Study On The Methodology Of Metagenomic Nanopore Sequencing In The Detection Of Pathogens

Background Infectious diseases remain leading causes of morbidity and mortality among all patient populations worldwide.Early identification of pathogens and their antibiotic resistance are essential for the management and treatment of patients affected by infectious diseases.However,the traditional methods for pathogen identification have some drawbacks,such as time-consuming,low sensitivity,narrow range of detection and so on.A delay or misidentification of pathogens has a significant effect on definitive antimicrobial therapy.Metagenomic nanopore sequencing has the potential to detect all pathogens by sequencing all nucleic acids from specimens,and this method also can real-time analyze long reads to achieve the aim of rapid and accurate identification of pathogens.However,there are still many challenges in the application of nanopore-based metagenomic sequencing(m NGS)in pathogen detection,such as the difficulty of undifferentiated access to DNA and different criteria for pathogen identification.Objective This study aimed to optimize the method of DNA extraction and compare the ability of sequencing indicators for pathogen identification in specimens of infectious disease,and develope a m NGS workflow for a detection of pathogens,and further evaluate the sensitivity,specificity and diagnostic speed of this m NGS method.Methods PartⅠDNA was extracted from 20 urine samples of patients with urinary tract infection by mechanical fragmentation method,enzymatic cleavage method and control method,and downstream nanopore sequencing was carried out.Three different DNA extraction methods were evaluated in terms of yield and integrity of the extracted DNA,identification of specific species and microbial diversity,and the most compatible DNA extraction method for nanopore sequencing was selected.PartⅠⅠendotracheal aspirate(ETA)samples from 63 patients with suspected ventilator-associated pneumonia(VAP)were sequenced by nanopore-based m NGS.The receiver operating characteristic(ROC)curves were established to compare the pathogen identification performance of target pathogen reads,reads percent of microbes and relative abundance,then the m NGS index with the highest diagnostic value was selected,and the threshold standard was determined according to the best Youden index.Next,the evaluation of the accuracy of the method was performed comparing with the microbial culture and the composite standard,respectively.Then,the pathogens detected by m NGS and microbial culture were compared byχ~2test.Finally,the drug resistance genes were analyzed by m NGS.Results PartⅠFor DNA yield,the concentration of DNA extracted by mechanical fragmentation method(1.1ng/μL,IQR 0.2-8.2ng/μL)was significantly lower than that by control method(4.0ng/μL,IQR 1.1-14.9ng/μL)(P<0.0001),but there was no significant difference between enzymatic cleavage method(2.9ng/μL,IQR0.9-23.3ng/μL)and control method(P>0.05).For DNA integrity,the average length of microbial reads obtained by enzymatic cleavage method(1045.7bp,IQR521.3-1488.3bp)was significantly longer than that of control method(564.6bp,IQR246.0-805.3bp)(P<0.0001)and mechanical fragmentation method(515.8bp,IQR338.9-1001.7bp)(P<0.01).Among all 20 samples,pathogens identified by culture in 15(75%)were identified by control method,14(70%)were identified by mechanical fragmentation method,and all of them were successfully identified by enzymatic cleavage method(100%).For the diversity of microbial community,compared with the control method and mechanical fragmentation method,the number of microbial species obtained by enzymatic cleavage method was more,and the Shannon index was also higher.PartⅠⅠ:The ROC curves showed that relative abundance has the highest diagnostic value(area under the curve=0.847),and the corresponding threshold for pathogen identification was 9.93%.Compared with the microbial culture,the sensitivity,specificity and accuracy of m NGS were 91.3%,78.3%and 86.5%,respectively.While compared with the composite standard,the sensitivity,specificity and accuracy of m NGS were 97.4%,100%and 98.1%,respectively.The detection rate of mixed infection by m NGS(47.6%)was significantly higher than that of microbial culture(27.0%)(P=0.017).The drug resistance genes detected by m NGS were consistent with the drug resistance phenotype.Patients with late-onset VAP had a significantly greater proportion of drug resistance genes in their respiratory microbiome compared to those with early-onset VAP(P=0.041).Moreover,the median turnaround time of m NGS was4.43 h,while that of routine microbial culture was 72.00 h.Conclusion The study indicated that enzymatic cleavage method had the highest compatibility with nanopore-based m NGS analysis,which was more conducive to accurate and reliable identification of pathogens.m NGS had high accuracy and short turnaround time for pathogen detection in VAP,indicating the method developed in this study can accurately identify VAP pathogens and enable to predict drug resistance within 5 h of ETA sample receipt by mNGS.