The Application Of Metagenomic Next-generation Sequencing To Identify Pathogens Causing Tuberculous Meningitis

BackgroundMetagenomic next generation sequencing(mNGS)is a kind of unbiased analysis method,which can theoretically identify all known pathogens by sequencing the total DNA or RNA in samples and then identifying bacteria,viruses,fungi,parasites and other pathogens through bioinformatics analysis software.In 2014,Wilson et al first reported the application of mNGS in the diagnosis of leptospirosis in patients with severe combined immunodeficiency.In 2015 Perlejewski et al and in 2016 Guan et al reported the application of mNGS detection in the diagnosis of central nervous system virus infection.In 2016,Yao et al reported the application of mNGS detection in the diagnosis of Listeria meningoencephalitis.In 2018,He et al used mNGS to diagnose Vibrio vulnificus infection in immunocompromised patients.In addition,in a small cohort study in 2017,Salzberg et al recruited 11 patients with suspected central nervous system(CNS)infection,and mNGS technology had been successfully applied in brain and spinal cord biopsies.In 2018,a retrospective analysis of the results of mNGS in cerebrospinal fluid of 99 children with bacterial meningitis revealed 10 different bacterial pathogens.In the same year,a large prospective study of 511 samples from patients with infectious diseases showed that mNGS technology was more sensitive to pathogens than pathogen culture,and its results were less affected by antibiotic use.Fan et al reported the application of mNGS in the diagnosis of neurobrucellosis and neurocysticercosis in 2018.In 2018,Ai et al simultaneously detected mNGS in liver tissue and peripheral blood samples,and finally diagnosed local hepatic tuberculosis.MethodmNGS technology is currently used in more and more infectious cases,but its application in the diagnosis of tuberculous meningitis(TBM)is still unclear.The traditional methods to diagnose TBM are:cerebrospinal fluid bacterial culture,acid-fast staining,PCR,Xpert MTB/RIF,mNGS and so on.The culture of Mycobacterium tuberculosis in cerebrospinal fluid is the gold standard for TBM diagnosis,but it takes two to four weeks to get the result.The application of mNGS in the diagnosis of TBM is still absent.From 2015 to 2018,130 specimens of cerebrospinal fluid(CSF)mNGS were examined in our center.Among them,17 cases were diagnosed as tuberculous meningitis.In addition of patients with TBM,patients with bacterial/cryptococcal meningitis were mNGS positive controls and a patient with autoimmue-encephalitis was an mNGS negative control.We reviewed the results of their mNGS and the conventional methods and compared their sensitivity,specificity,et al.ResultsThe Mycobacterium tuberculosis complex(MBTC)as shown in the results of mNGS test includes M.tuberculosis,M.africanum,M.bovis and M.canettii.Thirteen cases were found to be positive by mNGS test,of which 12 cases were found to be Mycobacterium tuberculosis complex.Traditional detection methods for tuberculous meningitis include culture of Mycobacterium tuberculosis(M.tuberculosis)in cerebrospinal fluid,acid-fast staining and PCR examination.Culture of M.tuberculosis was positive in only 1 case.The results of PCR were positive in 3 cases,acid-fast staining was positive in 8 cases,mNGS was negative in 4 cases with positive acid-fast staining.One patient was finally diagnosed as cryptococcal meningoencephalitis with TBM.ConclusionsmNGS is more sensitive than traditional tests and the combination of mNGS and acid-fast staining can improve the detection rate of infectious diseases in the central nervous system.This study suggests that the detection of mNGS is able to identify MTB in cerebrospinal fluid and can be used as a routine TBM diagnostic method.