Rapid And Highly Sensitive Detection Of Multiple Pathogens Directly From Whole Blood

Bloodstream infections(BSIs)are one of the serious clinical infections,and the incidence and mortality of bloodstream infection are increasing in recent years.Patients with suspected bloodstream infections need immediate treatment.Every minute counts.However,the current diagnosis standard,blood culture,cannot meets the urgent demands of rapid identification of pathogenic bacteria.Extraction and detection of pathogenic bacteria directly whole blood may break the diagnostic bottleneck.In this way,we can identify pathogenic bacteria of bloodstream infection rapidly,and guide antibiotics use and improve the survival rate of patients.In chapter one,we reviewed the current situation of bloodstream infections and the common diagnostic methods of bloodstream infection.Then the difficulties and solutions of direct whole blood detection are summarized.Finally,the purpose and content of this essay are put forward.In chapter two,we established a method for rapid enrichment of pathogenic bacteria and nucleic acid extraction from whole blood,which included four steps:selective lysis of human whole blood,bacterial filtration and recovery,lysis of bacteria and nucleic acid extraction,and the whole process took less than 1.5 h.This method can collect multiple pathogens as low as 10 CFU from 0.5 mL-5 mL whole blood.The recoveries were between 57%and 105%(50 CFU),and the human genome residue was about 0.01 ng/μL(0.5 mL whole blood).Compared with commercial kits,this method has three obvious advantages:short extraction time,high efficiency of bacterial nucleic acid recovery,and low residue of human genome.In chapter three,we applied the rapid nucleic acid extraction method of pathogens from whole blood to a highly sensitive and multiple detection system for common pathogens of bloodstream infection,forming a complete process of detecting multiple pathogens from whole blood.The overall time of the process was 5 h,and 21 pathogens of bloodstream infection were covered.The detection limits of six bacteria were obtained by using simulated whole blood samples,of which the detection limits of E.coli,K.pneumoniae,S.aureus,A.baumannii and E.faecium were 10 CFU,and the detection limits of P.aeruginosa were 50 CFU.The system could also detect mixed infection samples.37 patient cases(73 whole blood specimens)were tested by this procedure.A total of 14 cases were positive and 23 cases were negative,with a positive rate of 37.8%,which was significantly higher than the results of blood culture(5.4%).In addition,all positive blood culture specimens covered by this system can be detected correctly by this system.By the analysis of typical cases,it can be noted that the detection results of this system are consistent with the actual infection indications of patients,suggesting that this system can make up for the deficiency of low sensitivity of blood culture and vulnerability to be affected by antibiotics.This procedure can provide etiological basis for the timely treatment of patients with bloodstream infection.