Microbiologic Diagnosis of Diabetic Foot Infections Albert T. Sheldon, Jr. Ph.D. Microbiology Team...

Microbiologic Diagnosis of Microbiologic Diagnosis of Diabetic Foot InfectionsDiabetic Foot Infections

Albert T. Sheldon, Jr. Ph.D.Albert T. Sheldon, Jr. Ph.D.Microbiology Team LeaderMicrobiology Team Leader

Division of Anti-infective Drug ProductsDivision of Anti-infective Drug Products

28-Oct-200328-Oct-2003 Albert T. Sheldon, Jr. Ph.D. DAIDPAlbert T. Sheldon, Jr. Ph.D. DAIDP 22

Guidance for Industry- Guidance for Industry- Foot Infections in Patients with Foot Infections in Patients with

Diabetic mellitusDiabetic mellitusMicrobiological ConsiderationsMicrobiological Considerations– All patients should have pre-therapy cultures.All patients should have pre-therapy cultures.– Gram stain and cultures should be obtained from Gram stain and cultures should be obtained from

acceptable sources using acceptable methods.acceptable sources using acceptable methods.– We prefer cultures obtained by leading edge needle We prefer cultures obtained by leading edge needle

aspiration, soft tissue and joint aspiration, bone aspiration, soft tissue and joint aspiration, bone biopsy and/or surgical debridement.biopsy and/or surgical debridement.

– Microorganisms isolated should be assessed as true Microorganisms isolated should be assessed as true pathogens, colonizers, or contaminants.pathogens, colonizers, or contaminants.

– Only microorganisms designated as true pathogens Only microorganisms designated as true pathogens should be considered in determining microbiological should be considered in determining microbiological evaluability of enrolled subject.evaluability of enrolled subject.


Factors that Influence Infection Factors that Influence Infection RatesRates

Risk of Wound Infection varies Risk of Wound Infection varies according to the following equation:*according to the following equation:*

Dose of bacterial contamination x VirulenceDose of bacterial contamination x VirulenceResistance of the HostResistance of the Host

*Altemeire, W.A., & W.R. Culbertson (1965) Surgical Infection *Altemeire, W.A., & W.R. Culbertson (1965) Surgical Infection In In Moyer, C. et .al. (eds.):Moyer, C. et .al. (eds.):Surgery, Principles and Practices, 3Surgery, Principles and Practices, 3rdrd ed. Philadelphia, Lippincott Co. ed. Philadelphia, Lippincott Co.


Host Factors that Influence Host Factors that Influence Infection RatesInfection Rates

Diversity and abundance of Diversity and abundance of microorganisms present in the wound are microorganisms present in the wound are also influenced by host factors:also influenced by host factors:– Wound type, depth, location, and qualityWound type, depth, location, and quality– Presence of nonviable exogenous Presence of nonviable exogenous

contaminationcontamination– Peripheral blood insufficiencyPeripheral blood insufficiency– Immune competence of the hostImmune competence of the host


Manual of Clinical Microbiology*Manual of Clinical Microbiology*

““The use of specimens for bacteriological The use of specimens for bacteriological analysis requires that specific clinical analysis requires that specific clinical material be collected, stabilized, and material be collected, stabilized, and transported according to exacting transported according to exacting specifications to insure valid results.”specifications to insure valid results.”

**Murray, P. R., E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken. 2003. Murray, P. R., E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken. 2003. Manual of Clinical Microbiology, 8Manual of Clinical Microbiology, 8 thth Edition. ASM Press, Washington D.C. Edition. ASM Press, Washington D.C.


Clinical MicrobiologyClinical Microbiology

Implicit in this definition are two issues Implicit in this definition are two issues that are of interest to the discussion of that are of interest to the discussion of decubitus foot infections.decubitus foot infections.

1.1. The methods used to collect the clinical The methods used to collect the clinical sample andsample and

2.2. the validity of the results to assess the the validity of the results to assess the involvement of an organism in the etiology of involvement of an organism in the etiology of the disease.the disease.


1. Methods used in Collection of 1. Methods used in Collection of Microbiological Wound SamplesMicrobiological Wound SamplesDeep Tissue TechniquesDeep Tissue Techniques– Biopsy and/or surgically debrided tissueBiopsy and/or surgically debrided tissue**++

– Leading edge Needle aspirateLeading edge Needle aspirate**++

– Joint fluid or synoviumJoint fluid or synovium++

– Bone specimenBone specimen++

– BloodBlood++

Surface Sampling TechniquesSurface Sampling Techniques– SwabSwab**– CurettageCurettage**– DermabrasionDermabrasion– Velvet pad surface imprintVelvet pad surface imprint**Published methods used in decubitus ulcer sampling.Published methods used in decubitus ulcer sampling.++ Method recommended in Agency guidance document are deep tissue techniques. Method recommended in Agency guidance document are deep tissue techniques.


Comparison of Sampling MethodsComparison of Sampling MethodsSapico F.L., et. Al. (1984) Rev Infect Dis. 6:S171-S176Sapico F.L., et. Al. (1984) Rev Infect Dis. 6:S171-S176


Relationship Between Biopsy & Swab: Relationship Between Biopsy & Swab: Burn Wounds Burn Wounds

Thomson, P.D., (1994) Amer J Surgery 167:7S-10SThomson, P.D., (1994) Amer J Surgery 167:7S-10S


Relationship Between Relationship Between QuantitativeQuantitative Culture & SwabCulture & Swab

Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865 Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865 Study purpose:Study purpose:– To determine the relationship between the To determine the relationship between the

quantity of bacteria and infection in complex quantity of bacteria and infection in complex extremity wounds, and extremity wounds, and

– To compare the predictive value for wound To compare the predictive value for wound infection of quantitative cultures versus other infection of quantitative cultures versus other factors considered to have predictive value for factors considered to have predictive value for wound infection.wound infection.

I will focus on the latter purpose of the study.I will focus on the latter purpose of the study.


Relationship Between Quantitative Relationship Between Quantitative Culture & SwabCulture & Swab (cont) (cont)

Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865 Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865

Evaluated 50 patients with complex wounds-Evaluated 50 patients with complex wounds-defined as a soft-tissue defect that required flap defined as a soft-tissue defect that required flap for closure.for closure.Quantitative cultures (biopsy) compared to Quantitative cultures (biopsy) compared to – Clinical parameters (factors considered to have Clinical parameters (factors considered to have

predictive value in wound infection; wound position, predictive value in wound infection; wound position, mechanism of injury, fracture type)mechanism of injury, fracture type)

– Laboratory test (Swab culture)Laboratory test (Swab culture)

28 patients had quantitative cultures obtained 28 patients had quantitative cultures obtained after debridement and high pressure wash prior after debridement and high pressure wash prior to flap closure. 16 patients had swab cultures. 2-to flap closure. 16 patients had swab cultures. 2-5 samples obtained per wound.5 samples obtained per wound.


Criteria for Positive/Negative Test, Infection, Criteria for Positive/Negative Test, Infection, and Prevalenceand Prevalence

Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865

TestTest Positive Positive test test

criteriacriteria

InfectionInfection PrevalencePrevalence Negative Negative test test

criteriacriteria

InfectionInfection PrevalencePrevalence

QuantitativeQuantitative >10>1044 cfu/gcfu/g

8/98/9 89%89% <10<1044 cfu/gcfu/g

1/191/19 5%5%

SwabSwab PositivePositive 5/135/13 38%38% NegativeNegative 1/31/3 33%33%

No cultureNo culture ------ ------ ------ ------ 3/63/6 50%50%

Wound Wound positionposition

Lower Lower extremityextremity

7/137/13 54%54% Upper Upper extremityextremity

11/3711/37 30%30%

Mech. of Mech. of injuryinjury

Farm or Farm or lawn lawn

mowermower

4/114/11 36%36% Other Other than F or than F or

LM LM

14/3914/39 36%36%

Fracture Fracture typetype

Type IIIB Type IIIB or IIIC or IIIC

fracturefracture

10/2810/28 36%36% Type I, II, Type I, II, IIIA, or IIIA, or nonenone

8/228/22 36%36%


Criteria for Positive/Negative Test, Infection, Criteria for Positive/Negative Test, Infection, and Prevalenceand Prevalence

Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865Breidenbach W,C., & S. Trager (1994) Plats. Reconstr. Surg. 95:860-865

TestTest Positive Positive test test

criteriacriteria

InfectionInfection PrevalencePrevalence Negative Negative test test

criteriacriteria

InfectionInfection PrevalencePrevalence

QuantitativeQuantitative >10>1044 cfu/gcfu/g

8/98/9 89%89% <10<1044 cfu/gcfu/g

1/191/19 5%5%

SwabSwab PositivePositive 5/135/13 38%38% NegativeNegative 1/31/3 33%33%

No cultureNo culture ------ ------ ------ ------ 3/63/6 50%50%


Lower Lower extremityextremity

7/137/13 54%54% Upper Upper extremityextremity

11/3711/37 30%30%

Mech. of Mech. of injuryinjury

Farm or Farm or lawn lawn

mowermower

4/114/11 36%36% Other Other than F or than F or

LM LM

14/3914/39 36%36%

Fracture Fracture typetype

Type IIIB Type IIIB or IIIC or IIIC

fracturefracture

10/2810/28 36%36% Type I, II, Type I, II, IIIA, or IIIA, or nonenone

8/228/22 36%36%


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

TestTest PPVPPV NPVNPV 100-NPV100-NPV SensitivitySensitivity SpecificitySpecificity

Quantitative Quantitative cultureculture

89% (56-98)89% (56-98) 95% (75-99)95% (75-99) 5%5% 89%89% 95%95%

Swab cultureSwab culture 38% (18-64)38% (18-64) 67% (21-94)67% (21-94) 36%36% 83%83% 20%20%


54% (29-77)54% (29-77) 70% (54-83)70% (54-83) 33%33% 81%81% 19%19%

Mechanism Mechanism of injuryof injury

36%(15-65) 36%(15-65) 64% (48-77)64% (48-77) 30%30% 22%22% 78%78%

Grade III Grade III fracturefracture

36% (21-54)36% (21-54) 64% (43-80)64% (43-80) 36%36% 56%56% 44%44%

Note: Values following each % represent range of 95% confidence interval; PPV= positive predictive 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; value= posttest probability of a positive result (infection); NPV=negative predictive value;

100-NPV=posttest probability of a negative result (no infection).100-NPV=posttest probability of a negative result (no infection).


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

TestTest PPVPPV NPVNPV 100-NPV100-NPV SensitivitySensitivity SpecificitySpecificity

Quantitative Quantitative cultureculture

89% (56-98)89% (56-98) 95% (75-99)95% (75-99) 5%5% 89%89% 95%95%

Swab cultureSwab culture 38% (18-64)38% (18-64) 67% (21-94)67% (21-94) 36%36% 83%83% 20%20%


54% (29-77)54% (29-77) 70% (54-83)70% (54-83) 33%33% 81%81% 19%19%

Mechanism Mechanism of injuryof injury

36%(15-65) 36%(15-65) 64% (48-77)64% (48-77) 30%30% 22%22% 78%78%

Grade III Grade III fracturefracture

36% (21-54)36% (21-54) 64% (43-80)64% (43-80) 36%36% 56%56% 44%44%

Note: Values following each % represent range of 95% confidence limits. PPV= positive predictive 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; value= posttest probability of a positive result (infection); NPV=negative predictive value;

100-NPV=posttest probability of a negative result (no infection).100-NPV=posttest probability of a negative result (no infection).


2. Interpretation of Microbiologic 2. Interpretation of Microbiologic Diabetic foot infection Samples Diabetic foot infection Samples

(or Qualitative Microbiology)(or Qualitative Microbiology)

Diabetic foot ulcers are polymicrobic.*Diabetic foot ulcers are polymicrobic.*– S. aureusS. aureus is the predominant aerobic species followed is the predominant aerobic species followed

by by S. epidermidisS. epidermidis, , StreptococcusStreptococcus spp., spp., P. aeruginosaP. aeruginosa, , EnterococcusEnterococcus spp., and coliform bacteria. spp., and coliform bacteria.

– The predominant anaerobic species are The predominant anaerobic species are PeptostreptococcusPeptostreptococcus spp., spp., BacteroidesBacteroides spp., and spp., and PrevotellaPrevotella spp. spp.

Application of good microbiological techniques will allow Application of good microbiological techniques will allow isolation of anaerobes from up to 95% of decubitus ulcers. isolation of anaerobes from up to 95% of decubitus ulcers. However, such studies are not usually performed due to the However, such studies are not usually performed due to the labor intensive nature of anaerobic microbiological studies.labor intensive nature of anaerobic microbiological studies.

*Bowler, P.G., et. al. (2001) Clin Microbiol Rev 14:244-269.*Bowler, P.G., et. al. (2001) Clin Microbiol Rev 14:244-269.


Schools of Thought*Schools of Thought*

Although microorganisms are responsible for Although microorganisms are responsible for wound infections, there is controversy regarding wound infections, there is controversy regarding their role. Published literature is inconclusive!their role. Published literature is inconclusive!– The density of microorganisms is the critical factor in The density of microorganisms is the critical factor in

determining whether a wound is likely to heal.determining whether a wound is likely to heal.– The presence of specific pathogens is of primary The presence of specific pathogens is of primary

importance in delayed healing.importance in delayed healing.– Microorganisms are of minimal importance in delayed Microorganisms are of minimal importance in delayed

healing.healing.– Debate whether a wound should be sampled, the Debate whether a wound should be sampled, the

value of the results and the method that should be value of the results and the method that should be used.used.


ConclusionsConclusions

Widespread controversy still existsWidespread controversy still exists– Regarding the exact mechanisms by which Regarding the exact mechanisms by which

microorganisms cause wound infection, microorganisms cause wound infection, – Regarding the significance of microorganisms in Regarding the significance of microorganisms in

nonhealing wounds that do not exhibit signs of clinical nonhealing wounds that do not exhibit signs of clinical infection, andinfection, and

– Regarding the best microbiological techniques to Regarding the best microbiological techniques to monitor the microbiology of wounds.monitor the microbiology of wounds.

– ASM Manual Clinical Microbiology states “A swab is ASM Manual Clinical Microbiology states “A swab is not the specimen of choice…Since a swab specimen not the specimen of choice…Since a swab specimen of a decubitus ulcer provides no clinical information.”of a decubitus ulcer provides no clinical information.”


ConclusionsConclusions

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


Louis PasteurLouis Pasteur

“ “ The germ is nothing. It is the terrain in The germ is nothing. It is the terrain in which it is found that is everything.”which it is found that is everything.”

Pasteur, L. (1880) De l’attenuation virus du cholera des poules. CR Acad. Sci. Pasteur, L. (1880) De l’attenuation virus du cholera des poules. CR Acad. Sci. 91: 673-680.91: 673-680.

Microbiologic Diagnosis of Diabetic Foot Infections Albert T. Sheldon, Jr. Ph.D. Microbiology Team...

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