Bacterial DNA translocation in patients with cirrhosis: Reply
Authors: Rubén Francés, José Such
Journal: Journal of Hepatology (2008)

We thank Fujita and colleagues for their comments and this opportunity to discuss the questions raised regarding our recent investigation on bacterial DNA translocation in patients with cirrhosis. Our main line of research is focused on the study of bacterial translocation (BT) in patients with decompensated cirrhosis. Approximately, 30% of these patients show the simultaneous presence of bacterial DNA (bactDNA) in blood and ascitic fluid (AF) [1Such J. Frances R. Munoz C. Zapater P. Casellas J.A. Cifuentes A. et al.Detection and identification of bacterial DNA in patients with cirrhosis and culture-negative, nonneutrocytic ascites.Hepatology. 2002; 36: 135-141Crossref PubMed Scopus (237) Google Scholar, 2Frances R. Benlloch S. Zapater P. González J.M. Lozano B. Muñoz C. et al.A sequential study of serum bacterial DNA in patients with advanced cirrhosis and ascites.Hepatology. 2004; 39: 484-491Crossref PubMed Scopus (124) Google Scholar] and a recent study in an experimental model of CCl4-induced cirrhosis with ascites positively correlated this finding with the identification of bactDNA in mesenteric lymph nodes [[3]Guarner C. Gonzalez-Navajas J.M. Sanchez E. Soriando G. Francés R. Chiva M. et al.The detection of bacterial DNA in blood of rats with CCl(4)-induced cirrhosis with ascites represents episodes of bacterial translocation.Hepatology. 2006; 44: 633-639Crossref PubMed Scopus (86) Google Scholar]. However, and related to the first question, we recently conducted a study in which bactDNA detection and the associated inflammatory response was evaluated in patients with spontaneous bacterial peritonitis (SBP). In this study, a total series of 22 patients with SBP was analyzed. All 22 cases showed the presence of bacterial genomic fragments as revealed by PCR of the 16SrRNA gene. Identifications by DNA sequencing analysis in patients with culture-positive SBP corresponded to those isolated by microbiological culture in all cases, except for one patient that showed a Staphylococcus aureus by sequencing analysis and a Streptococcus pneumoniae by microbiological culture [[4]Francés R. Zapater P. González-Navajas J.M. Muñoz C. Caño R. Moreu R. et al.Bacterial DNA in patients with cirrhosis and noninfected ascites mimics the soluble immune response established in patients with spontaneous bacterial peritonitis.Hepatology. 2008; 47: 978-985Crossref PubMed Scopus (138) Google Scholar]. Regarding experimental procedures, control experiments are normally run over all DNA extraction Kit solutions as well as PCR mastermix reagents. Specificity experiments are always completed with a 10 pg-spike of Escherichia coli DNA to determine the presence of inhibitors in the media. In addition, all solutions and reagents are handled in sterile air-flow chambers previously irradiated with UV light, and sterile plastic material and filter tips are used all along the technique of DNA isolation and amplification. Finally, in all PCR reactions, two negative controls are always run: one with sterile water instead of sample templates and one with PCR mastermix alone. We are aware of the study by Heininger A et al. in which, as pointed out by Fujita T, 16.6% false-positive results are present when DNA is isolated from healthy volunteers with a commercially available kit [[5]Heininger A. Binder M. Ellinger A. Pfisterer J. Botzenhart K. Unertl K. et al.Effect of comprehensive validation of the template isolation procedure on the reliability of bacteraemia detection by a 16S rRNA gene PCR.Clin Microbiol Infect. 2004; 10: 452-458Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar]. DNA isolation kit used in that study (Puregene Whole Blood Kit, Biozym Diagnostics) is not the same as the one we usually handle (QiAmp Blood Minikit, QIAgen). Also, the set of primers used to perform PCR reactions are also different in that study. Therefore, it is difficult for us to compare or discuss on someone else’s reported contamination rates. A significantly increased cytokine response is present in patients with presence of bactDNA [6Frances R. Munoz C. Zapater P. Uceda F. Gascón I. Pascual S. et al.Bacterial DNA activates cell mediated immune response and nitric oxide overproduction in peritoneal macrophages from patients with cirrhosis and ascites.Gut. 2004; 53: 860-864Crossref PubMed Scopus (109) Google Scholar, 7Frances R. Gonzalez-Navajas J.M. Zapater P. Muñoz C. Caño R. Pascual S. et al.Bacterial DNA induces the complement system activation in serum and ascitic fluid from patients with advanced cirrhosis.J Clin Immunol. 2007; 27: 438-444Crossref PubMed Scopus (32) Google Scholar]. Despite this increment, values have never reached μg/ml levels. This is, as suggested, a typing mistake. All cytokine determinations were measured in pg/mL. We apologise for this confusion and thank Dr. Fujita and colleagues for their appreciation. Indeed, in the previously mentioned study on the inflammatory response in patients with advanced cirrhosis and ascites, extended results on several cytokine levels can be followed in different clinical settings. Endotoxin is a difficult to measure parameter. LPS traces can be detected in serum from patients with presence of Gram-positive microorganisms. We consider that reported differences in endotoxin levels simply reflect intrinsic characteristics of both subgroups of bacterial species and do not allow discussion about its biological activity. For this purpose lipopolysaccharide binding protein (LBP) measurement has been proposed instead [[8]Albillos A. de la Hera A. Gonzalez M. Moya J.L. Calleja J.L. Monserrat J. et al.Increased lipopolysaccharide binding protein in cirrhotic patients with marked immune and hemodynamic derangement.Hepatology. 2003; 37: 208-217Crossref PubMed Scopus (354) Google Scholar]. However, endotoxin, DNA or peptidoglycan, among others bacterial products, stimulate the innate immune system and induce the liberation of proinflammatory cytokines and nitric oxide, and these factors indeed have clinical consequences. The detection of high levels of endotoxin or the above-mentioned bacterial products identify a subgroup of patients with evidences of bacterial translocation, and this may affect prognosis [[9]Zapater P. Frances R. Gonzalez-Navajas J.M. Moreu R. Llanos L. Pascual S. et al.Presence of bacterial DNA is a new survival indicator in patients with cirrhosis and non-infected ascitic fluid.Hepatology. 2007; 46: 251AGoogle Scholar].