CLINICAL ORTHOPAEDICS AND RELATED RESEARCH
Number 414, pp. 69–88
Molecular and Antibiofilm Approaches to
Prosthetic Joint Infection
Andrej Trampuz, MD*; Douglas R. Osmon, MD, MPH*;
Arlen D. Hanssen, MD*,†; James M. Steckelberg, MD*; and
Robin Patel, MD*,**
The majority of patients with prosthetic joint re-
placement (arthroplasty) experience dramatic
relief of pain and restoration of satisfactory joint
function. In the United States, more than .5 mil-
lion people have a primary arthroplasty each
year. Less than 10% of prosthesis recipients
have complications develop during their life-
time, commonly as a result of aseptic biome-
chanical failure, followed by prosthetic joint in-
fection. The pathogenesis of prosthetic joint
infection is related to bacteria in biofilms, in
which they are protected from antimicrobial
killing and host responses rendering these infec-
tions difficult to eradicate. Current microbiology
laboratory methods for diagnosis of prosthetic
joint infection depend on isolation of a pathogen
by culture. However, these methods have neither
ideal sensitivity nor ideal specificity. Therefore,
culture-independent molecular methods have
been used to improve the diagnosis of prosthetic
From the *Division of Infectious Diseases, Department
of Internal Medicine; the **Division of Clinical Micro-
biology, Department of Laboratory Medicine and Pathol-
ogy; and the †Department of Orthopedic Surgery, Mayo
Clinic, Rochester, MN.
Supported by the Mayo Foundation, the Minnesota Chap-
ter of the Arthritis Foundation and the Swiss National
Science Foundation grant Number BS81-64248.
Reprint requests to Robin Patel, MD, Division of Infec-
tious Diseases, Mayo Clinic, 200 First St. S.W.,
Rochester, MN 55905. Phone: 507-255-6482; Fax: 507-
255-7767; E-mail: patel.robin@mayo.edu.
DOI: 10.1097/01.blo.0000087324.60612.93
69
© 2003 Lippincott Williams & Wilkins, Inc.
joint infection. In the research setting, detection
of 16S ribosomal deoxyribonucleic acid by poly-
merase chain reaction has been used in the mo-
lecular diagnosis of prosthetic joint infection.
Various antibiofilm strategies directed at dis-
ruption of adherent bacteria are the focus of in-
tense research to improve the detection of
biofilm organisms and their eradication. In this
article, molecular and antibiofilm approaches to
prosthetic joint infection are reviewed.
Prosthetic joint implantation has greatly im-
proved the quality of life for many individu-
als.48 Enthusiasm about the results of arthro-
plasty in elderly patients has led to gradual
expansion of indications for arthroplasty, such
that selected relatively young, active patients
now receive total joint replacements. In the
United States, more than 500,000 primary
arthroplasties are done annually and more than
1.3 million people live with an artificial
joint.36 Total joint replacement is one of the
most successful operations currently avail-
able; less than 10% of patients have complica-
tions develop during their lifetime for which
they require revision surgery.85 Aseptic bio-
mechanical loosening is the most common
cause of prosthetic joint failure, followed by
prosthetic joint infection. Although prosthetic
joint infection compromises the result in few
implant recipients, the economic impact, the

70 Trampuz et al
morbidity, and the emotional trauma of pros-
thetic joint infection is immense and devastat-
ing to the patient and society. The diagnosis
and the antimicrobial and surgical treatment of
prosthetic joint infection is complex, and the
recovery is arduous and prolonged.77
It would be ideal to diagnose prosthetic joint
infection reliably preoperatively to enable ap-
propriate surgical treatment and initial choice
of systemic antimicrobial agents and locally de-
livered antimicrobial agents in PMMA beads or
spacers. The suspicion of prosthetic joint infec-
tion when none is present may result in a delay
of several days before reimplantation of the
prosthesis, extending the hospital stay, and re-
quiring the use of an extra operating suite, ex-
tra anesthesia risk and expense, and a prolonged
period of patient immobilization and rehabilita-
tion. In addition, the consequences of failure to
recognize prosthetic joint infection may be sub-
stantial. In particular, implantation of the pros-
thesis into an infected surgical site, without
appropriate debridement and antimicrobial
treatment, may result in persistent infection,
and ultimately in implant failure. Furthermore,
a delay in the diagnosis and early treatment of
prosthetic joint infection can permit the infec-
tion to become established at the bone-implant
interface, thereby eliminating the possibility of
using a debridement procedure with antimicro-
bial treatment and prosthesis retention.13,89,108
In this study, we reviewed different mech-
anisms of orthopaedic implant failure with an
emphasis on prosthetic joint infection. We re-
viewed the importance of biofilm formation in
the pathogenesis of prosthetic joint infection
and discuss novel approaches to the diagnosis
of prosthetic joint infection directed at detect-
ing microorganisms in the biofilm environ-
ment. Finally, we reviewed molecular tech-
niques, such as detection of bacterial 16S
ribosomal DNA (rDNA), and their application
to diagnosis of prosthetic joint infection.
Mechanisms of Implant Failure
It is well-recognized that synthetic materials
used to construct implants will not last forever.
The functional implant life may be influenced
Clinical Orthopaedics
and Related Research
by its material and design (ceramic, stainless
steel, Ti, Ti alloy), technical aspects of device
implantation (cementing, creation of a porous
implant surface or HA cover for stimulating
bone apposition), antiadhesive implant coating
(antiseptic or bioactive surface properties),
quality of host bone, and extent of patient activ-
ity.5 Long-term implant stability depends
mainly on good initial fixation (osseointegra-
tion) and factors maintaining sufficient local
bone density around the prosthesis, which is the
net result of bone resorption (osteolysis) and
new bone formation. Table 1 shows different
mechanisms for implant failure. Aseptic biome-
chanical failure often is difficult to distinguish
from occult chronic prosthetic joint infection.78
Osteolysis induced by particulate wear debris
from implant materials is recognized as a major
cause of long-term prosthesis failure.41 Wear
particles become deposited in the space between
the implant and bone (or cement, if present), and
are phagocytosed by macrophages, forming a
granulomatous tissue layer or membrane. The
macrophages in turn release inflammatory me-
diators, which stimulate osteoclastic bone re-
sorption. Biopsy specimens from osteolytic le-
sions surrounding tissue often show only
acellular, necrotic debris, but biopsy specimens
from surrounding tissue may contain granular-
appearing macrophages associated with small
particles, foreign-body-type giant cells, and os-
teoclasts, the arthroplasty effect. Migration of
macrophages into lymphatic and blood vessels
ultimately may result in distant dissemination of
orthopaedic wear debris.95 Other aseptic mech-
anisms of implant failure include inappropriate
mechanical load, fatigue failure at bone-implant
interfaces, implant micromotions, and perhaps
synovial fluid hydrodynamic pressure.6
Infections occur in approximately 1% to 5%
of all primary arthroplasties.33,73,78 The inci-
dence of prosthetic joint infection is higher after
a revision arthroplasty, possibly because of the
long operating time, scar formation, or recrudes-
cence of unrecognized infection present at the
initial surgery. Other risk factors for prosthetic
joint infection include wound healing problems,
rheumatoid arthritis, diabetes mellitus, malig-
Number 414
September, 2003 Approaches to Prosthetic Joint Infection 71

TABLE 1. Septic and Aseptic Causes of Implant Failure

Mechanism Description

Aseptic biomechanical failure

A. Wear debris
B. Eccentric mechanical loads
C. Implant motion
D. Hydrodynamic pressure
Infection
Loss of material with generation of particles resulting from relative motion
between opposed surfaces (adhesive, abrasive or fatigue wear).
Inflammatory reaction to particles of orthopaedic wear debris causes
bone resorption
Misaligned arthroplasty alters the magnitude and direction of load
transmission through the prosthesis and causes implant loosening and
mechanical damage to the implant material or the interface between
implant and bone
Micromotion of loose prosthesis leads to progressive bone resorption.
Synovial fluid is compressed through defects of the prosthesis and host
bone. This effect is likely amplified in the presence of excessive wear
debris
Microorganisms colonize the prosthesis at the time of implantation or
postoperatively through hematogenous seeding or spread from a
contiguous infectious focus. Bacteria adhere to and grow on an implant
surface forming a biofilm protecting them from environmental influences
and immune responses

nancy, and high National Nosocomial Infec-
tions Surveillance (NNIS) System surgical pa-
tient risk index score and surgical site infection
not involving the prosthesis.9,47 Microorgan-
isms can colonize the prosthesis at the time of
implantation (either through direct inoculation
or as a result of airborne contamination of the
wound) or colonize the implant postoperatively
through hematogenous seeding or spread from a
contiguous infectious focus. Once microorgan-
isms attach to and grow on the surface of an or-
thopaedic implant, they begin to produce a
highly hydrated matrix of extracellular poly-
meric substances. The extracellular polymeric
substances together with embedded microor-
ganisms are collectively known as a biofilm.22
In large series, staphylococci are the most im-
portant microorganisms involved in prosthetic
joint infection, accounting for approximately
50% of infections overall, followed by strepto-
cocci, enterococci, Enterobacteriaceae, Pseudo-
monas aeruginosa, and anaerobes.30,36,78,85
Importance of Biofilm Formation
Prosthetic joint infection is a prototype of an in-
fection with a low organism burden, the patho-
genesis of which is related to bacteria in
biofilms. Existence within a biofilm represents a
basic survival mechanism of bacteria, by which
they are protected from external environmental
toxins (normal levels of antimicrobial agents)
and host immune responses (such as opsoniza-
tion, complement-mediated lysis, and phagocy-
tosis).20,109 As a consequence, prosthetic joint
infection is difficult to treat successfully and in
many patients removal of the implant ultimately
is necessary for microbiologic cure.
The basic structure unit of the biofilm is the
microcolony. Microcolonies are enclosed in a
highly hydrated polymeric matrix surrounded
by interstitial voids (water channels) in which
nutritients can circulate between bacterial cells.
Within biofilms bacterial cells develop into
organized and complex communities with
structural and functional heterogeneity that in
many ways resemble a multicellular organism
in which water channels with convective flow
serve as a rudimentary circulatory system.19
Release of cell-to-cell signaling molecules (a
rudimentary endocrine system) induces bacteria
in a population to respond in concert by chang-
ing patterns of gene expression, a phenomenon
called quorum sensing.22 At sufficient popula-
tion density, these signals reach concentrations
 Clinical Orthopaedics
72 Trampuz et al and Related Research

required for activation of genes involved in
biofilm differentiation. Furthermore, it has been
suggested that programmed cell death of dam-
aged cells may play an important role in bacte-
rial biofilms, similar to multicellular organ-
isms.49 In addition, proximity of cells within the
microcolony provides an ideal environment for
exchange of genes located on extrachromoso-
mal DNA (plasmids). Based on these charac-
teristics, the bacterial biofilm recently has been
defined as “ . . . a microbially derived sessile
community characterized by cells that are at-
tached to a substratum or interface or to each
other, are embedded in a matrix of extracellular
polymeric substances that they have produced,
and exhibit an altered phenotype with respect to
growth rate and gene transcription.”27
Figure 1 shows the evolution of biofilm
formation on a prosthesis surface. Attached
bacterial cells synthesize extracellular poly-
meric substances and aggregate into micro-
colonies.20 These multicellular communities
develop by bacterial surface motility, binary
division, and recruitment from the environ-
ment.88 During the formation of a biofilm, at-
tached bacterial cells release antigens thereby
stimulating the production of antibodies and
the attraction of phagocytes. In response,

A B

C D

Fig 1A–D. Evolution of biofilm formation on a prosthesis surface is shown. (A) Bacterial contamination
shows that planktonic bacteria are introduced into the prosthesis neighborhood. (B) Bacterial attach-
ment and microcolony formation shows that bacteria naturally adhere to the prosthesis surface and ag-
gregate into microcolonies. (C). Formation of ECM shows that attached bacteria synthesize extracellular
polymeric substances that protect them from antimicrobial agents and the host immune system,
whereas most planktonic bacteria are cleared by antibodies, phagocytes and/or antibiotics. Phagocytes
release enzymes, which can damage the surrounding host tissue. (D) Bacterial detachment shows that
mature sessile colonies detach from the periphery and disperse as planktonic bacteria.
Number 414
September, 2003
phagocytes release enzymes, which generally
are not able to destroy the biofilm completely,
but instead damage the surrounding host tis-
sue. With time, microcolonies differentiate
into mature thick biofilm structures. As bacte-
rial colonies mature, surface-associated ses-
sile bacteria on the periphery detach and dis-
perse as suspended planktonic bacteria that
can multiply rapidly.
Sessile (attached) bacteria in biofilms ex-
hibit dramatically increased resistance to
killing by antimicrobial agents as compared
with suspended (planktonic) bacteria.16 Sev-
eral reasons for increased antimicrobial resis-
tance have been postulated.86 Bacteria in bio-
films are enclosed within a negatively charged
exopolymer matrix that can physically restrict
the diffusion of large molecules, such as some
antimicrobial agents, enzymes, and comple-
ment components. More important, nutrient
depletion or waste product accumulation
within the biofilm causes some bacteria to en-
ter a nongrowing (stationary) state, in which
they are less susceptible to growth-dependent
antimicrobial killing. For example, glucose
and oxygen do not penetrate all the way
through the biofilm.1 Bacteria dispersed from
biofilms into medium quickly regain their an-
timicrobial susceptibility. A subpopulation of
bacteria may differentiate into a pheno-
typically resistant state or express biofilm-
specific resistance genes. Recently, a regula-
tory protein (PvrR) in Pseudomonas aerugi-
nosa that modulates the phenotypic switch
from antimicrobial-resistant to antimicrobial-
susceptible forms and also regulates biofilm
formation has been identified.28 This may ex-
plain why antimicrobial concentrations that
are sufficient to kill free-floating planktonic
bacterial cells such as those in synovial fluid,
may be inadequate to clear sessile (attached)
bacterial cells embedded in an established
biofilm such as in chronic prosthetic joint in-
fection. Such persisters seem to survive expo-
sure to antimicrobial agents and ultimately to
reform the biofilm when antimicrobial therapy
is discontinued; planktonic bacteria then will
be shed again. This construct explains the per-
Approaches to Prosthetic Joint Infection 73
sistent and recurrent nature of biofilm infec-
tions.50 Biofilm bacterial cells may be dis-
persed either by shedding of daughter cells,
detachment as a result of nutritient shortage or
quorum sensing, or shearing of biofilm aggre-
gates because of flow effects. The mode of
dispersal may affect the phenotypic character-
istics of the shed bacteria. Eroded or sloughed
aggregates from the biofilm are likely to retain
certain biofilm characteristics (such as antimi-
crobial resistance), whereas cells that have
been shed as a result of growth may revert to
the planktonic phenotype.26
Diagnosis of Prosthetic Joint Infections
The clinical presentation of prosthetic joint in-
fection is variable and sometimes difficult to
distinguish from that of aseptic implant fail-
ure. Early postoperative and hematogenous
prosthetic joint infection typically is caused by
relatively virulent organisms, such as Staphy-
lococcus aureus and often is characterized by
acute onset of symptoms and signs of infec-
tion. Conversely, late postoperative prosthetic
joint infection generally is chronic and insidi-
ous grade in nature and is more commonly
associated with relatively less-virulent organ-
isms, such as coagulase-negative staphylo-
cocci.78 As compared with early postoperative
prosthetic joint infection which in most cases
occurs in the first 3 months after surgery, late
postoperative prosthetic joint infection gener-
ally occurs thereafter and is characterized by
more subtle signs of inflammation, chronic
persistent postoperative pain, and/or early
loosening of the implant; differentiating late
postoperative prosthetic joint infection from
aseptic implant failure can be challenging.108
To date, no single routinely used clinical or
laboratory test has been shown to achieve
ideal sensitivity and specificity for the diag-
nosis of prosthetic joint infection. Table 2
summarizes available preoperative and intra-
operative tests for the diagnosis of prosthetic
joint infection. In most cases, the diagnosis of
prosthetic joint infection depends on a combi-
nation of clinical features, radiographic find-
ings, and laboratory results.92 Although the
 Clinical Orthopaedics
74 Trampuz et al and Related Research

TABLE 2. Routine Preoperative and Intraoperative Tests for Diagnosis of

Prosthetic Joint Infection
Category Description of the Diagnostic Tests

Preoperative
Clinical history and examination
Hematological tests
Synovial fluid aspiration
Radiographic imaging
Radionuclide imaging
Positron emission tomography
Intraoperative
Periprosthetic tissue
Explanted prosthesis
Persistent joint pain; fever, chills or rigors without known etiology;
erythema, warmth or effusion of the joint; sinus tract
Leukocyte count and differential; erythrocyte sedimentation rate;
C-reactive protein level
Leukocyte count and differential; Gram stain and culture
Plain radiography; computed tomography; prosthetic devices may
produce imaging artifacts
Scintigraphy by technetium (Tc99m), gallium citrate (Ga67) or indium
(In111) scan; accuracy improved by combination with labeled leukocyte
or monoclonal antigranulocyte antibody scan
Fluorine-18 fluorodeoxyglucose (F-18 FDG) positron emission tomography
Histopathology; Gram stain and culture
Culture

(Reprinted with permission from Trampuz A, Steckelberg JM, Osmon DR, et al: Advances in the laboratory diagnosis of prosthetic
joint infection. Rev Med Microbiol 14:1–14, 2003.)

presence of a sinus tract or frank purulence in
the joint aspirate or in tissue obtained at the
time of surgery is specific for defining pros-
thetic joint infection, these findings are not
sensitive.83 Findings supportive of infection
include joint pain; fever, chills or rigors with-
out a known cause; erythema, warmth or effu-
sion of the joint; and increased sedimentation
rate or C-reactive protein level. Routine hema-
tologic screening tests have not been particu-
larly helpful in diagnosing prosthetic joint in-
fection. Radiographic imaging is hampered by
artifact produced by prosthetic devices; scintig-
raphy has a low specificity.53
No standardized criteria exist for the diag-
nosis of prosthetic joint infection and different
investigators have used different defini-
tions.2,3,110 In one study, the diagnosis of pros-
thetic joint infection was made on the basis of
at least one of three criteria: (1) presence of an
open wound or a sinus tract communicating
with the joint; (2) systemic signs or symptoms
of infection with pain in the joint and puru-
lence surrounding the prosthesis at surgery; or
(3) three or more positive results of the fol-
lowing tests: ESR greater than 30 mm per
hour, C-reactive protein greater than 10 mg/L,
frozen section tissue histopathologic examina-
tion showing greater than 5 neutrophils per
high-power field, or at least one joint aspirate
or more than 1/3 of periprosthetic tissue speci-
mens culture positive.82 At our institution, the
following criteria are used for the definitive di-
agnosis of prosthetic joint infection: two or
more cultures of joint aspirates or intraopera-
tive tissue specimens positive for the same
microorganism; purulence surrounding the
prosthesis at the time of surgery; acute inflam-
mation consistent with infection on histo-
pathologic examination; or presence of a sinus
tract communicating with the prosthesis.9
Synovial fluid cell count and differential
frequently are used in evaluating joint disor-
ders but criteria for interpreting the associated
values have not yet been well defined in the
context of prosthetic joint infection. Gram
stain of synovial fluid generally is not helpful
because of low sensitivity (25%).12 A negative
culture result does not exclude prosthesis in-
fection; the sensitivity of synovial fluid culture
ranges from 50% to 93%.4,29,46,47,82,91 It has
been suggested that in patients with an ortho-
Number 414
September, 2003
paedic implant and an underlying noninflam-
matory disorder such as OA, a synovial fluid
leukocyte count less than 2000 per mL and a
differential of less than 50% neutrophils have
a 98% predictive value for the absence of in-
fection.43 In contrast, patients with an ortho-
paedic implant and an underlying inflamma-
tory disorder such as rheumatoid or crystal-
induced arthritis, may have leukocyte counts
between 2000 and 50,000 per mL and a dif-
ferential of greater than 50% neutrophils in the
absence of infection.43
Numerous studies have reported that the
presence of acute inflammation in a peripros-
thetic tissue specimen at the time of surgery
is specific for infection.23,52,67,82 However,
histopathologic studies do not identify the in-
fecting organism, an essential element in post-
operative treatment and selection of appropri-
ate antimicrobial therapy. Because of the focal
nature of prosthetic joint infection, sampling
errors can lead to false-negative results. In ad-
dition, acute inflammation has been variably
defined as from greater than or equal to 1 to
greater than or equal to 10 neutrophils per
high-power field (magnification,
400).
Histopathology studies are, by their nature,
subjective. Interobserver variability among
histopathology studies may be substantial,
even in specialized institutions.84 In addition,
histopathologic studies on tissue from patients
with underlying inflammatory joint disorders
(such as RA) may be indistinguishable from
histopathologic studies on tissues from pa-
tients with prosthetic joint infection.3 Gram
stain of periprosthetic tissue shows an ex-
tremely low sensitivity (range, 0%–23%) in
various studies.2,3,17,46,82 In contrast, intraop-
erative tissue cultures provide the most accu-
rate specimens, to date, for microbiologic cul-
ture and frequently have been used as the
reference standard for diagnosing prosthetic
joint infection.67,82 Cultures may be falsely
negative because of prior antimicrobial expo-
sure, a low number of organisms, inappropri-
ate culture media, or fastidious or atypical
organisms. In addition, bone cement impreg-
nated with antimicrobial agents may cause
Approaches to Prosthetic Joint Infection 75
false-negative culture results, even years later
when the cement is fractured during revision
operations.70 Conversely, cultures may be
falsely positive because of contamination
introduced in the operating room, during spec-
imen transport, or in the microbiology labora-
tory. For these reasons, multiple intraopera-
tive tissue specimens should be sent to the
microbiology laboratory for culture. In a
mathematic modeling study, culture of five or
six specimens was necessary to produce a di-
agnostic test that was sensitive and specific.3
If the implant is removed, the entire prosthesis
can be cultured in enrichment broth media.
The advantage of this approach is that the site
of infection is sampled directly. However,
care interpreting culture results is warranted,
because of the risk of contamination during
processing. Because prosthetic joint infection
is a biofilm-mediated infection, techniques
that sample bacteria in biofilms may improve
the diagnosis of prosthetic joint infection.
Methods to Dislodge Microorganisms
from Prosthesis Surfaces
Biofilm properties depend on attachment and
aggregation of bacterial cells into micro-
colonies. Antibiofilm diagnostic strategies are
directed at disruption of adherent bacteria and
their multicellular structure. By dispersing
adherent bacteria from the surface, the recov-
ery efficiency and therefore the sensitivity of
diagnostic assays may be increased. Free-
floating planktonic organisms as opposed to
their sessile surface-associated counterparts
are likely preferentially cultured using con-
ventional methods (synovial fluid and peripros-
thetic tissue culture). Various potential strate-
gies to dislodge sessile bacteria in biofilms
from foreign body surfaces, which may be of
diagnostic relevance, have been tested includ-
ing mechanical (sonication, vortexing, shock
wave treatment), biochemical (enzyme treat-
ment), and electrical approaches10,11,27,64,66
(Table 3). To date, mostly ultrasound has been
studied for improvement of recovery of adher-
ent bacteria from explanted orthopaedic de-
vices,25,61,94 and from vascular prosthetic
 Clinical Orthopaedics
76 Trampuz et al and Related Research

TABLE 3. Potential Antibiofilm Strategies to Disrupt Adherent Bacteria and Their

Multicellular Structure in Biofilms
Method Description

Ultrasound Formation of microscopic bubbles (cavitation) followed by their collapse

(implosion) releasing acoustic energy with scrubbing action at object
surface; enhancement of bacterial killing by antimicrobial agents
Shock wave Release of acoustic energy at boundary interface in the form of water jets and
high temperature by high pressure sonic pulse
Vortexing Mechanical disruption of biofilm structure by physical agitation, especially in
the presence of beads
Electric current Enhancement of bacterial killing by an antimicrobial agent
Enzymes Dissolution of extracellular matrix polymers disrupting the multicellular biofilm
architecture
Other chemical substances Blockage of biofilm formation by various mechanisms (e.g., stimulation of
bacterial motility, disruption of quorum-sensing signaling molecules)
Genes Regulation of gene expression responsible for biofilm formation

grafts, ureteric stents, and vascular and peri-
toneal catheters.32,42,66,79
Antibiofilm strategies have potential thera-
peutic application to enhance antimicrobial
killing of biofilm-associated bacteria; the syn-
ergistic effect of antimicrobial drugs and
ultrasound or electric current are known as
the bioacoustic or bioelectrical effect, respec-
tively. Because electrical, ultrasonic, and
shock wave approaches have been used to pro-
mote healing of nonunion of previous bone
fractures, such techniques theoretically are at-
tractive adjunctive approaches to the therapy
of prosthetic joint infection.24,55,97,107
Sonication
Ultrasound, transmitted at frequencies gener-
ally beyond the range of human hearing ( 20
kHz), has been used in many applications in
medicine and research. Although in diagnostic
and therapeutic applications high frequencies
(MHz range) are used, low frequencies (kHz
range) are used for bacterial detachment such as
the cleaning of medical instruments.14 The ul-
trasound waves radiate through a solution and
produce high and low pressure areas. During
the low-pressure stage, millions of microscopic
vapor bubbles are formed (a process referred to
as cavitation), which collapse during the high-
pressure stage releasing an enormous amount
of energy on the surface of the objects (a
process referred to as implosion). This agitation
causes a vacuum-scrubbing action by releasing
acoustic energy at the surface of objects.
Mild sonication has been used to dislodge
adherent bacteria from surfaces of orthopaedic
devices for diagnostic purposes. Dobbins et
al25 used ultrasound for 10 minutes (and vor-
texing for 30 seconds) on internal fixation de-
vices removed for reasons other than infec-
tion. Twenty of 26 (77%) sonicate cultures
were positive; in most cases (15 sonicates),
coagulase-negative staphylococci were cul-
tured. This was in contrast to swabs of
adjacent tissues, where coagulase-negative
staphylococci were isolated in only three of
26 cultures (12%). Unfortunately, no negative
controls were examined and the positive cul-
tures could represent contamination.
Tunney et al94 did mild sonication of ex-
planted hip prostheses followed by sonicate
culture, and by immunofluorescence micros-
copy and broad-range PCR amplification of
the explant sonicate.93 In the first study,94 mild
sonication was done on 120 removed hip pros-
theses. Sonicates were plated onto five aerobic
and five strict anaerobic blood agar plates.
Bacteria were cultured from 26 of 120 soni-
Number 414
September, 2003
cate cultures (22%), although the criteria used
for definition of a positive culture result were
not clear and varied between different publi-
cations by the same investigators describing
the same patients.93,94 Propionibacterium ac-
nes was isolated either alone or in association
with Staphylococcus species from 16 of 26 pa-
tients (62%) with positive sonicate cultures.
Review of the records for 18 of the 26 patients
with culture-positive implants revealed that
infection was suspected as the cause of loos-
ening in only six of 18 patients (33%) and in
all cases, histopathologic examination of the
periprosthetic tissue showed inflammatory
cells. Standard cultures of preoperatively aspi-
rated synovial fluid or intraoperatively sam-
pled periprosthetic tissue were positive in only
two patients. The limitations of this study
include the failure to document the diagnosis
of prosthetic joint infection using well-
formulated gold standard criteria and the in-
complete clinical and histopathologic data.
Nguyen et al61 treated 21 femoral compo-
nents of hip and knee prostheses with ultra-
sound removed from patients without clinical
evidence of infection. Swab cultures of the
prostheses grew no organisms. The explant
sonicate was passed through a 0.45-m pore
filter, and the filter residue was cultured aero-
bically. In one of 21 explant sonicates (5%) a
coagulase-negative Staphylococcus species
was cultured but the same organism also was
found in one of 21 sonicate cultures from ster-
ilized prostheses used in this study as a nega-
tive controls. Therefore, sonicate cultures
from aseptically failed prostheses did not
yield more organisms than those from nega-
tive controls.
Other investigators have shown that low-
frequency continuous and pulsed ultrasound
(28–67 kHz) for 6 to 24 hours enhances killing
by antimicrobial agents of biofilm microor-
ganisms (Pseudomonas aeruginosa and Es-
cherichia coli) in vitro and in animal mod-
els.40,69,71,74,75 The mechanism by which
ultrasound enhances antimicrobial action (the
bioacoustic effect) is unclear because ultra-
sound with a power density of 100 to 300
Approaches to Prosthetic Joint Infection 77
W/cm2 used in these studies has no inhibitory
or bactericidal activity against bacteria when
delivered alone. There are hypotheses to ex-
plain the mechanism of the bioacoustic effect,
including increased transport through the
membrane, a mechanical effect on the biofilm-
associated bacteria, or promotion of specific
gene expression.72 Whether the bioacoustic
effect could be applied to the therapy of pros-
thetic joint infection has not been determined.
Other Potential Antibiofilm
Defense Approaches
Similar to ultrasound, an acoustic shock
wave is a transient pressure disturbance that
propagates rapidly in three-dimensional space.
In contrast to ultrasound, a shock wave is a
sonic pulse with a high peak pressure (50–100
MPa) and a broad frequency spectrum (16
Hz–20 MHz). Shock waves produce high
stresses that act on boundary interfaces be-
tween two media (such as tissue and prosthe-
ses) generating cavitation bubbles. During col-
lapse of the bubbles a high amount of acoustic
energy is released in the form of water jets and
high temperature.64 Originally applied clini-
cally as lithotripsy to pulverize calcific de-
posits within the body (renal stones), shock
waves have been used increasingly in muscu-
loskeletal disorders (orthotripsy), especially in
treating enthesiopathies and delayed fracture
healing.63,76,90 The primary advantage of shock
wave therapy is its noninvasive nature. To
date, shock wave therapy has not been used for
diagnosis, prevention, or treatment of pros-
thetic joint infection.
Mechanical bacterial removal with vortex-
ing has been used in the diagnosis of infections
of vascular catheters and vascular grafts,
mostly as an adjunct to ultrasound treat-
ment.10,66,79 Vortex agitation with glass beads
is an efficient method to mechanically disrupt
and remove biofilm-associated bacteria.8
However, in diagnosis of prosthetic joint in-
fection, vortex agitation with beads may rep-
resent a risk for prosthesis contamination.
Low electric current combined with an an-
timicrobial agent has been shown to enhance

78 Trampuz et al
the killing of biofilm-associated bacteria as
compared with the antimicrobial agent alone
(the bioelectric effect).11,44,57,98 In particular,
the bioelectric effect reduces the very high con-
centrations of aminoglycosides or quinolones
needed to kill biofilm bacteria to levels close to
those needed to kill planktonic bacteria of the
same species. The mechanism of the bioelectric
effect is not defined completely. Possible mech-
anisms of the bioelectric effect include elec-
trophoresis, iontophoresis, electrolysis (result-
ing in increased delivery of oxygen to the
biofilm), and electrophoresis, which might over-
come biofilm biomass and cell wall barriers,
and metabolic activity and growth rate of the
bacteria themselves.38,87 To date, electric cur-
rent has not been used for dislodgment of bac-
teria associated with prosthetic joint infection.
A mixture of enzymes has been shown to
be bactericidal and effective in eradicating
biofilms of several different organisms by dis-
solving the matrix polymers of the biofilm. A
combination of streptokinase and streptodor-
nase was used by Nemoto et al.60 Other inves-
tigators tested the activity of enzymes against
bacterial cells in biofilms by fluorescence mi-
croscopy and an indirect conductance test in
which evolution of CO2 was measured.39 Ox-
idoreductases were bactericidal against bio-
film bacteria and a complex mixture of poly-
saccharide-hydrolyzing enzymes was able to
remove bacterial biofilm. Hatch and Schiller34
showed that alginate lyase allowed more ef-
fective diffusion of aminoglycosides through
alginate, the biofilm extracellular polymeric
substance of Pseudomonas aeruginosa.
Other chemical substances showed blockage
of biofilm matrix synthesis. For example, Ya-
suda et al105,106 showed that clarithromycin en-
hanced the therapeutic efficacy of other antimi-
crobial agents against infections caused by
clarithromycin-resistant strains of Pseudo-
monas aeruginosa and Staphylococcus epider-
midis. Lactoferrin, a ubiquitous constituent of
human secretions, was shown to block biofilm
development by Pseudomonas aeruginosa. By
chelating iron, lactoferrin stimulates twitching,
a specialized form of surface motility, causing
Clinical Orthopaedics
and Related Research
the bacteria to wander across the surface in-
stead of forming cell clusters and biofilms.80
Because the formation and detachment of
biofilms has been found to be controlled by
signaling molecules, a specific antibiofilm de-
fense mechanism might act on disruption of
quorum-sensing signaling molecules, involved
in biofilm architecture.22,68 At sufficient popu-
lation density, these signals reach concentra-
tions required for activation of genes involved
in biofilm differentiation. During biofilm for-
mation, different genes may be activated or re-
pressed. Upregulation and downregulation of
genes in bacteria provides an additional possi-
ble antibiofilm strategy through inhibition of
gene transcription.7,21,28,103 These studies re-
veal potential antibiofilm defense mecha-
nisms acting at a critical juncture in biofilm
development but to date, none of these meth-
ods have been used in the setting of prosthetic
joint infection.
Molecular Methods for Diagnosis of
Prosthetic Joint Infection
Advantages of Molecular Microbiology Testing
Molecular techniques are being used increas-
ingly in clinical microbiology laboratories
to establish the cause of infectious diseases.
Molecular diagnostic methods make it possi-
ble to detect bacterial nucleic acid in samples
from infected patients even when conven-
tional cultures are negative (because of un-
usual microbial growth requirements or fail-
ure to grow after antimicrobial exposure
or due to unfavorable environmental condi-
tions).102 Polymerase chain reaction (PCR) is
the most widely used nucleic acid amplifica-
tion technology, replicating a selected seg-
ment of DNA using a thermostable DNA poly-
merase enzyme.59 PCR-based assays can be
extremely sensitive. They are able to detect
one copy of bacterial DNA under optimal con-
ditions and can have a rapid turnaround time.
Timely microbiology results are critical to ef-
fective patient treatment and to reduction of
the overall cost of patient care. Such tech-
niques may be useful in the diagnosis of pros-
thetic joint infection.
Number 414
September, 2003
Specific Versus Broad-Range Polymerase
Chain Reaction
Polymerase chain reaction assays can be de-
signed to detect the specific (unique) DNA se-
quence of one microorganism using a specific
primer pair complementary to a known DNA
sequence unique to the target species. When
the infecting agent is not known, and the goal
is to identify the presence of any bacterial
pathogen, as in the case of prosthetic joint in-
fection, broad-range (universal) PCR amplifi-
cation can be used. In this case, universal
primers are used that anneal to regions of DNA
that are shared by most bacterial species. 16S
rDNA is the target that has been used most
commonly for broad-range bacterial PCR.45
16S rDNA of different bacteria contains alter-
nating regions of nucleotide conservation and
heterogeneity (Fig 2). The conserved region
makes it possible to amplify the target from all
or almost all bacterial species.56 The heteroge-
neous areas provide sequence polymorphisms
that can be used in subsequent sequence analy-
sis to classify the source bacterium. 16S rDNA
is present in multiple copies (generally be-
tween two and 11) in the genomes of most bac-
terial species but is not present in human, viral,
or fungal genomes. The presence of multiple
copies of this target in bacteria increases assay
sensitivity when applied to infected human
specimens.81 In addition to 16S rDNA, other
genetic targets, including other ribosomal tar-
gets (such as 23S rDNA, 16–23S rDNA inter-
genic spacer region), housekeeping genes
(such as groEL, rpoB, recA, gyrB, sodA), cit-
rate synthase gene, and heat shock protein
genes have been used for broad-range PCR,
but nucleotide sequence conservation among
them generally is lower than in ribosomal mol-
ecules. Another variation of PCR is multiplex-
ing, in which multiple specific PCR assays are
run simultaneously to test for multiple differ-
ent DNA templates. This approach may be
hampered by interference between primers
within the same reaction but well-designed as-
says targeted at the bacteria most frequently
implicated in prosthetic joint infection may be
useful in its molecular diagnosis.31
Approaches to Prosthetic Joint Infection 79
Conventional Versus Real-Time Polymerase
Chain Reaction
Since PCR first was described in the
mid1980s, several improvements have been
made resulting in faster, more contained reac-
tions, and real-time monitoring of amplified
DNA.101 Conventional PCR is based on block
thermocycling with subsequent detection and
identification of the PCR product (such as gel
electrophoresis, Southern blotting). This post-
amplification manipulation adds substantial
time and risk for contamination of subsequent
PCR reactions. In closed-system real-time
PCR assays, amplification and detection of
amplified DNA are done in the same reaction
vessel, providing an important control against
amplification product contamination. Addi-
tionally, an integrated microvolume thermal
cycler and fluorimeter allow simultaneous
(real-time) detection and quantification of the
fluorescently labeled PCR product in an auto-
mated and highly simplified manner. The op-
tical system excites fluorescent dyes incorpo-
rated during amplification and detects their
emissions at various wavelengths. The ampli-
fication procedure can be completed in 30 to
40 minutes, significantly reducing the assay
time of several hours to days for conventional
PCR and postamplification amplified product
detection. The log-linear fluorescence chem-
istry enables quantification of starting target
copy number using mathematical algorithms
(Fig 3A). Melting curve analysis at the end of
the amplification run results in improved PCR
specificity (Fig 3B). The amplified PCR prod-
uct has a characteristic sequence-dependent
melting temperature, at which half the DNA
strands become separated in solution, which
permits differentiation of amplified target
DNA from nonspecific amplification products
(such as primer dimers). Quantification and
melting curve analysis result in the ability to
differentiate specific from nonspecific (back-
ground) amplification products.
Identification of Amplification Products
Bacterial 16S rDNA amplification alone does
not identify the infecting bacteria. For defini-
 Clinical Orthopaedics
80 Trampuz et al and Related Research

Fig 2. This figure shows broad-range PCR. The double-stranded 16S rDNA first is heat denaturated,
followed by universal primer annealing to conserved regions, and synthesis of a complementary DNA
strand. The amplified segment contains a variable region allowing bacterial classification via sequence
analysis. Depending on the PCR efficiency, the PCR amplification product increases almost exponen-
tially with each temperature cycle. (Reprinted with permission from Trampuz A, Steckelberg JM, Os-
mon DR, et al: Advances in the laboratory diagnosis of prosthetic joint infection. Rev Med Microbiol
14:1–14, 2003.)
Number 414
September, 2003 Approaches to Prosthetic Joint Infection 81

100.000

Staphylococcus epidermidis (Number No specific template

of colony forming units per reaction): (negative controls):
Fluorescence (F1)

10.000

ix
rm

r
te
te

wa
as

ile
M
1.000

er
St
105 104 103 102 101

0.100
A 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Cycle Number

4.0

3.5
Staphylococcus epidermidis
(101 to 105 colony forming
Fluorescence -d(F1) /dT

3.0 units per reaction)

2.5

2.0

1.5 No template:
1.0 Mastermix
Sterile water
0.5

0.0 Nonspecific amplification (primer dimers and background (DNA)

-0.5
B 66.0 68.0 70.0 72.0 74.0 76.0 78.0 80.0 82.0 84.0 86.0 88.0 90.0 92.0 93.0

Temperature (˚C)

Fig 3A–B. The graph shows Staphylococcus epidermidis 16S rDNA PCR amplification using a se-
quence-independent double-stranded fluorescent dye (SYBR Green dye I) from different initial con-
centrations of target sequences (from 10 to 100,000 colony forming units per PCR reaction). (A) Quan-
tification curve analysis is shown. The negative controls (sterile water and mastermix) show a positive
signal at a high cycle number (28 cycles) representing a small amount of nonspecific (background)
DNA. (B) A melting curve analysis is shown. Staphylococcus epidermidis 16S rDNA amplified from dif-
ferent initial concentrations of target sequences yielded products exhibiting similar melting tempera-
tures (86.8 C). In contrast, products generated from negative controls containing no specific template
(sterile water and mastermix) exhibited a different melting temperature with lower fluorescence inten-
sity, presumably generated by primer dimers and nonspecific (background) DNA.

tive classification of bacteria, the amplifica- curacy of the data. Most DNA sequencing
tion products must be sequenced, and the se- methods are based on a modification of Sanger
quence data must be used to classify the source dideoxynucleotide triphosphate (ddNTP) chain
bacterium. Historically, DNA sequencing termination chemistry in which synthetic
methods were laborious, time-consuming, and ddNTP molecules that lack 3’ hydroxyl groups
had a relatively high error frequency.59 Ad- normally present in the natural molecules are
vances in sequencing instrument automation added to a DNA synthesis reaction. Whenever
and chemistry have decreased the turnaround a ddNTP molecule is incorporated into the
time to less than 24 hours and improved the ac- growing DNA strand in lieu of the usual de-

82 Trampuz et al
oxynucleotide triphosphate (dNTP), the syn-
thesis reaction is terminated. For dye-termina-
tor cycle sequencing, four different fluores-
cent dyes are incorporated into each of the
four ddNTPs (ddATP, ddCTP, ddGTP, and
ddTTP); the result is a collection of DNA frag-
ments of different lengths labeled with differ-
ent dyes depending on the terminal nucleotide.
Reaction end products are separated by elec-
trophoresis and excited and detected by a
laser. The resulting digital data are processed
into electronic DNA sequencing data. Se-
quence data are analyzed using special soft-
ware and displayed either as a linear string of
one-letter nucleotide codes or as a graphic
presentation (electropherogram). Bidirectional
sequencing (forward and reverse sequence of
each DNA strand) can improve the accuracy
of the data by resolving any nucleotide ambi-
guities present.
To classify the source bacterium, sequence
data are used to query a database. Public ge-
nomic databases (such as GenBank from the
National Center for Biotechnology Informa-
tion104) tend to contain large numbers of se-
quences but do not require verification of the
taxonomic classification of organisms or se-
quence data before submission. They can be
used in combination with a smaller but peer-
reviewed and well-validated private database
(such as MicroSeq® 16S 500 library, Applied
Biosystems, Foster City, CA). The analysis
focuses on the percent sequence identity with
the most closely related sequences. If more
than one genus is determined to be closely re-
lated by 16S rDNA sequence analysis, the
evaluation focuses on classifying the organ-
ism into the correct genus (or genera). If one
genus is determined to be closely related, the
average interspecies 16S rDNA sequence
variability of the organisms in the genus is de-
termined, and, if possible, species-level classi-
fication is made. Interpretation of 16S rDNA
sequence data may be problematic when close
homology among species limits resolution ca-
pabilities. For species (or genera) whose 16S
rDNA sequences are related closely but not
identical, there may be signature nucleotide
Clinical Orthopaedics
and Related Research
profiles that define species or genus level dif-
ferences; such profiles need to be defined for
each unique situation.
Contamination Issues with Polymerase
Chain Reaction
Polymerase chain reaction is capable of 106- to
107-fold amplification of one copy of template
DNA. Therefore, one of the most difficult chal-
lenges associated with PCR has been preven-
tion of exogenous nucleic acid contamina-
tion.18 Contaminating DNA may be introduced
in the surgical suites (airborne contamination),
during specimen transportation, or in the labo-
ratory. Sources of contamination in the labora-
tory include contamination introduced during
specimen processing (from the hands of labo-
ratory personnel, the laboratory environment,
or specimens containing large numbers of tar-
get molecules) and from manipulation of am-
plified nucleic acids. False-positive reactions
especially are challenging for broad-range
PCR targeting 16S rDNA. A positive PCR re-
action may contain more than a billion copies
of an amplified target sequence, each of which
may serve as a substrate for additional amplifi-
cation. Amplified nucleic acid may contami-
nate reagents, laboratory surfaces, ventilation
ducts, and skin, hair, and clothing of laboratory
personnel. The use of physically divided areas
for specimen processing, PCR setup, and
PCR product manipulations with unidirec-
tional flow from pre-PCR to post-PCR rooms
is essential.58 Furthermore, dedicated equip-
ment and instruments and the use of gloves and
gowns may help in preventing contamination.
For specimen processing, a Class II biologic
safety cabinet with high-efficiency particulate
air (HEPA)-filtered circulating air is recom-
mended and should not be used jointly for PCR
setup. In addition, safety cabinets should be
equipped with an UV lamp to degrade any ex-
ternal DNA before PCR amplification. Carry-
over contamination controls may be included
using deoxyuridine triphosphate (dUTP) and
enzymatic inactivation with uracil-N-glycosy-
lase (UNG).51 Deoxyuridine triphosphate is in-
corporated into the amplified PCR, in lieu of
Number 414
September, 2003
deoxythymidine triphosphate (dTTP), such
that the amplified target contains dUTP, and
native target will contain dTTP. Any dUTP-
containing amplification products present as a
contaminant in subsequent reactions will be
cleaved by UNG before amplification. The
UNG then is inactivated before proceeding
with PCR. Contamination between samples
containing large amounts of target DNA can be
minimized by frequent changing of gloves be-
tween handling of specimens, use of dedicated
equipment, avoiding aerosol generation, and
opening only one specimen at a time. Another
possible source of DNA contamination espe-
cially relevant for broad-range bacterial PCR is
reagents derived from a bacterial source, such
as DNA polymerase and UNG.15 During en-
zyme production, nucleic acids may be copuri-
fied and as such, enzyme may contain micro-
bial DNA (including rDNA). Numerous
methods have been used to eliminate back-
ground rDNA, including physical (UV irradia-
tion), photochemical (psoralens), or enzymatic
pretreatment of reagents (DNase I, restriction
endonucleases).18
Limitations of Polymerase Chain Reaction in
Diagnosis of Prosthetic Joint Infection
With some exceptions (such as methicillin re-
sistance in Staphylococcus aureus attributable
to the presence of mecA), amplification tech-
niques fail to provide antimicrobial suscepti-
bility of the pathogen. For prosthetic joint in-
fection, the antimicrobial susceptibility of the
pathogen is crucial in appropriate patient
treatment. Another disadvantage is the ex-
treme sensitivity of PCR, leading to the pos-
sibility of false-positive results, which may
complicate the interpretation of molecular re-
sults. In the case of prosthetic joint infection,
it is the commensal organisms on the skin that
most commonly infect prosthetic joints. This
renders it difficult to determine, simply from
the identity of the organism, whether it is clin-
ically significant or a contaminant derived
from the skin of the patient, the medical staff
obtaining the specimen, or the laboratory staff
processing the specimen. False-positive re-
Approaches to Prosthetic Joint Infection 83
sults may additionally arise from detecting
small quantities of bacterial DNA present
in specimens (such as normal background
DNA). It recently has been shown, for exam-
ple, that there is bacterial DNA in human spec-
imens (such as the blood of healthy subjects)
classically considered free of bacteria.62 Addi-
tionally, false-negative results may occur be-
cause of the presence of PCR inhibitors in
clinical samples, failure to extract bacterial
DNA (because of a thick cell wall of gram-
positive cocci), or loss of DNA during purifi-
cation. If a mixture of bacterial species is pre-
sent in a clinical specimen, interpretation of
multiple PCR products generated using broad-
range PCR is challenging and may only be
resolved by subjecting amplified DNA to
cloning, high performance liquid chromatog-
raphy, or denaturing- or temperature-gradient
gel electrophoresis to obtain a pure template
for sequencing of individual 16S rDNA. In ad-
dition, no standardized assays for 16S rDNA
PCR for the diagnosis of prosthetic joint in-
fection are available and results derived from
different laboratories may not be directly com-
parable.
DISCUSSION
Several studies have evaluated broad-range
PCR assays for diagnosis of joint infections
without implants,37,96,99,100 but only a few in-
vestigators have addressed the usefulness of
PCR in the diagnosis of prosthetic joint infec-
tion.35,54,93,94 Mariani et al54 compared bacter-
ial DNA detection by broad-range PCR in sy-
novial fluid with results of standard synovial
fluid and periprosthetic tissue cultures. Fifty
patients with symptoms after a total knee re-
placement were studied. Polymerase chain re-
action testing of synovial fluid from a preoper-
ative joint aspiration detected bacterial DNA in
32 of 50 specimens (64%). Six synovial fluid
cultures (12%) and 15 periprosthetic tissue cul-
tures (30%) were identified with bacterial
growth. All patients with positive cultures had
positive PCR results. This study suggests that
PCR may be more sensitive than conventional
 Clinical Orthopaedics
84 Trampuz et al and Related Research

cultures and that some cases of prosthetic joint
infection may be misclassified as aseptic loos-
ening using current methods. However, the
presence of prosthetic joint infection was not
defined by well-formulated standard criteria
and it is possible that some of the PCR results
were false-positive. In one instance, 16S rDNA
PCR was positive while Candida species was
cultured. This is likely a false-positive result
because Candida species are not known to har-
bor 16S rDNA. In addition, the investigators
did not sequence the amplified DNA to con-
firm that the amplified DNA was representa-
tive of the same organism found in culture (for
culture-positive cases) and to show the source
organisms for the amplified DNA (for culture-
negative cases).
Tunney et al93,94 did two studies that also
suggest that unrecognized infection may be
present in patients with aseptic loosening of the
prosthesis, and that ultrasound, immunofluo-
rescence microscopy, and broad-range PCR
may improve detection of subclinical prosthetic
joint infection. The first study was already pre-
sented. In the second study,93 the same explant
sonicates from 120 retrieved hip prostheses that
were used in the first study94 were examined us-
ing immunofluorescence microscopy and 16S
rDNA PCR amplification. A specific mono-
clonal antibody and polyclonal antiserum spe-
cific for Staphylococcus species were used. By
this method, coccoid or coryneform bacteria
were detected in all of the specimens that were
positive by culture and in 53% of the culture-
negative specimens. The total number of spec-
imens positive by immunofluorescence mi-
croscopy was 71 of 113 (63%). Bacterial DNA
was detected in 72% of sonicate specimens by
16S rDNA PCR. Unfortunately, Tunney et al93
did not determine the clinical significance of
the positive sonicate culture, immunofluores-
cence microscopy and PCR results in patients
without prosthetic joint infection. This could
have been accomplished by following up their
patients prospectively to determine whether
the cumulative probability of subsequent pros-
thetic joint infection was higher in the sonicate
culture, immunofluorescence microscopy, or
PCR positive group compared with the soni-
cate culture, immunofluorescence microscopy,
or PCR negative group. The investigators did
not sequence the amplified DNA to confirm
that the amplified DNA was representative of
the organism in culture (for culture-positive
cases) and to show the source organism for the
amplified DNA (for culture-negative cases).
Hoeffel et al35 attempted to reduce the
false-positive amplification of broad-range

Fig 4. The model shows prosthesis sonication and molecular diagnosis using explant sonicate. Ster-
ile Ringer’s salt solution (Oxoid Ltd, Basingstoke, Hampshire, England) is added to explanted pros-
thetic components. Biofilm-associated bacteria are dislodged from the surface by sonication in a bath
after which the explant sonicate is centrifuged. Deoxyribonucleic acid is extracted from the pellet and
subsequently amplified using broad-range PCR targeting 16S rDNA. The amplification product is se-
quenced and the microorganism classified by sequence analysis.
Number 414
September, 2003
PCR by developing genus-focused PCR
primers designed to detect a subgroup of
gram-positive cocci, but not Escherichia coli,
a common contaminant in their study. In the
preliminary testing of 21 specimens from 10
patients (five patients were thought to have
prosthetic joint infection and five patients
were deemed free of infection) the genus-
focused PCR was 91% sensitive and 100%
specific in comparison with culture. Addi-
tional evaluation of 108 specimens from 63
patients with total hip revision procedures
showed a sensitivity of only 71% and a speci-
ficity of only 49% compared with culture. In
this study, the investigators did not specify
which patient specimens were evaluated (sy-
novial tissue, periprosthetic tissue, or both)
and did not sequence the amplified DNA.
With newer amplification technologies
(quantitative, closed-system, real-time PCR)
and subsequent sequence analysis, the sensi-
tivity and specificity of 16S rDNA PCR for the
diagnosis of prosthetic joint infection poten-
tially can be improved, especially when com-
bined with antibiofilm strategies such as soni-
cation. Figure 4 shows a model of prosthesis
sonication followed by molecular diagnosis
using the explant sonicate being studied in our
laboratory. Whether such a system increases
sensitivity and specificity, and decreases turn-
around time has not been determined.
The clinical presentation of prosthetic joint
infection may be indistinguishable from that of
aseptic implant failure. Mo-lecular diagnostic
methods for the detection and identification of
infecting organisms in prosthetic joint infec-
tion are being developed. Attempting to iden-
tify bacterial DNA rather than culture entire
microorganisms potentially can improve pa-
tient care in terms of rapid, sensitive and accu-
rate diagnosis, reducing unnecessary addi-
tional tests and treatments, and lowering
medical expenses. However, before routine ap-
plication of these tests in the clinical microbi-
ology laboratory, additional research and de-
velopment is needed. Strategies to dislodge
bacteria attached to prosthesis surfaces repre-
sent areas of ongoing research.
Approaches to Prosthetic Joint Infection 85
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