The current pathogenic theory of spontaneous bacterial peritonitis (SBP) in patients with cirrhosis and ascites suggests that repeated episodes of bacterial translocation (BT) from intestinal lumen to mesenteric lymph nodes followed by systemic seeding are the key steps for the final development of infectious events. However, most of the episodes of systemic bacterial circulation remain undetected. Therefore, we investigated the hypothetical presence of bacteria in blood and/or ascitic fluid (AF) from patients with cirrhosis and sterile (culture negative) AF by means of bacterial DNA (bactDNA) detection and identification. Twenty-eight consecutively admitted patients with cirrhosis and presence of AF were included in the study. BactDNA was detected using a polymerase chain reaction (PCR)-based method. The corresponding bacteria were identified by nucleotide sequencing of purified PCR products. BactDNA was detected simultaneously in blood and AF in 9 patients (32.1%). DNA sequencing allowed the identification of Escherichia coli (n ‫؍‬ 7) and Staphylococcus aureus (n ‫؍‬ 2). In all cases, the similarity between the sequence found in AF and blood indicated that the bactDNA present in both locations originated from a single clone (single translocation event). Child-Pugh score and basic hemodynamic, clinical, endoscopic, and biochemical characteristics were similar among patients with or without the presence of bactDNA. In conclusion, we have detected bactDNA in serum and AF in 32% of all patients studied, and this likely represents single clone episodes of translocation and systemic seeding. E. coli is the most frequently identified bacteria. (HEPATOLOGY 2002;36:135-141.)

Bacterial translocation is currently considered the main pathogenic mechanism leading to spontaneous bacterial peritonitis in patients with advanced cirrhosis and ascites. However, to the authors' knowledge there is no information regarding the characteristics of this process in humans. The goals of the current study were to pursue partially identified bacterial DNA in blood (what the authors consider molecular evidence of bacterial translocation) through its relative quantification in a 72-hour study period by using real-time polymerase chain reaction (PCR). A consecutive series of 17 patients with advanced cirrhosis and culture-negative, nonneutrocytic ascites were studied. Therapeutic paracentesis was performed at the time of admission, and blood samples were obtained at baseline and every 8 hours in a 3-day period. S pontaneous bacterial peritonitis (SBP) is a severe infection developing in patients with advanced cirrhosis, in the absence of any intraabdominal, surgically treatable source of infection. 1 It is considered to be the final consequence of repeated episodes of bacterial translocation (BT) from the intestinal lumen and eventual arrival of bacteria in the ascitic fluid (AF). However, the predisposition to develop a SBP episode is related to its intrinsic bactericidal properties. [2][3][4] BT is an incompletely understood process by which intestinal bacteria can cross the epithelial wall, thereby reaching mesenteric lymph nodes and other organs. 5 BT has been studied extensively in cirrhotic rats, 6,7 but for obvious reasons it is difficult to study its incidence in patients with cirrhosis. 8 We recently reported the presence of bacterial DNA (BactDNA) in blood and AF in roughly 40% of patients with cirrhosis and culture-negative, nonneutrocytic ascites 9 and, although more experimental work is needed to confirm our hypothesis, the data available to date may represent molecular evidence of BT. This method allows the study of BT in patients without clinical evidence of infection, thus becoming a useful tool with which to investigate the steps preceding a fully developed infection.To our knowledge, to date it is not known whether bacteria translocate as the result of a "single pulse" event or, conversely, bacteria continuously are crossing the intestinal wall, and what is the rate of bacterial clearance from the systemic circulation. Although we previously reported that Escherichia coli is the most prevalent bacteria found to cause episodes of BT at the time of admission, 9 we do not know whether this finding may be different in the following hours or days.Therefore, the objectives of the current investigation were to explore the temporal pattern of BactDNA clear-

Background and aims: Translocation of intestinal bacteria to ascitic fluid is probably the first step in the development of episodes of spontaneous bacterial peritonitis in patients with cirrhosis. We have recently reported the detection of bacterial DNA in blood and ascitic fluid from patients with advanced cirrhosis, what we consider as molecular evidence of bacterial translocation. Several studies have shown the immunogenic role of bacterial DNA in vitro, and we hypothesised that the presence of bacterial DNA could activate the type I immune response in peritoneal macrophages from these patients, leading to greater cytokine synthesis (interleukin (IL)-2 and IL-12, tumour necrosis factor a, and interferon c) and effector molecules such as nitric oxide. Methods: Peritoneal macrophages obtained from patients with cirrhosis and culture negative nonneutrocytic ascitic fluid were collected and characterised by flow cytometry. Inducible nitric oxide synthase, nitric oxide levels, and cytokine production were measured by immunoenzymometric assays in basal and harvested conditions according to the presence/absence of bacterial DNA. Results: The ability of peritoneal macrophages to synthesise nitric oxide and levels of all cytokines were significantly increased in patients with bacterial DNA. There was a positive correlation between inducible nitric oxide synthase and nitric oxide levels. Conclusions: The presence of bacterial DNA in patients with decompensated cirrhosis is associated with marked activation of peritoneal macrophages, as evidenced by nitric oxide synthesising ability, together with enhanced cytokine production.

Background and aims: Bacterial infections are common complications in patients with acute pancreatitis, and translocation of bacteria from the intestinal lumen is probably the first step in the pathogenesis of these infections. As blood cultures in afebrile patients are usually negative, more sensitive methods to investigate this hypothesis in patients are needed. Our group has recently developed a method to detect the presence of bacterial DNA in biological fluids, and we aimed to detect bacterial DNA in patients with acute pancreatitis, as molecular evidences of bacterial translocation. Methods: Samples of blood were obtained on three consecutive days within the first six days after admission. Bacterial DNA was detected using a polymerase chain reaction based method, and an automated DNA nucleotide sequencing process allowed identification of bacteria species. Results: Thirty one consecutively admitted patients with acute pancreatitis were studied. Bacterial DNA was detected in six patients (19.3%), and the sequencing process allowed identification of Citrobacter freundii and Pseudomonas aeruginosa. In two patients the same bacteria detected at admission was detected 24 hours later (above 99.9% homology of nucleotide sequence). Basic clinical and biochemical characteristics were similar among patients with or without the presence of bacterial DNA. Conclusion: Detection of gram negative bacteria derived bacterial DNA in our series supports the contention that bacterial translocation is a systemic process in approximately 20% of patients with acute pancreatitis that does not seem to be related to the severity of the episode or immediate development of infection.

A longer time on the waiting list and referral by a general practitioner are factors associated with patients failing to keep their endoscopy appointment.