Rapid Molecular Diagnosis Of Major Pathogens In Bloodstream Infections

Bloodstream infections(BSIs)are a serious systemic infectious disease.The morbidity and mortality rates remain high over the past few years.Thus,rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for prompt initiation of appropriate therapy.The time-consuming blood culture-based approach,which often leads to the missed optimal time-window in clinical treatment,still represents a gold standard for the identification of pathogens.Howere,this problem could be overcome with the use of molecular diagnostic tools that often provide a more rapid diagnosis in septic patients.In chapter one,we briefly reviewed the background of bloodstream infections,microbiological etiology,and drug resistance of pathogens.Then we analyzed and compared several common diagnostic methods for the identification of pathogens in bloodstream infections.Finally,we proposed the purpose,content and methods of this thesis.In chapter two,we established a new molecular diagnostic method for detection of bloodstream infections pathogens based on MeltArray method.It is a real-time,high-throughput,fast and fully closed molecular method,and suitable for the identification of pathogens from blood cultures.This method can simultaneously detect 21 clinically common pathogens in bloodstream infections within 3 hours.The limit of detection of the system was 5-50 copies per reaction.It had a good specificity,precision and the ability to identify polymicrobial infections.We used 385 clinical blood-culture specimens for clinical evaluation of the system.We then compared the results of our method with the results obtained by MALDI-TOF MS,and Sanger sequencing was used as the reference method.The results showed that the overall concordance between our method and MALDI-TOF MS was 89.1%(kappa?0.603,P<0.001).If targets outside the detection range of this system were excluded,the overall concordance was increased to 95.3%(kappa=0.804,P<0.001).In addition,the consistency rates of all detected targets were greater than 98%(Kappa value was between 0.797 and 1,all P values were less than 0.001),and the specificity(98.6-100%)and sensitivity(88.9-100%)of all detected targets were high,indicating that the system established in this research had a good consistency with MALDI-TOF MS.The accuracy of MeltArray assay was 100%when referred to Sanger sequencing.The results showed that the newly developed assay is potentially an adaptable tool for the rapid detection of pathogens from blood cultures in clinical diagnosis with the advantages of high accuracy,simplicity,rapidity and automatic data processing.In the third chapter,based on the above MeltArray assay,we carried out research on pathogen nucleic acid pre-enrichment,in order to further improve the sensitivity of the system to meet the requirements of direct detection of whole blood samples.We introduced HAND system to a multiplex PCR for the nucleic acid pre-enrichment,and combined with the MeltArray to achieve the identification of 21 types of pathogens.By pre-enriching the nucleic acid,we increased the analytical sensitivity of the detection system to 5 copies per reaction.We also found that this established system had a good tolerance to human genomic DNA.In addition,we spiked bacteria of different concentrations into human whole blood collected from healthy donors to investigate the analytical sensitivity capabilities in blood,and even 20 CFU/mL loads of bacteria could be tested.Finally,we used the established method to detect 27 clinical blood specimens,and 4 positive specimens were detected,two more positive detected than blood culture.The two inconsistent samples were further verified by a TaqMan assay,which results were completely consistent with the high sensitive system.The above results showed that by using pathogen DNA pre-enrichment,direct detection of pathogen from whole blood could be achieved with even higher sensitivity than blood culture.