| Objective The purpose of this study is using Metagenomic Next-generation Sequencing(mNGS)for detecting pathogenic microorganisms in patients with sepsis to explore the effect and efficacy of mNGS collected from different infections(bloodstream infection,lung infection,etc.)and samples(blood,alveolar lavage fluid,etc.)by comparing them to common traditional pathogenic detection results.In the meantime,we explored the differences between the results of mNGS technology and traditional etiological diagnostic methods,to understand under which circumstances the use of mNGS is more conducive for improving the success rate of clinical diagnosis and treatment,so as to bring timely and effective treatment to patients with sepsis.Method This is a retrospective study,for which we collected 174 cases met the inclusion criteria by using mNGS sequencing technology for the pathogenic detection during the hospitalization in Wenzhou Central Hospital from January 2021 to September 2022.For each case,we collected the following three categories of information:1.Patient general information:age,gender,admission department,length of stay,chief complaint,main antibiotic treatment,discharge diagnosis;2.mNGS test-related information:test sample source,collection time,reporting time,test results(suspected pathogenic bacteria and non-pathogenic bacteria test results),drug resistance gene test status;3.Traditional testing methods related information:test items,sample sources,reporting time,test results.Statistical calculations were performed on the collected data by using SPSS(Version 26).We calculated the positive coincidence rate,sensitivity,specificity,and Kappa consistency test analysis of mNGS compared to traditional etiological testing.We analyzed the differences between the traditional etiological testing and the mNGS sequencing results in each sample sources.(The possible reasons for the positive coincidence rate,sensitivity,specificity calculation,and Kappa consistency test analysis were carried out between the mNGS sequencing results of whole blood and BALF-derived samples and the results of traditional etiology testing.)Results1.baseline characteristicsFor this study,174 patients hospitalized in Wenzhou Central Hospital from January 2021 to September 2022 and were screened by the inclusion and exclusion criteria.The average age was 58.83±18.16 years old,while the average length of stay was 14.35±17.64 days.The mean detection time of mNGS was 2.0±0.58 days.The average time of traditional culture detection was 2.31±0.62 days.According to the results of traditional etiology testing,the cases were divided into a group with positive findings in terms of pathogenic microorganisms,and a group with negative test results.A total of 73 cases were in the group with positive test results in the traditional etiology detection(hereinafter referred to as the positive transmission group),including 53 males(72.60%)and 20 females(27.40%).In contrast,101 cases were negative in the traditional etiological detection group(hereinafter referred to as the negative transmission group),including 65 males(64.36%)and 36 females(35.64%).In terms of age,the positive result group was in average 59.49±1 8.65 years old,while the group with negative results was 59.63±16.73 years old(P 0.959>0.05).In terms of hospitalization days,the mean stay of the group with positive results were 15.68±11.56 days,while the average stay in the negative group were 13.23±9.12 days(P 0.119>0.05).In terms of qSOFA score:the average score of the group with positive test results was 7(4.5,9),while those of the negative group was 6(4,8)(P 0.232>0.05).2.Pathogenic microorganisms detected by mNGSAmong the 174 sequenced samples,mNGS detected a total of 314 strains of pathogens that met clinical conditions,including 63 kinds of bacteria,a total of 143 strains;13 kinds of viruses,a total of 99 strains;and 10 types of fungi,a total of 42 strains.The rest also detected 5 kinds of special pathogens such as Question mark leptospira,Chlamydia psittacit,Rickettsia orteintalis,Mycoplasma pneumoniae,and Mycoplasma oralum,totaling 20 strains.In addition,2 cases of tumor genes were also detected,which was very important for the clinical decision making process.From the whole sample,top 10 bacteria strains were Pseudomonas aeruginosa,Klebsiella pneumoniae,Stenotrophomonas maltophilia,Acinetobacter baumannii,Streptococcus mitis,Streptococcus pneumoniae,Tuberculosis mycobacterium complex,Enterococcus faecium,Escherichia coli,Burkholderia polyphaga.The fungus was detected with Candida albicans,Pneumocystis jirovecii and Aspergillus fumigmoni more than others.Virus detections mainly included human herpesvirus type 4(Epstein-Barr virus),human herpesvirus type 5(CMV),human herpesvirus type 1 and human herpesvirus type 6B.Among the whole blood mNGS samples,60 cases were positive,19 different bacteria strains with a total of 25 strains,4 fungi strains with a total of 9 strains,10 virus strains with a total of 74 strains;4 special pathogens with a total of 15 strains were detected.From the 45 positive samples of alveolar lavage fluid,mNGS detected 37 bacteria with 75 strains in total,5 fungi strains with 18 strains in total,7 virus strains with 15 strains in total,and 2 strains of mycoplasma pneumoniae.3.The role of mNGS in detecting drug resistance of pathogenic microorganismsAmong the samples submitted for mNGS sequencing,a total of 6 sample cases were detected with drug resistance genes,namely Omp,Sul,OX A,SHV,Erm,and Tet.Among the six cases,Case 1:the drug-resistant gene Omp was detected in the first case,and the possible source was Klebsiella pneumoniae,which was in accordance with the clinical culture results.Drug-resistant genes indicated drug-resistant drugs were Carbapenems,Monocyclic lactams,cephalosporins,while the results of clinical culture drug sensitivity suggested drug resistances against cefuroxime,cefuroxime axetil,and cefuxime.Case 2:The Sul gene was detected in the case,and its possible sources include the possible pathogenic escherichia coli and the suspected commensal klebsiella pneumoniae detected by mNGS sequencing,so it cannot be determined whether it is the drug resistance carried by the pathogenic bacteria gene.Case 3:Several numbers of pathogenic bacteria were detected in the cases,including the sources of three drug-resistant genes,Sul,OXA,and SHV.Among them,the drug-resistant drugs included carbapenem antibiotics,but the clinical drug sensitivity results suggested that imipenem was sensitive,and the patient improved after anti-infection treatment with imipenem and cilastatin.Case 4:The source of the Sul gene of the case was in accordance with the actual detection of pathogenic bacteria,the clinical use of antibiotics and sequencing showed no conflict with drug-resistant drugs,while the treatment effect was positive.Case 5:The source of the Erm gene detected in the case was considered to be enterococcus faecium,but it was not detected in multiple clinical cultures,while the treatment with imipenem and cilastatin was effective.Case 6:The sample was BALF from a patient with interstitial pneumonia,and four drug-resistant genes Tet,Erm,OXA,and Sul were detected.The possibility of contamination by pathogenic microorganisms is high,and it is only for reference in clinical practice.4.Comparative analysis of mNGS technology and traditional etiological examination results in different samplesAmong the 174 samples in this study,131 cases were positive in the mNGS test which were also consistent with clinical findings,and 43 were negative;73 cases were positive and 101 were negative in traditional etiological examination;mNGS and traditional etiology.A total of 65 cases were tested positive,where 35 cases were negative tested.According to statistics,the coincidence rate of mNGS positive rate was 49.62%,the negative coincidence rate was 81.40%,the sensitivity was 89.04%,the specificity was 34.65%,and the kappa value was 0.213.In this study,the number of positive and negative cases obtained by traditional etiological examination and mNGS sequencing were compared with samples from 12 different sources.We found that the positive rate of mNGS sequencing results in whole blood samples and sputum samples was significantly higher than in traditional etiological examinations.For this reason,we further compared the number of pathogen strains obtained by mNGS sequencing with the number of pathogen strains obtained by traditional etiological detection,as well as the proportion of the number of different types of pathogen strains.Due to the analysis,we found this difference is partly resulted by the fact that mNGS has a greater advantage in the detection of viral infections.For example,in 76 whole blood samples,a total of 10 viruses and 74 strains were detected,accounting for 60.16%of the total number of pathogenic microorganism strains detected.However,the ability of the traditional etiology to diagnose viremia relies on the combination of various inspection methods,clinical symptoms and manifestations.Only 13 virus strains were detected,accounting for 38.26%of the total number of detected pathogenic microorganism strains.5.Comparative analysis of the test results of mNGS technology with bloodstream infection and traditional etiological examination in sepsis patientsA total of 76 whole blood mNGS samples were collected.According to statistics,the coincidence rate of mNGS positive rate was 36.67%,the negative coincidence rate was 75.0%,the sensitivity was 84.62%,and the specificity was 24.0%.The Kappa value was 0.066.6.Comparative analysis of the results of mNGS technology with pulmonary infection and traditional etiological examination in sepsis patientsA total of 66 cases of alveolar lavage fluid samples were collected.According to statistics,the positive coincidence rate of mNGS was 60.0%,the negative coincidence rate was 80.95%,the sensitivity was 87.10%,and the specificity was 48.57%.The kappa value was 0.348.Conclusion1.The mNGS sequencing technology has shown high sensitivity and positive rate,which could simultaneously sequence multiple types of pathogenic microorganisms indiscriminately in a short time.This provides a new option for sepsis patients who needs early clear etiological diagnosis.2.In mNGS,there are still cases of false positives caused by sample contamination,difficulties in nucleic acid extraction,or false negative results caused by improper storage and degradation.Therefore,the clinical decisions still do rely on the final judgment of the clinician.3.In terms of virus detection,mNGS has shown a higher positive detection rate than traditional etiological detection methods,which can provide a certain basis for clinical diagnosis and therapy.4.There are still great limitations in the application of mNGS in the detection of drug resistance genes.Currently,it is only for reference in clinical practice,wherefore there is more room for development in the future.5.For some special pathogens,there are difficulties to extract nucleic acids,as for example mNGS sequencing of mycobacterium tuberculosis.Even if the number of detected sequences is low,it may be possible to focus on the possibility of pathogenic microorganisms. |
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