A Comparative Methodological Study Of The Pathogenic Diagnosis Of Bloodstream Infections And A Preliminary Discussion Of The Value Of DdPCR In The Clinical Application Of Dynamic Monitoring Of Bloodstream Infections

Objective This study is a prospective study to evaluate the performance of droplet digital PCR(ddPCR)for pathogen identification in bloodstream infections by comparing it with metagenomic next-generation sequencing(NGS)and blood culture,and further explores the effect of ddPCR on the prognosis of BSI patients by monitoring pathogen DNA load and species changes in blood.Methods Patients with suspected bloodstream infections admitted to the********from April 2019 to January 2021 were recruited for this study.In 45 cases,blood specimens were collected for mNGS,blood culture analysis and droplet digital PCR technique for comparative methodological study of the pathogenic diagnosis of bloodstream infection.102 cases were studied for ddPCR dynamic monitoring.When BSI was suspected,whole blood was taken for both blood culture and ddPCR testing,followed by at least one follow-up blood culture.The 2nd and 3rd ddPCR tests were performed on days 3-4 and 6-7 of monitoring,respectively.Study data were analysed using SPSS 19.0 for data analysis(IBM Corporation,Armonk,NY,USA).Normally distributed continuous variables were expressed as mean ± standard deviation(x ± s)and analysed using Student’s t-test.Non-normally distributed continuous variables are expressed as median(M)and upper and lower quartile spacing(P25,P75)and analysed using the MannWhitney U test,and categorical variables are expressed as n(%)and analysed using Fisher’s exact test,with P < 0.05 being considered a statistically significant difference.Results In the methodological comparison study,45 patients with suspected bloodstream infection were included and 60 blood samples were collected,of which 50(83.3%)were positive by ddPCR,41(68.3%,without virus)by mNGS and 10(16.7%)by blood culture.Of the 10 positive blood cultures,nine cases were consistent between the mNGS and ddPCR methods.Head-to-head comparisons showed that ddPCR was faster(~4h vs ~2d)and more sensitive(88 vs 53 detectable pathogens)than mNGS in its own detection range,and mNGS detected a wider range of pathogens than ddPCR(126 vs 88 detectable pathogens,including viruses).ddPCR detected a total of 17 AMR genes,14 of which were bla KPC genes and 3 mec A genes.In another part,102 patients with suspected BSI were included in the study of ddPCR dynamic monitoring of bloodstream infection,of whom 16 were positive for both blood culture and ddPCR with consistent pathogenicity.ddPCR dynamic monitoring of these 16 cases was performed and found that compared to 28-day survivors,non-survivors were older and at the 2nd(3-4 days)and 3rd(6-7 days)The pathogen DNA content was higher in the ddPCR assay(P < 0.01).In the first three ddPCR assays,there was a decreasing trend in the change in pathogen DNA load for those who survived 28 days,while there was an increasing trend in pathogen DNA load for those who did not survive 28 days.In addition,the number of pathogenic species in BSI patients who survived 28 days decreased during effective antibiotic treatment.Conclusion ddPCR is faster and more sensitive than mNGS detection,and can be used to monitor changes in DNA load and type of pathogenic bacteria in blood,which has clinical significance for judging the efficacy of antibiotics and accurate prognosis assessment in BSI patients.