University of Groningen

Master of Medicine

Thesis:

The diagnostic application of sonication on heart

valves with suspected infective endocarditis

Student name: Kasper L. B. Bijker

Student number: 1956728

Supervisors: Mw. Anna Gomes, Dr. Marleen van Oosten, Dr. Greetje A.

Kampinga & Prof. Dr. Bhanu Sinha

Institution: University Medical Center Groningen

Department: Medical Microbiology

Date of Submission: 04-07-2016

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Abstract

Introduction: Infective endocarditis (IE) is a very serious disease. Although relatively

uncommon (5-11 per 100,000 people per year), it is associated with a high mortality and

morbidity. The infection is predominantly caused by bacteria. However, in 10-20% of cases

no causative organism is identified. This is a serious limitation to the ability of clinicians to

provide the afflicted patients with optimal treatment.

Infected heart valves may become damaged through the disease process, resulting in

impaired functioning. Valves affected by IE may require surgical replacement. An explanted

heart valve may provide important additional opportunity to obtain information pertaining to

the causative microorganism, particularly in cases of blood culture-negative IE. However,

standard microbiological tests on explanted heart valves frequently lead to negative results

(up to 49%). An increase in sensitivity for the detection of microorganisms would be

beneficial to the diagnosis and treatment of IE.

Sonication has demonstrated an increased microbiological yield when carried out on

various prosthetic materials that were suspect for infection, mainly in prosthetic joint

infections. The added value is particularly apparent in material cultures of patients that were

receiving prior antimicrobial therapy. It is hypothesized that similar added value may be seen

when applied to explanted (prosthetic or native) heart valves.

Methods: Through a prospective case-control study, the results obtained from the

experimental sonication protocol were compared to the results of the standard diagnostic

protocol for explanted cardiac material. Results were obtained through cultures on agar plates,

inoculation of aerobic and anaerobic blood culture bottles, and 16S PCRs. The sonication tests

were always done after the standard workup, avoiding interference with regular diagnostics

necessary for the clinics. Explanted valves with no suspicion of IE were used as negative

controls, in order to assess the reliability of the method.

Based on a power analysis for dependent samples with α=0.05, β=0.80, and d=0.3, the

necessary number of valves was determined to be n=16. Based on the UMCG’s records, the

expected time needed to collect 16 valves per study group would exceed 1 year. The project

was set up as a pilot study in order to evaluate and optimise the proposed study protocol, and

to start with the collection of patient data. The results presented in this report are therefore

limited to the number of valves collected within a 3 month period, between 01-04-2016 and

01-07-2016.

Results: Fourteen valves were included, of which 7 were suspected of IE. Polymerase chain

reaction (PCR) methods identified microbial deoxyribonucleic acid (DNA) from present

microorganisms on 4 of these valves. Cultures identified the presence of viable

microorganisms on 2 of these valves. All negative control valves showed no evidence of

microbial presence according the standard diagnostic method and the sonication protocol.

Notably, there was one clinical case that illustrates the potential for sonication as an

addition to the standard workup for explanted valves from patients with IE, by making a

significant contribution to the diagnosis and resulting therapy. Indeed, only sonication was

able to demonstrate the presence of viable Propionibacterium acnes (P. acnes) in this case of

culture-negative IE. The presence of P. acnes was confirmed by 16S PCR on the heart valve.

Conclusion: Based on the currently available data there is insufficient evidence to draw

definite conclusions, though initial results are promising. However, this project enabled me to

carefully formulate and validate a standard operating procedure (SOP) for the sonication of

heart valves in the microbiology laboratory. This method has demonstrated the ability to

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detect the presence of microorganisms. In one case sonication demonstrated additive value by

detecting the presence of a bacterium through cultures that was not cultured using the

standard diagnostic protocol, allowing for antibiotic susceptibility testing, which is crucial for

final patient treatment. This finding suggests that there seems to be benefit to the detection of

microorganisms by using sonication. Continuation of data collection will be needed to provide

conclusive evidence.

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Abstract (NL)

Introductie: Infectieuze endocarditis (IE) is een ernstige ziekte. Hoewel relatief zeldzaam (5-

11 gevallen per 100,000 personen per jaar), leidt deze ziekte tot een hoge mortaliteit en

morbiditeit. Deze cardiale endovasculaire infectie wordt voornamelijk door bacteriën

veroorzaakt. Echter, in 10-20% van de gevallen van IE wordt er geen veroorzakend organisme

geïdentificeerd. Het ontbreken van een verwekker is een serieuze beperking voor het

verstrekken van een optimale behandeling aan de betreffende patiënt.

Geïnfecteerde hartkleppen raken mogelijk beschadigd door het ziekteproces, wat leidt

tot verminderde klepfuntie. Kleppen die zijn aangedaan door IE moeten mogelijk chirurgisch

vervangen worden. Een verwijderde hartklep zou mogelijk een belangrijke bijdragen kunnen

leveren aan het identificeren van het micro-organisme in gevallen van IE, met name in

situaties waarbij bloedkweken negatief zijn. Echter, de huidige microbiologische onderzoeken

die worden toegepast op kleppen zijn niet altijd in staat om bacteriën aan te tonen. Tot 49%

van deze kleppen leiden tot negatieve testresultaten. Een toename van sensitiviteit bij de

detectie van micro-organismen zou bevorderlijk zijn voor het stellen van de diagnose en het

bepalen van de optimale behandeling bij IE.

Studies naar geïnfecteerd prothesemateriaal hebben aangetoond dat het toepassen van

sonicatie leidt tot een grotere microbiologische opbrengst, met namen bij infecties van

gewrichtsprothesen. De toegevoegde waarde van sonicatie is vooral duidelijk in gevallen

waarbij patiënten recent behandeld zijn geweest met antimicrobiële middelen. De hypothese

is dat er een mogelijk vergelijkbare toegevoegde waarde te behalen is bij verwijderde

(prothese of natieve) hartkleppen die verdacht zijn voor IE.

Methode: Door middel van een prospectieve case-control studie zullen de resultaten van het

huidige microbiologische diagnostische onderzoeken (standaard uitwerking) vergeleken

worden met deze onderzoeken plus sonicatie. De resultaten waren verzamend op basis van

kweken op platen, inoculatie van bloedkweekflesjes, en 16s PCR. Sonicatie en de

bijbehorende onderzoeken werden na de standard uitwerking uitgevoerd om niet de klinisch

belangrijke reguliere diagnostiek te beperken of anderszins te verstoren. Tevens werden

verwijderde hartkleppen van patiënten zonder verdenking op infectie (IE) gebruikt als

negatieve controles, om de betrouwbaarheid van de methode te controleren.

Een poweranalyse voor gepaarde steekproeven (dependent samples) met een α=0.05,

β=0.80, en d=0.3, gaf aan dat n=16 het aantal benodigde kleppen is per studiearm. Op basis

van beschikbare gegevens van het UCMG is het de verwachting dat het verzamelen van de

benodigde aantal kleppen langer dan 1 jaar zal duren. Dit project was opgezet als pilot studie

om het studieprotocol te ontwikkelen, evalueren en optimaliseren. Tevens wou ik gedurende

mijn stage een begin maken met het verzamelen van data. De resultaten die in dit verslag

gepresenteerd worden zijn op basis van de kleppen die zijn verzameld gedurende een periode

van 3 maanden, van 01-04-2016 tot 01-07-2016.

Resultaten: Veertien hartkleppen werden geïncludeerd, waarvan 7 verdacht waren voor IE.

PCR identificeerde microbiële desoxyribonucleïnezuur (DNA) van aanwezige micro-

organismen bij 4 van deze hartkleppen. Kweken leiden tot de detectie van micro-organismen

bij 2 van deze van IE verdachte kleppen. Alle negatieve controle hartkleppen toonden geen

aanwijzingen voor de aanwezigheid van micro-organismen, volgens de standaard diagnostiek

en het sonicatie protocol.

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Eén klinische casus illustreerde de potentiele waarde van sonicatie als toevoeging aan

de standard uitwerking voor verwijderde hartkleppen bij patiënten met IE. In dit geval, van

een patiënt met een bloedkweek negatieve IE, werd alleen d.m.v. kweken bij sonicatie de

aanwezigheid van levende Propionibacterium acnes (P. acnes) aangetoond. Dit was

bijdragend aan de diagnose en behandeling van deze patiënt. De aanwezigheid van P. acnes

werd bevestigd door 16S PCR uitgevoerd op hartklep materiaal.

Conclusie: Op basis van de momenteel beschikbare resultaten, is er onvoldoende bewijs om

concrete conclusies te trekken t.a.v. de toegevoegde waarde van sonicatie als onderdeel van

de standaard diagnostische verwerking van verwijderde hartkleppen bij patiënten met (de

verdenking voor) IE. Echter zijn de eerste resultaten veelbelovend. Tevens kon ik gedurende

deze stage het onderzoeksprotocol vormgeven en uitvoering valideren. De ontwikkelde

methode heeft aangetoond in staat te zijn om aanwezige micro-organismen aan te tonen. In

één geval was de sonicatiemethode in staat om een bacterie te kweken die niet werd gekweekt

door de standaard diagnostische kweken. De bevinding suggereert dat er inderdaad een

toegevoegde waarde zou zijn voor het toepassen van sonicatie bij de detectie van micro-

organismen op verwijderde hartkleppen. Verdere dataverzameling zal nodig zijn om met

harder bewijs uitsluitsel te geven aan dit vraagstuk.

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I hereby acknowledge and accept that this thesis will be included in the collection of

Rijksuniversiteit Groningen. I authorize the release of this thesis to any reader upon request.

With my signature I affirm that the work represented in this thesis is my own.

Kasper Bijker,

Student, Author

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Table of Contents

Abstract ................................................................................................................ 1

Abstract (NL) ....................................................................................................... 3

List of Abbreviations ........................................................................................ 8

Introduction ......................................................................................................... 9

1. Infective Endocarditis ................................................................................ 9

1.1 Definition .................................................................................................. 9

1.2 Pathophysiology ........................................................................................ 9

1.3 Epidemiology ............................................................................................ 9

1.4 Comorbidities .......................................................................................... 10

1.5 Clinical Presentation ............................................................................... 10

1.6 Diagnostics .............................................................................................. 10

1.7 Therapy ................................................................................................... 12

2. Sonication .................................................................................................. 12

2.1 Mechanism .............................................................................................. 12

2.2 Current Scientific Evidence .................................................................... 13

The aim of the study .......................................................................................... 14

3.1 The Aim .................................................................................................. 14

3.2 Hypotheses .............................................................................................. 14

3.3 The Aim of this Thesis ............................................................................ 15

Methods .............................................................................................................. 16

4. Study Design ............................................................................................. 16

5. Number & Inclusion of Patients ............................................................. 17

5.1 Numbers Needed ..................................................................................... 17

5.2 Valve Inclusion Criteria .......................................................................... 17

5.3 Expected Inclusion Rate.......................................................................... 18

6. Ethics & Consent...................................................................................... 18

7. Preliminary Work .................................................................................... 19

7.1 Orientation............................................................................................... 19

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7.2 Developing the Sonication Protocol ....................................................... 19

8. Data Collection ......................................................................................... 21

8.1 Patient Medical Data ............................................................................... 21

8.2 Heart Valve Data ..................................................................................... 21

9. Data Analysis ............................................................................................ 27

Results ................................................................................................................. 28

10. Included Valves ........................................................................................ 28

11. Culture Results ......................................................................................... 28

12. Case Report ............................................................................................... 30

Discussion ........................................................................................................... 31

13. Overall Findings ....................................................................................... 31

14. Case Report ............................................................................................... 32

15. Study Limitations ..................................................................................... 32

Conclusion .......................................................................................................... 34

16. What I have learned ................................................................................. 34

Acknowledgements ............................................................................................ 36

Bibliography ....................................................................................................... 37

Appendix A: modified Duke criteria ............................................................ 42

Appendix B: Patient Consent Form .............................................................. 44

Appendix C: Certificate Microbiological Safety Techniques ...................... 45

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List of Abbreviations

16S-rDNA

16S ribosomal deoxyribonucleic acid

AVR

aortic valve replacement

BA

blood agar

BBA

Brucella blood agar

CFU

colony forming unit

CHOC

chocolate agar

CT

computed tomography

DNA

deoxyribonucleic acid

HACEK

Haemophilus spp., Actinobacillus (Aggregatibacter)

actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens,

Kingella kingae/Kingella denitrificans

IE

infective endocarditis

IV

intravenous

MALDI-TOF

matrix-assisted laser desorption/ionization time-of-flight mass spectrometer

PCR

polymerase chain reaction

PET

positron emission tomography

RCF

relative centrifugal force

SOP

standard operating procedure

TEE

transoesophageal echocardiography

TTE

transthoracic echocardiography

T-streak

three-phase streaking pattern

UMCG

University Medical Center Groningen

WMO

Law: medical scientific research on humans

(wet medisch-wetenschapelijk onderzoek met mensen)

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Introduction

In order to comprehend the relevance of this study it is crucial have an understanding of the

context of the disease and the potential diagnostic role of sonication for patient suffering from

infective endocarditis. This section introduces infective endocarditis and the mechanism

behind sonication. The current application of sonication in diagnostics and the relevance to

infective endocarditis are briefly discussed.

1. Infective Endocarditis

1.1 Definition

Infective endocarditis (IE) is an endovascular infection of cardiovascular structures,

particularly involving cardiac valves, endocardium, major intrathoracic vessels, and

intracardiac foreign objects (prosthetic valves, pacemaker leads, etc.) [1,2].

1.2 Pathophysiology

The primary aetiology of infective endocarditis is based on two main components; the

presence of microorganisms in the bloodstream (bacteraemia) and an abnormal cardiac

endothelium that facilitates microbial adherence and proliferation [2-5]. The presence of

foreign material, such as a prosthetic heart valve, provides a surface that predisposes to

infiltration and infection by microorganisms [6].

The vast majority of IE cases are the result of extracellular bacteria [3].Streptococci and

staphylococci account for the majority of infections. However, infections can also be caused

by various fungi and several intracellular bacteria [7].

The most common causative organisms for IE:

- Staphylococcus aureus 20-31%

- Viridans group streptococci 17-50%

- Coagulase negative staphylococci 11%

- Enterococci 11%

- Streptococcus bovis (7%)

- HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans,

Cardiobacterium hominis, Eikenella corrodens, Kingella kingae/Kingella

denitrificans) 2%

- Fungal infection 2% [6,8]

In approximately 8-20% of suspected cases of IE, no causative organism is identified through

standard diagnostic tests [6,9]. These culture-negative cases may be the result of

antimicrobial therapy initiated prior to obtaining blood cultures [4].

1.3 Epidemiology

The incidence of IE in developed countries is reported to be in the range of 5-11 per 100,000

people per year [2,10].

Population characteristics:

- Age: more than 50% of cases are found in patients above the age of 60 years. A

steady increase of the median age has been measured during the previous decades

[11].

- Gender: men are more frequently affected than women; the male-to-female ratio is

reported to be as high as 9:1 [12].

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1.4 Comorbidities

There are a large number of factors and comorbidities that are associated with the

development of infective endocarditis.

- Prosthetic material: foreign material, such as prosthetic heart valves and pacemaker

leads, are at risk for bacterial colonization and the development of a subsequent

infection [13]. Patients with a prosthetic heart valve have up to 3% chance of

developing IE in the first year following implantation; up to 6% in the first 5 years

[14].

- Heart disease: structural heart disease, valvular heart disease, and congenital heart

disease are risk factors [6,15]. A prior episode of (infective) endocarditis increases the

probability of developing a second episode of IE.

- Oral cavity problems: Poor dental hygiene and undergoing procedures involving the

oral cavity is thought to increase the risk of developing IE [4,16].

- Intravenous drug use: intravenous (IV) drug users are a high-risk group. These

individuals are associated with endocarditis affecting the right side of the heart [17].

1.5 Clinical Presentation

Patients suffering from IE generally present with non-specific symptoms [18,19]. Pyrexia

(fever) is the most common sign; up to 90% of patients. Fever frequently occurs in

combination with malaise, fatigue, anorexia, dyspnoea, asthenia, arthralgia, abdominal pain,

headache, night sweats, and/or weight loss [20].

Careful physical examination is an important aspect of the clinical investigation in

patients with IE. Certain findings may help determine the diagnosis.

Common clinical signs associated with IE include [21]:

- Elevated body temperature (>90%)

- Cardiac murmur (80-85%)

- Embolic phenomenon (>50%)

- Splenomegaly (20-55%)

Janeway lesions, Osler nodes, and Roth spots are relatively uncommon (~2%) phenomena,

although highly indicative for IE [1].

If left untreated, infective endocarditis has a mortality of 100% [2].The in-hospital mortality

is approximately 20% and 30-40% after 1 year despite aggressive treatment [8,20,22].

Common complications of IE include [23]:

- Heart failure; usually resulting from valvular insufficiency

- Perivalvular abscess formation and/or pseudo-aneurysm formation; resulting from

local disease progression

- Pericarditis

- Septic embolization

o metastatic abscesses

o mycotic aneurysms

o Arterial insufficiency (kidney, spleen, etc.)

o cerebrovascular events

1.6 Diagnostics

It is important to be vigilant of possible cases of infective endocarditis, due to the serious

nature of the disease, the high mortality and severity of complications, and the fact that the

symptoms tend to be non-specific. This diagnosis if IE should always be considered in cases

with fever in combination with a new cardiac murmur, new valve insufficiency, certain pre-

11

existing heart conditions1, signs of heart failure, new vascular or immunological

phenomenon, peripheral abscess formation, chronic non-specific symptoms, and/or

unexplained (persistent) positive blood cultures [4].

The process of establishing the clinical diagnosis, in cases of (suspected) infective

endocarditis, regularly includes the use of a scoring-system based on standardised criteria.

The most commonly used system is the modified Duke criteria.2 This method of assessment

is based on pathological and clinical evidence for the presence of IE [24]. The clinical

evidence is composed of two “Major” criteria (positive blood cultures and evidence of

endocardial involvement) and several “Minor” criteria. Depending on the combination of

clinical major and/or minor criteria, the likelihood of the diagnosis IE is determined.

However, it is important to always bear in mind that the clinical criteria never completely

prove or refute the diagnosis. Pathological evidence, as defined in the criteria, is diagnostic

for IE if determined to be positive.

The diagnostic value of the modified Duke criteria is only achieved if all the

information required by the criteria is properly collected. Although the modified Duke criteria

do allow for systematic assessment of patients with potential signs of infective endocarditis,

thereby guiding the clinician to a (working) diagnosis, it is primarily a tool for grouping

patients according to degree of certainty for the diagnosis IE. The sensitivity and specificity

of this tool are both approximately 80% [24].This means that there is a significant role for an

expert panel in making, or rejecting, the diagnosis of infective endocarditis [18].

The microbiological culture is a cornerstone of the modified Duke criteria and crucial

for the treatment of (suspected) IE diagnosis. In order to provide appropriate antimicrobial

therapy, it is essential to determine the causative organism and its respective sensitivity and

resistance patterns [25]. The detection of microorganisms occurs in current clinical practice

through culturing of the patient’s blood; it is important that this is obtained before the start of

antimicrobial therapy.

Explanted valves from patients with (suspected) IE may also be used for

microbiological culture. Under certain clinical circumstances surgical replacement of the

affected valve may be necessary [26,27]. The use of heart valves for microbiological cultures

provides the ability to reinforce the diagnosis and assist in the process of determining the

appropriate therapy. Particularly in blood culture-negative IE this is potentially crucial. The

standard workup of heart valves includes a direct culture of explanted material on agar plates.

The diagnostic yield from cultures of heart valves is frequently limited. False-negative

microbiological results are found in 12-49% cases [28]. False negative cultures are regularly

associated with prior antibiotic use and biofilm-associated infection of implanted foreign

materials [25,29,30,31,32]. However, despite negative cultures, viable microorganisms may

still be present within the protective biofilm [28,33]. These organisms have the potential to be

cultured if they can be liberated from their biofilm [34].

UMCG:

In the University Medical Center Groningen (UMCG) a diagnostic protocol has been

implemented for guidance in the workup of cases suspected of IE. This guides the medical

professionals to take all the necessary steps in order to determine the validity of the suspicion.

This protocol includes the collection of information needed to properly employ the modified

Duke criteria.

1 Heart conditions associated with IE according to the BSAC: valvular heart disease, valve replacement, previous

infective endocarditis, hypertrophic cardiomyopathy, and structural (congenital) heart disease 2 Please refer to Appendix: A (modified Duke criteria)

12

The diagnostic microbiology laboratory uses a standard method in the processing of

explanted heart valves suspected for IE. In short, this standard diagnostic method involves

making cultures on solid growth medium plates through direct contact with the heart valves,

A glass slide is smeared with the heart valve and a gram staining is carried out, and a

polymerase chain reaction (PCR) analysis of valve tissue is done in order to detect the

presence of microbial ribosomal deoxyribonucleic acid (rDNA).

1.7 Therapy

The UMCG uses a multidisciplinary team in order to determine appropriate therapeutic steps

in cases of suspected infective endocarditis. This is reflected in the dedicated protocol used in

this hospital.

There are multiple extensive guidelines for the management of infective

endocarditis.[4,18] These suggest that the procedure for initiating antimicrobial therapy is

guided by the clinical presentation.[19,35]

In the situation of patients presenting with sub-acute illness it is recommended to first

perform the necessary diagnostic steps in order to confirm the diagnosis and determine the

causative organism. Based on these results the optimal therapy can consequently be selected

and provided to the patient.

Acute illness associated with (suspected) IE is an indication for the rapid start of

empiric antimicrobial treatment. This empiric therapy should only be administered after (at

least) three sets of blood cultures have been obtained [36,37]. Multiple sets of blood samples

increases the sensitivity for detection of microorganisms in the blood; improving the

probability of identifying the causative organisms. Additionally, positive blood cultures over

time intervals indicate persistent bacteraemia, which further suggests the presence of IE. The

therapy should be adjusted according the culture results.

The standard duration for antimicrobial therapy is 6 weeks, for ‘left-sided’ IE. In specific

cases of ‘right-sided’ IE this period may be shorter [4]. Treatments are routinely followed by

comprehensive reassessment. Alterations in the treatment may be subject to changes in the

clinical status of the patient. The pharmacological treatment of infections associated with

prosthetic heart valves are particularly challenging [38].

Certain clinical circumstances may indicate surgical treatment. All surgical

interventions require an individualized multidisciplinary approach [27]. Considerations for

surgery include signs of heart failure, annular or aortic abscess formation, infection due to a

difficult-to-treat pathogen, persistent infection despite appropriate antimicrobial therapy, and

emboli formation [19,26].

2. Sonication

2.1 Mechanism

Sonication is a technique that employs the treatment of materials with sound waves of an

ultrasonic frequency through a liquid medium, in order to improve the detection of

microorganisms. These ultrasonic vibrations result in pressure waves in the liquid medium

that form microscopic oscillating cavitation bubbles. The implosion of these bubbles results

in the release of energy in the form of local shockwaves and micro voltages. Sonication has

the capacity to selectively break down biofilm, while leaving cell structures sufficiently intact

to allow for culturing and analysis.

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The resulting suspension of microorganisms in the liquid medium is known as the

“sonicate”. This suspension of organisms may be used for microbiological cultures and

therefore a potentially valuable addition to diagnostics.

Figure 1: The mechanism of sonication in a liquid medium. Pressure (sound) waves of an ultrasonic frequency

cause the formation of microscopic “cavitation bubbles”. These bubbles implode, resulting in the release of

energy. (Source: modified image obtained from the website of EpiGentek)

2.2 Current Scientific Evidence

Culturing sonicate of (prosthetic) materials leads to a significantly higher microbiological

detection, in comparison to a direct tissue/material culture [39].

Improved detection of microorganisms through the use of sonication has been

demonstrated in explanted orthopaedic implanted prosthesis suspected for infection [31,40-

47]. Studies on explanted hip and knee prostheses demonstrated an increase in culture

sensitivity from 61% to 79%, through the use of sonication [31]. Similarly, in case of

suspected prosthetic shoulder infection, cultures of sonicate demonstrated a higher sensitivity

(67%) than cultures of periprosthetic tissue (55%) [48].Furthermore, sonication allowed for

the identification of the causative organism in 82% of patients with osteosynthesis-associated

infection, while in significantly less (39%) cases this was achieved through direct tissue

culture [49].

A notable difference in sensitivity is seen in patients who underwent

antimicrobial therapy in the 2 weeks prior to the surgical procedure; 45% detection with direct

cultures in comparison to 75% detection with the use of sonication [31]. In addition to this,

blood culture bottles inoculated with sonicate results in increased sensitivity and significantly

reduces the time to detection [50].

In recent studies sonication has also been utilized in diagnostics associated with

suspected infection of cardiac devices, demonstrating significantly higher detection of

microorganisms when compared to direct cultures [28-30,32,51,52]. The current application

of sonication to a range of materials and indications may be further extended to include

infected heart valves.

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The aim of the study

3.1 The Aim

This study aims to establish whether adding sonication to the standard diagnostic

microbiological analysis of explanted heart valves suspected of infective endocarditis

provides additional value for the identification of microorganisms. This will be achieved

through assessing the sensitivity and specificity of the addition of sonication to the standard

diagnostics.

Taking into consideration that the causative microorganisms are not always identified

with the standard diagnostic tests[4], the severity of the disease process[20], and the

importance of microbiological identification of microorganisms to the diagnosis and

subsequent therapy[18], it is important to further improve diagnostic capabilities for cases of

(suspected) infective endocarditis.

Explanted heart valves may be an invaluable addition to the diagnostic workup.

Identification of microorganisms on heart valves may provide further support for the

microbiological diagnosis in cases of IE and help to optimize treatment. The clinical additive

value of sonication has been demonstrated for infections of orthopaedic implants and

implanted cardiac devices. However, the diagnostic application of sonication in microbiologic

investigations of heart valves has not been demonstrated. The addition of sonication to the

microbiologic workup on explanted heart valves is hypothesized to increase the sensitivity for

the detection of microorganisms. Conversely, a relevant concern for the use of sonication is

the possibility for a reduced specificity; resulting from a potential increased risk for

contamination.

Particularly cases of IE, where blood cultures remain negative, may benefit from

sonication of explanted (prosthetic) heart valves. As with infected orthopaedic prosthesis,

(prosthetic) heart valves with prior exposure to antimicrobial treatment and biofilm formation

may result in false-negative results when the standard microbiological detection methods are

used. Therefore, it may be expected that sonication will be able increase the detection of

microorganisms in a similar fashion.

If sonication of explanted heart valves is able to demonstrate a significant increase in

sensitivity for microbial identification in cases of suspected IE compared to the standard

workup of heart valves, this technique will be considered for implementation as part of the

standard diagnostic workup for this material in the UMCG.

3.2 Hypotheses

H0: There is no significant increase in microbiological detection with the addition of

sonication to the standard diagnostic method on explanted heart valves suspected of infection,

in comparison to only the standard diagnostic method on its own.

H1: There is a significant increase in microbiological detection with the addition of sonication

to the standard diagnostic method on explanted heart valves suspected of infection, in

comparison to the standard diagnostic method on its own.

- This will be determined through assessing positive bacterial cultures

o The null-hypothesis is considered refuted if it can be demonstrated, with

statistically significant scientific probability (p ≤0.05) that the addition of

sonication leads to an improved overall sensitivity and specificity in

comparison to the standard diagnostic microbiological workup alone.

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3.3 The Aim of this Thesis

This thesis only covers the preliminary findings obtained during the period of my internship.

The presented work does not encompass the entire pilot study, as this would require more

time for data collection than was available during this internship (see section: “expected

Inclusion Rate”). The aim of this internship was to set up the project and test the study

procedures. The results that have been obtained are included and used to make an initial

assessment of the application of sonication in the diagnostic processing of explanted heart

valves.

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Methods

4. Study Design

Study type: prospective case-control study This is an observational study, in which two arms are compared for outcome. The standard

diagnostic culture method for explanted heart valves is compared to the standard culture

method plus sonication. In order to test the specificity of sonication, the protocol will also be

carried out using negative controls.

The primary outcome is microbiological yield, measured in terms of the presence of

microbial growth through cultures. The yield resulting from standard diagnostic method plus

sonication will be compared to the results from the current standard microbiological method

alone.

A secondary comparison will be made through 16S PCR using Sanger sequencing,

measured as positive/negative and in terms of the organism that is identified through this

technique. PCR results from a direct tissue sample is compared to results from PCR on

sonicate. This PCR technique detects evidence of microbial (ribosomal) DNA in a sample.

This may guide the identification of the organism and therefore the diagnosis if no organisms

can be cultured.

The study will be carried out on all types of heart valves. This includes prosthetic heart

(both mechanical and biological) valves and native heart valves (Figure 2). There is evidence

in the literature, as stated in the introduction, for the benefit of using sonication in cases of

infected prosthetic materials, particularly orthopaedic. The hypothesized application of

sonication on prosthetic valves may be regarded as an extrapolation of the currently available

evidence. Although there is no clear evidence suggesting that native valves may also benefit

from sonication, further extrapolation of the evidence does not exclude this possibility.

Therefore, it would be interesting to include these valves as an exploratory investigation into

the use of sonication of non-prosthetic materials.

Furthermore, this study will assess the specificity of sonication by carrying out the

same diagnostic process on explanted heart valves that are not suspect for IE; these valves are

negative controls. The indication for the removal of these valves is commonly valvular

stenosis or insufficiency. Detection of microorganisms from these valves will indicate

possible false-positive results. False-positives may suggest contamination of the sample(s) at

some point in the surgical, collection, and/or diagnostic process. Although an increase in

positive results through sonication would suggest increased sensitivity for the detection of

microorganisms, false-positive results would reduce the method’s specificity. High specificity

is of particular of importance. A reduced certainty that the identified organism is in fact the

causative organism leads to difficulty in making clinical decisions; this will have

consequences for the optimal treatment of the patient. The patient may be exposed to the

consequences of an unnecessary and/or wrong therapy.

17

Figure 2: Simplified overview of project flowchart. Explanted heart valves are packaged in sterile containers.

These containers are sent to the in-hospital diagnostic laboratory for microbiology. The valves are processed

within 24hours after removal from the patient. All valves are subjected to the standard diagnostic tests.

Subsequently these valves are processed with sonication.

5. Number & Inclusion of Patients

5.1 Numbers Needed

The target for this pilot study is to include a total of 40 prosthetic heart valves. This includes

20 prosthetic valves from patients suspected for having IE and 20 control valves (prosthetic

valves from patients with no suspicion for IE). Mechanical and biological valve prostheses are

included in these groups.

A power analysis indicates that each of these two groups requires at least an n=16, for

statistical analysis to be possible. This calculation was carried out for α=0.05 and β=0.80, for

dependent samples. An expected difference of 30% (d=0.3) between the standard and

sonication method is based on results from studies concerning the use of sonication on

infected orthopaedic joint prosthesis [31]. Although 16 valves per group would be sufficient,

it was decided to set a higher target in order to ensure a sufficiently large sample size.

Although not the primary focus of this study, this same method will be applied to

native heart valves. This includes 40 native valves; 20 with suspected IE and 20 control native

valves, explanted for other reasons than IE.

5.2 Valve Inclusion Criteria

General inclusion criteria:

- Age ≥12 years; regarding a person’s legal ability to provide consent

- Clinical indication for valve removal/replacement

- Valves processed according to the research protocol <24 hours after surgical removal

Inclusion criteria for valves suspected IE:

18

- Suspicion of IE according to the modified Duke criteria and expert opinion

Inclusion criteria for negative control valves:

- No indication of infection and/or bacteraemia at the time of surgical intervention;

based on the patient medical information and indication for surgical intervention.

5.3 Expected Inclusion Rate

The availability of heart valves determines the time frame that is required to attain the desired

number of heart valves. The collection of heart valves will occur within the UMCG. It is

possible to estimate the expected availability of heart valves based on several hospital’s

databases:

- According to the department of thoracic surgery, approximately 450 heart valves are

explanted in 1 year. This is approximately 37 per month. The removed heart valves are

predominantly native heart valves.

- The department of medical microbiology has received 92 heart valves in the past two

years, with the suspicion of infective endocarditis. This includes 61 native valves and

31 prosthetic heart valves; 2.5 and 1.3 heart valves per month respectively.

Based on the category of valves with the lowest known incidence, namely prosthetic valves

suspected for infection, it would take slightly more than 1.3 years to collect the desired

numbers (n=20). Taking the minimum number required for statistical analysis (n=16), data

collection would take approximately 12 months.

6. Ethics & Consent This study is carried out in accordance with the principles described in the Declaration of

Helsinki. The proposed protocol for this research project does not fall within the regulations

set by the Netherland’s law “Wet medisch-wetenschapelijk onderzoek met mensen” (WMO)

as this project will not require any special effort from or actions by the participating persons.

This research is conducted with approval of the local medical ethical committee3.

The explanted heart valves will be subject to additional testing. These specific tests

will not hinder or otherwise interfere with the standard diagnostics tests or therapy required

for the delivery of optimal care. Removal of heart valves will only be carried out based on

medical indication, as determined by the treating physician and multidisciplinary team of

medical experts.

Furthermore, medical information relevant to infective endocarditis and/or the health

situation of the patient in the period leading up to the surgical removal of their heart valve will

be collected. This information will be handled with maximum patient confidentiality and

safety in mind.

Informed consent will be obtained from all participating individuals (or legal

representatives) for the use of the heart valve and medical information. In this decision the

patient is free to refuse or provide consent4. The patient has the ability to revoke consent at

any point.

3 “Medisch Ethische Toetsingsingscommissie” (METc)

4 Please refer to Appendix: B (Patient Consent Form)

19

7. Preliminary Work

This section will address the process through which the methodology and protocols for this study were

developed and optimized. Additionally it will briefly describe several personal learning experiences

that contributed to my ability to help shape this project.

7.1 Orientation

It was important to further expand the necessary knowledge and gain experience with

practices relevant to this project. Through this scientific internship experience was gained in

practical research, working with and creating of protocols, and the proper use of techniques in

a microbiology laboratory.

Prior to the commencement of the internship this pilot study was still in a conceptual

phase. The goals and general outline of the research process was known. The project was

approved by the regulatory bodies; including the medical ethics committee. However, there

was no concrete methodology or protocol in place to allow for the study to be carried out. An

integral aspect of the internship was the further developed the proposed project.

In order to be able to work in the microbiology laboratory of the UMCG a certificate for

Microbiological Safety Techniques5 was obtained, permitting work in a Biosafety Level 2

(BSL-2) laboratory6. Furthermore, an intensive 1-week introductory course was followed on

diagnostic microbiology. This lead to the ability to work professionally in the laboratory

environment and gave the necessary insight into the methods that were used in

microbiological diagnostics.

In order to gain insight into the clinical cases and the challenges faced by medical

professionals in the diagnosis and treatment of infective endocarditis, various

multidisciplinary meetings were attended every week. Furthermore, the opportunity was given

to observe multiple surgical procedures in which heart valves were removed. Understanding

of the procedures and investing in direct communication with the department of thoracic

surgery was useful to organising the logistics surrounding the collection of heart valves.

Without the willing cooperation of the surgical department this project would not have been

possible.

7.2 Developing the Sonication Protocol

In order to provide answers to the aim of this pilot study, a clear and validated research

protocol needed to be developed. The UMCG’s standard diagnostic protocol for

microbiological investigation of explanted materials was the foundation for the project’s

methodology. The specific protocol outlining the procedures associated with sonication (see

below) was derived from a protocol for sonication of orthopaedic materials, as published by

Trampuz et al.[31] The applicability and validity of this protocol was extensively tested

experimentally and adjusted according to our own findings.

Three notable experiments included investigation into the effects of sonication on the

viability of bacteria, the use of a centrifuge to increase yield, and a “spike sample”

experiment. In summary, these experiments consisted of:

o Does sonication not reduce bacterial viability? Bacteria in suspension, of a

known ‘density’ (number of bacteria per unit of volume), were exposed to

intensive sonication. The number of bacterial colonies counted on culture

plates from the suspension that was sonicated showed no difference to the

5 Please refer to Appendix: C (Certificate Microbiological Safety Techniques)

6 In Dutch: Microbiologisch laboratorium klasse II (“ML-II”)

20

number of colonies counted on plates from the same suspension that did not

undergo sonication.

o Does centrifuging sonicate increases the yield? As sonication results in a

sonicate, culturing only a small portion (100 µL) of this fluid means that only a

small proportion of present bacteria actually cultured. The risk exists that with

limited numbers of bacteria, the culture will be false-negative. Centrifuging

50mL of sonicate, at 2500 RCF7 for 15 minutes, leads to the formation of a

concentrated “pellet”. Re-suspending the pellet in 1mL and culturing 100uL of

this suspension leads to a >10 fold increase in the number of CFU counted on

plates. In this process several different centrifuge parameters (namely RCF and

time) were investigated.

o “Spiked sample” shows promising results.

Mechanical heart valve prostheses were artificially infected with a

range of bacteria in vitro. Bacteria that are typical causal agents for IE

(see introduction) were selected for this purpose. The strains used for

this purpose were clinically obtained; these frozen isolates were

cultured from cases of IE and orthopaedic prosthesis infections. Strains

with resistance to multiple antibiotics were not used.

The bacteria were stimulated to produce biofilm.[34,53-59] A broth

was made using colony was suspended in tryptic soy broth (TSB) with

2.5% glucose concentration (“TSB+2.5%”) and cultured overnight. A

small volume of broth was added to a container with TSB+2.5% and a

mechanical heart valve. This container was incubated overnight at 35°C

and 5%CO2. The ‘infected’ valve was transferred to a new container

with TSB+2.5% and incubated overnight; this process was repeated a

few times. After a total of 72-96 hours of incubation the valves were

inspected for visible signs of biofilm formation. Strains that were able

to produce biofilm were further investigated.

Infected valves were treated in various methods (including: saline,

ethanol, and antibiotics), in order to reduce the number of viable

microorganisms on the surface of the valves. The goal of this

experiment was to reproduce circumstances in which only organisms

contained within a biofilm would remain viable for possible culturing.

This included a 24 hour treatment with antibiotics (vancomycin

20mg/L + gentamicin 10mg/L); a regimen in accordance with the

standard antimicrobial protocol for empirical treatment of prosthetic

valve infections in vivo.

Mechanical valves that were infected with biofilm forming bacteria and

treated with antibiotics were processed according to the final research

protocol; including the use of sonication.

Due to the many challenges associated with producing biofilm in vitro,

and attempts to reduce the number of viable bacteria outside of the

biofilm, this experiment has not been concluded yet8.

7 Relative Centrifugal Force; expressed as multiple of the gravitational force of the earth (“g” = 9.81 m / sec

2)

8 This experiment is an on-going process with the goal to provide supportive evidence for the use of sonication

based on experimental evidence. It aims to validate of the protocol for sonication in the microbiological

laboratory and use in clinical practice.

21

8. Data Collection

8.1 Patient Medical Data

Information is collected from the patients’ medical files. The collected data is limited to what

is relevant for charting the study population, the diagnosis endocarditis, and the indication for

the surgical removal of their heart valve.

General demographical data is used to describe the patient population. For each

individual case the patient’s past medical history, history of the present illness, recent

antimicrobial therapy, and results from relevant diagnostic tests are collected in order to

assess the circumstances in which the heart valve is removed.

8.2 Heart Valve Data

This section outlines the methodology that is used for collecting data (see Figure 2). The

process of collecting data from heart valves is divided into 4 sequential steps. The steps

mentioned in the overview below are further explained in the coming section.

Overview of the 4 steps:

1. Obtaining heart valves

2. Processing the heart valves

A: Standard diagnostics – Protocol

- Summary of the steps in the standard diagnostic protocol.

- In accordance with the current standard operating procedure employed

by the diagnostics department of medical microbiology of the UMCG.

B: Sonication diagnostics – Protocol

- Summary of the steps in the sonication protocol

- Designed specifically for this study. Literature sources, research

publications, and the extensive validation experiments were used to

motivate specific choices made in the in the creation of this protocol

(see: “Producing the Protocol”).

3. Data collection

A: Standard diagnostics

- Overview of the types of data that is collected from the standard

diagnostic method.

- The manner of data collection is in accordance with the standard

operating procedure employed by the diagnostics department of

medical microbiology of the UMCG.

B: Sonication diagnostics:

- Overview of the types of data that is collected from the sonication

method.

4. Culture identification

Summary of the additional steps in order to identify microorganisms

Any culture that demonstrates growth is further analysed

1: Collection of heart valves:

Based on clinical indication, the patient undergoes cardiac surgery in order to replace a

defective and/or infected valve. The operating surgeon removes the aforementioned heart

valve and packages it in a sterile container.

The container with the valve is sent to the diagnostic microbiology laboratory. The

valve is fully processed <24hours after surgical removal. All valves are subjected to the

standard diagnostic tests. Subsequently, these valves are processed with sonication (Figure 2).

22

2A: Summary of the Standard Diagnostic Protocol:

1. Cultures: A direct culture is made on 5% sheep blood agar9 (BA), Brucella blood

agar10

(BBA), and chocolate agar11

(CHOC) plates using the explanted heart valve.

This selection of carriers is been chosen as they facilitate the culturing of most major

groups of bacteria. The plates were purchased at Mediaproducts BV, Groningen, The

Netherlands.

o The valve is carefully streaked along one side of the plat, making the first

segment of the three-phase streaking pattern (T-Streak) (Figure 3).

o Using a sterile inoculation loop, the second and third phases of the streak are

made.

o The BA and CHOC plates are placed in an incubator at 35°C with an

atmosphere containing 5.0% CO2.

o The BBA plate is placed in an air-tight container with an oxygen-free

atmosphere and incubated at 35°C.

o The plates are incubated for 9 days, checked daily for growth by an

experienced laboratory analyst.

2. Gram stain: Under sterile conditions suspect areas of the heart valve will be carefully

smeared on to a glass slide. A gram staining is carried out on the slide; an automated

system is used for this purpose

3. PCR: Using a sterile scalpel a small piece of suspect material on the valve is removed

and placed into a sterile 2mL tube. This tube is submitted to the department of

molecular microbiology for PCR investigation.

o The material is used to perform a 16S PCR according to Sanger sequencing;

for the detection of microbe-specific ribosomal DNA. This method allows for

any present bacteria to be identified based on their genetic material.

Figure 3: The T-Streak Method. The material (in this case the explanted heart valve) is streaked along the surface

of the agar plate in “Zone 1”. A sterile inoculation loop is used to create the streak lines for “Zone 2”, followed

by “Zone 3”. After incubation the analyst observes in which zones bacterial growth is present.

9 BA is an enriched non-selective medium; able to facilitate the growth of most types of bacteria.

10 BBA is used for the culturing of obligate anaerobic microorganisms.

11 CHOC is a medium used for growth fastidious microorganisms that are unable to lyse red blood cells

23

Figure 4: Data collected from the standard diagnostic tests

3A: Collection of data from the Standard Diagnostic Protocol:

(See Figure 4)

- Cultures: the plates are examined by an experienced laboratory analyst and scored

according to the streak phase that has been reached (Figure 4)

o 0 = no growth in any of the streak zones.

o 1 = growth limited to the primary streak.

o 2 = growth in both the 1st and 2

nd streak zones.

o 3 = growth in all three streak zones.

- Gram staining: The slide is examined by an experienced microbiology laboratory

analyst for the presence of leukocytes, erythrocytes, and any/all present microbes.

o Microbes will be differentiated according to gram stain (“positive” or

“negative”), morphology, and grouping.

- 16S PCR: The result of this test will either be “negative” or “positive”

o Negative = no detection of microbial rDNA

o Positive = detection of microbial rDNA

Positive results will include supplementary information, namely the

identified organism.

24

Figure 5: Simplified overview of the steps within the sonication process. The valve, in a container with medium

(Ringer lactate), undergoes sonication for 1 minute. The medium fluid after sonication (“sonicate”) is used for

plate cultures and inoculation of blood culture bottles. 50mL of the remaining sonicate is centrifuged at a RCF of

2500g for a duration of 15 minutes. The supernatant is siphoned off and the pellet is re-suspended in 1mL. The

concentrated suspension is used for culturing on plates, PCR, and a gram stain.

2B: Summary of the Sonication Protocol:

(See Figure 5)

This protocol directly followings the Standard Diagnostic Protocol

All the following steps are carried out under sterile conditions where possible.

1. The valve is placed in a sterile container

2. Sterile Ringers lactate solution is added to the container until the valve this is covered

for ≥90% but <110% and the container is carefully sealed.

3. The container is vortexed for 30 seconds

4. The container is placed in the sonication bath (“BactoSonic” by Bandelin, Berlin,

Germany)

5. The container with the heart valve and Ringer solution is exposed to sound waves of

an ultrasonic frequency (40±2 kHz at a power density of 0.22±0.04 W/cm2) during 1

minute

6. The container is vortexed during 30 seconds

7. Using a pipet a BA, BBA, and CHOC agar plates are inoculated with 100uL of the

fluid (“sonicate”). Sterile Drigalski spatulas are used to evenly spread the fluid across

the plates; this is the “spread plate method” (Figure 6).

o The BA and CHOC plates are placed in an incubator at 35°C with an

atmosphere containing 5.0% CO2.

o The BBA plate is placed in an air-tight container with an oxygen-free

atmosphere and incubated at 35°C.

o The plates are incubated for 9 days, checked daily for growth and possible

visible changes by an experienced laboratory analysis.

8. Two 10mL syringes are filled with sonicate and used to inoculate one aerobic and one

anaerobic blood culture bottle. This is processed by an automated growth detection

system (“BD BACTECTM

FX” by Becton Dickinson, Franklin Lakes, United States of

America). These are cultured for 9 days or until growth is detected.

25

o Positive cultures are further processed by an experienced analyst.

9. A sterile conical tube is filled with 50mL of sonicate. This is centrifuged at 2500RCF

for a duration of 15 minutes. This produces a concentration of potential present cells

and microorganisms at the bottom of the tube in a ‘pellet’.

10. 49mL of fluid (supernatant) is carefully siphoned off. The pellet is re-suspended in the

remaining 1mL.

11. Using a pipet a BA, BBA, and CHOC agar plates are inoculated with 100uL of the

concentrated sonicate. Sterile Drigalski spatulas are used to evenly spread the fluid

across the plates; this is the “Spread Plate Method”.

o The BA and CHOC plates are placed in an incubator at 35°C with an

atmosphere containing 5.0% CO2.

o The BBA plate is placed in an air-tight container with an oxygen-free

atmosphere and incubated at 35°C.

o The plates are incubated for 9 days, checked daily for growth and possible

visible changes by an experienced laboratory analysis.

12. 200uL concentrated sonicate is transferred to a sterile 2mL tube. This tube is

submitted to the department of molecular microbiology for PCR investigation.

o The material is used to perform a 16S PCR according to Sanger sequencing;

for the detection of microbe-specific ribosomal DNA. This method allows for

any present bacteria to be identified based on their genetic material

13. 50uL concentrated sonicate is transferred to a sterile glass slide.

o A gram staining is carried out on the slide; an automated system is used for this

purpose.

Figure 6: The Spread-Plate Method. Liquid (in this case 100uL of sonicate) is pipetted onto the surface of the

agar plate in. A Drigalski spatula is used to spread the sample of sonicate across the entire plate, without

touching the edges (due to the risk for contamination). After incubation the analysist observes the presence of

growth on the plates and counts the number of colony forming units (CFU’s).

26

Figure 7: Data collected from diagnostic tests used in the sonication protocol.

3B: Collection of data from the Sonication Protocol:

(See Figure 7)

- Cultures: Following incubation, the number of colonies (CFU’s) is counted on each

plate. This number is registered.

o This procedure is also carried out for the concentrated sonicate following

centrifuge. The number of colonies in the concentrate are compared to those in

the culture of the original sonicate.

- Inoculated blood culture bottles: The automated growth detection system (BACTEC)

will signal the presence of microbial growth.

o The time to positivity is measured.

Based on literature (see: Introduction), inoculation of blood culture

bottles may facilitate a more rapid detection of bacterial growth.

- 16S PCR: The result of this test will either be “negative” or “positive”

o Negative = no detection of microbial rDNA

o Positive = detection of microbial rDNA

Positive results will include supplementary information, namely the

identified organism.

- Gram staining: The slide is examined by an experienced microbiology laboratory

analyst for the presence of leukocytes, erythrocytes, and any/all present microbes.

o Microbes will be differentiated according to gram stain (“positive” or

“negative”), morphology, and grouping.

27

4: Culture Identification

All cultures that are positive and demonstrate growth are assessed and analysed by an

experienced microbiological laboratory analyst.

- Colonies are identified and classified based on macroscopic colony morphology and

standard identification techniques.

- A purification culture is made for each morphologically different colony.

o A single colony is used to make a T-Streak on a new agar plate; if all the

colonies from this culture are morphologically identical it is considered a

‘pure’ culture

o Purified cultures are used to determine resistance patterns.

- A clinical microbiologist determines the need for any further tests if an indication is

present.

o Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer

(MALDI-TOF) was generally used to identify the micro-organism.

9. Data Analysis The primary outcome data for this study will be analysed through non-parametric tests,

namely the “Wilcoxon matched pairs rank sum test”.

28

Results

10. Included Valves

Table 1: Overview of included valves

Number of valves

Total 14

Native 10 IE: 5

Control: 5

Prosthetic

Mechanical: 2 IE: 1

Control: 1

Biological: 2 IE: 1

Control: 1

Table 2: Overview of the demographics of the patient population

Average Age

(years)

Suspected IE cases: 51 Range: 14-76

Negative control: 67 Range: 48-78

Total: 58 Range: 14-78

Sex

Suspected IE cases: 7 Female: 1

Male: 6

Negative control: 7 Female: 2

Male: 5

During the time of my internship 14 valves were included, originating from 13 different

patients (Table1). Ten of these heart valves were native; five were suspected of having IE and

five were negative controls. Four heart valves were prosthetic; two mechanical valve and two

biological valve. There were two prosthetic valves from patients that were not suspect for

having IE.

The basic population demographics include gender and age (Table 2). The 13 included

patients were predominantly male; 10 men and 3 women. The overall average age was 58

years. The youngest patient included was 14 years old while the oldest was 78 years.

11. Culture Results

Seven of the included heart valves were suspect of having IE (Table 3). Five of these were

native heart valves of which only one resulted in positive cultures in the standard diagnostic

method. This valve (“B” = positively cultured native heart valve) was positive for

Staphylococcus aureus in both the standard diagnostic method as the sonication method.

Direct culture of the valve on plates demonstrated growth into the 3rd

streak zone of

the three-phase streaking method that was used for this culture. The cultures from the

sonication protocol demonstrated similar results. The numbers of colony forming units

(CFU’s) on these plates were too many to count; it was not possible to differentiate between

the numbers of CFU’s from sonicate and the number from concentrated sonicate (Table 4).

The blood culture bottles inoculated with sonicate were positive after 10 hours; the cultures

on plates indicated growth at earliest after approximately 24 hours. The 16S PCRs that were

carried out the tissue and concentrated sonicate of native valves were consistent with the

culture results.

29

Table 3: Overview of the culture results from the standard diagnostics and from sonication

Valves Valve Types

Standard

Culture

Positive

Sonicate

Direct Culture

Positive

Sonicate

Inoculated Blood

Culture Bottle

Positive

Suspected IE: 7 Prosthetic = 2 0 1

+ 1

+

Native = 5 1* 1* 1*

Native Control: 7 Prosthetic = 2 0 0 0

Native = 5 0 0 0 + Valve “A” = positively cultured prosthetic heart valve with P. acnes through sonication

*Valve “B” = positively cultured native heart valve with S. aureus in both standard culture and sonication.

Of the two prosthetic valves suspect for IE, one was found to show microbial growth

(Table 4). This valve (“A” = Positively cultured prosthetic heart valve through sonication)

showed no growth on the cultures made through the standard diagnostic method. The standard

16S PCR of valve material did demonstrate the presence of Propionibacterium acnes (P.

acnes).

Sonication of valve “A” showed evident growth. Approximately 180 hours (7.5 days)

after carrying out sonication the anaerobic blood culture bottle, inoculated with sonicate,

turned “positive”. After 9 days the BBA (anaerobic) plates were also found to be positive. In

this case the concentrated sonicate demonstrated considerably more growth (Table 4). Fifty-

eight CFU’s were counted on the plate cultured with the concentrate; four CFU’s were

cultured from the standard sonicate. PCR carried out on the concentrated sonicate was

positive, indicating the presence of P. acnes. The positive cultures were further assessed.

MALDI-TOF analysis of the cultures confirmed the organism to be P. acnes. Resistance

analysis showed sensitivity to all the standard tested antibiotics, namely amoxicillin,

ceftriaxone, clindamycin, or rifampicin.

Two valves suspected for IE (1 native and one 1 mechanical prosthesis), originating

from the same patient, were negative for all the cultures according to both the standard and

sonication methods. Conversely, 16S PCR analysis in both the standard diagnostic protocol

and the sonication protocol indicated the presence of rDNA associated with Streptococcus

spp. Multiple blood cultures, taken several weeks prior to the surgical intervention, identified

the presence of Streptococcus parasanguinis. The patient subsequently received extensive

antimicrobial therapy.

The remaining heart valves suspected for IE (4 native valves) were negative in all

performed analysis.

Table 4: Overview of results from valves with positive cultures

Valve Standard (Streak #) Sonicate (CFU) Concentrated

Sonicate (CFU) Organism

A 0 4 58 P. acnes

B 3 >1000 >1000 S. aureus

The negative-control valves were all negative. The cultures showed no growth, gram

staining did not reveal bacteria, and the PCRs carried out on valvular material and sonicate

did not suggest the presence of microorganisms.

30

12. Case Report Patient “A” was a 30+ year old male, admitted to the department of cardiology with a

suspicion of infective endocarditis. The clinical presentation and findings upon examination

were non-specific. His main complaints were general malaise and heavy night-sweats, leading

to a very broad differential diagnosis. The most notable finding was a new and prominent

systolic murmur. The patient had a history of congenital aortic stenosis that was treated in the

past with a “ROSS procedure”12

followed by an aortic valve replacement (AVR) using a

biological valve prosthesis13

. A few months prior to the onset of symptoms the patient had

been extensively evaluated by a cardiologist as part of his regular follow-up; no abnormalities

were identified that could suggest the presence of IE.

Initial blood cultures were negative and echocardiography14

was not conclusive.

However, this does not rule out infective endocarditis and the suspicion of IE maintained as

part of the differential diagnosis, prompting empiric therapy for infective endocarditis. This

therapy consisted of a combination of vancomycin and gentamicin.

Modified Duke Criteria

Major Minor

Blood cultures:

(“typical” organisms)

Echocardiographic evidence:

Negative

Inconclusive

Predisposition:

Fever:

Vascular phenomenon:

Immunological phenomenon:

Blood cultures (other):

Yes

No

No

No

Negative

During the course of admission additional investigation was carried out, leading to

stronger indications for the diagnosis endocarditis. 18F-FDG-PET/CT showed clear signs of

infection at the site of the aortic valve. Repeated transoesophageal echocardiography

eventually identified a large mobile structure associated with the aortic valve. This confirmed

the diagnosis endocarditis. However, the causative organism(s) remained unknown.

A “Bentall procedure”15

was carried out in order to remove the infected bio-prosthetic

heart valve. The explanted heart valve was analysed by the diagnostic microbiology

laboratory. Standard diagnostics showed no growth from direct tissue cultures. PCR detected

microbial DNA associated with Propionibacterium acnes (P. acnes), but due to the negative

culture, no resistance patterns could be assessed. Sonication of the heart valve, directly

following the standard diagnostic tests, revealed growth of P. ances in inoculated blood

culture bottles after 179 hours (7.5 days) and on culture plates after 9 days. PCR of the

sonicate also confirmed the presence of P. acnes.

The positive cultures of the sonication allowed for further analysis of the causative

micro-organism. The resistance pattern was determined, revealing full sensitivity to all

antibiotics tested. As a result, the therapy was adjusted to a combination of vancomycin and

ceftriaxone. Postoperatively, the blood cultures remained negative and repeated assessment of

the heart showed no signs of infection.

12

Ross procedure: the aortic valve is replaced by the patient’s pulmonary valve. The transplanted pulmonary

valve is replaced by a homograft (a heart valve from a human donor). 13

Biological valve prostheses (“bio-valves”) are usually manufactured using valve leaflets obtained from animal

donors; usually bovine or porcine. 14

Transthoracic Echocardiography (TTE) and Transoesophageal Echocardiography (TEE) 15

Bentall procedure: a surgical procedure involving the combined replacement of the aortic valve, aortic root,

and ascending aorta using a graft.

31

Discussion

13. Overall Findings The general demographics of the patient population included in this study are consistent with

what may be expected from known epidemiological data. The mean age of patients with

(suspected) IE, was 51 years. This is fairly consistent with the available literature; the median

age is found to be 60 years [11]. It must be noted that the study group of patients with IE

contained one case of a 14 year old patient; this is considerably lower than the group average

and therefore a possible outlier. The group of negative control patients have a higher average

age (67 years) and a narrower range. The male-to-female ratio among the (suspected) IE

patients was 6:1, which is consistent with figure stated in literature [12].

All of the negative control valves produced negative results. Cultures and 16S-rDNA

PCR from the standard diagnostic method and the sonication method were unable to indicate

the presence of microorganisms. This suggests that both the standard diagnostic method and

the sonication method are not vulnerable to contamination. The absence of false-positive

results indicates that the specificity is no reduced through the addition of sonication.

The sonication method is able to demonstrate the presence of microorganisms. Of the

seven cases of heart valves with (suspected) IE, only two valves resulted in positive cultures.

It is impossible to state whether if these two valves were the only valves carrying viable

bacteria. Therefore it is not possible to state whether the used methods lead to false-negative

cultures. The standard diagnostic method was able to produce cultures from one heart valve.

Sonication of that heart valve lead to positive cultures with the same microorganism. Identical

results from both methods show that sonication is able to produce positive cultures; this may

be seen as a positive-control for the sonication method. It can be stated that sonication works.

Sonication has shown a higher sensitivity than the standard diagnostic method, in

terms of the ability to culture bacteria. Sonication was able to detect and culture an

microorganism that was not cultured through the standard diagnostic method (see section:

“12. Case Report”). The fact that the sonication method was able to culture an organism that

would otherwise not have been found (through the standard diagnostic method) suggest that

sonication has a higher sensitivity. However, more valves would need to be included in order

to reliably establish the overall increase in sensitivity through the addition of sonication to the

standard diagnostic method.

The inoculation of blood culture bottles with sonicate leads to a more rapid detection

of microorganisms than cultures on solid growth mediums on plates. As described in the case

report, P. acnes was first detected through the inoculated blood culture bottles. Approximately

179 hours (7.5 days) after the valve was processed the automated growth detection system

indicated the presence of the bacteria. The culture plates with sonicate were found to be

positive after 9 days. In this case sonication and the inoculation of blood culture bottles with

sonicate lead to a more rapid growth detection. This result is consistent with other studies

[50]. Further data collection would be required in order to establish whether this reduced

detection time is also found in more cases and with different microorganisms.

32

14. Case Report The presented case importantly underlines the potential value of sonication for diagnostic

purposes of identifying microorganisms from prosthetic heart valves affected by IE. Although

a single case is not sufficient evidence to draw any clear conclusions from, it demonstrates the

concept and is an indicator of the possible added value of sonication. The sonication protocol

was able to produce viable P. acnes colonies, where the standard diagnostics was unable to do

so. Sonication led to direct identification of the organism and the ability to determine

resistance patterns. Furthermore, these positive cultures were obtained from a patient with

culture-negative IE. Culture-negative IE cases are hypothesized to benefit the most from

sonication, as the identification of a (previously unknown) causative microorganism would

have a considerable clinical impact. Identifying viable cultures is crucial to the diagnosis and

subsequent patient care.

15. Study Limitations This pilot study, as presented here, faces a number of limitations that should be considered

before any conclusions are drawn.

The most prominent limitation is the low number of valves that were processed and

included as part of this research project. Although there are a few interesting results, the

numbers are insufficient to perform the statistical analysis needed to draw clear and data-

supported conclusions. The primary reason for the low inclusion numbers was the limited

time available for data collection. The insufficient amount of time was not a surprise, as it was

predicted at the start of the project. The preliminary work that was needed to properly

organize and validate the research protocol, through various experiments, comprised a

considerable portion and important aspect of my internship. The logistics surrounding the

setting up of a successful cooperation between the various specialists that needed to

coordinate the collection and processing of heart valves, demanded a considerable effort.

With more time to include the desired number of valves, it is acceptable to assume that

sufficient data will be obtained in order to draw more substantiated conclusions.

Native valves are an interesting addition to this project. Sonication has shown to be of

additive value in infected orthopaedic prosthetic materials, besides regular tissue culture.[31]

Although prosthetic heart valves have never before been processed with sonication according

to the currently available literature, the added value can be reasoned through extrapolation of

the available evidence for other prosthetic materials. Although there is no evidence that would

suggest that diagnostics for native heart valves would potentially find benefit form sonication,

this exploratory arm of this study may provide an indication whether there may be a possible

role for sonication on materials beyond prosthetics. Additionally, this method may be able to

suggest evidence for the presence of biofilm formation on native valves.

The manner in which cultures are created through the standard diagnostic protocol and

the sonication protocol are not identical and therefore prevent any detailed comparison. The

primary goal of this project was to determine whether sonication could produce positive

cultures in cases where the standard diagnostic method was unable to do so. This was

demonstrated in the case of valve “A”. However, beyond this negative-positive distinction it

is difficult to compare the cultures. The standard protocol makes use of a T-streak, while the

sonication protocol uses the spread-plate method. A T-streak may be able to provide a rough

estimate of the amount of bacteria that are present. Arguably, the spread-plate method would

be more useful, as the number of individual colonies may be counted; reflecting the

concentration of organisms in a fixed volume of fluid. Therefore, a compromise was made to

use the optimal technique based on the possibilities determined by the material.

33

The sonication protocol is quite elaborate and therefore considerably vulnerable for

mistakes and contamination. The clear benefit of the way this project is organised, is that it

does not interfere with the standard diagnostics that are necessary for clinical purposes.

Therefore, the patient is not subject to possible negative consequences that could result from

sub-optimal diagnostics due to the use of an experimental method that had not been proven to

provide precise and accurate results. Sonication is a possible addition to the standard

diagnostic test that may provide useful information that would otherwise not have been

obtained. However, the inherent risk of contamination is particularly problematic as this may

lead to obscured or false-positive results. Therefore, it would be advisable to remain sceptical

of any positive sonication results, until the method has proven its reliability. Notably, no cases

of contamination were found in the limited number of analysed heart valves in this project.

34

Conclusion The inability to identify the causative microorganism in IE, as seen in up to 20% cases, is a

considerable limitation in the ability to provide those patients with optimal care. Sub-optimal

care leads to a greater risk for a sub-optimal outcome. As IE is a very serious disease with

potentially fatal consequences, it is of paramount importance to further improve the diagnostic

capabilities in order to improve therapeutic options and improve the disease outcome.

Sonication of explanted heart valves from patients with (suspected) IE is a potential

improvement to the diagnostic detection of causative microorganisms. An increase in

sensitivity for the detection of microorganism is needed to reduce the number of culture-

negative cases of IE. Standard microbiological tests frequently lead to negative results.

Sonication has been demonstrated to achieve an increased sensitivity when applied to

orthopaedic prosthetic materials that were suspected of infection. We hypothesise, that it may

be possible to demonstrate a similar added value in the diagnostic approach for explanted

(prosthetic) heart valves.

Based on the currently available data, this pilot study was unable to statistically prove

a clear benefit for the use of sonication as part of the standard diagnostic work-up of

explanted heart valves from patients suspected for endocarditis. The low numbers of included

heart valves was the primary limitation in our ability to establish proof. Despite this

limitation, there was one particular case that nicely illustrated the potential for sonication by

making a significant contribution to the diagnosis and resulting therapy. Although one case

bears limited value as scientific evidence, it does provide an indication that the concept and

methodology used in this study may lead to potentially significant data once more valves are

included. The research will be continued by the department of Medical Microbiology.

Even if sonication does eventually prove to have a significantly higher detection

(sensitivity) for the presence of microorganism on heart valves suspected of IE, the results

must always be examined carefully. The increased risk of contamination associated with the

sonication protocol implies that there may be an increase in false-positive results; thereby

lowering the specificity. On the other hand, sonication can enhance overall specificity by

confirming the findings of the regular culture, which might otherwise be interpreted as

contamination and vice versa. In the current situation, sonication should only be considered to

be a supplementary step in the diagnostic process; sonication is capable of providing crucial

additional information relevant to the diagnosis and treatment of patients. An expert with the

ability to determine clinical value and consequences should always review the results prior to

translating into clinical decisions.

With continued collection of data, it will be possible to make a meaningful

contribution to the diagnostics of infective endocarditis through the application of sonication.

This potential contribution would provide patients with culture-negative endocarditis, with

improved chances for a proper diagnosis and optimized treatment.

16. What I have learned This internship has given me an extensive insight into the practical aspects associated with

organising and carrying out scientific research in a medical setting. I learned to critically

appraise scientific literature, particularly in the field of IE. In order to design, perform, and

optimize experiments (with the goal of validating the methodology used in this study) I was

stimulated to think creatively and in a structured manner. The project required me to work in a

multidisciplinary setting, involving the logistical coordination of material collection and the

inclusion of patients for this study. Direct interaction with patients allowed me to develop an

understanding of the disease and the impact on people afflicted with IE. I learned to work

35

safely at a ML-II laboratory, and I have learned a variety of laboratory techniques. I was

tasked with performing the sonication protocol, that I helped design. Additionally, scientific

writing techniques were practiced during the creation of this report. Furthermore, towards the

end of my internship I was able to present the project and preliminary results to a group of my

peers. These various personal and professional competences, within the context of medical

research, have developed considerably during my internship.

I believe that it is important to mention that the exposure I had to the clinical setting

and my personal interaction patients with infective endocarditis allowed me to place the work

I was doing in a clear context. To me, this underlined the significance and importance of

scientific research in the continual process of improving the quality of health care.

36

Acknowledgements

My scientific research internship in the department of medical microbiology would not have

been possible with the support of many people. I am grateful for the opportunity that I was

given to learn and experience the ‘world of research’. I thank my supervisors Anne Gomes,

Dr. Marleen van Oosten, Dr. Greetje Kampinga, and Prof. Dr. Bhanu Sinha. I would also like

to extend my thanks to the entire department and all the people the contributed to making the

project possible.

There are several people in particular that I would like to thank for their role in contributing to

an educational and enjoyable time during this period.

- Anne Gomes: PhD student and my direct supervisor. Anne was responsible for guiding

me through this internship. The energy and time she invested in as supervisor must not

be understated. I recognise that without Anne this internship would not have been

possible.

- Marleen van Oosten: Researcher and Dr in the department of medical microbiology.

Marleen was essential in supervising my activities in and around the laboratory. She

was invaluable to the process of developing the project’s protocols and various

experiments I carried out.

- Kathleen Boiten & Elisa Salomon: Analysts in the diagnostic microbiology laboratory.

These two individuals taught me the various laboratory techniques and process that

were crucial to my work. They were indispensable in the process of creating a

workable sonication protocol. Kathleen and Elisa, along with this rest of the

‘sonication team’, were responsible for processing and analysing the heart valves used

for this study. I consider myself lucky to have been able to rely on such a dedicated

team of analysts.

37

Bibliography

[1] Sexton D, Fowler V. Clinical manifestations and evaluation of adults with suspected native valve

endocarditis. 2016; Available at: http://www.uptodate.com/contents/clinical-manifestations-and-

evaluation-of-adults-with-suspected-native-valve-

endocarditis?source=machineLearning&search=modified+duke+criteria&selectedTitle=1%7E150&se

ctionRank=1&anchor=H4084836#H973176202. Accessed Mar, 2016.

[2] Kumar P, Clark M. Cardiovascular disease. Clinical Medicine. 7th ed.: Elsevier; 2009. p. 769-73.

[3] Schoen F, Mitchell R. The Heart. In: Kumar V, Abbas A, Fausto N, Mitchell R, editors. Robbins

Basic Pathology. 8th ed. United States of America: Elsevier; 2007. p. 406-7.

[4] Gould F, Denning D, Elliott T, Foweraker J, Perry J, Prendergast B, et al. Guidelines for the

diagnosis and antibiotic treatment of endocarditisin adults: a report of the Working Party of the British

Society for Antimicrobial Chemotherapy. J Antimicrob Chemother 2012 Feb;67(2):269-89.

[5] Peels C. 42 Endocarditis. In: Van der Wall E, Van der Werf F, Zijlstra F, editors. Cardiologie. 2nd

ed. Houten: Bohn Stafleu van Loghum; 2008. p. 369-77

[6] Sexton D. Epidemiology, risk factors, and microbiology of infective endocarditis. 2015; Available

at: http://www.uptodate.com/contents/epidemiology-risk-factors-and-microbiology-of-infective-

endocarditis?source=see_link. Accessed Mar, 2016.

[7] Selton-Suty C, Célard M, Le Moing V, Doco-Lecompte T, Chirouze C, Iung B, et al. Preeminence

of Staphylococcus aureus in infective endocarditis: a 1-year population-based survey. Clin Infect Dis

2012 May;54(9):1230-9.

[8] Murdoch D, Corey G, Hoen B, Miró J, Fowler V, Bayer A, et al. Clinical Presentation, Etiology,

and Outcome of Infective Endocarditis in the 21st Century. Arch Intern Med 2009 Mar;169(5):463-73.

[9] Watkin R. Cardiovascular infections. In: Elliot T, Casey A, Lambert P, Sandoe J, editors. Medical

Microbiology and Infection Lecture Notes. 5th ed. United Kingdom: Wiley-Blackwell; 2011. p. 238-

40.

[10] Tleyjeh I, Abdel-Latif A, Rahbi H, Scott C, Bailey K, Steckelberg J, et al. A systematic review of

population-based studies of infective endocarditis. Chest 2007;132(3):1025.

[11] Durante-Mangoni E, Bradley S, Selton-Suty C, Tripodi M, Barsic B, Bouza E, et al. Current

features of infective endocarditis in elderly patients: results of the International Collaboration on

Endocarditis Prospective Cohort Study. Arch Intern Med 2008;168(19):2095.

[12] Lerner PI, Weinstein L. Infective endocarditis in the antibiotic era. N Engl J Med

1966;274(4):199.

[13] Karchmer A. Epidemiology, clinical manifestations, and diagnosis of prosthetic valve

endocarditis. 2015; Available at: http://www.uptodate.com/contents/epidemiology-clinical-

manifestations-and-diagnosis-of-prosthetic-valve-endocarditis?source=see_link. Accessed Mar, 2016.

[14] Piper C, Körfer R, Horstkotte D, Valve disease: Prosthetic valve endocarditis, Heart [Internet],

2001 may [Accessed 2016 Jun]; 85:590–593, Available from:

http://heart.bmj.com/content/85/5/590.full.pdf

38

[15] Sun BJ, Choi SW, Park KH, Jang JY, Kim DH, Song JM, et al. Infective endocarditis involving

apparently structurally normal valves in patients without previously recognized predisposing heart

disease. J Am Coll Cardiol [Internet] 2015 Jan [Accessed 2016 Jun];65(3):307-9, Available from:

http://content.onlinejacc.org/article.aspx?articleid=2090119.

[16] Lockhart P, Brennan M, Thornhill M, Michalowicz B, Noll J, Bahrani-Mougeot F, et al. Poor oral

hygiene as a risk factor for infective endocarditis-related bacteremia. J Am Dent Assoc 2009

Oct;140(10):1238-44.

[17] Sexton D, Chu V. Infective endocarditis in injection drug users. 2015 Jun; Available at: Available

at: http://www.uptodate.com.proxy-ub.rug.nl/contents/infective-endocarditis-in-injection-drug-

users?source=see_link. Accessed Jun, 2016.

[18] Habib G, Lancellotti P, Antunes M, Bongiorni M, Casalta J, del Zotti F, et al. 2015 ESC

Guidelines for the management of infective endocarditis. Eur Heart J 2015 Nov;36(44):3075-128.

[19] Baddour L, Wilson W, Bayer A, Fowler V, Tleyjeh I, Rybak M, et al. Infective Endocarditis in

Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications: A Scientific Statement

for Healthcare Professionals From the American Heart Association. Circulation 2015;132(15):1435.

[20] Cahill T, Prendergast B. Infective endocarditis. Lancet [Internet] 2016 Feb 27 [cited 2016

May];387(10021):882-93 Available from: http://www.thelancet.com/pdfs/journals/lancet/PIIS0140-

6736(15)00067-7.pdf.

[21] Bennett J, Dolin R, Blaser M. Mandell, Douglas and Bennett's Principles and Practice of

Infectious Diseases. 8th ed. London, United Kingdom: Elsevier Health Sciences; 2014.

[22] Thuny F, Giorgi R, Habachi R, Ansaldi S, Le Dolley Y, Casalta J. Excess mortality and morbidity

in patients surviving infective endocarditis. Am Heart J 2012;164(1):94-101.

[23] Spelman D, Sexton D. Complications and outcome of infective endocarditis. 2015; Available at:

http://www.uptodate.com/contents/complications-and-outcome-of-infective-

endocarditis?source=see_link. Accessed Mar, 2016.

[24] Li J, Sexton D, Mick N, Nettles R, Fowler V, Ryan T, et al. Proposed Modifications to the Duke

Criteria for the Diagnosis of Infective Endocarditis. Clin Infect Dis 2000 Apr;30(4):633-8.

[25] Rohacek M, Weisser M, Kobza R, Schoenenberger A, Pfyffer G, Frei R, et al. Bacterial

Colonization and Infection of Electrophysiological Cardiac Devices Detected With Sonication and

Swab Culture. Circulation 2010 Apr;121(15):1691-7.

[26] Karchmer A, Suri R. Surgery for prosthetic valve endocarditis. 2016; Available at:

http://www.uptodate.com/contents/surgery-for-prosthetic-valve-endocarditis?source=see_link.

Accessed Apr, 2016.

[27] Sexton D, Suri R. Surgery for left-sided native valve endocarditis. 2015; Available at:

http://www.uptodate.com/contents/surgery-for-left-sided-native-valve-endocarditis?source=see_link.

Accessed May, 2016.

[28] Inacio RC, Klautau GB, Murça MAS, da Silva CB, Nigro S, Rivetti LA, et al. Microbial

diagnosis of infection and colonization of cardiac implantable electronic devices by use of sonication.

Int J Infect Dis 2015 Jul;38(2015):54-9

39

[29] Nagpal A, Patel R, Greenwood-Quaintance K, Baddour L, Lynch D, Lahr B, et al. Usefulness of

Sonication of Cardiovascular Implantable Electronic Devices to Enhance Microbial Detection. Am J

Cardiol 2015 Apr;115(7):912-7.

[30] Mason P, Dimarco J, Ferguson J, Mahapatra S, Mangrum J, Bilchick K, et al. Sonication of

Explanted Cardiac Rhythm Management Devices for the Diagnosis of Pocket Infections and

Asymptomatic Bacterial Colonization. PACE 2011 Feb;34(2):143-9.

[31] Trampuz A, Piper K, Jacobson M, Hanssen A, Unni K, Osmon D, et al. Sonication of Removed

Hip and Knee Prostheses for Diagnosis of Infection. N Engl J Med 2007 Aug;357(7):654-63.

[32] Oliva A, Nguyen B, Mascellino M, D'Abramo A, Iannetta M, Ciccaglioni A, et al. Sonication of

Explanted Cardiac Implants Improves Microbial Detection in Cardiac Device Infections. J Clin

Microbiol 2013 Feb;51(2):496-502.

[33] Costerton J, Stewart P, Greenberg E. Bacterial biofilms: a common cause of persistent infections.

Science 1999;284(5418):1318.

[34] Donlan R. Biofilm formation: a clinically relevant microbiological process. Clin Infect Dis

2001;33(8):1387.

[35] Habib G, Hoen B, Tornos P, Thuny F, Prendergast B, Vilacosta I, et al. Guidelines on the

prevention, diagnosis, and treatment of infective endocarditis (new version 2009). Eur Heart J 2009

August 27;30(19):2016 Apr 5.

[36] Sexton D. Antimicrobial therapy of native valve endocarditis. 2016; Available at:

http://www.uptodate.com/contents/antimicrobial-therapy-of-native-valve-

endocarditis?source=see_link. Accessed Apr, 2016.

[37] Martí-Carvajal A, Dayer M, Conterno L, Gonzalez Garay A, Martí-Amarista C, Simancas-

Racines D, et al. A comparison of different antibiotic regimens for the treatment of infective

endocarditis. Cochrane Database Syst Rev 2016 Apr 19:2016 Jun-Avaialble from:

http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009880.pub2/full.

[38] Karchmer A. Antimicrobial therapy of prosthetic valve endocarditis. 2016; Available at:

http://www.uptodate.com/contents/antimicrobial-therapy-of-prosthetic-valve-

endocarditis?source=see_link. Accessed Apr, 2016.

[39] Holinka J, Bauer L, Hirschl A, Graninger W, Windhager R, Presterl E. Sonication Cultures of

Explanted Components as an Add-On Test to Routinely Conducted Microbiological Diagnostics

Improve Pathogen Detection. J Orthop Res 2011 Apr;29(4):617-22.

[40] Sampedro M, Huddleston P, Piper K, Karau M, Dekutoski M, Yaszemski M, et al. A Biofilm

Approach to Detect Bacteria on Removed Spinal Implants. Spine 2010 May;35(12):1218-24.

[41] Portillo M, Corvec S, Borens O, Trampuz A. Propionibacterium acnes: An Underestimated

Pathogen in implant-Associated Infections. Biomed Res Int 2013 Oct;2013(2013):2016 May.

[42] Vergidis P, Greenwood-Quaintance K, Sanchez-Sotelo J, Morrey B, Steinmann S, Karau M, et al.

Implant sonication for the diagnosis of prosthetic elbow infection. J Shoulder Elbow Surg 2011

Dec;20(8):1275-81.

[43] Tunney M, Patrick S, Gorman S, Nixon J, Anderson N, Davis R, et al. Improved detection of

infection in hip replacements: a currently underestimated problem. J Bone Joint Surg 1998

Jul;80(4):568-72.

40

[44] Dapunt U, Lehner B, Burckhardt I, Zimmermann S, Hänsch G, Ewerbeck V. Evaluation of

implant Sonication as a diagnostic tool in implant-associated infections. J Appl Biomater Funct Mater

2014 Dec;12(3):135-40.

[45] Monsen T, Lövgren E, Widerström M, Wallinder L. In Vitro Effect of Ultrasound on Bacteria and

Suggested Protocol for Sonication and Diagnosis of Prosthetic Infections. J Clin Microbiol 2009

Aug;47(8):2496-501.

[46] Rak M, KavčIč M, Trebše R, CőR A. Detection of bacteria with molecular methods in prosthetic

joint infection: sonication fluid better than periprosthetic tissue. Acta Orthopaedica, Acta Orthop 2016

Apr 28:2016 Jun.

[47] Wengrovitz M, Spangler S, Martin L. Sonication provides maximal recovery of staphylococcus

epidermidis from slime-coated vascular prosthetics. Am Surg 1991 Mar;57(3):161-4.

[48] Piper K, Jacobson M, Cofield R, Sperling J, Sanchez-Sotelo J, Osmon D, et al. Microbiologic

diagnosis of prosthetic shoulder infection by use of implant sonication. J Clin Microbiol 2009

Jun;47(6):1878-84.

[49] Yano M, Klautau G, da Silva C, Nigro S, Avanzi O, Mercadante M, et al. Improved Diagnosis of

Infection Associated with Osteosynthesis by Use of Sonication of Fracture Fixation Implants. J Clin

Microbiol 2014 Dec;52(12):4176-82.

[50] Portillo M, Salvadó M, Trampuz A, Siverio A, Alier A, Sorli L, et al. Improved diagnosis of

orthopedic implant-associated infection by inoculation of sonication fluid into blood culture bottles. J

Clin Microbiol 2015 May;53(5):1622-7.

[51] Oliva A, Belvisi V, Iannetta M, Andreoni C, Mascellino M, Lichtner M, et al. Pacemaker Lead

Endocarditis Due to Multidrug-Resistant Corynebacterium striatum Detected with Sonication of the

Device. J Clin Microbiol 2010 Dec;48(12):4669-4671.

[52] Rohacek M, Erne P, Kobza R, Pfyffer G, Frei R, Weisser M. Infection of Cardiovascular

Implantable Electronic Devices: Detection with Sonication, Swab Cultures, and Blood Cultures.

PACE 2015 Feb;38(2):247-53.

[53] van Oosten M, Schäfer T, Gazendam J, Ohlsen K, Tsompanidou E, de Goffau M, et al. Real-time

in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently

labelled vancomycin. Nat Commun 2013 Oct;2013(4):2016 Feb.

[54] Calà C, Amodio E, Di Carlo E, Virruso R, Fasciana T, Giammanco A. Biofilm production in

Staphylococcus epidermidis strains isolated from the skin of hospitalized patients: genetic and

phenotypic characteristics. New Microbiol 2015 Oct;38(4):521-9.

[55] Elkhatib W, Khairalla A, Ashour H. Evaluation of different microtiter plate-based methods for

the quantitative assessment of Staphylococcus aureus biofilms. Future Microbiol 2014;9(6):725-35.

[56] Fey P, Olson M. Current concepts in biofilm formation of Staphylococcus epidermidis. Future

Microbiol 2010 Jun;5(6):917-33.

[57] Hou W, Sun X, Wang Z, Zhang Y. Biofilm-Forming Capacity of Staphylococcus epidermidis,

Staphylococcus aureus, and Pseudomonas aeruginosa from Ocular Infections. Invest Ophthalmol Vis

Sci 2012 Aug;53(9):5624-31.

41

[58] Kierek‐Pearson K. Biofilm Development in Bacteria. Advances in Applied Microbiology Volume

57: Elsevier; 2005. p. 79-111.

[59] McBain A. Chapter 4 In Vitro Biofilm Models: An Overview. Advances in Applied

Microbiology Volume 69: Elsevier; 2009. p. 99-132.

Images:

EIT health, Vector_UMCGlang_FC [image on the Internet], 2015 Jun 30 [cited 2016 Jun 8]. Available

from: https://eithealth.eu/wp-content/uploads/2015/10/Logolangenaam_ENG.jpg

Rijksuniversiteit Groningen, rugr_logonl_rood_rgb [image on the Internet], 2016 May 23, [cited 2016

Jun 8]. Available from: http://www.rug.nl/about-us/how-to-find-

us/huisstijl/logobank/corporatelogo/corporatelogorood/rugr_logonl_rood_rgb.png

EpiGentek, Epigentec Group inc., schematic_example_small [image on the Internet], 2016 [cited 2016

Jun 20], Available from:

http://www.epigentek.com/catalog/images/products/episonic1100/schematic_example_large.gif

42

Appendix A: modified Duke criteria

Source:

UpToDate, Modified Duke criteria for diagnosis of infective endocarditis - Table A, 2016 [accessed 2016 Mar],

Available from:

http://www.uptodate.com/contents/image?imageKey=ID/73023&source=graphics_search&rank=0&search=mod

ified+duke+criteria

43

Source:

UpToDate, Modified Duke criteria for diagnosis of infective endocarditis - Table B, 2016 [accessed 2016 Mar],

Available from:

http://www.uptodate.com/contents/image?imageKey=ID/53004&source=graphics_search&rank=0&search=mod

ified+duke+criteria

44

Appendix B: Patient Consent Form

TOESTEMMINGSVERKLARING

Voor deelname aan onderzoek: Sonicatie voor de diagnose hartklepinfectie

Bij u is recent / zal binnenkort een hartklep worden verwijderd. Wij willen u vragen of wij deze verwijderde hartklep mogen gebruiken voor onderzoek naar infectie van het hart. U hoeft voor dit onderzoek niks extra’s te doen. Er verandert niks aan de medische zorg die zal worden verleend. In ons onderzoek zullen wij een techniek testen die de oorzaak (vaak een bacterie) van een hartklepinfectie beter kan vaststellen. Dit is cruciaal voor het goed behandelen van deze infecties. Om dit onderzoek uit te kunnen voeren hebben wij hartkleppen nodig. Naast geïnfecteerde kleppen, hebben wij ook hartkleppen nodig die niet geïnfecteerd zijn. Er zal bij alle kleppen altijd eerst de standaardtechniek worden gebruikt die het laboratorium al jaren toepast. Als toevoeging zullen wij ook de nieuwe techniek (“sonicatie”) uitvoeren. Deze nieuwe techniek is al ingevoerd bij infecties van gewrichtsprothesen, bijvoorbeeld bij een kunstheup of kunstknie. Onze vragen aan u: 1) Mogen wij uw verwijderde hartklep gebruiken voor het testen van deze nieuwe techniek? 2) Mogen wij aanvullende informatie rondom de operatie uit uw medisch dossier halen?

- Ik ben goed over het onderzoek geïnformeerd. Ik heb bovenstaande informatie gelezen. Ik kon

aanvullende vragen stellen. Mijn vragen zijn voldoende beantwoord. Ik heb goed over deelname aan het onderzoek kunnen nadenken.

- Ik weet dat meedoen aan dit onderzoek helemaal vrijwillig is. Ik weet dat ik op ieder moment kan beslissen om toch niet mee te doen. Daarvoor hoef ik geen reden te geven. Ik weet dat aan meedoen geen negatieve consequenties voor de behandeling verbonden zijn.

- Ik stem ermee in dat relevante nieuwe gegevens, betreffende mijn gezondheid wordt gemeld aan mijn behandelend arts.

- Ik weet dat mensen betrokken bij dit onderzoek mijn medische gegevens kunnen inzien. Ik geef toestemming om deze gegevens te gebruiken, voor de doelen die hierboven omschreven staan.

- Ik geef toestemming om de onderzoeksgegevens 15 jaar na afloop van dit onderzoek te bewaren, zoals vereist in het toetsingsreglement nWMO plichtig onderzoek van het UMCG.

- Ik stem toe met deelname aan dit onderzoek. Naam: Geboortedatum: Handtekening: Datum:

Ondergetekende verklaart dat de hierboven genoemde persoon zowel schriftelijk als mondeling over het bovenvermelde onderzoek geïnformeerd is. Hij/zij verklaart tevens dat een voortijdige beëindiging van de deelname door bovengenoemde persoon van geen enkele invloed zal zijn op de zorg die hem of haar toekomt. Naam : Functie : Handtekening : Datum:

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Appendix C: Certificate Microbiological Safety Techniques