Cerebrospinal fluid MinION sequencing of 16S rRNA gene for rapid and accurate diagnosis of bacterial meningitis
Authors: Nguyen Thi Thu Hong, Ho Dang Trung Nghia, Tran Tan Thanh, Nguyen Phu Huong Lan, Nguyen Thi Han Ny, Nghiem My Ngoc, Vu Thi Ty Hang, Le Thi My Chau, Van Xuan Quynh, Le Thi Diem, Bui Thi Bich Hanh, Nguyen Ho Hong Hanh, Du Trong Duc, Dinh Nguyen Huy Man, James Campbell, Pham Kieu Nguyet Oanh, Jeremy Day, Nguyen Hoan Phu, Nguyen Van Vinh Chau, Guy Thwaites, Le Van Tan
Journal: Journal of Infection (2019)

We read with interest recent articles in this journal regarding the utility of next-generation sequencing for the diagnosis bacterial meningitis.[1]Guo L.Y. Li Y.J. Liu L.L. Wu H.L. Zhou J.L. Zhang Y. et al.Detection of pediatric bacterial meningitis pathogens from cerebrospinal fluid by next-generation sequencing technology.J Infect. 2019; 78: 323-337Abstract Full Text Full Text PDF Scopus (25) Google Scholar,[2]Zhang J.Z. Zheng P. Sun H.M. Dong J.J. Li S.L. Fan S.Y. et al.Next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a chinese tertiary pediatric neurology center.J Infect. 2019; 78: 409-421Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar Bacterial meningitis causes substantial morbidity and mortality worldwide.[3]Costerus J.M. Brouwer M.C. Bijlsma M.W. van de Beek D. Community-acquired bacterial meningitis.Curr Opin Infect Dis. 2017; 30: 135-141PubMed Google Scholar Rapid identification of the microorganisms is essential for early initiation of appropriate antimicrobial therapy, thereby improving clinical outcome. Yet routine diagnostic methods fail to identify the bacteria in the majority of patients. Over the last decade, advanced sequencing technologies have greatly improved our capacity to detect the causative agents of infectious diseases in clinical samples.[4]Kai S. Matsuo Y. Nakagawa S. Kryukov K. Matsukawa S. Tanaka H. et al.Rapid bacterial identification by direct pcr amplification of 16S rRNA genes using the MinION™ nanopore sequencer.FEBS Open Bio. 2019; 9: 548-557Crossref PubMed Scopus (68) Google Scholar,[5]Moon J. Kim N. Lee H.S. Shin H.R. Lee S.T. Jung K.H. et al.Campylobacter fetus meningitis confirmed by a 16S rRNA gene analysis using the MinION nanopore sequencer, South Korea, 2016.Emerg Microbes Infect. 2017; 6: e94PubMed Google Scholar Of these, the single molecule real-time sequencing developed by Oxford Nanopore Technologies (ONT) is a promising tool for diagnostic setting because of its short turnaround time. In late April 2019, a 59-year old seller of fish-noodles was referred to our hospital with a 1-day history of headache, fever and vomiting. He had a history of heavy alcohol use and hepatitis C infection, and had cirrhosis and diabetes mellitus. On admission, he was unconsciousness (a Glasgow Coma Scale of 8), with a body temperature of 40 °C, a blood pressure of 140/80 mmHg and neck stiffness. Initial Gram-stain and microscopy of CSF showed Gram-positive cocci, 8449 white cells/uL with 91% neutrophils, elevated protein and low glucose level, and high lactate concentration (Fig. 1A). Routine bacterial culture, plus Streptococcus pneumoniae and S. suis PCRs were all negative. He was diagnosed with bacterial meningitis, and given a combination of ceftriaxone (2 g/12 h) and dexamethasone (0.4 mg/kg/12 h). His clinical condition steadily improved. His second and third CSF samples became negative by Gram stain. The other CSF parameters also improved, except the glucose, which remained low (Fig. 1A). On day 20 of hospitalization, the patient suddenly became unconsciousness with fever. Brain magnetic resonance imaging showed bifrontal abscesses (Fig. 1B). After consulting a local neurosurgeon, aspiration of the brain abscesses was not advised and the patient was treated empirically with meropenem (2 g/8 h) and vancomycin (1 g/8 h). Due to continued diagnostic uncertainty, we performed 16S rRNA sequencing of the admission CSF, stored as part of an going clinical study (Supplementary Materials), using an established Sanger-sequencing based 16S rRNA method.[6]Tytgat B. Verleyen E. Obbels D. Peeters K. De Wever A. D'Hondt S. et al.Bacterial diversity assessment in antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional Pyrosequencing and cultivation.PLoS One. 2014; 9: e97564Crossref PubMed Scopus (50) Google Scholar Subsequently, analysis of the obtained sequences revealed evidence of S. agalactiae (Supplementary Figure 1). Given this new diagnostic result of the admission CSF and because the patient had recovered clinically, the patient was given 24 million units of penicillin G for every 4 h. After day 43 of hospitalization, all CSF parameters had normalised (Fig. 1A). Likewise, on CT scan the brain abscess was now significantly improved (Fig. 1C). The patient was discharged with full clinical recovery. Additionally, MinION sequencing of complete 16S rRNA gene was retrospectively carried out on the extracted nucleic acid of the admission CSF yielded a total of 14,848 reads after 100 min of sequencing run. Of these, 11,556 reads (79%) were successfully aligned to S. agalactiae (Fig. 1D). The remaining reads were assigned to other Streptococcus species (mostly S. dysgalacticiae (n = 2.145, 14%)), likely attributed to a combination of the high level of sequence similarities of the 16S rRNA region between them and the sequencing errors introduced by the MinION systems. Analysis of sequencing data generated during the 25, 50 and 75 min of sequencing run time also yielded the same results (Supplementary Figure 2). Details about the MinION procedure are presented in Supplementary Materials. To further assess of the utility of CSF MinION sequencing of 16S rRNA gene for the detection of bacterial meningitis pathogens, six CSF samples from patients with confirmed bacterial meningitis enrolled in the abovementioned clinical study were tested (Table 1). Analysis of the MinION reads obtained after two hours of the sequencing run showed that the majority of reads were correctly assigned to the corresponding bacterial species (S. pneumoniae and S. suis) or genus (Neisseria) found in the CSF samples by diagnostic work up of the clinical study (Fig. 1E and Table 1). Additional analysis of the obtained reads generated at two earlier time points (20 min and 60 min) of the sequencing run generated the same results (Table 1).Table 1Demographics and clinical outcome of the additional six patients included for MinION Nanopore sequencing analysis of 16S rRNA gene.Patient 1Patient 2Patient 3Patient 4Patient 5Patient 6Demographics Age (years)336523295341 GenderMaleMaleFemaleMaleMaleFemale OriginBPBTBPNTBTTNIllness day at enrollment (days)5311542Length of hospital stay (days)1751215^1315Clinical signs/symptoms Body temperature ( °C)3738383737.237 Cranial nerve palsyNNNYNN Hemiplegia/paresisNNNNNN Paraplegia/paresisNNNNNN Tetraplegia/paresisNNNNNN Generalized convulsionsNNNNNN Localized convulsionsNNNNNN Neck stiffnessYYYYYN GCS at enrolment14141313912CSF examinations CSF white cell counts51,81016093111112616,7444760 CSF neutrophils (%)789594606588 CSF lymphocytes (%)2256403512 CSF/blood glucose ratio0.110.640.0140.420.320.028 CSF lactate11.49.2112.455.8213.9415.62 Total protein1.331.1334.7311.373.8615.746Routine microbial investigations ZN smearNDNDNegativeNegativeNDND India Ink stainNegativeNDNegativeNegativeNDND Cryptococcal antigen testNDNDNDNegativeNDND Gram stainGram-positive cocciGram-positive cocciNegativeNegativeNegativeNegative Bacterial cultureS. pneumoniaeS. suisN. meningitidisNegativeNegativeNegative Bacterial PCRS. pneumoniaeS. suisN. meningitidisS. pneumoniaeS. suisN. meningitidisMinION 16S rRNA sequencing 20 minS. pneumoniaeS. suisNeisseriaS. pneumoniaeS. suisNeisseria 1 hS. pneumoniaeS. suisNeisseriaS. pneumoniaeS. suisNeisseria 2 hS. pneumoniaeS. suisNeisseriaS. pneumoniaeS. suisNeisseriaGCS at discharge151415141415Notes to Table 1: GCS: Glasgow Coma Score, BT: Ben Tre, BP: Binh Phuoc, TN: Tay Ninh, NT: Ninh Thuan, HCMC: Ho Chi Minh City, BM: bacterial meingitis, TBM: tuberculous meningitis; N: no, Y: yes; ND: not done. Open table in a new tab Notes to Table 1: GCS: Glasgow Coma Score, BT: Ben Tre, BP: Binh Phuoc, TN: Tay Ninh, NT: Ninh Thuan, HCMC: Ho Chi Minh City, BM: bacterial meingitis, TBM: tuberculous meningitis; N: no, Y: yes; ND: not done. Collectively, we report the first application of MinION sequencing of 16S rRNA gene to detect bacterial meningitis causing pathogens in CSF samples from a low and middle-income country. The assay was able to detect the bacterial causes in all of the seven tested CSF samples. Meanwhile, Gram stain and culture, the two most commonly used methods in clinical microbiology laboratories worldwide, were negative in 3/7 samples. (Fig. 1 and Table 1). In addition to CSF samples described in the present study and a recent pilot study from Korea,[7]Moon J. Kim N. Kim T.J. Jun J.S. Lee H.S. Shin H.R. et al.Rapid diagnosis of bacterial meningitis by nanopore 16S amplicon sequencing: a pilot study.Int J Med Microbiol. 2019; 309151338Crossref PubMed Scopus (27) Google Scholar successful detections of Haemophilus influenzae in sputum and Campylobacter fetus in culture materials by MinION sequencing of 16S rRNA have recently been reported.[8]Moon J. Jang Y. Kim N. Park W.B. Park K.I. Lee S.T. et al.Diagnosis of haemophilus influenzae pneumonia by nanopore 16S amplicon sequencing of sputum.Emerg Infect Dis. 2018; 24: 1944-1946Crossref PubMed Scopus (20) Google Scholar Together, the data suggest that MinION sequencing of 16S rRNA is a sensitive method for rapid and accurate detection of pan-bacterial pathogens, including unexpected microorganisms, in clinical samples. Additionally, the bacterial species information generated by the analysis of 16S rRNA sequences can be useful for disease surveillance and vaccine evaluation. Thus, the application of the method would be relevant for both patient management and epidemiological research. Indeed, to the best of our knowledge the present study represents the first report of S. agalactiae associated meningitis in Vietnam. Because the incidence of invasive diseases (including meningitis) caused by S. agalactiae has been reported with increased frequency in recent years,[9]Francois Watkins L.K. McGee L. Schrag S.J. Beall B. Jain J.H. Pondo T. et al.Epidemiology of invasive group b streptococcal infections among nonpregnant adults in the united states, 2008-2016.JAMA Intern Med. 2019; 179: 479-488Crossref PubMed Scopus (86) Google Scholar S. agalactiae should be considered as an important differential diagnosis for patients presenting with acute CNS infections in Vietnam. Owing to the unavailability of the reagents at the time of patient admission, we were not able to perform real-time diagnosis using MinION sequencing on the collected CSF samples. However, same day diagnosis is theoretically achievable, because the current workflow takes 5 – 6 h to operate. Prospective study is urgently needed to assess its translational potential in the diagnosis of bacterial meningitis. Since September 2017, a prospective observational study aiming at exploring the utility potential of next-generation sequencing in patients presenting with central nervous system (CNS) infections has been conducted in the brain infection ward of the Hospital for Tropical Diseases (HTD) in Ho Chi Minh City, Vietnam. HTD is a tertiary referral hospital for patients with infectious diseases from southern provinces of Vietnam, serving a population of >40 million. Any patient (≥16 years) with an indication for lumbar puncture was eligible for enrolment. Patient was excluded if no written informed consent was obtained. As per the study protocol, CSF, plasma and urine samples were collected at presentation alongside demographic, meta-clinical data and results of routine diagnosis. After collection, all clinical specimens were stored at −80 °C until analysis. The clinical study received approvals from the Institutional Review Board of the HTD and the Oxford Tropical Research Ethics Committee of the University of Oxford. Written informed consent was obtained from each study participant or relative (if the patient was unconsciousness). Sequencing of complete 16S rRNA gene was retrospectively performed using MinION Nanopore sequencer (ONT), following the manufacturer's instructions. In brief, amplification of the complete 16S rRNA gene and library preparation were carried out on extracted nucleic acid using 16S Barcoding Kit (SQK-RAB204, ONT) and primers (27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R 5′-GGTTACCTTGTTACGACTT-3′), followed by the sequencing of the amplified product using R9.4 Flow cells (ONT). MinION reads were first basecalled using Albacore v2.1.7 (ONT), followed by demultiplexing using Porechop (https://github.com/rrwick/Porechop). Determination of bacterial genus/species composition in the obtained reads was then carried out using Epi2Me interface (Metrichor, Oxford, UK), a platform for cloud-based analysis of MinION data. Overall, the whole procedure of MinION sequencing of 16S rRNA gene takes 5–6 h to complete (Supplementary Figure 3). We, the author of the submitted manuscript declare that we do not have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership, relevant patents, or research funding). We thank Le Kim Thanh, Le Nguyen Truc Nhu, and Lam Anh Nguyet for their logistic support. We are indebted to patients for their participations in this study. This study was funded by the Wellcome Trust of Great Britain (106680/B/14/Z and 204904/Z/16/Z). 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