al. (2001) Clin Microbiol Rev 14:244-269.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Schools of Thought*

- Although microorganisms are responsible for wound infections, there is controversy regarding their role. Published literature is inconclusive!
- The density of microorganisms is the critical factor in determining whether a wound is likely to heal.
- The presence of specific pathogens is of primary importance in delayed healing.
- Microorganisms are of minimal importance in delayed healing.
- Debate whether a wound should be sampled, the value of the results and the method that should be used.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Conclusions

- Widespread controversy still exists
- Regarding the exact mechanisms by which microorganisms cause wound infection,
- Regarding the significance of microorganisms in nonhealing wounds that do not exhibit signs of clinical infection, and
- Regarding the best microbiological techniques to monitor the microbiology of wounds.
- ASM Manual Clinical Microbiology states “A swab is not the specimen of choice...Since a swab specimen of a decubitus ulcer provides no clinical information.”

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Conclusions

- A regulatory agency must require microbiological methods that provide us with confidence and data necessary to assess the response of antimicrobials for their intended uses.
- We describe, in our guidance document, what we consider to be relevant methods. These are the deep tissue techniques discussed in a previous slide.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Louis Pasteur

- “ The germ is nothing.  It is the terrain in which it is found that is everything.”
- Pasteur, L. (1880) De l’attenuation virus du cholera des poules. CR Acad. Sci. 91: 673-680.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Obtaining a Swab Culture

- Before taking the specimen
- Cleanse the wound with water or saline
- Remove excess necrotic debris
- Compress edges to elicit new drainage
- Use swab tip to swab the healthiest tissue
- Do not swab exudate, pus, eschar, or heavily fibrous tissue.
- Gram stain helps define quality of specimen as determined by polymorphonuclear cells, squamous epithelial cells and bacteria on gram stain.
- 	ASM Manual Clinical Microbiology states “A swab is not the specimen of choice...Since a swab specimen of a decubitus ulcer provides no clinical information.”

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Microbiology of noninfected wounds

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Microbiology of Infected Wounds

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003

## Useful Wound Definitions 

- Contamination-Presence of bacteria on the wound surface.
- Colonization-Presence of and multiplication of surface microbes/bacteria contaminants without infection.
- Infection-Invasion and multiplication of microorganisms in body tissues resulting in local cellular injury.
- 	Distinction between definitions not so clear in the clinical setting.

- Albert T. Sheldon, Jr. Ph.D. DAIDP

- 28-Oct-2003