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54
eD
*Consider further moleular diagnostics such as next-generation sequencing
F. DaRindeLorenzo
Major Criteria (at least on of the following)
Two positive growths of the same organism using standard culture methods.
Sinus tract with evidence of communication to the joint or visualization of the prosthesis
Minor Criteria
CRP (mg/L) 100
D-D Dimer (
Elevated Serum ESR (mm/hr)
Elevated Synovial WB
(cell/
or
Leukocyte Esterase
or
Positive Alpha-defensin (signal/
cutoff)
Elevated Synovial PMN (%)
Signle Positive Culture
Positive Histology
Positive Intraoperative Purulence
*This criteria were never validated on acute infection. No role in suspected adverse local tissue reaction.
g/L)
C
L)
Unknown
No rule
10.000
++
1. 0
90
V
Acute
Thershold
€
10 2
860
30
3,000
++
1. 0
70
Chronic
Scor
1
3
2
2
3
3
Fig. 6.6 Proposed 2018 ICM criteria for PJI
Decision
Infected
ecision
Combined preoperative
and postoperative
score:
6 Infected
3 to 5 Inconclusive*
<3 Not Infected
the use of molecular diagnostics for a prompt
septic arthritis/PJI diagnosis. Nucleic acid amplication techniques, such as polymerase chain
reaction (PCR) and Next Generation Sequencing
(NGS), represent the most appealing for the scientic community because of the capability to
accelerate the diagnosis, identify causative
agents, and direct targeted antibiotic therapy [59,
60]. NGS provides comprehensive pathogen
detection, supporting accurate and targeted treatment decisions in PJI management. NextGeneration Sequencing (NGS) for Periprosthetic
Joint Infections (PJI) involves a detailed process
to identify pathogens:
1. Sample Collection and Preparation: NGS
begins with collecting a sample, typically
synovial uid or tissue, from the infected joint.
2. DNA Extraction: DNA is extracted from the
collected sample. This DNA includes genetic
material from the patient and any microbes
present in the sample.
3. Library Preparation: The extracted DNA is
processed to prepare a sequencing library.
This involves fragmenting the DNA into
smaller pieces and adding specialized adapters for sequencing.
4. Sequencing: The library is then sequenced.
NGS allows for the simultaneous sequencing
of millions of DNA fragments, producing a
vast amount of genetic data in a single run.
5. Data Analysis: The sequenced data is analyzed using bioinformatics tools. This involves
aligning the sequences against known microbial genomes to identify the specic pathogens present in the sample.
6. Pathogen Identication: By comparing the
sequenced data to reference databases, the
specic pathogens causing the infection are
identied.
7. Report Generation: A report is generated that
lists the identied pathogens, which helps in
guiding the treatment plan.
This molecular diagnostic tool (Biore Joint
Infection panel, Biomerieux, Salt Lake City, USA)
is capable to identify, in less than an hour, 39
microorganisms, including 14 Gram-negative bacteria and two yeasts and eight antimicrobial resis-
6 “Diagnosis ofBone andJoint Infections fromSample toCulture”
55
tance (AMR) genes from synovial uid specimens,
allowing for rapid microbiological identication
and subsequently targeted adequate antimicrobial
therapy. In a recent multicenter study, this panel
has shown a sensitivity of 90.9% and a specicity
of 98.5% [61]: the major limitation of this diagnostic technology is the lack of coagulase-negative
staphylococci (i.e., S. Epidermidis) and Grampositive rods (i.e., Cutibacterium Acnes) in its
diagnostic spectrum, butbeyond these limitations,
the molecular testappears to be the ideal diagnostic tool for acute joint infections, both in native
joints as well as in PJI: in fact, acute joint infections are mainly caused by S. aureus and Grambacilli (Escherichia coli, Enterobacter cloacae
complex, Klebsiella, Pseudomonas aeruginosa)
which are included in the panel.
In chronic infections, where coagulasenegative staphylococci play a major role, next
generation sequencing (NGS, which can quickly
sequence DNA, represents a modern and innovative diagnostic methodology, especially in
culture- negative scenarios. Recent reports have
highlighted that the NGS technique, especially in
its metagenomic variation (mNGS), had a 95.9%
sensitivity and a 95.2% specicity [62]. The limitation of NGS stays in its very high sensitivity
[63]: in a recent study, surgeons from the
American Association of Hip and Knee Surgeons
(AAHKS), using NGS in a study on culture negative PJI, reported pathogen identication in
65.9% of cases; more than 90% of those PJI were
polymicrobial [64]. This last nding raised a
major, still unanswered, question: does the
threshold of reads for distinguishing real pathogens from background signals (knee microbioma?) [65] need to be established before the
widespread use of NGS as the most emerging
technology for microorganism identication in
septic arthritis and PJI scenarios?
The diagnosis of septic arthritis and PJI in the
setting of equivocal test results, negative cultures,
or chronic, indolent infection can raise signicant
dilemmas, especially without a single, “gold
standard” diagnostic test available. Molecular
diagnostics represent revolutionary technologies
that could disrupt the traditional diagnostic paradigm of musculoskeletal infections.
Another series of tests that can be requested
consists of radiological ones, which range from
the least demanding to those that have a greater
impact on the patient.
The X-ray (Figs.6.7, 6.8 and 6.9) Is perhaps
the rst test done when faced with the symptoms
of a painful prosthesis and can give us some
information, such as the relationship between the
prosthesis and the bone structure. Lytic areas or
periosteal reactions can be detected, but all these
images are nonspecic because they can also
concern, for example, aseptic loosening. It should
be considered that lysis occurs when there is signicant bone loss (at least 30%), so in the initial
stages, it is difcult to establish a diagnosis.
Fig. 6.7 X-ray of a cemented anatomical shoulder prosthesis, where a separation between the cement and the
bone is visible but without other signs and tests it is not
possible to say whether it is an infection or an aseptic
loosening
56
Fig. 6.8 Another example of an X-ray of a cemented
reverse shoulder replacement, where you can see a notch
in the proximal humerus but without other signs and tests
you can't tell if it's an infection or aseptic loosening
Therefore, if it were negative, it would not
exclude that there is an ongoing PJI [66, 67].
In Figs.6.7 , 6.8, and 6.9.There are two X-rays;
an anatomical prosthesis and a reverse prosthesis,
both infected and showing no radiographic sign of
a septic diagnosis. Although in the case on the
anatomical prosthesis we notice a gap between
the cement and the prosthesis, nothing tells us
whether it is a septic loosening or not septic.The
situation is similar for the knee x-rays. It should
be noted that they were all septic forms.
An associated exam is stulography (Fig.6.10)
[68] if, obviously, there is a stula as in the image
below. It is an exam that is performed without
anesthesia and uses a buttoned needle and a contrast medium, in our case lipiodol, which is made
up of 1ml of solution containing active ingredients: mixture of ethyl esters of iodinated fatty
acids of plant origin equal to 480mg of iodine.
Density: 1280 at 15° C.
F. DaRindeLorenzo
Viscosity: 65 centipoises at 15° C.
Iodine content: lg contains 0. 38g of iodine—1ml
contains 0.48g of iodine Lipiodol Ultra Fluid
does not contain excipients. In general, the
dose administered varies from 1 to 20 ml,
according to the needs of the different radiological investigations carried out. Administer
Lipiodol Ultra Fluid using an adequate glass
syringe and a cannula, by slow injection or
cannulation.
Lipiodol Ultra Fluid must not be administered
intravenously and intra-arterialbut only locally.
Fistulography can give us indications on the
main focus or on any abscess collections, useful
for, possibly, under ultrasound control, to do an
aspiration that can help us nd the germ responsible for the infection and, consequently, the
antibiogram.
Another test that can be easily carried out, with
low invasiveness and low cost is ultrasound
(Fig.6.11) [69, 70], a test used in PJI to identify
collections above or below the fascia which,
always under ultrasound control, can be evacuated to perform, above all, the search for any
pathogen. It has the advantage of being able to be
done dynamically to evaluate any prosthetic and/
or joint impingement and any existing relationships with the vascular system. Therefore, it does
not have the diagnostic capabilities of a PJI or differentiation but guides in ne needle aspirates and
biopsies without the use of ionizing radiation.
MRI (Fig.6.12) [71–73] as in the case below,
can help us highlight foci that would not have
been highlighted on X-ray, having a sensitivity
for the soft parts. The presence of metal has
always been a contraindication, but the arrival
of less ferromagnetic materials and the technical growth of equipment has made it possible to
reduce artifacts. One fact is that this system
does not use ionizing radiation and, therefore, in
patients who need repeated tests this may be a
possibility. The possibility of performing it in
open environments should be considered, especially for those who suffer from
claustrophobia.
6 “Diagnosis ofBone andJoint Infections fromSample toCulture”
Fig. 6.9 Another
example where the
X-ray does neither show
any sign of apparent
infection, nor other signs
of loosening
57
Fig. 6.10 An example of stolography of an
infection of the greater trochanter which, thanks to
the examination, made us understand that the joint
had not been affected by the infection. It is a test
whose importance has been lost but in some cases, it
is useful and is not complicated to perform
Computed Tomography (CT) [74, 75] This
exam allows us to better analyze the relationships
between bone and prosthesis. It is a test that
hardly helps us in making a diagnosis of PJI, as
the manifestations of the infection are evident
when the infection is already established, and,
58
F. DaRindeLorenzo
therefore, in the initial stages, it is of little help.
The presence of the prosthesis, then, creates artefacts, even if today it is possible to perform image
subtraction CT scans, which avoid artefacts.
The Triphasic Bone Scan [76–78] is a test that
must be requested when at least 2 years have
passed since the implantation of a hip prosthesis,
while at least 4years after the implantation of a
knee and shoulder prosthesis, in how long the
bone manipulation takes place and would certainly give false positives. It is a test that is performed by injecting a bisphosphonate analogue
Fig. 6.11 Aspiration of a septica area under ultrasound
guidance
labeled with metastable Technetium-99 (99mTc)
into the blood. Similarly, to unlabeled bisphosphonates, this radiopharmaceutical is incorporated into the crystals of hydroxyapatite of the
newly afxed bone matrix, providing information relating to osteoblastic metabolism. In the
suspicion of an infection, the most appropriate
bone scan is the one acquired in triphasic mode,
i.e., exploring three fundamental functional
moments of the infectious process: vascularization, permeability vascular, and bone metabolism. In the presence of an infection, in fact, all
three of these phases are enhanced and therefore
positive on the triphasic bone scan. It should be
considered that the increased metabolism is typical of other forms in addition to the infectious
one, such as aseptic loosening, tumors, and osteometabolic pathologies. Therefore, its negativity
(negative predictive value) indicates the nonpresence of a septic pathology. Therefore, high
sensitivity but low specicity.
Scintigraphy with Autologous Labeled
Leukocytes (Fig. 6.13) [79–81] Should be
requested, possibly associated with SPECT/CT
(Fig.6.14) method or spinal cord scintigraphy, in
cases in which serological tests and those on
Fig. 6.12 An example of MRI that highlights endo-epiphyseal foci of the humerus that cannot be seen on a simple
X-ray
6 “Diagnosis ofBone andJoint Infections fromSample toCulture”
Fig. 6.13 An example of a scan with autologous labeled leukocytes in a case of periprosthetic knee infection
59
Fig. 6.14 The SPECT/CT is considered the best technique that can contribute to the evaluation of the presence
and extent of the infection both in the diagnostic phase, in
the case of suspected infection, and as a control after
appropriate medical and/or surgical treatment, to exclude
the persistence of the infection
60
F. DaRindeLorenzo
More than 2 years after prosthesis implant
3-phase bone scan or FDG-PET
Negative
Suspicion of acute
infection: WBC
2
scan
(or AGA scan2)
Negative Negative
Fig. 6.15 Nuclear Medicine Flow chartfor diagnosing
infection after prosthesis implant. Signore, LM
Sconenza, O. Borens, AW.J.M. Glaudemans, VC
Pullicino, A Trampuz, H. Winkler, O. Gheysen,
FM.H.M. VanhoenackerN Petrosillo, P C. Jutte
1
Positive
Suspicion of chronic
infection: AGA scan
(or WBC scan2)
Positive
Within 2 years after prosthesis implant
WBC scan (with or w/out
bone-marrow scan
2
Positive
Infection
“Consensus document for the diagnosis of prosthetic joint
infections: a joint paper by the EANM, EBJIS, and ESR
(with ESCMID endorsement)” A.Signore et Al. European
Journal of Nuclear Medicine and Molecular Imaging
https://doi.org/10.1007/s00259- 019- 4263- 9
2
No infectionNo infection
synovial uid are not sufcient to conrm or
exclude the diagnosis of infection. Scintigraphy
with leukocytes labeled with 111-Indium (111In)
or 99m-Technetium (99mTc) is still the “gold
standard” nuclear medical test for the diagnosis
of infectious processes and is therefore indicated
in the majority of infectious pathologies, if performed and interpreted according to the guidelines provided by the European Society of
Nuclear Medicine (EANM) (Fig. 6.15).
Therefore, in compliance with the pathophysiology of infections, it is necessary to acquire
images at multiple times (30min–1h; 2–4h; and
20–24h after the administration of the labeled
autologous leukocytes) in order to verify the
trend of radioactivity. In case of increased uptake
in extent and/or extent of uptake, we speak of
infection, on the contrary, if the uptake shows a
reduction/stability over time, it is a sterile
inammation. Adding SPECT/CT provides better localization of the septic process and its
extension by differentiating between peripros-
thetic-bony localization and soft parts, with an
accuracy that, in some cases, reaches 100%. This
test is also valid in low infections where the bone
tournover is not very high. Another fact, especially for the acute forms, both hematogenous
and post- prosthesis surgery, is that the exam can
be done approximately a month later and that it
is not conditioned by the use of antibiotics even
if some authors recommend their use. Suspension
10–15days before the exam.
PET (Positron Emission Tomography)/CT
with F-FDG (uorodeoxyglucose) [82–
84] Another, but invasive, test is the biopsy
(Fig.6.16), which is performed under amplioscopic control and under local anesthesia, but it
must also be performed intraoperatively.It consists of taking at least in 6 samples from the
bone- prosthesis or bone-cement-prosthesis
space.Each withdrawal must be indicated for
the headquarters made as the septic process is
6 “Diagnosis ofBone andJoint Infections fromSample toCulture”
sampling coincided in 86% and 100% of cases
[86, 87]. At this point, we have made the diagnosis of PJI or we still have doubts but we have
decided that we must operate on the patient by
removing the prosthesis. We may not even have a
positive culture test or we may nd ourselves
faced with the doubt that it is an aseptic loosening. I will not go into the merits of the various
surgical techniques of removal and/or reimplantation with passage through an antibiotic spacer
but I will focus on the diagnostic part of this surgical act which involves some gestures that can
help us identify, in a safer way, the responsible
germ of the pathology or give certainty about the
diagnosis of PJI:The microbiological examina-
tion must be done regardless of the preoperative
Fig. 6.16 Material for biopsy sampling both under
closed amplioscopic control and in the open surgical
phase
diagnosis and consists of 3–5 samples in the
areas of doubtful appearance due to infection.
Each sample must be done by changing the sam-
not uniform in the whole articulation. Culture
research on the germ and a histological examination can be carried out on the sample which
should evaluate the presence of neutrophilic
polymorphonuclear leukocytes indicative of
infection which should be at least ve neutrophils per eld at high magnication (400x). As
you can easily understand, it's evaluation of
several tests that together contribute to the
dignosis of infection.The experience and relationship with other specialists (radiologists,
microbiologists, infectious disease of infection,anatomical pathologists, plastic surgeons)
is crucial.
ple material and must be placed in a sterile test
tube describing the site of the sample itself.
Material must be subjected to aerobic and
anaerobic culture for at least 15days. The number of samples is also given by the clinical situation. In the suspicion of a low infection,
samples should be more than 5. The material
collected may consist of synovial tissue, the
membrane that surrounds the prosthesis, in the
bone and, if necessary, cement always close to
the prosthesis and the bone. The samples must
be taken to the laboratory as soon as possible to
prevent the killer bacteria from attacking the
pathogenic bacteria, lysing them. The sample
must be stored at −4°C.The removed prosthe-
Another test that can be done in situations of
high fever and/or sepsis is the blood culture
which can give us information on the circulating
germ. Some authors consider it of little signicance as the isolation of the bacterium(s) is much
more important batteries in the joint eld. But the
most important thing is that if we are faced with
septicemia it is very important to treat, regardless
of the germ found in the joint, also the blood culture germ, even if the positivity of the blood culture in the context of a PJI varies from 4% to 25%
[85]. Some works have shown that the isolated
germs in blood culture and from intraoperative
sis can follow two paths, both perfectly similar
in terms of results and both use a system to
break the biolm [88–90] which is not only
present on the prostheses but also in the periprosthetic tissues, and, therefore, all the material taken must be sent for analysis with the
following methods, and thus allow the identication of the pathogenic germ and the subsequent sensitivity to the antibiotics tested and
are:
• Sonication (physical method) [91]; this consists of subjecting both cellular suspensions to
the action of ultrasound, to cause the rupture
61
62
F. DaRindeLorenzo
of biological membranes and recover the
intracellular contents, and proteins or DNA, to
obtain fragments of uniform molecular mass.
In our case it serves to break the biolm of the
germs that are positioned on the prosthesis but
also in the periprosthetic tissues.
Dithiothreitol (DTT) (chemical method) [92]
is chemically classied as a thiol. It is a solid,
white compound, very soluble in water, with a
typical “rotten egg” odor, an odor that is accentuated when the compound passes into solution,
especially if concentrated. It belongs to the family of reducing chemical compounds. It is one of
the strongest reducing agents known in the biological and laboratory elds, capable of breaking disulde bridges of both most proteins and
organic compounds, even at room temperature.
It was used in the past to break up the sputum in
the search for mycobacterium in pulmonary
tuberculosis and this ability is used today to
break up the biolm both on the prosthesis and
on the tissues.The only clinical nding of puru-
lent material (Fig. 6.17) on intra-operative
examination, although highly suggestive of PPI,
cannot be considered in itself a certain sign of
periprosthetic infection, therefore it is still recommended to complete the diagnosis with a
microbiological and anatomical-pathological
examination. “Pus” is a dense uid with a
Fig. 6.17 Pus leakage during PJI surgery
creamy appearance and a yellowish-white color,
which forms during particular types of inammation. It is made up of decaying white blood
cells, which can be mixed with bacteria (live
and/or dead), in this case representing a sign of
infection. However, the accumulation of white
blood cells and the formation of purulent material can also be caused by “aseptic” inammation therefore in the absence of infecting
microorganisms; on the basis of this observation, authoritative experts have highlighted the
limits of clinical evaluation both regarding the
subjectivity of the denition of “purulent material” and regarding the impossibility of clinically appreciating the true cause of a purulent
collection; in fact, only the microscopic examination and the possible isolation of the pathogenic agent can allow us to unequivocally
establish the septic or aseptic nature of a purulent collection [93]. Purulent material can also
be found in the presence of metallosis [94,
95],pseudotumors due to wear of biomaterials
[96], some rheumatic diseases such as gout [97]
or other crystal deposition diseases [98], aseptic
loosening or other bone pathologies such as, for
example, osteonecrosis [98].
As the conclusion of this chapter, I would like
to draw attention to what EBJIS has proposed
with a new denition of PJI, trying to give a new
value to infections given that there is not yet, to
date, a valid test to dene the presence of an
infection and therefore a further differentiation
was made by dividing them into unlikely infections, probable infections, and conrmed infections [99]. Based on the above, Shohat and A.A
[100]. Combined a stepwise clinical algorithm
that could collect all the characteristics we have
described. Patients who had a low-virulence
infection and patients who were culture-negative
were considered. Developed a stepwise clinical
algorithm based on the revised 2018 MSIS denition. They applied the algorithm to a multicentered cohort of patients and demonstrated
high sensitivity (96.6%) and specicity (99.5%)
(Fig.6.18).
6 “Diagnosis ofBone andJoint Infections fromSample toCulture”
Sinus Tract
Not present
LOW
+
Serum markers all
negative
Does not meet PJI
definition
No PJI
Possible infection/Dry
Tap
No revision TJA
planned
Repeat aspiration
Dry Tap
Advanced Testing
• Nuclear Medicine Scan
• PET scan
• Biopsy
Does not meet PJI
definition
Clinical Suspicion
Serum Tests
• ESR
• CRP
• D-dimer
–
lntraoperative Tests/Findings
• Histology
• Purulence
• Synovlal Fluid Culture
• Next Generation Sequencing
Synovial Fluid Tests
• Synovial WBC
• Positive LE
• Synovial PMN% or Alpha
Defensin
• Synovial Fluid Culture
Revision TJA Planned
HIGH
+
Any positive serum
markers
+
Meets PJI
Definition
PJI
63
Fig. 6.18 Like all algorithms, this one can also have
some negativity but it is an indication that the whole world
is taking action to ght this terrible pathology.(Stepwise
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