An Investigation On The Diagnosis Of Sepsis Based On The Next-generation Sequencing

Part one:Study on the application of Next-generation Sequencing in the diagnosis of bloodstream infectionObjectiveTo explore the sensitivity,specificity and accuracy of Metagenomic Next-generation Sequencing(m NGS)for the diagnosis of bloodstream infection,and whether it can provide new ideas and methods for the diagnosis of patients with bloodstream infection,so as to bring timely and effective treatment for the treatment of patients with bloodstream infectionMethods and materialsBased on the inclusion and exclusion criteria,data were collected from all ICU patients who were admitted to Changzheng Hospital of Nave military medical university from February 1st 2018 to June 30 th 2019,and a total of 74 ICU patients with sepsis were enrolled.Separate blood samples of 5 ml were collected from ICU patients for blood cultures and metagenomic next-generation sequencing.The basic information of each patient was collected,including gender,age(years),time of admission,time of discharge,time of each blood culture and retention of blood samples sequenced by m NGS,blood culture results(specific pathogenic microorganisms),microorganisms measured by m NGS and relevant pathogenic microorganisms results of culture in other parts within 24 hours before and after the sampling time.Statistical calculations were performed in R(version3.5.0).According to the abundance value of pathogens obtained from the sequencing of m NGS,the sequence was carried out.The sensitivity,specificity,positive coincidence rate,negative coincidence rate and Kappa consistency analysis of m NGS compared with blood culture were calculated.Then,the sensitivity,specificity,positive coincidence rate and negative coincidence rate of three kinds of bacteria commonly used in ICU were selected to calculate with m NGS,and kappa consistency test analysis was carried out.Results1、Demographic characteristic:This study included patients who were suspected of having bloodstream infection in ICU ward of Changzheng Hospital affiliated to Naval Medical University from February2018 to July 2019.According to the inclusion criteria,118 samples from 74 patients wereincluded in this study.Based on the results of blood culture(herein after referred to as BC),the current "gold standard" for the diagnosis of bloodstream infection,the patients were divided into BC negative group and BC positive group.The baseline is shown in Table 1,there were 38 males(64%)and 21 females(36%)in BC negative group,12 males and 3females(20%)in BC positive group(P > 0.05).The average age of BC negative group was 58.07 ±16.32 years,and in BC positive group the average age was 58.27 ±16.49 years(P > 0.05).In terms of ICU hospitalization days,the hospitalization days were 20(11-36.5)days in BC negative group and 29(18-65)days in BC positive group(P > 0.05).With regard to the outcome of the patients,27(46%)died in the BC negative group,32 survived(54%),12(80%)died in BC positive group,and 3(20%)survived(P > 0.05).2、Pathogenic microorganisms identified by NGSA total of 1438 date elements were acquired from 118 blood samples,including 13 m NGS-negative samples.In addition,in positive results,totally 205 bacteria,62 fungi,and20 virus were identified.3、Comparison of results between blood culture and m NGSEighteen positive samples,were identified,including seventeen m NGS-positive sample.In 100 negative samples based on blood cultures,m NGS produced 88 positive samples,with the sensitivity of 94.44%,specificity of 12%,positive coincidence rate of16.19% and negative coincidence rate of 92.315.the Kappa value was 0.018963.4、Comparison of three most frequently common G-bacteria in ICU between blood culture and m NGSIn positive blood culture results,we found 6 samples with Klebsiella pneumoniae,one with Acinetobacter baumannii,and none with pseudomonas aeruginosa,whereas for m NGS results,25 samples were found to be infected with Klebsiella pneumoniae,32 with pseudomonas aeruginosa,and 11 with Acinetobacter baumannii.For the diagnosis of Klebsiella pneumoniae,the sensitivity was 100%,the specificity was 77.68%,and the positive coincidence rate was 19.35%.The negative coincidence rate was 100%.Kappa value was 0.261392..For pseudomonas aeruginosa,the specificity was 72.88% and the negative coincidence rate was 100%.For Acinetobacter baumannii,the sensitivity was100%,specificity was 90.60%,and the positive coincidence rate was 8.33%.The negative coincidence rate was 100%.Kappa value was 0.140397.5 、 Comparison of the positive results of three most frequently common G-bacteria between all parts culture and m NGSWe made a comparison of positive results from Klebsiella pneumoniae,Acinetobacter baumannii,and pseudomonas aeruginosa based on infection site samples(blood,Sputum,urine,purulent secretion,alveolar lavage fluid,focus drainage fluid,and so on)between culture and m NGS.For Klebsiella pneumonia,the sensitivity was 47.22%,the specificity was 82.93%,the positive coincidence rate was 54.84%,and the negative coincidence rate was 78.16%,with Kappa value was 0.313712.For pseudomonas aeruginosa,the sensitivity was 33.33%,the specificity was 73.79%,the positive coincidence rate was 15.63%.,and negative coincidence rate was 88.37%,with Kappa value was 0.047972.For acinetobacter baumannii,the sensitivity was 21.95%,the specificity was 96.10%,the positive coincidence rate was 75%,and the negative coincidence rate was 69.81%,with Kappa value was 0.216319.ConclusionCompared with the blood culture technology,Next-generation sequencing has the advantages of high sensitivity and specificity,short time and wide detection range.Moreover,this technology can provide the etiology basis for clinicians in the early treatment of patients suspected of bloodstream infection but negative blood culture detection,and provide a new idea for early diagnosis and treatment of bloodstream infection.However,in the process of diagnosing bloodstream infection,m NGS can not avoid environmental pollution,antibiotic interference and other confounding factors,so the diagnosis of bloodstream infection can not rely on a single detection technology,but also combined with blood culture results and clinical indicators.Part two :To explore the impact of positive virus infection and bacterial diversity on sepsis patients based on m NGSObjectiveTo investigate the performance of m NGS-based diagnostic technique for the identification of potential bacterial and viral infections and the effect of viral infection on the survival rate of intensive care unit(ICU)sepsis patients,and the correlation between bacterial diversity and severity of sepsis patients.Methods and materialsBased on inclusion criteria,a total of 74 ICU patients with sepsis were enrolled.Separate blood samples of 5 ml were collected from ICU patients for blood cultures and metagenomic next-generation sequencing,when the patients’ body temperature was higher than 38.5 ℃.The demographic data,including gender,age,duration,ICU scores,and laboratory results,of the patients,were recorded.Statistical calculations were performed in R,including univariate and multivariate Cox proportional hazards models,to identify independent prognostic factors for 90-day mortality.Analyses of correlation between bacteria strain diversity and sepsis severity were performed using linear regression with coefficient of determination(R2).Results1、Demographic characteristic:Patients were divided into two groups,the ‘virus positive group’ and the ‘virus negative group,according to the presence or absence,respectively,of concomitant viral infection confirmed by NGS.There were 27 male and 10 female patients in the virus negative group,whereas there were 23 male and 14 female patients in the virus positive group(P>0.05).The mean age of the virus negative group(53.86±17.03 years)was lower than that of the virus positive group(62.35±14.43 years)(P<0.05).Patients with concomitant viral infection experienced a longer period of ICU hospitalization than those in the virus negative group: 39.11±28.18 days vs.24.62±28.05 days,(P<0.05).Patients in the virus positive group had higher APACHE 2 and SOFA scores than those in the virus negative group(both P<0.01).However,no statistically significant differences were observed in the laboratory infection results.2、Correlation of viral infection with sepsis severity identified by Cox regression analysisWe employed Cox regression analysis.In the entire cohort of 74 patients with sepsis,six variables(sex,WBC count,C-reactive protein level,lactic acid level,SOFA score,and concomitant viral infection)were observed to correlate with the severity of sepsis based on Cox univariate regression analysis.Further,to correct for possible confounding factors,data for these six variables were adjusted based on the Cox multivariate regression model.In all patient groups,virus label was closely associated with increased hazard ratio for sepsis severity.3、Correlation of viral infection with survival rate identified by Cox regression analysisThe impact of concomitant viral infection on the survival rate was significantly obvious(P<0.05).In addition,from 25 days after admission,the survival rate decreased continuously for those in the virus positive group,whereas it did not change significantly in the virus negative group.These results suggest that concomitant viral infection correlates closely with sepsis severity and has a negative impact on the survival of patients with sepsis.4、Relationship between bacterial species and severity of sepsisWe first investigated the correlation between the bacteria varieties and sepsis severity.Patients with less than ten types of bacteria,and for patients with more than 10 types of bacteria,no significant correlation was observed between the bacteria varieties and sepsis severity in either laboratory results or ICU scores.Taken together,these results indicate that bacteria variety did not correlate with the severity of sepsis.ConclusionThe present study revealed that concurrent viral load correlated closely with sepsis severity and the survival rate of sepsis patients in the ICU.In addition,bacteria varieties did not correlate with sepsis severity.