Predictable Value Of PCR Detecting Bacterial DNAs In Rat's Blood And Ascites For Early Diagnosis Of Intestinal Anastomotic Leakage

Background: Anastomotic leakage is one of the most importantcomplication specific to intestinal surgery. The incidence rate is different inreportings.The course of this complication develops quickly once takingplace.The delay in diagnosis and treatment will worsen the intestinal injuryand often lead to serious morbidity including bowel necrosis,secondaryperitonitis,sepsis,systemic inflammatory response syndrome(SIRS),and evenmultiple organ failures(MOF) which threaten the life.Usually,intestinalanastomotic leakage is diagnosed by the typical clinical features in laterstage because of its delitescence and lack of good diagnostic methods inearly stage.So the early diangnosis and treatment of anastomotic leakage isvitally important to the prognosis of this complication. Numerousresearchers were focusing on searching a kind of convenient, quick, preciseand objective methods to diagnose anastomotic leakage early.OBJECTIVE: This study was designed to assess the value of detectingbacterial DNA in rat's blood and ascites with the polymerase chain reaction(PCR) technique for early diagnosis of anastomotic leakage.Methods: 65 healthy female Wistar rats were divided into sevenrandom groups:Group A(n=5) sham operation group;Group B(n=10)colonic anastomosis group,Group C(n=10) colonic anastomotic leakagegroup;Group B and C,rats had standardized colon resection 3cm away from the ileocecal junction 10cm,Group B rats had a complete anastomosis(end-to-end single layer anastomosis with 0~# silk sutures),while Group C ratshad an anastomosis leaving a 5mm opening in colonic anterior wall.GroupD(n=10) jejunal anastomosis group,Group E(n=10)jejunal anastomoticleakage group,Group F(n=10) ileal anastomosis group, Group G(n=10) ilealanastomotic leakage group.Group D and E rats had standardized jejunumresection 3cm away from the Treitz ligament 15cm.Group F and G rats hadstandardized ileaum resection 3cm above the ileocecal junction 15cm.GroupD and F rats had a complete anastomosis (end-to-end single layeranastomoses with 0~# silk sutures).Group E and G rats had an anastomosiswith a 5mm opening in intestinal anterior wall. Group B and C constitutedcolon operation group,markedⅠ,group D,E,F,G are combined to be jejunumand ileum operation group,markedⅡ. 1ml and 3ml venous blood sampleswere collected from Group A and GroupⅠ,at the third and sixth daypostoperative.Collected 1ml venous blood samples postoperative 36 hours,3ml venous blood and 1-2ml ascites postoperative 72 hours from GroupⅡ.DNAs were extracted from these blood and ascites samples and PCRtechniques were used to amplify lacZ genes from Escherichia coli and 16Sribosomal RNA genes(16SrRNA genes).The data were analysed by chisquare test.Specimens of the experimental intestine were HE stained forpathological studies in order to view the degree of injury.Results: 1. The positive ratio of expressing lacZ genes in peripheral blood (PB) with PCR in Group C were significantly higher than in GroupB(P＜0.05),but there were no differences between the two groups inexpressing 16SrRNA genes(P＞0.05). 2. When comparing Group D withGroup E, or Group F with Group G:there were no differences in positiveratio of lacZ genes (P＞0.05),but in ascites samples the positive expressingratioes in Group E and Group G were higher than Group D and GroupF(P＜0.05);the positive ratioes of expressing 16SrRNA genes in PB andascites in Group E and Group G were significantly higher than in group Dand Group F respectively (P＜0.05). 3.Comparing with the total positiveratioes of lacZ genes and 16SrRNA genes in PB in jejunal anastomoticleakage and ileal anastomotic leakage group,the former is obviously lowerthan the latter(P＜0.05).But there were no significantly differencesexpressing in ascites. 4. Histopathological changes of mucosal necrosis,surface epithelial disruption, lamina propria congestion and hemorrhage,infiltration of inflammatory cell were heavily present in the mucosa duringanastomotic leakage periods in Group C,E,G.Conclusions: 1.PCR could be a useful tool for diagnosis of colonicanastomotic leakages in detecting lacZ genes of Escherichia coli fromperipheral blood(PB),but no much significant in detecting 16SrRNAgenes.2.Detecting lacZ genes of Escherichia coli from PB with PCR have nomuch significant in diagnosing jejunal anastomotic leakage and ilealanastomotic leakage,but it's useful in detecting lacZ genes from ascites.3.Detecting bacterial 16SrRNA from PB and ascites with PCR are alluseful in diagnosing jejunal anastomotic leakage and ileal anastomoticleakage.But there were no significant difference between from PB orascites.4.In the aspect of diagnosing jejunal anastomotic leakage and ilealanastomotic leakage, detecting 16SrRNA genes from BP with PCR isobviously more sensitive than detecting lacZ genes. But in the ascitessamples,there were no significant difference in diagnostic rate by detectingthe two target genes.In the aspect of diagnosing colonic anastomotic leakage,lacZ genes is more suitable than 16SrRNA genes with PCR.