Microbiologic Diagnosis of Diabetic Foot Infections

1
Microbiologic Diagnosis of Diabetic Foot
Infections

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

2
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.

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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.

4
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

5
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.

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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.

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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.

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Comparison of Sampling MethodsSapico F.L., et.
Al. (1984) Rev Infect Dis. 6S171-S176
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Comparison of Sampling MethodsSapico F.L., et.
Al. (1984) Rev Infect Dis. 6S171-S176
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Relationship Between Biopsy Swab Burn Wounds
Thomson, P.D., (1994) Amer J Surgery 1677S-10S
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Relationship Between Quantitative Culture Swab
Breidenbach W,C., S. Trager (1994) Plats.
Reconstr. Surg. 95860-865

• 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.

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Relationship Between Quantitative Culture Swab
(cont) Breidenbach W,C., S. Trager (1994)
Plats. Reconstr. Surg. 95860-865

• 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.

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Criteria for Positive/Negative Test, Infection,
and PrevalenceBreidenbach W,C., S. Trager
(1994) Plats. Reconstr. Surg. 95860-865
Test Positive test criteria Infection Prevalence Negative test criteria Infection Prevalence
Quantitative gt104 cfu/g 8/9 89 lt104 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
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Criteria for Positive/Negative Test, Infection,
and PrevalenceBreidenbach W,C., S. Trager
(1994) Plats. Reconstr. Surg. 95860-865
Test Positive test criteria Infection Prevalence Negative test criteria Infection Prevalence
Quantitative gt104 cfu/g 8/9 89 lt104 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
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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) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence interval PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence interval PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence interval PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence interval PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence interval PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection).
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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) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence limits. PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence limits. PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence limits. PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence limits. PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection). Note Values following each represent range of 95 confidence limits. PPV positive predictive value posttest probability of a positive result (infection) NPVnegative predictive value 100-NPVposttest probability of a negative result (no infection).
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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
  14244-269.

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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.

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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 choiceSince a swab
  specimen of a decubitus ulcer provides no
  clinical information.

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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.

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Louis Pasteur

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

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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 choiceSince a swab
  specimen of a decubitus ulcer provides no
  clinical information.

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Microbiology of noninfected wounds
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Microbiology of Infected Wounds
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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.