A Study On Evaluation Of Bacterium Coding16S Ribosomal RNA Genes By PCR In Ascites Of Patients With Liver Cirrhosis

Liver cirrhosis patients complicated with ascites, especially with severe liver dysfunction, are prone to the occurrence of bacterial translocation which easily leading to ascites infection. In addition, bacterial translocation can activate the immune system through a series of mechanisms leading to the hyperdynamic circulatory state, futher causing portal hypertension and hepatorenal syndrome. Therefore, the study of bacterial translocation is of great significance.ObjectiveTo evaluate polymerase chain reaction (PCR) detecting bacterium coding16S ribosomal RNA (16S rRNA) genes in ascites of patients with liver cirrhosis and the risk facters resulting bacterial translation(BT). In addition, the application of antibiotics was also analysed.Methods1. We collected ascites samples from48patients with liver cirrhosis in Tianjin infectious diseases hospital from August2011to February2012.2.48ascites of liver cirrhosis patients were amplified with PCR with a set of universal primers designed basing on conservative regions of bacterial16S rRNA genes. Amplified products were identified by agarose gel electrophoresis. Postive, negetive and blank control were also amplified with PCR. By using human genome DNA and HBV-DNA as comparison, the specificity of primers was tested. The sensitivity of test was done by the method of gradual dilution. The corresponding bacteria were identified by nucleotide sequencing of purified PCR products.3. The clinical data of patients was also collected. Serum and ascites routine, biochemical index, complications, past history and so on between DNA positive and negative groups were analysed by T test and chi-square test to find the risk factors of BT. In addition, the application of antibiotics was also analysed.Results1.370bp DNA fragmens were detected from15of48ascites (31.25%) by PCR with a set of universal primers designed basing on conservative regions of bacterial16S rRNA genes. Genes sequencing and BLAST contrast of PCR products of the bacterial DNA positive samples allowed the identification of bacterium. They are respectively Escherichia coli (n=10), Enterobacter Cloacae (n=l), Klebsiella pneumoniae (n=1), Acinetobacter lwoffii (n=1), Pasteurella (n=1) and staphylococcus aureus (n=1).2. Serum and ascites routine, biochemical index, complications, past history and so on between DNA positive and negative groups were analysed. There were significant differences in white blood cell (WBC), neutrophils (NEUT), sodium (Na), C-reactive protein (CRP), polymorphonuclear ratio (PMN%), upper gastrointestinal hemorrhage ratio, SBP history and Child-Pugh score between groups with and without bacteria DNA in ascites (P<0.05).3. If ascites PMN≥250/mm3was the diagnostic criteria of ascites infection, the specificity and sensitivity of positive ascites DNA detecting ascites infection were72.50%,50.00%; positive ascitic culture were87.50%,12.50%.4.370bp DNA fragments were amplified in all postive control bacterial Strains, while negetive and blank control bacterial strains were not amplified by this method. The sensitivity could be improved to lOpg bacterial DNA by the method of gradual dilution.5.27in48were using antibiotics, and91.90%were single drug. The most were piperacillin or cefoperazone complicated with enzyme inhibitors, the third generation cephalosporin and carbapenems (imipenem/cilastatin). The effective rate of the antibiotics treating SBP(PMN>250/mm3) was87.50%.Conclusion1. PCR amplification of bacterial16S rRNA genes with a set of universal primers designed basing on the conservative regions of bacterial16S rRNA genes was available and could be used to detect bacterial DNA in patients with liver cirrhosis. Identified bacteria were mostly Escherichia coli. Compared with the bacteria cultures, this method might be more efficient for rapid diagnosis of ascites infection and pathogenic bacteria.2. Compared with DNA negative group, WBC, NEUT, CRP, ascites PMN ratio, Child-Pugh integral, upper gastrointestinal bleeding rate, past SBP history and blood sodium levels of DNA postive group were statistically significant. 3. The most antibiotics on usage were piperacillin or cefoperazone complicated with enzyme inhibitors. The effective rate of antibiotics treating SBP (PMN>250/mm3) was87.50%.