Rajiv Gandhi University of Health Sciences, Karnataka s8

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE CANDIDATE : DR. RAVI KUMAR . R

ADDRESS : DEPARTMENT OF BIOCHEMISTRY ST. JOHN’S MEDICAL COLLEGE SARJAPUR ROAD BANGALORE– 560034.

2. NAME OF THE INSTITUTION : ST. JOHN’S MEDICALCOLLEGE

3. COURSE OF STUDY AND : MD BIOCHEMISTRY SUBJECT

4. DATE OF ADMISSION TO : 25-03-2010 COURSE

5. TITLE OF THE TOPIC : ASCITIC FLUID BIOMARKERS FOR THE EARLY DIAGNOSIS OF SPONTANEOUS BACTERIAL PERITONITIS.

1 6. BRIEF RESUME OF THE INTENDED WORK .

6.1: NEED FOR THE STUDY

Ascites is the most common complication of cirrhosis in patients with liver disease.1 Ascitic fluid can become infected without any apparent intra-abdominal source of infection, a condition called Spontaneous bacterial peritonitis.2

Spontaneous bacterial peritonitis (SBP) is an important cause of morbidity and mortality in patients with cirrhosis and ascites. SBP is identified in 10% - 30% of patients hospitalized with ascites3, and mortality can approach 30%.4

The typical clinical symptoms suggestive of SBP in a patient with ascites are pain abdomen and fever which are seen in few patients only.5 Patients may present with altered gastro-intestinal motility, hepatic encephalopathy, abdominal tenderness, ileus and hypothermia whereas some patients may be completely asymptomatic or there may be minor symptoms only (especially in the initial stages).6 Hence, the clinical diagnosis of SBP requires a high degree of suspicion. Therefore, a diagnostic paracentesis is recommended for all cirrhotic patients with ascites at the time of hospital admission to investigate for the presence of SBP.1,3

Currently, spontaneous bacterial peritonitis is diagnosed when, (a) The ascitic fluid culture grows pathogenic bacteria (almost always pure growth of a single type of organism), (b) The ascitic fluid polymorpho nuclear (PMN) cell count >= 250 cells /mm3 and (c) There is no evidence of surgically treatable intra-abdominal sources of infection.6

But conventional culture of ascitic fluid to confirm the diagnosis may be negative in upto 60% of patients with SBP.3 Hence, according to the current guidelines,3,4 the detection of a PMN count greater than 250 cells/mm3 is highly suggestive of SBP and provides an indication to initiate empirical antibiotic therapy. For this analysis, the ascitic fluid is transported to the laboratory and the number of PMNs in the ascitic fluid is

2 measured manually by a laboratory technician.2 This method is presently considered “gold standard” for evaluation of ascitic PMN count, but this procedure is laborious and time consuming.5 This results in the laboratory often providing the PMN count too late in the day, or sometimes even on the next day.5 In practioners’ offices or small clinics without a laboratory, longer time is required2 by the time which patient might have progressed from an early stage to the fatal stage of infection. This often delays the initiation of empirical therapy and may expose the patient to a higher risk of morbidity and mortality.5 Moreover manual measurement of ascitic fluid PMN count is operator dependent which makes quality control difficult.7 Further, lysis of PMNs during transport to the laboratory may lead to false negative results.2,7 An early start of empirical antibiotic therapy is important for the successful treatment of SBP and has been shown to reduce mortality and improve survival.2

On the basis of these considerations, considerable efforts have been made in recent years to develop an alternative test for a more rapid diagnosis of SBP. This represents an interesting and promising area of investigation, which could determine the further optimization of SBP management and further improvement in its prognosis.5 Ideally such a test should be performed at the bedside at the time of paracentesis and should have a high sensitivity and a low false-positive rate. An important study done by Parsi et al2 evaluated the novel use of ascitic fluid lactoferrin as a biomarker for SBP and showed that measurement of ascitic fluid lactoferrin could provide a reliable biomarker for early diagnosis of SBP.

But studies evaluating the utility of lactoferrin for the diagnosis of SBP are few. Therefore, there is a need for more studies to validate the same. Hence, this study is being done to evaluate whether ascitic fluid lactoferrin can serve as useful markers for early diagnosis of SBP.

3 6.2: REVIEW OF LITERATURE

Ascites is the accumulation of excess fluid in the peritoneal cavity. Majority (75%) of patients who present with ascites have cirrhosis. Spontaneous bacterial peritonitis (SBP) is defined as an infection of initially sterile ascitic fluid (AF) without a detectable, surgically treatable source of infection.9 It is a frequent and severe complication of cirrhotic ascites, first described in the middle of the 1960s.10 Spontaneous infection of ascites is divided into subgroups:

(1) Spontaneous bacterial peritonitis (SBP) is defined as a positive bacterial finding in ascites, together with increased polymorphonuclear leukocytes in ascites (> 250 cells/mm3). Microorganisms responsible for SBP are isolated in 60%-70% of cases.

(2) Culture negative neutrocytic ascites (CNNA) - ascites is sterile, bacterial infection is not demonstrable by culturing; only an increased number of polymorphonuclear leukocytes above the limit of 250 cells/mm3 is revealed. It is necessary to eliminate other causes of increased leukocytes in ascites, e.g. previous antibiotic therapy, hepatocellular carcinoma, peritoneal carcinomatosis or tuberculosis, pancreatitis or bleeding into ascitic fluid.

(3) Monomicrobial non-neutrocytic bacterascites (or only bacterascites) has rarely been described. In this disorder, positive bacterial cultivation is present without increased leukocytes. It is usually seen in Child-Pugh class A patients. Recovery from bacterascites can be spontaneous (in 60%-80%), or it can develop into typical SBP.9

SBP and CNNA are identical, both from the clinical point of view and the therapeutic approach. Therefore, the consensus conference of the International Ascites Club3 has recommended not to differentiate between these two entities, as SBP and CNNA are identical both from the clinical point of view and the therapeutic approach.

4 Earlier, mostly retrospective, studies described SBP in about 8% of patients with ascites; later prospective trials revealed SBP in 10%-30% of patients with ascites admitted to hospital.5 SBP is found in about 5% of non-selected outpatients.5 Older studies reported 80%-100% mortality connected with SBP,5 which may partly be due to worse therapeutic possibilities in cirrhotic patients and lack of availability of effective antibiotics. But better results [20%-40% as reported in later studies4,5,11] are, to a certain extent, due to increased awareness, early diagnosis and treatment. High mortality is not primarily associated with the severity of the infection, and patients do not die of sepsis. An infection only worsens the changes present in cirrhotic patients, especially blood supply and renal function.5 But mortality is still very high.

In some of the Indian studies, Amarapurkar DN et al11 reported similar prevalence of SBP as 22% in hospitalized patients. The prevalence of SBP depends on severity of liver dysfunction, being higher in advanced liver disease. Jain et al,12 reported that the prevalence of SBP was 34.92% out of 63 patients. All patients who had SBP were in child Pugh class C. Puri AS et al,13 reported 21 out of 70 i.e.30% had SBP or its variants and 77% of the patients were in Pugh class C. All these studies are done in a tertiary care hospital where all facilities are available and most of the cases are reported. The problem could be much worse in a general set up.

The mean age at the diagnosis of SBP in Indian studies is found to be in the range of 39 – 44 years.14

Great variations in symptoms and signs presented by patients with SBP have been reported. Minhas et al,15 reported fever 54%, pain abdomen 57% and Hepatic encephalopathy 67%. In other study, Pelletier et al,16 found 89% of patients were having fever, UGI bleed (42%), pain abdomen (53%) and hepatic encephalopathy in 50% of cases. Completely asymptomatic cases have been reported between 14% to 100%.13

The clinical picture is non-specific. SBP is frequently manifested only by the occurrence or deepening of symptoms that accompany the course of liver cirrhosis -

5 increased ascites and failure of diuretic therapy, worsening encephalopathy, vomiting, etc.3 Therefore, an active search for the ascites infection is necessary. Diagnostic paracentesis is recommended in all patients with ascites admitted to hospital as well as in cirrhotics (whether in hospital or not) with worsened ascites.1 An active approach to the SBP diagnosis is extraordinarily important even from the prognostic point of view.9

After paracentesis, the ascitic fluid is investigated for the diagnosis of SBP. Spontaneous bacterial peritonitis is diagnosed when, (a) The ascitic fluid culture grows pathogenic bacteria OR (b) The ascitic fluid polymorpho nuclear (PMN) cell count ≥250 cells /mm3 AND (c) There is no evidence of surgically treatable intra-abdominal sources of infection.6 In clinical practice, however, a positive bacterial culture is obtained only in a minority of patients with SBP and culture may be negative in upto 60% of patients with SBP.2,3

SBP causes an inflammatory reaction resulting in the elevation of polymorphonuclear leukocyte (PMN) count in ascitic fluid, which represents evidence of failure of the first line of defense, i.e. the peritoneal macrophages, to kill invading bacteria.17 Guidelines currently available,1,3 suggest that the diagnosis of SBP should be based on PMN cell count in ascitic fluid. Hence, detection of PMN counts greater than 250 cells/mm3 is highly suggestive of SBP and is an indication for empirical antibiotic therapy. Studies have shown that this early start of empirical antibiotic therapy has reduced the mortality and improved the survival in SBP patients.1,2

Unfortunately, in clinical practice, ascitic fluid PMN cell count is not always possible within few hours, thus causing an unacceptable delay in diagnosis and treatment of this potentially lethal infection.5 On the basis of these considerations, considerable effort has been made in the recent years to develop an alternative test for a more rapid diagnosis of SBP. Various parameters of ascitic fluid such as total protein, pH, LDH, albumin, total cholesterol, lactate, glucose, leucocyte esterase and lactoferrin were evaluated for immediate diagnosis of SBP.

6 Studies done by Norman Gitlin et al18 and W Stassen et al19 showed that ascitic fluid pH estimation was the best and simplest test for the immediate diagnosis of infected ascitis. Golam Mustafa et al20 concluded that ascitic fluid total protein level is significantly reduced in patients with ascites who developed SBP. The urinary reagent strips have been recently proposed for rapid diagnosis of SBP.2 The urinary strips identify leukocytes by detecting their esterase activity via colorimetric reaction.5 However, a large multicenter study suggested a lack of sensitivity of strip tests for diagnosis of SBP and indicated an absence of diagnostic efficacy for this test.21

Lactoferrin is an iron-binding protein that is found mainly in the external secretions such as breast milk and in PMN leucocytes. Lactoferrin is synthesized during the transition of neutrophils from promyelocyte to myelocyte and stored in the secondary granules. When neutrophils come in contact with foreign antigens, they degranulate and release lactoferrin and other antibacterial peptides. The bactericidal effect of lactoferrin is either by sequestering free iron or by the effects of lactoferricin, an antibacterial peptide generated by proteolytic cleavage of lactoferrin.2 Thus the presence of lactoferrin in body fluids is proportional to the flux of PMN leucocytes.22 It has been shown that fecal lactoferrin estimation can be used to distinguish inflammatory from non-inflammatory conditions of GIT with high sensitivity and specificity.23 Lactoferrin in ascitic fluid is also remarkably stable and resistant to degradation when left at room temperature for extended periods of time.24 These properties make this marker attractive for clinical use. The study by Parsi et al2 showed that asctic fluid lactoferrin level >= 242 ng/dl had a sensitivity of 96% and specificity of 98% to identify a PMN count > 250/mm3. This shows that measurement of ascitic fluid lactoferrin could provide a reliable biomarker for early diagnosis of SBP.

The pH estimation of pleural fluid has diagnostic value in differentiating exudates and transudates, which is widely used in the evaluation and management of pleural effusions.18 Ascitic fluid pH of SBP patients was significantly lower than ascitic fluid pH of patients with sterile ascitis.18,19,25 Also estimation of pH is easy , rapid and highly sensitive for diagnosis of SBP.18 The pH of ascitic fluid is the best and simplest

7 single test for the diagnosis of infected ascitic fluid.19 Studies from France, California and Cleveland18,19,25 have shown that the ascitic fluid pH of less than 7.31 is diagnostic of SBP. The total protein concentration in ascitic fluid correlates directly with the risk of developing SBP in cirrhotic patients.17 It is also been shown recently that the ascitic fluid total protein level in patients who developed SBP (1.1gm +/- 0.3) is significantly lower than those without SBP (1.5+/- 0.5).20 Ascitic fluid protein estimation is an easy biochemical test and can be done in any standard laboratory.20

It is therefore important to conduct further studies particularly with regard to ascitic fluid lactoferrin estimation in patients with cirrhosis. This will help in the early diagnosis and initiation of treatment of SBP and thereby minimize morbidity and mortality of SBP patients.

8 6.3 : OBJECTIVES OF THE STUDY.

1. To estimate the lactoferrin concentration, pH and total protein concentration in ascitic fluid of patients with SBP and without SBP.

2. To compare these three parameters in SBP and non-SBP group of patients with ascites.

3. To correlate these three parameters with PMN cell count for their usefulness in the diagnosis of SBP.

7. MATERIALS AND METHODS.

7.1: Source of data:

(1) The study will be conducted on the cirrhotic patients with ascites admitted in the Gastroenterology ward of St John’s hospital, Bangalore.

(2) Operational definitions  Cases: The cases are defined as cirrhotic patients with ascites > 18 years of age with PMN count > 250 cells/mm3.

 Controls: The controls are defined as cirrhotic patients with ascites > 18 years of age with a PMN count < 250 cells/mm3.

9 (3) Subjects who satisfy the inclusion criteria and give consent will be included in the study.

(a) For cases:

Inclusion criteria: 1. Age: > 18 years. 2. Patients with cirrhosis diagnosed clinically, radiologically or histologically. 3. Presence of ascites with PMN cell count > 250cells/mm3. 4. Not on any antibiotic treatment on admission.

Exclusion criteria: 1. Age: < 18 years. 2. Undergone abdominal surgery within 3 months of study entry. 3. Presence of other causes of neutrocytic ascites such as pancreatitis, appendicitis, TB, peritoneal carcinomatosis, haemorrhagic ascites. 4. On antibiotic therapy during current admission.

(b) For controls:

Inclusion criteria: 1. Age : > 18 years 2. Patients with cirrhosis diagnosed clinically, radiologically or histologically.

3. Presence of ascites with pmn count < 250cells/mm3. 4. Not on any antibiotic treatment on admission.

10 Exclusion criteria :

1. Age : < 18 years. 2. Undergone abdominal surgery within 3 months of study entry. 3. Presence of other causes of neutrocytic ascites such as pancreatitis, appendicitis, TB, peritoneal carcinomatosis, haemorrhagic ascites. 4. On antibiotic therapy during current admission.

7.2: Method of collection of data.

7.2 a: Method of sample collection

All the cirrhotic patients with ascitis admitted in the Gastroenterology ward of St. John’s hospital between November 2010 – March 2012 who fit into the criteria mentioned above will be included in this study after obtaining an informed written consent. The following data will be collected from the case record form. 1) Demography- age, sex, weight, height, body mass index. 2) Clinical history, examination and diagnosis. 3) Relevant investigations. 4) Ascitic fluid analysis including total and differential leucocyte count.

All patients included in the study will have to undergo a diagnostic paracentesis under aseptic precautions. The ascitic fluid will be collected for the study in empty plain vacutainers. The pH of the sample will be measured immediately. The sample will be centrifuged and cell free supernatant will be aliquoted and stored at –200C until assayed.

11 7.2 b: Sample size :

Thirty cirrhotic ascitic patients with SBP and thirty cirrhotic ascitic patients without SBP admitted in the Gastroenterology ward of St Johns hospital, Bangalore during the period from November 2010 – March 2012.

7.2 c: Type of study : Cross-sectional study.

7.2 d: Statistical analysis:

Data collected will be will be analyzed for any significance in variation In the values among the biochemical parameters studied using,

1. Student ‘t’ test. 2. ANOVA analysis 3. Chi square test 4. SPSS (16.0) Software will be used for statistical analysis.

7.3 Does this study require any investigations or interventions to be conducted on patients or other human or animals? If so, please describe briefly.

The study requires the following investigations to be done on humans: A) ASCITIC FLUID LACTOFERRIN LEVEL. B) ASCITIC FLUID pH ESTIMATION. C) ASCITIC FLUID TOTAL PROTEIN ESTIMATION.

12 (A) ASCITIC FLUID LACTOFERRIN LEVEL.

LACTOFERRIN (LTF) is estimated by enzyme-linked immunosorbent assay (ELISA) method. Lactoferrin is captured by a monoclonal antibody (Mab) that is coated on wells of a sectional microplate. A second LTF- Mab labeled with biotin is added to the well and binds with the captured LTF forming a “sandwich”. A solution of streptavidin-peroxidase is then added. Streptavidin has a high affinity for biotin and once bound, its peroxidases label is available for colour development by addition of substrate, o- phenylenediamine. This colour development is proportional to the quantity of LTF in sample.

(B) ASCITIC FLUID pH ESTIMATION.

The pH of the ascitic fluid sample is measured using ELICO LI613 pH METER. The glass electrode of the ph meter is dipped into the ascitic fluid sample which causes the flow of only hydrogen ions across the thin glass membrane. This generates a potential difference between two sides of the glass membrane depending on the hydrogen ion concentration (pH) of the test solution. This potential difference is used to calculate the ph by the potentiometer.

(C) ASCITIC FLUID TOTAL PROTEIN ESTIMATION.

Total Protein estimation by Folin-Ciocalteu (Lowry) method in ascitic fluid:

When Biuret reagent is added to protein solution, the peptide bonds react with Cu2+ ions in alkaline solutions to form a violet colored compound. Intensity of the colour produced is proportional to the number of peptide bonds and to the concentration of protein When Folin-Ciocalteu reagent is added to a protein solution, the amino acids, tyrosine and tryptophan present in the protein molecules reduce phosphotungstic - phosphomolybdic acid (Folin-Ciocalteu) reagent to give a blue colour. The intensity of the colour is proportional to the number of tyrosine and tryptophan and to the concentration of protein.

7.4 Has ethical clearance been obtained from your institution?

Yes (consent form enclosed).

8. REFERENCES.

14 1. Runyon BA. Management of adult patients with ascitis due to cirrhosis. Hepatology 2004;39:841-56.

2. Parsi MA, Saadeh SN, Zein NN, Davis GL, Lopez R, Boone J, et al. Ascitic fluid lactoferrin for diagnosis of spontaneous bacterial peritonitis. Gastroenterology 2008;135:803-807.

3. Rimola A, García-Tsao G, Navasa M, Piddock LJ, Planas R, Bernard B, et al. Diagnosis, treatment and prophylaxis of spontaneous bacterial peritonitis: a consensus document. International Ascites Club. J Hepatol 2000;32:142-153.

4. Thuluvath PJ, Morss S, Thompson R. Spontaneous bacterial peritonitis-in-hospital mortality, predictors of survival, and health care costs from 1988 to 1998. Am J Gastroenterol 2001;96:1232-6.

5. Riggio O, Angeloni S. Ascitic fluid analysis for the diagnosis and monitoring of spontaneous bacterial peritonitis. World J Gastroenterol 2009;15(31):3845-3850.

6. Syed VA, Ansari JA, Karki P, Regmi M, Khanal B. Spontaneous bacterial peritonitis (SBP) in cirrhotic ascites: A prospective study in a tertiary care hospital, Nepal. Kathmandu University Medical Journal (2007),Vol. 5,No. 1,Issue 17,48-59.

7. Runyon BA. Stripes and tubes: improving the diagnosis of spontaneous bacterial peritonitis. Hepatology 2003;37:745-747.

8. Parsi MA, Manteuffel L, Zein NN. Ascitic fluid lactoferrin: a novel marker for rapid diagnosis of spontaneous bacterial peritonitis. Gastroenterology 2004;126:A386.

9. Lata J, Stiburek O, Kapocova M. Spontaneous bacterial peritonitis: A severe complication of liver cirrhosis. World J Gastroenterol 2009;5(44):505-5510.

10. Conn HO. Spontaneous peritonitis and bacteremia in Laennec's cirrhosis caused by enteric organisms. A relatively common but rarely recognized syndrome. Ann Intern Med 1964;60:68- 580.

11. Amarapurkar DN, Viswanathan N, Parikh SS, Kalro RH, Desai HG. Prevalence of Spontaneous Bacterial Peritonitis. J Assoc Physician India 1992; 0(4):236−238

12. Jain AP, Chandra LS, Gupta S, et al. Spontaneous Bacterial Peritonitis in liver cirrhosis with ascites. J Assoc Physicians India 1999;47 (6):619-621.

13. Puri AS, Puri J, Ghoshal UC. Frequency, microbial spectrum & outcome of Spontaneous Bacterial Peritonitis. Indian J Gastroenterol 1996;15(3):86-89.

15 14. Bhatnagar MK, Rawat N. To assess the role of serial ascitic fluid cell count in the treatment of Spontaneous Bacterial Peritonitis. J Assoc physicians India 2006:53:350-352.

15. Mihas AA, Toussaint J, Hsu HS, Dotherow P, Achord JL. Spontaneous bacterial peritonitis in cirrhosis: clinical and laboratory features, survival and prognostic indicators. Hepatogastroenterology. 1992 Dec;39(6):520-522.

16. Pelletier G, Lesur G, Ink O, Hagege H, Attali P,Buffet C, et al. Asymptomatic bacterascites: is it spontaneous bacterial peritonitis? Hepatology 1991;14:112-115.

17. Arroyo V, Navasa M. Ascites and spontaneous bacterial peritonitis. In : Sciff ER, Sprrel MF, Maddrey (eds) Sciff’s diseases of the liver, 10th edn. Philadelphia: Lippincott Williams and Wilkins; 2007.p.527-568.

18. Gitlin N, Stauffer JL, Silvestri RC. The ph of ascitic fluid in the diagnosis of spontaneous bacterial peritonitis in alcoholic cirrhosis. Hepatology 1982;2:408-411.

19. Stassen W, Bacon BR, McCullough AJ, Gutnik S, Kalhan S, Tavill AS, Superior diagnostic value of ascitic fluid ph and lactate levels as immediate indicators of infected ascites. Hepatology 1984;4:A1341.

20. Mustafa GM et al. Study on ascitic fluid protein level in cirrhotic patients with spontaneous bacterial peritonitis. Bangladesh Med Res Counc Bull 2009;35:41-43.

21. Nousbaum JB, Cadranel JF, Nahon P, Khac EN, Moreau R, Thevenot T,et al. Diagnostic accuracy of the multistix 8 SG reagent strip in diagnosis of spontaneous bacterial peritonitis. Hepatology 2007;45:1275-1281.

22. Martins CA, Fonteles MG, Barrett LJ. Correlation of lactoferrin with neutrophilic inflammation in body fluids. Clin Diag Lab Immunol 1995;16:417-419.

23. Parsi MA, Shen B, Achkar JP. fecal lactoferrin for diagnosis of symptomatic patients with ileal pouch-anal anastomosis. Gastroenterology 2004;126:1280-1286.

24. Kayazawa M, Saitoh O, Kojima K. lactoferrin in whole gut lavage fluid as a marker for disease activity in inflammatory bowel disease: comparison with other neutrophil-derived products. Am J Gastroenterol 2002;97:360-369.

25. Attali P, Turner K, Pelletier G, Ink O, Etienne JP, Ph of ascetic fluid: diagnostic and prognostic value in cirrhotic and non-cirrhotic patients. Gastroenterology 1986;90:1255-1260.

9. Signature of the candidate :

16 10. Remarks of the guide :

11. Name and designation of

11.1 Guide : DR. ANITA . R. BIJOOR MD, PhD. PROFESSOR, DEPARTMENT OF BIOCHEMISTRY, ST. JOHN’S MEDICAL COLLEGE, BANGALORE.

11.2 Signature :

11.3 Co-guide : DR. HARSHAD DEVARBHAVI MD, DNB, DM, DNB. PROFESSOR AND HEAD, DEPARTMENT OF GASTROENTEROLOGY, ST. JOHN’S HOSPITAL, BANGALORE.

11.4 Signature :

11.4 Head of the : DR. SULTANA FURRUQH Department MD. PROFESSOR AND HEAD, DEPARTMENT OF BIOCHEMISTRY ST. JOHN’S MEDICAL COLLEGE, BANGALORE.

11.5 Signature :

12.1 Remarks of the chairman and principal :

12.2 Signature:

PATIENT INFORMATION AND CONSENT FORM

STUDY OF USE OF ASCITIC FLUID LACTOFERRIN, pH AND TOTAL PROTEIN IN THE SCREENING OF SPONTANEOUS BACTERIAL PERITONITIS

Investigator : DR. RAVI KUMAR. R

Institutional address : St. John’s Medical College and Hospital, Sarjapur road, Koramangala, Bangalore- 560034.

Introduction:

You are requested to take part in a research study of lactoferrin, pH and total protein in cirrhosis. Lactoferrin, pH and total protein levels will be assessed in your ascitic fluid sample as a part of the study. Before agreeing to participate in this study, you will receive oral and written information about this study. Your participation in this study will be in the form of giving your ascitic fluid sample during the study period.

Purpose of the study:

The purpose of this study is to assess the levels of lactoferrin, pH and total protein in cirrhotic patients with ascitis with SBP and compare it to the non-SBP state. This will help in the identification of lactoferrin as an early marker of SBP. Subsequently early treatment may be initiated to reduce mortality and morbidity.

Study procedures:

If you choose to participate in the study, your ascitic fluid sample will be collected for lactoferrin, pH and total protein estimation.

Risks:

For most people, PARACENTESIS for drawing ascitic fluid does not cause any serious problems. Rarely, it may cause bleeding, bruising, discomfort, infections, and/or pain at the needle site or dizziness or injury of any organ. However, this will be done under aseptic precaution under guidance.

Participation in the study:

Your taking part in the study is entirely voluntary. You may refuse to take part in the study.

Questions:

If you have any questions regarding this study or your rights, you can contact Dr.Ravi Kumar.R, Department of Biochemistry, St. John’s Medical College, Sarjapur road, Koramangala, Bangalore-560034. Ph: 080-22065050.

Confidentiality:

The study doctor and staff will handle your personal health information in a confidential manner. Your health information will be used and disclosed in accordance with the Data Privacy Statement. - The study doctor and staff will use your medical records and information created or collected during the study to conduct the study. - The data will be used for research purposes to support the scientific objectives of this study, for better understanding of the disease included in the study, or to improve the design of future studies. - Study data that does not identify you may be published in medical journals or shared with others as part of scientific discussions. - Your medical records may be held and processed on computers.

You have the right to see and copy your personal health information related to the research study. However, to ensure scientific integrity of the study, you will not be able to review some of the study information until after the study has been completed.

You may cancel your authorization at any time by providing written notice to the study doctor.

20 PATIENT INFORMATION AND CONSENT FORM

ATTACHMENT 2 SIGNATURE PAGE

To become part of this study and to authorize use and disclosure of your personal health information, you or your legal representative must sign and date this page.

By signing this page, you are conforming the following:

1. You have read all of the information in this Patient Information and Consent form, and you have had time to think of it. 2. All of your questions have been answered to your satisfaction. 3. You voluntarily agree to be a part of this research study. 4. You may refuse to participate or freely choose to stop being a part of this study at any time. 5. You allow the study doctor to use and disclose your personal health information as described in this document. 6. You agree that your sample can be used for any other studies.

------Signature of the patient (dd/mm/my)

------Patient name Patient Initials and Number

------Signature of Principal investigator (dd/mm/yy)

------Name of Principal investigator

I hereby state that the study procedures were explained in detail and all questions were fully and clearly answered to the above-mentioned participant.

------Name of Individual Conducting Informed Consent

------Signature of Individual Conducting Informed Consent (dd/mm/yy)