JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2006, p. 628–631
0095-1137/06/$08.00⫹0 doi:10.1128/JCM.44.2.628–631.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 2
Sonication of Explanted Prosthetic Components in Bags for Diagnosis of
Prosthetic Joint Infection Is Associated with Risk of Contamination
Andrej Trampuz,1 Kerryl E. Piper,1 Arlen D. Hanssen,1,3 Douglas R. Osmon,1
Franklin R. Cockerill,2 James M. Steckelberg,1 and Robin Patel1,2*
Division of Infectious Diseases, Department of Internal Medicine,1 Division of Clinical Microbiology,
Department of Laboratory Medicine and Pathology,2 and Department of Orthopedic Surgery,3
Mayo Clinic College of Medicine, Rochester, Minnesota
Received 14 November 2005/Accepted 16 November 2005
Prosthetic joint implantation has improved the quality of life
for many individuals by restoring satisfactory, pain-free joint
function (8). The most common complication of joint replacement is aseptic failure, followed by prosthetic joint infection
(PJI) (5, 6). Since treatment strategies are fundamentally different, it is important to accurately distinguish these two clinical entities (11). Periprosthetic tissue is the standard specimen
cultured intraoperatively for the diagnosis of PJI but does not
sample the prosthesis surface, to which microorganisms are
attached and grow in biofilms (2).
Tunney et al. used sonication of explanted hip prostheses to
dislodge adherent bacteria (9, 10). Their study was limited by
the lack of a well-formulated definition of PJI, incomplete
clinical and histopathologic data, and missing information on
antimicrobial treatment prior to revision arthroplasty. Nevertheless, they suggested that the incidence of PJI is underestimated by current culture detection methods. We therefore
performed a study to determine whether their sonication
method, aimed at detecting bacteria in biofilms on the prosthesis surface, can improve the diagnosis of PJI.
Between July 1998 and August 2003, patients at the Mayo
Clinic, Rochester, MN, undergoing total knee or hip prosthesis
removal for aseptic failure or presumed infection were prospectively enrolled. Subjects were excluded if prosthetic components were incompletely removed, obvious contamination of
an explanted component occurred in the operating room,
fewer than two periprosthetic tissue specimens were collected
for culture, or fungal infection occurred. The study was approved by the Mayo Clinic Institutional Review Board; all
patients provided informed consent.
Prosthetic joint infection was diagnosed if at least one of the
following was present (1): (i) visible purulence of synovial fluid
or area surrounding the prosthesis (as determined by the surgeon), (ii) acute inflammation on histopathologic examination
of permanent periprosthetic tissue sections (as determined by
the clinical pathologist), or (iii) a sinus tract communicating
with the prosthesis. Aseptic failure was defined as prosthesis
failure not meeting criteria for PJI. Organisms were defined as
causative if the same organism was cultured from a patient
meeting the definition of PJI from at least two periprosthetic
tissue specimens (with the exception of Staphylococcus aureus,
which was considered causative even when isolated from a
single specimen) or from synovial fluid. Prior antimicrobial
therapy was defined as receipt of antimicrobials for at least 2
weeks, completed within 3 days before surgery. The status of
the newly implanted prostheses was assessed 2 years after
implantation.
Preoperatively, synovial fluid was aspirated at the discretion of
the surgeon. Intraoperatively, tissue specimens with the most obvious inflammatory changes were collected for microbiological
and histopathologic studies. Removed prosthetic components
were placed in sterile 12- by 12-inch polyethylene bags (Bitran PE
3 Mil; COM-PAC International, Carbondale, IL). The tibial component and tibial tray (from knee prostheses) or acetabular component and liner (from hip prostheses) were placed in one bag
and the femoral component (from knee and hip prostheses) and
patellar button (from knee prostheses) in another. The bags were
placed into an anaerobic jar and processed within 4 h of prosthesis removal. Prior to usage, polyethylene bags were sterilized with
1 kGy in a self-contained 137Cs gamma-irradiator (Mark I; J. L.
Shepherd, San Fernando, CA).
In the microbiology laboratory, 100 ml of Ringer’s solution
(25% [vol/vol]) containing L-cysteine (0.05% [wt/vol]) was
added to each bag. Sonication of double-bagged prosthetic
components was performed with a continuous sinusoidal wave
ultrasound cleaner (Aquasonic 750T; VWR Scientific, West
Chester, PA) for 5 min at room temperature. Ultrasound parameters were measured using a calibrated hydrophone (type
8103; Brüel and Kjær, Naerum, Denmark). No differences in
frequency (40 ⫾ 5 kHz) or power density (0.22 ⫾ 0.05 W/cm2)
were observed over the study period at various locations within
the ultrasound bath, including inside and outside the bags.
During sonication, the temperature of the tank water increased
⬍0.5°C.
Synovial fluid was inoculated in aliquots of 0.1 ml to aerobic
* Corresponding author. Mailing address: Division of Infectious Diseases, Mayo Clinic College of Medicine, 200 First St. S.W., Rochester,
MN 55905. Phone: (507) 255-6482. Fax: (507) 284-9066. E-mail: patel
[email protected].
628
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Explanted orthopedic implants from 54 patients with aseptic failure and 24 patients with prosthetic knee or
hip infection were sonicated in polyethylene bags. The sensitivities of periprosthetic tissue and sonicate fluid
cultures for the diagnosis of prosthetic joint infection were 54% and 75%, whereas the specificities were 98%
and 87%, respectively. Sonication in bags improved bacterial recovery from the surface of orthopedic implants;
however, it lacked specificity, due to bag leakage.
VOL. 44, 2006
NOTES
629
TABLE 1. Characteristics of study patients and results of microbiological studies
Characteristic a
Median age, yr (range)
Male gender
Revision arthroplasty
Median age of prosthesis, mo (range)b
Result for patients with:
P
Aseptic failure (n ⫽ 54)
PJI (n ⫽ 24)
71.5 (40–88)
29 (54)
7 (13)
71 (1.4–307.3)
71 (50–89)
13 (54)
8 (33)
18 (0.3–170.7)
Presence of sinus tractc
Visible purulence of synovial fluid (n ⫽ 63)c
Visible purulence at implant sitec
Acute inflammation in periprosthetic tissue (n ⫽ 74)c
0
0/46
0
0/52
Laboratory findings
Synovial fluid leukocytes, ⬎1.7 ⫻ 109/liter d
Synovial fluid differential, ⬎65% neutrophilsd
5/30 (17)
1/30 (3)
0/47
5 (9)
1 (2)
7 (13)
15/15 (100)
15/15 (100)
13/17 (77)
18 (75)
13 (54)
18 (75)
0.0005
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
a
Data are numbers (percentages) of patients, unless otherwise indicated. Where the number of subjects is shown, data were not available for all study subjects.
Time between last surgery at the implant site and removal of the implant.
Considered a diagnostic criterion for PJI.
d
Cutoff taken from reference 7.
b
c
blood, chocolate, and anaerobic blood agar and into thioglycolate broth (BD Diagnostic Systems, Sparks, MD). Residual volumes of synovial fluid ⬎0.5 ml were inoculated into a
BACTEC Peds Plus/F bottle and incubated in a BACTEC
9240 instrument (BD Diagnostic Systems) for 5 days, as previously described (3). Tissue specimens were individually homogenized in 3 ml brain heart infusion broth for 1 min (using
a mortar and pestle, if bone was present), and the homogenate
was inoculated in aliquots of 0.5 ml to aerobic blood, chocolate, and anaerobic blood agar and into thioglycolate broth.
For purposes of comparing quantitative yields of sonicate fluid
versus those of tissue cultures, one periprosthetic tissue specimen was subjected to quantitative culture. Aliquots of 0.5 ml
sonicate fluid were plated onto each of five aerobic and five
anaerobic blood agar plates. A positive sonicate fluid culture
was defined as growth of any organism on at least four of five
plates from any bag (9). All aerobic cultures were incubated at
35 to 37°C in 5 to 7% CO2 for 5 days and anaerobic cultures at
35 to 37°C in anaerobic conditions for 7 days and examined
daily. Each unique colony was classified using routine microbiological techniques. Comparisons between variables were
performed by the Wilcoxon rank sum, 2, or Fisher exact tests,
as appropriate, using the statistical software package JMP (version 6.0; SAS Institute, Inc., Cary, NC).
After exclusion of patients with incomplete implant removal
(n ⫽ 3), fewer than two periprosthetic tissue specimens submitted (n ⫽ 2), and Sporothrix schenckii infection (n ⫽ 1), 78
patients with total knee (n ⫽ 68) or hip prostheses (n ⫽ 10)
were studied; 54 had aseptic failure and 24 PJI (Table 1). The
groups were similar in terms of age, gender, type of prosthesis,
and frequency of radiographic loosening. The predominant
reasons for primary arthroplasty had been osteoarthritis (n ⫽
64), bone fracture (n ⫽ 9), inflammatory joint disease (n ⫽
3), and avascular necrosis (n ⫽ 2). The sensitivities of peri-
prosthetic tissue (considering at least two specimens positive)
and sonicate fluid cultures were 54% (95% confidence interval
[CI], 33 to 75%) and 75% (95% CI, 53 to 95%), respectively,
whereas the specificities of the same specimens were 98%
(95% CI, 90 to 100%) and 87% (95% CI, 75 to 95%), respectively. For patients with PJI, all six negative sonicate fluid
cultures were obtained from those patients receiving antimicrobial agents, whereas only 7 of 18 patients (39%) with positive sonicate fluid cultures had taken antimicrobial agents (P ⬍
0.02). Excluding patients who had received antimicrobial
agents, the sensitivities of sonicate fluid, periprosthetic tissue,
and synovial fluid culture were 100% (95% CI, 81 to 100%),
73% (95% CI, 48 to 94%), and 90% (95% CI, 66 to 100%),
respectively.
Causative microorganisms were isolated from synovial fluid
and/or periprosthetic tissue cultures from 18 patients (75%)
with PJI. Among patients with aseptic failure, bacteria were
isolated in cultures from five patients (coagulase-negative
Staphylococcus species [n ⫽ 3], Corynebacterium species [n ⫽
1], and Propionibacterium species [n ⫽ 1]). No synovial fluid
culture was positive for patients with aseptic failure. The microbiology of sonicate fluid cultures was concordant with that
of synovial fluid and periprosthetic tissue cultures where these
were positive.
In patients with aseptic failure, seven sonicate fluid cultures
were positive (Propionibacterium species [n ⫽ 2] and nonfermenting gram-negative bacilli [n ⫽ 5]). None of these results
correlated with results of tissue cultures. The nonfermenting
gram-negative bacilli appeared to relate to contamination with
waterborne microorganisms as a result of bag leakage, as suggested by growth of the same organisms from water in the
ultrasound bath. Visible bag leakage was apparent in several
cases, presumably due to penetration of the bags by sharp bone
and cement fragments and/or the effects of irradiation and/or
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Cultures
Synovial fluid (n ⫽ 64)
Periprosthetic tissue (ⱖ1 specimen positive)
Periprosthetic tissue (ⱖ2 specimens positive)
Sonicate fluid
6 (25)
10/17 (59)
21 (88)
16/22 (73)
0.953
0.970
0.059
0.0006
630
NOTES
J. CLIN. MICROBIOL.
TABLE 2. Culture results of study subjects with PJI
Result for cultures froma:
Patient group
Sample
no.
Synovial
fluid
No. positive/
no. taken
CFU/g tissueb
Organism
CFU/ml
sonicatec
ND
CNS
CNS
ND
ND
Neg
Corynebacterium sp.
CNS
CNS
CNS
Corynebacterium sp.
S. aureus
2/4
1/3
6/6
3/3
1/3
1/3
4
⬎200
11
2
5
38
Corynebacterium sp.
CNS
CNS
CNS
Neg
Neg
250
⬎2,000
⬎2,000
⬎2,000
0
0
7
8
9
10
11
12
13
Neg
S. aureus
Neg
S. aureus
ND
ND
ND
Neg
S. aureus
CNS
S. aureus
Neg
Neg
Neg
0/5
3/3
4/4
1/3
0/4
0/3
0/3
0
⬎200
9
36
0
0
0
Neg
S. aureus
CNS
S. aureus
Neg
NF-GNB
Neg
0
⬎2,000
150
⬎2,000
0
850
0
14
15
16
17
18
CNS
VGS
CNS
S. aureus
CNS
CNS
Neg
CNS
S. aureus
Neg
3/3
0/3
3/4
3/3
0/2
20
0
2
⬎200
0
CNS
VGS
CNS
S. aureus
CNS
⬎2,000
⬎2,000
750
⬎2,000
⬎2,000
19
20
21
22
23
24
CNS
CNS
CNS
S. aureus
Neg
ND
CNS
CNS
CNS
S. aureus
CNS
CNS
3/3
3/3
3/3
3/3
1/3
3/4
ND
17
⬎200
⬎200
2
50
CNS
CNS
CNS
S. aureus
CNS
CNS
⬎2,000
⬎2,000
⬎2,000
110
16
⬎2,000
1
2
3
4
5
6
a
Abbreviations: Neg, negative; CNS, coagulase-negative Staphylococcus sp.; VGS, viridans group Streptococcus sp.; NF-GNB, nonfermenting gram-negative bacillus;
ND, not done.
b
Mean CFU per gram of periprosthetic tissue.
c
Mean CFU per ml of sonicate fluid.
sonication on the bags. In patients with PJI, the number of
CFU of bacteria from positive sonicate fluid cultures (per ml
sonicate fluid) was greater than the number from the respective periprosthetic tissue culture (per gram tissue) (P ⫽ 0.002)
(Table 2). At follow-up 2 years after arthroplasty, none of the
five aseptic-failure patients with positive periprosthetic tissue
cultures and none of the seven aseptic-failure patients with
positive sonicate fluid cultures had developed PJI or aseptic
failure.
Results of this study suggest that sonication of the removed
implants improves microbial recovery in comparison to conventional periprosthetic tissue culture. Sonicate fluid cultures
detected infecting organisms in higher numbers than did
periprosthetic tissue cultures (Table 2). Importantly, all six
false-negative sonicate fluid cultures occurred with patients
taking antimicrobial agents, emphasizing the importance of
discontinuation of antimicrobial therapy prior to specimen collection. For 5 of 18 patients with PJI, sonicate fluid cultures
grew at least 10-fold-greater numbers of bacteria from one bag
than from the other (data not shown), suggesting either that
PJI can be a focal infection involving only some component or
that trapping of air between the double bags hindered transmission of ultrasound to the implant surface.
Unfortunately, bag sonication was suboptimal because of
false-positive cultures, apparently related to bag leakage. The
finding of Propionibacterium species in sonicate fluid cultures
from two aseptic-failure cases (one hip and one knee prosthesis) is concordant with the findings of Tunney et al. (9), although in their study this organism was found more frequently.
As has been suggested by others (4), the role of Propionibacterium species in the pathogenesis of aseptic implant failure
remains to be determined.
In conclusion, methods aimed at detecting biofilm bacteria
on prosthesis surfaces improve bacterial recovery in comparison to conventional tissue cultures. However, sonication of
prosthetic components in bags lacks specificity due to bag
leakage and subsequent risk of microbial contamination. We
are currently performing a study processing removed orthopedic implants in solid containers.
We thank John G. Hughes, Don Baltes, and Dean S. Strain for
their technical assistance, Jayawant Mandrekar for statistical support, and K. Krishnan Unni for reviewing the histopathology of the
periprosthetic tissue sections.
This study received financial support from the Mayo Foundation,
Rochester, MN; the Orthopaedic Research and Education Foundation; the Swiss National Science Foundation (grant number
BS81-64248); and the Roche Research Foundation.
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Subjects who did not receive
antimicrobial therapy
before surgery
Sonicate fluid
Organism
Organism
Subjects who received
antimicrobial therapy
before surgery
Periprosthetic tissue
VOL. 44, 2006
NOTES
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