Dataset Information

Diagnosis of pathogens causing bacterial meningitis using Nanopore sequencing in a resource-limited setting.

ABSTRACT:

Aim

The aim of the present study is to compare the performance of 16S rRNA Nanopore sequencing and conventional culture in detecting infectious pathogens in patients with suspected meningitis in a resource-limited setting without extensive bioinformatics expertise.

Methods

DNA was isolated from the cerebrospinal fluid (CSF) of 30 patients with suspected bacterial meningitis. The isolated DNA was subjected to 16S sequencing using MinION™. The data were analysed in real time via the EPI2ME cloud platform. The Nanopore sequencing was done in parallel to routine microbiological diagnostics.

Results

Nanopore sequencing detected bacterial pathogens to species level in 13 of 30 (43%) samples. CSF culture showed 40% (12/30) positivity. In 21 of 30 patients (70%) with suspected bacterial meningitis, both methods yielded concordant results. About nine of 30 samples showed discordant results, of these five were false positive and four were false negative. In five of the culture negative results, nanopore sequencing was able to detect pathogen genome, due to the higher sensitivity of the molecular diagnostics. In two other samples, the CSF culture revealed Cryptococcus neoformans and Streptococcus pneumoniae, which were not detected by Nanopore sequencing. Overall, using both the cultures and 16S Nanopore sequencing, positivity rate increased from 40% (12/30) to 57% (17/30).

Conclusion

Next-generation sequencing could detect pathogens within six hours and could become an important tool for both pathogen screening and surveillance in low- and middle-income countries (LMICs) that do not have direct access to extensive bioinformatics expertise.

SUBMITTER: Pallerla SR

PROVIDER: S-EPMC9443622 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Diagnosis of pathogens causing bacterial meningitis using Nanopore sequencing in a resource-limited setting.

Pallerla Srinivas Reddy SR Van Dong Do D Linh Le Thi Kieu LTK Van Son Trinh T Quyen Dao Thanh DT Hoan Phan Quoc PQ Trung Ngo Tat NT The Nguyen Trong NT Rüter Jule J Boutin Sébastien S Nurjadi Dennis D Sy Bui Tien BT Kremsner Peter G PG Meyer Christian G CG Song Le Huu LH Velavan Thirumalaisamy P TP

Annals of clinical microbiology and antimicrobials 20220905 1

<h4>Aim</h4>The aim of the present study is to compare the performance of 16S rRNA Nanopore sequencing and conventional culture in detecting infectious pathogens in patients with suspected meningitis in a resource-limited setting without extensive bioinformatics expertise.<h4>Methods</h4>DNA was isolated from the cerebrospinal fluid (CSF) of 30 patients with suspected bacterial meningitis. The isolated DNA was subjected to 16S sequencing using MinION™. The data were analysed in real time via the ...[more]

PMID: 36064402

Similar Datasets

Project description:Meningitis is a complex disease which can be caused by infection with either viral or bacterial pathogens. Viral meningitis is usually a sterile self-limiting disease with a good clinical prognosis, while bacterial meningitis is a potentially more serious disease with a higher mortality rate. Early diagnosis of bacterial meningitis is of paramount importance, as intervention with antimicrobial therapy increases the likelihood of a favourable clinical outcome. Routine diagnosis in many laboratories is still dependent to some degree on traditional methods e.g. culture, though molecular methods have been developed which can give a shorter time to diagnosis. However, there is not as yet a single test format that can detect all bacterial pathogens capable of causing meningitis. In addition, many tests e.g. real-time PCR have a finite limit for multiplexing and do not provide additional information such as strain or serogroup which is useful during outbreaks and for retrospective epidemiological surveillance. To this end we have developed a microarray probe set for detection of meningitis-associated bacterial pathogens including those in the N. meningitidis serogroups. Here we demonstrate utility of this array in specific detection of represented bacterial species and strains and in detection of pathogen signals in cerebrospinal fluid samples from patients with suspected bacterial meningitis. This method shows promise for development as a diagnostic tool; however, we discuss the technical issues encountered and suggest mechanisms to improve resolution of pathogen-specific signals in complex clinical samples. We designed as part of a larger pan-pathogen microarray a sub-set of probes to meningitis-associated bacterial pathogens. We present here data confirming the pathogen-specificity of many of these probes and their potential use in clinical diagnosis through testing on a small number of patient clinical samples using human DNA and no added nucleic acid controls. These data are from single channel Cy3-labelled nucleic acids. Four technical replicates for each feature are included on the array.

Project description:PurposeThe application of metagenomic next-generation sequencing (mNGS) in the diagnosis of tuberculous meningitis (TBM) remains poorly characterized. Here, we retrospectively analyzed data from patients with TBM who had taken both mNGS and conventional tests including culture of Mycobacterium tuberculosis (MTB), polymerase chain reaction (PCR) and acid-fast bacillus (AFB) stain, and the sensitivity and specificity of these methods were compared.MethodsWe retrospectively recruited TBM patients admitted to the hospital between December 2015 and October 2018. The first collection of cerebrospinal fluid (CSF) samples underwent both mNGS and conventional tests. In addition, patients with bacterial/cryptococcal meningitis or viral meningoencephalitis were mNGS positive controls, and a patient with auto-immune encephalitis was an mNGS negative control.ResultsTwenty three TBM patients were classified as 12 definite and 11 clinical diagnoses, which were based on clinical manifestations, pathogen evidence, CSF parameters, brain imaging, and treatment response. The mNGS method identified sequences of Mycobacterium tuberculosis complex (MBTC) from 18 samples (18/23, 78.26%). In patients with definite TBM, the sensitivity of mNGS, AFB, PCR, and culture to detect MTB in the first CSF samples were 66.67, 33.33, 25, and 8.33%, respectively. The specificity of each method was 100%. Among the four negative mNGS cases (4/23, 17.39%), three turned out positive by repeated AFB stain. The agreement of mNGS with the total of conventional methods was 44.44% (8/18). Combination of mNGS and conventional methods increased the detection rate to 95.65%. One patient was diagnosed as complex of TBM and cryptococcal meningitis, in which AFB stain and cryptococcal antigen enzyme immunoassay were positive and the DNA of Cryptococcus neoformans was detected by mNGS.ConclusionOur study indicates that mNGS is an alternative method to detect the presence of mycobacterial DNA in CSF samples from patients with TBM and deserves to be applied as a front-line CSF test.

Dataset Information

Diagnosis of pathogens causing bacterial meningitis using Nanopore sequencing in a resource-limited setting.

Aim

Methods

Results

Conclusion

Publications

Diagnosis of pathogens causing bacterial meningitis using Nanopore sequencing in a resource-limited setting.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets