shake’n bake...2015/04/17 · 1. to review the epidemiology, pathogenesis, and microbiology of...

SHAKE’N BAKE

(SHAKE, RATTLE AND ROLL)

What’s New in the Diagnosis of

Device-Associated Infection?

Robin Patel, M.D.

Professor of Medicine and Microbiology

Mayo Clinic College of Medicine

[email protected]

mailto:[email protected]

Disclosures I am a consultant St. Jude, Thermo Fisher Scientific,

Curetis

I have received payment from a

commercial organization. (including gifts

or other consideration or ‘in kind’

compensation)

Up-To-Date (royalties), Mayo Clinic (my

employer), ASM (reimbursement for

travel, editor’s stipend)

My institution has received grants or

instrument evaluations from a

commercial organization

Pfizer, Pradama, Tornier, Astellas,

Pocared, nanoMR, BioFire, Curetis,

Check-Points, Actavis, 3M, Cubist,

Hutchison Biofilm Medical Solutions,

Accelerate Diagnostics (Instrument

Evaluations: bioMérieux, Bruker, Abbott,

Nanosphere, Siemens, BD)

I hold a patent for a product referred to

in the CME/CPD program or that is

marketed by a commercial organization

B. pertussis PCR, anti-biofilm substance,

device/method for sonication

I am currently participating in or have

participated in a clinical trial within the

past 2 years

Curetis, nanoMR, Accelerate Diagnostics

Objectives

1. To review the epidemiology,

pathogenesis, and microbiology of

device-associated infection

2. To determine appropriate specimen

types and methods for culture for

diagnosing device-associated infection

3. To understand the role of molecular

diagnostics in the diagnosis of device-

associated infection

Total Hip & Knee Replacement Procedures

United States National Hospital Discharge Survey http://www.cdc.gov/nchs/about/major/hdasd/listpubs.htm


Total knee

Total hip

Prosthetic Joint Infection Rates

Years*

0-2 2-10

Prosthetic knee infection 1.55% 0.46%

Prosthetic hip infection 0.78% 0.33%

Kurtz et al. Clin Orthop Relat Res 2010:468:52

Ong et al. J Arthroplasty 2009;24:105

*Medicare Population

Primary Elective Arthroplasty

1997-2006

Prosthetic Hip and Knee Infections

United States

0

1,000

2,000

3,000

4,000

5,000

6,000

7,000

1989 1991 1993 1995 1997 1999 2001 2003 2005

Infected total knee arthroplasty

Infected total hip arthroplasty

Kurtz et al. J Arthroplasty 2008;23:984

Slide courtesy of Rizwan Sohail

Prosthetic Joint Infection

Tande and Patel. Clin Microbiol Rev 2014;27:302

Prosthetic Joint Infection Microbiology

Hip and Knee Hip Knee Shoulder Elbow

All time

periods Early

Number of joints 2435 637 1979 1427 199 110

Staphylococcus

aureus 27 38 13 23 18 42 Coagulase negative

staphylococci 27 22 30 23 41 41

Streptococcus species 8 4 6 6 4 4

Enterococcus species 3 10 2 2 3 0 Aerobic gram negative

bacilli 9 24 7 5 10 7

Anaerobic bacteria 4 3 9 5 Propionibacterium

acnes

24 1

Other anaerobes 3 0

Culture negative 14 10 7 11 15 5

Polymicrobial 15 31 14 12 16 3

Other 3


Cardiovascular Implantable Electronic

Device Infection - Microbiology

Culture negative, 7% Polymicrobial,

7%

Fungal, 2%

Gram negative

bacilli, 9% Other Gram

positive cocci, 4%

Staphylococcus aureus, 29%

Coagulase-negative

staphylococci, 42%

Sohail et al. J Am Coll Cardiol 2007;49:1861

Definitions of Prosthetic Joint Infection 2011

Musculoskeletal

Infection Society1

2013

Infectious Diseases

Society of America2

2013

International

Consensus3

Definitive

evidence

Supportive

evidence

Definitive

evidence

Supportive

evidence

Definitive

evidence

Supportive

evidence

Sinus tract communicating with the prosthesis x x x

Identical microorganism isolated from ≥2 cultures x x x

Purulence surrounding the prosthesis x x

Acute inflammation of periprosthetic tissue x x x

A single culture with any microorganism x x

A single culture with a virulent microorganism x

Elevated synovial fluid leukocyte count x x

Elevated synovial fluid neutrophil percentage x x

Elevated serum ESR and CRP x x

PJI Definitions

1Parvizi et al. Clin Orthop Relat Res 2011;469:2992 2Parvizi & Gehrke. Proceedings of the International Consensus Meeting on Periprosthetic Joint Infection 2013 3Osmon et al. Clin Infect Dis 2013:56:e1

Musculoskeletal Infection Society (MSIS) and

Infectious Diseases Society of America (IDSA)

Diagnostic Criteria for Prosthetic Joint Infection

Melendez et al. IDWeek poster 2014

MSIS

Aseptic

Failure

Prosthetic

Joint Infection

IDSA

Aseptic

Failure 297 1

Prosthetic

Joint

Infection

4 69


p=0.56

Musculoskeletal Infection Society (MSIS) and

International Consensus Meeting (ICM) Diagnostic

Criteria for Prosthetic Joint Infection


MSIS

Aseptic

Failure

Prosthetic

Joint Infection

ICM

Aseptic

Failure 298 0

Prosthetic

Joint

Infection

3 70


p=0.65

Infectious Diseases Society of America (IDSA) and

International Consensus Meeting (ICM) Diagnostic

Criteria for Prosthetic Joint Infection


IDSA

Aseptic

Failure

Prosthetic

Joint Infection

ICM

Aseptic

Failure 294 4

Prosthetic

Joint

Infection

4 69


p=0.9

Test Joint Threshold value Sensitivity Specificity + LR - LR Cost Comments

PERIPHERAL BLOOD

WBC

Hip and knee

11,000 x 109/L 45 87 3.5 0.6 $ 1796 patients in 15 studies

CRP 10 mg/L 88 74 3.4 0.2 $ 3225 patients in 23 studies

ESR 30 mm/hr 75 70 2.5 0.4 $ 3370 patients in 25 studies

IL-6 10 pg/mL 97 91 10.8 0.0 $ 432 patients in 3 studies

Procalcitonin 0.3 ng/mL 33 98 16.5 0.7 $ 78 patients in single study

IMAGING

Plain radiograph Hip

Lucency or

periosteal new

bone formation 75 28 1.0 0.9 $ 65 patients in single study

Triple phase bone scan Late hip

Increased uptake

on all 3 phases 88 90 8.8 0.1 $$$ 46 patients in single study

Bone scan/labeled

leukocyte scan

Late hip and

knee

Incongruent

images 64 70 2.1 0.5 $$$ 166 patients in single study

FDG-PET scan Hip and knee 82 87 6.1 0.2 $$$$$ 635 patients in 11 studies

SYNOVIAL FLUID

Cell count Knee

1100 cells/µL 91 88 7.6 0.1 $$ 429 patients in single study

Neutrophil percentage 64% 95 95 17.9 0.1 $$

Cell count Hip

4200 cells/µL 84 93 12.0 0.2 $$ 201 patients in single study Neutrophil percentage 80% 84 82 4.7 0.2 $$

Cell count Knee

(<6 weeks)

27,800 cells/µL 84 99 84.0 0.2 $$ 146 patients in single study

Neutrophil percentage 89% 84 69 2.7 0.2 $$

Culture Hip and knee 72 95 14.4 0.3 $$ 3332 patients in 34 studies

Test Characteristics and Relative Costs

of Preoperative Tests for PJI Diagnosis

Tande and Patel. Clin Microbiol Rev 2014;27:302 © 2015 Mayo Foundation for Medical Education and Research

Synovial Fluid Leukocyte/Differential

Prosthetic Knee

(>6 months from index surgery)

*

*

*

*

Trampuz et al. Am J Med 2004;117:556 © 2015 Mayo Foundation for Medical Education and Research

Sensitivity, 94%

Specificity, 88%

Sensitivity, 97%

Specificity, 98%

Synovial Fluid

Leukocyte/Differential WBC

(cells/l)

Sensitivity Specificity Neutrophil

percentage

Sensitivity Specificity Joint

type

Time

from

index

surgery

Trampuz Am J Med

2004;117:556

1,700 94 88 65 97 98 Knee

(133)

>6

months

Ghanem JBJS

2008;90:1637

1,100 91 88 64 95 95 Knee

(429)

Varied

Schinsky JBJS

2008;90:1869

4,200 84 93 80 84 82 Hip

(201)

Varied

Bedair CORR

2011;469:34

10,700 95 91 89 84 69 Knee

(146)

<6 weeks

Cipriano JBJS

2012;94:594

Non-inflammatory

arthritis

3,450 91 93 78 96 87 Hip/Knee

(810)

Unknown

Cipriano JBJS

2012;94:594 Inflammatory arthritis

3,444 88 80 75 100 82 Hip/Knee

(61)

Unknown

Zmistowski J

Arthroplasty

2012;27:1589

3,000 93 94 75 93 83 Knee

(150)

Unknown

Dinneen Bone &

Joint J 2013;95:554

1,590 90 91 65 90 87 Hip/Knee

(75)

Unknown

Natural Progression of Synovial

Fluid Leukocytes in TKA

Christensen et al. JBJS 2013;95:2081

WBC, Neutrophil Percentage and Total Neutrophil Count by Time Period*

First 45 Days Days 46 to 90 3 Months to 1

Year

1 to 2 Years

WBC count/µL 3037±3786 1119 ±1325† 474 ±894‡

428±706

Neutrophil % 69±27 49±23† 41±27 28±32‡

Neutrophils µL 2533±3483 649±871† 270±598‡ 241±552

*Values are means and standard deviations

†Significantly different from the first 45 days (p<0.05)

‡Significantly different from days 46 to 90 (p<0.05)

Novel Synovial Fluid Biomarkers Biomarker Cutoff Sensitivity (%) Specificity (%)

-Defensin 4.8 µg/mL 100 100 Deirmengian et al. Clin Orthop

Rel Res 2014;472:3254

(95 subjects; 29 PJI; hip/knee) Neutrophil elastase 2 2.0 µg/mL 100 100

Bactericidal/permeabiity-increasing protein 2.2 µg/mL 100 100

Neutrophil gelatinase-associated lipocalin 2.2 µg/mL 100 100

Lactoferrin 7.5 µg/mL 100 100

IL-8 6.5 ng/mL 100 95

CRP 12.2 mg/L 90 97

Resistin 340 ng/mL 97 100

-Defensin 5.2 mg/L 97 96 Deirmengian et al. JBJS

2014;96:1439 (149 subjects; 37

PJI; hip/knee; did not report

WBC)

CRP 3 mg/L 97 79

-Defensin if positive, CRP 97 100

-Defensin 5.2 mg/L

100 100 Deirmengian et al. CORR

2015;473:198 (46 subjects; 23


WBC performance)

-Defensin 7720

ng/mL

95 100 Bingham et al. CORR

2014;472:4006 (57 subjects; 19


%neutrophils)

-Defensin 0.48 S/CO 63 95 Frangiamore et al. J Shoulder

Elbow Surg 2015 In Press (33

cases; 11 PJI; shoulder)

IL-6 359.3

pg/mL

87 90 Frangiamore et al. J Bone Joint

Surg Am 2015:97:63 (35

subjects; 15 PJI; shoulder)

Intraoperative Frozen Section Histopathology Reference Specimen Joint #PMN* n Sensitivity Specificity PPV**

Feldman et al. JBJS(Am) 1995;77:1807 JC, IM Hip/knee 5 33 100 100 100

Athanasou et al. JBJS(Br) 1995;77:28 JC, IM Hip/knee 1 106 90 96 88

Lonner et al. JBJS(Am) 1996;78:1553 JC, IM, ASPI Hip/knee 5 175 84 96 70

10 84 99 89

Pace et al. J Arthroplasty 1997;12:64 JC, IM Hip/knee 5 18 82 93 82

Abdul-Karim et al. Mod Pathol

1998;11:427

IM, ST, UDT Hip/knee 5 64 43 97 60

Banit et al. CORR 2002;401:230 JC, ASPI Knee 10 55 100 96 82

Hip 10 63 45 92 55

Musso et al. Postgrad Med J

2003;79:590

JC, IM, ASPI Hip/knee 5 45 50 95 60

Wong et al. J Arthroplasty

2005;20:1015

JC, IM, SS Hip/knee 5 33 93 77 68

10 86 85 75

Ko et al. J Arthroplasty 2005;20:189 JC, IM, ASPI Hip/knee 5 40 67 97 86

Frances Borrego et al. Int Orthop

2007;31:33

PST Hip 10 63 67 90 80

Knee 83 50 100 100

Nunez et al. Acta Orthop 2007;78:226 JC, IM, ASPI Hip 5 136 86 87 79

Tohtz et al. CORR 2010;468:762 IM Hip 10 52 87 200 100

*Some studies used >, others ≥ the number shown, **Positive predictive value

JC, joint pseudocapsule; IM, interface membrane; ASPI, any area that appears suspicious for possible infection; ST, synovial

tissue; SS, synovial surface; UDT, unusually discolored tissue; PST, periprosthetic soft tissue


Propionibacterium acnes PJI

Butler-Wu et al. J Clin Microbiol 2011;49:2490

Histopathology 2 Cultures positive for P. acnes

Positive 6

Negative 9

All 15


Butler-Wu et al. J Clin Microbiol 2011;49:2490


• ~6 month period, 2011

• Fluids and tissues placed in anaerobic

fluid or tissue vials in operating room

• Tissues homogenized in 3 ml brain heart infusion broth

– 0.1 ml tissue homogenate/fluid onto CDC anaerobic sheep

blood agar, incubated anaerobically @ 37°C for 14 days

• Examined Monday, Wednesday, and Friday for first

week, then on days 7 and 14 or until positive

– 1 ml tissue homogenate/fluid inoculated into

anaerobically prereduced thioglycolate broth, closed,

incubated @ 37°C for 14 days

• Examined daily or until positive Shannon et al. J Clin Microbiol 2013;51:731 © 2015 Mayo Foundation for Medical Education and Research


• 14 subjects ≥2 shoulder bone/joint culture P. acnes

• 72 anaerobic plate or broth cultures (3 to 13/event)

• P. acnes growth

– 28 plates (27 within 7 days)

– 53 broths (52 within 7 days)

• All events 2 positive broth cultures by 7 days, but 3

negative plate cultures and another 3 single positive

plate cultures even with 14 days of incubation

• No events plate positive only

• Broth more likely to be positive than plate culture by

day 7 and overall (P, 0.0001)

Shannon et al. J Clin Microbiol 2013;51:731 © 2015 Mayo Foundation for Medical Education and Research

Periprosthetic Tissue Culture

• Multiple (5 or 6) specimens cultured

• or >2 operative specimens indistinguishable

organism

• IDSA Guideline - At least 3 and optimally 5 or 6

periprosthetic intraoperative tissue samples or the

explanted prosthesis itself should be submitted for

aerobic and anaerobic culture at the time of surgical

débridement or prosthesis removal to maximize the

chance of obtaining a microbiologic diagnosis (B-II).

Atkins et al. J Clin Microbiol 1998;36:2932

Osmon et al. Clin Infect Dis 2013:56:1

Periprosthetic Tissue Culture

Blood Culture Bottles


Periprosthetic Tissue Culture Blood

Culture Bottle Study (Oxford #1)

• Tissue + sterile glass beads (Ballotini) 5 ml saline (shaking)

– 1 ml each into Robertson’s cooked meat broth, fastidious

anaerobic broth, BACTEC Anaerobic/F & Aerobic/F bottles

– 0.25 ml each onto chocolate, aerobic & anaerobic (2) blood agars

– 5 day incubation

– 141 elective joint revisions (mean, 4.9 specimens/case)

– 23 PJI cases

Sensitivity/

Specificity

(≥2 positive)

Direct

plates

Fastidious

anaerobic

broth

Cooked meat

broth

BACTEC

blood culture

bottles

Sensitivity, % 39 57 83 87

Specificity, % 100 100 97 98

Hughes et al. Clin Microbiol & Infect 2011;10:1528

Periprosthetic Tissue Culture Blood

Culture Bottle Study (Oxford #2) • Tissue + 3 ml saline + sterile glass beads vortexed 15 sec

– 0.5 ml BACTEC Lytic/10 Anaerobic/F and Plus Aerobic/F bottles

– 14 day incubation

• 322 patients (mean, 4 specimens/case)

• 66/79 PJI culture-positive (sensitivity, 84%)

– All positive within 3 days of culture except 1 (detected at 8 days)

• 7/243 non-PJI culture-positive (specificity, 97%)

• Propionibacterium species, 30 isolates

– 67 samples positive

– 6 patients - 2 PJI, detected at 3 and 8 days

– Detected, median of 5 days (1 day for other bacterial species)

– Sub-culturing negative bottles after 14-days’ incubation detected

only a single (clinically insignificant) additional Propionibacterium

isolate from 1000 bottles

Minassian et al. BMC Infect Dis 2014;14:233

Periprosthetic Tissue

Blood Culture Bottle Study

(Mayo Clinic) • Patients undergoing revision arthroplasty, Mayo Clinic, 8/2013–4/2014

– 369 subjects - 138 hip, 160 knee, 49 shoulder, 22 elbow arthroplasties

– 117 (32%) met IDSA diagnostic criteria for PJI

• Tissues collected in anaerobic tissue vials

• Tissues homogenized using a Seward Stomacher 80 Biomaster in 5 ml of

brain heart infusion broth for 1 minute

– 0.1 ml inoculated onto sheep blood and chocolate agar and incubated

aerobically in 5% CO2, 5 days

– 0.1 mL inoculated onto CDC anaerobic blood agar and incubated

anaerobically, 14 days

– 1 mL inoculated into enriched thioglycollate broth, incubated 14 days

– 1 mL inoculated into BACTEC Plus Aerobic/F & BACTEC Lytic/10

Anaerobic/F blood culture bottle; placed on a BACTEC 9240

instrument, incubated 14 days


Periprosthetic Tissue Blood Culture Bottle Study (Mayo Clinic)

Bayesian Latent Class Modelling

Media Combination Sensitivity Specificity

Aerobic and anaerobic agars 48.9 99.7

Aerobic and anaerobic agars and thioglycollate broth 62.6 98.1

Aerobic and anaerobic blood culture bottles 92.1 99.7

Aerobic and anaerobic blood culture bottles and thioglycollate broth 92.1 98.8

Aerobic and anaerobic blood culture bottles and aerobic agar 94.6 99.7

Aerobic and anaerobic blood culture bottles and anaerobic agar 96.8 99.8

Aerobic and anaerobic blood culture bottles and aerobic and anaerobic agars 99.1 99.7

All media combined 99.1 97.3


Sensitivity and Specificity using PJI (IDSA Criteria) as the Gold Standard

Media Combination Sensitivity Specificity

Aerobic and anaerobic agars 33.3 100.0

Aerobic and anaerobic agars and thioglycolate broth 44.4 98.8

Aerobic and anaerobic blood culture bottles 60.7 98.8

Aerobic and anaerobic blood culture bottles and thioglycolate broth 63.3 98.8

Aerobic and anaerobic blood culture bottles and aerobic agar 62.4 98.8

Aerobic and anaerobic blood culture bottles and anaerobic agar 62.3 98.0

Aerobic and anaerobic blood culture bottles and aerobic and anaerobic agars 64.1 98.0

All media combined 67.5 96.8

Periprosthetic Tissue Blood Culture Bottle Study (Mayo Clinic)

Time-to-Detection


0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7

Nu

mb

er o

f ti

ssu

e sp

ecim

ens

Days until detection

Pathogen

Contaminant

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Nu

mb

er o

f ti

ssu

e sp

ecim

ens

Days until detection

Pathogen

Contaminant

MALDI TOF MS and Prosthetic Joint Infection

• May 2012 - May 2013

• 178 PJI, 82 aseptic failure (AF) cases with positive cultures

• 770 organisms

• Median 3/subject

• MALDI TOF MS used to identify 455 organisms (59%)

• 89% identified to species level

• Staphylococcus aureus, Staphylococcus caprae always associated with infection

• Staphylococcus epidermidis, Staphylococcus lugdunensis pathogens or contaminants

• All other coagulase-negative staphylococci more frequent as contaminants

• Most streptococcal & Corynebacterium isolates pathogens

Peel et al. Diagn Microbol Infect Dis 2015;81:163

Staphylococcus epidermidis Biofilm on Polycarbonate Coupons

Scanning Electron Microscopy

Soaking Scraping Sonication


Sonicate Tissue


Current Orthopaedic Implant Processing - Mayo Clinic

Sonicate

5 minProsthesis Placed

in Container

(Operating Room)

400 ml

Ringer’s

Solution

Added

Vortex

30 sec

Vortex

30 secCentrifuge

5 min

Aspiration

Plating


Comparison of Sonicate Fluid and Tissue Culture for

Diagnosis of Infection Associated with Orthopedic

Implants

Implant type Sonicate

fluid

Periimplant

tissue

p value Reference

Our studies

Prosthetic hip/knee

joint

Sensitivity 79% 61% <0.001 Trampuz et al, NEJM 2007

Vol 357;654 Specificity 99% 99%

Prosthetic shoulder

joint

Sensitivity 67% 55% 0.046 Piper et al, JCM 2009

Vol 47:1878 Specificity 98.0% 95%

Prosthetic elbow

joint

Sensitivity 89% 55% 0.18 Vergidis et al, JSES 2011

Vol 20;1275 Specificity 100% 93%

Spine implant Sensitivity 91% 73% 0.046 Sampedro et al, Spine 2010

Vol 25:1218 Specificity 97% 93%


Sonication versus Vortexing for

Prosthetic Joint Infection Diagnosis

• 135 removed prostheses

– 35 PJI

– 100 aseptic failure

• Using cut-off of 50 CFU/ml

– Sonication more sensitive than vortexing (60%

versus 40%)

• Specificity was 99% for both methods

© 2015 Mayo Foundation for Medical Education and Research Portillo et al. J Clin Microbiol 2013:51:591

Sonication of Orthopedic Implants Particularly

Helpful for Delayed Implant Infection

Puig-Veridié et al. Bone Joint J 2013;95-B:244-9.

Type of failure Sensitivity Specificity

Sonicate Tissue p-value Sonicate Tissue p-value

Overall (n = 317) 90 67 <0.001 99 99.5 1

Suspected septic

failure (n = 79)

86 76 0.057 100 100 1

Unsuspected septic

failure (n = 238)

100 49 <0.001 99 99.5 1

p-value 0.032 0.007 0.864 1

Early (<3 months)

(n = 43)

93 85 0.625 94 100 1

Delayed (>3 months)

(n = 240)

88 68 <0.001 99 100 1

p-value 0.723 0.013 0.169 1

Sonication Provides “Rapid” Results

• 231 explanted prostheses (AF, 162; PJI, 69)

– Sensitivity/specificity

• Sonicate fluid culture 81/99%, tissue culture 61/100%

Portillo et al. J Infect 2014;69:35-41

Cardiovascular Implantable

Electronic Device Infection

Dababneh and Sohail Cleveland Clinic J Med 2011;8:529

Sohail et al. Expert Rev Anti-infect Ther 2010;7:831

Cardiovascular Implantable Electronic Device

Cultures

• High degree of device

colonization and/or

culture contamination

in absence of infection

o 33-42% of generator

pockets positive1,2

• Negative cultures from

those with device

infection

o 31%1

1Dy Chua J, et al. Pacing Clin Electrophys. 2005;28:1276 2Kleemann T, et al. Europace. 2010;12:58-63

Cardiovascular Implantable

Electronic Device Infection

Guidelines for Diagnosis*

*Routine microbiologic studies should not be performed on devices removed for non-infectious reasons

**Tissue and lead tip should be cultured for fungi and mycobacteria if the initial Gram stain is negative

***British Heart Rhythm Society, British Cardiovascular Society, British Heart Valve Society, British Society for Echocardiography

****Collected via syringe +/- needle from a discharging wound

1Baddour et al. Circulation 2010;121:458 2Sandoe et al. J Antimicrob Chemother 2015;70:325

Blood

Cultures

Generator Pocket Lead tip

culture

Device

culture Tissue

Gram

stain

Tissue

culture

Swab Pus or

fluid****

American Heart

Association1

**

British Society for

Antimicrobial

Chemotherapy2***

(ideally

distal and

proximal)

and lead

vegetation

Merits

further study


Sonication (Sapienza Università Study)

40 patients

20 without

infection

20 with

infection

Generator only

removed

Generator and

leads removed

Inoculated in

TSB, 24 hours

Vortex, sonication, vortex,

centrifuge

Pocket swab,

blood cultures

Cutoff of 2 CFU/mL

Oliva et al. J Clin Microbiol 2013;51:496

Positive

sonicate

fluid culture,

n(%)

Positive

broth

incubation

culture,

n(%)

Non-infected

components

(n=20)

8 (40) 4 (20)

Infected

components

(n=60)

46 (77) 36 (60)

p=0.001

Sonication, Swab and Blood Cultures

17 Subjects With Infected Cardiovascular

Implantable Electronic Devices

(Swiss Study)

Rohacek et al. PACE 2015:38:247

CIED Site of Infection Echocardiography Sonication

(CFU/mL)

Swab Culture

(Growth of Bacteria)

Blood Cultures

(Positive Bottles)

PM Pocket and definite IE TTE: vegetations on lead and

valve MSSA (>1,000) MSSA (strong) MSSA (8/8)

CRT Pocket and possible IE TTE: no vegetation MSSA (>1,000) MSSA (strong) MSSA (3/4)

PM Pocket and possible IE TTE: no vegetation MSSA (>1,000),

P. aeruginosa (>1,000)

MSSA (strong),

P. aeruginosa

(strong)

MSSA (10/10)

PM Pocket and possible IE TEE: no vegetation S. lugdunensis (710) S. lugdunensis

(few) S. lugdunensis (6/6)

PM Pocket TTE: no vegetation MSSA (>1,000) MSSA, 2 MT

(strong) Sterile

PM Pocket TTE: no vegetation MSSA (>1,000) MSSA (moderate) Sterile

ICD Pocket No echocardiography MSSA (>1,000, >1,000), P.

acnes (750) MSSA (few) Sterile

PM Pocket TEE: no vegetation CNS (50) CNS (few) Sterile

PM Pocket No echocardiography P. acnes (700) P. acnes (few) Sterile

PM Pocket No echocardiography CNS (>1,000) Sterile CNS (1/2)

PM Definite IE TTE: vegetation on lead and

valve MSSA (790) Sterile

MSSA (4/4),

S. mitis (1/4)

PM Pocket and definite IE TTE: vegetation on lead B. cereus (>1,000) Not performed MSSA (10/10)

PM Pocket No echocardiography P. acnes (>1,000), CNS (110) Sterile Sterile

PM Pocket TEE: no vegetation P. acnes (700) Sterile Sterile

PM Pocket No echocardiography CNS (>1,000) Sterile Sterile

PM Pocket TTE: no vegetation MSSA (>1,000) Sterile Sterile

ICD Pocket TTE: no vegetation Sterile Sterile Sterile

Sonication and Swab Cultures

115 Subjects With Non-Infected Cardiovascular

Implantable Electronic Devices

(Swiss Study)

• ≥10 CFU/mL of sonication fluid from 21 devices (18%)

• Pocket swab cultures positive 30 of 112 devices cultured (27%)

– Moderate in 2 cultures

– Few in 20 cultures

– After enrichment in 7 cultures

• Propionibacterium acnes and CNS most common

• 6 subjects with >150 CFU/mL in sonication fluid

(4 Propionibacterium acnes, 2 CNS) 100% concordance with

swabs

• 2 subjects with detection of CNS in sonication and swab culture

developed clinical infection with CNS 3 weeks and 4 months later

Rohacek et al. Circulation 2010;121:1691


Sonication (Mayo Clinic Study)

• Determine whether device vortexing-

sonication followed by culture of

resulting sonicate fluid enhances

microbial detection compared with

swab or pocket tissue cultures – 42 subjects with noninfected + 35 with infected

devices enrolled over 12 months

– One swab each from device pocket & device

surface, pocket tissue & device per patient

– Swabs, tissues cultured using routine methods

– Device processed in Ringer's solution using

vortexing-sonication resultant fluid

semiquantitatively cultured

Nagpal et al. American Journal of Cardiology, 2015;115:912

Sensitivity and Specificity of Culture For

Cardiovascular Implantable Electronic

Device Infection by Specimen Type

(Mayo Clinic Study)


Test Proportion

Sensitivity

(95% CI) P-value∗ Proportion

Specificity

(95% CI) P-value∗

Device Swab

(≥2+)

3/35 9 (2-23%) <0.001 41/42 98 (87-100%) 0.317

Pocket Swab

(≥2+)

7/35 20 (8-37%) 0.001 41/42 98 (87-100%) 0.317

Tissue (≥2+) 3/35 9 (2-23%) <0.001 40/42 95 (84-99%) 1.000

Sonicate Fluid

(≥20 CFU/10 ml)

19/35 54 (37-71%) 40/42 95 (84-99%)

Device Swab

(any)

10/35 29 (15-46%) <0.001 40/42 95 (84-99%) <0.001

Pocket Swab

(any)

15/35 43 (26-61%) 0.008 39/42 93 (81-99%) <0.001

Tissue (any) 16/35 46 (29-63%) 0.004 28/42 67 (50-80%) 0.366

Sonicate Fluid

(any)

26/35 74 (57-88%) 25/42 60 (43-74%)

*p-value testing for a difference in performance rate relative to sonicate fluid culture via McNemar's test

Culture Sensitivity Based on Specimen Types

Studied Among Patients With Cardiovascular

Implantable Electronic Device Infection

(Mayo Clinic Study)


Proportion Rate, % (95% CI) P-value∗

Device Erosions/Pocket Infections (n=16)

Device Swab 2/16 13 (2-38%) 0.003

Pocket Swab 5/16 31 (11-59%) 0.034

Tissue 2/16 13 (2-38%) 0.003

Sonicate Fluid 11/16 69 (41-89%)

Lead or Valve associated Endocarditis (n=19)

Device Swab 1/19 5 (0-26%) 0.008

Pocket Swab 2/19 11 (1-33%) 0.014

Tissue 1/19 5 (0-26%) 0.008

Sonicate Fluid 8/19 42 (20-67%)

*p-value testing for a difference in performance rate relative to sonication via McNemar's test.

Intravascular Catheter

Roll Plate Culture Method

• Most commonly used method is

semiquantitative roll plate method

o Peripheral (Maki 1977)

o Pediatric umbilical catheters (Adam 1982)

o Short term/long-term CVCs (Bouza 2005)

oDialysis catheters (Brodersen 2007)

• Simple, requires no special equipment

oMay not detect intraluminal colonization?

Intravascular Catheter Culture

Comparative Methods Evaluation

Erb S, et al. Clin Infect Dis 2014;59:514

Roll plate method Sonication

88 85

Likelihood of detection of catheter colonization, using 15 cfu/tip

(roll plate method) and 100 cfu/tip (sonication and vortex)

4x

975 nontunneled central venous catheters cut into 2 equal 5 cm-

sized segments (subcutaneous/tip); 217 had significant colonization

Randomized order

Long Term Intravascular Catheters

• Random order of roll plate vs sonication

– 313 Hickman catheters

• Roll plate 21%/sonication 17%

• 89 CRBSI sensitivity roll plate 35%/sonication 45% Slobbe et al. J Clin Microbiol 2009;47:885

– 149 tunneled long term catheters

o39 colonized

• Roll plate 95%/sonication 44% (p<0.001) Guembe et al. J Clin Microbiol 2012;50:1003

3-4X vs.

• 223 Port-A-Caths

– 53 colonized

– 17 bloodstream infection (BSI)

Vascular Access Ports (VAP)

Method Sensitivity

(port

colonization)

Sensitivity/

specificity (VAP-

associated BSI)

Tip Roll plate 57 59/90

Tip Sonication 32 47/96

Port Aspirate (before sonication) 36 53/95

Port Sonication fluid 57 77/92

Port Aspirate (after sonication) 51 59/92

Port Internal surface swab 70 94/90

Tip + Port Roll plate + sonication + internal surface swab 94 100/84

Best single

method

Bouza et al. Diagn Microbiol Inf Dis 2014;78:162

Molecular Diagnostics

• Promises:

– Increased diagnostic yield (e.g., in setting of

prior antimicrobial therapy)

– Rapid

• Broad range (e.g., 16S rRNA gene) PCR

o Lack of specificity; requires sequencing or

additional step for identification; difficult to

detect polymicrobial infection

• Panel PCR

o Limited to organisms included in panel

Prosthetic Joint Sonication and

Broad-Range PCR

- No Improvement Over Culture

© 2015 Mayo Foundation for Medical Education and Research Gomez et al. J Clin Microbiol 2012:50:3501

Test Sensitivity

135 PJI

Specificity

231 Aseptic Failure Accuracy

% (95% Confidence Interval)

Tissue culture 70.4 (64.5-76.3) 98.7 (97.2-100) 88.3 (84.2-92.4)

Sonicate fluid culture 72.6 (66.8-78.4) 98.3 (96.6-100) 88.8 (84.7-92.9)

Sonicate fluid broad-range PCR 70.4 (64.5-76.3) 97.8 (95.9-99.7) 87.7 (83.5-91.9)

Combination of two tests above 83.0 (78.2-87.8) 95.7 (93.1-98.3) 91.0 (87.3-94.7)

Sonicate fluid culture plus PCR 78.5 (73-2-83.8) 97.0 (94.8-99.2) 90.2 (86.4-94.0)

Synovial fluid culture 64.7 (56.5-72.9) 96.9 (93.9-99.9) 84.1 (77.8-90.4)

Sonicate fluid PCR - lower cutoff

(CP <27.59 cycles) 80.0 (74.8-85.2) 90.9 (87.2-94.6) 86.8 (82.5-91.3)

Lack of Sensitivity of Periprosthetic

Tissue Broad-Range PCR • 2-year period

• 5 tissues/patient

• Culture, 16S rRNA gene

PCR/sequencing

• 264 suspected cases of PJI

– PJI confirmed (215)

• Culture positive, 192 (89%)

• PCR positive, 151 (73%)

– Non-PJI (49)

• PCR positive, 2 (specificity,

96%)

Bémer et al. J Clin Microbiol 2014:52:3583

Test Aseptic failure

(290)

PJI

(144)

Sensitivity Specificity PPV NPV

No. of patients with positive

specimens

% (95% confidence interval)

Synovial-fluid culture 5/161 59/89 66.3 (55.5-76.0) 96.9 (92.9-99.0) 92.2 (82.7-97.4) 83.9 (77.8-88.8)

Tissue culture

Any growth 45 119 82.6 (75.4-88.4) 84.5 (79.8-88.5) 72.6 (65.1-79.2) 90.7 (86.6-93.9)

≥2 positive tissues (same organism) 6 101 70.1 (62.0-77.5) 97.9 (95.6-99.2) 94.4 (88.2-97.9) 86.9 (82.7-90.3)

Sonicate fluid culture 5 105 72.9 (64.9-80.0) 98.3 (96.0-99.4) 95.5 (89.7-98.5) 88.0 (83.9-91.3)

Sonicate fluid PCR (10 assay panel)

Any positive result

Staphylococcus sp

S. aureus

Coagulase-negative staphylococci

Streptococcus sp

Enterococcus/Granulicatella/Abiotrophia sp

Enterobacteriaceae

Gram-positive anaerobic cocci

Propionibacterium sp

P. aeruginosa

Corynebacterium sp

C. jeikeium/urealyticum

Non-jeikeium sp

Proteus sp

B. fragilis group

6

2

0

2

3

0

1

0

0

0

0

0

0

0

0

111

75

28

47

11

11

8

8

8

5

4

0

4

1

0

77.1 (69.3-83.7) 97.9 (95.6-99.2) 94.9 (89.2-98.1) 89.6 (85.7-92.7)

Prosthetic Joint Sonication – PJI PCR Panel

Cazanave et al. J Clin Microbiol 2013;51:2280 © 2015 Mayo Foundation for Medical Education and Research

Summary “Device-Related Infections Require Device-Specific Diagnostics”

• Prosthetic joint infection

– Cultures – synovial fluid and periprosthetic tissue (blood

culture bottles), sonicate fluid

– Novel synovial fluid markers?

– Panel PCR

• Cardiovascular implantable electronic device infection

– Avoid culturing devices removed for non-infectious reasons

– Vortexing-sonication with semiquantitative

aerobic/anaerobic culture

– Tissue Gram stain?

• Intravascular catheter and port infection

– Roll plate method (intravascular catheter tips)

– Culture both tip and port (vascular access ports)

Acknowledgments and Funding Kerryl Greenwood-Quaintance, MS

Melissa Karau

Suzannah Schmidt, MS

Charles Cazanave, MD

Marta Fernandez-Sampedro, MD

Trisha Peel, PhD

Andrej Trampuz, MD

Aaron Tande, MD

Paolo Melendez, MD

Eric Gomez-Urena, MD

Cassandra Brinkman, Ph.D.

Mark Rouse

Awele Maduka-Ezeh, MD

Jin Won Chung, MD

Jose del Pozo, MD

Trisha Peel, MD

Seong Yeol Ryu, MD

Larry Baddour, MD

Rizwan Sohail, MD

Harmony Tyner, MD

Paschalis Vergidis, MD

Matt Thoendel, MD, PhD

Douglas Osmon, MD

James Steckelberg, MD

Elie Berbari, MD

Franklin Cockerill, MD

Jayawant Mandrekar, PhD

Arlen Hanssen, MD

David Lewallen, MD

Robert Trousdale, MD

Mark Pagnano, MD

Miguel Cabanela, MD

David Jacofsky, MD

Franklin Sim, MD

Daniel Berry, MD

Michael Stuart, MD

Robert Cofield, MD

Paul Huddleston, MD

John Sperling, MD

Joaquin Sanchez-Sotelo, MD

Mark Dekutoski, MD

Bradford Currier, MD

Mike Yaszemski, MD

Youlonda Loechler

Krishnan Unni, MD

James Greenleaf, PhD

James Uhl

Scott Cunningham, MS

Clinical Microbiology Bacteriology and IP Staff

Mayo Clinic patients

Quantitative Considerations in

Sonication Cultures

Janz et al. J Orthop Res 2013;31:2021

• Inoculated sonicate fluid onto blood, chocolate, McConkey,

& Sabouraud agars and into thioglycolate broth (+/- others)

– Sonicate fluid culture versus tissue culture

• 23 PJI, 36 AF

• Sensitivity 91% versus 75%

• Specificity 81% versus 100%

Janz et al. Internat Orthoped 2013;37:931

• 37 PJI, 65 AF Sonicate Cultures Sensitivity Specificity PPV NPV Accuracy

Single 89% 72% 80% 92% 78%

Multiple (≥1 positive) 100% 70% 71% 100% 83%

Multiple (≥2 positive) 100% 100% 100% 100% 100%

Inoculation of Sonication Fluid

into Blood Culture Bottles

Portillo et al. J Clin Microbiol In Press

• 30 ml sonicate fluid centrifuged; sediment resuspended in 2 ml Ringer’s solution; 1 ml

inoculated into BACTEC Plus Aerobic/F and Anaerobic Lytic/F bottles; incubated 5 days

• PJI (n = 50); aseptic failure (n = 60)

• 10 ml sonicate fluid inoculated into aerobic and anaerobic BacT/Alert FAN blood culture

bottles with antimicrobial removal systems, incubated on BacT/Alert system for 5 days

• 45 joint prostheses and 30 fracture fixation devices

• 39 orthopedic implant-associated infections

Diagnostic method Sensitivity, %

(95% CI)

Specificity, %

(95% CI)

PPV, %

(95% CI)

NPV, %

(95% CI)

Intraoperative tissue

samples

59 (42-74) 100 (90-100) 100 (85-100) 69 (55-81)

Conventional

sonication fluid

87 (73-96) 100 (90-100) 100 (90-100) 88 (74-96)

Inoculation of

sonication fluid in

blood culture bottles

100 (91-100) 100 (90-100) 100 (91-100) 100 (90-100)

Diagnostic

method

Sensitivity, %

(95% CI)

Specificity, %

(95% CI)

PPV, %

(95% CI)

NPV, %

(95% CI)

Sonicate fluid 88 (76–95) 87 (75–94) 85 (72–93) 90 (79–96)

Synovial fluid 64 (49–77) 98 (91–100) 97 (84–100) 77 (66–86)

Shen et al. J Clin Microbiol 2015;53:777

Intravascular Catheters

IDSA Guidelines 2009 • >15 CFU (roll-plate) or >102 CFU (quantitative sonication or luminal flushing) reflects

catheter colonization (A-I)

o Qualitative broth cultures not recommended (A-II)

• Culture tip not the subcutaneous portion (B-III)

• Short term and arterial catheters – roll-plate (A-II)

• Long term catheters - unclear which method is superior

• Port - culture catheter tip + qualitative culture of the port reservoir (B-II)

• Catheter-related bloodstream infection

– Same organism grow from at ≥1 percutaneous blood culture and a catheter culture

(A-I), or 2 blood samples (catheter hub and peripheral vein) meet CRBSI criteria for

• Quantitative blood cultures (A-II)

– Colony count of microbes grown from blood obtained through the catheter hub is ≥3-

fold greater than colony count from blood obtained from peripheral vein

• Differential time to positivity (A-II)

– Growth of microbes from blood sample drawn from catheter hub ≥2 h before

microbial growth detected in blood sample obtained from peripheral vein

Mermel LA, et al. Clin Infect Dis 2009;49:1-45

http://www.idsociety.org/

Cerebrospinal Fluid Shunt Cultures • Specific microbiologic methods

o 474 pediatric shunt surgeries

o Shunt tip in TSB; CSF in anaerobic blood culture bottle, BA, chocolate

agar, and ABA

o Tip and bottle incubated 7-10 days; agar 2-4 days

o 39 proven or suspected infections

o S. epidermidis, S. aureus, and P. acnes most frequent

o Culture for 7 days and use of anaerobic culture increased diagnosis

from 28 to 39 infections

o No increase with incubation to 10 days

o Culture CSF from suspected infected shunts aerobically and

anaerobically Arnell et al. J Neurosurg Pediatr 2008;1:366-72

• Positive shunt component cultures in absence of infection o 10.9%, using shunt components in thioglycollate broth

o Coagulase negative staphylococci and P. acnes

o Routine culture of shunt components removed during shunt revision

not indicated Steinbok et al. J Neurosurg 1996;84:617-23

• 6% of positive cultures occurred after 7 days

• True infection vs contaminant not specified

Central Nervous System Shunts

Duration of Culture Incubation

• CSF cultures drawn

through hardware

• BA, chocolate, and

thioglycollate broth; 10

days

• 158 of 1011

specimens had growth

– 127 had + culture with

clinical findings of

infection

Desai et al. J Neurosurg Pediatr 2009;4:184-9.

9 of 158 (6%)

Sonication of Catheter Tips from

Ventriculoperitoneal Shunts

Patient Age, sex Dx Shunt (days)

Sonication VCT

(organism/CFU)

Sonication PC

(organism/CFU)

CSF culture

(organism/

quantity) Meningitis

1 75, M Infection 24 CoNS >100 CoNS 57 CoNS ++ Yes

2 20, M Infection 283 CoNS >20 CoNS >1000 CoNS ++ Yes

3 62, M Infection 552 E. coli >14000 E. coli >105

K. Oxytoca >105

E. coli +++ Yes

4 50, M Infection NA P. aeruginosa 400 NA P. aeruginosa

+++

Yes

5 60, M Infection 8 E. coli >100 NA E. coli + Yes

6 43, F Infection NA CoNS >1000 CoNS >1000 NA Yes

7 28, M Dysfunction 32 S. aureus >1000 NA Sterile No

8a, 8b 70, M Dysfunction 1085, 105 CoNS (+) NA Sterile No

9 83, M Dysfunction 365 CoNS (+) NA Sterile No

10 67, F Dysfunction 264 Sterile NA Sterile No

11 35, M Dysfunction NA CoNS (+) NA NA No

12 70, F Pinealis cyst 107 P. acnes >1000 NA Sterile No

Jost et al. J Clin Neurosci 2014;21:578

Breast Implants

Bacteria and Capsular Contracture

No. of

implants/no.

of patients

Microbiological methods

No. of implants with positive

culture/total no. of implants (%)

Predominant isolated

microorganism Reference

Capsular

contracture group

Non-

capsular-

contracture

group

55/40 Prolonged incubation with

continuous agitation of a

portion of the implant

15/27 (55) 5/28 (18) Staphylococcus

epidermidis

Virden et al. aesthetic

Plast Surg 1992;16:173

150/87 Prolonged incubation with

continuous agitation of a

portion of the implant

62/82 (75) 19/68 (28) S. epidermidis Dobke et al. Ann Plast

Surg 1995:34:562

139/72 Standard culture of a portion

of the implant

7/66 (10) 7/73 (9) Propionibacterium acnes Ahn et al. Plast Reconstr

Surg 1996;98:1225

48/27 Sonication for 20 min of a

portion of the implant or the

tissue capsule

24/48 (50)

(implants), 17/19

(89) (tissue culture)

1/8 (12) S. epidermidis Pajkos et al.

2003;111:1605

45/29 Sonication (positive ≥20

CFU/10 ml)

9/37 (33%) 1/18 (5%) CNS, Propionibacterium

species

Del Pozo et al. J Clin

Microbiol 2009:1333

89/? Sonication (positive, any

growth, including in broth)

36/68 (53%) 4/21 (19%) CNS, P. acnes

Rieger et al. Br J Surg

2013;100:768

Breast tissue expanders – sonicated 52/321 (16%) ≥20 CFU/10 ml

Karau et al. BioMed Res Int 2013;article ID 254940:1

www.implantforum.com/capsular-contracture

Diagnosis of Infections of Miscellaneous

Devices Summary

• CSF shunt cultures should be incubated for at

least 7 days

oUnclear if incubation >7 days is necessary

• Broad range and multiplex PCR assays may

be useful, particularly with prior antimicrobials o Source specific assays should be used

Polymerase Chain Reaction-Electrospray Ionization

Mass Spectrometry – Sonicate Fluid

• 431 subjects

– Knee (n=270), hip (n=161)

prostheses

– 152 PJI, 279 aseptic failure

• Sensitivity 77.6% for PCR-ESI/MS

and 69.7% for culture (p=0.0105)

• Specificity 93.5% for PCR-ESI/MS

and 99.3% for cultures (p=0.0002)

Greenwood-Quaintance et al. J Clin Microbiol 2014;52:642


Polymerase Chain Reaction-Electrospray Ionization

Mass Spectrometry – Synovial Fluid, Knee

• 103 subjects

– 21 PJI, 82 aseptic failure

• Sensitivity 81% for PCR-ESI/MS

and 86% for culture (p=0.56)

• Specificity 95% for PCR-ESI/MS

and 100% for cultures (p=0.045)

Melendez et al. J Clin Microbiol 2014;52:2202 © 2015 Mayo Foundation for Medical Education and Research

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