Clinical Study On The Prevention Of Recurrent Choledocholithiasis By Saline Irrigation After ERCP Operation And Exploring The Role Of Biliary Microenvironment In The Stone Formation Process Of Recurrent Common Bile Duct Stones

In this thesis,we focused on two important topics in the role of saline irrigation in preventing the recurrence of CBDS after ERCP and the relationship between the microenvironment of the biliary tract and the recurrence of CBDS.The thesis consisted of three parts of the study.Part ⅠBackground: Common Bile Duct Stones(CBDS)is the clinic’s most common benign disease of the biliary system.Endoscopic retrograde cholangiopancreatography(ERCP)combined with endoscopic sphincterotomy(EST)is the first-line treatment for CBDS.Still,the recurrence rate of CBDS is high,the risk factors for the recurrence of CBDS are not clear,and there are no effective preventive measures.For small stones or stone fragments remaining after ERCP,cholangiography is easy to miss,which often leads to the recurrence of CBDS.Removing the small residual stones or stone fragments may have the effect of preventing the recurrence of stones.Objective: To determine the effect of saline irrigation after ERCP to prevent the recurrence of CBDS and the risk factors of the recurrence of CBDS.Methods: In this study,a retrospective cohort study was adopted,and patients who underwent ERCP for CBDS were included in the study and divided into the saline irrigation group and the control group(no irrigation)to analyze the relationship between normal saline irrigation and CBDS recurrence and the risk factors of stone recurrence.Results: A total of 226 patients who received ERCP for CBDS at the Surgical Endoscopy Center of the First Hospital of Lanzhou University from May 2016 to June2017 were included in the clinical study.According to the inclusion and exclusion criteria,113 patients were finally included in the study,and 6-36 months of follow-up were completed.There was no significant difference in age,gender,BMI,comorbidity,chronic constipation,history of biliary surgery and total bilirubin between the two groups.During the follow-up period,CBDS recurrence was observed in 27 of 113 patients with common bile duct stones,accounting for 23.89%.7 of 49 patients(14.3%)in the saline irrigation group had recurrence of CBDS,and 20 of 64 patients(31.3%)in the control group had recurrence of stones.Kaplan-Meier curve showed that the recurrence rate of CBDS in the saline irrigation group was significantly lower than that in the control group(log-rank p=0.0184).There was no significant difference in surgery-related complications between the two groups.The median time of the saline irrigation group was 44.4 ± 8.0 min,and that of the control group was 41.9 ± 7.6 min.The operation time of the saline irrigation group was slightly longer than that of the control group,but there was no statistical difference(p<0.10).There were no significant differences in choledochal diameter(p=0.13),maximum stone diameter(p=0.59),number of stones(p=0.07),mechanical lithotripsy(p=0.08),PAD(p=0.80),the median length of stay after ERCP(p=0.95)and hospitalization cost(p=0.46)between the saline irrigation group and the control group.Univariate analysis showed that PAD,common bile duct diameter,maximum stone diameter,and the number of stones were correlated with the recurrence of common bile duct stones.Saline irrigation is a protective factor for the prevention of choledocholithiasis recurrence.Multivariate risk analysis showed PAD(Odds ratio [OR] 4.11;95% CI,1.14-16.90,p=0.004),choledochal diameter(OR1.21;95% CI,1.02-1.44,p=0.027),maximum stone diameter(OR 1.28;95% CI,1.09-1.51,p=0.003)and mechanical lithotripsy(OR 8.52;95% CI,2.37-37.61,p=0.002)were the independent risk factors for choledocholithiasis recurrence.Saline irrigation was the protective factor for preventing choledocholithiasis recurrence(OR 0.11;95%CI,0.025-0.40,p=0.001)Conclusions: Saline irrigation after ERCP may prevent the recurrence of CBDS by removing residual stones.PAD,common bile duct diameter and maximum stone diameter and mechanical lithotripsy were independent risk factors for the recurrence of CBDS.Part ⅡBackground: Mechanical lithotripsy is a commonly used technique for the endoscopic treatment of large or complex stones,which makes the stones crushed and easy to be removed.It produces small stones or stone fragments that are not easily detected by choledocholithiasis,which is a potential cause of choledocholithiasis recurrence.Our previous study confirmed that intermittent irrigation with 100 ml saline resulted in a degree of biliary duct cleaning in 94% of cases.However,the prevention of CBDS recurrence by saline irrigation has not been confirmed by prospective randomized controlled trials.Objective: The purpose of this study was to determine whether 100 ml saline irrigation after mechanical lithotripsy can reduce the recurrence rate of CBDS and analyze the risk factors for the recurrence of common bile duct stones.Methods: This study was an investigator-initiated,parallel,single-center,openlabel,1:1,randomized controlled clinical trial.Patients with CBDS who underwent ERCP and required mechanical lithotripsy were included in the study.To compare the effect of saline irrigation and no irrigation on the recurrence rate of CBDS after mechanical lithotripsy.To evaluate the effect of saline irrigation on preventing stone recurrence after ERCP and to analyze the risk factors of CBDS recurrence.This trial is registered at https://clinicaltrials.gov.(Trial registration No: NCT03937037)and is independently monitored by the Data Monitoring Committee.Results: 180 patients were enrolled from May 10,2019,to December 31,2020,including 90 in the saline irrigation group and 90 in the control group.There was no significant difference between the two groups in age,gender,BMI,comorbidity,chronic constipation or previous history of biliary surgery.At a median follow-up time of 35.6 months(quartile range 26.0-40.7),43 of the 180 patients had stone recurrence(24%).The frequency of recurrence of CBD stones was 12.22% in the saline irrigation group and 35.56% in the control group,with a difference of 23.33% between the two groups(95% CI: 11.35%-35.32%,p<0.001).Kaplan-Meier curve also showed that the recurrence rate of CBDS in the saline irrigation group was significantly lower than that in the control group(log-rank p< 0.001).There was no significant difference in surgeryrelated complications between the two groups.The median time of the saline irrigation group was 47.8±7.5 min,and that of the control group was 43.5±7.6 min.The operation time was approximately 4 minutes longer in the saline irrigation group compared to the control group(p< 0.001).The diameter of the common bile duct(p=0.99),the maximum diameter of CBDS(p=0.99),There were no significant differences in the number of stones(p=0.70),length of EST incision(p=0.98),balloon dilation diameter(p=0.18),PAD(p=0.72),the median length of stay after ERCP(p=0.88),and hospitalization cost(p=0.90).Multivariable Cox proportional hazards analyses showed that constipation(HR 2.82;95% CI,1.41-5.64,p=0.003)and periampullary diverticulum(PAD)(HR 3.34;95% CI,1.77-6.31,p<0.001)were independent risk factors for the recurrence of CBDS.Saline irrigation was the preventive factor for the recurrence of CBDS(HR 0.20;95% CI,0.10-0.40,p<0.001).Conclusions: For patients with CBDS requiring mechanical lithotripsy,100 ml saline irrigation effectively reduces the recurrent rate of CBDS after endoscopic stone removal.Given this method’s efficiency,simplicity,low cost,and safety,saline irrigation is recommended as a routine operation procedure after endoscopic lithotripsy and stone extraction.Chronic constipation and PAD were independent risk factors for choledocholithiasis recurrence.Part ⅢBackground: CBDS is the most common clinical biliary tract disease,and ERCP combined with EST is the first-line treatment for CBDS.A high postoperative recurrence rate and reoperation rate is a difficult clinical problem.Some studies have reported that EST is the risk of recurrence of CBDS,and EST causes partial loss of duodenal papilla function,leads to enterobiliary reflux,and changes the biliary microenvironment,which may be the potential cause of CBDS recurrence.Objective: To reveal the diversity and structural changes of biliary microflora in patients with recurrent choledocholithiasis and to clarify the relationship between the change of the biliary microenvironment and the recurrence of choledocholithiasis.Methods: In this study,patients with choledocholithiasis and noncholedocholithiasis treated with ERCP were included using the case-control method.Clinical diagnosis and treatment data were collected.Bile samples were extracted after successful intubation with ERCP,and high-throughput sequencing was performed with Illumina Nova 6000.Biliary microflora diversity and species differences at different taxonomic levels between the two groups were analyzed,and PICRUSt was used to predict and analyze the different pathways.Results: 86 patients were enrolled from May 2019 to December 2020,including43 patients with recurrent CBDS(recurrence group)and 43 without choledocholithiasis(control group).There were no significant differences in age,gender,BMI,comorbidity,previous biliary surgery history,preoperative biochemical indexes and other clinical characteristics between the two groups.The α diversity index analysis showed that the observed index(p =0.034),Chao1 index(p =0.029),ACE index(p =0.023),Shannon index(p =0.039),Simpson index(p =0.033)and Plelou index(p =0.048)were higher in the stone recurrence group than in the control group.β-diversity analysis showed significant differences in the structure of biliary tract microflora between the two groups.The variation of PCo1 was 17.32%,and the PCo2 was 10.71%.Adonis multivariate analysis of variance found that biliary tract microorganisms in the stone recurrence and control groups could be significantly distinguished by PCo A analysis(R2=0.058,p =0.0001).Regarding the distribution of biliary tract bacteria,at the phylum level,pActinobacteri and P-firmicutes in the stone recurrence group were significantly higher than those in the control group(p < 0.05).In contrast,P-Fusobacteria and PBacteroidetes in the control group were significantly higher than in the recurrence group(p < 0.05).At the species level,g-Streptococcus,g-Klebsiella,g-Porphyromonas,g-Blautia,g-Bilophila,g-Dorea,g-Romboutsia,g-Catenibacterium,g-Pseudomonas and g-Serratia were significantly higher in the stone recurrence group than in the control group.In the control group,g-Bacteroides,g-Fusobacterium,g-Lachnospira,g-Hungatella,g-Lactobacillus,g-Sutterella,g-Roseburia and g-Lachnoclostridium were significantly higher than stone recurrence group(p < 0.05).PICUSt predicted the possible KEGG pathways at all levels and their abundance values in 16 S sequencing samples,showing that the stone recurrence group,Biosynthesis of amino acids,ABC transporters,Taurine and hypotaurine metabolism,Secondary bile acid biosynthesis,Cysteine and methionine metabolism,Biosynthesis of secondary metabolites,Fatty acid metabolism,Metabolic The enrichment of pathways,Flagellar assembly,Calcium signaling pathway was higher than that of the control group.The difference was statistically significant(p < 0.05).In the stone recurrence group,Protein digestion and absorption,Adipocytokine signaling pathway,Bile secretion,and Protein processing in the endoplasmic reticulum were less abundant in the bile duct than the control group,and the difference was statistically significant(p <0.05).Conclusions: After endoscopic stone extraction,the biliary tract microbial composition of patients in the recurrence group was more complex and diverse.Common intestinal pathogenic bacteria such as Streptococcus,Bilophila,and Porphyromonas were enriched in the biliary tract.In contrast,the abundance of probiotics or opportunistic pathogens such as Lactobacillus,Lachnospira and Roseburia decreased.Intestinal biliary reflux changes the biliary microenvironment,resulting in the imbalance of biliary microflora.It may be closely related to the formation and recurrence of choledocholithiasis.