infection in CAPD patients

Transcription

1 Rapid detection and identification of infection in CAPD patients Irina Maribel Villacrés Granda Masters of Infectious Diseases School of Pathology & Laboratory Medicine Perth,

2 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. ii

3 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 10 6 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. iii

4 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. iv

5 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 Peritoneal Dialysis and Continuous Ambulatory peritoneal Dialysis Peritoneal dialysis (PD) Peritoneal dialysis system Categories of peritoneal dialysis Continuous ambulatory peritoneal dialysis (CAPD) Continuous ambulatory peritoneal dialysis system Advantages and disadvantages of CAPD system CAPD-ASSOCIATED INFECTION Peritonitis Prevalence of peritonitis Pathogenesis Microorganisms isolated in CAPD-associated peritonitis Treatment Prevention Epidemiology Worldwide infection rates of peritonitis v

6 Infection rates of peritonitis in Australia and New Zealand LABORATORY DIAGNOSIS OF CAPD PERITONITIS Conventional bacterial identification Molecular techniques for bacterial identification Bacteria identification by matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) MALDI-TOF MS system Advantages and disadvantages of MALDI-TOF MS system AIMS OF THE PROJECT MATERIALS AND METHODS SAMPLE COLLECTION MALDI-TOF MS SAMPLE PREPARATION AND ANALYSIS: DIRECT EXTRACTION FROM CAPD DIALYSATE CONCENTRATION OF BACTERIA: ASSAYS USING POSITIVE CAPD DIALYSATE SAMPLES Time and centrifugation assay: use of different times and velocities of centrifugation in 1.5 ml of sample Time and centrifugation assay: use of different times and velocities of centrifugation in 25 ml of sample DIFFERENT TYPES OF WATER: ASSAY FOR WASHING CAPD DIALYSATE SAMPLES MALDI-TOF MS SAMPLE PREPARATION: MODIFIED METHODS FOR EXTRACTION FROM PELLET SAMPLE MALDI-TOF MS: modifications to the sample preparation for direct transfer method MALDI-TOF MS: modifications to the sample preparation using FA extraction method vi

7 2.6. MINIMUN BACTERIAL CONCENTRATION IN CAPD DIALYSATE McFarland scale assay Serial dilutions assays MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED INFECTIONS WITH A SINGLE ORGANISM CAPD induced infection with Gram- positive bacterium CAPD induced infection with Gram- negative bacteria MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED POLYMICROBIAL INFECTIONS CAPD induced polymicrobial infection with Gram- positive bacteria CAPD induced polymicrobial infection with Gram- negative bacteria CAPD induced polymicrobial infection with Gram positive and Gramnegative bacteria MOLDI-TOF MS READINGS RESULTS SAMPLE COLLECTION MALDI-TOF MS SAMPLE ANALYSIS FOR DIRECT EXTRACTION FROM CAPD DIALYSATE CONCENTRATION OF BACTERIA IN POSITIVE CAPD DIALYSATE SAMPLES Time and centrifugation assay in 1.5 ml of sample Time and centrifugation assay in 25 ml of sample ANALYSIS OF DIFFERENT TYPES OF WATER USED FOR WASHING CAPD DIALYSATE SAMPLES MALDI-TOF MS SAMPLE ANALYSIS OF MODIFIED METHODS FOR EXTRACTION FROM PELLET SAMPLE MINIMUM BACTERIAL CONCENTRATION IN CAPD DIALYSATE vii

8 3.7. MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED INFECTIONS WITH A SINGLE ORGANISM MINIMUM BACTERIAL CONCENTRATION IN CAPD INDUCED POLYMICROBIAL INFECTIONS DISCUSSION LIMITATIONS OF THE STUDY, FURTHER EXPERIMENTS REQUIRED AND CONCLUSIONS LIMITATIONS OF THE STUDY FURTHER EXPERIMENTS REQUIRED CONCLUSIONS APPENDIX REFERENCES viii

9 LIST OF TABLES Table 1.1 Microorganisms identified in CAPD-associated peritonitis Table 1.2 Treatment of peritonitis according to the causative organism Table 1.3 Incidence of peritonitis in different countries Table 3.1 Samples of CAPD dialysate obtained from PathWest Laboratory Medicine Western Australia Table 3.2 MALDI-TOF MS reading results for direct extraction method from CAPD dialysate Table 3.3 Size of recovered pellets using different times and centrifugation velocities in 1.5 ml of CAPD dialysate sample 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 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 Table 3.6 Size of recovered pellets using different times and centrifugation velocities in 25 ml of CAPD dialysate sample Table 3.7 Size of recovered pellets after washing step and using different times and centrifugation velocities in 25 ml of CAPD dialysate sample Table 3.8 MALDI-TOF MS reading results for assay using different times and centrifugation velocities protocols in 25 ml of CAPD dialysate sample Table 3.9 MALDI-TOF MS readings results for assays using different types of water to wash CAPD dialysate samples Table 3.10 MALDI-TOF MS reading results for assay using modified method for extraction from pellet samples Table 3.11 MALDI-TOF MS readings results for different McFarland suspensions ix

10 Table 3.12 MALDI-TOF MS readings results for serial dilutions made from 1 McFarland suspension 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 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 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) x

11 LIST OF FIGURES Figure 1.1 Graphic description of CAPD process... 6 Figure 1.2 Schematic illustration of MALDI-TOF MS system Figure 1.3 Graphic scheme of bacterial identification by MALDI-TOF MS protein mass detection method xi

12 LIST OF ABBREVIATIONS ACN APD API API Staph CAPD CCPD CFU FA g HCCA L MALDI-TOF MS Acetonitrile Automated peritoneal dialysis Analytical profile index Analytical profile index for Staphylococci Continuous ambulatory peritoneal dialysis Continuous cycling peritoneal dialysis Colony-forming unit Formic acid gravity α-cyano-4- hydroxycinnamic acid Litre Matrix-assisted laser desorption-ionization time of flight mass spectrometry Max MIC Min Ml MRSA NIPD PCR Maximum Minimum inhibitory concentration Minimum Millilitre Methicillin-resistant Staphylococcus aureus Nocturnal intermittent peritoneal dialysis Polymerase chain reaction xii

13 LIST OF ABBREVIATIONS (CONTINUATION ) PD PVC RNA rpm TPD μl Peritoneal dialysis Polyvinyl chloride Ribonucleic acid Revolutions per minute Tidal peritoneal dialysis Microliter C Degrees Celsius xiii

14 1. INTRODUCTION 1

15 1. INTRODUCTION REVIEW OF THE LITERATURE 1.1. PERITONEAL DIALYSIS AND CONTINUOUS AMBULATORY PERITONEAL DIALYSIS 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. 2

16 Peritoneal dialysis system The basic PD system consists of a Polyvinyl chloride (PVC) bag containing 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). 3

17 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) 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 4

18 represents a continuous portable dialysis system which allows patients to continue with daily activities (Popovich et al. 1978) 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). 5

19 Figure 1.1. Graphic description of CAPD process (from Fresenius Medical Care AG & Co 2013) 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). 6

20 1.2. CAPD-ASSOCIATED INFECTION 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/mm 3 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) 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- 7

21 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) 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 Gramnegative 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 Gramnegative 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). 8

22 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) 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 9

23 found in approximately 5% of Gram-positive peritonitis episodes (Troidle & Finkelstein 2006). Streptococcus viridans and other streptococci are found in lower incidence in singleorganism 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 singleorganism 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 10

24 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 Mycobacterium Staphylococcus epidermidis Escherichia coli Staphylococcus aureus Pseudomonas spp. Staphylococcus aureus (MRSA) Gram-negative Klebsiella spp. Viridans streptococci bacteria Enterobacter spp. Non viridans streptococci Acinetobacter spp. Enterococcus spp. Serratia spp. Listeria monocytogenes Proteus spp. M. tuberculosis Candida albicans Non- albicans M fortuitum species Fungal M. avium Rhizopus spp. M. abscessus Aspergillus flavus M kansasii Paecilomyces spp Treatment The development of antimicrobial resistance may be due to the empirical use of extendedspectrum cephalosporins and quinolones. Vancomycin resistant enterococci, vancomycin intermediate sensitive and methicillin-resistant staphylococci and multi-drug resistant Gramnegative organisms have all been reported to cause dialysis related peritonitis (Troidle et al. 1996; Zelenitsky et al. 2000). 11

25 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 singleorganism 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). 12

26 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 13

27 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 1 st generation Catheter replacement if there is epidermidis cephalosporins biofilm formation Anti-staphylococcal Staphylococcus penicillin, 1 st generation aureus cephalosporin MRSA Vancomycin Enterococcus spp. Ampicillin + vancomycin + aminoglycoside If vancomycin resistance treat with linezolid or quinupristin/dalfopristin Escherichia coli Aminoglycoside, cedtazime, Klebsiella spp. cefepime, carbapenem Proteus spp Pseudomonas Ceftazidime/cefepime, trobramycin or Oral quinolone aeruginosa piperacillin Mycobacterium Rifampin, isoniazid, tuberculosis pyrazinamide, ofloxacin Nontuberculosis No established Requires individual protocols base on susceptibility testing Mycobacterium Fungal Caspofungin, fluconazole, or Amphotericin B + voriconazole (used to can be used to flucytosine treat Candida and filamentous fungi). MRSA: Methicillin-resistant Staphylococcus aureus 14

28 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) 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). 15

29 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 Davenport 2009 Hong Kong Szeto et al Sudan Abu- Aisha et al India Keithi-Reddy et al Colombia Pecoits-Filho et al Canada Mujais 2006 France Verger et al United States Mujais 2006 Korea Han et al 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. 16

30 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) LABORATORY DIAGNOSIS OF CAPD PERITONITIS 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 17

31 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) 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). Broadrange 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 18

32 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) 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). 19

33 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) 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). 20

34 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. 21

35 Figure 1.3 Graphic scheme of bacterial identification by MALDI-TOF MS protein mass detection method (from Kliem & Sauer 2012) 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). 22

36 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. 23

37 2. MATERIALS AND METHODS 24

38 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 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. 25

39 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 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 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 26

40 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 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 27

41 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 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 28

42 - 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 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 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. 29