Rapid detection and identification of infection in CAPD ...repositorio.educacionsuperior.gob.ec/bitstream/28000/2118/1/T... · 1 Rapid detection and identification of infection in

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Rapid detection and identification of

infection in CAPD patients

Irina Maribel Villacrés Granda

Masters of Infectious Diseases

School of Pathology & Laboratory Medicine

21243178

Perth, 2014

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SUMMARY

Continuous ambulatory peritoneal dialysis (CAPD) is a category of peritoneal dialysis used

for the treatment of chronic renal failure as an alternative to hemodialysis.

Peritonitis is a common complication of CAPD technique. There are various routes of

infection during peritoneal dialysis. Commonly Gram- positive bacteria are the source of

infection followed by Gram-negative and fungal microorganisms.

Laboratory methods used for diagnosing CAPD-associate peritonitis are based on culture of

the dialysate by using enrichment media. In addition, some molecular methods as PCR or

DNA microarrays are used for bacterial identification. MALDI-TOF- MS is a mass

spectrometry technique that detects analytes and is used in clinical laboratories for the

diagnosis of infections.

Modifications to MALDI-TOF MS extraction protocols were made in order to use CAPD

dialysate as an initial sample and develop a new protocol that can be used to rapid detection

of infection in CAPD. The result showed that CAPD positive sample have a bacterial

concentration that is not high enough for the machine to read. Centrifugation protocols were

used to concentrate bacteria in the dialysate samples. Maximum velocity of centrifugation for

20 minutes was the protocol that gave better results. Depending on the centrifuge used and

the quantity of liquid in the assay, two different velocities were determined: 14,500 rpm in

1.5 ml and 4, 400 rpm in 25 ml.

Different types of water were tested in order to determinate the difference between their use

in washing steps. The highest scores value (2.305 and 2.303) were obtained in MALDI-TOF

reading using deionized water in washing steps.

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Minimum concentration of bacteria was determinated using induced infections, a. Both

Gram-positive and Gram- negative infections were estimated to have a threshold of 1

McFarland scale (3.0 x 106 CFU/mL). In addition, polymicrobial infections were induced in

negative CAPD dialysate. The result showed that MALDI-TOF MS cannot determinate

organisms that are in polymicrobial infections, only one microorganism was diagnoses in

every assay.

The obtained results showed that bacteria concentration in CAPD dialysate is not high

enough to perform a direct extraction for diagnosis by MALDI-TOF MS. It is suggested that

MALDI-TOF MS complements diagnosis by culture techniques and the study of other

methods to concentrate bacteria in CAPD dialysate therefore a direct, rapid detection can be

achieved.

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ACKNOWLEDGMENTS

I am extremely grateful to Professor Tim Inglis for initially suggesting this project and

successively supervising me and sharing your knowledge and experience.

I also would like to express my gratitude to Dr. Aron Chakera for your help as co supervisor

on this project.

Heartfelt thanks to all the staff in the bacteriology area of PathWest especially to Paul Healy

and Barbara Henderson for their unconditional assistance in the realization of this project.

To Fern Smith, Julie-Ann De Bond, Kaylee Anderson and Esteban Orellana thank you for

taking your time to answer my questions and help me with my dissertation.

To my friends in Ecuador and my friends in “por qué no los dos” group, thanks you guys for

being supportive and help me during this time.

To my family in Ecuador, thank you for keeping an eye on me and sending messages and

greetings of good luck.

Finally, I am very thankful with my parents and brother for being always there for me and

always cheering me up. Thank you for all the love and encouragement. I love you all you are

always in my thoughts.

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TABLE OF CONTENTS

SUMMARY ........................................................................................................................... ii

ACKNOWLEDGMENTS ..................................................................................................... iv

TABLE OF CONTENTS ....................................................................................................... v

LIST OF TABLES ................................................................................................................ ix

LIST OF FIGURES ............................................................................................................... xi

LIST OF ABBREVIATIONS ............................................................................................... xii

LIST OF ABBREVIATIONS (CONTINUATION…) ......................................................... xiii

1. INTRODUCTION ............................................................................................................. 1

1.1. Peritoneal Dialysis and Continuous Ambulatory peritoneal Dialysis .............................. 2

1.1.1. Peritoneal dialysis (PD) .......................................................................................... 2

1.1.1.1. Peritoneal dialysis system .............................................................................. 3

1.1.1.2. Categories of peritoneal dialysis .................................................................... 4

1.1.2. Continuous ambulatory peritoneal dialysis (CAPD) ................................................ 4

1.1.2.1. Continuous ambulatory peritoneal dialysis system ......................................... 5

1.1.2.2. Advantages and disadvantages of CAPD system ........................................... 6

1.2. CAPD-ASSOCIATED INFECTION ............................................................................. 7

1.2.1. Peritonitis ............................................................................................................... 7

1.2.1.1. Prevalence of peritonitis ................................................................................ 7

1.2.2. Pathogenesis ........................................................................................................... 8

1.2.2.1. Microorganisms isolated in CAPD-associated peritonitis ............................... 9

1.2.3. Treatment ............................................................................................................. 11

1.2.4. Prevention ............................................................................................................ 15

1.2.5. Epidemiology ....................................................................................................... 15

1.2.5.1. Worldwide infection rates of peritonitis ....................................................... 16

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1.2.5.2. Infection rates of peritonitis in Australia and New Zealand .......................... 16

1.3. LABORATORY DIAGNOSIS OF CAPD PERITONITIS .......................................... 17

1.3.1. Conventional bacterial identification .................................................................... 17

1.3.2. Molecular techniques for bacterial identification .................................................. 18

1.3.3. Bacteria identification by matrix-assisted laser desorption-ionization

time of flight mass spectrometry (MALDI-TOF MS) ....................................... 19

1.3.3.1. MALDI-TOF MS system ............................................................................ 20

1.3.3.2. Advantages and disadvantages of MALDI-TOF MS system ........................ 22

1.4. AIMS OF THE PROJECT .......................................................................................... 23

2. MATERIALS AND METHODS ..................................................................................... 24

2.1. SAMPLE COLLECTION ........................................................................................... 25

2.2. MALDI-TOF MS SAMPLE PREPARATION AND ANALYSIS: DIRECT

EXTRACTION FROM CAPD DIALYSATE ............................................................. 25

2.3. CONCENTRATION OF BACTERIA: ASSAYS USING POSITIVE CAPD

DIALYSATE SAMPLES ............................................................................................ 26

2.3.1. Time and centrifugation assay: use of different times and velocities of

centrifugation in 1.5 ml of sample .................................................................... 26


centrifugation in 25 ml of sample ..................................................................... 27

2.4. DIFFERENT TYPES OF WATER: ASSAY FOR WASHING CAPD

DIALYSATE SAMPLES ............................................................................................ 28

2.5. MALDI-TOF MS SAMPLE PREPARATION: MODIFIED METHODS

FOR EXTRACTION FROM PELLET SAMPLE ........................................................ 29

2.5.1. MALDI-TOF MS: modifications to the sample preparation for direct

transfer method ................................................................................................ 29

2.5.2. MALDI-TOF MS: modifications to the sample preparation using FA

extraction method ............................................................................................ 30

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2.6. MINIMUN BACTERIAL CONCENTRATION IN CAPD DIALYSATE ................... 30

2.6.1. McFarland scale assay .......................................................................................... 30

2.6.2. Serial dilutions assays ........................................................................................... 31

2.7. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED

INFECTIONS WITH A SINGLE ORGANISM .......................................................... 32

2.7.1. CAPD induced infection with Gram- positive bacterium ...................................... 32

2.7.2. CAPD induced infection with Gram- negative bacteria ......................................... 33


POLYMICROBIAL INFECTIONS ............................................................................. 33

2.8.1. CAPD induced polymicrobial infection with Gram- positive bacteria ................... 34

2.8.2. CAPD induced polymicrobial infection with Gram- negative bacteria .................. 34

2.8.3. CAPD induced polymicrobial infection with Gram positive and Gram-

negative bacteria .............................................................................................. 35

2.9. MOLDI-TOF MS READINGS .................................................................................... 35

3. RESULTS........................................................................................................................ 36

3.1. SAMPLE COLLECTION ........................................................................................... 37

3.2. MALDI-TOF MS SAMPLE ANALYSIS FOR DIRECT EXTRACTION

FROM CAPD DIALYSATE ....................................................................................... 37

3.3. CONCENTRATION OF BACTERIA IN POSITIVE CAPD DIALYSATE

SAMPLES .................................................................................................................. 41

3.3.1. Time and centrifugation assay in 1.5 ml of sample ................................................ 41

3.3.2. Time and centrifugation assay in 25 ml of sample................................................. 42

3.4. ANALYSIS OF DIFFERENT TYPES OF WATER USED FOR

WASHING CAPD DIALYSATE SAMPLES ............................................................. 49

3.5. MALDI-TOF MS SAMPLE ANALYSIS OF MODIFIED METHODS FOR

EXTRACTION FROM PELLET SAMPLE ................................................................ 49

3.6. MINIMUM BACTERIAL CONCENTRATION IN CAPD DIALYSATE .................. 52

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INFECTIONS WITH A SINGLE ORGANISM .......................................................... 52


POLYMICROBIAL INFECTIONS ............................................................................. 53

4. DISCUSSION.................................................................................................................. 61

5. LIMITATIONS OF THE STUDY, FURTHER EXPERIMENTS REQUIRED AND

CONCLUSIONS ............................................................................................................. 67

5.1 LIMITATIONS OF THE STUDY ............................................................................... 68

5.2 FURTHER EXPERIMENTS REQUIRED .................................................................. 68

5.3 CONCLUSIONS ......................................................................................................... 69

6. APPENDIX ..................................................................................................................... 70

7. REFERENCES ................................................................................................................ 73

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LIST OF TABLES

Table 1.1 Microorganisms identified in CAPD-associated peritonitis............................... 11

Table 1.2 Treatment of peritonitis according to the causative organism. .......................... 14

Table 1.3 Incidence of peritonitis in different countries. .................................................. 16

Table 3.1 Samples of CAPD dialysate obtained from PathWest Laboratory Medicine

Western Australia. ........................................................................................... 38

Table 3.2 MALDI-TOF MS reading results for direct extraction method from CAPD

dialysate. .......................................................................................................... 40

Table 3.3 Size of recovered pellets using different times and centrifugation velocities

in 1.5 ml of CAPD dialysate sample. ................................................................ 43

Table 3.4 Size of recovered pellets after washing step and using different times and

centrifugation velocities in 1.5 ml of CAPD dialysate sample. ......................... 44

Table 3.5 MALDI-TOF MS reading results for assay using different times and

centrifugation velocities protocols in 1.5 ml of CAPD dialysate sample. .......... 45


in 25 ml of CAPD dialysate sample. ................................................................. 46


centrifugation velocities in 25 ml of CAPD dialysate sample. .......................... 47


centrifugation velocities protocols in 25 ml of CAPD dialysate sample. ........... 48

Table 3.9 MALDI-TOF MS readings results for assays using different types of water

to wash CAPD dialysate samples. .................................................................... 50

Table 3.10 MALDI-TOF MS reading results for assay using modified method for

extraction from pellet samples. ......................................................................... 51

Table 3.11 MALDI-TOF MS readings results for different McFarland suspensions. ......... 54

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Table 3.12 MALDI-TOF MS readings results for serial dilutions made from 1

McFarland suspension. ..................................................................................... 55

Table 3.13 MALDI-TOF MS reading results for serial dilutions made from 0.5

McFarland scale from CAPD dialysate samples with Gram- positive and

Gram- negative induced infection. ................................................................... 56

Table 3.14 MALDI-TOF MS reading results for serial dilutions made from 1

McFarland scale from CAPD dialysate samples with Gram- positive and

Gram- negative induced infection. ................................................................... 58

Table 3.15 MALDI-TOF MS reading results for serial dilutions made from 1 McFarland

scale using CAPD dialysate samples with an induced polymicrobial infection

(mix of Gram- positive and Gram- negative). ................................................... 60

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LIST OF FIGURES

Figure 1.1 Graphic description of CAPD process ................................................................. 6

Figure 1.2 Schematic illustration of MALDI-TOF MS system ........................................... 20

Figure 1.3 Graphic scheme of bacterial identification by MALDI-TOF MS protein

mass detection method. ...................................................................................... 22

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LIST OF ABBREVIATIONS

ACN Acetonitrile

APD Automated peritoneal dialysis

API Analytical profile index

API Staph Analytical profile index for Staphylococci

CAPD Continuous ambulatory peritoneal dialysis

CCPD Continuous cycling peritoneal dialysis

CFU Colony-forming unit

FA Formic acid

g gravity

HCCA α-cyano-4- hydroxycinnamic acid

L Litre

MALDI-TOF MS Matrix-assisted laser desorption-ionization time of flight mass

spectrometry

Max Maximum

MIC Minimum inhibitory concentration

Min Minimum

Ml Millilitre

MRSA Methicillin-resistant Staphylococcus aureus

NIPD Nocturnal intermittent peritoneal dialysis

PCR Polymerase chain reaction

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LIST OF ABBREVIATIONS (CONTINUATION…)

PD Peritoneal dialysis

PVC Polyvinyl chloride

RNA Ribonucleic acid

rpm Revolutions per minute

TPD Tidal peritoneal dialysis

μl Microliter

°C Degrees Celsius

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1. INTRODUCTION

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1. INTRODUCTION – REVIEW OF THE LITERATURE

1.1. PERITONEAL DIALYSIS AND CONTINUOUS AMBULATORY

PERITONEAL DIALYSIS

1.1.1. Peritoneal dialysis (PD)

Peritoneal dialysis is the third most common method of renal replacement therapy.

Approximately 120,000 individuals with end-stage renal disease are undergoing this

treatment worldwide (Blake & Daugirdas 2007; Mehrotra & Boeschoten 2009).

In early 1900’s, Thomas Graham developed the theoretical basis of PD as a form of renal

replacement therapy by the discovery of laws of diffusion of gases, investigation of osmotic

force, and separation of chemical or biological fluids by dialysis (Gottschalk & Fellner 1997;

McBride 2005). In 1959, Morton Maxwell started the modern era of peritoneal dialysis

through the introduction of a semi-rigid nylon peritoneal catheter with a curved tip and

promotion of the commercial production of standard dialysis solution in 1 litre sterile glass

bottles (McBride 1984; Negoi & Nolph 2009). Many investigators have since tried to

improve the technology used for PD.

In 1983, Umberto Buoncristiani generated the Y system which decreased the number of

peritonitis episodes (Buoncristiani et al. 1983). Later, in 1991 a commercially introduced

double bag system based on the Y principle was used. This system used an empty bag and

one with dialysis solution which further reduced the number of disconnects and connections

(Balteau et al. 1991). These advances have reduced the infection rates and have improved the

quality of life in patients undergoing PD.

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1.1.1.1. Peritoneal dialysis system

The basic PD system consists of a Polyvinyl chloride (PVC) bag containing 1.5–3.0 litres of

dialysate, a transfer set, and a catheter access to the peritoneal cavity (Mehrotra &

Boeschoten 2009).

The peritoneal membrane is a single layer of mesothelial cells overlying layers of connective

tissues. It has two important properties: allows substances of certain sizes to move from an

area of greater concentration to lower concentration (semi-permeable membrane), and it is a

bidirectional membrane where substances move in either direction across the membrane

(Shrestha et al. 2010). Both properties allow the process of peritoneal dialysis. The dialysate

is introduced into the peritoneal cavity where it comes into contact with capillaries

surrounding the peritoneum and viscera. Solutes diffuse from blood in the capillaries into the

dialysate and are discarded (Shrestha et al. 2010).

Successful development of PD depends on the removal of solute and the fluid exchange that

occurs between peritoneal capillary blood and dialysis solution in the peritoneal cavity (Levy,

Morgan & Brown 2004). Transport of waste products and excess fluid from blood across the

peritoneal membrane is possible by three transport processes taking place simultaneously:

diffusion, ultrafiltration, and absorption (Blake & Daugirdas 2007).

The composition of dialysis solution in the peritoneal cavity can vary but the main goal is to

maximize diffusive solute loss from blood. Typically, the peritoneal dialysate contains

sodium, chloride, lactate or bicarbonate, and a carbohydrate osmotic component (Blake &

Daugirdas 2007; Levy, Morgan & Brown 2004; Mallappallil 2010).

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1.1.1.2. Categories of peritoneal dialysis

Different types of PD have evolved according to the social convenience of the patient and to

maximize the efficiency of PD (Levy, Morgan & Brown 2004).

The main categories of PD are manual versus automated dialysate delivery (Mallappallil

2010). In manual peritoneal dialysis, also known as continuous ambulatory peritoneal

dialysis (CAPD), the dialysis solution is constantly present in the abdomen and it is changed

four times daily (Heimbürger & Blake 2007). The automated dialysate delivery is termed

automated peritoneal dialysis (APD) and it can be continuous cycling peritoneal dialysis

(CCPD), nocturnal intermittent peritoneal dialysis (NIPD) or tidal peritoneal dialysis (TPD)

(Heimbürger & Blake 2007; Blake & Daugirdas 2007; Mehrotra & Boeschoten 2009). APD

uses an automatic cycling device to perform rapid exchanges of dialysate overnight (Levy,

Morgan & Brown 2004).

1.1.2. Continuous ambulatory peritoneal dialysis (CAPD)

The concept of CAPD was described for the first time in 1976 by Popovich, Moncrief,

Decherd, Bomar, and Pyle. The technique was introduced for treatment of chronic renal

failure as a viable alternative to hemodialysis (Mehrotra & Boeschoten 2009). CAPD is

currently a widely accepted treatment for end-stage renal disease that might be caused by

chronic glomerulonephritis, pyelonephritis, hypertension, some immunological diseases, and

toxic or ischemic damage to the kidney (Nissenson et al. 1986).

The acceptance of CAPD has increased since its introduction due to its simplicity,

convenience, and relatively low cost (Blake & Daugirdas 2007). CAPD essentially

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represents a continuous portable dialysis system which allows patients to continue with daily

activities (Popovich et al. 1978).

1.1.2.1. Continuous ambulatory peritoneal dialysis system

CAPD uses the continuous presence (24 hours a day, 7 days a week) of peritoneal dialysis

solution in the peritoneal cavity, except for periods of drainage and instillation of fresh

solution three to five times per day (Popovich et al. 1978).

The dialysate is instilled into the peritoneal cavity via a trans-abdominal catheter entering

through the anterior abdominal wall, piercing the parietal peritoneum and with its tip sited in

the pelvis. The peritoneal membrane is then utilized for the exchange of electrolytes, glucose,

urea, albumin and other small molecules from the blood (Goldstein, Carrillo & Ghai 2013).

Drainage of dialysate and inflow of fresh dialysis solution are performed manually, using

gravity to move fluid into and out of the peritoneal cavity (Heimbürger & Blake 2007). At

the end of the procedure, the patient is disconnected from all tubing, the indwelling peritoneal

catheter is capped and the patient is free to participate in his usual daily activities (Popovich

et al. 1978) (Figure 1.1).

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Figure 1.1. Graphic description of CAPD process (from Fresenius Medical Care AG & Co 2013)

1.1.2.2. Advantages and disadvantages of CAPD system

Although many potential complications occur more frequently with CAPD than other renal

replacement techniques, its advantages include the absence of need for a highly skilled

operator and lack of need for anticoagulation (Cochran et al. 1997).

Disadvantages of the system include electrolyte/ acid-base imbalance, infection and surgical

or mechanical catheter related problems; however, the most frequent complication of CAPD

is the occurrence of peritonitis associated with a high risk of mortality and morbidity

(Cochran et al. 1997; Gould & Casewell 1986; Males, Walshe, & Amsterdam 1987; Dalaman

et al. 1998).

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1.2. CAPD-ASSOCIATED INFECTION

1.2.1. Peritonitis

Peritonitis is a common complication and a leading cause of technique failure in patients

undergoing CAPD (Prasad et al. 2003; Guo & Mujais 2003). It can be associated with severe

pain leading to hospitalization, catheter loss and a risk of death (Bender, Bernardini &

Piraino 2006).

Clinically, CAPD-peritonitis is diagnosed according to three criteria: cloudy or turbid

peritoneal dialysate containing >100 white blood cells/mm3 of which 50% or more are

polymorphonuclear leukocytes; indications of peritoneal inflammation, such as abdominal

tenderness, nausea, vomiting and fever; and a microbiologically positive fluid culture

(Peterson, Matzke & Keane 1987; Troidle & Finkelstein 2006; Popovich et al. 1978).

1.2.1.1. Prevalence of peritonitis

Although 18% of the infection-related mortality in PD patients is the result of peritonitis,

only 4% of peritonitis episodes result in death (Akoh 2012). In the last 15 years, techniques

and technology have reduced the number of peritonitis infections from 1 in 11 to 1 in 24 or

more patient months on treatment (Daly et al. 2001).

The incidence of peritonitis in CAPD patients depends on different risk factors, for example,

age, race, educational background, environment, poor nutrition, immunosuppression, and

organisms isolated (Fried et al. 1996; Chow et al. 2005). Some studies show that prior

antibiotic use is also a risk factor for fungal peritonitis (Goldie et al. 1996; Johnson et al.

1985; Huang et al. 2000), while the use of gastric inhibitors increases the risk of Gram-

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negative bacterial peritonitis (Caravaca, Ruiz-Calero & Dominguez 1998). Besides these

factors, it is considered that the strongest dialysis related factors are the type of connection

system and staphylococcal nasal carriage (Fried & Piraino 2009).

Peritonitis episodes are defines as recurrent or relapsing if the same organism with the same

susceptibility pattern is isolated within a four week period after the completion of a standard

two week course of antimicrobial therapy (Troidle & Finkelstein 2006). 60 to 90% of

peritonitis episodes are resolved with antibiotic therapy. However, in some cases, the

removal of the catheter and transfer to hemodialysis is necessary due to technique failure or

peritoneal membrane failure because of severe and prolonged peritonitis (Fried & Piraino

2009; Woodrow, Turney & Brownjohn 1997; Tranaeus, Heimburger & Lindholm 1989).

1.2.2. Pathogenesis

There are various routes of microorganism entry during peritoneal dialysis (Fried & Pirano

2009). The most common contamination route is the intraluminal route. At the time of the

fluid exchange, improper catheter connection technique allows bacteria from the patient’s

skin to gain access to the peritoneal cavity (Leehey, Cheuk-Chun & Li 2007). The resulting

infection is predominantly caused by Gram-positive bacterial skin flora (Vas 1981). However

some patients have Gram-negative bacteria colonizing their skin which can lead to Gram-

negative peritonitis (Fried & Piraino 2009). Contamination from mouth and nose organisms

can also occur in individuals who do not wear a protective face mask during connections (De

Vecchi & Scalamogna 2001).

The intestinal flora might cause peritonitis by an enteric or transmural route where Gram-

negative bacteria are more predominant. Infection may be caused by abdominal perforation,

diarrheal states, instrumentation of the colon and strangulated hernia (Rotellar et al. 1992;

Leehey, Cheuk-Chun & Li 2007).

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Ascending infections from a gynecological source (transvaginal route) may also lead to

peritonitis (Bailey et al. 2002; Leehey, Cheuk-Chun & Li 2007).

Biofilms formation has been reported after several months of peritoneal catheter use. The

intraperitoneal portion of almost all permanent peritoneal catheters becomes covered with a

bacteria-laden slime predisposing to relapsing Pseudomonas and staphylococcal peritonitis

(Finkelstein et al. 2002).

1.2.2.1. Microorganisms isolated in CAPD-associated peritonitis

Although detection of microorganisms in CAPD-associated peritonitis is common, some

studies have reported up to 20% of episodes may be culture-negative (Lye et al. 1994; Akoh

2012); Consequently, the importance of adequate culturing techniques cannot be overstated

(Piraino et al. 2005).

There is no significant difference in causative agents between home and hospital acquired

peritonitis (Nakwan, Dissaneewate & Vachvanichsanong 2008). It has been determined that

episodes of peritonitis can be polymicrobial or caused by a single microorganism; and can be

due a wide spectrum of microorganisms (Ghali et al. 2011; Akoh 2012).

The typical spectrum of isolates includes Gram-positive organisms (62.6%), Gram-negative

organisms (28.9%), fungal (5.7%) and mycobacteria (2.8%) (Troidle & Finkelstein 2006;

Akman et al. 2009; Vikrant et al. 2013; Ghali et al. 2011).

The most frequently isolated Gram-positive bacteria from CAPD-associated peritonitis are

the coagulase negative and coagulase positive staphylococci. Staphylococcus epidermidis and

Staphylococcus aureus account for approximately 40% to 50% of the isolates (Sharma et al.

2010; Troidle & Finkelstein 2006). Methicillin-resistant Staphylococcus aureus (MRSA) is

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found in approximately 5% of Gram-positive peritonitis episodes (Troidle & Finkelstein

2006). Streptococcus viridans and other streptococci are found in lower incidence in single-

organism peritonitis episodes as well as polymicrobial episodes (Levy, Morgan & Brown

2004).

Enterococcal infections are uncommon cause of peritonitis episodes (7-8%) (Ghali et al.

2011; Vikrant et al. 2013). Usually, Enterococcus spp. is prevalent in polymicrobial

peritonitis and it has been associated with older age, renovascular disease and coronary artery

disease (Edey et al. 2010; Akoh 2012).

Among the Gram-negative bacteria isolated in peritoneal infections, Escherichia coli is the

most frequently isolated organism, followed by Pseudomonas, Klebsiella, and Enterobacter;

Acinetobacter spp., and other Gram-negative bacteria are identified in lower incidence (Ghali

et al. 2011; Vikrant et al. 2013).

Mycobacterial infections are infrequent, occurring in 1% to 3% of polymicrobial or single-

organism episodes (Ghali et al. 2011; Vikrant et al. 2013). However, a recurrence of

mycobacterial infection has been reported in CAPD patients with reduced cellular immunity

(Goldstein, Carrillo & Ghai 2013). Mycobacterium tuberculosis or non-tuberculosis

mycobacteria, such as Mycobacterium fortuitum, Mycobacterium avium, Mycobacterium

abscessus, Mycobacterium kansasii and Mycobacterium chelonae can be found causing an

infection (Akoh 2012).

Fungal infections are caused in the majority (69–85%) by Candida spp. (Wang et al. 1998).

Other causes of fungal peritonitis include Rhizopus spp., Aspergillus flavus and

Paecilomyces species (Wright et al. 2003; Vikrant et al. 2013). Risk factors predisposing to

fungal peritonitis include prior antibiotic use, and patient malnutrition, particularly in patients

http://ovidsp.tx.ovid.com/sp-3.11.0a/ovidweb.cgi?targetFrame=1&S=EDMBFPLCILDDGNABNCMKCCGCDHMMAA00&Book+Content=BIB.22%7c1%7c00000000%7c%7c/CT%7b06b9ee1beed59419ef23ac3ebca478776cda56a76f91e4a226ebe40cba4ffc150aa4eea55437b1d70a2846659489f909%7d/OVIDBOOK%5b1%5d/TXTBKBD%5b1%5d/DIVISIONA%5b3%5d/CHAPTER%5b7%5d#RU43-24

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with low serum albumin level (Leehey, Cheuk-Chun & Li 2007). Mortality due to fungal

peritonitis ranged from 14.3 to 46% (Wang et al. 1998; Chan et al. 1994).

Table 1.1 (adapted from Troidle & Finkelstein 2006; Akman et al. 2009; Vikrant et al. 2013;

Ghali et al. 2011) provides a summary of the microorganisms causing CAPD-associated

peritonitis.

Table 1.1 Microorganisms identified in CAPD-associated peritonitis

Gram- positive

bacteria

Staphylococcus epidermidis

Gram-negative

bacteria

Escherichia coli

Staphylococcus aureus Pseudomonas spp.

Staphylococcus aureus

(MRSA) Klebsiella spp.

Viridans streptococci Enterobacter spp.

Non viridans streptococci Acinetobacter spp.

Enterococcus spp. Serratia spp.

Listeria monocytogenes Proteus spp.

Mycobacterium

M. tuberculosis

Fungal

Candida albicans

M fortuitum Non- albicans

species

M. avium Rhizopus spp.

M. abscessus Aspergillus flavus

M kansasii Paecilomyces spp.

1.2.3. Treatment

The development of antimicrobial resistance may be due to the empirical use of extended-

spectrum cephalosporins and quinolones. Vancomycin resistant enterococci, vancomycin

intermediate sensitive and methicillin-resistant staphylococci and multi-drug resistant Gram-

negative organisms have all been reported to cause dialysis related peritonitis (Troidle et al.

1996; Zelenitsky et al. 2000).

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It has been determined that patients with Gram-negative peritonitis generally have a worse

clinical outcome than patients with Gram-positive peritonitis (Troidle et al. 1998). However,

the mortality due to fungal peritonitis can be as high as 46% (Sahu et al. 2000).

Polymicrobial peritonitis infections are associated with higher rates of hospitalization,

catheter removal, permanent hemodialysis transfer, and mortality compared with single-

organism infections (Edey et al. 2010; Barraclough et al. 2010).

Treatment of peritonitis depends on the microorganism isolated in the peritoneal dialysate

and the clinical history of the patient. Awareness of microbiologic profiles, local antibiotic

resistance patterns, and local peritonitis rates are important in guiding medical treatment

(Ghali et al. 2011).

When treating dialysis – associated peritonitis, the abdomen should be drained and the

effluent carefully inspected and sent for cell count and white blood cell differential, Gram

stain, and culture (Piraino et al. 2005). To prevent delay in treatment, antibiotic therapy

should be initiated as soon as a cloudy effluent is seen.

Empiric antibiotics must cover both Gram-positive and Gram-negative organisms. Systemic

vancomycin and ciprofloxacin administration is used as first-line protocol for antibiotic

therapy (Goffin et al. 2004). Another therapy that has been effective is the use of

intraperitoneal antibiotics in peritoneal dialysis solution concentrations such as gentamicin,

cephalotin, and ampicillin (Popovich et al. 1978).

Peritonitis caused by coagulase negative staphylococci, including S. epidermidis, is generally

a minor form of peritonitis and can be treated with first- generation cephalosporins; although,

in some cases, coagulase-negative Staphylococcus can lead to relapsing peritonitis due to

biofilm involvement, and catheter replacement is advised (Leehey et al. 2007; Read et al.

1989).

13

Staphylococcus aureus is associated with catheter infection or colonization (Amato et al.

2001; Piraino, Bernardini & Sorkin 1987). Therefore, peritoneal infections are treated with

anti-staphylococcal penicillins or first generation cephalosporins (Piraino et al. 2005). In the

case of MRSA infection, the use of vancomycin is recommended (Mulhern et al. 1995).

Enterococcal peritonitis can be treated with ampicillin or vancomycin plus an

aminoglycoside are generally employed. However, in cases where sensitivity testing indicates

vancomycin resistance, the use of linezolid or quinupristin/dalfopristin is recommended

(Leehey et al. 2007).

Treatment for Gram-negative bacteria such as Escherichia coli, Klebsiella and Proteus can

be provided with an aminoglycoside, ceftazidime, cefepime, or carbapenem. Peritonitis

caused by Pseudomonas aeruginosa is generally severe, and difficult to eliminate (Piraino et

al. 2005). Usually an oral quinolone can be given but alternative drugs including ceftazidime,

cefepime, tobramycin, or piperacillin can be used (Leehey et al. 2007).

Mycobacteria are an uncommon cause of peritonitis and can be difficult to diagnose. The

current treatment for Mycobacterium tuberculosis is with four drugs: rifampin, isoniazid,

pyrazinamide, and ofloxacin while the treatment protocol for non-tuberculous mycobacterial

peritonitis is not well established and requires individualized protocols based on

susceptibility testing (Piraino et al. 2005).

When a fungal microorganism is diagnosed as causing peritonitis, immediate catheter

removal and the application of amphotericin B and flucytosine as initial therapy is indicated

(Piraino et al. 2005). Caspofungin, fluconazole, or voriconazole may replace amphotericin B,

based on species identification and minimum inhibitory concentration (MIC) values. For

14

filamentous fungi and Candida peritonitis it is recommended, as an alternative, the use of

voriconazole (Piraino et al. 2005).

Table 1.2 summarizes the different organisms and the treatment applied in CAPD -

associated peritonitis.

Table 1.2 Treatment of peritonitis according to the causative organism.

Microorganism Treatment Additional/Alternative treatment

Staphylococcus

epidermidis

1st generation

cephalosporins

Catheter replacement if there is

biofilm formation

Staphylococcus

aureus

Anti-staphylococcal

penicillin, 1st generation

cephalosporin

---------------

MRSA Vancomycin ---------------

Enterococcus

spp.

Ampicillin + vancomycin +

aminoglycoside

If vancomycin resistance treat with

linezolid or quinupristin/dalfopristin

Escherichia coli Aminoglycoside, cedtazime,

cefepime, carbapenem --------------- Klebsiella spp.

Proteus spp.

Pseudomonas

aeruginosa Oral quinolone

Ceftazidime/cefepime, trobramycin or

piperacillin

Mycobacterium

tuberculosis

Rifampin, isoniazid,

pyrazinamide, ofloxacin ---------------

Non-

tuberculosis

Mycobacterium

No established Requires individual protocols base on

susceptibility testing

Fungal Amphotericin B +

flucytosine

Caspofungin, fluconazole, or

voriconazole (used to can be used to

treat Candida and filamentous fungi).

MRSA: Methicillin-resistant Staphylococcus aureus

15

1.2.4. Prevention

Preventing infections in PD patients is very important as this is one of the biggest causes of

technique failure and resulting change from PD to hemodialysis.

An effective method of infection control is the instruction of patients on aseptic and proper

hand washing techniques, including the use of alcohol hand washing before exchanging the

bag (Piraino et al. 2005).

Prophylactic antibiotics are used to avoid infections in certain cases; for example, if dialysis

solution was infused after contamination or if the catheter has been exposed. CAPD patients

requiring extensive dental procedures should receive amoxicillin two hours before the

procedure to avoid transient bacteremia (Fried, Bernardini & Piraino 2000; Piraino et al.

2005)

In the case of relapsing or repeated episodes of peritonitis it is recommended to replace the

PD catheter to avoid constant infections (Finkelstein et al. 2002).

1.2.5. Epidemiology

The utilization of PD in different countries since its development has increased. There are

149,000 patients undergoing PD worldwide (Nolph 1996; Mallappallil 2010). In 2000, the

use of continuous cyclers in North America was 54%, while in Australia usage has increased

from 4% in 1995 to 42% in 2004 (Brown et al. 2013). By 2009, over 90% of all renal patients

in North America, Europe, Australia, and New Zealand used continuous cyclers (Mehrotra &

Boeschoten 2009).

16

1.2.5.1. Worldwide infection rates of peritonitis

Even though, development of new technology and prevention of infections has increased and

has improved since the first use of PD, peritonitis remains a significant complication of

chronic PD (Akoh 2012). Table 1.3 describes the incidence of peritonitis in different

countries ranging from 0.82 episodes per patient-year in the United Kingdom to less than

0.29 episodes in Korea.

Table 1.3 Incidence of peritonitis in different countries.

Country Infection rate

(episodes/patient-year) Year Reference

United Kingdom 0.82 2002-2003 Davenport 2009

Hong Kong 0.68 1995-2003 Szeto et al. 2005

Sudan 0.66 2006 Abu- Aisha et al. 2006

India 0.62 2000-2004 Keithi-Reddy et al. 2007

Colombia 0.46 1994-2003 Pecoits-Filho et al. 2007

Canada 0.43 1996-2005 Mujais 2006

France 0.41 2000- 2007 Verger et al. 2006

United States 0.37 1998-2004 Mujais 2006

Korea 0.29 2004-2005 Han et al. 2007

1.2.5.2. Infection rates of peritonitis in Australia and New Zealand

In Australia and New Zealand, peritonitis is the major cause of PD technique failure,

accounting for up to 40% of cases (Brown et al. 2013). Ghali et al. (2011) determined in an

overall peritonitis rate of 0.60 episodes per patient in a contemporaneous cohort of all

Australian patients treated with PD.

17

The Australian and New Zealand Dialysis and Transplant Registry (ANZDATA), in its 2013

report, reported 406 of the 2227 patients undergoing PD acquired peritonitis of which 25 died

(Brown et al. 2013).

1.3. LABORATORY DIAGNOSIS OF CAPD PERITONITIS

1.3.1. Conventional bacterial identification

Commonly, the number of causative microorganisms in the peritoneal dialysate is low in

CAPD peritonitis. In order to increase the concentration of bacteria in the dialysate, methods

such as enrichment media culture or concentration by centrifugation are used in clinical

laboratories (Sauer & Kliem 2010; Males et al 1986).

Procedures such as Gram staining, catalase, latex agglutination, and the catalase and oxidase

tests have been used as a first line strategy to identify bacteria. Secondary phenotypic tests,

such miniaturized biochemical tests or automated identification systems are able to fully

identify the organism (Carbonnelle et al. 2011; Males et al 1986).

Although the culture media used to identify peritoneal dialysate samples varies between

clinical laboratories, chocolate blood agar, sheep blood agar; MacConkey agar, electrolyte

deficient agar, lysed blood agar, anaerobic and aerobic blood culture bottles, and

thioglycollate broth are the most commonly used (Dekker & Branda 2011; Knight et al.

1982; Fenton 1982).

The analytical profile index (API) method is a miniaturized commercial method which allows

rapid identification of bacteria according to its biochemical profile. Although it is considered

rapid method, the analysis takes several hours and in some cases can be inaccurate in

18

assigning bacteria to a species. This method is especially useful for the identification of

Enterobacteriaceae and Staphylococcus which are identified by using the API 20E method

and API Staph assays respectively (Carbonnelle et al. 2011; Fenton 1982; Brown et al. 1991).

While some of these tests are performed within minutes, identification can only be completed

and reported around 24 hours after isolation (Cherkaoui et al. 2010). The amount of time can

be increased if the organism has a slow growth rate and/or requires specialized culture media

(Carbonnelle et al. 2011).

1.3.2. Molecular techniques for bacterial identification

The use of molecular techniques is being increasingly employed in clinical laboratories to

complement and enhance the use of conventional culture methods (Kim et al. 2012). Broad-

range polymerase chain reaction (PCR) and sequencing are commonly used molecular

methods because most microorganisms can be detected regardless of their species and

specific culture conditions, (Sontakke et al. 2009; Fenollar, Lévy & Raoult 2008).

PCR is a rapid and highly sensitive and specific test for the identification of microorganisms.

The technique allows the identification of slow-growing and non-cultivable organisms

(Shankar et al. 1990). PCR assays used for bacterial identification rely on the amplification of

conserved genes such as those encoding for ribosomal ribonucleic acid (RNA) (Goldenberger

et al. 1997; Xu et al. 2003), RNA polymerase (rpoB) (Drancourt & Raoult, 2012) or

elongation factors (Schneider, Gibb & Seemuller 1997).

One of the most frequently used PCR assays used in laboratory diagnostics is the 16s rRNA-

PCR (Cherkaoui et al. 2010). The technique uses universal primers that amplify the highly

conserved 16S rRNA in prokaryotes. In addition, sequencing of the amplicons and

19

comparison with open access gene databases allows the identification of causative organisms

in many infectious diseases (Dekker & Branda 2011).

DNA chips and DNA microarrays have also been implemented to amplify and detect

multiple DNA sequences simultaneously (Cherkaoui et al. 2010). However, cost and

workload requirements limit their routine use in clinical diagnosis laboratories (Couzinet et

al. 2005).

1.3.3. Bacteria identification by matrix-assisted laser desorption-ionization time of flight

mass spectrometry (MALDI-TOF MS)

MALDI-TOF MS is a new and powerful technique that has emerged for rapid identification

of microorganism in the clinical microbiology laboratory (Sauer et al. 2008; Ho & Reddy

2010; Carbonnelle et al. 2011). The method is used to analyze intact proteins extracted from

whole microorganisms, without extensive sub fragmentation, and subsequent release of

protein mass spectra (Dekker & Branda 2011).

Although the current emphasis of MALDI-TOF MS technique is on bacterial detection, it

may be used to analyze many types of samples including solutions of organic molecules,

nucleic acids and proteins (Kliem & Sauer 2012). Furthermore, the method can be used for

the identification of fungi (Cassagne et al. 2011), mycobacteria (El Khechine et al. 2011), and

yeast (Marklein et al. 2009).

Commercialization of MALDI-TOF MS began in the twenty-first century (Martiny et al.

2013). Since then, many new strategies to perform rapid detection of microorganisms by

MALDI-TOF MS have been evaluated (Gaibani et al 2009; Croxatto, Prod’hom & Greub

2012).

20

Research by Holland et al. (1996) was the first report of bacterial identification based on

MALDI-TOF MS analysis without undergoing any treatment before the analysis. In the same

year, spectral fingerprints of pathogenic species such as Bacillus anthracis, Brucella

melitensis, Yersinia pestis, and Francisella tularensis were obtained by (Krishnamurthy,

Ross & Rajamani 1996).

Research in MALDI-TOF MS method has resulted in the development of extensive

microorganism databases and the refinement of the technique (Martiny et al. 2013).

1.3.3.1. MALDI-TOF MS system

Figure 1.2 shows the classic MALDI-TOF MS system. It consists of three principal

components: specimen ionization chamber where the laser-based vaporization of the

specimen takes place, a time of flight mass analyzer, and a particle detector (Dekker &

Branda 2011).

Figure 1.2 Schematic illustration of MALDI-TOF MS system (from Dekker & Branda 2011).

21

The first step of the technique is to place the sample onto a MALDI-TOF MS target plate

with a chemical matrix (Hortin 2006). This process causes the formation of a crystal between

the sample and the organic matrix. The matrix has two major functions: absorption of energy

from the laser and isolation of the biopolymer molecules. The matrices most commonly used

are 2, 5-dihydroxybenzoic acid (gentisic acid), 3, 5-dimethoxy-4-hydroxycinnamic acid

(sinapinic acid), and α-cyano-4- hydroxycinnamic acid (α-CHCA) (Fenselau & Demirev

2001).

After the sample and matrix have been applied to the target plate, it is inserted and loaded

into the specimen ionization chamber of the MALDI-TOF MS machine where the target is

pulsed with an ultraviolet nitrogen laser (337 nm). Vibrational excitation of the sample is a

result of the laser irradiation which creates positively charged analyte cations in the gas

phase. Once desorbed, the matrix molecules are pulsed into a flight tube where the gas

analyte cations are accelerated across an electric field within the ionization chamber to a

velocity that depends on the mass-to-charge (m/z) ratio of the analyte (Dekker & Branda

2011; Carbonnelle et al. 2011).

At this point, a mass spectrum is generated based on the time of flight and the m/z ratio of

each particle. The results obtained are compared with suitable mass spectral fingerprints

databases that are available in commercial software packages developed by the machine

manufacturer (Dekker & Branda 2011; Sauer et al. 2008; Klien & Sauer 2012).

Figure 1.3 shows a graphic scheme of bacterial identification using the MALDI-TOF MS

technique.

22

Figure 1.3 Graphic scheme of bacterial identification by MALDI-TOF MS protein mass detection

method (from Kliem & Sauer 2012).

1.3.3.2. Advantages and disadvantages of MALDI-TOF MS system

One of the advantages of the MALDI-TOF MS method applied to clinical diagnosis is that

whole bacterial cells can be processed with minor amounts of work and low-cost

consumables (Bizzini & Greub 2010). The cost is estimated to be reduced by three to five

times compared to conventional and biochemical identification systems (Dekker & Branda

2011). In addition, parallel analysis of 10 isolates can be performed in less than 15 min, and

as a result, the transmission of result to the physicians is faster and accurate treatment to

patients can be applied more quickly (Carbonnelle et al. 2011; Cherkaoui et al. 2010).

Although MALDI-TOFMS is considered an accurate technique for bacterial identification,

species that are similar at the proteomic level can be misidentified (Dekker & Branda 2011).

As an example, Shigella species can be identified as Escherichia coli and Streptococcus

species as Streptococcus pneumoniae, Streptococcus mitis and Streptococcus viridans

(Carbonnelle et al. 2011). Also polymicrobial cultures are difficult to identify due to mix

spectra analysis (Dekker & Branda 2011).

23

1.4. AIMS OF THE PROJECT

Currently, there are few studies involving the laboratory diagnosis of CAPD peritonitis using

MALDI-TOF MS technique directly from peritoneal dialysates. For this reason, this project

is important for the scientific community involved in the research for treatment of CAPD

peritonitis.

The aims of this project are:

- To develop a MALDI-TOF method to identify bacteria directly from CAPD fluids in a

routine clinical microbiology laboratory.

- To determinate the sensitivity and specificity of the MALDI-TOF method against a range

of microorganism frequently encounter in CAPD- associated peritonitis.

- To determinate the usefulness of the methods in polymicrobial CAPD- associated

peritonitis.

24

2. MATERIALS AND METHODS

25

2. MATERIALS AND METHODS

2.1. SAMPLE COLLECTION

The present research includes 20 samples of peritoneal dialysate obtained from patients

undergoing CAPD. The samples were collected in PathWest Laboratory Medicine Western

Australia from March to May of 2014 and stored at 4 °C.

Every sample was processed by cleaning the CAPD bag with 70% ethanol and preparing two

aliquots of 50 ml each in plastic containers. Aliquots were used in order to avoid

contamination of the original sample.

Number of the samples, date of collection and diagnostic are detailed in table 3.1

2.2. MALDI-TOF MS SAMPLE PREPARATION AND ANALYSIS: DIRECT

EXTRACTION FROM CAPD DIALYSATE

Positive samples 7 and 11 of CAPD dialysate were used in order to perform PathWest

MOLDI-TOF MS sample preparation and analysis laboratory method: direct extraction

method using Formic acid (FA) (Appendix one).

Modifications in step “a” from the current protocol were made by using 1 ml of peritoneal

dialysate instead of 1 ml of a positive blood culture fluid.

26

2.3. CONCENTRATION OF BACTERIA: ASSAYS USING POSITIVE CAPD

DIALYSATE SAMPLES

Samples 7 and 11 which were diagnosed as positive (table 3.1) were used to perform these

assays. The different quantities of CAPD dialysate were tested in order to determine if the

quantity of bacteria recovered is different, and if there is a variation on the MALDI-TOF MS

reading.


centrifugation in 1.5 ml of sample

1.5 ml of CAPD dialysate were taken from the aliquots of samples 7 and 11 and placed in 1.5

ml eppendorf tubes respectively. Different times and velocities were applied to each sample

as follow:

1) 3,000 rpm for 10 minutes






Eppendorf Minispin plus F-45-12-11 centrifuge (Max velocity: 14,500 rpm (14,100 x g)) was

used to perform this assay at room temperature. After samples were centrifuged, supernatant

was removed and 1 ml of saline water 0.85% was added in order to wash and resuspend the

obtained pellet. Centrifugation steps described above were repeated, supernatant was

discarded and pellet was resuspended with 1 ml of saline water 0.85%. Once concentration

and washing steps were finished, the samples were prepared according to the MALDI-TOF

27

MS: protocol for sample preparation using FA extraction method (Appendix one). As

modification to the protocol, the final samples obtained in this assay were used instead of the

positive blood culture that is used in step “a” of the original protocol.

Data about the presence and size of the obtained pellets were collected after the first and

second centrifugation steps.


centrifugation in 25 ml of sample

25 ml of CAPD dialysate were taken from the aliquots of samples 7 and 11 and placed in 25

ml CAPD plastic tubes. Different times and velocities were applied to each sample as

follow:







Eppendorf 5702 A-4-38 centrifuge (Max velocity: 4,400 rpm (3,000 x g)) was used at room

temperature to perform this assay. After samples were centrifuged, supernatant was

discarded, 5 ml of saline water 0.85% were added and the pellet obtained was resuspended by

pipetting. Subsequently, centrifugation steps were repeated, supernatant was discarded and

pellet was resuspended with 1 ml of saline water 0.85%. 1 ml of the sample was poured in

1.5 ml eppendorf tubes and MALDI-TOF MS: protocol for sample preparation using FA

28

extraction method (Appendix one) was performed with modifications in step “a” where

samples obtained in this assay were used instead of positive blood culture fluid.

Data about the presence and size of the pellet was obtained after the first and second

centrifugation steps.

2.4. DIFFERENT TYPES OF WATER: ASSAY FOR WASHING CAPD

DIALYSATE SAMPLES

Positive samples 7 and 11 were used to perform this assay. Additionally, negative samples 4

and 12 were inoculated with Staphylococcus aureus and Streptococcus dysgalactiae

respectively to establish a bacterial infection and used as positive control.

Bacteria previously incubated in blood agar were diluted in 5 ml of saline water until change

in the turbidity was observed. Induced infection was obtained by adding 1 ml each bacterium

suspension in 24 ml of negative samples 4 and 12 of CAPD dialysate.

Different types of water were used in the washing steps to establish differences in results

obtained after performing the reading in the MALDI TOF MS machine.

The types of water used were: saline water 0.85 %, deionized water, and distilled water.

The protocol used, in every sample, to perform this assay was as follows:

- Put 25 ml of each CAPD dialysate to be tested in 25 ml CAPD plastic tubes

- Centrifuge at 4,400 rpm for 20 min using eppendorf 5702 A-4-38 centrifuge

- Discard the supernatant

- Add 5 ml of saline water 0.85 %, deionized water and distilled water in the respective

tube

- Centrifuge at 4,400 rpm for 20 min using eppendorf 5702 A-4-38 centrifuge

29

- Discard the supernatant

- Add 1 ml of saline water 0.85 %, deionized water and distilled water in the

corresponding tube

- Put 1 ml of the samples in 1.5 eppendorf tubes respectively

- Continue with the MALDI-TOF MS: protocol for sample preparation using FA

extraction method (Appendix one) from step “b”

2.5. MALDI-TOF MS SAMPLE PREPARATION: MODIFIED METHODS FOR

EXTRACTION FROM PELLET SAMPLE

Positive CAPD dialysate samples 11 and 16 were used in this assay. In addition, induced

infection with Streptococcus dysgalactiae in negative sample 4 was used as positive control

and sample 12 (negative) was used as negative control.

Induced infection was performed by using bacterial isolate previously incubated in blood

agar. Bacterial sample was suspended in 5 ml of saline water until change in the turbidity was

observed. 24 ml of negative CAPD dialysate sample 4 was inoculated with 1 ml of bacterial

suspension.

2.5.1. MALDI-TOF MS: modifications to the sample preparation for direct transfer

method

25 ml of each sample were centrifuged at 4,400 rpm for 20 minutes using eppendorf 5702 A-

4-38 centrifuge. After discarding the supernatant, the pellet obtained was placed in the steel

MALDI target with the help of a bacteriological loop and toothpick. Later, MALDI-TOF

MS: protocol for sample preparation direct transfer method (Appendix two) was performed

with the modification in step 1 where the sample’s pellet was used instead of bacterial smear.

30

2.5.2. MALDI-TOF MS: modifications to the sample preparation using FA extraction

method

25 ml of each CAPD dialysate sample were centrifuged at 4,400 rpm for 20 minutes using

eppendorf 5702 A-4-38 centrifuge. After discarding the supernatant, pellet was placed in 1.5

eppendorf tube and MALDI-TOF MS: protocol for sample preparation using FA extraction

method (Appendix one) was performed starting from step “b” where 200 μl of lysis buffer

were added directly to the pellet obtained.

2.6. MINIMUN BACTERIAL CONCENTRATION IN CAPD DIALYSATE

Induced infection was made using CAPD dialysate 4 (negative) and Staphylococcus

epidermidis sample previously incubated in blood agar to determinate the minimum

concentration of bacteria in CAPD dialysate necessary to obtain an accurate reading from the

MALDI-TOF MS machine.

2.6.1. McFarland scale assay

Determination of 0.5, 1, 2 and 3 McFarland suspensions were made using BioMerieux Vitek

Colorimeter No. 52-1210. 2ml of negative CAPD dialysate were placed in glass tubes. Using

a cotton swab, Staphylococcus epidermidis colonies were taken from a blood agar plate and

diluted in the tube containing CAPD dialysate sample until reaching the corresponding

McFarland suspension.

1.5 ml of each sample was placed in 1.5 ml eppendorf tubes and centrifuged for 20 minutes at

14,500 rpm using Eppendorf Minispin plus F-45-12-11 centrifuge. The supernatant was

discarded and the pellet obtained was resuspended by adding 1 ml of deionized water.

31

Afterwards, MALDI-TOF MS: protocol for sample preparation using FA extraction method

(Appendix one) was performed starting from step b where 200 μl of lysis buffer were added

to the sample.

2.6.2. Serial dilutions assays

Determination of 1 McFarland suspension was made using BioMerieux Vitek Colorimeter

No. 52-1210. 2ml of negative CAPD dialysate were placed in glass tubes. Using a cotton

swab, Staphylococcus epidermidis colonies were taken from a blood agar plate and diluted in

the tube containing CAPD dialysate sample until reaching the corresponding McFarland

suspension.

Starting from 1 McFarland suspension, serial dilutions were made according to the 1:10 scale

starting with 1 ml of the initial sample and continuing the serial dilutions until obtaining

1:1000 dilution. In addition, the scale 1:2 was performed starting with 1 ml of the initial

sample and continuing the serial dilutions until obtaining the final dilution 1:16.

Following, 1.5 ml of each sample were placed in 1.5 ml eppendorf tubes and centrifuged for

20 minutes at 14,500 rpm using Eppendorf Minispin plus F-45-12-11 centrifuge. The

supernatant was discarded and 1 ml of deionized water was added to each sample in order to

wash and resuspend the obtained pellet. Afterwards, MALDI-TOF MS: protocol for sample

preparation using FA extraction method (Appendix one) was performed starting from step b

where 200 μl of lysis buffer were added to the sample.

32


INFECTIONS WITH A SINGLE ORGANISM

For this assays negative CAPD dialysates 6 and 9 were used to perform the induced infection.

Determination of 0.5 and 1 McFarland suspensions was made using BioMerieux Vitek

Colorimeter No. 52-1210. 2ml of each negative CAPD dialysate were placed in glass tubes.

Using a cotton swab, the corresponding bacterial isolates used in each assay were taken from

a blood agar plate and diluted in the tube containing the CAPD dialysate sample until

reaching the corresponding McFarland suspension.

Dilutions made for each sample of CAPD dialysate were as follow:

- Initial sample McFarland scale: 0.5 and 1

- Dilution 1:10

- Dilution 1:2

After obtaining every suspension and dilution, 1.5 ml of each sample was placed in 1.5 ml

eppendorf tubes and centrifuged for 20 minutes at 14,500 rpm using Eppendorf Minispin plus

F-45-12-11 centrifuge. The supernatant was disposed and 1 ml of deionized water was added

to each sample resuspending the obtained pellet by pipetting. Afterwards, MALDI-TOF MS:

protocol for sample preparation using FA extraction method (Appendix one) was performed

starting from step “b” where 200 μl of lysis buffer were added to the sample.

2.7.1. CAPD induced infection with Gram- positive bacterium

Four different Gram-positive isolates, previously incubated in blood agar, were used to

execute this assay: Streptococcus dysgalactiae ATCC 12394, Staphylococcus epidermidis

33

ATCC 12228, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC

29212.

McFarland suspensions were obtaining as described in numeral 2.7. Each dilution was

produced using 10 ml plastic tubes. After obtaining the required samples 1.5 ml were taking

from each tube and placed in 1.5 eppendorf tubes. Consequently, steps described in numeral

2.7 were performed for each bacterial sample.

2.7.2. CAPD induced infection with Gram- negative bacteria

Gram- negative bacteria previously incubated in blood agar were used in this assay. Three

different isolates were used: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC

27853, and Klebsiella pneumonia ATCC 700603.

Initial suspension 1 and 0.5 McFarland were obtained as described in numeral 2.7.

Thereafter, 10 ml plastic tubes were used to perform the dilutions for each sample followed

by centrifugation steps, washing and MOLDI-TOF FA extraction method as detailed in

numeral 2.7.


POLYMICROBIAL INFECTIONS

CAPD dialysate number 15 (negative) was used to perform the induced infection for these

assays. 1 McFarland suspensions were prepared using BioMerieux Vitek Colorimeter. 2 ml

of negative CAPD dialysate were placed in glass tubes. Using a cotton swab, the

corresponding bacterium isolate, described for each assay in the following sections, was

34

taken from a blood agar plate and diluted in the tube containing the CAPD dialysate sample

until reaching the chosen McFarland suspension.

Suspensions at 1 McFarland were chosen as the initial sample. Serial dilution 1:2 of each

sample was performed. 1 ml of each sample (initial sample and dilution respectively) was

taken and mixed in a separated 10 ml plastic tube. 1.5 ml of the final suspension of each mix

was pour in 1.5 ml eppendorf tube respectively and centrifuged for 20 minutes at 14,500 rpm

using Eppendorf Minispin plus F-45-12-11 centrifuge.

The supernatant was discarded and 1 ml of deionized water was added to each sample

resuspending the obtained pellet by pipetting. Afterwards, MALDI-TOF MS: protocol for

sample preparation using FA extraction method (Appendix one) was performed starting from

step “b” where 200 μl of lysis buffer were added to the sample.

2.8.1. CAPD induced polymicrobial infection with Gram- positive bacteria

This assay was performed by using the same four Gram- positive isolates described in

numeral 2.7.1 and following the process described in numeral 2.8 for each bacterium.

2.8.2. CAPD induced polymicrobial infection with Gram- negative bacteria

This assay was performed by using the same three Gram- negative bacteria described in

numeral 2.7.2 and following the process described in numeral 2.8 for each bacterium.

35

2.8.3. CAPD induced polymicrobial infection with Gram positive and Gram- negative

bacteria

The preparation of a polymicrobial induced infection with Gram-positive and Gram-negative

bacteria was performed by using the following bacteria:

- Escherichia coli ATCC 25922

- Pseudomonas aeruginosa ATCC 27853

- Staphylococcus epidermidis ATCC 12228

- Enterococcus faecalis ATCC 29212

After obtaining the aimed mix, procedure described in numeral 2.8 was followed.

2.9. MOLDI-TOF MS READINGS

All MALDI-TOF MS readings were performed using Bruker Daltonics MALDI-TOF MS

microflex LT system in addition with MALDI Biotyper Real Time Classification (RTC) and

FlexControl-microflex platforms.

Positive or negative readings were classified according to the score value obtained in each

reading as follow:

- 2.3 - 3: highly probable specie identification

- 2 – 2.299: secure genus identifications, probable specie identification

- 1.7 – 1.99: probable genus identification

- 0 – 1.69: not reliable identification

36

3. RESULTS

37

3. RESULTS

3.1. SAMPLE COLLECTION

From the 20 samples of CAPD dialysate obtained, 6 (30%) were diagnosed as positive while

14 (70%) were negative. The appearance of the fluid was noted as 9 (45%) samples visibly

clear while 11 (55%) were cloudy.

Culture positive samples had white cell count from 1,080 x 106

cells/L to 90,000 x 106 cell/L

whereas negative diagnostic is given for samples between <1 x 106

cells/L and 990 x 106

cells/L.

Table 3.1 lists the samples obtained of CAPD dialysate, the diagnostic and the fluid

characteristics.

3.2. MALDI-TOF MS SAMPLE ANALYSIS FOR DIRECT EXTRACTION FROM

CAPD DIALYSATE

The results obtained are presented in table 3.2. All samples were read as negative. The

MOLDI-TOF MS machine did not find peaks to compare with the databases.

38


Western Australia.

CAPD

Sample

Number

Collection

date

White Cells

count

Cells/L

Fluid culture diagnostic Fluid description

1 07/04/14 73 x 106 No growth

- Bright yellow

- Cloudy

2 07/04/14 13 x 106 No growth

- Bright yellow

- Clear

3 27/03/14 540 x 106 No growth

- Dark yellow

- Cloudy

- Presence of blood

4 08/04/14 205 x 106 No growth

- White

- Clear

5 26/03/14 990 x 106 No growth

- Bright yellow

- Cloudy

6 19/03/14 810 x 106 No growth

- White

- Clear

7 25/03/14 5,940 x 106 Bacillus sp.

- Dark yellow

- Cloudy

8 08/04/14 29 x 106 No growth

- Bright yellow

- Clear

9 08/04/14 540 x 106 No growth

- Dark yellow

- Cloudy

- Presence of blood

10 04/04/14 100 x 106 No growth

- Dark yellow

- Clear

11 31/03/14 5,500 x 106 Streptococcus mitis

- Dark yellow

- Cloudy

39


Western Australia (Continuation…).

CAPD

Sample

Number

Collection

date

White Cells

count

Cells/L

Fluid culture diagnostic Fluid description

12 08/04/14 <1 x 106 No growth - White

- Clear

13 17/04/14 1080 x 106 Candida tropicalis - Dark yellow

- Cloudy

14 18/04/14 140 x 106 No growth - White

- Cloudy

15 18/04/14 140 x 106 No growth - White

- Clear

16 05/05/14 90,000 x 106 Pseudomonas

oryzihabitans

- Dark yellow

- Cloudy

17 22/04/14 5,940 x 106 Bacillus cereus - Dark yellow

- Clear

18 28/04/14 14 x 106 No growth - Dark yellow

- Clear

19 26/04/14 ------- Streptococcus salivarius - Dark yellow

- Cloudy

20 07/05/14 290 x 106 No growth - Bright yellow

- Cloudy

40

Table 3.2 MALDI-TOF MS reading results for direct extraction method from CAPD

dialysate.

Analyte ID/Sample

number

Organism (best match) Score Value

7 No peaks found <0

7 No peaks found <0

11 No peaks found <0

11 No peaks found <0

41

3.3. CONCENTRATION OF BACTERIA IN POSITIVE CAPD DIALYSATE

SAMPLES

3.3.1. Time and centrifugation assay in 1.5 ml of sample

The different protocols applied for each sample revealed that the best centrifugation velocity

was 14,500 rpm for 20 minutes in 1.5 ml of sample.

The size of pellets was graded visually by comparing between the different sizes of pellets

obtained in each assay.

Absence of pellet was observed in CAPD dialysate sample 7 using protocols 1 to 4. Using

protocol 5 a small pellet was obtained while a medium size pellet was collected with protocol

6. After the second centrifugation, a small pellet was recovered using protocol 5 and a pellet

of medium size was present using protocol 6.

In the case of sample 11 of CAPD dialysate, centrifugation velocities and times used in

protocols 1 to 3 were not enough to recover a pellet, but with protocols 4 and 5 a small pellet

was obtained. Moreover, a medium size pellet was recovered with protocol 6. After the

washing and centrifugation steps, no pellet was collected using protocols 1 to 4; however, a

small pellet was recovered using protocol 5 while protocol 6 was useful to recover a medium

size pellet

Table 3.3 shows the size of pellet recovered after applying protocols 1 to 6 for the first time

and table 3.4 shows the size of pellet recovered after the washing step and applying protocols

1 to 6 for the second time in 1.5 ml of sample.

MALDI-TOF MS readings are shown in table 3.5. Even though pellet was recovered in some

samples, the readings were negative for all of them.

42

3.3.2. Time and centrifugation assay in 25 ml of sample

Results showed that the protocol that allowed to obtain a large size pellet in 25 ml of CAPD

dialysate sample is protocol number 6 (4,400 rpm for 20 minutes).

Table 3.6 shows the amount of pellet recovered after applying protocols 1 to 6 for the first

time and table 3.7 shows the amount of pellet recovered after the washing step and applying

protocols 1 to 6 for the second time.

Using protocols 1 and 2 a small pellet was obtained for sample 7 of CAPD dialysate. Also a

medium size pellet was recovered with protocols 3, 4 and 5. Large pellet was recovered with

protocol 6. After the second centrifugation a small pellet was observed using protocol 1 and

2, a medium pellet was recovered using protocol 3 and a large size pellet was recovered using

protocols 4, 5 and 6.

Protocols 1, 2 and 3 were effective to recover a medium size pellet from CAPD dialysate

sample 11 while protocols 4, 5 and 6 allowed the recovering of large size pellets. After the

washing and centrifugation steps, a medium pellet was recovered using protocol 1 and 2

while protocols 3, 4, 5 and 6 were useful to recover a large size pellet.

Large pellets were recovered in during these assays but MALDI-TOF MS readings were

negative for all the samples. These results are shown in table 3.8.

43

Table 3.3 Size of recovered pellets using different times and centrifugation velocities in 1.5

ml of CAPD dialysate sample.

Sample

number

Times and velocities of centrifugation protocols

1 2 3 4 5 6

7 - - - - + ++

11 - - - + + ++

- Absence of pellet

+ Small size pellet

++ Medium size pellet

44


centrifugation velocities in 1.5 ml of CAPD dialysate sample.

Sample

number


1 2 3 4 5 6

7 - - - - + ++

11 - - - - + ++

- Absence of pellet

+ Small size pellet


45


centrifugation velocities protocols in 1.5 ml of CAPD dialysate sample.

Analyte ID/Sample

number Organism (best match) Score Value

7.1 No peaks found <0
























46


in 25 ml of CAPD dialysate sample.

Sample

number


1 2 3 4 5 6

7 + + ++ ++ ++ +++

11 ++ ++ ++ +++ +++ +++

+ Small size pellet


+++ Large size pellet

47


centrifugation velocities in 25 ml of CAPD dialysate sample.

Sample

number


1 2 3 4 5 6

7 + + ++ +++ +++ +++

11 ++ ++ +++ +++ +++ +++

+ Small size pellet


+++ Large size pellet

48


centrifugation velocities protocols in 25 ml of CAPD dialysate sample.

Analyte ID/Sample

number Organism (best match) Score Value

























49

3.4. ANALYSIS OF DIFFERENT TYPES OF WATER USED FOR WASHING

CAPD DIALYSATE SAMPLES

The results presented in table 3.9 show that there is not much difference between the types of

water used to do the washing steps of the samples. Although readings from the MALDI-TOF

MS machine were negative for samples 7 and 11, results for samples 4 and 11 with induced

infections showed that the use of deionized water permits the acquisition of higher value

scores (2.305 and 2.303) in the readings while maximum score values obtained in the

readings with saline water are 2.179 and 1.206 and with distilled water are 2.073 and 2.067.

3.5. MALDI-TOF MS SAMPLE ANALYSIS OF MODIFIED METHODS FOR

EXTRACTION FROM PELLET SAMPLE

Negative results were obtained for samples prepared using the MALDI-TOF MS method for

direct transfer in CAPD dialysates 11 and 16. However, sample 4 (induced infection) showed

a 2.012 value score and a positive reading for Streptococcus dysgalactiae.

Using the MOLDI-TOF MS FA extraction method directly to the pellet, negative results were

obtained for samples 11 and 16. For sample 4 (induced infection), a score value of 1.867 and

a Streptococcus dysgalactiae positive reading were obtained.

Results of the MALDI-TOF MS reading results are shown in detail in table 3.10.

50

Table 3.9 MALDI-TOF MS readings results for assays using different types of water to

wash CAPD dialysate samples.

Analyte ID/Sample number Organism (best match) Score Value

7S No peaks found <0


7W No peaks found <0

7W No peaks found <0

7D No peaks found <0

7D Not reliable identification 1.143



11W Not reliable identification 1.273

11W Not reliable identification 1.045

11D No peaks found <0

11D Not reliable identification 1.113

4S Streptococcus dysgalactiae 2.179

4S Streptococcus dysgalactiae 2.164

4W Streptococcus dysgalactiae 2.038

4W Streptococcus dysgalactiae 2.067

4D Streptococcus dysgalactiae 2.306

4D Streptococcus dysgalactiae 2.246

12S Staphylococcus aureus 1.153

12S Staphylococcus aureus 1.206

12W Staphylococcus aureus 2.045

12W Staphylococcus aureus 2.073

12D Staphylococcus aureus 2.303

12D Staphylococcus aureus 2.225

S Saline water 0.85%

W Distilled water

D Deionized water

51

Table 3.10 MALDI-TOF MS reading results for assay using modified method for

extraction from pellet samples.

Analyte ID/Sample

number


11p No peaks found <0


11e No peaks found <0






4p Streptococcus dysgalactiae 2.012

4p Streptococcus dysgalactiae 1.046

4e Streptococcus dysgalactiae 1.848

4e Streptococcus dysgalactiae 1.867





p MOLDI-TOF MS: sample preparation for direct transfer modified method

e MOLDI-TOF MS: sample preparation using FA extraction method with modifications

52

3.6. MINIMUM BACTERIAL CONCENTRATION IN CAPD DIALYSATE

Assays using an induced infection with S. epidermidis showed that MALDI-TOF MS

machine displays accurate readings in concentrations of bacteria from 1 McFarland scale (3.0

x 108 CFU/mL). Readings at 0.5 McFarland (1.5 x 10

8 CFU/mL) were not accurate enough to

be considered as threshold. Table 3.11 shows the results obtained from the readings with the

MALDI-TOF MS machine using the different suspensions.

Table 3.12 shows the results obtained in the readings of different dilutions made from 1

McFarland scale in order to determinate a threshold for MALDI-TOF MS machine using

CAPD dialysate. Positive results were obtained in suspension 1 McFarland (3.0 x 108

CFU/mL) and dilution 1:2 (1.5 x 108 CFU/mL), however, one of the repetitions showed a not

reliable identification in dilution 1:2. For the rest of the samples, no peaks were found.


INFECTIONS WITH A SINGLE ORGANISM

Using two different samples of CAPD dialysate and different Gram positive and Gram

negative bacteria, a threshold of 3.0 x 108 CFU/mL (1 McFarland scale) was determined for a

single organism infection.

Readings on the MALDI-TOF MS machine showed not reliable results (score values from

1.441 to 1.655) in 0.5 McFarland scale suspensions (1.5 x 108 CFU/mL); also negative results

were shown in dilutions 1:10 (1.5 x 107 CFU/mL) and 1:2 (7.5 x 10

7 CFU/mL) made from

0.5 McFarland scale. These results were the same in both samples of CAPD dialysate.

Complete information of the reading results of this assay is described in table 3.13.

53

MALDI-TOF MS results for suspension 1 McFarland scale (3.0 x 108 CFU/mL) showed a

positive reading in all induced infection samples with score values from 1.975 to 2.286.

Readings in dilution 1:10 (3.0 x 107 CFU/mL) did not show peaks in any of the tested

samples. In addition, readings in dilution 1:2 (1.5 x 108 CFU/mL) showed positive results in

induced infection with S. epidermidis, E. faecalis and K. pneumoniae in dialysate sample 6

while in sample 9, positive reading were obtained for S. epidermidis, S. aureus, E. faecalis,

K. pneumoniae. Table 3.14 shows detailed information of the results obtained with all

samples.


POLYMICROBIAL INFECTIONS

MALDI-TOF MS readings for Gram- positive polymicrobial infection was positive for only

one bacterium in the mix. In this case S. aureus was recognized with 1.887 score value in 1

McFarland scale suspension (3.0 x 108 CFU/mL). For dilution 1:2 (1.5 x 10

8 CFU/mL) not

reliable results with 1.68 score value were shown.

For Gram- negative polymicrobial infection, positive result was shown for K. pneumoniae

with 1.993 score value in 1 McFarland scale suspension (3.0 x 108 CFU/mL). Also not

reliable results with 1.663 score value were establish for dilution 1:2 (1.5 x 108 CFU/mL).

Results for sample with a mix between Gram –positive and Gram- negative bacteria showed

that the machine recognized as positive only one bacterium. In this case E. faecalis was

recognized with 2.098 score value in 1 McFarland scale suspension (3.0 x 108

CFU/mL). For

dilutions 1:2 (1.5 x 108 CFU/mL) not reliable results with 1.6054 score value were shown.

The results obtained in this assays for all samples are detailed in Table 3.15.

54

Table 3.11 MALDI-TOF MS readings results for different McFarland suspensions.

Analyte ID/McFarland

scale


0.5 Not reliable identification 1.121

0.5 Not reliable identification 1.088

1 Staphylococcus epidermidis 2.113






55

Table 3.12 MALDI-TOF MS readings results for serial dilutions made from 1 McFarland

suspension.

Analyte ID/McFarland

scale/dilution Organism (best match) Score Value



1:10 No peaks found <0




1:1,000 No peaks found <0






1:2 Staphylococcus epidermidis 2.221

1:2 Not reliable identification 1.141







56

Table 3.13 MALDI-TOF MS reading results for serial dilutions made from 0.5 McFarland scale from CAPD dialysate samples with Gram- positive

and Gram- negative induced infection.

CAPD

Sample Induced infection

McFarland/Dilution Gram

Classification 0.5 0.5 1:10 1:10 1:2 1:2

6

Streptococcus dysgalactiae Not reliable

identification

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Gram +

Score value 2.242 1.655 <0 <0 <0 <0 <0

Staphylococcus epidermidis Not peaks

found

Not reliable

identification

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Score value 2.237 <0 1.554 <0 <0 <0 <0

Staphylococcus aureus Not reliable

identification

Not reliable

identification

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Score value 2.427 1.64 1.625 <0 <0 <0 <0

Enterococcus faecalis Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Score value 2.392 <0 <0 <0 <0 <0 <0

Escherichia coli Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Gram -

Score value <0 <0 <0 <0 <0 <0 <0

Pseudomonas aeruginosa Not peaks

found

Not reliable

identification

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Score value 2.386 <0 1.536 <0 <0 <0 <0

Klebsiella pneumoniae Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Not peaks

found

Score value 2.306 <0 <0 <0 <0 <0 <0

57

Table 3.13 MALDI-TOF MS reading results for serial dilutions made from 0.5 McFarland scale from CAPD dialysate samples with

Gram- positive and Gram- negative induced infection (Continuation…).

CAPD



Classification 0.5 0.5 1:10 1:10 1:2 1:2

9

Streptococcus dysgalactiae No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

Gram +

Score value 2.116 <0 <0 <0 <0 <0 <0

Staphylococcus epidermidis No peaks

found

No reliable

identification

No peaks

found

No peaks

found

No peaks

found

No peaks

found

Score value 2.338 <0 1.624 <0 <0 <0 <0

Staphylococcus aureus No reliable

identification

No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

Score value 2.476 1.475 <0 <0 <0 <0 <0

Enterococcus faecalis No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

Score value 2.403 <0 <0 <0 <0 <0 <0

Escherichia coli No reliable

identification

No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

Gram -

Score value 2.358 1.653 <0 <0 <0 <0 <0

Pseudomonas aeruginosa No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

Score value 2.109 <0 <0 <0 <0 <0 <0

Klebsiella pneumonia No reliable

identification

No peaks

found

No peaks

found

No peaks

found

No peaks

found

No peaks

found

Score value 2.302 1.441 <0 <0 <0 <0 <0

58

Table 3.14 MALDI-TOF MS reading results for serial dilutions made from 1 McFarland scale from CAPD dialysate samples with Gram- positive and

Gram- negative induced infection.

CAPD



Classification 1 1 1:10 1:10 1:2 1:2

6

Streptococcus

dysgalactiae S. dysgalactiae S. dysgalactiae

Not peaks

found

Not peaks

found Not peaks found

Not reliable

identification

Gram +

Score value 2.242 1.775 1.732 <0 <0 <0 1.532

Staphylococcus

epidermidis S. epidermidis S. epidermidis

Not peaks

found

Not peaks

found S. epidermidis

Not reliable

identification

Score value 2.237 2.286 2.216 <0 <0 2.139 1.503

Staphylococcus aureus S. aureus S. aureus Not peaks

found

Not peaks

found

Not reliable

identification

Not reliable

identification

Score value 2.427 1.726 1.742 <0 <0 1.485 1.531

Enterococcus faecalis E. faecalis E. faecalis Not peaks

found

Not peaks

found E. faecalis

Not reliable

identification

Score value 2.392 1.863 1.791 <0 <0 1.977 1.541

Escherichia coli E. coli E. coli Not peaks

found

Not peaks

found

Not reliable

identification

Not reliable

identification

Gram -

Score value <0 1.954 1.975 <0 <0 1.414 1.531

Pseudomonas

aeruginosa P. aeruginosa P. aeruginosa

Not peaks

found

Not peaks

found Not peaks found

Not reliable

identification

Score value 2.386 1.943 1.934 <0 <0 <0 1.608

Klebsiella pneumoniae K. pneumoniae K. pneumoniae Not peaks

found

Not peaks

found K. pneumoniae

Not reliable

identification Score value 2.306 1.958 1.881 0 <0 1.753 1.381

59

Table 3.14 MALDI-TOF MS reading results for serial dilutions made from 1 McFarland scale from CAPD dialysate samples with Gram-

positive and Gram- negative induced infection (Continuation…).

CAPD



Classification 0.5 0.5 1:10 1:10 1:2 1:2

9

Streptococcus

dysgalactiae S. dysgalactiae S. dysgalactiae

No peaks

found

No peaks

found No peaks found

No peaks

found

Gram +

Score value 2.116 2.223 2.256 <0 <0 <0 <0

Staphylococcus

epidermidis S. epidermidis S. epidermidis

No peaks

found

No peaks

found

Not reliable

identification S. epidermidis

Score value 2.338 2.103 2.071 <0 <0 1.567 1.766

Staphylococcus aureus S. aureus S. aureus No peaks

found

No peaks

found S. aureus

Not reliable

identification

Score value 2.476 2.186 2.204 <0 <0 1.837 1.554

Enterococcus faecalis E. faecalis E. faecalis No peaks

found

No peaks

found

Not reliable

identification E. faecalis

Score value 2.403 2.425 2.342 <0 <0 1.322 2.139

Escherichia coli E. coli E. coli No peaks

found

No peaks

found

Not reliable

identification

Not reliable

identification

Gram -

Score value 2.358 2.034 2.188 <0 <0 1.529 1.565

Pseudomonas

aeruginosa P. aeruginosa P. aeruginosa

No peaks

found

No peaks

found

Not reliable

identification

Not reliable

identification

Score value 2.109 1.962 2.104 <0 <0 1.489 1.497

Klebsiella pneumoniae K. pneumoniae K. pneumoniae No peaks

found

No peaks

found K. pneumoniae

Not reliable

identification

Score value 2.302 1.944 1.819 <0 <0 1.959 1.442

60

Table 3.15 MALDI-TOF MS reading results for serial dilutions made from 1 McFarland scale using CAPD dialysate samples with an induced

polymicrobial infection (mix of Gram- positive and Gram- negative).

Induced infection McFarland /Dilution

1 1 1:2 1:2

Gram +

- Streptococcus dysgalactiae

- Staphylococcus epidermidis

- Staphylococcus aureus

- Enterococcus faecalis

Identified

bacterium S. aureus S. aureus S. aureus

Not reliable

identification

Score Value 1.887 1.847 1.715 1.681

Gram -

- Escherichia coli

- Pseudomonas aeruginosa

- Klebsiella pneumoniae

Identified

bacterium K. pneumoniae K. pneumoniae

Not reliable

identification

Not reliable

identification

Score value 1.993 1.975 1.663 1.625

(Gram + ) + (Gram - )

- Staphylococcus epidermidis

- Enterococcus faecalis

- Escherichia coli

- Pseudomonas aeruginosa

Identified

bacterium E. faecalis E. faecalis

Not reliable

identification

Not reliable

identification

Score value 2.098 1.965 1.457 1.604

61

4. DISCUSSION

62

4. DISCUSSION

Continuous ambulatory peritoneal dialysis (CAPD) is considered an effective therapy for

patients with end-stage renal disease (Whaley-Connell et al. 2005). Peritoneal fluid is filled

and drained to and from the abdominal cavity, using the peritoneum as a dialysis membrane

(Breborowicz & Oreopoulos, 1996). Although it is a well-used method, peritonitis is still the

leading cause of technique failure. Contamination sources are various and despite the

advances in CAPD connection systems, contamination at the time of the dialysate exchange

is the major cause of peritonitis (Vikrant et al. 2013).

The diagnosis, effective treatment and outcome of peritonitis are dependent on clinical

evaluation of the patient and correlation of this with laboratory examination of the dialysate

which includes the determination of total leukocyte count and the recovery and identification

of microorganism (Peer et al. 1992). The diagnosis of peritonitis in CAPD is established

when there is a cloudy or turbid effluent containing > 100 x 106 white cells/L, symptoms and

signs of peritoneal inflammation and a positive fluid culture (Peterson, Matzke & Keane

1987; Troidle & Finkelstein 2006). In this study 20 CAPD dialysate samples where

recovered, from them 6 samples were diagnosed as positive. All positive samples had a white

cell count higher than 100 x 106 cells/L and were characterized with a dark yellow colour and

a cloudy turbidity (Table 3.1). Causes of presence of cloudy or turbid dialysate can be

different from microorganism growth, for example rapid migration of polymorphonuclear

leukocytes (PMNs), and symptoms and signs of peritoneal irritation may precede the

development of turbid effluent (Fried & Piraino 2009). In this research, 13 samples

characterized as dark yellow in colour and cloudy with a white cell count higher than 100 x

106 cells/L were diagnosed as culture negative.

Gram-positive peritonitis followed by Gram–negative peritonitis is the leading cause of

CAPD- related peritonitis (Vikrant et al. 2013; Dalaman et al. 1998). Likewise, in this study

63

6 different organisms were isolate from different samples. 4 (66%) isolates were Gram-

positive: Bacillus sp., Bacillus cereus, Streptococcus mitis and Streptococcus salivarius; 1

(17%) isolate was Gram-negative: Pseudomonas oryzihabitans; and 1 isolate (17%) was

fungal: Candida tropicalis.

The Gram-positive spectrum of bacteria among patients with CAPD-related peritonitis

includes Staphylococcus aureus, coagulase negative staphylococcus, viridans and no viridans

streptococci, Enterococcus faecalis and Listeria monocytogenes (Levy, Morgan & Brown

2004); Vikrant et al. 2013). Among Gram-negative bacteria, the most commonly seen in

peritoneal infections are Escherichia coli, Pseudomonas spp., Klebsiella spp., Enterobacter

spp. and Acinetobacter spp (Ghali et al. 2011; Vikrant et al. 2013). Induced infections were

performed according to the finding in the literature. Commonly reported bacteria were used

to perform the induced infections in the assays that required these samples. Streptococcus

dysgalactiae and Staphylococcus aureus were isolated from urine samples processed in

PathWest while ATCC samples Streptococcus dysgalactiae ATCC 12394, Staphylococcus

epidermidis ATCC 12228, Staphylococcus aureus ATCC 29213, Enterococcus faecalis

ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and

Klebsiella pneumonia ATCC 700603 were obtained from blood culture and used as control in

the diagnosis of infections in PathWest laboratories.

Samples with differences in colour, white cell count, turbidity and presence of

microorganism were taken to perform the assays described in this research project. The

variability in the samples was important in order to standardize the assays and ensure their

replicability. Samples 7, 11 and 16 (previously diagnosed as positive) were used in assays

that required positive samples. On the other hand, samples 4, 6, 9, and 12, previously

diagnosed as negative, were used as negative controls and also as base liquid to perform the

induced infection and McFarland standards required in assays 2.4 to 2.8.

64

Various techniques have been used to enhance recovery of microorganisms from CAPD

dialysate (Males, Walshe & Amsterdam 1987). Among the techniques used are the

processing of large volumes of dialysis effluent by centrifugation, total volume culture or

filtration, chemical or physical disruption of phagocytes in dialysate sediment for recovery of

sequestered organism, and the removal of antibiotics from dialysate by washing the sample

(Peer et al. 1992). However, it has been suggested that the large volume of dialysate in the

peritoneal cavity dilutes the organism to such a degree that it may escape detection,

particularly by conventional methods (Vas & Law 1985). In this study, assay 2.2 was

performed in order to prove the sensitivity of the MALDI-TOF MS machine in identifying

microorganisms directly from CAPD dialysate using the protocol currently used in the

laboratory diagnosis in PathWest. The results showed negative readings from the MALDI-

TOF MS machine which means that the concentration of the bacteria was not high enough to

be read by the machine. Likewise, assays 2.3, 2.4 and 2.5 were negative even though

methods for concentration of bacteria were used, also in this assays, an induced infection

using negative dialysate was prepared as positive control in order to determinate if the

problem was the number of bacteria or some compound in the CAPD dialysate. As a result,

MALDI-TOF MS achieved to read the positive controls (induced infections) while normal

positive samples were not read. Comparing the results obtained in this assays with the

literature, it was proved that the bacterial concentration in the positive sample that we had

was not high enough to be detected by MALDI-TOF MS.

Laboratory methods developed for analysing CAPD dialysate samples usually use around 10

ml to 50 ml of dialysate for the assays (Walshe et al. 1986; Fenton 1982). After acquiring the

sample from the dialysate bag, a centrifugation step is performed in order to precipitate the

bacteria and followed by Gram staining and culturing in media (Ferreira et al. 2010). In this

research, 1.5 ml and 25 ml of dialysate were used in the corresponding assays. 1.5 ml were

used because according to the MALDI-TOF MS protocol (Appendix one) that is the initial

65

amount of sample that is used for beginning the extraction. This protocol was performed with

a modification therefore CAPD dialysate was used instead of blood fluid. This change in the

protocol was made because one of the objectives of the project is to implement a protocol

where the detection of bacteria can be made directly from the CAPD dialysate sample. On the

other hand, 25 ml sample were used to prove if there was a difference between this amount of

sample comparing with the 1.5 ml sample used in assays. In addition, 25 ml is the amount of

CAPD dialysate that is taken from the bag and used in the conventional protocol used in

PathWest for clinical diagnosis of infection.

Even though there was a difference in size of the pellet between samples 1.5 ml and 25 ml,

the maximum velocity of the centrifuges played an important role in the assays. This was

expected because there were difference between centrifugation velocity apply to each sample.

It was identified that at maximum velocity (14,500 rpm in 1.5 ml sample and 4,400 rpm in 25

ml sample) for 20 minutes, the pellet obtained was larger in all samples. Centrifugation at

maximum velocity was used in order to separate the bacteria and cell from the rest of the

liquid. As described by Ludlam et al. (1990) centrifugation can be used for concentrate the

bacteria in CAPD liquid. Moreover, the use of deionized water for washing the samples after

the first centrifugation step, allows eliminating substances that can interfere with the readings

(Smirnov et al. 2004). Assays made in 1.5 ml did not reveal a difference between pellet size

after washing the sample (Tables 3.3 and 3.4) but in assays with 25 ml of sample, it is clear

that after washing the sample, a bigger pellet was obtained. After obtaining these results, the

following assays using positives samples were conducted by using 25 ml of sample. In

addition, it was seen that the use of deionized water was more effective in obtaining bacteria.

This can be because the use of this water plus the velocity of centrifugation allows the

leukocytes to break down and free the bacteria inside them which at the end allowed the

extraction of more bacterial proteins. As seen in Table 3.9, higher score values were obtained

using deionized water.

66

MALDI-TOF-MS has been adapted to generate a protein mass spectrum from whole bacteria

and other microorganisms (Dekker & Branda 2011). The sensitivity of the machine has been

reported in several studies. Ferreira et al. (2010) used MALDI-TOF MS for diagnosis of

urinary tract infections. Bacterial identification was positive in urine samples with >1.0 x 105

CFU/mL. In another study, Hsieh et al. (2008) used pure strains that were recognized when 5

x 103

CFU/mL were present and 3 x 104 CFU/mL in strains mixtures. In this research, the

bacterial threshold was established at 1 McFarland scale (3.0 x 108

CFU/mL) which is higher

than the threshold reported in assays using other body fluids. Some samples had a positive

reading at 0.5 McFarland (Tables 3.13 and 3.14), but because there was a difference between

bacteria, the establishment of the threshold was made in order to be the same for every

bacterium. In addition, polymicrobial infections reading resulted in only one bacterium read

(Table 3.15). MALDI-TOF MS correct identification depends on the proportion between

populations in the sample; therefore, the machine will recognize the microorganism that is in

higher quantity in the sample (Hsieh et al. 2008). Even though the established protocol tried

to maintain an equitable concentration of each bacterium in the sample, because of pipetting

technique or distribution of the bacteria in the tube, the minimal variation in number could

affect the result.

67

5. LIMITATIONS OF THE STUDY,

FURTHER EXPERIMENTS

REQUIRED AND CONCLUSIONS

68

5.1 LIMITATIONS OF THE STUDY

The collection of CAPD samples is one of the limitations in this study. CAPD-associate

peritonitis has decreased in the last years. For this reason few samples were analyzed in this

study.

The difference between the samples made difficult to standardize some assays. Some samples

had mucoid sediments that got mix with the pellet after centrifugation. The sticky nature of

this structure made difficult to remove it from the sample without taking the pellet obtained.

The quantity of bacteria present in the samples made difficult to obtain positive result. As

described in the literature, CAPD dialysate contains a diluted quantity of bacteria which

makes difficult the direct diagnosis.

5.2 FURTHER EXPERIMENTS REQUIRED

In future experiments, other techniques can be applied for achieving a good concentration of

bacteria in CAPD-associated infections.

For using MALDI-TOF- MS, assays with filtration of the entire bag of CAPD liquid or a

much sensitive protein extraction method can be analyzed to apply to the reading with this

machine.

In additions technology as flow cytometry or rtPCR for CAPD fluids can be standardized in

order to perform a rapid diagnosis of CAPD- associated infections.

69

5.3 CONCLUSIONS

The cellular and chemical composition of the CAPD dialysate does not interfere with the

mechanism of MALDI-TOF MS. The negative readings obtained in previously diagnosed

positive samples were due to the low concentration of microorganisms in the dialysate.

The minimal bacteria that should be present in the sample are 3.0 x ^6 CFU/mL. This

number was established as a threshold and it is the accurate number of bacteria that the

MALDI-TOF machine will identify.

MALDI-TOF MS is a very important and useful machine for the diagnostic laboratory. It

reduces the time of diagnosis and it is more accurate than culture techniques. Unfortunately

there are still some failures in the technique due to sensitivity of the machine. For example, in

polymicrobial infections, the machine was not able to read all bacteria that were in the

sample, it only reads one bacteria per sample. Also, bacteria that are closely related can be

misidentified.

70

6. APPENDIX

71

APPENDIX ONE MALDI-TOF MS: PROTOCOL FOR SAMPLE PREPARATION

USING FORMIC ACID (FA) EXTRACTION METHOD (PathWest laboratory manual,

2014)

a) Put 1 ml of a positive blood culture fluid in a reaction tube

b) Add 200 μl lysis buffer

c) Vortex approximately 10 seconds

d) Centrifuge 1 minute at 13 000 rpm

e) Remove supernatant by pipetting and discard

f) Resuspend pellet with 1 ml washing buffer thoroughly by pipetting up and down

g) Centrifuge 2 minutes at 13 000 rpm

h) Remove supernatant by pipetting and discard

i) Add 300 μl deionized water, resuspend and add 900 μl ethanol 100%

j) Proceed with standard Ethanol/Formic acid extraction

k) Centrifuge 2 minutes at 13 000 rpm and remove the supernatant with a fine tip pipette

l) Repeat step k until all the ethanol is removed

m) Add 50 μl of 70% FA to the pellet and perform vigorous mixing by pipetting

n) Add 50 μl of ACN and mix the suspension by pipetting up and down, followed by

vortexing

o) Centrifuge 2 minutes at 13 000 rpm

p) Place 1 μl of supernatant on Steel MALDI target and allow to dry

q) Add 1 μl of HCCA matrix solution and allow to dry

r) Proceed to place the target with the sample in the MALDI-TOF machine to start the

analysis

72

APPENDIX TWO MALDI-TOF MS: PROTOCOL FOR SAMPLE PREPARATION

FOR DIRECT TRANSFER METHOD (PathWest laboratory manual, 2014)

1. Smear the bacterial colony to be tested as a thin film directly onto the respective

analyte position on a cleaned stainless steel MALDI target using a toothpick. All

isolates are to be performed in duplicate.

2. Allow it to dry at room temperature

3. Add 1 μl of 70% Formic acid and allow to dry

4. Add 1 μl of HCCA matrix and allow to dry

5. Proceed to place the target with the sample in the MALDI-TOF machine to start the

analysis.

73

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