Accurate Diagnosis Of Pathogen Of Peritoneal Dialysis Associated Peritonitis

Objective: 1.To observe the pathogenic distribution of bacteria in Peritoneal dialysis-associated peritonitis(PDAP).2.Exploring the application of Droplet Digital PCR,ddPCR)to detect and identify pathogenic bacteria in PDAP.Methods: The article included two parts.Part one: Retrospectively analyzed distribution of pathogenic bacteria in318 peritonitis from January 2009 to September2016 in PD center.Part two: 1.Primary bacteria which are epidermis staphylococcus aureus,staphylococcus aureus,escherichia coli,and enterococcus faecalis were found out according to pathogenic bacteria distribution of 318 PDAP in eight years in PD center.2.Four specific primers and probes were designed according to references.Target fragments were amplified and verified by PCR after extracted DNA of standard strains.3.PD fluids specimens were collected in duplicate in 30 PDAP patients,one for traditional bacterial culture,another for subsequent ddPCR detection and kept in-80°C refrigerator.The DNA extract of standard strains and sterile double distilled water(ddH2O)were positive control and negative control respectively.Results: Part one: 185 cases were positive cultured in 318 PDAP(58.1%),a total of194 strains of pathogenic bacteria were identified,included 131 Gram-positive strains(67.5%),49 Gram-negative strains(25.2%)and 14 fungi(7.2%).Among Gram-positive strains,staphylococcus epidermidis,staphylococcus aureus and enterococcus faecalis were leading pathogens,25.9%,10.6% and 8.4%respectively.Escherichia coli was major pathogens(40.8%)in gram-negative strains.Part two:(1).Bacterial culture results: the positive rate of 30 PDAP specimens culture was 43.3%(13/30).3 cases of staphylococcus aureus,2 cases by staphylococcus epidermidis and 1 case of e.coli,2 cases of staphylococcus hemolysis in,1 case of walter type staphylococcus aureus,1 case of verdigris false single spore fungus,1case of nearly smooth candida,1 case of e.sakazakii,1 case of microbe wereidentified in 13 positive cultured specimens.(2).digital PCR results: the detection results of specific probe primers in 4 kinds of bacteria were same as traditional culture.One case of staphylococcus aureus and enterococcus faecalis were identified in culture negative specimens by digital PCR.(3).The time for traditional bacterial culture is 82.57±14.13 hours,the detection time of digital PCR is about 6.5 hours.Conclusion: 1.The main pathogenic bacteria are staphylococcus epidermidis,staphylococcus aureus and enterococcus faecalis in Gram-positive bacteria and escherichia coli in gram-negative strains.2.Digital PCR method is a timeliness,sensitivity and specificitymethod compared to traditional culture.