microbiological profile of peritoneal dialysis fluid...
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MICROBIOLOGICAL PROFILE OF PERITONEAL
DIALYSIS FLUID IN ACUTE AND CHRONIC RENAL
FAILURE PATIENTS IN A TERTIARY CARE HOSPITAL
Dissertation submitted to
THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY
In partial fulfillment of the regulations
for the award of the degree of
M.D.(MICROBIOLOGY)
BRANCH –IV
MADRAS MEDICAL COLLEGE,
THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY
CHENNAI – TAMILNADU.
MAY 2018
CERTIFICATE
This is to certify that this dissertation titled “MICROBIOLOGICAL
PROFILE OF PERITONEAL DIALYSIS FLUID IN ACUTE AND
CHRONIC RENAL FAILURE PATIENTS IN A TERTIARY CARE
HOSPITAL” is a bonafide record of work done by Dr.M.SORNARANJANI,
during the period of her Postgraduate study from 2015 to 2018 under the guidance
and supervision in the Institute of Microbiology, Madras Medical College and
Rajiv Gandhi Government General Hospital, Chennai-600003, in partial
fulfillment of the requirement for M.D.MICROBIOLOGY Degree Examination of
The Tamilnadu Dr.M.G.R.Medical University to be held in May 2018.
Prof. Dr. C.P.RAMANI, MD., (Guide)
Professor,
Institute of Microbiology
Madras Medical College
Chennai-600 003.
Prof. Dr. ROSY VENNILA. MD.,
Director & Professor,
Institute of Microbiology
Madras Medical College
Chennai-600 003.
DEAN
Madras Medical College and
Rajiv Gandhi Government General Hospital,
Chennai - 600 003.
DECLARATION
I declare that the dissertation titled “MICROBIOLOGICAL PROFILE
OF PERITONEAL DIALYSIS FLUID IN ACUTE AND CHRONIC RENAL
FAILURE PATIENTS IN A TERTIARY CARE HOSPITAL” submitted by
me for the degree of M.D. is the record work carried out by me during the period
of April 2016 to March 2017 under the guidance of professor DR.C.P.RAMANI,
M.D, Institute of Microbiology, Madras medical college, Chennai. This
dissertation is submitted to the Tamilnadu Dr.M.G.R. Medical University,
Chennai, in partial fulfillment of the University regulations for the award of
degree of M.D.Microbiology (branch IV) examinations to be held in May 2018.
Place: Chennai
Date: Dr. M.SORNARANJANI
Signature of the Guide
Prof. Dr. C.P. RAMANI, M.D., Professor,
Institute of Microbiology Madras Medical College, Chennai-600 003.
ACKNOWLEDGEMENT
I wish to express my sincere thanks to the Honorable Dean
Dr.R.NARAYANA BABU, MD, DCH, Madras Medical College &RGGGH,
Chennai for permitting me to use the resources of this Institution for my study.
I express my sincere thanks to Dr.ROSYVENNILA M.D.,Director,
Institute of Microbiology, Madras Medical College & RGGGH, Chennai.
I also express my thanks and gratitude to our former Professor
Dr.MANGALA ADISESH M.D., for her guidance and support.
I feel fortunate and indebted to be under the guidance of
Dr.C.P.RAMANI, Professor, Institute of Microbiology, for suggesting the topic
for my dissertation and for her valuable advice, guidance in preparing and
compilation of my work. She is a source of inspiration in my endeavors.
My sincere thanks to Prof.Dr.N.GOPALAKRISHNAN M.D.,
D.M.,MRCP, FRCP.,Director., Institute of Nephrology for permitting me to
carry out my study.
I extend my sincere thanks to our Professors Dr. S.THASNEEM BANU
M.D., Dr.U.UMA DEVI M.D., Dr.R.VANAJA M.D., for their support,
guidance and valuable advices.
I extend my whole hearted gratitude and special thanks to my Senior
Assistant Professor, Dr.C.S.SRIPRIYAM.D.,for her valuable guidance and
support in doing my study.
I express my sincere thanks to our Assistant Professors Dr. R.DEEPA
M.D., Dr.N.RATHNAPRIYA M.D., Dr.K.USHAKRISHNAN M.D.,
Dr.K.G.VENKATESH M.D., Dr.N.LAKSHMIPRIYA M.D.D.C.H.,
Dr. DAVID AGATHA M.D., and Dr.B.NATESANM.D., for their support in my
study.
I would like to thank all my colleagues, my junior postgraduates and all
staff of Institute of Microbiology, Madras Medical College for their support and
cooperation.
I would like to thank the faculty and staff of the Institute of Pathology for
their support and cooperation. I would like to thank the Institutional Ethical
Committee for approving my study.
I feel indebted to my parents who had been a solid pillars of everlasting
support and encouragement and for their heartful blessings.
I affectionately thank my husband Dr.S.Karuppasamy M.S, for taking great
care, support and encouragement without which this work would not have been
possible.
Last but not the least I am very grateful to all the patients without whom
this study would not have been completed.
TABLE OF CONTENTS
SI. NO TITLE PAGE
No.
1 INTRODUCTION 1
2 AIMS & OBJECTIVES 6
3 REVIEW OF LITERATURE 7
4 MATERIALS & METHODS 43
5 RESULTS 66
6 DISCUSSION 79
7 SUMMARY 86
8 CONCLUSION 88
9 BIBLIOGRAPHY
APPENDIX-I ABBREVATIONS
APPENDIX-II STAINS,REAGENTSAND MEDIA
ANNEXURE-I CERTIFICATE OF APPROVAL
ANNEXURE-II PROFORMA
ANNEXURE-III PATIENTS CONSENT FORM
ANNEXURE-IV MASTER CHART
LIST OF TABLES
S. NO TITLE PAGE
NO.
1 GENDER DISTRIBUTION OF CASES 66
2 AGE WISE DISTRIBUTION OF THE PATIENTS 66
3 CLINICAL DIAGNOSIS OF DIALYSIS PATIENTS 67
4 MODE OF DIALYSIS 68
5 PATHOGENS ISOLATED FROM THE EFFLUENT OF PERITONEAL DIALYSIS 68
6 PATHOGENS ISOLATED FROM PATIENTS WITH INTERMITTENT PERITONEAL DIALYSIS 69
7 PATHOGENS ISOLATED FROM PATIENTS WITH CONTINUOUS AMBULATORY PERITONEAL DIALYSIS 70
8 SYMPTOMS ASSOCIATED WITH CULTURE POSITIVE PATIENTS ON DIALYSIS 71
9 CORRELATION OF CELL COUNT WITH CULTURE POSITIVITY 71
10 FREQUENCY OF SYMPTOMS OF PERITONITIS WITH CULTURE POSITIVITY 72
11 RISK FACTORS ASSOCIATED WITH PATIENTS UNDERGOING DIALYSIS 73
12 ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF GRAM POSITIVE ISOLATES 74
13 ANTIBIOTIC SUSCEPTIBILITY PATTERN OF GRAM NEGATIVE ISOLATES 75
14 VANCOMYCIN SUSCEPTIBILITY AMONG STAPHYLOCOCCUS SPECIES 76
15 COLISTIN SUSCEPTIBILITY AMONG NON FERMENTERS 77
16 MOLECULAR DETECTION OF RESISTANT GENES IN CLINICAL ISOLATES 78
17 ANTIFUNGAL SUSCEPTIBILITY OF CANDIDA SPECIES 78
LIST OF CHARTS
S.NO TITLE PAGE. NO
1 AGE WISE DISTRIBUTION OF THE PATIENTS 67
2 DISTRIBUTION OF PATHOGENS AMONG DIALYSIS PATIENTS 69
3 DISTRIBUTION OF PERITONEAL INFECTION IN PATIENTS UNDERGOING DIALYSIS 72
4 RISK FACTORS ASSOCIATED WITH PATIENTS UNDERGOING DIALYSIS 73
5 RESISTANCE PATTERN IN STAPHYLOCOCCUSSPECIES 75
6 EXTENDED SPECTRUM BETA LACTAMASE PRODUCING ENTEROBACTERIACEAE 76
7 METALLOBETA LACTAMASE PRODUCING NON FERMENTERS 77
CERTIFICATE – II
This is to certify that this dissertation work titled
“MICROBIOLOGICAL PROFILE OF PERITONEAL DIALYSIS FLUID
IN ACUTE AND CHRONIC RENAL FAILURE PATIENTS IN A
TERTIARY CARE HOSPITAL” of the candidate
Dr.M.SORNARANJANIwith registration Number 201514004 for the award of
M.D. in the branch of MICROBIOLOGY. I personally verified the urkund.com
website for the purp`11ose of plagiarism Check. I found that the uploaded thesis
file contains from introduction to conclusion pages and result shows 4 percentage
of plagiarism in the dissertation.
Guide & Supervisor sign with Seal.
Introduction
1
INTRODUCTION
Chronic Kidney Disease(CKD) affects 10% of the population worldwide
and millions die each year as they do not have access to affordable treatment.
According to the 2010 Global Burden of Disease study, CKD was ranked 18th in
the list of causes of total number of deaths worldwide and according to the World
Health Organizationin the year 2005, there were 58 million deaths worldwide with
35 million attributed to CKD.(1)
Chronic kidney Disease refers to irreversible kidney damage for more than
three months withstructural and/or functional abnormalities with or without
decreased Glomerular Filtration Rate(GFR)(2).In CKD stage V (End Stage Renal
Disease), there will be a complete cessation of effective kidney function occurs
and renal replacement therapy such as Hemodialysis or Peritoneal dialysis or
Kidney transplantation is required(3).Another condition called, Acute Renal
Failure where rapid loss of renal function occurs that leads to rapid decline in
GFR and daily rise in Serum creatinine, Blood urea nitrogen. This condition will
be reversed if the underlying causes are resolved(4).
Peritoneal Dialysis is an effective and widely used(15-50%) form of renal
replacement therapy.Peritoneal Dialysis does not require direct access to the
circulation and placement of a peritoneal catheter allows infusion of a dialysate
solution into the abdominal cavity5. Many modalities exist for Peritoneal Dialysis
now. They are broadly classified as continuous or intermittent dialysis.
2
In Continuous Ambulatory Peritoneal Dialysis(CAPD), patient himself
performs manual exchanges of dialysate. Here the dialysate is continuously
dwelling inside the peritoneal cavity and interrupted only by drainage and
infusion of new dialysate. 2-3 L bags are exchanged usually 4 times a day in
CAPD. In Automated Peritoneal Dialysis, a cycler machine is used which
automatically performs exchanges at night. In Manual Intermittent Peritoneal
Dialysis, dialysis is carried out usually two to three times a day in a week
intermittently in a hospital. In Night Intermittent Peritoneal Dialysis there will be
no fluid in the abdomen in the daytime (6).
The longevity of Peritoneal Dialysis is markedly reduced by the risk of PD-
related infections. Peritonitis is the main complication of Peritoneal Dialysis.
Peritonitis remains a cause of hospitalisation, transfer to Hemodialysis
andmortality rates varying from 2 to 25% (6).The incidence of Peritonitis in acute
dialysis is 0.5% to 4%.In CAPD, by the end of 6 months of treatment the
probability of developing at least one episode of Peritonitis is 30%, 50% by the
end of 1 year, 70% by 2 years and 80% by 3 years of uninterrupted therapy (7).
Peritonitis has been diagnosed when at least 2 of the following are present (8)
1. Clinical features consistent with Peritonitis (abdominal pain and/or cloudy
dialysis effluent)
2. Dialysis effluent white cell count>100/µl with >50% polymorphonuclear
3. Positive dialysis effluent culture.
3
The predominant clinical manifestations occurring in Peritonitis are
abdominal pain (73-95%) followed by vomiting( 25-35%), diarrhea(6-9%),
cloudy effluent( 86-98%) and fever (36%)(7).
Peritoneal dialysis infections arise from direct contamination of the
catheter or intra abdominal focus or from the catheter exit site or from the
subcutaneous tunnel or from the Peritoneum(9).Microbes enter the peritoneal
cavity via Transluminal(30-40%), or Periluminal(20-30%),or Transmural(25-
30%),or Hematogenous(5-10%),or by Ascending route(2-5%)(10).
Gram positive organisms are the predominant cause of peritonitis.
Coagulase negative Staphylococci (40-65%)is the predominant one followed by
Staphylococcus aureus (10-25%), Streptococcal species(8-15%), Enterococci(3-
7%) and Corynebacterium or Bacillus species (1-4%). Gram negative bacteria
account for 20-30% of all episodes.Among them, Escherichia coli (7-12%)is more
commonly isolated. Klebsiellaspecies(2-4%) is the second most common
followed by other Enterobacteriaceae(1-7%) and Pseudomonas species(5-
9%).Isolation rate of Anaerobic bacteriais less than 5%(7).
Another major complication of peritoneal dialysis is Fungal Peritonitis.1%-
15% of episodes of peritonitisare caused by fungal infections.The incidence being
0.2-1.7 episodes per 12 patient months of dialysis. Overall, 89.3% of episodes are
caused by Candida species.Among Candida species 53.6% are Candida non
albicans species , particularly Candida parapsilosis accounting for one third of
4
Fungal Peritonitis. 10.7% of episodes are caused by Dematiaceous fungi. In
addition to that Aspergillus species, Zygomycetes, Paecilomyces species also have
been reported in recent studies.
The criteria for diagnosing Fungal peritonitis is the same as bacterial
peritonitis.Cloudy dialysate,fever, abdominal pain,nausea, diarrhea, constipation
and poor dialysate outflow are the main features of fungal peritonitis.The major
route of fungal pathogens is periluminal, ascending and transvaginal. Fungal
Peritonitis occurs more frequently in patients receiving broad spectrum antibiotics
within 3 months(74%) and 87% within 6 months.
Mortality rate of Fungal peritonitis varies from 5% to 53%., failure to
resume PD occurs in 40% of patients and death in 16% of patients(8). Abdominal
pain, abdominal pain with fever, catheter in situ are the most common risk factors
for mortality and technique failure. Mortality rates are high withCandida 47% and
non-candida species 37%. Mortality rate will be reduced if the catheter is
removed within 24 hours and increased if it is delayed.(10).
ThoughQuality standards demand an infection rate less than 0.67
episodes/patient/year on dialysis but the reported overall rate of Peritoneal dialysis
associated infection is 0.24-1.66 episodes/patient/year, it is estimated that for
every 0.5 per year increase in Peritonitis rate, the risk of death increases by 4%
and 18% of the episodes resulted in removal of Peritoneal Dialysis catheter and
3.5% resulted in death(8).
5
So that routine PD fluid culture and sensitivity should be done after the
completion of the procedure and appropriate antibiotics should be prescribed at
the earliest before any significant clinical problem occurs.
This study is focused to determine the microbiological profile and their
drug susceptibility pattern to identify the peritoneal dialysis related infections and
to find out the risk factors associated with peritonitis.
Aims & Objectives
6
AIMS AND OBJECTIVES
AIM
To identify the microbiological profile of Peritoneal Dialysis fluid in Acute &
Chronic Renal Failure patients in a Tertiary care hospital.
OBJECTIVES
• To identify aerobic bacterial & fungal isolates in effluent of Peritoneal
dialysis.
• To study the risk factors associated with Peritoneal Dialysis.
• To determine the antimicrobial susceptibility pattern of the isolates.
• Characterization of the resistant bacterial isolates by phenotypic and
genotypic methods.
Review of Literature
7
REVIEW OF LITERATURE
ANATOMY OF KIDNEY
Kidneys are a pair of excretory organs which are situated on the posterior
abdominal wall, one on each side of the vertebral column, behind the peritoneum.
They remove excess of water and salts from the blood and waste products of
metabolism from the body.
The kidneys occupy the epigastric region, hypochondriac region, lumbar
and umbilical regions. Vertically the kidneys extend from the upper border of
twelfth thoracic vertebra to the centre of the body of third lumbar vertebra. The
right kidney is slightly lower than the left. The left kidney is nearer to the median
plane than the right.
Kidneys are bean shaped and they have upper and lower poles, medial and
lateral borders, anterior and posterior surfaces. The kidneys are covered by fibrous
capsule, perirenal fat and renal fascia. The coronal section of the kidney shows,
outer reddish brown cortex, inner pale medulla and a space called renal sinus.
Anterior and posterior branches of renal artery supply blood to the kidneys. They
obtain nerve supply from the renal plexus(11).
FUNCTIONS OF KIDNEY:
The primary function of kidneys is to maintain the homeostasis of the
body. Kidneys regulate various activities in the body. Excretion of waste products
(Urea, Uric acid, Creatinine, Bilirubin and other products of metabolism),
8
Maintenance of water balance, Maintenance of electrolyte balance, maintenance
of acid base balance are the major functions of kidneys. Kidneys stimulate the
production of erythrocytes by secreting a specific hormone called Erythropoietin.
Thrombopoietin, Renin, 1, 25-dihydroxycholecalciferol and Prostaglandins are the
other hormones secreted by the kidneys. They also regulate blood pressure by
regulating the volume of extracellular fluid and through Renin-Angiotensin
mechanism(11).
RENAL FAILURE
Renal failure is the loss of renal function that leads to fall in Glomerular
filtration Rate to below 80ml/min1 and accumulation of urea, creatinine and other
nitrogenous wastes. Renal failure is classified into mild, moderate and severe
based on Glomerular Filtration Rate (GFR) and serum creatinine level .
Type of Renal failure Glomerular
Filtration Rate (ml/min)
Serum creatinine level (mg/dl)
Mild renal failure 50-79 ≤2.5
Moderate renal failure 20-49 2.6-6
Severe renal failure <20 >6
9
In mild renal failure, there will be no symptoms. Nocturia and anemia are
the main symptoms of moderate renal failure. In severe renal failure, uremia,
hypertension, pulmonary congestion, peripheral edema and metabolic acidosis are
the major complications.
Based on the progression of disease, renal failure is also classified as
Acute, Subacute and Chronic renal failure. In Acute renal failure, there will be
rapid loss of renal function that leads to daily rise in serum creatinine and Blood
Urea Nitrogen. Usage of nephrotoxic drugs is the most common cause of Acute
Renal Failure. This condition is reversible if the underlying causes are resolved. In
Subacute renal failure, there will be a rise in serum creatinine from one week to
another. In Chronic Renal Failure, the loss of renal function is slow and
progressive in nature. But the rise in serum creatinine is not apparent from one
week to another and becomes evident over months to years. The renal damage is
irreversible in this condition (3).
10
CHRONIC KIDNEY DISEASE
Definition
1. Kidney damage persists for more than three months, as defined by structural or
functional abnormalities of the kidney, with or without decreased GFR,
manifest by either;
Pathological abnormalities
Markers of kidney damage, including abnormalities in the composition
of the urine or blood or abnormalities in imaging tests.
2. GFR<60ml/min/1.73 m2 for ≥ 3 months, with or without kidney damage.
CKD is classified into five stages based on the level of estimated GFR
normalized to body surface area. In stage I, the GFR will be 90 ml/min/1.73m2, in
stage II 60-90 ml/min/1.73m2,in stage III 30-59 ml/min/1.73m2,in stage IV 15-29
ml/min/1.73m2,and in stage V <15 ml/min/1.73m2. In Chronic Kidney Disease
stage V otherwise called as End Stage Renal Disease, complete cessation of
effective kidney function occurs and hence, renal replacement therapy such as
Hemodialysis or peritoneal dialysis or Kidney transplantation is required(2).
11
EPIDEMIOLOGY
According to 2010 Global burden of Disease study, Chronic Kidney
Disease(CKD) was 27th in the list of causes of total number of deaths worldwide
in 1990 and it raised to 18th in 2010.Worldwide 10% of population are affected by
CKD and millions die every year as they are not affordable to the treatment. Two
million people worldwide currently receive treatment with dialysis or kidney
transplantation to stay alive. Among them the majority are treated in only five
countries including United States, Japan, Germany, Brazil and Italy. More than
80% of all patients who receive treatment are in affluent countries with universal
access to health care system. It is estimated that number of cases of renal failure
increasing disproportionately in developing countries like India and China where
elderly people are more in number. In middle income countries, treatment with
dialysis or renal transplantation creates a high financial burden. In these countries
many people cannot afford treatment at all, resulting in the death of over one
million. It is estimated that one in five men and one in four women are affected by
CKD in 65 to 74 years of age. According to World Health Organization,
approximately 58 million people died worldwide due to CKD in 2005 and 35
million attributed to chronic disease.
The prevalence of CKD Stages II–V has continued to increase nowadays
and the prevalence of diabetes and hypertension, are respectively etiologic in 40%
and 25% of cases. According to NHANES data, the prevalence of CKD in 2003-
2006 was 15.2% and it was decreased from 15.9% which was estimated from
1999 to 2002 in United states. This reduction was seen in CKD Stage I only and
12
for Stage III, the prevalence was increased to 6.5% from 2003 to 2006. The
prevalence of CKD Stages 4 and 5 has been increased twice from 1988 to1999,
but has remained stable from 2002 at 0.6%.
Among the four stages the prevalence of Stage I is 4.1%, Stage II is 3.2%,
Stage III is 6.5% and Stages IV and V is 0.6%. All CKD stages were more
prevalent in persons aged 60 years and above (39.4%) than in 40–59 years
(12.6%) and 20–39 years (8.5%) of age group. Also, CKD prevalence was higher
in diabetics (40.2%), cardiovascular disease (28.2%), Hypertension (24.6%) than
in those without these diseases. In 2009, the prevalence of ESRD was greater than
in 2005.The various etiologic factors associated with newly-initiated ESRD
patients are diabetes (37.5%), hypertension (24.4%), glomerulonephritis(14.8%),
cystic disease of kidney(4.7%) and others (18.6%).
DIALYSIS
Dialysis is used as an artificial replacement for lost kidney function in acute
kidney injury or chronic renal failure. Initiation of dialysis depends on a
combination of the patient’s symptoms, comorbid conditions, and laboratory
parameters. Dialytic options include Hemodialysis(HD) and peritoneal dialysis
(PD).(5)
Absolute indications for dialysis include severe volume overload refractory
to diuretic agents, severe hyperkalemia and/or acidosis, and pericarditis or
other serositis(5).
13
Additional indications for dialysis include symptomatic uremia (e.g.,
intractable fatigue, anorexia, dysgeusia, nausea, vomiting, pruritus, difficulty
maintaining attention and concentration) and protein-energy
malnutrition/failure to thrive without other overt causes..(5).
HEMODIALYSIS
Hemodialysis requires direct access to the circulation via a native
arteriovenous fistula at the wrist, an arteriovenous graft madeup of
polytetrafluoroethylene; a large bore intravenous catheter; or a subcutaneous
device attached to intravascular catheters. Here blood is pumped through hollow
fibers of the Dialyzer and bathed with a solution of favorable chemical
composition (Isotonic, low potassium, absence of urea and nitrogenous
compounds). Toxins and waste products are removed from the circulation by
diffusion and Ultra filtration.(5)
PERITONEAL DIALYSIS
Peritoneal Dialysis does not require direct access to the circulation(5).
Peritoneal catheter is placed, through which dialysate solution is infused into the
abdominal cavity. Here Peritoneal membrane acts as an artificial kidney and
allows transfer of solutes like urea, potassium, uremic molecules across it. (5)
14
HEMODIALYSIS VERSUS PERITONEAL DIALYSIS
Hemodialysis was first used for the treatment of Acute Renal Failure in the
1940s. The selection of PD or HD will be based on patient’s desire, education,
motivation, and geographic distance from an HD unit. Collins et al studied the
risk of death among the patients underwent HD and PD in Medicare from 1994-
1996. In his study he observed that there was a significant lower risk of death in
all patients on PD except diabetic patients (>/=55years). Danish study showed that
there was a increased survival advantage of PD over HD in the first two years. (3)
PERITONEAL DIALYSIS
Peritoneal Dialysis is an established form of therapy in the management of
End stage Renal Disease(6).Dialysis solution composed of balanced salts and
various concentrations of glucose. Dialysate is infused into the peritoneal cavity
by means of a catheter. Retained metabolites traverse the peritoneum from the
blood stream to the solution by difference in osmolality.(7)
Chronic Peritoneal Dialysis
Chronic Intermittent Peritoneal Dialysis
Continuous Ambulatory Peritoneal Dialysis
Continuous Cycling Peritoneal Dialysis
15
ACUTE PERITONEAL DIALYSIS
Acute peritoneal dialysis is limited to the patients with acute renal failure
or other circumstances in which dialysis is anticipated for only a few days. A rigid
catheter is inserted into the peritoneal cavity via a small incision and manual
exchanges are performed every 1 to 3 hours. This procedure has some
complications like bowel perforation and infection. Infection is common in
cannulations persisting for more than a few days. The reasons for infection
include same location of entry and exit site, lack of an implanted cuff barrier
which prevents the entry of microorganisms, migration of the catheter with
resultant serosal injury, and frequent exchanges.(7)
CHRONIC PERITONEAL DIALYSIS
Chronic renal failure patients require maintenance peritoneal dialysis to
alleviate symptoms of uremia and to correct other metabolic abnormalities.
Tenckhoff catheter is frequently used for dialysis today. Repeated insertions not
required. This catheter is composed of pliable silicon and two extra peritoneal
Dacron cuffs. Chronic peritoneal dialysis can be performed either intermittently or
continuously.(7)
CHRONIC INTERMITTENT PERITONEAL DIALYSIS (CIPD)
Dialysis is performed for prolonged periods permitting at least 48 hours of
freedom from dialysis. A closed automated dialysis system is used to deliver
dialysate to the patient. Automated peritoneal dialysis system was developed to
simplify the process.(7)
16
CONTINUOUS AMBULATORY PERITONEAL DIALYSIS (CAPD)
CAPD is a form of closed system continuous dialysis and machine free.
CAPD patients manually exchange the dialysate, usually four times daily, by
using dialysate delivered by gravity into the peritoneal cavity. Fluid from the last
exchange retains in the peritoneal cavity overnight.(7)
CONTINUOUS CYCLING PERITONEAL DIALYSIS (CCPD)
CCPD combines the principles of continuous automated dialysis during the
night and prolonged dwell time dialysis during the day by use of a machine cycler
which will allow frequent exchanges. Advantages of CCPD include eliminating
active dialysis during the day, reducing number of exchanges, and reducing rate of
peritonitis. Disadvantages include cost, machine dependency, and lack of
portability.(7)
PERITONITIS
Peritonitis is defined as inflammation of the peritoneum, which lines the
inner wall of the abdomen. It may occur during the procedure via infected PD
catheter (periluminal or Intraluminal spread). It may also occur as a result of intra
abdominal infections like perforated viscera (Transmural spread). The usage of
Henry Tenckhoff catheter significantly reduces the incidence of peritonitis
though patients undergoing CAPD with this catheter have peritonitis rates of more
than six episodes per patient-year.(12) This rate has been decreased by introduction
of collapsible plastic bags, improved adapters (Y system), and better techniques.
Peritonitis remains the major cause for peritoneal catheter removal,
17
discontinuation of peritoneal dialysis, and switch to hemodialysis. (13) Peritonitis
occurs at a rate of about one episode per patient year (range, <0.5 to ≥3). 45% of
CAPD patients having peritonitis at least once during their initial 6 months and
60% to 70% during the first year.(14) Now the infection rate has been declined that
fewer than one episode in patient-months and as low as one episode in 60 patient-
months in various centers(15). Due to faulty sterile technique by the patients during
self-administration of CAPD (touch contamination) leads to increased rate of
infection. CAPD patients need to perform an increased number of sterile
exchanges are compared to continuous cycling peritoneal dialysis (CCPD)
patients. In CCPD, the rate of peritonitis will be lower because less frequent
manipulations are required during the procedure. (16)
TYPES OF PERITONITIS
REFRACTORY PERITONITIS
Clinical symptoms and signs persist beyond four or five days of therapy.(8)
RELAPSING PERITONITIS
Peritonitis occurs within four weeks of completion of therapy of a prior
episode with the same organism or one sterile episode.(8)
REPEAT PERITONITIS
Peritonitis occurs more than four weeks after completion of therapy of a
prior episode with the same organism.(8)
18
RECURRENT PERITONITIS
Peritonitis occurs within four weeks of completion of therapy of a prior
episode but with a different organism.(8)
DIAGNOSIS OF PERITONITIS
Peritonitis has been diagnosed when at least 2 of the following are present
1) Clinical features consistent with Peritonitis(abdominal pain and/or cloudy
dialysis effluent)
2) Dialysis effluent white cell count>100/µl with >50% polymorphonuclear
3) Positive dialysis effluent culture.(8)
CLINICAL FEATURES OF PERITONITIS
Abdominal pain and tenderness (60% to 80% of patients),
Nausea and vomiting (30% to 50%),
Fever (25% to 50%)
Diarrhea (10%). (29,33)(8)
ABDOMINAL PAIN
Abdominal Pain in Peritoneal dialysis patients, starts from the epigastric
region then spreads to the entire abdomen. No correlation has been observed
between intensity of the pain with the cloudiness of the effluent. Some patients
have clear fluid and increased pain and some have cloudy fluid with little pain.
Severity of the pain also differs among the causative microorganisms. For
19
example Streptococcal peritonitis associated with more pain. Pain may also be
secondary to primary diseases in other abdominal organs. Hence other causes of
abdominal pain should be ruled out before diagnosing PD related Peritonitis.(3)
CLOUDY EFFLUENT
The effluent can be considered as cloudy if it contains more than 50% of
neutrophils and total white blood cell count of more than100 cells/µl. But in some
cases the rise in WBC count can be delayed. Also cell count lower than 100/ µl do
not rule out the diagnosis of peritonitis. Repeat cell count after an exchange often
shows increase in number. Leukocyte esterase reagent strips can be used as an
alternative method. Cloudy peritoneal effluent may also appear in
hemoperitoneum, due to capillary rupture. In this condition the effluent has a red
discoloration and few white blood cells can be seen under microscopy. Chylous
effluent will be seen due to the breach in lymphatic vessels. The opaline
appearance of the effluent increases after a meal. In this condition, higher number
of lymphocytes and triglycerides will be seen in the effluent. To avoid these
problems, one liter of peritoneal dialysis fluid should be infused and allowed to
dwell for a minimum of 1-2 hours in the peritoneal cavity before examining for
turbidity and cell count.(3)
20
MICROBIOLOGICAL ANALYSIS OF PERITONEAL DIALYSIS
EFFLUENT
CELL COUNT
In Bacterial Peritonitis, the dialysate looks cloudy, and the leukocyte count
will be greater than 100 cells/mm3with neutrophils predominating. Number of
effluent cells also depends on the length of the dwelling time. A low leukocyte
count can be seen in tunnel infection. Increased number of lymphocytes seen in
fungal and mycobacterial infections. Eosinophilic peritonitis occurs due to the
placement of the Tenckhoff catheter and may represent allergy to the tubing.
Peritoneal eosinophilia may also occur due to fungal and parasitic peritonitis or
due to chemical and drug (i.e., vancomycin) effects or due to the presence of
icodextrin in the dialysate. (17)
GRAM STAIN AND CULTURE
Gram stain is positive in 9% to 50% of cases.(18) and blood cultures are
rarely positive. Peritonitis with negative cultures occurs in 5% to 10% of cases.
Negative cultures occur due to the constant flow of dialysis fluid into and out of
the peritoneal cavity diluting the microbial density to be low. It may also occur
due to infection with fastidious organisms, or due to previous antimicrobial
treatment, or due to inadequate culture techniques (e.g., the collection of too little
effluent). Recovery rate will be enhanced by collecting 50 ml of dialysate and
centrifuging the dialysate before culture or culturing 10 ml of dialysate in blood
culture bottles.(19) All cultures should be bone aerobically and Fungal,
21
mycobacterial, and anaerobic cultures should be performed if clinically indicated
(e.g., negative aerobic cultures) .
ETIOLOGIC AGENTS:
Peritonitis mostly occurs due to contamination of the catheter by common
skin commensals(18).Peritoneal contamination with other enteric pathogens may
also occur due to alterations of skin flora (54). Skin commensals of the patients
mainly responsible for CAPD associated Peritonitis are Staphylococcus
epidermidis, followed by Staphylococcus aureus, Streptococcus species and
Diphtheroids. High incidence of peritonitis is seen in patients who are carriers of
nasal staphylococcus aureus. Staphylococcal isolates able to grow on polymer
surfaces and subsequently produce an extracellular slime substances or biofilm.
The biofilm protect staphylococcus bacteria from host defenses (55). The
antimicrobial therapy of CAPD- associated staphylococcus peritonitis includes
oxacillin or methicillin, cephalosporins, aminoglycosides, vancomycin or
trimethoprim-sulfamethoxazole. Vancomycin-resistant enterococci is now more
frequently recognized in peritonitis cases.
Gram negative pathogens contribute 15% to 30% of peritonitis.
Escherichia coli is the most commonly isolated, followed by Klebsiella species,
Enterobacter species, Proteus species, and Pseudomonas species. Peritonitis
caused by gram-negative organisms have been increased
nowadays(56)Acinetobacter species, Candida albicans, and anaerobic bacteria are
less commonly isolated. Atypical mycobacteria (usually Mycobacterium
22
chelonaeor Mycobacterium fortuitum), M. tuberculosis, Candida parapsilosis,
Aspergillus fumigatus, Nocardia asteroides complex, and Fusarium spp are rarely
isolated. Polymicrobial peritonitis mostly occurs secondary to a primary intestinal
process (e.g., bowel perforation) and requires surgical exploration. Since O2 in the
peritoneal dialysis fluid is very high, the anaerobic organisms are rarely isolated.
Coagulase negative Staphylococcus
Among Coagulase negative Staphylococci, 80% are Staphylococcus
epidermidis. Infections produced by Staphylococcus epidermidis is milder and
respond well to therapy. Infection most commonly occurs due to touch
contamination.(8)
Staphylococcus aureus
Nasal carriers of Staphylococcus aureus are at high risk for exit site
infections and peritonitis. Staphylococcus aureus peritonitis occurs due to touch
contamination or from catheter infection. Vancomycin resistant isolates are
frequently isolated now.(8)
Enterococci
Vancomycin resistant enterococci are recently isolated from Peritonitis
patients especially those who are heavily exposed to antimicrobials. (8)
Enterobacteriaceae
These are most frequently isolated from exit site infections which may be
the source of peritonitis. Hand and cutaneous carriage is the main source
23
compared to transmural migration. Chronic ill patients are heavily colonized by
these microorganisms. Dialysate is more frequently contaminated with these
microorganisms.(8)
Pseudomonas species
Pseudomonas species normally colonize the skin of chronically debilitated
patients. It will contaminate the water sources like pool water or even portable
water.(8)
ROUTES OF INFECTION
i) Intraluminal transmission of microorganisms (microorganisms enter
through the infusion system.
ii) Periluminal infections ( infection of the catheter site)
iii) Transmural infections(Intestinal injury, perforation or transmigration of
microorganisms)
iv) Hematogenous spread (from site of infection elsewhere)(7)
INTRALUMINAL SPREAD
Intraluminal contamination occurs during the connect-disconnect
manipulations by means of loose-fitting connectors or malfunctioning clamps, or
any defects in the plastic tubing or bags. Many studies reveal that a major cause of
peritonitis in patients on chronic dialysis is poor technique or observed breaches
in technique. CIPD or CCPD associated with fewer manipulations and hence there
will be fewer infections. Incorporating devices or procedures which prevent touch
contamination have resulted in reduced infections rate.(7)
24
EXIT SITE INFECTION
Infection of the catheter site is the second common cause for peritonitis and
also it is the leading cause of exit-site infections in patients on chronic peritoneal
dialysis. Implanted catheter never forms a complete sealed junction with the skin,
so that microorganisms present in the exit site can produce infection. The
superficial embedded Dacron cuff is a reasonable barrier limiting the migration of
microorganisms deeper into the abdominal wall or to the peritoneum, but its
efficacy is not 100%. 17% of patients who develop an exit-site infection have
concurrent peritonitis. Staphylococcus aureus carriers are at high risk developing
an exit-site infection. Chronic dialysis patients who have exit site infections are
having two fold increased risk of getting peritonitis. (7)
CONTAMINATED DIALYSATE
Intrinsic contamination of dialysate may result in infective peritonitis.
Sterile peritonitis results from delivery of endotoxin. Commercially available
dialysate does not support the growth of staphylococci but some gram-negative
microorganisms proliferate readily if introduced. Mycobacterium chelonae and
Pseudomonas species have caused outbreaks of contaminated dialysate associated
peritonitis in patients on CIPD.(7)
TRANSMURAL INFECTION
Enteric bacteria may enter by transmural migration through an intact
intestinal wall after the introduction of hypertonic solutions into the peritoneum.
Transmural infections occur due to abdominal perforation or injury, or any
25
inflammation of the serosal surfaces. Rates of peritonitis due to intestinal
microorganisms are higher in patients with preexisting diverticular disease.
Infection by hematogenous route is uncommon. Vaginal leak may also
serve as a source of peritonitis in rare occasions. Polymicrobial infection with
fecal organisms occurs due to bowel perforation during catheter placement.(7)
IMMUNO PATHOGENESIS OF PERITONITIS
Alterations in peritoneal defenses increase the risk of peritonitis. Peritoneal
fluid contains up to 200 cells/mm3 of which >80% are mononuclear cells
predominantly macrophages. Macrophages are the primary cellular barrier against
infection. Patients who are prone to infection may have fewer macrophages.
Microorganisms are effectively removed by opsonins. Deficiency with IgG or C3
may predispose the patients to infection and the concentrations of these
opsonizing agents are inversely related to the frequency of peritonitis.(19)Delivery
of dialysate into the peritoneal cavity has a direct adverse effect on host defense
mechanisms because of the effects of low pH and hyperosmolarity of the
dialysate. Acidity and hyperosmolarity of the dialysate reduce the ability of
polymorphonuclear leucocytes to phagocytize and kill the microorganisms. The
presence of excess of fluid in the peritoneal cavity during dialysis results in a
marked dilutional effect on both cellular and humoral protective factors, resulting
in fewer leucocytes/ml and a relative opsonic deficiency(7). Newer peritoneal
dialysis fluids which contain glucose polymers (e.g., icodextrin) have less effect
on macrophages and polymorphonuclear leukocytes activity. The formation of
26
biofilm on the catheter is one of the cause of relapsing or recurrent infection, as
well as decreased therapeutic responses and development of antimicrobial
resistance. Indwelling peritoneal catheter acts as a conduit between the outside
environment and the peritoneum. The catheter may also act as a foreign body,
initiating inflammatory changes which predispose to infection and also can serve
as a substrate on which colonization is established. Sialistic catheters are less
thrombogenic than polyurethane catheters. All catheters will be coated with a
fibrin sheath at the end. Microorganisms can become embedded in the fibrin
sheath or in the biofilm produced by them, resulting in proliferation and infection.
This protective environment may be responsible for the difficulty in eradicating
infection.(7)
TREATMENT OF PERITONITIS
Initial antimicrobial therapy should be based on the results of Gram
staining or, if the Gram stain is not helpful, directed against the most likely
pathogens. Adequate levels of antimicrobial agents should be obtained in the
peritoneal fluid by either the systemic or intraperitoneal route. Intraperitoneal
route is more effective than intravenous route.(57)This has allowed most patients
to be treated on an ambulatory basis. Hospitalization is indicated for patients who
are unable to manage the administration of intraperitoneal antibiotics at home.
Initial doses followed by maintenance doses should be given to achieve
therapeutic level (i.e. Greater than the MIC of the Pathogen). Otherwise
intermittent dosing regimens (antimicrobials given once daily) and continuous
dosing regimens (given in each exchange) can also been given.(57) During
27
intermittent dosing, the antimicrobial agents should dwell for at least 6 hours.
And also Physicians should be very cautious when reviewing the MIC and
minimal bactericidal concentration data because these concentrations will be
increased when peritoneal dialysis effluent is used as the in vitro growth medium.
According to International Society for Peritoneal Dialysis, the following
treatment regimens are recommended;
Coagulase negative Staphylococcus
Intraperitoneal Cephalosporins or Vancomycin is advised based on
antimicrobial susceptibility for a period of 2 weeks. Relapsing Coagulase
negative Staphylococcus peritonitis suggests PD catheter colonization. In this
condition, catheter removal is advised with antimicrobials.(8)
Enterococcus species
Enterococcal peritonitis should be treated with intraperitoneal Vancomycin
for three weeks. In severe enterococcal peritonitis intraperitoneal aminoglycoside
should be added. For Vancomycin resistant Enterococcus, intraperitoneal
Ampicillin should be given for 3 weeks if the organism is susceptible to
Ampicillin. Otherwise Linezolid, Quinupristin/ Dalfopristin, Daptomycin or
Teicoplanin can be given.(8)
Streptococcal species
Intraperitoneal Ampicillin should be given for 2 weeks.(8)
28
Staphylococcus aureus
Staphylococcus aureus peritonitis should be treated with effective
antibiotics for 3 weeks. For Methicillin sensitive Staphylococcus aureus, first
generation cephalosporin is the drug of choice. If the isolate is methicillin
resistant, Intra peritoneal Vancomycin is the drug of choice. But Teicoplanin and
Daptomycin can also be used as alternatives. Prolonged treatment with
Vancomycin may predispose to the emergence of Vancomycin resistant
Staphylococcus aureus. For patients with concomitant exit site or tunnel infection,
Catheter should be removed.(8)
Corynebacterium
Corynebacterium Peritonitis should be treated with effective antibiotics
for 3 weeks. For refractory Corynebacterium Peritonitis catheter should be
removed within 1 week after the onset of peritonitis. For patients with
concomitant exit site or tunnel infection, Catheter should be removed.(8)
Pseudomonas
Pseudomonas Peritonitis should be treated with two antibiotics with
different mechanisms of action (E.g.Intra peritoneal Gentamicin or Oral
Ciprofloxacin with Intra peritoneal Ceftazidime or Cefepime for 3 weeks).In
Pseudomonas Peritonitis with concomitant exit site or tunnel infection, Catheter
should be removed.(8)
29
Other Gram negative bacteria
Non Pseudomonas gram negative bacteria should be treated with effective
antibiotics for 3 weeks.(8)
Polymicrobial
If multiple gram negative or mixed gram negative/gram positive organisms
are isolated, surgical evaluation should be obtained. Patients should be treated
with Metronidazole with Intra peritoneal Vancomycin or Intra peritoneal
Ceftazidime for 3 weeks. If multiple gram positive isolates are isolated, effective
antibiotics should be given for 3 weeks.(8)
Culture Negative Peritonitis
Negative effluent cultures should be reevaluated for WBC count and
differential count on day 3. If the culture negative peritonitis is resolving at day 3,
first generation Cephalosporins or Vancomycin should be given for 2 weeks. If
the culture negative peritonitis is not resolving at day 3, special culture techniques
should be done to identify the unusual organisms.(8)
30
EMERGENCE OF MULTIDRUG RESISTANT PATHOGENS:
The most common microorganisms associated with inadequate empiric
therapy are multidrug resistant (MDR) bacteria. Although Pseudomonas
aeruginosa and Methicillin resistant Staphylococcus aureus(MRSA) are the two
most common organisms, other MDR bacteria include penicillin resistant
Streptococcus pneumoniae, Vancomycin resistant Enterococci and extended-
spectrum β lactamases (ESBL) containing gram negative bacteria.
METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA)
The isolation of MRSA is increased nowadays from CAPD peritonitis
patients. It leads to Increased duration of hospital stay (67%), Catheter removal
(23%), Hemodialysis transfer(18%) and death(2.2%) The risk for recurrent
peritonitis in Staphylococcus aureus is 60%.These patients are treated with
vancomycin and cephazolin. The prevalence of MRSA has been increased
steadily. In many hospitals, 40-50% of staphylococcus aureus isolates are
resistant to methicillin, and to all cephalosporins. Many MRSA isolates are
resistant to other antimicrobial families, including aminoglycosides, quinolones
and macrolides.
Jevons first reported methicillin resistant Staphylococcus aureus in 1961.
Soon after the introduction of methicillin, with subsequent isolation of similar
strains reported from Centres throughout U.K & Europe.In the mid 1970s MRSA
was recognized as a significant problem in the U.S with documentation of several
31
outbreaks in tertiary care centre. Methicillin and aminoglycosides resistance in
staphylococcus aureus were reported in Australia(MRSA) and London.
MRSA are resistant to many antibiotics and it is very difficult to eradicate
them from patients as well as carriers. The therapeutic options are limited and the
spectrum of resistance is worrying.
The source of MRSA could be community acquired or hospital acquired.
The later could be from infected patients or hospital staff. Emergence of MRSA as
an important nosocomial pathogen due to outbreaks in various nursing homes and
tertiary care institutions lead to the focus upon carriers amongst the hospital staff.
In nososcomial as well as community acquired MRSA infections, nasal carriage
rate seems to be the main source of infection. Apart from nasal carriage perineal,
axillary and subungual carriages play a role in transmission of MRSA.The crucial
strategy in avoiding this is through hand disinfection and the therapeutic regimen
includes mupirocin nasal ointment combined with parenteral vancomycin
administration.
TREATMENT OF MRSA INFECTION
Strains of MRSA vary in their degree of resistance to various antibiotics.
Nosocomial MRSA strains are usually multidrug resistant, most of them are
resistant to a number of antibiotics except Glycopeptide antibiotics. Vancomycin
remains the drug of choice, since resistance to this drug has not been reported in
vitro but however recently MRSA with reduced susceptibility to glycopeptides
has been reported. Treatment of carriers may include chlorhexidine gel and 2%
32
mupirocin to the nose. G.V.paint 1% is also effective in treating carriers and a
more fastidious adoption of the hand washing practice is recommended.
EXTENDED SPECTRUM ΒETA LACTAMASES (ESBL)
Extended spectrum β lactamases are a particular concern because they are
plasmid mediated and may spread rapidly from strain to strain, can be transferred
between gram negative bacilli, even of other species. Extended spectrum β
lactamase pathogens are often cross resistant to other class of drugs such as
fluoroquinolones, aminoglycosides and sulphamethoxazole trimethoprim.
METALLO ΒETA LACTAMASE
Carbapenams are often used for treating infection due to multidrug
resistant gram negative bacilli, because they are stable even response to extended-
spectrum and AmpC lactamase. In the recent years there has been an increased
prevalence of carbapenam resistant occurs due to metallo β lactamase(MBLs)
production and reported markedly for pseudomonas aeruginosa and acinetobacter
species. So rapid detection of MBL producing Gram-negative bacilli is necessary
to control the spread of resistance.
PROPHYLACTIC TREATMENT
Vancomycin in combination with an aminoglycoside can be given as an
initial empirical regimen. Vancomycin is preferable to a cephalosporin in
Methicillin Resistant Staphylococci. Alternatively, ceftazidime, cefepime, a
carbapenem, or a fluoroquinolone can also be used instead of aminoglycoside for
empirical coverage of gram-negative organisms. Initial antibiotic choices can be
33
modified after culture results .In Pseudomonas aeruginosa peritonitis there will
be an increased rate of treatment failure and relapses occur. So it may be treated
best with a combination of agents active against the infecting strain, in addition to
catheter removal. For vancomycin-resistant enterococci , linezolid or daptomycin
should be administered.(26)The minimal length of duration for therapy ranges
from 10 days to 3 weeks. Some cases exhibit clinical improvement within 48 to
96 hours after initiation of antimicrobial therapy. Persistence of symptoms after
96 hours of therapy indicates of resistant pathogens, unusual organisms (e.g.,
mycobacterial, fungal), should be considered.(17)
OTHER NON ANTIMICROBIAL INTERVENTIONS
Routine peritoneal lavage, usage of fibrinolytic agents, and the instillation
of intraperitoneal immunoglobulins have no role in the management of peritoneal
dialysis-associated peritonitis. Dialysis catheter should be removed in the
following conditions like persistent skin exit site infection or tunnel infection,
fungal peritonitis, fecal peritonitis, mycobacterial peritonitis, Pseudomonas
aeruginosa peritonitis, persistent peritonitis despite 5 days of treatment, recurrent
peritonitis with the same organism, catheter malfunction (e.g., poor flow),and
intraperitoneal abscess. Oral or intraperitoneal antibiotics have no value in
preventing peritonitis during peritoneal dialysis .But if the specific antibiotic is
given just before placement of the peritoneal catheter, may decrease the incidence
of peritonitis and wound infection. Antibiotic prophylaxis should be given for
dialysis patients before extensive dental procedures and before colonoscopy with
polypectomy .In addition, topical application of mupirocin on the exit site and
34
intranasal application of mupirocin have markedly reduced the nasal carriage rate
with Staphylococcus aureus and also have been shown to reduce exit-site
infections. But it does not significantly reduce the incidence of CAPD-related
peritonitis.(27)Usage of titanium adapters, connector systems with disinfectant, and
in-line filters, may decrease the frequency of peritonitis.(17)
FUNGAL PERITONITIS
Fungal peritonitis is one of the serious complications of Peritoneal
dialysis.1%-15% of peritonitis episodes are caused by fungi. It is associated with
high morbidity and mortality ranging from 5% to 53% and failure to resume PD
occurs in 40% of patients.
The inflammatory process in fungal peritonitis is irreversible and cause
subsequent dropout from peritoneal dialysis therapy.(10)
CAUSATIVE ORGANISMS IN FUNGAL PERITONITIS
Fungal peritonitis is most commonly caused by Candida
species(89.3%)(38-43,44,45) and 10.7% by dematiaceous fungi. Among Candida
species 53.6% were Candida non albicans and 35.7% were Candida albicans.
Aspergillus, Paecilomyces species, Zygomycetes, Curvularia, Exophiala
jeanselmeihave also been reported(10).
PORTAL OF ENTRY OF ORGANISMS
The major route of entry of fungal pathogens is periluminal, ascending,
transvaginal.(10)
35
RISK FACTORS OF FUNGAL PERITONITIS
Previous antibiotic therapy mainly for bacterial peritonitis
Immunosuppression
Malnutrition
Bowel perforation
Diverticulitis
Presence of Comorbid conditions such as diabetes mellitus and
malignancy. (10)
CLINICAL MANIFESTATIONS OF FUNGAL PERITONITIS
Clinical features are similar to bacterial peritonitis.(28,29,30,31,32) and consist
of fever, abdominal pain, nausea, diarrhea, Constipation, Cloudy dialysate and
poor dialysate flow are the major clinical manifestations of Fungal peritonitis.
Peritoneal eosinophilia and blood eosinophilia were observed in Paecilomyces
variotii and Aspergillus niger infection.(33,34).
Fungal peritonitis mainly occurs in patients who are under prolonged
antibiotic therapy. The median duration of hospital stay in Fungal peritonitis
patients is 18 days.(36,31)The signs and symptoms do not differ from primary
peritonitis in recurrent infection.(31)
36
DIAGNOSIS OF FUNGAL PERITONITIS
Peritonitis has been diagnosed when at least 2 of the following are present
1) Symptoms of Peritoneal inflammation.
2) Dialysis effluent white cell count>100/µl with >50% polymorphonuclear
3) Positive dialysis effluent culture or positive Gram stain.(7)
If the cell count is highly elevated and the differential is predominantly
lymphocytes or mononuclear cells means it indicates fungal infection. Number of
cells in the effluent depends on the duration of dwell.
FACTORS PREDICTING MORTALITY IN FUNGAL PERITONITIS
Mortality rate of Fungal peritonitis varies from 5% to 53%.,failure to
resume PD occurs in 40% of patients and death in 16% of patients.(10) Abdominal
pain, abdominal pain with fever, catheter in situ are the most common risk factors
for mortality and technique failure.(39,31) Mortality rates are high with Candida
species (47%) when compared to non-candida species (37%). Mortality rate will
be reduced if the catheter is removed within 24 hours and increased if it is
delayed.
MANAGEMENT25
According to International Society for Peritoneal Dialysis, Catheter should
be immediately removed if fungi are isolated, and appropriate antifungal therapy
should be given for 2 weeks after catheter removal. Initial therapy should be
started with combination of Amphotericin B and Flucytosine(10). Intra peritoneal
Amphotericin B is an effective choice but it causes Chemical peritonitis and pain
37
in the patients. In addition if Flucytosine is used, regular monitoring of serum
concentration is mandatory to avoid bonemarrow toxicity. Fluconazole,
posaconazole, voriconazole ,echinocandin can also be used. Azole resistance is
increasing nowadays. Echinocandins are widely used for Aspergillus species and
non-candida albicans species.(37,38,39).Caspofungin is used alone or in combination
with amphotericin B(37,38). Posaconazole and Voriconazole are widely used for
Filamentous fungi. Catheter removal improves outcome and reduces mortality.(40-
45). Antifungal therapy should be continued after catheter removal for 2 weeks and
around one third of patients could return PD.
PREVENTION OF FUNGAL PERITONITIS
Prolonged intake of antibiotics is the main cause of Fungal peritonitis.
Fungal Peritonitis occurs more frequently in patients receiving broad spectrum
antibiotics within 3 months(74%) and 87% within 6 months. Antifungal
prophylaxis along with antibiotics have markedly reduced fungal
peritonitis.(46,47,48,49,50). Oral nystatin 500000 U four times per day is given as
prophylactic antifungal agent.
OTHER NOVEL DIAGNOSTIC TECHNIQUES FOR THE DIAGNOSIS
OF PERITONITIS
Leucocyte esterase reagent strips
Biomarker assays(Matrix metalloproteinase -8 and -9, neutrophil gelatinase
associatedlipocalin and procalcitonin)(53)
38
Polymerase chain reaction for Bacterial derived DNAfragments
Matrix assisted laser desorption –time of flight
Pathogen specific Fingerprints
Nitric oxide can also be used as a non specificmarker(51,52).
PREVENTION AND CONTROL OF PERITONEAL DIALYSIS
ASSOCIATED PERITONITIS
According to International Society for Peritoneal Dialysis the following
prevention modalities are suggested;
CATHETER PLACEMENT
Prophylactic antibiotics should be administered immediately before
catheter insertion.
Catheter placement technique has no significance in the prevention of
infections.(8)
CATHETER DESIGN
Design of the catheter has no significance in the prevention of
infections.(8)
CONNECTION METHODS
Y-connection systems with the “flush before fill” design has less infection
rate compared to the traditional spike systems .Although the risk of peritonitis has
reduced by the Y-systems, the rate of catheter-related infections have not reduced
39
in many trials. In some randomized controlled trials , they compared the double-
bag system with the Y-connection systems (both with the “flush before fill”
design),but there is no significant difference in the rate of catheter-related
infections (8).
TRAINING PROGRAMS
Peritoneal Dialysis patients and their caregivers should be taught about
sterile techniques to reduce the infection rate. PD training should be given by
nursing staff with experience and appropriate qualifications.(8)
TOPICAL ANTIBACTERIAL AND ANTISEPTIC AGENTS
Daily topical application of antibiotic cream or ointment to the catheter
exit site will prevent the infection rate. No cleansing agent has been shown to be
superior to others with respect to preventing catheter-related infections.
Topical Antibacterials, Antiseptics, and Cleansing Agents which have been
widely used for the Prevention of Catheter-Related Infections are, povidone-
iodine, chlorhexidine solution, Amuchina solution/hypochlorite solution,
mupirocin cream, gentamicin cream ciprofloxacin otologic solution,
polysporin triple ointment and polyhexanide(8).
OTHER ASPECTS OF EXIT-SITE CARE
Exit site should be cleansed at least twice weekly and every time after a
shower.(8)
40
OTHER ANTIMICROBIAL APPROACHES
Screening for nasal Staphylococcus aureus carriers should be done for all
Peritoneal Dialysis patients. If nasal carriage of Staphylococcus aureus is found in
PD patients, they should be treated by topical nasal application of mupirocin.(8)
OTHER MODIFIABLE RISK FACTORS
Poor glycemic control is one of the most important risk factor of catheter
related infections (54). Diabetic patients should achieve a reasonable glycemic
control while undergoing Peritoneal Dialysis. Patients undergoing PD in an area
of high air pollution had a higher infection rate than those with low exposure (55).
So patients should be advised to perform PD and exit-site care in a clean
environment.
CONTINUOUS QUALITY IMPROVEMENT
A Continuous Quality Improvement (CQI) program has been proposed for
reducing infection in PD patients (56,57). The CQI team includes nephrologists,
nurses, social workers, and dietitians, and they should arrange regular meetings to
examine all PD-related infections to identify the root cause of each episode. If any
infection develops, the team should investigate and plan interventions to rectify
the problem. CQI programs reduce peritonitis infection rate markedly(57,58–60).
MICROBIOLOGICAL INVESTIGATIONS
Microorganisms present in the normal skin flora, such as Coagulase
negative Staphylococcus aureus, Corynebacteria can cause exit-site and tunnel
infections (61,62). Microbiological examination should be done at the end of the
41
procedure. Samples should be sent to the laboratory using transport media. The
Gram stain of exit-site drainage and microbiological culture findings may help to
guide the subsequent therapy. Both aerobic and anaerobic culture should be done
for the samples and Antimicrobial sensitivity testing is important to determine the
specific antibiotic therapy.
EXIT-SITE CARE
Exit sites should be cleansed at least daily during exit-site infection.(8)
EMPIRICAL ANTIBIOTIC TREATMENT
Empiric oral antibiotic treatment for Exit-site infections with appropriate
Staphylococcus aureus coverage such as a penicillinase-resistant penicillin (e.g.
dicloxacillin or flucloxacillin) or first-generation cephalosporin should be given to
the patients. If the patient had a prior history of infection or colonization with
methicillin-resistant S. aureus (MRSA) or Pseudomonas species they should
receive a glycopeptide or clindamycin, or appropriate anti-pseudomonal
antibiotic, respectively.(8)
MONITORING AND DURATION OF THERAPY
Exit-site infection, except episodes caused by Pseudomonas species,
should be treated with at least 2 weeks of effective antibiotics. Exit-site infection
caused by Pseudomonas species and any tunnel infection should be treated with at
least 3 weeks of effective antibiotics.(8)
42
CATHETER REMOVAL AND REINSERTION
Refractory exit-site or tunnel infection defined as failure to respond after 3
weeks of effective antibiotic therapy. Simultaneous removal and reinsertion of the
dialysis catheter with a new exit site under antibiotic coverage should be done for
Refractory exit-site infections. Removal of the dialysis catheter is recommended
for, PD patients with exit-site infections that progress to, or occur simultaneously
with, peritonitis. Reinsertion of a PD catheter should be performed at least 2
weeks after catheter removal and complete resolution of peritoneal symptoms.
Possible Indications for Catheter Removal in Catheter-Related Infections
are, Catheter infections occurring simultaneously with peritonitis episodes,
Catheter infections leading to subsequent peritonitis episodes and Refractory
catheter infections.(8)
OTHER CATHETER INTERVENTIONS
Cuff-shaving is an effective alternative to catheter replacement for
persistent tunnel infection (63,64,65). The un-roofing technique, with or without en
bloc resection of the skin and tissues around the peripheral cuff, has also been
used as an alternative but is associated with considerable risk of
peritonitis(64,65,66,67,68).Partial catheter re-implantation (69) and catheter diversion
procedure with exit-site renewal (70-75) have been considered for catheter salvage.
This technique has been extensively reported as an alternative to catheter removal
for refractory exit-site or tunnel infections. (66,72,75,76).
Materials & Methods
43
MATERIALS AND METHODS
This cross sectional study was conducted in the Institute of Microbiology
and Institute of Nephrology in the Rajiv Gandhi Government General Hospital.
Total no of 100 patients under dialysis who satisfied the inclusion criteria were
included in the study from April 2016 to March 2017.
STUDY DESIGN
Cross-sectional study
STUDY PERIOD
This study was conducted for one year from April 2016 to March 2017.
PLACE OF STUDY
This study was conducted in the INSTITUTE OF MICROBIOLOGY in
association with INSTITUTE OF NEPHROLOGY at Rajiv Gandhi Govt General
Hospital, Chennai.
STUDY POPULATION
A total of 100 samples were collected from patients who attended
Peritoneal Dialysis in RGGGH and satisfied the inclusion criteria.
ETHICAL CONSIDERATION
Ethical clearance was obtained from Institutional Ethics Committee before
starting this study. Informed consent was obtained from the study population.
44
Patients those who were satisfying the inclusion criteria were included in this
study. Study population was interviewed by a structured questionnaire.
INCLUSION CRITERIA
Patients above 18 years of age were included in this study.
Acute and Chronic Renal Failure patients on Peritoneal Dialysis were
included.
Patients on Continuous and Intermittent Peritoneal Dialysis were included
in this study.
EXCLUSION CRITERIA
Patients below 18 years of age were excluded from this study.
Patients already on antibiotics treated for peritonitis were not included in
this study.
DATA COLLECTION
Data collection included patient’s name, age, IP number, occupation,
Address,date of admission, Clinical diagnosis at admission, Presenting
complaints, Type of Dialysis, Frequency of Dialysis, Prior antibiotic therapy,
Comorbid conditions.
STATISTICAL ANALYSIS
Statistical analysis were carried out using SPSS software.
45
SAMPLE COLLECTION, TRANSPORT AND PROCESSING
Under aseptic precautions, the Dialysate was collected from the dialysate
bag and transported immediately to the Laboratory and Processed as per standard
guidelines.
SAMPLES
Peritoneal Dialysis effluent from Acute & Chronic Renal Failure patients.
COLLECTION OF PERITONEAL DIALYSIS EFFLUENT
Samples were collected from the dialysate bag. Under sterile aseptic
precautions, 25 ml of effluent was aspirated by a sterile needle and syringe from
the bag and5 ml of effluent was sent to the pathology department for leucocyte
count.(9)
MACROSCOPIC EXAMINATION
Appearance of Peritoneal Dialysis fluid was examined and noted.
-Clear, turbid or cloudy.
SAMPLE PROCESSING
5 ml of dialysate was sent to the pathology department for Direct
Leukocyte count, which was done in a counting chamber. Leucocytes>100/ml,
was considered as a developing infection.
25 ml of dialysate was aspirated and centrifuged at 1500g for 5 minutes.
Then the supernatant discarded and 0.5 ml deposit was taken.10 ml sterile distilled
water was added to the centrifuged deposit and mixed by vigorous shaking for
46
30s.Again the fluid was centrifuged at 1500g for5 mins.(9)The deposit was used
for preparation of Potassium hydroxide [KOH mount]mount, Gram’s staining and
for aerobic bacterial and fungal culture.
DIRECT MICROSCOPIC EXAMINATION
GRAM’S STAIN
Smears were prepared from the sediment. They were allowed to air dry and
heat fixation was done. Gram’s stain was performed and the smears were
observed for the presence of polymorphonuclear leucocytes, bacteria and fungal
elements.
POTASSIUM HYDROXIDE MOUNT
Principle
The purpose of using KOH is to clear out any background scales or cell
membranes that may be confused with hyphal elements. Clearing can be
accelerated by gently heating the mount over the flame of a Bunsen burner.(77)
Procedure
This mount was done by emulsifying the sediment in a drop of 10% KOH
on a microscope slide.A coverslip was applied over the mount. Then it was
examined in low and high power in light microscope for the presence of fungal
elements.(77)
47
AEROBIC BACTERIAL CULTURE
The sediment was plated on 5% Sheep blood agar, Chocolate agar,
MacConkey agar and incubated at 37°C for 24-48 hours. Chocolate agar plates
were incubated in candle jar at 37°C.The sediment was also inoculated in Brain
Heart Infusion broth and incubated at 37°C. BHI broth was monitored for 1 week
for turbidity and subculture was done.(78)
FUNGAL CULTURE
The effluent was streaked on two Sabouraud’s Dextrose agar media and
incubated at 25°C and37°C for four weeks and examined periodically for growth.
INTERPRETATION OF BACTERIAL CULTURES
Bacterial isolates were identified by their colony morphology, Gram
staining, Motility and Biochemical reactions by standard microbiological
techniques recommended by Clinical and Laboratory Standards Institute(CLSI)
2016 and 2017.(79)
The following tests were done for the Gram positive cocci,
Catalase test
Slide coagulase test
Tube coagulase test
Differential discs like Optochin 5 µg, Bacitracin 0.04U, Polymyxin B 300 U,
Novobiocin 5 µg.
Bile Esculin agar test
48
For Gram positive cocci in clusters, Catalase test, Slide and Tube
coagulase test, Novobiocin, Polymyxin B discs test were done to identify
Staphylococcus species.
For Gram positive cocci in pairs and short chains, Catalase test, Optochin
test, Bacitracin test, Bile solubility test and Bile Esculinagar test were done to
identify Streptococcus and Enterococcus species.(79)
The following tests were done for Gram negative bacilli to identify the pathogen;
Motility
Catalase test
Oxidase test
Nitrate reduction test
Huge Leifson’s Oxidative Fermentative test
Test for Indole production
Methyl red test
Vogesproskauer test
Citate utilization test
Urease test
Sugar fermentation test
Lysine decarboxylase test
Ornithine decarboxylase test
Arginine dihydrolase test.(79)
49
ANTIMICROBIAL SUSCEPTIBILITY TESTING:
Antibiotic susceptibility testing was done by Kirby Bauer Disc Diffusion
method on Mueller Hinton agar according to CLSI guidelines 2016 and 2017.
The diameters of Zones of inhibition were interpreted according to CLSI
standards. Media and discs were checked for Quality control by using standard
strains.
ANTIMICROBIAL SUSCEPTIBILITY BY KIRBY- BAUER DISC
DIFFUSION METHOD:
1) Identical colonies from the culture plate were taken by a sterile
bacteriological loop and transferred to a tube containing a suitable broth
medium.
2) The broth suspension was incubated at 37°C until it matches the turbidity
of 0.5McFarland standards.
3) A sterile swab was dipped into the adjusted broth suspension and rotated
several times. Then it was pressed firmly on the inside wall of the test tube
above the fluid level to remove excess fluid.
4) Mueller Hinton agar plate was inoculated by streaking the swab three times
over the entire agar surface rotating the plate approximately 60 degrees to
ensure an even distribution. Then the plate was allowed to dry for 3 to 5
minutes not more than 15 minutes before adding the antibiotic discs.
5) Appropriate antibiotic discs were placed on the surface of the agar by using
sterile forceps and the plate was incubated at 37°C overnight.
50
6) After incubation, Zone diameters were measured from the edge of the disc
to the Zone edge with a ruled template on the agar surface.(80)
PANEL OF ANTIBIOTICS FOR GRAM POSITIVE COCCI
Antibiotics Disc content
Inhibition Zone in mm
Resistance Intermediate Sensitive
Penicillin Staphylococcusspecies Enterococcus species
10U
≤28 ≤14
- -
≥29 ≥15
Ampicillin 10 µg ≤16 - ≥17
Amikacin 30µg ≤14 15-16 ≥17
Erythromycin 15µg ≤13 14-22 ≥23
Trimethoprim Sulfamethoxazole 1.25/3.75µg ≤10 11-15 ≥16
Ciprofloxacin 5µg ≤15 16-20 ≥21
Cefoxitin 30µg ≤21 - ≥22
Linezolid 30µg ≤20 21-22 ≥23
Chloramphenicol 30µg ≤ 12 13-17 ≥18
Clindamycin 2 µg ≤14 15-20 ≥21
51
PANEL OF ANTIBIOTICS FOR GRAM NEGATIVE BACILLI
Antibiotics Disc content Inhibition Zone in mm Resistance Intermediate Sensitive
Amikacin 30 µg ≤14 15-16 ≥17
Ampicillin 10 µg ≤13 14-16 ≥17
TrimethoprimSulfamethoxazole 1.25/23.75µg ≤10 11-15 ≥16
Ciprofloxacin 5µg ≤15 16-20 ≥21
Cefotaxime 30µg ≤22 23-25 ≥26
Ceftazidime 30 µg ≤14 15-17 ≥18
PiperacillinTazobactam Acinetobacter species Pseudomonas aeruginosa Enterobacteriaceae
100/10 µg ≤ 17 ≤ 14 ≤17
18-20 15-20 18-20
≥21 ≥21 ≥21
Tetracycline 30µg ≤11 12-14 ≥15
Imipenem Acinetobacterspp Pseudomonas species Enterobacteriaceae
10 µg
≤18 ≤15 ≤19
19-21 16-18 20-22
≥22 ≥19 ≥23
DETECTION OF ANTIMICROBIAL RESISTANCE CAUSING
ENZYMES
Phenotypic assessment of Extended Spectrum Beta Lactamase(ESBL),
Metallo beta lactamase resistance pattern among the bacterial pathogens were
performed.
52
DETECTION OF EXTENDED SPECTRUM BETA LACTAMASE
PRODUCTION
Extended Beta Lactamases are classified under Bush class A beta
lactamases, which are capable of hydrolyzing penicillins,
Oxyiminocephalosporins, monobactams and inhibited by Beta lactamase
inhibitors (Clavulanic acid, Sulbactam and tazobactam) and also have no activity
against cephamycins or carbapenems.(Imipenem, Meropenem).
ESBLSCREENING METHOD
Standard disc diffusion method using Ceftazidime 30µg or Cefotaxime
30µg or Ceftriaxone 30 µg disc was done as initial screening test for ESBL
producer.
For Klebsiellapneumoniae, Klebsiellaoxytoca and Escherichia coli:
Cefotaxime ≤27 mm-ESBLproducer
Ceftazidime ≤22 mm- ESBLproducer
Ceftriaxone ≤25 mm-ESBLproducer
For Proteus mirabilis:
Ceftazidime ≤22 mm- ESBLproducer
Cefotaxime ≤27 mm- ESBLproducer(81)
53
PHENOTYPIC CONFIRMATORY TEST
COMBINED DISC METHOD
Ceftazidime (30 µg), Ceftazidime-Clavulanate (30/10 µg),Cefotaxime 30
µg and Cefotaxime-Clavulanate 30/10 µg disc were used for this test. All these
disc were placed at a distance of 20mm from the centre to centre of the other disc
on Mueller Hinton Agar and incubated at 37°C for 20-24 hours. A ≥5 mm
increase in a zone diameter around the inhibitor combination disc than around
Cefotaxime and Ceftazidime alone was considered as ESBL producer.(81)
METALLO-BETA-LACTAMASE DETECTION
Isolates which showed Imipenem resistance by Disc diffusion method in
Mueller Hinton Agar plate were taken for this test. Two Imipenem(10µg) discs
were placed on the Mueller Hinton Agar plate at a distance of 20mm apart from
each other. Then 10µl of EDTA solution was added to one disc and kept in
incubator at 35°C for 16-18 hrs. After incubation, Zone of inhibition of the two
discs were compared. If the increase in inhibition zone with the Imipenem+
EDTA disc was ≥7mm than the Imipenem disc alone, it was detected as Metallo-
beta-lactamase positive.(81)
54
DETECTION OF METHICILLIN RESISTANCE IN STAPHYLOCOCCUS
AUREUS
1. DISC DIFFUSION METHOD:
Methicillin resistance was detected by adding a Cefoxitin 30µg disc in
Mueller Hinton Agar which was inoculated with the testing organism. ATCC
Staphylococcus aureus 25923 was used as Quality Control strain.
PROCEDURE
0.5 McFarland standards suspension of the Staphylococcal isolate was
made and lawn culture was done on Mueller Hinton Agar plate. Then the plates
were incubated at 37°C for 18-24 hours and zone diameters were measured.
For Staphylococcus aureus,≥22mm-Methicillin sensitive.
≤ 21mm-Methicillin resistant.
For Coagulase negative Staphylococcus aureus, ≥25 mm- Methicillin sensitive
≤24mm-Methicillin resistant.(81)
E-TEST FOR DETECTING VANCOMYCIN RESISTANCE
The Ezy MCTM strip of Vancomycin was used to detect MIC. Four or five
identical colonies were inoculated into 5ml of Trypticase soya broth and
incubated for 2 hours. The suspension was matched with 0.5 McFarlands
standards.Then lawn culture was made on the Mueller Hinton Agar plate.
EzyVancomycin strip was taken by using a sterile applicator and placed on the
Mueller Hinton Agar plate. The plate was incubated at 37°C for 48 hrs.
55
ATCC Staphylococcus aureus 25923 was used as the control.The
Minimum Inhibitory Concentration was read, where the eclipse intersects the
growth. The interpretation was done according to CLSI guidelines 2016.
MIC of Vancomycin: ≤2µg/ml-Susceptible
4-8 µg/ml-Intermediate
≥16µg/ml-Resistant.(81)
E test for detecting Colistin MIC
Four or five identical colonies were inoculated into 5ml of peptone water
and incubated for 2 hours. The suspension was matched with 0.5 McFarlands
standards. Then lawn culture was made on the Mueller Hinton Agar plate.
Biomerieux E strip was taken by using a sterile applicator and placed on the
Mueller Hinton Agar plate. The plate was incubated at 37°C for 48 hrs. The
Minimum Inhibitory Concentration was read, where the eclipse intersects the
growth. The interpretation was done according to CLSI guidelines 2016 and 2017.
MIC of Colistin : ≤2µg/ml-Sensitive
≥4µg/ml-Resistant.(81)
INTERPRETATION OF FUNGAL CULTURE
Sabouraud’s Dextrose agar slants were inspected daily during the first
week and twice weekly during the next three weeks for growth. The growth rate,
56
temperature requirement & colony characteristics of the fungi grown were
noted.(79)
MACROSCOPIC APPEARANCE OF YEAST
Fungal colonies were Creamy white colored, pasty ,smooth and grown
within 3-4 days at 37°C.
MICROSCOPY
Gram stain was performed from the smooth creamy colonies. Gram stain
showed Gram positive ovoid budding yeast cells with and without pseudohyphae.
GERM TUBE TEST
Germ tube test was done to differentiate Candida albicans from Candida
non albicans. Production of germ tubes within 2 hours were considered as
Candida albicans.
PROCEDURE:
Single colony was emulsified in 0.5 ml of sterile serum (pooled human
serum or fetal calf serum). Now the inoculum contains 105to 106 cells/ml. Then
the suspension was incubated at 37°C for two hours. After that, wet mount was
prepared and observed under low power and high power of light microscope to
identify germ tube formation. Presence of Germ tube indicates initiation of hyphal
growth, arising directly from the yeast cell. They are not constricted at the point of
origin.(77)
57
CHROM AGAR MEDIA
Candida species were differentiated by their color production in Chrom
agar media. This media is based on direct detection of specific enzymatic
activities by adding the fluorochrome substrates to media.
Chromagar media shows the following Colored colonies at 30°C for 48-72 hrs.
Candida albicans-Light Green
Candida dubliniensis-Dark green
Candida glabrata-Pink to Purple.
Candida krusei–Pink
Candida parapsilosis-Cream to Pale pink
Candida tropicalis-Blue with Pink halo(82)
CORN MEAL AGAR
This is an enriched media used to identify the Candida species by rapid and
abundant chlamydospore formation. With a sterile loop, the test organism was
taken and streaked on the corn meal agar plate to make a well inoculum. Then
dilution strokes were made by cutting into the agar with the edge of the loop. A
glass coverslip was sterilized and kept on the agar to cover part of the well and
some of the dilution strokes. Then the plate was incubated at 22°C for 48 hrs and
examined in low power and High power of Light microscope.(82)
58
Candidaspecies Cultural characteristics on Cornmeal agar
Candida tropicalis
Single or small clusters of Blastoconidia formed
along pseudohyphae. True hyphae may also be
present.
Candida parapsilosis
Single or small clusters of Blastoconidia formed
along curved pseudohyphae, large hyphal elements
called giant cells seen.
Candida lusitaniae
Single or small clusters of Blastoconidia formed
along pseudohyphae. True hyphae may also be
present.
Candida guilliermondii Short pseudohyphae with clusters of blastoconidiaat
septa seen. True hyphae not seen.
Candida glabrata Small oval yeast cells with single terminal budding
seen.pseudohyphae not seen.
Candida krusei
Pseudohyphae with elongated blastoconidia
resembling cross-match sticks or tree like
appearance.
SUGAR FERMENTATION TEST
Sugars such as 2% Dextrose, 2% Lactose, 2% Maltose, 2% Sucrose were
taken in sterile test tubes and 0.1 ml of test organism was added to each tube.
Then the test tubes were kept in incubator at 25°C for 48-72 hrs for the production
of acid and gas.(82)
59
No Candida species Glucose Maltose Sucrose Lactose
1 Candida albicans AG AG _ _
2 Candida tropicalis AG AG AG _
3 Candida keyfr AG AG AG _
4 Candida guilliermondii AG _ AG _
5 Candida parapsilosis AG _ _ _
6 Candida krusei AG _ _ _
7 Candida glabrata AG _ _ _
A-Acid, G-Gas
DETECTION OF ANTIFUNGAL SUSCEPTIBILITY
DISK DIFFUSION TEST
Mueller Hinton Agar with 2% glucose and 0.5 µg/ml Methylene Blue dye
was used for this test.
Inoculum preparation
Five distinct colonies were picked from a 24 –hour-old culture of Candida
species and suspended in 5 ml of sterile saline (0.85%). Then the suspension was
vortexed for 15 seconds and its turbidity was adjusted to 0.5 McFarland standards.
Inoculation of Test plates
Within 15 minutes, a sterile cotton swab was dipped into the suspension
and rotated several times. Then the swab was pressed against the inside wall of
the tube above the fluid level to remove the excess fluid. The dried surface of a
60
sterile Mueller Hinton+Glucose Methylene blue agar plate was inoculated by
evenly streaking the swab over the entire agar surface. This method is repeated by
streaking two more times, rotating the plate approximately 60°each time for even
distribution. Then the lid was closed and allowed to dry for three to five minutes
not more than 15 minutes.
The Commercial kit named Hexa Antimyco-01(HX104-1PK)(Himedia)
was used to detect antifungal susceptibility. Hexa disc were dispensed onto the
surface of the inoculated agar plate and kept in incubator at 35°C for 24-48 hours.
Reference strains were used.
It contains the following drugs
Amphotericin-B 100U, Clotrimoxazole-10 µg, Fluconazole-25
µg,Itraconazole-10 µg, Ketoconazole-10 µg, Nystatin-100 U(83)
ANTIFUNGAL DRUGS SENSITIVE INTERMEDIATE RESISTANT
Fluconazole(25 µg) ≥19 14-19 ≤14
Ketoconazole(10 µg) ≥20 10-20 ≤10
AmphotericinB(100U) ≥15 10-14 ≤10
Nystatin(100 U) ≥15 10-14 ≤10
Clotrimoxazole(10 µg) ≥20 12-19 ≤11
Itraconazole(10 µg) ≥23 14-22 ≤13
61
MOLECULAR METHODS TO IDENTIFY DRUG RESISTANT GENES IN
THE BACTERIAL ISOLATES
Genes responsible for Methicillin Resistance in Staphylococcus aureus and
Carbapenem resistance in non fermenters were detected by Polymerase Chain
Reaction.
POLYMERASE CHAIN REACTION
PCR is the widely used technique in molecular biology to amplify a single
or few copies of a segment of DNA to generate millions of copies.
Principle
Basic steps in PCR are;
DNA extraction
Amplification of extracted DNA(Denaturation, Annealing,
Extension)
Gel electrophoresis of amplified product
Materials & Methods
PureFast® Bacterial DNA minispin purification kit [Kit contains
Lysozyme, Binding buffer, Wash Buffer-1, Wash Buffer-2,Lysozyme digestion
buffer, Proteinase-K, Spin columns with collection tube and elution buffer.
HELINI 2X ReDdye PCR Master Mix, Agarose gel electrophoresis consumables
and Primers from HELINI Biomolecules, Chennai were used.
62
Components of master mix
2U ofTaq DNA polymerase, 10 X Taqreaction buffer,2mM MgCl2, 1 µl of
10mM dNTPs mix and Red dye PCR additives.
Components of agarose gel electrophoresis
Agarose, 50 XTAE buffer,6Xgel loading buffer and Ethidium bromide.
Requirements of PCR
HELINI Ready to use OXA-23 gene Primer mix(5µl/reaction)and PCR
Product(453bp),
HELINI Ready to use blaVIM gene Primer mix(5µl/reaction) and PCR
Product(480bp).
HELINI Ready to use mecA gene primer mix(5µl/reaction) and PCR
Product(533bp)
BACTERIAL DNA PURIFICATION
1ml of overnight culture was centrifuged at 6000rpm for 5min.
Then the supernatant was discarded
Pellet was suspended in 0.2ml of Phosphate buffer saline.
Then, 180µl of Lysozyme digestion buffer and 20µl of Lysozyme
[10mg/ml] added.
Then the mixture was incubated at 37C for 15min.
Then 400µl of Binding buffer, 5µl of internal control template and 20µl of
Proteinase K were added and mixed well by inverting several times.
63
Then the mixture was incubated at 56ºC for 15min.
After that 300µl of ethanol was added and mixed well.
Then the entire sample was transferred into the PureFast® spin column
and centrifuged for 1 min. The flow-through was discarded and the
column was placed back into the same collection tube.
Then 500μlof Wash buffer-1 was added to the PureFast® spin column.
Again centrifuged for 30-60 seconds and the flow-through was discarded.
Then the column was placed back into the same collection tube.
Then 500μl of Wash buffer-2 was added to the PureFast® spin column
and centrifuged for 30-60 seconds and the flow-through was discarded and
again the column was placed back into the same collection tube.
Then the flow-through was discarded and centrifuged for an additional 1
min. This step was done to avoid residual ethanol.
Then the mixture was transferred the PureFast® spin column into a fresh
1.5 ml micro-centrifuge tube.
Then 100μl of Elution Buffer was added to the center of PureFast® spin
column membrane and incubated for 1 min at room temperature and
centrifuge for 2 min.
Then the column was discarded and the purified DNA was stored at -
20°C. Quality and Quantity of extracted DNA was checked by loading in
1% agarose gel and 5µl of extracted DNA was used for PCR
amplification.
64
PCR PROCEDURE
Quantity of the components
HELINI RedDye, HELINI Ready to use PCR Master mix 10µl, OXA23
gene Primer Mix / VIMgene primer mix /mecA gene primer mix 5µl ,Purified
Bacterial DNA 5µl Total volume 20µl.The mixture was mixed gently and spin
down briefly and Placed into PCR machine and program started as follows;
Initial Denaturation was done at 94ºC for 5 mins.Then denaturation was
done at 94ºC for 30sec followed by annealing at 58ºC for 30sec and extension at
72ºC for 30sec.This was repeated for 35 cycles and final extension was done at
72º C for 5 minutes.
LOADING IN AGAROSE GEL
2% agarose gel (2gm of agarose +100 ml of 1X TAE buffer)was prepared
and the electrophoresis was run at 50V till the dye reaches three fourth distances
and the bands were observed in UV Transilluminator.
AGAROSE GEL ELECTROPHORESIS
2% agarose was prepared by mixing, 2gmagarose in 100ml of TAE buffer
and melted using micro oven.
When the agarose gel temperature was around 60ºC, 5µl of Ethidium
bromide was added.
65
Warm agarose solution was poured slowly into the gel platform.
Then the gel was kept undisturbed till the agarose solidifies.
1XTAE buffer was poured into submarine gel tank.
The gel was carefully placed into the tank and the tank buffer was
maintained 0.5cm above the level than the gel.
PCR Samples were loaded after mixed with gel loading dye along with
10µl HELINI 100bp DNA Ladder. [The Ladder contains 100bp, 200bp,
300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp and 1500bp]
The electrophoresis was run at 50V till the dye reaches three fourth
distance of the gel and the gel was viewed in UV Transilluminator and the
bands pattern were observed.
Results
66
RESULTS
This cross sectional study was conducted in the Institute of Microbiology
and Institute of Nephrology in the Rajiv Gandhi Government General Hospital.
Total number of 100 patients underwent dialysis who satisfied the inclusion
criteria were included in the study from April 2016 to March 2017.
Table 1: GENDER DISTRIBUTION OF CASES(n=100)
GENDER NUMBER OF PATIENTS PERCENTAGE
Male 63 63%
Female 37 37%
Total 100 100%
TABLE 2: AGE WISE DISTRIBUTION OF THE PATIENTS(n=100)
AGE NO OF PATIENTS PERCENTAGE
18-30 years 22 22%
31-40 years 25 25 %
41-50 years 21 21%
51-60 years 18 18%
>60 years 14 14%
Total 100 100%
67
Table 1 & 2: Among the total patients 63% were males and 37% were
females.Majority of the patients in the study belonged to the age group 31-40
years. The patients had a mean age of 44.15±13.89.
CHART 1: AGE WISE DISTRIBUTION OF THE PATIENTS
Table 3: CLINICAL DIAGNOSIS OF DIALYSIS PATIENTS (n=100)
22
25
21
18
14
0
5
10
15
20
25
30
18-30 yrs 31-40yrs 41-50yrs 51-60yrs more than 60yrs
No
of p
atie
nts
DIAGNOSIS NUMBER OF PATIENTS PERCENTAGE (%)
CKD 92 92
AKI 8 8
Total 100 100
68
Table 4: MODE OF DIALYSIS (n=100)
TYPE OF DIALYSIS
NUMBER OF PATIENTS PERCENTAGE
IPD 96 96.0
CAPD 4 4.0
Total 100 100.0
In the present study,92% of the patients had Chronic Kidney Disease and
only 8% had Acute Kidney Injury and 96% were under Intermittent Peritoneal
dialysis and 4% were under Continuous Ambulatory Peritoneal Dialysis .
Table 5: PATHOGENS ISOLATED FROM THE EFFLUENT OF
PERITONEAL DIALYSIS (n=28)
PATHOGENS NUMBER PERCENTAGE(%)
Gram negative isolates 13 46.4
Gram positive isolates 10 35.7
Fungal isolates 5 17.9
Out of 100 samples, 28 samples were culture positive. Among them 13
(46.4%) were Gram negative, 10 (35.7%) were Gram positive and 5 (17.9%) were
Fungal isolates.
69
CHART 2: DISTRIBUTION OF PATHOGENS AMONG DIALYSIS
PATIENTS(n=28)
Table 6: PATHOGENS ISOLATED FROM PATIENTS WITH
INTERMITTENT PERITONEAL DIALYSIS(n=25)
ISOLATED PATHOGENS NO OF ISOLATES PERCENTAGE
Acinetobacterbaumannii 5 20%
Staphylococcus aureus 4 16%
Staphylococcus epidermidis 4 16%
Escherichia coli 3 12%
Klebsiellaoxytoca 2 8%
Klebsiellapneumoniae 1 4%
Enterococcus faecalis 1 4%
Candida non albicans 5 20%
Gram positive bacteria
46%Gram negative bacteria
36%
Fungal isolates18%
Gram positive bacteria
Gram negative bacteria
Fungal isolates
70
Among the 28 culture positive patients 89.2% were isolated from
Intermittent Peritoneal Dialysis. Among them the majority were
Acinetobacterbaumannii (20%) and Candida non albicans(20%)followed by
Staphylococcusaureus(16%), Coagulase negative Staphylococcus(16%),
Klebsiellaoxytoca(8%),Klebsiellapneumoniae(4%), Enterococcus faecalis(4%).
Table 7: PATHOGENS ISOLATED FROM PATIENTS WITH
CONTINUOUS AMBULATORY PERITONEAL DIALYSIS (n=3)
ISOLATED PATHOGENS NO OF ISOLATES PERCENTAGE
Klebsiellapneumoniae 1 33.3%
Pseudomonas aeruginosa 1 33.3%
Escherichia coli 1 33.3%
Out of 28 isolates,3(10.7%) were isolated from Continuous Ambulatory
Peritoneal Dialysis patients.The pathogens isolated from CAPD were
Klebsiellapneumoniae(33.3%),Pseudomonasaeruginosa(33.3%), Escherichia
coli(33.3%).
71
Table 8: SYMPTOMS ASSOCIATED WITH CULTURE POSITIVE
PATIENTS ON DIALYSIS (n=28)
SYMPTOMS NUMBER OF PATIENTS PERCENTAGE
Diffuse abdominalpain 8 28.5%
Fever 5 17.8%
Vomiting 5 17.8%
Diarrhea 2 7.1%
Commonest symptom associated with infection was abdominal pain 8
(28.5%) followed by fever 5 (17.8%), Vomiting 5(17.8%) and diarrhea 2(7.1%).
Table 9: CORRELATION OF CELL COUNT WITH CULTURE
POSITIVITY
CULTURE CELL COUNT NUMBER OF PATIENTS PERCENTAGE
POSITIVE (n=28)
<100/µl 12 43%
>/=100/µl 16 57%
NEGATIVE
(n=72)
<100/µl 61 85%
>/=100/µl 11 15%
*p value-0.0001
Among the 28 culture positive patients, 43% had cell count <100/µl and
57% patients had cell count ≥100/µl. Out of 72 culture negative patients, 85%
had cell count <100/µl and 15% patients had cell count ≥100/µl.
72
Table 10: FREQUENCY OF SYMPTOMS OF PERITONITIS WITH
CULTURE POSITIVITY
CULTURE SYMPTOMS OF PERITONITIS
NUMBER OF PATIENTS PERCENTAGE
POSITIVE(n=28)
Present 10 35.7%
Absent 18 64.2%
NEGATIVE(n=72)
Present 12 16.6%
Absent 60 83.3%
*p value-0.0001
Out of 28 culture positive patients, 10 patients (35.7%)had symptoms of
peritoneal infection and 18 patients (64.2%) had no symptoms. In 72 culture
negative patients, 12 patients (16.6%) had symptoms of peritoneal infection and
60 patients (83.3%) didn’t have.
CHART 3: DISTRIBUTION OF PERITONEAL INFECTION IN
PATIENTS UNDERGOING DIALYSIS:
65%
70%
75%
80%
85%
90%
95%
Intermittent PeritonealDialysis
Continuous ambulatoryPeritoneal Dialysis
89%
75%
73
Among the 28 culture positive patients 89% were under Intermittent
Peritoneal Dialysis and 75% were under Continuous Ambulatory Peritoneal
Dialysis.
Table 11: RISK FACTORS ASSOCIATED WITH PATIENTS
UNDERGOING DIALYSIS(n=100)
COMORBID CONDITIONS
DIALYSIS(n=100) PERCENTAGE IPD CAPD IPD CAPD
TYPE II DM 32 3 32% 3%
CARDIAC FAILURE 9 0 9% 0%
DCLD 5 1 5% 1%
NO COMORBIDITY 49 1 49% 1%
CHART 4: RISK FACTORS ASSOCIATED WITH PATIENTS
UNDERGOING DIALYSIS
23%
3%
6%1%67%
Diabetes
Cardiac failure
combination
Decompensated liver disease-2
No comorbidity
74
Out of 100 patients underwent dialysis, the majority 23% were diabetic
followed by 3% had Cardiac Failure, 1%had Decompensated Liver Disease and
6% patients all these diseases.
Table 12: ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF GRAM
POSITIVE ISOLATES(n=10)
Antibiotics Coagulase Negative
Staphylococcus
Staphylococcus aureus
Enterococcus feacalis
Penicillin (1)25% 0(0%) 1(100%)
Ampicillin _ _ 1(100%)
Amikacin (3)75% 4(100%) 1(100%)
High Level Gentamicin _ _ 1(100%)
Erythromycin 4(100%) 4(100%) 1(100%)
Trimethoprim sulfamethoxazole 2(50%) 4(100%) 0(0%)
Ciprofloxacin 1(25%) 2(50%) 0(0%)
Clindamycin 4(100%) 4(100%) 1(100%)
Cefoxitin 2(50%) 3(75%) _
Vancomycin 1(100%) 1(100%) _
Linezolid 4(100%) 4(100%) 1(100%)
Chloramphenicol 4(100%) 4(100%) 1(100%)
75
Table 13: ANTIBIOTIC SUSCEPTIBILITY PATTERN OF GRAM
NEGATIVE ISOLATES(n=13)
Antibiotics Escherichia coli
Acinetobacter baumannii
Klebsiella pneumoniae
Klebsiella Oxytoca
Amikacin 2(75%) 2(40%) 2(100%) 2(100%)
Ampicillin 3(75%) _ _ _ Trimethoprim
sulfamethoxazole 1(25%) 2(40%) 2(100%) 2(100%)
Ciprofloxacin 1(25%) 1(20%) 1(50%) _
Cefotaxime 3(75%) _ 1(50%) 1(50%)
Ceftazidime _ 1(20%) _ _
Imipenem 4(100%) 4(80%) 2(100%) 2(100%)
PiperacillinTazobactam 4(100%) 4(80%) 2(100%) 2(100%)
Tetracycline 4(100%) 4(80%) 2(100%) 2(100%)
Colistin _ 1(100%) _ _
CHART 5RESISTANCE PATTERN IN STAPHYLOCOCCUSSPECIES(n=8)
25% of Staphylococcus aureus and 25% ofCoagulase negative
Staphylococcusaureus were Methicillin resistant.
100% 100%
25% 25%
0
20
40
60
80
100
120
Staphylococcus aureus CoNS
Staphylococcus species
Methicillin resistance
76
Table 14: VANCOMYCIN SUSCEPTIBILITY AMONG
STAPHYLOCOCCUS SPECIES(n=2)
VANCOMYCIN SENSITIVE MIC≤2
INTERMEDIATE MIC=4-8
RESISTANT MIC≥16
Methicillin Resistant Staphylococcus
aureus (n=1)
Sensitive(MIC=0.75) _ _
Methicillin resistant Coagulase negative
Staphylococcus(n=1) Sensitive(MIC=0.5) _ _
All Methicillin resistant Staphylococcus species(n=2) were sensitive to
Vancomycin which was tested by Vancomycin E-Strip. MIC for both isolates
were less than two.
CHART 6: EXTENDED SPECTRUM BETA LACTAMASE PRODUCING
ENTEROBACTERIACEAE(n=8)
100% 100% 100%
50% 50%
0
20
40
60
80
100
120
Klebsiella pneumoniae Klebsiella oxytoca Escherichia coli
Total
ESBL
77
Among the Klebsiella isolates 50% of Klebsiellapneumoniae and
Klebsiellaoxytoca were ESBL producers and Out of 4Escherichia coli isolates, 1
(25%) was ESBL producer.
CHART 7: METALLOBETA LACTAMASE PRODUCING NON
FERMENTERS(n=6)
Among the non fermenters, 100% of Pseudomonas aeruginosa and 20% of
Acinetobacterbaumannii were Metallo beta-lactamase producers and sensitive to
colistin.
. Table 15: COLISTIN SUSCEPTIBILITY AMONG NON FERMENTERS:
MBL PRODUCING NON FERMENTERS
SENSITIVE MIC ≤ 2
INTERMEDIATE _
RESISTANT MIC ≥ 4
Pseudomonas aeruginosa (n=1) Sensitive(MIC=1) _ _
Acinetobacterbaumannii (n=1) Sensitive(MIC=1) _ _
0
20
40
60
80
100
120
Pseudomonas aeruginosa Acinetobacter baumannii
Total isolates (%)
MBL (%)
78
Table 16: MOLECULAR DETECTION OF RESISTANT GENES IN
CLINICAL ISOLATES(n=4)
RESISTANT ISOLATES GENES TESTED
NUMBER OF POSITIVES PERCENTAGE
Staphylococcus aureus mecA gene 1 100%
Staphylococcus epidermidis mecA gene 1 100%
Pseudomonas aeruginosa VIM gene 0 0%
Acinetobacterbaumannii OXA23 gene 1 100%
mecA gene was detected in Methicillin resistant Staphylococcus species
and OXA23 gene was detected in MBL producing Acinetobacterbaumannii by
Polymerase Chain Reaction.
Table 17:ANTIFUNGAL SUSCEPTIBILITY TESTING OF CANDIDA
SPECIES:
CANDIDA SPECIES Fluconazole Itraconazole Amphotericin
B Clotrimoxazole Ketoconazole Nystatin
Candida tropicalis
(n=2) 50% 100% 100% 100% 50% 50%
Candida glabrata(n=1) 100% 100% 100% 100% 100% 100%
Candida parapsilosis
(n=2) 50% 100% 100% 100% 100% 100%
Discussion
79
DISCUSSION
Peritoneal Dialysis is an effective mode of therapy in Acute and Chronic
Renal Failure patients. Peritonitis is the major complication of Peritoneal Dialysis
and it remains a cause of hospitalisation, Catheter removal, discontinuation of
peritoneal dialysis and switch to hemodialysis. Mortality rates varying from 2 to
25% in bacterial peritonitis and 5% to 53% in fungal peritonitis. Hence, routine
PD effluent culture at the end of the procedure should be done which helps to
identify the patients at risk of developing peritonitis at an early stage. Thereby, the
longevity of Peritoneal Dialysis can be enhanced.
100 patients who underwent Intermittent and Continuous Peritoneal
Dialysis were included in the present study. There was a male predominance
among the patients included and 31-40 years was the most common age group.
Among Dialysis patients, 24% were diabetic, 12% Cardiac failure,6% Coronary
Artery Disease, 2% Decompensated Liver Disease and 56% without any
comorbid conditions.
Among the culture positive cases 78.6% were males and 21.4% were
females. Most of them were in the age group of 51-60 years which is similar to
the study by Sharon J et al in 2009. This could be due to the high prevalence of
immunosuppression and End Stage Renal Disease in elderly patients.
Among the Dialysis patients 92% had Chronic Kidney Disease and only
8% with Acute Kidney Injury. Culture positivity was seen in 30.4% of CKD
80
patients. This indicates that the risk of getting infection is high in patients who
had been in dialysis for longer duration.
In the present study, among culture positive patients, 29% had abdominal
pain, 18% with fever and vomiting and 7% had diarrhea. Shalinie et al study
reported that abdominal pain was seen in 48%, fever 36%, vomiting 14% and
diarrhea6%.(6)
Among the culture positive cases, 26.04% was in Intermittent Peritoneal
Dialysis and 75% Continuous Ambulatory Peritoneal Dialysis. This is similar to
the studies of Sharma et al and Soham Gupta et al who showed 30% culture
positivity among IPD patients and 60% among CAPD patients. This variation may
be due to faulty sterile technique and increased number of exchanges in CAPD
compared to IPD. (16)
Lysis centrifugation technique yields 28% culture positivity in this study.
However, in Sewell et al study, the culture positivity increases with different
methods like Bactec (94%) and large volume dialysate culture (88%)(84); Lye et al
reported 75% in Bactec and 58% (P=0.05) in large volume culture method.(85)
Culture positivity could be increased if large volume (50 ml) of the effluent and
various other processing techniques have been used.(86)
In the present study Gram's stain positivity in relation to culture was
17.8%. The study by S.Vikrant et al who showed 8.4% positive gram stain(87).
81
In this study Clinical Peritonitis was seen in 7% of patients similar to the
findings of Bonnie et al 11%(88). Meanwhile, 15% showed culture negative
though the cell count was >100/µl, correlated well with the similar studies where
10-50% culture negative was reported(89,90,19,91,92).This non specific rise in effluent
cell count may be due to other extra peritoneal infections like exit site infection,
abdominal surgery, diverticulitis. Culture negative peritonitis may be due to
constant flow of dialysis fluid into and out of the peritoneal cavity diluting the
microbial density to be low or due to infection with fastidious organisms like
fungi, mycobacteria, Legionella, Campylobacter, Ureaplasma species,
Mycoplasma or enteroviruses.(19) or noninfectious causes like chemical
irritation(by icodextrin), chylous ascites or effluent eosinophilia.(93)This can be
differentiated from true infection by performing a differential count.
Polymorphonuclear leucocytes predominate during peritonitis whereas in the
absence of infection, the normal cellular response is monocytic.
Among the culture positive cases 46.4% were Gram negative bacteria and
Gram positive bacteria constitutes 35.7% .Similar findings were reported by
Verbrugh et al in 1984 showed Gram negative bacteria as the predominant
isolates(92). The study by Prasad N et al (2013) also reported that 60% were Gram
negative and only 30% were Gram positive organisms (94). This is because of,
commercially available dialysate not supporting the growth of gram positive
organismsand easily contaminated with Gram negative bacteria. (20)
82
In the present study Acinetobacter baumannii constitutes 20% in
Intermittent Peritoneal Dialysis cases, similar findings were observed in the study
by Sharma et al, in which they showed 21.5% of Acinetobacter
baumannii.(95).This may be due to the hygiene breaks and failure to perform sterile
exchange procedures.(96)
Among the culture positive isolates, Klebsiella species and Escherichia
coli were isolated in 12% each in IPD patients. The study by Sharma et al showed
Escherichia coli 4% and Klebsiella species 1% respectively(97).This may occur
due to transmural migration or from contaminated dialysate or from exit site
infections. Peritoneal contamination with enteric pathogens may also occur due to
alterations of skin flora (20).
But in Gram positive isolates, Staphylococcus aureus was 16% in the
present study correlated well with the studies of Sharma et al, where
Staphylococcus aureus constitutes 25%. Infection with Staphylococcus aureus
occurs due to touch contamination or from catheter infection or the patient may be
a nasal carrier of Staphylococcus aureus. Hence this can be prevented by nasal
screening and sterile techniques.(8)Also Staphylococcus epidermidis was reported
in 16% of cases and Enterococcus faecalis was in 4% in IPD patients. Similar
study by Shalinie et al reported 13% Staphylococcus epidermidis and
Enterococcus feacalis6% respectively. Infections with Staphylococcus
epidermidis may occur due to touch contamination. This can be prevented by
sterile exchanges during dialysis.(8)
83
In the present study among the CAPD patients, Klebsiella pneumoniae,
Pseudomonas aeruginosa and Escherichia coli were isolated 33.3% each. The
study by A K Balla et al reported 8% of Escherichia coli and Klebsiella
pneumoniae, and 5% of Pseudomonas aeruginosa . As Pseudomonas species
normally colonize the skin of chronically debilitated patients it can easily produce
infection among chronic dialysis patients. Pseudomonas species have also caused
outbreaks of contaminated dialysate associated peritonitis in patients on CIPD.(8)
But in Previous studies ,Gram positive organisms were more commonly
isolated(96). This recent change may be due to transmural migration of intestinal
organisms and contamination with fecal organisms.
In the present study 17.9% of fungal pathogens were also isolated. The
study by KV Kumar et al also reported 14% fungal pathogens(98). All isolates were
found to be Candida non albicans. Similar findings were reported by Jasmin
levallois et al which was done in 2012 showed, Candida non albicans as the
predominant causative agent(27). Sogam Gupta et al study which was done in 2011
reported Candida albicans as the fungal etiological agent. This recent changing
trend from Candida albicans to non albicans have been reported in most of the
studies. Change in the epidemiology of fungal isolates from Candida albicans to
Candida non albicans occurs in the post prophylactic era and after usage of
fluconazole.(98).
Candida tropicalis and Candida parapsilosis were 40% each among the
Candida non albicans in the present study. This similar distribution was seen in
84
Upma Narain et al study, Candida tropicalis (13.8%) and Candida parapsilosis
(12.3%)(99)
In our study, among the culture positive patients all had Chronic Kidney
Disease and 86% were diabetic and 75% were under Continuous Ambulatory
Peritoneal Dialysis. This might be due to immunocompromised state in CKD and
diabetic patients. This high percentage in CAPD patients may be due to unsterile
technique or inadequate nursing.
Among the Staphylococcal isolates 25% Staphylococcus aureus and
Staphylococcus epidermidis were found to be methicillin resistant. The source of
MRSA could be community acquired or hospital acquired. The later could be
from infected patients or hospital staff. In nososcomial as well as community
acquired MRSA infections, nasal carriage rate seems to be the main source of
infection. Apart from nasal carriage perineal, axillary and subungual carriages
play a role in transmission of MRSA. Therefore the crucial strategy in avoiding
this is through hand disinfection and the therapeutic regimen includes mupirocin
nasal ointment combined with parenteral vancomycin administration.(8)
In the present study, 50% of Klebsiella species and 25% of Escherichia
coli were ESBL producers. This similar findings were also observed in kashinath
Prasad study who showed 54.3% were ESBL producers. Uses of cephalosporins
and gastric acid inhibitors have been associated with ESBL production.
Prevention by judicious use of antibiotics and infection prevention measures like
hand wash, proper exit site care will be the most efficacious step.(100)
85
In the present study, MBL production was observed in Pseudomonas
aeruginosa 100% and Acinetobacter baumannii 20%. The KashiNath Prasad et al
study also reported that 23.5% of Acinetobacter species and 11.5% of
Pseudomonas aeruginosa were metallo-β-lactamase (MBL) producers.(100)
OXA 23 gene was responsible for carbapenamase production in
Acinetobacter baumanniiin this study. The study by Pragasam et al , OXA gene
was identified in 98% of Carbapenamase producing Acinetobacter baumannii
isolates.(101)mecA gene was detected in 100% of Methicillin resistant
Staphylococcus species in our study similar to the study of C.L.C.Wielders et
al,95% of MRSA isolates possess mecA gene (102)
In the present study, Antifungal susceptibility of Candida isolates,
resistance to Fluconazole, Itraconazole and Ketoconazole are more common than
Amphotericin B and Nystatin.The study by Levallois J et al also showed Azoles
showed more resistance in non albicans Candida species.(27)
Summary
86
SUMMARY
In the present study 100 patients undergoing Peritoneal dialysis were analysed
during the one year study period.
Majority of the patients were in the age group of 31-40 years with male
preponderance.
Culture positive was observed in 28%. Among them 46.4% were Gram
negative, 35.7% Gram positive and 17.9% fungal isolates.
89.3% of isolates were from Intermittent Peritoneal Dialysis. Only 10.7% from
CAPD.
Among culture positive patients 35.7% of them had symptoms of peritoneal
infection and 64.2% had no symptoms.
Diabetes mellitus (86%) was the predominant comorbid condition seen in
culture positive patients.
Abdominal pain (28.5%) was the predominant symptom seen in culture
positive patients.
57% of the patients had culture positive with cell count ≥100/µl and 15% of
patients had cell count ≥100/µl but culture was negative.
Klebsiella pneumoniae,Pseudomonas aeruginosa, Escherichia coli each
33.3% were isolated in CAPD patients.
ESBL production was seen in 50% in Klebsiella pneumoniae.
Acinetobacter baumannii constitute 20% among IPD patients.
87
MBL production was observed in 100% of Pseudomonas aeruginosa and 20%
of Acinetobacter baumannii.
Methicillin resistance was found in 25% of Staphylococcus aureus and
Coagulase negative Staphylococcus. All are sensitive to Vancomycin.
mecA gene was detected in Methicillin resistant Staphylococcus species and
OXA23 gene was detected in MBL producing Acinetobacter baumannii.
Candida non albicans(20%) was the predominant fungal pathogen. All the
isolates were sensitive to Itraconazole, Amphotericin B and Clotrimoxazole.
Conclusion
88
CONCLUSION
In this cross sectional study, majority of the patients were affected with
Chronic Renal Failure. Peritoneal infections were more common in patients with
longer duration of dialysis and diabetes mellitus. CAPD patients were having
higher risk of infections compared to IPD. This can be prevented by advising the
patients to maintain proper glycemic control and to follow the sterile techniques
during exchanges.
Cell count analysis of effluent has a significant role in identifying the
infection. Also by processing larger volume of fluid enhances the detection of
pathogens. Gram negative isolates were predominantly isolated in bacterial
culture. Candida non albicans was the main fungal pathogens identified.
Acinetobacter baumannii was the most common Gram negative pathogen.
This can be decreased by technique reeducation and sterility maintenance. MRSA,
ESBL, MBL producing strains were also isolated in this study. This can be
prevented by encouraging the use of first line antibiotics based on antimicrobial
susceptibility and avoiding or postponing the use of higher antibiotics.
As Peritoneal dialysis associated infections are being increased, routine PD
fluid microbiological analysis after the completion of the procedure will be useful
to improve patient care by using appropriate antibiotic at the earliest before any
significant clinical problem occurs.
COLOUR PLATES
METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS
E-TEST DETECTING VANCOMYCIN MIC FOR MRSA
E-TEST DETECTING VANCOMYCIN MIC FOR METHICILLIN
RESISTANT STAPHYLOCCUS EPIDERMIDIS
ESBL PRODUCING ESCHERICHIA COLI
METALLO BETA LACTAMASE PRODUCING PSEUDOMONAS
AERUGINOSA
E-TEST FOR DETECTION OF COLISTIN MIC
MOLECULAR CHARACTERISATION OF VIM & OXA23 GENES
AMONG NON FERMENTERS
L1-VIM NTC L2-SAMPLE1 L3-DNA LADDER L4-OXA NTC L5-
SAMPLE 2
MOLECULAR CHARECTERISATION OF mecA GENE AMONG
STAPHYLOCOCCUS SPECIES
L1-mecA NTC L2-MOLECULAR LADDER L3-SAMPLE3 L4-
SAMPLE4 L5-POSITIVE CONTROL
CANDIDA SPECIES ON SABOURAUD DEXTROSE AGAR
CANDIDA NON ALBICANS ON CHROM AGAR PLATE
ANTIFUNGAL SUSCEPTIBILITY TESTING OF CANDIDA NON
ALBICANS
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APPENDIX-I
ABBREVIATIONS
CKD - Chronic Kidney Disease
AKI - Acute Kidney Injury
CAPD - Continuous Ambulatory Peritoneal Dialysis
IPD - Intermittent Peritoneal dialysis
CIPD - Chronic Intermittent Peritoneal dialysis
CCPD - Continuous Cycling Peritoneal Dialysis
NHANES - National Health and Nutrition Examination Surveys
ESRD - End Stage Renal Disease
PD - Peritoneal Dialysis
HD - Hemodialysis
ISPD - International Society for Peritoneal Dialysis
MRSA - Methicillin Resistant Staphylococcus aureus
ESBL - Extended Spectrum Beta Lactamase
MBL - Metallo Beta Lactamase
CLSI - Clinical & Laboratory Standards Institute
ATCC - American Type Culture Collections
APPENDIX-II
A. STAINS AND REAGENTS
I. Gram staining: Methyl violet(2%) 10g Methyl violet in 100ml
absolute alcohol in 1 litre of distilled water(Primary stain)
Grams iodine 10 g iodine in 20 g KI (fixative) Acetone Decolourising agent Carbolfuchsin 1% secondary stain
II. Lactophenol cotton blue stain:
Lactic acid 20ml Phenol 20ml Cotton blue (dye) 0.5g Glycerol 40ml Distilled water 20ml
III. 10% KOH:
Potassium hydroxide 10g Glycerol 10ml Distilled water 80ml
B. MEDIA USED: 1. MacConkey agar:
Peptone 20g Sodium taurocholate 5g Distilled water 1 ltr Agar 20g 2% neutral red in 50% ethanol 3.5ml
10% lactose solution 100ml Dissolve peptone and taurocholate in water by heating. Add agar and dissolve it in steamer. Adjust pH to 7.5. Add lactose and neutral red shake well and mix. Heat in free steam (100°C) for 1 hour, then autoclave at 115°c for 15 minutes. 2. 5% Sheep blood agar: Peptone 10g Nacl 5g Distilled water 1 litre Agar 10g
Dissolve ingredients in distilled water by boiling and add 5% sheep blood (sterile) at 55°C and adjust pH to 7.4. 3. Chocolate agar:
Sterile defibrinated blood 10 ml Nutrient agar (melted) 100ml
When the temperature was about 75°C sterile blood was added with constant agitation. After adding of blood, kept in water bath and heating was continued till the blood changed to chocolate color. Cooled to about 50°C and poured about 15ml in to petri dishes sterile precaution. 4. Sabouraud dextrose agar:
Dextrose 40g Peptone 10 g Agar 20g Distilled water 1000ml pH=5.5 5. Mueller-Hinton Agar: Beef infusion 300ml Casein hydrolysate 17.5g Starch 1.5g Agar 10g Distilled water 1 ltr pH =7.4
Sterilize by autoclaving at 121°C for 20 mins. C. MEDIA RECQUIRE FOR BIOCHEMICAL IDENTIFICATION: 1. Oxidase reagent: Tetra methyl p-phenylenediaminedihydrochloride – 1% aqueous solution. 2. Catalase reagent: 3% hydrogen peroxide. 3. Indole reagent: Amyl or isoamyl alcohol 150 ml, Para dimethyl amino benzaldehyde 10g, concentrated hydrochloric acid 50ml.Dissolve the aldehyde in the alcohol and slowly add the acid. Prepare in small quantities and store in the refrigerator. Shake gently before use.
4. Christenson’s Urease test medium: Peptone 1g Sodium chloride 5g Dipotassium hydrogen phosphate 2g Phenol red 6 ml Agar 20g
Distilled water 1 ltr 10% sterile solution of glucose 10ml Sterile 20% urea solution Sterilize the glucose and urea solution by filtration. Prepare the basal medium without glucose and urea, adjust to pH 6.8-6.9 and sterilize by autoclaving in a flask at 121°C for 30min. cool to about 50°C, add the glucose and urea, and tube the medium as slopes. 5. Simmon’s Citrate Medium: Koser’s medium 1 ltr Agar 20g Bromothymol blue 0.2% 40ml Dispense, autoclave at 121°C for 15 min and allow to set as slopes. 6. Triple sugar iron medium: Beef extract 3g Yeast extract 3g Peptone 20g Glucose 1g Lactose 10g Sucrose 10g Ferric citrate 0.3g Sodium chloride 5g Sodium thiosulphate 0.3g Agar 12g Phenol red 0.2% solution 12ml Distilled water 1 ltr Heat to dissolve the solids, add the indicator solution, mix and tube. Sterilize at 121°C for 15 min and cool to form slopes with deep butts. 7. Glucose phosphate broth: Peptone 5g Dipotassium hydrogen phosphate 5g Water 1 ltr Glucose 10% solution 50 ml
Dissolve the peptone and phosphate and adjust the Ph to 7.6. Filter dispense in 5 ml amounts and sterilize at 121°C for 15 min. Sterilize the glucose solution by filtration and add 0.25ml to each tube. 8. Peptone water fermentation test medium:
To the basal medium of peptone water, add sterilized sugars of 1% indicator bromothymol blue with Durham’s tube.
Basal medium peptone water sugar solutions: Sugar 1ml Distilled water 100ml pH = 7.6 9. Sugar fermentation medium: Peptone 15g Andrade’s indicator 10ml Sugar to be tested 20g Water 1 ltr Andrade’s indicator is prepared from 0.5% aqueous acid fuchsin to which sufficient 1M sodium hydroxide has been added to turn the color of the solution yellow. Dissolve the peptone and andrade’s indicator in 1 litre of water and add 20g of the sugar; sugars to be tested generally include glucose, sucrose, lactose and maltose. Distribute 3ml amounts in standard test tubes containing an inverted Durham’s tube. Sterilize by steaming at 100°C for 30 min on 3 consecutive days.
ANNEXURE – I
ANNEXURE-II PROFORMA
Name : IP NO:
Age: Ward:
Date of admission:
Sex:
Occupation:
Address:
Clinical diagnosis:
Presenting complaints:
Type of Dialysis:
Frequency of dialysis:
Prior antibiotic therapy:
Co-morbid conditions if any:
Investigation:
Peritoneal fluid analysis:
Macroscopic observation of fluid:
Leucocyte count:
Direct gram staining :
KOH mount :
Bacterial culture :
Fungal culture :
Organisms isolated :
Antibiotic sensitivity pattern :
ANNEXURE – III
CONSENT FORM
STUDY TITLE:
Microbiological profile of peritoneal dialysis fluid in acute and chronic renal
failure patients in a tertiary care hospital.
I_______________________ here by give consent to participate to the study
conducted by Dr.M.Sornaranjani, Madras medical college, Chennai and to use my
personal clinical data and the result of investigations for the purpose of analysis and to
study nature of diseases. I also give consent to give my peritoneal dialysis effluent for
further investigations. I also learn that there is no additional risk in this study. I also give
consent for my investigator to publish the data in any forum or journal.
Signature/ thumb impression Place Date of the parent/relative
Patient name & address:
Signature of the investigator:
S,No Name Age Gender Diagnosis Type of Dialysis Comorbid conditions Macro scopic cell count Gram stain KOHmount Fungal culture Bacterial cultureDuration of dialysis in
years
Symptoms of Peritonitis
No of exchange
s
1 Ranjith kumar 33 M CKD IPD Type II DM,DCLD,cardiac failure. clear 100 No pus cells,No organisms seen No fungal elements seen No growthStaphylococcus
epidermidis4.08 absent 22
2 karthick 39 M CKD IPD NIL clear 600 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 48
3 Pazhani 62 M CKD CAPD Type II DM Turbid 2200Few pus cells seen,No
organisms seenNo fungal elements seen No growth
Klebsiella pneumoniae
4.25 present 18
4 Dhinagaran 56 M CKD IPD NIL clear 500 No pus cells,No organisms seen No fungal elements seen No growth Escherichia coli 1 absent 18
5 Chandrabharathy 48 F AKI IPD NIL clear 100 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 18
6 Poongavanam 50 M CKD IPD NIL clear 100 No pus cells,No organisms seen Oval budding yeast cells seen C.tropicalis No growth 2 absent 16
7 Aravindhan 49 M CKD IPD NIL clear 800 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 16
8 Nagappan 30 M CKD IPD NIL clear 100 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 20
9 Sugunabai 40 F CKD IPD Type II DM Turbid 1500Few pus cells,No organisms
seenNo fungal elements seen No growth Klebsiella oxytoca 4.41 present 15
10 Selvi 59 F CKD IPD Type II DM clear 400 No pus cells,No organisms seen No fungal elements seen No growthStaphylococcus
epidermidis4.66 absent 18
11 Manikandan 19 M CKD CAPD NIL clear 2500 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 12
12 Nelson 30 M CKD IPD NIL clear 800 No pus cells,No organisms seen No fungal elements seen No growthAcinetobacter
baumannii4 present 20
13 Ramesh 38 M CKD IPD NIL clear 600 No pus cells,No organisms seen No fungal elements seen No growth Escherichia coli 3.83 present 18
14 Murugesan 31 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 19
15 Vidhya 24 F CKD IPD Cardiac failure clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 20
16 Nirmala 50 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2.33 absent 20
17 Sekar 55 M CKD IPD NIL clear 100 No pus cells,No organisms seen No fungal elements seen No growthKlebsiella
pneumoniae4.05 present 18
18 Pandiyan 46 M CKD IPD NIL clear 0 No pus cells,No organisms seen Oval budding yeast cells seen C.parapsilosis No growth 3 absent 18
19 Prasad 42 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 18
20 Veeramani 30 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 20
21 Sathish kumar 28 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 18
22 Diliraj 35 M CKD IPD Type II DM clear 2200 No pus cells,No organisms seen No fungal elements seen No growthAcinetobacter
baumannii4.33 present 16
23 Sridhar 61 M CKD CAPD Type II DM clear 1400Few pus cells,No organisms
seenNo fungal elements seen No growth Escherichia coli 4 present 20
24 Kuzhabthai velu 30 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 18
25 Rajammal 35 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 20
26 Kokila 45 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 15
27 Kumaresan 60 M CKD IPD NIL clear 0 No pus cells,No organisms seen Oval budding yeast cells seen C.tropicalis No growth 4.58 absent 20
28 Mariappan 58 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 15
ANNEXURE IV- MASTER CHART
29 Annammal 45 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 14
30 Akash 25 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 20
31 Seetha 38 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 18
32 Kalasalingam 42 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 16
33 Govindharaju 46 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 18
34 Ganesh 42 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 18
35 Sathish kumar 30 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 20
36 Kesavan 58 M CKD IPD NIL clear 0 No pus cells,No organisms seen Oval budding yeast cells seen C.glabrata No growth 4.91 present 16
37 Kalaiselvi 60 F AKI IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 18
38 Mahesh 45 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 20
39 Govindhammal 32 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 22
40 Ganesan 50 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 4 absent 16
41 Rameenabee 55 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 18
42 Muthukumar 40 M CKD IPD NIL clear 800 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 20
43 Velu 63 M CKD IPD NIL clear 400 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 28
44 Mubeena 36 F CKD IPD Type II DM clear 100 No pus cells,No organisms seen No fungal elements seen No growthAcinetobacter
baumannii4.33 present 32
45 Kanaga 28 F CKD IPD NIL clear 0Few pus cells,No organisms
seenNo fungal elements seen No growth Klebsiella oxytoca 4.66 present 18
46 Nasar 35 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growthStaphylococcus
aureus5 present 18
47 Loganathan 61 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growthStaphylococcus
aureus5.16 present 20
48 Sukumar 55 M CKD CAPD Type II DM,DCLD clear 0 No pus cells,No organisms seen No fungal elements seen No growthPseudomonas
aeruginosa5 present 16
49 Raja 45 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 18
50 Nazeer 52 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 16
51 Rasukutti 18 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 6
52 Madhiyazhagan 53 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 16
53 Muniyammal 38 F CKD IPD Cardiac failure clear 0 No pus cells,No organisms seen No fungal elements seen No growth Escherichia coli 5 present 30
54 Kaliyaperumal 60 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 18
55 Kaliammal 60 F CKD IPD Type II DM clear 0 No pus cells,No organisms seen Oval budding yeast cells seen C.parapsilosis No growth 2 present 20
56 Murugan 45 M AKI IPD NIL clear 100 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 18
57 Sudhan 28 M AKI IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 16
58 Santhosh 30 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 32
59 Vellammal 58 F CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 16
60 Dhinesh 30 M AKI IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 18
61 Anandhi 63 F CKD IPD Type II DM, Cardiac failure clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 18
62 Murugesan 50 M CKD IPD Type II DM, Cardiac failure clear 0 No pus cells,No organisms seen No fungal elements seen No growthStaphylococcus
aureus5.66 present 37
63 Nagaraj 39 M CKD IPD DCLD clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 20
64 Boopathy 51 M CKD IPD Cardiac failure clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 30
65 Muthamizh 84 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growthStaphylococcus
aureus4.41 present 18
66 Menaka 24 F CKD IPD Type II DM.DCLD clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 18
67 Ramajayam 53 F CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 16
68 Arjunan 23 M CKD IPD NIL clear 200 No pus cells,No organisms seen No fungal elements seen No growthAcinetobacter
baumannii5 present 18
69 Durai 63 M CKD IPD Type II DM clear 0Few pus cells seen,No
organisms seenNo fungal elements seen No growth
Staphylococcus epidermidis
4.05 absent 20
70 Ethiyan 67 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growthStaphylococcus
epidermidis4.58 absent 22
71 Thilagavathy 24 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 19
72 Devarajan 30 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growthAcinetobacter
baumannii4 present 1
73 Kadhar moideen 65 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 11
74 Gnanasekar 47 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 16
75 Asha jothi 45 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 18
76 Kamala kannan 41 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 20
77 Manimegalai 35 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 12
78 Rajeshwari 35 F CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 14
79 Banu priya 24 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2.58 absent 10
80 Venkatesan 59 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2.33 absent 7
81 Vethagiri 50 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growthEnterococcus
faecalis4 present 14
82 Vasantha 68 F CKD IPD NIL clear 100 No pus cells,No organisms seen No fungal elements seen No growth No growth 2.66 absent 10
83 Amudha 55 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2.41 absent 14
84 Murugesan 40 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3 absent 14
85 Ramesh 35 M AKI IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 12
86 Pravin 25 M AKI IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 16
87 Krishnaveni 40 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1 absent 18
88 Parvathy 21 F AKI IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1.58 absent 19
89 Kalaiselvi 35 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1.83 absent 20
90 Murugan 35 M CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 12
91 Lakshmi 38 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3.83 absent 17
92 Kalaivani 60 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2.91 absent 20
93 Pazhaniammal 40 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 1.41 absent 22
94 Mary 50 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 13
95 Anandhavalli 63 F CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3.66 absent 19
96 Murugaiah 65 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3.83 absent 20
97 Rajeshwari 35 F CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2.91 absent 16
98 Sangeetha 24 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 3.58 absent 18
99 Mugilvannan 60 M CKD IPD Type II DM clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2.66 absent 12
100 Vasantha 68 F CKD IPD NIL clear 0 No pus cells,No organisms seen No fungal elements seen No growth No growth 2 absent 10
sex Bacterial culture PenAmpic
illinAmika
cinHLG Erythro Cotri Cipro Clinda Cefoxitin Vanco Linezolid Chloram Cefotaxime Ceftazidime Imipenem PT Tetra Colistin Fungal culture Flu Itra AmpB Clotri Keto Nystatin
Ranjith kumar 33 MStaphylococcus
epidermidisR _ S _ S S R S S _ S S _ _ _ _ S _ No growth
karthick 39 M No growth _ _ _ _ _ _ _ _ _ _ S _ _ _ _ _ _ _ No growth
Pazhani 62 M Klebsiella pneumoniae _ _ S _ _ S S _ _ _ _ _ R _ S S S _ No growth
Dhinagaran 56 M Escherichia coli _ _ S _ _ R R _ _ _ _ _ S _ S S S _ No growth
Chandrabharathy 48 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Poongavanam 50 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ C.tropicalis R S S S R R
Aravindhan 49 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Nagappan 30 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Sugunabai 40 F Klebsiella oxytoca _ _ S _ S S R _ _ _ _ _ R _ S S S _ No growth
Selvi 59 FStaphylococcus
epidermidisS _ S _ S R S S _ _ S S _ _ _ _ S _ No growth
Manikandan 19 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Nelson 30 MAcinetobacter
baumannii_ _ S _ _ S S _ _ _ _ _ _ R S S S _ No growth
Ramesh 38 M Escherichia coli _ S S _ _ R R _ _ _ _ _ S _ S S S _ No growth
Murugesan 31 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Vidhya 24 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Nirmala 50 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Sekar 55 M Klebsiella pneumoniae _ _ S _ _ S R _ _ _ _ _ S _ S S S _ No growth
Pandiyan 46 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ C.parapsilosis R R S S S S
Prasad 42 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Veeramani 30 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Sathish kumar 28 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Diliraj 35 MAcinetobacter
baumannii_ _ S _ _ R R _ _ _ _ _ S S S S S _ No growth
Sridhar 61 M Escherichia coli _ S S _ _ S S _ _ _ _ _ S _ S S S _ No growth
Kuzhabthai velu 30 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Rajammal 35 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Kokila 45 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Kumaresan 60 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ C.tropicalis S S S S S S
Mariappan 58 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Annammal 45 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Akash 25 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Seetha 38 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Kalasalingam 42 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Govindharaju 46 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Ganesh 42 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Sathish kumar 30 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Kesavan 58 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ C.glabrata S S S S S S
Kalaiselvi 60 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Mahesh 45 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Govindhammal 32 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Ganesan 50 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Rameenabee 55 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Muthukumar 40 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Velu 63 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Mubeena 36 FAcinetobacter
baumannii_ _ R _ _ S R _ _ _ _ _ _ R S S S _ No growth
Kanaga 28 F Klebsiella oxytoca _ _ S _ _ S R _ _ _ _ _ S _ S S S _ No growth
Nasar 35 M Staphylococcus aureus R _ S _ S R R S S _ S S _ _ _ _ S _ No growth
Loganathan 61 M Staphylococcus aureus R _ S _ S R S S S _ S S _ _ _ _ S _ No growth
Sukumar 55 MPseudomonas
aeruginosa_ _ R _ _ _ R _ _ _ _ _ _ R R R R S No growth
Raja 45 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Nazeer 52 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Rasukutti 18 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Madhiyazhagan 53 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Muniyammal 38 F Escherichia coli _ R R _ _ R R _ _ _ _ _ R _ S S S _ No growth
Kaliyaperumal 60 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Kaliammal 60 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ C.parapsilosis S R S S S S
Murugan 45 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Sudhan 28 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Santhosh 30 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Vellammal 58 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Dhinesh 30 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Anandhi 63 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Murugesan 50 M Staphylococcus aureus R _ S _ S R R S R S S S _ _ _ _ _ _ No growth
Nagaraj 39 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Boopathy 51 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Muthamizh 84 M Staphylococcus aureus R _ S _ S R S S S _ S _ _ _ _ _ _ _ No growth
Menaka 24 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Ramajayam 53 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Arjunan 23 MAcinetobacter
baumannii_ _ R _ _ R R _ _ _ _ _ _ R S S S _ No growth
Durai 63 MStaphylococcus
epidermidisR _ S _ S R R S R S S S _ _ _ _ S _ No growth
Ethiyan 67 MStaphylococcus
epidermidisR _ R _ S S R S S _ S S _ _ _ _ _ _ No growth
Thilagavathy 24 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Devarajan 30 MAcinetobacter
baumannii_ _ R _ _ R R _ _ _ _ _ _ R R R R S No growth
Kadhar moideen 65 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Gnanasekar 47 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Asha jothi 45 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Kamala kannan 41 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Manimegalai 35 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Rajeshwari 35 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Banu priya 24 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Venkatesan 59 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Vethagiri 50 M Enterococcus faecalis S S S S S _ R S _ _ S S _ _ _ _ _ _ No growth
Vasantha 68 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Amudha 55 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Murugesan 40 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Ramesh 35 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Pravin 25 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Krishnaveni 40 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Parvathy 21 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Kalaiselvi 35 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Murugan 35 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Lakshmi 38 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Kalaivani 60 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Pazhaniammal 40 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Mary 50 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Anandhavalli 63 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Murugaiah 65 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Rajeshwari 35 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Sangeetha 24 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Mugilvannan 60 M No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
Vasantha 68 F No growth _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ No growth
KEY TO MASTER CHART
M - Male
F - Female
CKD - Chronic Kidney Disease
AKI - Acute Kidney Injury
IPD - Intermittent Peritoneal Dialysis
CAPD - Continuous Ambulatory Peritoneal Dialysis
DM - Diabetes Mellitus
DCLD - Decompensated Liver Disease
KOH - Potassium Hydroxide
S - Sensitive to drug
R - Resistant to drug
Pen - Penicillin
Ampi - Ampicillin
Amik - Amikacin
HLG - High Level Gentamicin
Erythro - Erythromycin
Cotri - Trimethoprim sulfamethoxazole
Cipro - Ciprofloxacin
Clinda - Clindamycin
Vanco - Vancomycin
Chloram - Chloramphenicol
PT - PiperacillinTazobactam
Tetra - Tetracycline
Flu - Fluconazole
Itra - Itraconazole
AmpB - AmphotericinB
Clotri - Clotrimoxazole
Keto - Ketoconazole
Nystatin - Nystatin
C.trpoicalis - Candida tropicalis
C.parapsilosis - Candida parapsilosis
C.glabrata - Candida glabrata