Rapid detection and identification of infection in CAPD...
Transcript of Rapid detection and identification of infection in CAPD...
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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
2.3.2. Time and centrifugation assay: use of different times and velocities of
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
2.8. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED
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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3.7. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED
INFECTIONS WITH A SINGLE ORGANISM .......................................................... 52
3.8. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED
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
Table 3.6 Size of recovered pellets using different times and centrifugation velocities
in 25 ml of CAPD dialysate sample. ................................................................. 46
Table 3.7 Size of recovered pellets after washing step and using different times and
centrifugation velocities in 25 ml of CAPD dialysate sample. .......................... 47
Table 3.8 MALDI-TOF MS reading results for assay using different times and
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
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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).
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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.
2.3.1. Time and centrifugation assay: use of different times and velocities of
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
2) 3,000 rpm for 15 minutes
3) 3,000 rpm for 20 minutes
4) 14,500 rpm for 10 minutes
5) 14,500 rpm for 15 minutes
6) 14,500 rpm for 20 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.
2.3.2. Time and centrifugation assay: use of different times and velocities of
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:
1) 3,000 rpm for 10 minutes
2) 3,000 rpm for 15 minutes
3) 3,000 rpm for 20 minutes
4) 4,400 rpm for 10 minutes
5) 4,400 rpm for 15 minutes
6) 4,400 rpm for 20 minutes
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
2.7. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED
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.
2.8. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED
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
Table 3.1 Samples of CAPD dialysate obtained from PathWest Laboratory Medicine
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
Table 3.1 Samples of CAPD dialysate obtained from PathWest Laboratory Medicine
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
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.
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
45
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.
Analyte ID/Sample
number Organism (best match) Score Value
7.1 No peaks found <0
7.1 No peaks found <0
7.2 No peaks found <0
7.2 No peaks found <0
7.3 No peaks found <0
7.3 No peaks found <0
7.4 No peaks found <0
7.4 No peaks found <0
7.5 No peaks found <0
7.5 No peaks found <0
7.6 No peaks found <0
7.6 No peaks found <0
11.1 No peaks found <0
11.1 No peaks found <0
11.2 No peaks found <0
11.2 No peaks found <0
11.3 No peaks found <0
11.3 No peaks found <0
11.4 No peaks found <0
11.4 No peaks found <0
11.5 No peaks found <0
11.5 No peaks found <0
11.6 No peaks found <0
11.6 No peaks found <0
46
Table 3.6 Size of recovered pellets using different times and centrifugation velocities
in 25 ml of CAPD dialysate sample.
Sample
number
Times and velocities of centrifugation protocols
1 2 3 4 5 6
7 + + ++ ++ ++ +++
11 ++ ++ ++ +++ +++ +++
+ Small size pellet
++ Medium size pellet
+++ Large size pellet
47
Table 3.7 Size of recovered pellets after washing step and using different times and
centrifugation velocities in 25 ml of CAPD dialysate sample.
Sample
number
Times and velocities of centrifugation protocols
1 2 3 4 5 6
7 + + ++ +++ +++ +++
11 ++ ++ +++ +++ +++ +++
+ Small size pellet
++ Medium size pellet
+++ Large size pellet
48
Table 3.8 MALDI-TOF MS reading results for assay using different times and
centrifugation velocities protocols in 25 ml of CAPD dialysate sample.
Analyte ID/Sample
number Organism (best match) Score Value
7.1 No peaks found <0
7.1 No peaks found <0
7.2 No peaks found <0
7.2 No peaks found <0
7.3 No peaks found <0
7.3 No peaks found <0
7.4 No peaks found <0
7.4 No peaks found <0
7.5 No peaks found <0
7.5 No peaks found <0
7.6 No peaks found <0
7.6 No peaks found <0
11.1 No peaks found <0
11.1 No peaks found <0
11.2 No peaks found <0
11.2 No peaks found <0
11.3 No peaks found <0
11.3 No peaks found <0
11.4 No peaks found <0
11.4 No peaks found <0
11.5 No peaks found <0
11.5 No peaks found <0
11.6 No peaks found <0
11.6 No peaks found <0
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
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
11S No peaks found <0
11S No peaks found <0
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
Organism (best match) Score Value
11p No peaks found <0
11p No peaks found <0
11e No peaks found <0
11e No peaks found <0
16p No peaks found <0
16p No peaks found <0
16e No peaks found <0
16e No peaks found <0
4p Streptococcus dysgalactiae 2.012
4p Streptococcus dysgalactiae 1.046
4e Streptococcus dysgalactiae 1.848
4e Streptococcus dysgalactiae 1.867
12p No peaks found <0
12p No peaks found <0
12e No peaks found <0
12e No peaks found <0
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.
3.7. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED
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.
3.8. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED
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
Organism (best match) Score Value
0.5 Not reliable identification 1.121
0.5 Not reliable identification 1.088
1 Staphylococcus epidermidis 2.113
1 Staphylococcus epidermidis 2.052
2 Staphylococcus epidermidis 2.275
2 Staphylococcus epidermidis 2.191
3 Staphylococcus epidermidis 2.248
3 Staphylococcus epidermidis 2.283
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 Staphylococcus epidermidis 2.171
1 Staphylococcus epidermidis 2.303
1:10 No peaks found <0
1:10 No peaks found <0
1:100 No peaks found <0
1:100 No peaks found <0
1:1,000 No peaks found <0
1:1,000 No peaks found <0
1:10,000 No peaks found <0
1:10,000 No peaks found <0
1 Staphylococcus epidermidis 2.172
1 Staphylococcus epidermidis 2.254
1:2 Staphylococcus epidermidis 2.221
1:2 Not reliable identification 1.141
1:4 No peaks found <0
1:4 No peaks found <0
1:8 No peaks found <0
1:8 No peaks found <0
1:16 No peaks found <0
1:16 No peaks found <0
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
Sample Induced infection
McFarland/Dilution Gram
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
Sample Induced infection
McFarland/Dilution Gram
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
Sample Induced infection
McFarland/Dilution Gram
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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