Diagnosis of Spontaneous Bacterial Peritonitis by Identification of 16s rrna Genes in Liver Cirrhosis Patients

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1 Med. J. Cairo Univ., Vol. 83, No. 2, December: , Diagnosis of Spontaneous Bacterial Peritonitis by Identification of 16s rrna Genes in Liver Cirrhosis Patients MONA A. AMIN, M.D.; AHMED E. EL-BADRY, M.D.; DINA SABRY, M.D.* and AHMED YOUSSEF, M.Sc. The Departments of Internal Medicine and Medical Biochemistry*, Faculty of Medicine, Cairo University 209 Introduction SPONTANEOUS bacterial peritonitis (SBP) is a common and severe complication in patients with cirrhosis and ascites, occurring in 10-25% of patients, with in-hospital mortality rate ranging from 20 to 40% [1,2]. Early bacterial detection and characterization would allow targeted intervention to prevent the development of SBP, or where peritonitis is already present, treatment with the most appropriate therapy. Culture-based analysis of ascitic fluid has shown that a wide range of bacterial species can be isolated. Despite this, even where the most modern practice is applied, ascites fluid culture is negative in approximately 20% of patients with clinical manifestations suggestive of SBP, including elevated ascites polymorphonuclear leukocyte (PMN) counts. Where there is evidence of infection, delaying treatment until the ascitic fluid culture grows bacteria may result in the death of the patient from overwhelming infection [3]. Therefore, empirical antibiotic treatment for SBP is initiated when objective evidence of a local inflammatory reaction is present without prior knowledge of the causative organisms or their antibiotic susceptibility [4]. In contrast to culture-based methodologies, the rapid and accurate identification of bacteria in clinical samples using molecular, culture independent approaches has led to the rapid proliferation of the use of such strategies. Their particular strength is in their ability to detect bacterial species, regardless of whether these species can be grown readily in vitro [5,6]. In the study presented here, we investigated the potential of culture-independent strategy to

2 210 Diagnosis of Spontaneous Bacterial Peritonitis by Identification characterize the bacterial content of ascites fluid. Our strategy is the confirmation of species identities through 16S ribosomal gene clone sequence analysis. Patients and Methods Between October 2013 and June 2014, we prospectively enrolled 100 consecutive patients with liver cirrhosis and ascites diagnosed as SBP or clinically suspected at Internal Medicine Department Kasr El-Aini Hospital. Cirrhosis was diagnosed using physical examination, standard laboratory and radiological findings. SBP diagnosis was established by clinical manifestations and ascitic fluid neutrophil count is 250/mm 3 with no evidence of an intraabdominal source of infection. After approval of our faculty ethical committee informed consent was obtained from each participant or a responsible family member after fully explaining the possible complications of the diagnostic procedures. All patients were subjected to thorough history and clinical evaluation with special emphasis on conscious level, temperature, abdominal pain, tenderness & previous history of SBP. Laboratory investigations were done in form of: Complete blood count (CBC), serum bilirubin, AST, ALT, prothrombin time and concentration, serum albumin, Creatinine and BUN. Paracentesis performed under aseptic conditions. Ascitic fluid samples examined for total leucocytic count (TLC) and polymorphnuclear leucocytes (PMN), culture and sensitivity of ascitic fluid. 16s rrna genes by polymerase chain reaction quantitative method in ascitic fluid. Inclusion criteria were patients with liver cirrhosis and ascites with following criteria: 1- Patients with SBP diagnosed by clinical manifestations and PMN cell count in ascitic fluid is more than 250 cells/mm Patients with culture-negative nonneutrocytic ascites (CNNNA) (ascitic fluid neutrophil count <250/mm 3 and negative culture) with manifestation suggestive of SBP. 3- Sudden deterioration of general condition in cirrhotic patients. The exclusion criteria included the following: 1- Patients with an alternative cause for increased ascitic fluid neutrophil count (i.e. bloody ascites, pancreatitis or tuberculosis). 2- Upper gastrointestinal bleeding or intake of antibiotics in the preceding 2 weeks before study. 3- Hepatocellular carcinoma, or portal vein thrombosis and transjugular intrahepatic portosystemic shunt (TIPS). Statistical analysis: Pre-coded data was entered on the computer using "Microsoft Office Excel Software" program (2010) for windows. Data was then transferred to the Statistical Package of Social Science Software program, version 21 (SPSS) to be statistically analyzed. Receiver operating characteristics (ROC) curve analysis was conducted to explore the discriminant ability of Gene expression 16s RRNA in predicting positive ascetic cultures. p-values less than 0.05 were considered statistically significant, and less than 0.01 were considered highly significant [7]. Results From the 100 patients we studied, 77 patients were confirmed SBP (clinical symptoms, signs and ascitic fluid neutrophil count is 250/mm 3 ) and 23 patients were clinically suspected SBP (clinical symptoms, signs or Sudden deterioration of general condition with ascitic fluid neutrophil count <250/mm 3 ) (Fig. 1). Ascitic fluid culture was positive only in 4 patients and 96 patient had negative cultures. Gene expression 16s RRNA in ascitic fluid was positive in all patients with a mean level (3.3±3.8). All the studied cases had positive 16s rrna gene expression (100%) and its levels were not correlated with any of the laboratory parameters (p-value >0.05), its levels were only correlated with results of ascitic fluid culture where 16s rrna level was significantly higher in culture positive cases when compared to those with culture negative cases (p-value = 0.002). (Table 1), (Fig. 2). In predicting positive cultures area under the curve for 16s RRNA gene expression [0.909, 95% confidence interval (CI) ]. At a 16s rrna cut-off value of 5.1 the test achieved a a PPV of 22.2%, sensitivity 100%, specificity 85.4%, NPV 100% and total accuracy of 84% (Table 2), (Fig. 3). According to the results of ascitic fluid culture and sensitivity the patients were divided into 2 groups culture positive (no=4) and culture negative (no=96). There was no statistically significant difference in the different laboratory parameters between those with culture positive and those with culture negative results (p-value >0.05). The only difference between both groups was the level of 16s rrna which was higher in culture positive

3 Mona A. Amin, et al. 211 when compared to those with culture negative (pvalue = 0.002) (Table 3) (Fig. 2). According to the results of ascitic fluid polymorphnuclear leucocytes (PMN) the patients were divided into 2 groups proved SBP (PMN 250/ mm 3 ) and clinically suspected SBP (PMN <250/ mm 3 ). There was no statistically significant difference in different laboratory parameters between those proved SBP and those suspected SBP (pvalue >0.05), the only difference between both groups was in ascitic total leucocytic count which was higher in those with proved SBP compared to those suspected SBP (p-value <0.001) (Tables 4,5). Suspected SBP 23% SBP Suspected SBP Confirmed SBP 77% Fig. (1): Percentage of patients diagnosed as SBP and those clinically suspected. 1.0 Gene expression 16s RRNA 9.1 Table (1): Correlation between the relative gene expression 16s RRNA and the other laboratory parameters. Tested variable Gene expression 16s RRNA Relative gene expression 16s RRNA r p-value Hb (g/dl) WBC (/µl) Plt (10 9 /L) PC INR AST (IU/L) ALT (IU/L) Albumin (g/dl) Urea (mg/dl) Creatinine (mg/dl) Total Bil (mg/dl) Direct Bil (mg/dl) TLC (ascitic) PMN (ascitic) Table (2): Area under the curve & cut off point for 16s RRNA gene expression. AUC 95% CI p-value Tested variable Gene expression 16s RRNA Cut off point Specificity Sensitivity PPV NPV Accuracy % 85.4% 22.2% 100.0% 84.0% 0.0 +VE Culture VE Fig. (2): Relation between the relative gene expression 16s RRNA and culture results. Sensitivity ROC Curve Specificity Fig. (3): ROC curve analysis to explore the discriminant ability of Gene expression 16s RRNA in predicting +VE cultures. Table (3): Comparison between laboratory parameters according to culture results. +VE (n=4) Culture VE (n=96) p value Hb (g/dl) 9.10± ± WBC (/µl) 6.00± ± Plt (10 9 /L) ± ± PC 51.50± ± INR 2.05± ± AST (IU/L) 55.75± ± ALT (IU/L) 63.75± ± Albumin (g/dl) 2.13± ± Urea (mg/dl) 39.75± ± Creatinine (mg/dl) 1.15± ± Total Bil (mg/dl) 1.53± ± Direct Bil (mg/dl) 1.10± ± TLC (ascitic) ± ± PMN (ascitis) ± ±

4 212 Diagnosis of Spontaneous Bacterial Peritonitis by Identification Table (4): Comparison of laboratory parameters according to ascitic fluid PMN leucocytic count. PMN Clinically suspected SBP <250 (n=23) Proved SBP 250 ( n=77) p value Hb (g/dl) 8.57± ± WBC (/µl) 7.20± ± Plt (10 9/L) ± ± PC 51.00± ± INR 1.97± ± AST (IU/L) 37.52± ± ALT (IU/L) 39.70± ± Albumin (g/dl) 2.17± ± Urea (mg/dl) 35.83± ± creatinine (mg/dl) 1.03± ± Total Bil (mg/dl) 1.30± ± Direct Bil (mg/dl) 0.80± ± TLC (ascitic) ± ± <0.001 Gene expression 3.38± ± s RRNA Table (5): Comparison of ascitic fluid culture results according to ascitic fluid PMN leucocytic count. PMN C&S Clinically suspected Proved SBP p (ascitic) SBP <250 (n=23) 250 ( n=77) value N % N % +VE VE NS Discussion Bacterial identification by sequencing of the 16S ribosomal RNA (rrna) gene is a universal bacterial identification method. Bacterial taxonomists have used this technology for a number of years as a measure of DNA similarity between isolates. More recently 16S rrna gene amplification and sequencing has been used to detect and identify fastidious bacterial pathogens [8]. In our study which was done on 100 patients presented with symptoms such as low grade fever, rapidly accumulating ascites, unexplained deterioration of general condition and some of them had abdominal pain. They were admitted to the hospital suspected of having SBP and diagnostic paracentesis was done for all patients. PMN was found <250 cells/mm 3 in 23 patients and 250 c ells/mm 3 in 77 patients. Ascitic fluid cultures were found negative in 96 patient and positive only in 4 patients accordingly 23 patients were diagnosed as culturenegative non-neutrocytic ascites (ascitic fluid neutrophil count <250/mm 3 and negative culture) (CNNNA). 16s rrna gene expression in ascitic fluid was positive in all 100 patients suspected to have SBP even in CNNNA, culture negative or patients had PMN <250 cells/mm 3. There was no correlation between 16s rrna levels and the different laboratory parameters of our cases, it is only correlated with the results of ascitic fluid culture where its levels was higher in culture positive compared to those with culture negative. According to the results of ascitic fluid culture and sensitivity the patients were divided into 2 groups culture positive (no=4) and culture negative (no=96) with no significant difference between both groups in all laboratory parameters except 16s rrna levels. According to the results of ascitic fluid polymorphnuclear leucocytes (PMN) the patients were divided into 2 groups proved SBP (PMN 250/mm 3 ) and clinically suspected SBP (PMN <250/mm 3 ) with no significant difference in the laboratory parameters between both groups except for ascitic fluid TLC. Upon clinical suspicion of SBP diagnostic paracentesis was done and start Cefotaxime IV 2gm every 8 hours for 5 days. Our patients actually had clinical improvement 2-3 days after starting the antibiotic. We initiate empiric therapy as soon as possible to maximize the patient's chance of survival. These recommendations are consistent with a 2009 guideline issued by the American Association for the Study of Liver Diseases (AASLD). Accordingly 23% of patients CNNNA and 96% of patients with negative ascitic fluid cultures had already pathogenic bacteria in ascitic fluid causing SBP but undiagnosed. So ascitic fluid 16s RRNA gene expression can detect ascitic fluid pathogenic bacteria and SBP in CNNNA and culture negative SBP patients which represent large percentage of patients we meet in our daily practice. Previous studies reported the culture-positive rate of SBP ascites to be high, namely ranging between 72% and 90% of cases [9,10]. On the other hand, the rate of culture-positive cases was obviously low in our study despite the fact that ascitic fluid cultures were performed by the standard (culture-bottle) method. However, this low culturepositive rate of SBP ascites has been also observed in several reports, with a proportion between 39% and 59% and they suggested that the low rate probably depended on an earlier diagnosis of the infection [11,12].

5 Mona A. Amin, et al. 213 Our results are in accordance with the conclusion of Zapater et al. [13] and Bruns et al. [14] where both studies agree that identification of ascitic Bacterial DNA is an appropriate alternative to bacterial ascites culture for pathogen identification in patients at risk for SBP. Mohamed et al. [15] concluded that identification of bacterial DNA in serum and ascitic fluid has its significant clinical and diagnostic relevance in suspicion of SBP and its prompt therapy especially in CNNNA. It might be an alternative diagnostic tool to ascitic bacterial culture in early diagnosis and prompt treatment with subsequent improvement of survival which needs further investigations. Here, we propose that it is more likely lowlevel colonization exist in the absence of a neutrophil response. The current use of the ascitic PMN to base clinical decisions with help from occasional positive cultures may therefore be a vast oversimplification with the data from this initial study suggesting that this needs to be revised subsequently. Conclusion: Identification of bacterial 16s RRNA in ascitic fluid has its significant clinical and diagnostic relevance in suspicion of SBP and its prompt therapy especially in CNNNA. It might be an alternative diagnostic tool to ascitic fluid bacterial culture and PMN in early diagnosis and prompt treatment with subsequent improvement of survival. Recommendations: From our study we recommend Reassessment of ascitic 16s rrna in patients with ascites due to other causes (not liver cirrhosis), Trying to add ascitic fluid 16s rrna gene expression to the diagnostic criteria of SBP in patient with liver cirrhosis. References 1- THULUVATH P.J., MORSS S. and THOMPSON R.: Spontaneous bacterial peritonitis in-hospital mortality, predictors of survival, and health care costs from 1988 to Am. J. Gastroenterol., 96: , PARK Y.H., LEE H.C., SONG H.G., JUNG S., RYU S.H., SHIN J.W., CHUNG Y.H., LEE Y.S. and SUH D.J.: Recent increase in antibiotic-resistant microorganisms in patients with spontaneous bacterial peritonitis adversely affects the clinical outcome in Korea. J. Gastroenterol. Hepatol., 18: , RUNYON B.A.: AASLD Practice Guidelines Committee Management of adult patients with ascites due to cirrhosis: an update. Hepatology, 49: , RIMOLA A., GARCÍA-TSAO G., NAVASA M., PID- DOCK L.J., PLANAS R., BERNARD B. and INADOMI J.M.: Diagnosis, treatment and prophylaxis of spontaneous bacterial peritonitis: A consensus document. International Ascites Club. J. Hepatol., 32: , WHELEN A.C. and PERSING D.H.: The role of nucleic acid amplification and detection in the clinical microbiology laboratory. Annu. Rev. Microbiol., 50: , ESPY M.J., UHL J.R., SLOAN L.M., BUCKWALTER S.P., JONES M.F., VETTER E.A., YAO J.D., WENGEN- ACK N.L., ROSENBLATT J.E., COCKERILL F.R. 3 rd and SMITH T.F.: Real-time PCR in clinical microbiology: Applications for routine laboratory testing. Clin. Microbiol. Rev., 19: , DAWSON B. and TRAPP R.G.: Basic and clinical biostatistics; Mcgraw-Hill Inc. Third edition., RELMAN D.A., SCHMIDT T.M., MacDERMOTT R.P. and FALKOW S.: Identification of the uncultured bacillus of Whipple s disease. N. Engl. J. Med., 327: , MOORE K.P. and AITHAL G.P.: Guidelines on the management of ascites in cirrhosis.gut, 55: vi1-vi12, RUNYON B.A., HOEFS J.C. and MORGAN T.R.: Ascitic fluid analysis in malignancy-related ascites. Hepatology, 8: 1104, GINES P., ANGELI P., LENZ K., et al.: EASL clinical practice guidelines on the management of ascites, spontaneous bacterial peritonitis, and hepatorenal syndrome in cirrhosis. J. Hepatol., 53: , FERNANDEZ J., NAVASA M., GOMEZ J., COLMEN- ERO J., VILA J., ARROYO V., et al.: Bacterial infections in cirrhosis: Epidemiological changes with invasive procedures and norfloxacin prophylaxis. Hepatology, 35: , ZAPATER P., FRANCÉS R., GONZÁLEZ-NAVAJAS J.M., de la HOZ M.A., MOREU R., PASCUAL S., et al.: Serum and ascitic fluid bacterial DNA: A new independent prognostic factor in noninfected patients with cirrhosis. Hepatology, 48 (6): , BRUNS T., SACHSE S., STRAUBE E., ASSEFA S., HERRMANN A., HAGEL S., LEHMANN M. and STALLMACH A.: Identification of bacterial DNA in neutrocytic and non-neutrocytic cirrhotic ascites by means of a multiplex polymerase chain reaction. Liver Int., 29 (8): , MOHAMED A. RADY, NASHWA SHEBLE, MONA M. HASSOUNA, MONA A. EL SHAFIE, GHADA R. EL HENDAWY and DOA ZKARIA: Significance of Serum and Ascitic Fluid Bacterial DNA in Culture Negative Non-Neutrocytic Ascites. Journal of American Science, 8 (2): , 2012.

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