Ascitic infection is a frequent complication in patients with portal hypertension secondary to chronic liver disease, especially during advanced stages or extrahepatic causes such as Budd-Chiari syndrome. This infection seems to result from several mechanisms associated with portal hypertension and liver dysfunction (1–3). It is not known how intestinal bacteria reach the ascitic fluid; this phenomenon has been attributed to the translocation of viable bacteria from mesenteric lymph nodes to blood and then to ascitic fluid (2,4). Ascitic infection is associated with great morbidity and mortality (5,6) and therefore requires prompt diagnosis and treatment.
Two types of ascitic infection are identified in the absence of secondary causes such as bowel perforation or focal inflammatory processes: bacterascites (BA) and spontaneous bacterial peritonitis (SBP). The former usually indicates bacterial colonization and is diagnosed in the presence of a positive ascites culture without inflammatory peritoneal response (7). Conversely, SBP is diagnosed on basis of polymorphonuclear (PMN) cell counts greater than 250 cells/μL in ascitic fluid, regardless the result of the ascites culture (7). The low rates of positivity of bacterial culture in ascites samples is possibly caused by the relatively small concentration of bacteria in ascitic fluid, such as occurs in cerebrospinal fluid (7,8). Runyon et al have demonstrated that inoculation of ascites directly into blood culture bottles at the bedside increases the sensitivity of bacterial culture to 90% (9). However, in general practice, ascites culture is negative in approximately 40% of patients with SBP (10–12).
Polymerase chain reaction (PCR) is a molecular diagnostic technique originally described by Mullis et al that is based on the enzymatic amplification of a specific sequence of DNA with production of millions of copies (13); thus, this technique has the potential increase for sensitivity (14–16). The use of universal PCR primers targeting conserved DNA regions in bacteria, such as 16S ribosomal RNA (rRNA) sequences, is a promising means for identifying bacteria in clinical samples (16–20). We have decided to use a universal primer because, although there is a high prevalence of enteric bacteria causing ascites infection in adults, the prevalence of bacteria causing ascites infection in children is not well established. The main potential applications of PCR include the detection of microorganisms that are not identified by conventional methods, such as slow-growing bacteria and fastidious bacterial pathogens, as well as the detection of bacteria in low concentrations (14,15,21).
To our knowledge, only 1 study so far (22) has focused on the amplification of 16S rRNA to investigate the presence of bacteria in ascitic fluid. The authors found bacterial DNA in 9 of 28 asymptomatic patients with culture-negative, non-neutrocytic ascites (CNNNA). Therefore, the aim of the present study was to evaluate 16S rRNA gene amplification in the diagnosis of ascites infection in a group of pediatric patients with portal hypertensive ascites.
PATIENTS AND METHODS
From March 1995 to June 2002, 31 patients with portal hypertensive ascites were enrolled in the study. All of the patients were attending the pediatric gastroenterology clinic at Hospital de Clínicas de Porto Alegre, Brazil, a university hospital in southern Brazil. A serum-to-ascites albumin gradient of at least 1.1 g/dL (blood and ascitic fluid obtained simultaneously) was used to diagnose portal hypertensive ascites.
The diagnosis of cirrhosis was based on clinical, biochemical, ultrasonographic, and/or histological criteria. Cryptogenic cirrhosis was diagnosed when the following causes were ruled out: α-1-antitrypsin deficiency, inborn errors of metabolism, sclerosing cholangitis, viral hepatitis B and C, congenital infective disease, use of hepatotoxic drugs, presence of autoantibodies to autoimmune hepatitis types 1 and 2, and Wilson disease. The severity of the underlying liver disease was assessed according to the Pediatric End-stage Liver Disease (PELD) score (23).
Diagnostic paracentesis was performed on hospital admission in all of the patients with ascites. Criteria for admission included ascites associated with fever and/or clinical worsening defined as an increase in ascites volume detected by ultrasound, a worsening of jaundice, signs and symptoms suggesting encephalopathy (eg, conscious alterations, irritability, or electroencephalography findings), and worsening of laboratory liver function test results. Patients submitted to therapeutic paracentesis were not included.
Exclusion criteria were the use of antibiotics in the week before the paracentesis procedure, therapeutic paracentesis, paracentesis procedures performed 48 hours after diagnosis of SBP or BA, and refusal to sign the informed consent form. Patients using diuretic drugs were not excluded from the study. Blood was obtained for bacterial culture and for investigation of total and unconjugated bilirubin, serum albumin, prothrombin time, International Normalized Ratio, and activated thromboplastin time. The study was approved by the research ethics committee from our institution.
Ascites Samples and Bacterial Culture
Paracentesis was performed in all patients under standard aseptic conditions. Nine patients were submitted to more than 1 paracentesis procedure, resulting in 40 ascites samples. Ascitic fluid (10 mL) was submitted for the following analyses: Gram stain; aerobic and anaerobic cultures; total and differential cell count; determination of total protein and albumin concentrations, pH, glucose, and cholesterol levels; determination of lactic acid dehydrogenase activity; cytology; and determination of amylase and adenosine deaminase levels according to the standard operational procedures of the clinical pathology laboratory from our hospital. Although this study was originally designed to evaluate the clinical and laboratory features (ie, biochemical and cytology) of infected and noninfected ascites (24), we already had the intention to study the bacterial DNA content of the fluid. Therefore, 10 to 30 mL of ascitic fluid were stored at −20°C for posterior bacterial DNA analysis.
Bacterial aerobic and anaerobic cultures were performed by bedside inoculation of 10 mL of ascitic fluid into culture bottles and incubation in the BACTEC 9240 culture system (Becton Dickinson, Sparks, MD). Diagnosis of SBP was based on a PMN cell count greater than 250 cells/μL (7). BA was identified in the presence of a positive culture and PMN cell count no greater than 250 cells/μL (7). CNNNA was identified in the presence of a negative culture and PMN cell count no greater than 250 cells/μL.
Detection of Bacterial DNA
After defrosting at room temperature, 10 mL of ascitic fluid was centrifuged at 2000 rpm for 20 minutes and 9 mL of supernatant was removed. The pellet was mixed by vortexing, and a volume of 0.5 mL was used for DNA extraction. The remaining fluid was kept frozen for further analysis. DNA extraction was performed using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
Bacterial DNA was detected using 16S rRNA gene primers, a universal bacterial probe, as described by Such et al. (22). Briefly, 2 μL of DNA were mixed to 1X KCl buffer, 0.2 mmol/L of dNTP mix, 1.5 mmol/L of MgCl2, 0.5 U of Taq DNA polymerase, and 40 pmol of each primer, for a total volume of 50 μL. All reagents were obtained from Invitrogen. The reaction was performed in a thermocycler (MJ Research, Waltham, MA) for 27 cycles at an annealing temperature of 45°C. The expected 540-bp fragment was visualized after electrophoresis in 1.5% agarose gels stained with ethidium bromide.
Serial dilutions of ATCC 25922 Escherichia coli (108 to 101 cfu/mL) were submitted to PCR as described earlier to establish the detection limits of the method. PCR for all of the dilutions was performed in triplicate.
The statistical analysis was performed with SPSS software (version 10.0; SPSS, Chicago, IL). The baseline characteristics of the patients are presented as medians and interquartile range. For continuous variables with non-Gaussian distribution, the Mann-Whitney U test was used. To assess the association among categorical variables, the χ2 test was used. P < 0.05 was considered to indicate statistical significance.
The median age of patients was 2.9 years (interquartile range, 0.8–8.5). Sixteen patients were males. Cirrhosis of several etiologies was present in 24 patients. Causes of portal hypertension not related to cirrhosis were venocclusive disease, hepatoblastoma, Budd-Chiari syndrome, mucopolysaccharidosis VII, and hepatic congenital fibrosis. The clinical and laboratory features of the patients are described in Table 1. Median PELD score was 18.5 (interquartile range, 10.0–27.5).
According to the criteria of PMN cell count and culture, 8 ascitic fluid samples were classified as SBP and 4 as BA. Culture was positive in 4 of 8 cases of SBP. The following bacteria were identified: E coli (2 of 4), Klebsiella pneumoniae (1 of 4), and Staphylococcus aureus (1 of 4). The bacteria cultured in the 4 samples classified as BA were E coli, Salmonella species, S pneumoniae, and a Gram-positive rod. CNNNA was observed in 28 of 40 ascitic fluid samples (Table 2).
Amplification of bacterial DNA was positive in 7 of 8 cases of SBP, 3 of 4 cases of BA, and 8 of 28 cases with CNNNA (Fig. 1). Bacterial DNA was not detected in any of the negative controls (Fig. 1). All of the samples were positive for amplification of human genomic DNA (25), and this ruled out the presence of PCR inhibitors. PCR detection limits were 105 ufc/mL. The bands that indicate positive DNA amplification are those of similar size to the control, despite the presence of other bands (ie, nonspecific bands). The band indicative of positive DNA amplification was marked with an arrow in Figure 1. For patients with BA, faint bands were considered positive for DNA amplification as indicated by sample number. Moreover, patients with higher numbers of leukocytes (as shown by higher numbers of PMN) tended to present nonspecific bands upon DNA amplification (as seen in lane 5). However, only a bright band of similar size to the control was considered positive.
Patients with CNNNA with or without bacterial DNA were compared in terms of severity of liver disease estimated by PELD score, serum to albumin ascites gradient, rate of mortality in 3 months, and laboratory features such as PMN blood cell count, International Normalized Ratio, serum and ascitic albumin levels, serum total bilirubin level, serum creatinine level, and serum and ascitic total protein. There was no statistically significant difference between these 2 groups (Table 3).
We evaluated bacterial 16S rRNA gene amplification to diagnose ascites infection. Patients were not screened for clinical features suggestive of ascites infection as a result of low specificity of these criteria (7,24).
The culture of ascites using the BACTEC system yielded positive results in 50% of the ascitic fluid samples with a PMN cell count greater than 250 cells/μL. Gram-negative rods from the Enterobacteriaceae family (E coli and K pneumoniae) were the most prevalent bacterium cultured. These results are similar to those obtained with conventional blood culture bottles.
The results of BACTEC and 16S rRNA gene amplification were in agreement in 7 samples (87.5%). 16S rRNA gene amplification did not identify 1 case with negative bacterial culture classified as SBP and another case classified as BA that was caused by a Gram-positive rod. According to the literature, bacteria such as Gram-positive species and Mycobacterium species may not be detected by 16S rRNA gene amplification (15,20,21). This is probably due to the difficulty in disrupting the cell wall of these organisms, with a consequent failure in extracting DNA. Two measures used to try to solve this problem are the addition of lytic enzymes to the reaction mixture and mechanical disruption of cell walls (15,20). Because the addition of enzymes increases the risk of contamination, mechanical disruption seems to be the best solution (15). We have not attempted to compare different methods to extract DNA in this study.
In our study 8 of 28 patients with CNNNA (28.5%) presented positive results of amplification of bacterial DNA, which could be considered false-positive results. Sample contamination is the main cause for false-positive results in PCR (20,21). In our study we tried to avoid contamination by performing DNA extraction in airflow chambers and including a negative control in every PCR procedure. Conversely, Such et al (22) found bacterial DNA in the ascitic fluid samples of 9 of 28 patients (32.0%) with CNNNA. However, bacterial DNA was also observed in the blood of these patients, and molecular sequencing of these bacteria identified the same species in all positive samples (22). According to the authors, this rules out the possibility of contamination.
In our study 3 of 8 patients with positive bacterial DNA and CNNNA died after 3 months, and 1 was listed to receive a liver transplant. However, when we compared the data of these CNNNA patients with our CNNNA patients without bacterial DNA in ascites in terms of PELD score, serum to albumin ascites gradient, mortality at 3 months, count of PMN cells (blood and ascites), International Normalized Ratio, serum and ascites albumin levels, total bilirubin level, serum creatinine level, and ascites total protein level, no statistical difference was observed (Table 3). Similarly, Child-Pugh classification and endoscopic and clinical features were not different in the patients studied by Such et al (22) in relation to DNA positivity.
The results of this study, as well as those reported by Such et al (22), suggest that the presence of bacterial DNA in patients who do not meet cytological criteria of infection has no effect on their outcomes, at least in the short term. We agree with Such et al (22) that these positive results may signal the transitory presence of bacteria in ascitic fluid. Recently, Francés et al demonstrated that the presence of bacterial DNA in ascites samples of patients with decompensated cirrhosis was associated with activation of peritoneal macrophage immune response suggested by the production of nitric oxide and synthesis of cytokine (26).
The consequences of the presence of bacteria will depend on the bactericidal capacity of this fluid. Detection of BA in a symptomatic patient may be of interest because patients with BA may develop SBP within a few days (27) and the BACTEC culture system is unable to identify all of these patients as a result of its low sensitivity.
In the present study some patients seemed to have bacterial DNA in ascites even though they did not present clinical disease. This leads to the perception that amplification of bacterial DNA does not distinguish patients with ascitic fluid infection from those with ascites colonized by bacteria.
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