| Objective:Spontaneous bacterial peritonitis(SBP)is the most common infection complication in patients with cirrhosis,which is closely related to the prognosis of patients with decompensated cirrhosis.The delayed treatment will increase the incidence of hepatorenal syndrome,or even progress to sepsis or septic shock.The treatment cost and the mortality rate are high.At present,the conventional diagnostic criteria for SBP are low in sensitivity,and it is difficult to identify SBP early.In recent years,metagenomic next-generation Sequencing(mNGS),as an emerging method for detection of infectious diseases,has attracted more and more attention of clinicians,b but there are few reports on its application in SBP diagnosis.This study aims to evaluate the positive detection rate of pathogens through mNGS,compared with ascites culture,in patients with cirrhotic ascites and the significance of mNGS in the diagnosis of SBP,and to further evaluate the relationship between the results of ascites mNGS and the prognosis of patients with cirrhotic ascites.Methods:Prospectively enrolled 60 patients with cirrhotic ascites who underwent ultrasound-guided abdominal puncture in the Department of Hepatology,the First Hospital of Jilin University,from July 11,2020 to December 15,2020.Their ascites specimens were obtained under aseptic conditions and sent Check ascites routine,ascites biochemistry,ascites culture,and ascites mNGS.At the same time,blood samples were collected to test blood routine,liver function,kidney function,coagulation routine,procalcitonin,and C-reactive protein.Record the patient’s general clinical information,etiology of cirrhosis,basic diseases,and antibiotic use in recent 2weeks.According to the clinical data,whether the patients were complicated with hepatorenal syndrome,hepatic encephalopathy,infection of other sites and upper gastrointestinal bleeding were identified,and the aspartate aminotransferase/platelet ratio(APRI)index,Fibrosis 4 score(FIB-4),Model for End-stage Liver Disease(MELD)score,Model for end-stage liver disease with the incorporation of serum sodium(MELD-NA)score and Child-Pugh score were calculated.Primary study endpoint:(1)The positive detection rate of ascites mNGS was compared with that in ascites culture(2)the incidence of adverse events and fatality at 28 and 90days.Secondary study endpoints:(1)The types and quantities of pathogens detected by ascites mNGS and those detected by ascites culture were compared.(2)The differences in the characteristics and prognosis of patients with positive ascites mNGS and positive ascites culture.Results:1.Analysis of the basic situation of the research objects(1)The composition ratio of age,gender and etiological of cirrhosis were analyzed in the ascites mNGS positive group and the ascites mNGS negative groupAccording to the results of ascites mNGS,60 patients were divided into ascites mNGS positive group and mNGS negative group.There were 24 patients in the ascites mNGS positive group,Age 55.38±8.67 years old,including 15 males,accounting for 62.5%,9 females,accounting for 37.5%,7 patients with hepatitis B cirrhosis,accounting for 29.2%,2 patients with hepatitis C cirrhosis,accounting for8.3%,and 8 patients with alcoholic cirrhosis,accounting for 33.3%,3 patients with Primary biliary cholangitis(PBC),accounting for 12.5%,3 patients with unexplained liver cirrhosis,accounting for 12.5%,1 patients with hepatitis B cirrhosis combined with alcoholic cirrhosis,accounting for 4.2%.There were 36 patients in the ascites mNGS-negative group,Age 51.92±9.74 years old,29 males,accounting for 80.6%,7females,accounting for 19.4%.13 patients with hepatitis B cirrhosis,accounting for36.1%,2 patients with hepatitis C cirrhosis,accounting for 5.6%,and 14 patients with alcoholic cirrhosis,accounting for 38.9%,2 patients with PBC,accounting for 5.6%,2 patients with unexplained liver cirrhosis,accounting for 5.6%,3 patients with hepatitis B cirrhosis combined with alcoholic cirrhosis,accounting for 8.3%.There were no significant differences between the two groups in terms of composition ratio of age,gender and etiological of cirrhosis(P>0.05).(2)Analysis of laboratory indexes of ascites mNGS negative group and ascites mNGS positive groupThe prothrombin(Z=-2.793,P=0.005),international standardized ratio(Z=-2.438,P=0.014),procalcitonin(Z=-2.367,P=0.018)and c-reactive protein(Z=-2.038,P=0.042)of the mNGS-positive group were significantly higher than those of the ascites mNGS-negative group,and the serum total protein(Z=-2.143,P=0.032)and Na~+(Z=-2.249,P=0.024)were significantly lower than those of the ascites mNGS-negative group.There were no significant differences between the two groups in terms of the total number of serum white blood cells,the absolute value of neutrophils,the absolute value of lymphocytes,the absolute value of neutrophil-lymphocytes,hemoglobin,platelet count,alanine aminotransferase,aspartate aminotransferase,total bilirubin,albumin,creatinine,urea nitrogen,prothrombin activity,fibrinogen,total number of ascites white blood cells,ascites Polymorphonuclear leukocyte count,ascites total protein(P>0.05).(3)Analysis on the basic situation of pathogens detected by ascites mNGS and ascites culture60 enrolled patients with cirrhotic ascites were tested for ascites mNGS.24patients were positive.A total of 50 strains were detected,including 18 Gram-positive bacteria,accounting for 36%;22 Gram-negative bacteria,44%;4 strains of fungi,accounting for 8%(aspergillus,etc.);6 strains of viruses(including parvovirus,Epstein-Barr virus,human herpes virus,and hepatitis B virus),accounting for 12%.The most gram-positive strains are Staphylococcus,and the most gram-negative bacteria are Escherichia coli and Acinetobacter.There were 14 positive strains in ascites culture,including 9 gram-positive bacteria,accounting for 64.3%;4gram-negative bacteria,28.6%;1 fungus,accounting for 7.1%.Further comparing the results of ascites culture and ascites mNGS detection,in ascites mNGS results,the strain types are more abundant than ascites culture,and the strain classification is more detailed than ascites culture.2.The value of ascites mNGS results in the diagnosis of SBP in patients with cirrhosisAmong the 60 patients with cirrhotic ascites included in this study,the classification of cirrhotic ascites was based on the results of ascites mNGS results.There are 3 patients with typical SBP,1 patients with culture-negative neutrophilic ascites(CNNA),21 patients with bacterial ascites,and 35 1 patients with aseptic ascites.Classification of cirrhosis ascites based on the results of ascites culture.A total of 0 cases of typical SBP,4 cases of CNNA,11 cases of bacterial ascites,45 cases of aseptic ascites were detected.Patients with cirrhotic ascites was diagnosed as SBP in25 cases(41.67%)by combining ascites PMN count with ascites mNGS detection,and in 15 cases(25%)by combining ascites PMN count with ascites culture.The positive rate(41.67%)of ascites PMN count combined with ascites mNGS detection in the diagnosis for SBP in patients with cirrhosis was significantly higher than that(25%)of ascites PMN count combined with ascites culture(χ~2=13.333,P=0.000).The sensitivity and specificity of ascites PMN count combined with mNGS in the diagnosis for SBP in patients with cirrhosis were 86.7%and 78.9%.3.The value of ascites mNGS results in the prognosis of patients with cirrhotic ascites(1)Comparison of the incidence of adverse events and mortality rate in ascites mNGS-positive group and ascites mNGS-negative group at 28 and 90daysThe incidence of adverse events in the ascites mNGS-positive group at 28 days was significantly higher than those in the ascites mNGS-negative group(58.3%vs22.2%,χ~2=7.239,P=0.007).The mortality in the ascites mNGS-positive group at28 days was significantly higher than those in the ascites mNGS-negative group(16.7%vs 0,χ~2=6.429,P=0.022).The incidence of adverse events in the ascites mNGS-positive group at 90 days was significantly higher than those in the ascites mNGS-negative group(66.7%vs 38.9%,χ~2=4.444,P=0.035).The mortality in the ascites mNGS-positive group at 90 days was significantly higher than those in the ascites mNGS-negative group(29.2%vs 2.7%,χ~2=8.678,P=0.003).(2)Comparison of the proportion of pre-ACLF patients in ascites mNGS-positive group and negative ascites mNGS-negative group at 28 days and90 daysDuring the 28-day follow-up period,the proportion of pre-ACLF patients in the ascites mNGS-positive group was significantly higher than that in the ascites mNGS-negative group(50%vs 5.6%,χ~2=15.901,P=0.000).During the 90-day follow-up period,the proportion of pre-ACLF patients in the ascites mNGS-positive group was significantly higher than that in the ascites mNGS-negative group(54%vs11.1%,χ~2=13.146,P=0.000).4.Analysis on the correlation between ascites mNGS results and complications of cirrhosisIn the ascites mNGS-positive group,6 patients were complicated with hepatic encephalopathy,accounting for 25%;5 patients were complicated with renal dysfunction,accounting for 20.8%,4 patients were complicated with other infections,accounting for 16.7%;4 patients were complicated with upper gastrointestinal bleeding,accounting for 16.7%.In the ascites mNGS-negative group,3 patients were complicated with hepatic encephalopathy,accounting for 8.3%;5 patients were complicated with renal dysfunction,accounting for 13.9%,4 patients were complicated with other infections,accounting for 11.1%;2 patients were complicated with upper gastrointestinal bleeding,accounting for 5.6%.The incidence of cirrhosis complications was compared between the ascites mNGS-positive group and the ascites mNGS-negative group.There were no significant differences in the incidence between the two groups in terms of combined with hepatic encephalopathy,renal dysfunction,other infections and upper gastrointestinal bleeding(P>0.05).5.Analysis on the correlation between ascites mNGS results and Cirrhosis related score systemThere were no significant differences between the two groups in terms of APRI index,FIB-4 index reflecting the degree of portal hypertension(P>0.05).The Child-Pugh score(11 vs 9.5,Z=-2.520,P=0.011),MELD score(18 vs13,Z=-2.313,P=0.020)and MELD-Na score(23.79 vs 14.60,T=-3.024,P=0.004)of the ascites mNGS-positive group were significantly higher than those of the ascites mNGS-negative group.6.The influence of pathogens in the ascites mNGS results on the prognosis for patients with cirrhotic ascites(1)Relationship between pathogen species in ascites mNGS positive results and prognosisThe incidence of 28-day adverse events in patients with ascites mNGS results containing 1 pathogen was significantly higher than the ascites mNGS-negative group(χ~2=11.517,P=0.001).The 28-day mortality rate of patients with ascites mNGS results containing 1 pathogen was significantly higher the ascites mNGS negative group(χ~2=7.527,P=0.043).The incidence of 90-day adverse events in patients with ascites mNGS results containing 1 pathogen was significantly higher than that in the ascites mNGS-negative group(χ~2=8.178,P=0.004).The 90-day mortality rate of patients with ascites mNGS results containing1 or 2 pathogen species was significantly higher than that in the ascites mNGS-negative group(χ~2=7.305,8.969,P=0.007,0.003).(2)Relationship between Gram stain types of bacterias in ascites mNGS positive results and prognosisThe incidence of adverse events and mortality at 28 days and the incidence of adverse events at 90 days in patients with ascites mNGS results containing Gram-positive bacteria,Gram-negative bacteria or mixed-bacteria infections were not significantly different from those in the ascites mNGS-negative group(P>0.05).The90-day mortality rate of the three types of patients was significantly higher than that of the ascites mNGS-negative group(χ~2=4.375,4.375,8.474,P=0.036,0.036,0.004).(3)Relationship between pathogen types in ascites mNGS positive results and prognosisThe incidence of adverse events in patients with ascites mNGS results containing simple virus-positive 28 days was significantly higher than that of the ascites mNGS-negative group(χ~2=8.273,P=0.018),and the 28-day mortality rate of patients with ascites mNGS results containing simple bacteria positive was significantly higher than that of the ascites mNGS-negative group(χ~2=7.163,P=0.025).The 90-day mortality rate of patients with ascites mNGS results containing simple bacteria positive or simple virus positive was significantly higher than that of the ascites mNGS-negative group(χ~2=6.294,15.919,P=0.012,0.000)Conclusions:1.Compared with ascites culture,ascites mNGS detection in patients with cirrhotic ascites can increase the positive detection rate of pathogens.2.The positive rate of ascites PMN count combined with ascites mNGS in diagnosis for SBP in patients with cirrhosis was significantly higher than that of ascites PMN count combined with ascites culture.3.The ascites mNGS detection results of patients with cirrhotic ascites are more comprehensive than ascites culture and guide the application of antibiotics earlier.4.Positive ascites mNGS results is related to the poor prognosis of patients with cirrhotic ascites.Compared with ascites culture,ascites mNGS results can more accurately predict the incidence of adverse events in patients with cirrhotic ascites During the 28-day and 90-day follow-up period. |
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