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fhg – Center for Health Professions Tyrol GmbH FH
Bachelor's Degree Program in Biomedical Analysis
Comparison of
microbiological diagnostics
of tissue samples using solid
cultures and blood cultures
in periprosthetic and implant
infections
Bachelor thesis
Author: Rana Ebru Özgün
Supervisor: OA Dr. Harald Dirschmid
Institute: Institute of Pathology at Feldkirch Regional Hospital
Innsbruck, June 2024
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Abstract
This study compares the effectiveness of microbiological diagnostics of tissue
samples using agar plates and blood culture methods in the context of
periprosthetic and implant infections. A total of 64 tissue samples from 24 patients
were processed. Of the 24 patients, 13 samples without infection and 11 samples
with infection were identified using both the solid culture method and the liquid
culture method. However, four additional contaminated samples were identified in
the solid culture method. The results show that the incubation period for blood
cultures can be reduced from 14 to seven days based on the average time to
positivity (TTP). The most frequently identified pathogens were S. aureus, followed
by S. epidermidis and E. faecium. In addition, 74% of all blood culture bottles were
positive after 24 hours of incubation. The study emphasizes the advantages of the
blood culture method, including high sensitivity and specificity as well as its
potential for routine use with a shorter incubation period. Further research is
needed to optimize diagnostic techniques and reduce contamination rates in
general.
Keywords (PubMed): periprosthetic infections, microbiological diagnostics, blood
culture, agar plates, time to positivity, C. acnes, S. aureus
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Abstract
This study compares the effectiveness of microbiological diagnostics of tissue
samples using agar plates and blood culture methods in the context of
periprosthetic and implant infections. A total of 64 tissue samples from 24 patients
were processed. Of the 24 patients, 13 samples were identified as non-infected
and 11 samples as infected using both the solid culture method and the liquid
culture method. However, the solid culture method also yielded four contaminated
samples. The results show that the incubation time for blood cultures can be
reduced from 14 to seven days based on the average time to positivity (TTP). The
most frequently identified pathogens were S. aureus, followed by S. epidermidis
and E. faecium. In addition, 74% of all blood culture bottles were positive after 24
hours of incubation. The study highlights the advantages of the blood culture
method, including high sensitivity and specificity, as well as its potential for routine
use with a shortened incubation period. Further research is needed to optimize
diagnostic techniques and reduce contamination rates in general.
Keywords: periprosthetic infections, microbiological diagnostics, blood culture,
agar plates, time to positivity, C. acnes, S. aureus
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Table of contents
1. Introduction ................................................................................................6
1.1 Types of prostheses...................................................................................7
1.2 Symptoms ..................................................................................................7
1.3 Common microorganisms causing implant infections ................................8
1.4 Risk factors ................................................................................................9
1.5 Prevention................................................................................................10
1.6 Diagnostics ..............................................................................................11
1.6.1 Blood biomarkers .....................................................................................11
1.6.2 Synovial fluid cytology..............................................................................12
1.6.3 Synovial fluid biomarkers .........................................................................12
1.6.4 Microbiology.............................................................................................13
1.6.5 Errors in diagnosis ...................................................................................14
1.7 Therapy....................................................................................................14
1.8 Overview of further steps in the future.....................................................15
1.9 Research question and hypotheses ........................................................16
2 Materials and methods ............................................................................17
2.1 Equipment................................................................................................17
2.1.1 SpinAX.....................................................................................................19
2.1.2 Bactec-FX ................................................................................................21
2.1.3 Incubator and incubation room.................................................................22
2.1.4 MALDI-TOF mass spectrometry ..............................................................22
2.1.5 Previ Color Gram .....................................................................................25
2.1.6 BactoSonic...............................................................................................27
2.2 Preparation of tissue samples..................................................................27
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2.3 Incubation of the liquid culture .................................................................29
2.4 Transition from liquid culture to solid culture............................................30
2.5 Identification.............................................................................................32
2.6 Sonification with BactoSonic ....................................................................34
2.7 Evaluation ................................................................................................35
3 Results.....................................................................................................36
4 Conclusion ...............................................................................................45
5 Bibliography .............................................................................................47
6 List of abbreviations .................................................................................48
7 List of figures............................................................................................49
8 List of tables.............................................................................................51
9 Affidavit ....................................................................................................52
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1. Introduction
Arthroplasty-associated infection or periprosthetic joint infections (PJI) are a rare
condition involving interactions between various microorganisms, the implant, and
the immune system. In addition to classic pathogenic bacteria such as
Enterobacterales or S. aureus, microorganisms that can cause such infections
also include apathogenic bacteria from the skin flora, such as coagulase-negative
staphylococci, corynebacteria, or Cutibacterium acnes, and, in rare cases, fungi.
Only a small number of microorganisms are necessary for the manifestation of PJI.
The bacteria and fungi, see chapter 1.3 for examples, adhere to the surface of the
prosthesis and form biofilms there, which protect them from antibiotics and
antifungal agents as well as the immune system. (Patel, 2023, p. 251)
The idea for this work arose from the problem that, in cases of suspected implant
infections, the wide range of possible pathogens in microbiological diagnostics
means that the solid cultures inoculated with tissue samples must be incubated for
at least seven days as part of routine procedures and inspected manually by
laboratory staff on a daily basis. This carries the risk of laboratory contamination
by skin germs such as coagulase-negative staphylococci or cutibacteria, which
can lead to false positive results or, in case of doubt, unnecessary treatment. By
inoculating the blood culture bottles with the patients' tissue samples, incubation
takes place in a closed system and further processing only occurs if the blood
culture incubator detects growth in the bottle. The aim of this work is therefore to
prove that blood culture bottles inoculated with tissue samples detect the same
pathogens as the conventional solid culture method, as well as to show
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that the risk of contamination by laboratory personnel is minimized or completely
prevented.
The following chapters will explain in more detail the most common joint
prostheses, the symptoms of PJI, the causative microorganisms, the diagnosis,
and the treatment of PJI.
1.1 Types of prostheses
The most common joint prostheses include knee and hip joint prostheses,
shoulder and elbow joint prostheses, and ankle, hand, metacarpal, and
interphalangeal joint prostheses. (Patel, 2023, p. 252)
In the US, knee joint prostheses are the most commonly used, followed by hip joint
prostheses. The other joints listed above are replaced less frequently. (Patel,
2023, pp. 251-253)
1.2 Symptoms
As with any infection, a distinction can be made between acute and chronic
infections. Chronic infections are those that persist over a longer period of time
and typically have slower and less intense symptoms. In contrast, acute infections
are characterized by a sudden onset and rapid deterioration of clinical signs. Both
types of infection require different diagnostic and therapeutic approaches. Acute
PJIs are often characterized by intense signs of inflammation such as redness,
swelling, joint warmth, fistula formation, and pain, while chronic PJIs may cause
more subtle clinical symptoms such as persistent pain or loss of function. In
addition, the treatment of chronic PJIs is more complex and protracted, often due
to the formation of biofilms that can impair the effectiveness of antibiotics. (Patel,
2023, p. 251) (McNally, et al., 2021, p. 21)
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1.3 Common microorganisms causing implant infections
(Patel, 2023, p. 253)
Figure 1: Microorganisms frequently detected in implant infections. These
pathogens were most commonly detected in knee and hip joint prostheses.
(Patel, 2023, p. 253)
Figure 1 shows a table listing the most common bacteria found in hip and knee
joint infections. It shows that aerobic Gram-positive bacteria are the most common
(82%), followed by coagulase-negative staphylococci (37%) and S. aureus (24%).
Other common pathogens are streptococci (14%), enterococci (8%), and
Corynebacterium species (5%). Aerobic Gram-negative bacteria cause 11% of
cases. The most common are enterobacteria and Pseudomonas species.
Anaerobic bacteria are involved in 13% of cases. Cutibacterium occurs in 8% of
infections. Other pathogens are less common. The distribution of pathogens may
vary depending on the facility. (Patel, 2023, p. 253)
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1.4 Risk factors
(Patel, 2023, p. 254)
Figure 2: Risk factors for PJI. These factors, some of which are preventable,
increase the risk of PJI. (Patel, 2023, p. 254)
Figure 2 shows a table of risk factors for PJI. These risk factors are in
different categories : potentially
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Modifiable preoperative risk factors, non-modifiable preoperative risk factors,
operative risk factors, and postoperative risk factors. Potentially modifiable
preoperative risk factors include anemia, intravenous drug use, malnutrition,
obesity, intra-articular injections in the last three months, and tobacco use. Non-
modifiable preoperative risk factors include cardiovascular disease, diabetes,
immunodeficiency, inflammatory arthritis, kidney or liver disease, male gender,
mental illness, previous joint infections and
previous revision arthroplasties. Surgical risk factors include
allogeneic blood transfusions, prolonged operating times, and simultaneous
bilateral arthroplasties. Postoperative risk factors include discharge in
a rehabilitation or nursing facility, prolonged
hospital stays, S. aureus bacteremia and
wound healing complications. (Patel, 2023, pp. 253-254)
1.5 Prevention
There are not only risk factors that can be reduced, but also other ways to prevent
PJI. In general, it is recommended that treatment be carried out in specialized
centers. Preoperative screening of S. aureus carriers with appropriate
decolonization or universal decolonization is beneficial. Patients with knee or hip
joint prostheses also have an increased risk of other general infections. The type
of decolonization is irrelevant in this case. There are several different antibiotics
that serve as prophylaxis. Cefazolin, for example, should be administered within
60 minutes before surgery. Compared to vancomycin and clindamycin, cefazolin
is an antibiotic with a rather lower effect. In patients with MRSA, cefazolin is
replaced by the active ingredient vancomycin. The duration of antibiotic
prophylaxis varies. Nevertheless, it is recommended that antibiotic prophylaxis
be discontinued after the closure of the incision .
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The primary source and associated risk of contamination is the operating room. To
minimize the risk of such contamination, it is necessary to keep traffic in the
operating room to a minimum. This means that only essential personnel should be
present and unnecessary movement or activity should be avoided. Reducing the
number of people present in the operating room and the movements taking place
there leads to a significant reduction in the risk of contamination. This results in
improved outcomes and a reduction in the infection rate. Aggressive
anticoagulation should also be avoided. Tranexamic acid, a drug used to inhibit
the fibrinolysis system, reduces the rate of PJI in the hip and knee. (Patel, 2023,
pp. 254-255)
1.6 Diagnostics
The diagnosis of joint prosthesis infection is very important, as the treatment of PJI
differs from that of a non-infected implant, such as a loosened implant.
Microbiological diagnosis plays a major role in this. The microbiological findings
are a building block and provide surgeons with the results they need to decide on
the further course of action and, if necessary, select the appropriate antimicrobial
agents. (McNally, et al., 2021, pp. 19-21)
1.6.1 Blood biomarkers
The most important and widely used blood markers are the erythrocyte
sedimentation rate (ESR), the white blood cell count, the percentage of
polymorphonuclear neutrophils (PMN), and C-reactive protein (CRP). The
percentage of PMN refers to the proportion of these specific white blood cells in
relation to the total number of white blood cells in a blood test. All of these tests
indicate general inflammation and are not specific to bacterial PJI. CRP can be
helpful in determining the severity of an infection. If the CRP
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has a value above 10 mg/ml, PJI cannot be ruled out. Nevertheless, the CRP
value is not sufficient for a definitive diagnosis. It is important to note that a normal
CRP does not rule out infection. (McNally, et al., 2021, p. 21)
1.6.2 Cytology of synovial fluid
Synovial fluid aspiration is a viscous fluid found in the cavities of movable joints.
This aspiration is an important part of the diagnosis of PJI. It is important for
assessing the extent of inflammation. When performing aspiration analyses, both
sensitivity and specificity are close to 90%. If the leukocyte count and PMN
percentage from the synovial fluid are below the detection limit, infection is
unlikely. On the other hand, values exceeding this limit indicate infection,
especially in PJI of the knee. The detection limits for leukocytes are between 1,500
and 4,000 cells/µl. Those for the PMN percentage are between 65% and 80%. A
cut-off value of 3,000 cells/µl and a PMN above 80% indicate a periprosthetic
infection of the hip and knee. The limits mentioned may be influenced by other
diseases and are only relevant for knee and hip prostheses. (McNally, et al.,
2021, p. 21) (Patel, 2023, pp. 256-257)
1.6.3 Synovial fluid biomarkers
Alpha defensin tests are very accurate and can be beneficial, for example, after
prior antibiotic administration or in the case of microorganisms with low virulence.
The alpha defensin result is not altered or influenced by other pathogens. Due to
its high specificity, infections can be suspected using this biomarker. Nevertheless,
it is not possible to rule out non-infected prostheses. (McNally, et al., 2021, pp. 21-
22)
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1.6.4 Microbiology
Examination of joint fluid prior to surgery is not suitable for ruling out joint infection.
However, a positive result indicates that the joint is infected. During intraoperative
surgery, at least five reliable tissue samples should be taken and sent to the
laboratory immediately. It is particularly important to take tissue samples from the
membrane at the interface between the bone and the implant. The patient should
not take antibiotics for at least two weeks prior to surgery. Antibiotic prophylaxis
prior to surgery is not necessary. Blood cultures should be incubated for at least
14 days. If the same bacteria with the same antibiotic resistance pattern are
found in all positive samples or in at least two different samples, this is an
indication of infection. A single positive culture result must be considered in
conjunction with other findings. If rare bacteria or viruses such as S. aureus or
Gram-negative rods are detected, this is an indication of infection. A single positive
culture result does not necessarily mean that patients have been infected with a
pathogen. Further investigations must be carried out. It is best to examine the
implant as well, using sonication (see section 2.6). This can reveal whether a
biofilm has formed there. Any positive culture from the sonication fluid must be
considered a potential infection. More than 50 colony-forming units per milliliter
(CFU/mL) confirm an infection. If a concentration technique is used, the
threshold for confirming an infection is 200 CFU/mL. Histologically, inflammatory
cells can be seen in the tissue if PJI is present. This can be determined from
biopsies or tissue samples. At least three deep samples must be taken for this
purpose. Certain stains in the tissue can also be used to detect microorganisms
that are otherwise invisible. This is particularly important for bacteria that cannot
otherwise be detected. (McNally, et al., 2021, p. 22) (Patel, 2023, pp. 255-257)
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1.6.5 Errors in diagnosis
It is very important to treat an infection quickly. No time should be wasted by
waiting and hoping that the clinical symptoms will improve or even disappear on
their own. The DAIR method (see section 1.7) should be performed surgically as
soon as possible, as the effectiveness of this form of therapy decreases with
increasing waiting time. Microorganisms attach themselves to the implants within
seconds and begin to surround themselves with a polysaccharide matrix. After
about three weeks, this biofilm is difficult to break down using antimicrobial
substances. (Li, Renz, Trampuz, & Ojeda-Thies, 2019, pp. 3-4)
Intraoperative swabs of periprosthetic tissue should be avoided. Previous reports
have shown that cultures from swabs have a low sensitivity of 53-76%. (Li, Renz,
Trampuz, & Ojeda-Thies, 2019, p. 4)
Since CRP and ESR can be analyzed quickly, some surgeons prefer these
parameters when PJI is suspected. These two analytes are highly recommended
in the 2010 guidelines of the American Association of Orthopaedic Surgeons
(AAOS). Nevertheless, as already explained in section 1.6.1, these inflammatory
markers have low sensitivity, as values within the normal reference range do not
rule out infection. In particular, infections caused by low-virulence pathogens
(mainly skin bacteria) do not necessarily go hand in hand with elevated CRP. (Li,
Renz, Trampuz, & Ojeda-Thies, 2019, p. 4)
1.7 Therapy
The treatment of PJI is both complicated and expensive. Where possible, such
treatment should be carried out in specialist centers. The objectives of PJI therapy
include maintaining the function of the joint prostheses, alleviating pain, and, in the
best case, restoring joint function.
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Curing the infection. Antibiotic therapies are usually unsuccessful without surgical
intervention. One well-known form of therapy is the DAIR method. DAIR stands for
debridement, antibiotics, and implant retention. This refers to the removal of dead
material and contaminated tissue from a wound, antibiotic therapy, and implant
retention. Performing a DAIR in cases of acute PJI of hip and knee prostheses is
important. In cases of chronic PJI, the affected implant is removed. This can be
done in a single stage, i.e., removal of the infected prosthesis and reimplantation
of a new prosthesis in the same session, or in a two-stage revision. In a two-stage
revision, antibiotic-releasing joint spacers are used to maintain function during the
prosthesis-free interval. If patients do not want surgical intervention, antimicrobial
suppression can be performed. However, this method does not usually cure the
infection. For this reason, patients often have to take the prescribed antibiotics for
the rest of their lives. If all these different methods have no positive effect,
resection arthroplasty with the creation of pseudoarthrosis can be performed for
hip infections, or arthrodesis or amputation (as a last resort) for knee joint
infections. The treatment is lengthy and usually lasts at least 12 weeks. (Patel,
2023, pp. 257-258)
1.8 Overview of future steps
Over the past two decades, significant progress has been made in the diagnosis of
PJI, but most of the data available relates only to hip and knee prostheses. More
data is needed for other joints. Further research is needed to determine whether
genetic predisposition plays a role in joint infections. It is important to identify
pathogens that form biofilms on prostheses. It is also often mentioned that surgery
for joint prostheses or the treatment of a
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PJI should be treated at specialized centers. It is also important to determine the
best surgical approaches and to conduct a medical assessment. Both antibiotic
and non-antibiotic therapies, as well as the duration and method of
administration, still need to be investigated in more detail. Nevertheless, antibiotics
are always administered for diagnosed infections. (Patel, 2023, p. 259)
1.9 Research question and hypotheses
The guiding principle of this work is the question of whether there is a difference in
the diagnosis of periprosthetic infections using blood culture or conventional solid
culture incubation and whether blood culture bottles can be used to better
differentiate between contamination and infection.
My research questions are as follows: Which of the two methods, blood culture or
solid culture incubation, is better suited for differentiating between contamination
and actual infection in patients with suspected periprosthetic and implant
infections? And can the incubation time of blood culture bottles be reduced from a
maximum of 14 days to seven days?
The expected results include, on the one hand, the finding that both cultures,
aerobic and anaerobic bottles, will have approximately the same TTP (see section
2.3). In most cases, both bottles will be positive. With the exception of anaerobes,
as these can only be cultivated in anaerobic blood culture bottles. The same
applies to aerobic pathogens, which can only be cultivated in aerobic bottles. It is
also to be expected that the results of the blood culture method will be similar or
identical to those of the conventional method.
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2 Materials and methods
This chapter explains the equipment used and describes the technical
background. It also outlines the process from sample collection to the results
obtained using the methods employed. In addition, it lists the materials used that
are relevant to this work. Finally, it explains the conventional method currently
used by the Feldkirch microbiology laboratory.
2.1 Equipment
Table 1 provides information about the device names and respective
manufacturers used in this study. A technical explanation of the devices
mentioned is provided in the following chapters. It should be noted that the BMAs
in the pathology laboratory do not require SpinAX or BACTEC-FX for the original
method.
Table 1: List of devices used
Device Manufacturer Reference number
SpinAX AxonLab, Baden-Dättwil, 22553
Switzerland
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Bactec-FX Becton, Dickinson and Top FT4952
Company, Sparks, USA Top + Bottom FT4939
Incubator MEMMERT, Schwabach, Germany O3070015
INC-246
Incubation Smeva B.V., Valkenswaard, 411812
chamber Netherlands
XR110C
MALDI Becton, Dickinson and 860467404819
Biotyper Company, Sparks, USA; Bruker
Daltonik GmbH, Bremen,
Germany
Previ Color Biomérieux, Maryy-L´Etoile, France 414292212875
V2 Gram
Bactosonic Bandelin Electronic GmbH & 277003.000062174.009
BS14 Co. KG, Berlin, Germany
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2.1.1 SpinAX
(AxonLab AG, n.d., p. 7)
Figure 3: SpinAX. The structure and function keys of the SpinAX are shown
here. (AxonLab AG, n.d., p. 7)
The SpinAX (see Figure 3) consists of a tube holder for ProbeAX Evolution tubes
(see Figure 4), a start/stop button, a reset/save button, a time display, and
separate minute and second buttons. (AxonLab AG, n.d., p. 9)
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(AxonLab AG, n.d., p. 9)
Figure 4: ProbeAX Evolution Tubes. The ProbeAX Evolution tube consists of
a rasp and a cylinder. (AxonLab AG, n.
D., p. 9)
The ProbeAX Evolution Tubes (see Figure 4) are equipped with a lid containing a
metal rasp and an exposed ceramic cylinder. (AxonLab AG, n.d., p. 9)
These tubes are filled with 5 ml of 0.85% sodium chloride (NaCl). Various tissue
types can be dispersed and homogenized using these tubes in the SpinAX. To do
this, the sample is placed in the tube and, after carefully closing the tube with the
cap, placed upside down in the SpinAX tube holder. Within one minute, the tissue
pieces are dispersed and homogenized by a grinding motion between the ceramic
block and the metal rasp. (AxonLab AG, n.d., p. 9)
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2.1.2 Bactec-FX
The BACTEC-FX from BD is used for the rapid detection of bacteria and fungi
from blood culture bottles and is suitable for the incubation and detection of
microorganisms. (Becton, Dickinson and Company, 2021, p. 9)
(Becton, Dickinson and Company, 2021, p. 10)
Figure 5: BACTEC-FX operating principles. This figure shows how the
BACTEC-FX checks cultures for microorganisms. (Becton, Dickinson and
Company, 2021, pp. 9–10)
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Figure 5 illustrates how the BACTEC-FX works. After the blood culture bottles
have been inoculated with a sample, they are loaded horizontally into the devices.
This causes the blood culture bottles to dock with the bottom of the device. If
microorganisms are present in the bottles, the nutrients in the culture medium of
the bottle are metabolized and carbon dioxide (CO2) is released. When CO2 is
released, the dyes at the bottom of the bottles react with the CO2 . The device
contains light-emitting diodes (LEDs). These activate the light sensors located at
the bottom of the bottles so that the photodetectors can measure the fluorescence.
The light value corresponds to the amount of CO2 released. These text analyses
are performed every ten minutes. Positive blood culture bottles appear in red on
the indicator light, a warning tone is emitted, and they also appear on the LCD
display. (Becton, Dickinson and Company, 2021, pp. 9-10)
2.1.3 Incubator and incubation room
The incubator and incubation room are used daily at the Institute of Pathology in
Feldkirch and are an important part of the laboratory's inventory. They are heated
by all-round heating and CO2 or nitrogen (N2) is introduced via sterile filters. These
are fed into the interior from above. Turbulence-free interior ventilation ensures
even distribution of the gases. (Memmert GmbH + Co KG., 2014, p. 11)
2.1.4 MALDI-TOF mass spectrometry
The principle of matrix-assisted laser desorption/ionization time-of-flight (MALDI-
TOF) is based on the mass-to-charge ratio (m/z) of the molecular ions of
microorganisms. This ratio can be used to identify all ionized biological molecules.
The mass spectrum is interpreted in the spectrometer and molecules are
identified. (Calderaro & Chezzi, 2024, p. 3)
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MALDI requires samples that have been incubated on agar plates, a lyophilized
matrix, diluted formic acid, and a biotarget. All materials required for MALDI can be
found in Table 6. The preparation steps for these materials can be found in
Chapter 2.5. First, part of a colony is applied to a free position on the biotarget
using the tip of a toothpick, dried at room temperature, and covered with 1 µl of
70% formic acid. After drying, 1 µl of matrix is added to each position. The
samples crystallize together with the matrix. After drying, the biotarget is placed in
the MALDI and a vacuum is created in the device, as shown in Figure 6. The
molecules are then ionized using short laser pulses. The matrix protects the
samples during this process. This process creates ions that fly through a flight
tube. The ions are accelerated by electromagnetic fields. The time of flight is
measured in this tube. This flight time then produces a spectrum that is compared
with other spectra from various databases. The database used in the microbiology
laboratory is MBT Compass HT IVD, version 5.2.300. The software with reference
number 1877017 contains Library V12. The additional MBT HT Subtyping IVD
module with reference number 1877010 is part of this database. The score
determined from this, a number of points corresponding to the number of similar
spectra found, enables the species to be identified. (Calderaro & Chezzi, 2024, pp.
3-4)
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(Calderaro & Chezzi, 2024, p. 5)
Figure 6: Principle of MALDI-TOF. Laser pulses ionize the molecules. These
ions are accelerated in the flight tube, producing spectra that are necessary for
the identification of microorganisms. (Calderaro & Chezzi, 2024, pp. 3-4)
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2.1.5 Previ Color Gram
(bioMérieux SA, 2021, pp. 3-6)
Figure 7: Structure and components of the Previ Color Gram. The Previ
Color Gram consists of several components and is used for the efficient and
environmentally friendly staining of sample smears. (bioMérieux SA, 2021, pp.
1-1, 1-2, 3-1, 3-6)
The Previ Color Gram is an automated Gram staining device for staining and
analyzing samples. Figure 7 shows that the Previ uses various reagents (6) and
has a spray mechanism that distributes the staining solution evenly on the slides.
This ensures that the samples are stained uniformly and reproducibly. The
centrifuge also has a high volume staining capacity (1 + 2). This allows many
samples (12 to 30 samples) to be stained quickly. The device has a cytocentrifuge
(4) that prepares the samples , and a screen (5), with the the
device
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can be controlled. In addition, different staining programs can be created, edited,
and saved on the Previ. The system works according to these preprogrammed
protocols. This means that the staining and destaining times are always identical.
This leads to clearly differentiated bacterial images and reliable diagnoses. Special
nozzles and pumps distribute the reagents evenly. A barcode reader helps to
manage the reagents listed in Table 2 and use them optimally to minimize waste.
The system is not only more efficient but also more environmentally friendly than
manual staining methods. (bioMérieux SA, 2021, pp. 1-1, 1-2, 3-1, 3-6)
Table 2: List of reagents for the Previ Color Gram
Material Manufacturer Reference number
Reagent A – Fuchsin or Biomérieux, Maryy- Acetone
Safranin (with or without L´Etoile, France fuchsin/safranin:
acetone) 295521/29582
Fuchsin/safranin:
29585/29583
Reagent B – Iodine Biomérieux, Maryy- 29586
L'Etoile, France
Reagent C - Biomérieux, Maryy- 29587
Crystal violet L'Etoile, France
Reagent D –
Distilled water
Reagent E – approved SAV Liquid Production ETO-10000-99-1
alcohol GmbH, Flinntsbach am
Inn, Germany
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2.1.6 BactoSonic
BactoSonic ultrasonic baths utilize the effect of cavitation. Piezoelectric vibration
systems are mounted under the bottom of the vibration tank, whose energy is
transferred to the bath liquid in the form of mechanical vibrations at ultrasonic
frequency. This constantly generates microscopic bubbles in the bath liquid, which
release energy when they implode and create local microcurrents. This process is
called cavitation. Compared to other ultrasonic baths, the sonication in the
BactoSonic is based on low-frequency ultrasound, whose low intensity is at the
cavitation threshold. The adhesion of the biofilm to the implant surface is reduced
to such an extent by the microcurrents, shear forces, and oscillating cavitation
bubbles that it detaches. However, the cavitation events that occur are still so low
in energy that there is no significant destruction of cell structures and the detached
microorganisms are available as sample material for subsequent analysis.
(Bandelin electronic GmbH & Co. KG, 2020, p. 6)
2.2 Preparation of tissue samples
If prosthetic infections are suspected, tissue samples are taken intraoperatively
from the affected patients. These are delivered from all over Vorarlberg to the
microbiology laboratory of the Feldkirch pathology department. The tissue samples
relevant to this work come exclusively from orthopedics and trauma surgery. Three
tissue samples are always taken per patient and removal, which arrive at the
laboratory from Monday to Friday. Samples that arrived over the weekend were
not used for this work. If more than three tissue samples were available, three
were selected at random and then used for this work. Care was taken to ensure
that the samples had a maximum diameter of one centimeter. Not only were
samples from patients used, at those a suspicion of an
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infection, but also from patients who have, for example, a loose prosthesis. The
analysis of these tissue samples also serves as a follow-up during or after
therapy.
The tissue was processed by the BMAs in the microbiology laboratory under a
fume hood using sterile scissors, sterile tweezers, or a disposable scalpel in a
sterile mortar. After the tissue had been cut to size, one piece was placed in each
ProbeAX Evolution tube. In the best case scenario, three ProbeAX tubes were
filled per patient. These tubes were further processed as described in section 2.3
below. The rest of the sample was ground in a mortar for routine processing. Two
different plates were smeared in a circular motion using the mortar itself and
incubated overnight. The rest was mixed with the broth liquid in the mortar and
added to the broth tubes as a whole. The materials required for processing are
listed in Table 3.
Table 3: List of materials used for the preparation of tissue samples
Material Manufacturer Reference number
SampleAX Evolution AxonLab, Baden-Dättwil, AL0510
Switzerland
Tubes
disposable scalpels Troge Medical GmbH, Hamburg, 146210
Germany
Sterile mortar
Sterile scissors
Sterile tweezers
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2.3 Incubation of liquid culture
Further processing was carried out independently. After the ProbeAX Evolution
tubes had been filled by the BMAs, they were then homogenized and dispersed in
the SpinAX for one minute at 2200 rpm. Meanwhile, the membrane of the aerobic
and anaerobic blood culture bottles was disinfected with a cellulose swab and
Sterillium. Using a syringe, one milliliter from each ProbeAX vessel was injected
into one aerobic and one anaerobic bottle. Finally, three sets consisting of three
aerobic and three anaerobic blood culture bottles were available for each patient.
These were incubated in the BACTEC-FX for a maximum of 14 days. In the case
of positive blood cultures, the time-to-positivity (TTP) is important. The TTP refers
to the time span in which the bottles become positive, namely the period between
inserting the bottle into the BACTEC-FX and the time at which the device was able
to detect microorganisms. The positive blood culture bottles are removed and
further processed as described in section 2.4 below. The materials used for these
steps can be found in Table 4.
Table 4: List of materials used for the BACTEC-FX
Material Manufacturer Reference number
BACTEC Plus Becton, Dickinson and 442023
Aerobic/F Culture Company, Sparks, USA
Vials
BACTEC Lytic/10 Becton, Dickinson and 442021
Anaerobic/F Culture Company, Sparks, USA
Vials
Injekt Luer Solo (10 B Braun, Melsungen, Germany 4606108N
ml)
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Blunt Fill Needle Becton, Dickinson and 303129
Company, Sparks, USA
Sterillium pure BODE Chemie GmbH, 981534
Hamburg, Germany
Zelletten Lohmann & Rauscher 13 356
International GmbH,
Rengsdorf, Germany
2.4 Transfer from liquid culture to solid culture
Since the BACTEC-FX cannot accurately identify microorganisms, positive blood
culture bottles must be processed manually. The materials required for this are
listed in Table 5. Mueller-Hinton chocolate agar (PVX) and Schaedler agar with
vitamin K1 (SK1) are required for further analysis.
Mueller-Hinton chocolate agar (PVX)
PVX plates are enriched, non-selective media used for the isolation and
cultivation of both fastidious and non-fastidious bacteria, as well as bacteria from
normal flora. Fastidious bacteria are those that do not grow, or grow poorly, on
sheep blood-based primary isolation media. These plates are incubated at 35-
37°C. According to the manufacturer, the culture plates can be read for the first
time after 18-24 hours. They are then incubated further and read again after 42-48
hours. PVX plates are also suitable for sensitivity testing of N. gonorrhoeae. H.
influenzae, N. gonorrhoeae, and N.
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meningitidis grow on this medium. (Becton, Dickinson and Company, 2011, pp. 1-
5)
Schaedler agar with vitamin K1 and 5% sheep blood (SK1)
SK1 medium is nutrient-rich and is used for the non-selective isolation of
anaerobes and the selective isolation of both Gram-negative and Gram-positive
anaerobic rods. The addition of vitamin K1 and hemin forms the basis for several
selective media. These plates are incubated anaerobically for 48–72 hours at 35–
37°C. B. fragilis, B. thetaiotaomicron, C. perfringens, F. nucletum, and others
grow on SK1 plates.
P. anaerobius, P. levii, but also facultative anaerobes such as E. coli, S. aureus,
and P. mirabilis. (Becton, Dickinson and Company, 2016, pp. 1–5)
One drop from each positive blood culture bottle was applied to a PVX and SK1
plate. Before the applicator with the adapter was placed on the membrane of the
bottle, it had to be disinfected with a cellulose swab and Sterillium to prevent
contamination. All the materials required can be found in Table 4. The positive
culture bottles were streaked using the three-eyelet method with a swab. As
already mentioned, the SK1 plate must be incubated anaerobically. For this
purpose, the plate was placed in a plastic bag with a gas-generating bag. While
the PVX plate was incubated in the incubator, the SK1 medium was incubated in
the incubation chamber.
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Table 5: List of materials used for solid culture incubation
Material Manufacturer Reference number
Mueller-Hinton chocolate Biomérieux, Maryy- 43109
agar (PVX) L´Etoile, France
Schaedler agar with Becton, Dickinson and 254084
vitamin K1 (SK1) Company, Sparks, USA
AnaeroGen Compact Thermo Fischer Scientific, AN0020D
Massachusetts, USA
Applicator plastic, Becton, Dickinson and 09-812-8052
viscose tip, 2.5x150mm Company, Sparks, USA
Vacutainer Multiple Becton, Dickinson and 367300
Sample Luer Adapter Company, Sparks, USA
Inoculation loop Nerbe Plus, Winsen/Luhe, 09-401-5002
Germany
PS stick with small Nerbe Plus, Winsen/Luhe, 09-812-8052
viscose tip Germany
2.5 Identification
Ideally, colonies were visible on the culture plates the next day. If this was not the
case, they had to be incubated until colonies were present. Both the conventional
method and the liquid culture method require MALDI for pathogen identification
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. It should be noted that the laboratory's BMAs did not create liquid cultures using
the conventional routine method. Therefore, they performed a MALDI directly after
streaking and incubating the agar plates.
As already mentioned, various solutions are required for MALDI. The organic
solvent (OS) is enriched so that it can be used immediately. The matrix is
lyophilized and was dissolved with 250 µl OS. The 100% formic acid was diluted to
70% with ultrapure water. After preparing the solutions, the colonies were removed
with a toothpick and smeared onto a Biotarget. After further processing steps,
which are explained in more detail in section 2.1.4, a wide variety of
microorganisms that have grown on the plates can be identified using mass
spectrometry. For personal verification, Gram stains (see section 2.1.5) were
prepared and viewed under a microscope. The materials required for the
identification of microorganisms are listed in Table 6.
Table 6: List of materials used for pathogen identification
Material Manufacturer Reference number
Menzel Superfrost Plus Gerhard Menzel, J1800AMNZ
slides Braunschweig, Germany
microscope slides
Solution Organic Solvent VWR International 89449.180
(OS) Chemicals, Pennsylvania,
USA
Formic acid 98/100% for Bernd Kraft, Duisburg, 05314.2010
analysis ACS Germany
IVD Matrix HCCA Bruker Daltonik GmbH, 8290200
lyophilized portioned Bremen, Germany
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Water, Chromasolv LC-MS Honeywell Riedel-de 39253-1L
Haen, Seelze, Germany
MBT Biotarget 96 Bruker Daltonik GmbH, 8267615
Bremen, Germany
10µl sterile filter tips Greiner Bio-One, 771261
Kremsmünster, Austria
2.6 Sonication with BactoSonic
Sonification is an ultrasonic treatment in which the biofilm on the surface of the
implant is removed by high-frequency sound waves. This procedure was
performed by the BMAs in the laboratory. There are a few things to keep in mind
when preparing for sonification. An oscillating tank was filled 1/3 full with water.
A surfactant-containing preparation was added and the tank was refilled with water
up to the mark. In the next step, the liquid had to be degassed by turning on the
ultrasound for 10 minutes. After all the materials listed in Table 7 had been
prepared and the sterile workbench was ready for use, ear protection and sterile
gloves had to be put on. The bag filled with physiological saline solution was
opened and a transfer device was inserted into the bag as far as it would go. The
sample, which was in a box, had to be covered with sterile saline solution to at
least 90%. The box was then closed and shaken vigorously for at least 30
seconds. The sonication itself runs at 100% power in the BactoSonic for one
minute. It is important to note that the box must not touch the bottom and the
walls of the bath not . After the
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Sonification: The box was shaken again. Then, 50 ml of the sonicate had to be
transferred to a test tube. From this liquid, 1 ml each was placed on a cooked
blood, Columbia, and Schädler plate. Likewise, 6 ml each was injected into an
aerobic and anaerobic blood culture bottle. The rest of the sonicate was then
emptied from the box and the sample was shaken vigorously in the BactoSonic
again for at least 30 seconds. Finally, the box was sealed and sent to the central
reception. After 24 hours of incubation, the culture plates were examined. The
results were evaluated as colony-forming colonies (CFU). The number of CFU per
plate was given in CFU/ml of sonication fluid. A significant result is obtained when
the number is greater than or equal to 50 CFU/ml. The result of the sonication is
decisive and confirms the presence of an infection. (Bandelin electronic GmbH &
Co. KG, 2020, pp. 16-18)
Table 7: List of materials used for the BactoSonic
Material Manufacturer Reference number
free flex, 0.9% physiological saline Fresenius Ref. no.: 1-16417
solution, 500 ml Kabi,
(infusion solution) + 250 ml bag Austria
Transfer device Fresenius 8500432
Kabi,
Austria
2.7 Evaluation
The results of both the agar plate and blood culture methods, as well as the
names and dates of birth of the patients, were collected in an Excel file. The date
of submission and the date of processing were documented. The results of the
conventional method used by the Feldkirch pathology department were recorded
and compared with the method used in this study. In the following chapter 3, all
results are presented graphically and discussed.
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3 Results
This chapter presents all results in graphical and tabular form. Both methods will
be compared. The tissue types used for this work, the results of both methods,
and their differences will be explained. In addition, the positive samples will be
listed in more detail. Furthermore, meaningful data on the TTPs will be presented
and conspicuous samples will be discussed.
In the period from February 19, 2024, to April 12, 2024, a total of 64 tissue
samples from the immediate vicinity of implants from 21 different patients were
processed and analyzed for this work. Tissue samples were taken twice at
different times from three of these patients. Nevertheless, these samples are
considered individual samples. The results of this study therefore refer to 24
patients or 24 samples. Three tissue samples were used for the blood culture
method from each patient in most cases. With the exception of six patients, from
whom only two or one tissue sample was received in some cases.
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Implants submitted for diagnosis
16
14
12
10
8
6
4
2
0
Figure 8: Implants submitted for diagnosis. This diagram shows the types of
joint tissue used for this study.
The state hospitals in Vorarlberg sent in the most tissue samples from hip joints,
followed by knee joints. The exact data can be found in Figure 8. One sample
each from a shoulder, lower leg, and lumbar spine was also processed.
Number of negative cases in
comparison
Solid culture
method
Blood culture
method
0 2 4 6 8 10 12 14
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Figure 9: Number of negative cases in comparison. This bar chart shows
the number of negative samples from both the solid culture method and the
blood culture method.
As can be seen in Figure 9, there is no difference in the number of negative
samples. The results m a t c h 100%. Both in routine and in the blood culture
method, the negative results always match. It follows that both methods have
identical specificity.
Comparison of the number of
positive cases
Solid culture
method
Blood culture
method
0 2 4 6 8 10 12
Figure 10: Comparison of the number of positive cases. These two bars
show the number of positive samples obtained using the conventional method
and the blood culture bottle method.
Similarly, the positive results of both methods in Figure 10 match 100%, and there
were no false positive results. Thus, the sensitivity is identical for both methods.
To explain the results of this study, Table 8 is probably the most informative. All
samples are listed in ascending order. The order was determined based on the
date of submission of the samples and in the following tables
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continued to be handled in this way. Each patient has his or her own number. The
three columns next to the sample number are for the results of the conventional
method and the blood culture method, respectively. In addition, the last column is
for comments.
Table 8: The results of both methods and comments
Sample number Solid culture Liquid culture Comments
method method
Sample 1 Negative Negative -
Sample 2 Negative Negative -
Sample 3 Positive Positive Patient double
(see sample 13)
Sample 4 Negative Negative -
Sample 5 Negative Negative -
Sample 6 Positive Positive -
Sample 7 Negative Negative -
Sample 8 Negative Negative Contaminants on
solid culture method
Sample 9 Negative Negative -
Sample 10 Negative Negative Contaminants on
solid culture method
Sample 11 Negative Negative -
Sample 12 Negative Negative -
Sample 13 Positive Positive -
Sample 14 Positive Positive -
Sample 15 Positive Positive -
Sample 16 Positive Positive Contaminants on
solid culture method
Sample 17 Positive Positive Patient double
(see sample 21)
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Sample 18 Negative Negative Contaminant using solid
culture method and
patient double (see
Sample 20)
Sample 19 Negative Negative -
Sample 20 Positive Positive -
Sample 21 Negative Negative -
Sample 22 Positive Positive -
Sample 23 Positive Positive -
Sample 24 Positive Positive -
Samples numbered three, 17, and 18 are from patients from whom tissue samples
were taken twice at different times. While several contaminations, four to be
precise, occurred with the conventional solid culture method, there were no false
positive cases with the blood culture method. Contamination occurs when, in
addition to the main pathogen with a high bacterial count, additional
microorganisms with a low bacterial count are detected. It can therefore be
concluded that the blood culture method did not produce any false positive
results. Samples that show a further discrepancy between the methods are cases
14 and 16. The two samples produced different results in both methods. Table 9
shows that two of four tissue samples from sample 14 and two of three tissue
samples from sample 16 were positive. In both cases, sonication yielded a positive
result. The liquid culture method showed a positive result in only one of three
sample sets. However, sonication must be taken into account. It can therefore be
concluded that both samples confirm an infection in both methods.
Table 9 lists all positive samples from the conventional method, the number of
tissue samples submitted, and the number of positive tissue samples. For a case
to be classified as positive
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, the same pathogen must be detectable in at least two samples. With the liquid
culture method, the same pathogen also had to be present in two out of three
sets in order to be classified as positive. However, the sonification result is
decisive in both methods. For example, if fewer than two out of three tissue pieces
or sets were positive, the result of sonication determined whether the case had to
be evaluated as positive or negative. Sonication was not performed on most
samples. Four sonications (samples 14, 16, 22, and 23) yielded a positive result.
Table 9: Number of positive tissues and results of sonication of
the solid culture method
Sample number Number of tissues Of which positive Sonification
Sample 4 4 -
Sample 6 1 1 -
Sample 13 4 4 -
Sample 14 4 2 Positive
Sample 15 2 2 -
Sample 16 3 2 Positive
Sample 17 5 5 -
Sample 20 3 3 -
Sample 22 2 2 Positive
Sample 23 3 2 Positive
Sample 24 5 4 -
Table 10 below lists both the number of blood culture bottle sets used and the
number of positive sets. One set corresponds to one aerobic and one anaerobic
bottle. The same table also shows the shortest times to a positive result. The
minimum time is only two hours, 59 minutes, and ten seconds, while the
maximum time is four days, 22 hours, and 20 minutes. This illustrates that the time
span to a positive result
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Bottle is highly variable. However, it is noteworthy that none of the cases reached
the 14-day mark. It is also striking that all samples became positive within less
than seven days, regardless of whether they were anaerobic or aerobic bottles.
Even with anaerobes, such as sample 23, positive results were achieved within
four days, 22 hours, and 20 minutes. Data from Table 10 show that aerobic bottles
tended to turn positive faster than anaerobic ones. After 24 hours of incubation,
the BACTEC-FX evaluated 60.87% of anaerobic bottles and 90% of aerobic
bottles as positive. After 24 hours of incubation, 74% of all sets were evaluated as
positive.
Table 10: Number of positive sets using the blood culture bottle method,
shown with the shortest TTP
Sample Sets of which Positive TTP TTP aerobic
bottles
number positive anaerobic
Sample 3 3 3 6/6 00d 06h -
14 min 16
sec
Rehearsal 6 1 1 1/1 00d 10h -
06min
00sec
Sample 13 3 3 5/6 - 00d 15h
15 min 44
sec
Rehearsal 14 3 1 2/6, - 00d 16h
Sonification 38min
positive 27sec
Sample 15 2 2 4/4 00d 12h -
13min
42sec
Sample 16 3 1 2/6, - 01d 03h
Sonification 44min
positive 58sec
Sample 17 3 3 6/6 - 00d 07h
38min
39 sec
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Rehearsal 3 3 6/6 - 00d 02h
20
59 min 10
sec
Rehearsal 1 1 1/1 - 00d 17h
22
03min
11 sec
Rehearsal 3 3 3/6 04d 22h Negative
23
20min
00sec
Sample 24 3 3 5/6 00d 19h -
07min
34sec
The results of the MALDI are shown in the last table 11. The results of both
methods are consistent.
Table 11: Microorganisms identified in the positive samples using both
methods
Sample number Solid culture method Blood culture method
Sample 3 E. faecium E. faecium
Sample 6 S. aureus S. aureus
Sample 13 E. faecium E. faecium
Sample 14 S. haemolyticus S. haemolyticus
Sample 15 S. epidermidis S. epidermidis
Sample 16 S. lugdunensis S. lugdunensis
Sample 17 S. aureus S. aureus
Sample 20 E. clocae complex E. clocae complex
Sample 22 S. epidermidis S. epidermidis
Sample 23 C. acnes C. acnes
Sample 24 S. aureus S. aureus
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Results of pathogen identification using both
4 methods
3
2
1
0
E. faecium S. aureus S. haemolyticus S. epidermidis S. lugdunensis E. clocae C. acnes
Complex
F i g u r e 11: Frequency of pathogens identified by both methods. This
bar chart shows all pathogens identified using MALDI.
The most frequently detected bacterium is S. aureus. S. epidermidis and E.
faecium bacteria occur with equal frequency. All other bacteria were detected only
once in samples. These results are shown in Figure 11.
The results of sample 23 allow us to conclude that the initially recommended
incubation period of 14 days can be reduced to a maximum of seven days.
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4 Conclusion
This study examined 24 patients, analyzing a total of 64 tissue samples. Using the
blood culture method, 13 of the 24 samples tested were negative and 11 were
confirmed infections, which was also confirmed by the conventional solid culture
method. However, four contaminations occurred with the solid culture method.
The TTPs achieved indicate that the incubation period can be reduced from a
maximum of 14 days to a maximum of 7 days. The most frequently identified
pathogen was S. aureus, followed by E. faecium and S. epidermidis.
It should be noted that the blood culture method has several positive aspects. On
the one hand, no samples were contaminated, which increases the reliability of the
results. The liquid culture method enabled rapid diagnosis, as approximately 74%
of samples were positive after just 24 hours. The blood culture method is very
sensitive, meaning that infections are not overlooked. No false-negative or false-
positive results occurred in the present study. The results of the blood culture
method are consistent with the results of routine tests, confirming its reliability. The
diagnostic process can be accelerated with the blood culture method, as it would
generally be sufficient to incubate the BC bottles for a maximum of seven days
instead of 14 days.
The results of this work provide valuable insights into optimizing incubation times
and improving methods for identifying pathogens in clinical samples.
The examination of blood cultures will be important for further development . It
requires further research. A central
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focus should be on shortening incubation times, especially for anaerobic bacteria.
New technologies should also be developed to improve sampling and thus prevent
contamination. Overall, different areas of research offer opportunities to improve
the diagnosis and treatment of infections.
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5 Bibliography
AxonLab AG. (n.d.). SpinAX Dispersing and Homogenizing Device. Polling,
Switzerland: AxonLab AG. Retrieved June 7, 2024
Bandelin electronic GmbH & Co. KG. (2020). bandelin.com. Retrieved on
May 28, 2024, from bandelin.com: https://bandelin.com/wp-
content/manuals/BS 6366-006_EN_BANDELIN.pdf
Becton, Dickinson and Company. (2011). www.bd.com. Retrieved February 27,
February 27, 2024, from www.bd.com:
https://www.bd.com/resource.aspx?IDX=8624
Becton, Dickinson and Company. (2016). www.bd.com. Retrieved February 27,
February 27, 2024, from www.bd.com:
https://www.bd.com/resource.aspx?IDX=25602
Becton, Dickinson and Company. (February 2021). www.static.bd.com.
Retrieved February 28, 2024, from www.static.bd.com:
https://static.bd.com/documents/eifu/8005110_ZLU_D_SD_8005110_
DE.pdf
bioMérieux SA. (May 2021). PREVI® COLOR GRAM and cytocentrifuge rotor.
Lyon, France: bioMérieux SA. Retrieved June 7, 2024, from 07.
Calderaro, A., & Chezzi, C. (February 3, 2024). pubmed.ncbi.nlm.nih.gov.
doi:10.3390/microorganisms12020322
Li, C., Renz, N., Trampuz, A., & Ojeda-Thies, C. (October 22, 2019).
pubmed.ncbi.nlm.nih.gov. doi:10.1007/s00264-019-04426-7 McNally,
M., Sousa, R., Wouthuyzen-Bakker, M., Chen, A., Soriano, A.,
Vogely, H., . . . Trebše, R. (January 2021). pubmed.ncbi.nlm.nih.gov.
doi:10.1302/0301-620X.103B1.BJJ-2020-1381.R1
Memmert GmbH + Co KG. (November 2014). www.memmert.com.
Retrieved February 28, 2024, from www.memmert.com:
https://www.memmert.com/index.php?eID=dumpFile&t=f&f=2771&tok
en=420bb30982313abf3a3b5d10e5f2f04ff48f8287
Patel, R. (January 19, 2023). pubmed.ncbi.nlm.nih.gov.
doi:10.1056/NEJMra2203477
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6 Index
Abbreviati
on
AAOS Association of Orthopedic Surgeons
BSG Blood sedimentation rate
CFU colony forming units
CNA Columbia nalidixic acid agar
CO2 carbon dioxide
CRP C-reactive protein
DAIR Debridement, antibiotics, and implant retention
LED Light-emitting diodes
MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
MRSA Methicillin-resistant Staphylococcus aureus
NaCl sodium chloride
OS Organic solvent solution
PJI Periprosthetic joint infection
PMN polymorphonuclear cell
PVX Mueller-Hinton chocolate agar
SK1 Schaedler agar with vitamin K1
TTP Time-to-positivity
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7 List of figures
Figure 1: Microorganisms frequently detected in implant infections. These
pathogens were most commonly detected in knee and hip joint prostheses.
(Patel, 2023, p. 253).................................................................................................8
Figure 2: Risk factors for PJI. These factors, some of which are preventable,
increase the risk of PJI. (Patel, 2023, p. 254) ..........................................................9
Figure 3: SpinAX. The structure and function keys of the SpinAX are shown here.
(AxonLab AG, n.d., p. 7) ........................................................................................19
Figure 4: ProbeAX Evolution Tubes. The ProbeAX Evolution tube consists of a
rasp and a cylinder. (AxonLab AG, n.d., p. 9) . 20 Figure 5: Procedural
principles of the BACTEC-FX. This shows how the BACTEC-FX checks cultures
for microorganisms. (Becton, Dickinson and Company, 2021, pp. 9-10) ..............21
Figure 6: Principle of MALDI-TOF. Laser pulses ionize the molecules. These
ions are accelerated in the flight tube, producing spectra that are necessary for the
identification of microorganisms. (Calderaro & Chezzi, 2024, pp. 3-4) .................24
Figure 7: Structure and components of the Previ Color Gram. The Previ Color
Gram consists of several components and is used for the efficient and
environmentally friendly staining of sample smears. (bioMérieux SA, 2021, pp. 1-1,
1-2, 3-1, 3-6) ..........................................................................................................25
Figure 8: Implants submitted for diagnosis. This diagram graphically shows the
types of joint tissue used for this study. .................................................................37
Figure 9: Comparison of the number of negative cases. This bar chart shows
the number of negative samples obtained using the solid culture method and the
blood culture method. ............................................................................................38
Figure 10: Comparison of the number of positive cases. These two bars
provide information about the number of positive samples from the conventional
method and the blood culture bottle method. .........................................................38
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Figure 11: Frequency of pathogens identified by both methods. This bar chart
graphically illustrates all pathogens identified using MALDI...................................44
50
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8 List of tables
Table 1: List of equipment used .........................................................................17
Table 2: List of reagents for the Previ Color Gram...........................................26
Table 3: List of materials used for preparing tissue samples .........................28
Table 4: List of materials used for BACTEC-FX ................................................29
Table 5: list of materials used for the for solid
culture incubation ................................................................................................32
Table 6: List of materials used for pathogen
identification 33
Table 7: List of materials used for the BactoSonic...........................................35
Table 8: Results of both methods and comments
...............................................................................................................................39
Table 9: Number of positive tissues and results of sonication of the solid
culture method .....................................................................................................41
Table 10: Number of positive sets of the blood culture bottle
method shown with the shortest TTP ................................................................42
Table 11: Microorganisms identified in positive samples using both methods
...............................................................................................................................43
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9 Affidavit
Student data
Last name First name
Özgün, Rana Ebru
Title
Personal identification 2110496015
number
Degree program/course Biomedical Analysis
Year, class BMABsc_2021
I hereby declare that I have written my bachelor's thesis on the topic
Comparison of microbiological diagnostics of tissue
samples using solid cultures and blood cultures in
periprosthetic and implant infections
……………………………………………………………………………………………
Prepared as part of the course
FLB6P - Research-based learning in the professional field, bachelor's thesis
independently and have not used any sources or aids other than those indicated,
and have identified all passages taken verbatim or in substance from other texts
as such. This applies to printed texts as well as to texts taken from the Internet,
audiovisual media, audiobooks, and image credits.
Innsbruck, June 7, 2024
Place, date Signature of the student
52