| Background and Objectives:Bacterial meningitis(BM)is an acute or chronic inflammatory disease of central nervous system(CNS),caused by various bacterial infection,with a high morbidity,mortality and disability rate.In recent years,the infectious disease patterns undergo a series of changes as the use of vaccines and the alter of bacterial resistance,that the morbidity of BM increased gradually.Traditional methods,including isolation,culture and identification of bacteria from blood or cerebrospinal fluid(CSF),are time-consumed and high operation intensity with lower positive rate.Although molecular biological diagnostic techniques can improve the proportion of pathogens detected,they have limitations in clinical application.The aim of this study:(1)To evaluate the potential applied value of metagenomic next-generation sequencing(mNGS)technology in the diagnosis of adults BM.(2)To inspect the diagnostic effectiveness of mNGS technology in detecting pathogen of CSF when compared with traditional clinical pathogen detection methods and comprehensive pathogen evidences of CSF.Methods:(1)The subjects:In a retrospective case-control study,20 adult patients who diagnosed with probable BM were selected in Henan Provincial People’s Hospital from March,2017 to March,2019.All their medical history,physical examination,laboratory tests and ancillary examination results were recorded in detail.(2)Diagnostic criteria:There exist clinical symptoms or signs of meningitis or encephalitis;The routine leukocytes of CSF is greater than or equal to 500×10~9/L,with or without evidence of bacteriologic in blood or CSF.(3)Research methods:The CSF specimens(2-3 ml)were collected and stored at-80°C within 30 min after the routine lumbar examination.Genomic DNA was isolated from 300μl of CSF samples,then the extracted DNA was fragmented to a DNA smear of 200–300bp by using ultrasonic waves.High-quality sequencing data were generated following filtering of low-quality data,the linker and primer sequences were filtered out,followed by rolling circle amplification of magnitude to generate DNA Nano Ball(DNB).DNA sequencing was performed using the BGISEQ-500 sequencing platform following loading of the prepared DNB nanospheres onto the array of silicon cores.Subsequently,the remaining data were aligned using microbial genome database after the human genome sequences were excluded.Data Analysis:Statistical analysis was performed using SPSS22.0 software.Calculators information can be described by“the number of cases”or“rate”.We also characterized the sensitivity and specificity of each methods.Comparison of diagnositic methods between paired samples were tested using the McNemar test.P values of<0.05 was considered to be statistically significant.Results:1.Among the 20 adult patients with probable BM,5 pathogens were detected positive,including 2 listeria monocytogenes(LM)by using blood culture,1 klebsiella pneumoniae by using CSF culture,and 2 mycobacterium tuberculosis complex in cerebrospinal fluid by using Xpert MTB/RIF assay.Under the condition of bacteria genome sequence greater than 1,8 CSF samples were sequenced to be positive by using NGS after filtered the sequences of the background.2.Taking clinical diagnosis of probable BM as gold standard,which had CSF leukocytes≥500×10~6/L.The sensitivity of CSF culture,CSF comprehensive pathogen evidences,clinical overall pathogen evidences and NGS are 5%(1/20),15%(3/20)and 25%(5/20)of,and 40%(8/20).3.McNemar test indicated there had no difference(P=0.375>0.05)in the pathogens detection rate of CSF between mNGS and clinical overall pathogen evidences.4.McNemar test indicated there had no difference(P=0.219>0.05)in the pathogens detection rate of CSF between mNGS and CSF comprehensive pathogen evidences.Conclusion:The application of mNGS in adult patients with probable BM,of which CSF routine cell count≥500×10~6/L,can not be considered to improve the proportion of pathogen detected,for the limited sample size.Further research need to base upon more large survey sample. |
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