Dear Editor,
We read with interest recent articles in this journal regarding the utility of next-generation
sequencing for the diagnosis bacterial meningitis.
1
,
2
Bacterial meningitis causes substantial morbidity and mortality worldwide.
3
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
,
5
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
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.
Fig. 1
Clinical profile of the S. agalactiae patient, and result of CSF MinION sequencing
of 16S rRNA gene. (A) results of CSF investigations over the course of illness; (B)
MRI showing bifrontal brain abscesses; (C) follow-up CT scan performed on 5th June
2019 showing the improvement of the brain abscesses; (D) results of MinION sequencing
of the admission CSF samples taken on 25th April 2019. A total of 14,848 reads were
obtained after 100 min of the sequencing procedure, of which1,556 (78%) reads were
successfully aligned S. agalactiae; (E) result of MinION sequencing of 16S rRNA gene
analysis of the six additional CSF samples alongside routine diagnostic yields. The
appearances of specific symbols indicate the success of the corresponding methods
in detecting the pathogens in the tested samples.
Note to Figure 1: The sampling dates of the second, third and fourth CSF samples were
27th April 2019, 17th May 2019, and 5th June 2019, respectively.
Fig 1
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 1
Demographics and clinical outcome of the additional six patients included for MinION
Nanopore sequencing analysis of 16S rRNA gene.
Table 1
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Demographics
Age (years)
33
65
23
29
53
41
Gender
Male
Male
Female
Male
Male
Female
Origin
BP
BT
BP
NT
BT
TN
Illness day at enrollment (days)
5
3
1
15
4
2
Length of hospital stay (days)
17
5
12
15^
13
15
Clinical signs/symptoms
Body temperature ( °C)
37
38
38
37
37.2
37
Cranial nerve palsy
N
N
N
Y
N
N
Hemiplegia/paresis
N
N
N
N
N
N
Paraplegia/paresis
N
N
N
N
N
N
Tetraplegia/paresis
N
N
N
N
N
N
Generalized convulsions
N
N
N
N
N
N
Localized convulsions
N
N
N
N
N
N
Neck stiffness
Y
Y
Y
Y
Y
N
GCS at enrolment
14
14
13
13
9
12
CSF examinations
CSF white cell counts
51,810
1609
3111
1126
16,744
4760
CSF neutrophils (%)
78
95
94
60
65
88
CSF lymphocytes (%)
22
5
6
40
35
12
CSF/blood glucose ratio
0.11
0.64
0.014
0.42
0.32
0.028
CSF lactate
11.4
9.21
12.45
5.82
13.94
15.62
Total protein
1.33
1.133
4.731
1.37
3.861
5.746
Routine microbial investigations
ZN smear
ND
ND
Negative
Negative
ND
ND
India Ink stain
Negative
ND
Negative
Negative
ND
ND
Cryptococcal antigen test
ND
ND
ND
Negative
ND
ND
Gram stain
Gram-positive cocci
Gram-positive cocci
Negative
Negative
Negative
Negative
Bacterial culture
S. pneumoniae
S. suis
N. meningitidis
Negative
Negative
Negative
Bacterial PCR
S. pneumoniae
S. suis
N. meningitidis
S. pneumoniae
S. suis
N. meningitidis
MinION 16S rRNA sequencing
20 min
S. pneumoniae
S. suis
Neisseria
S. pneumoniae
S. suis
Neisseria
1 h
S. pneumoniae
S. suis
Neisseria
S. pneumoniae
S. suis
Neisseria
2 h
S. pneumoniae
S. suis
Neisseria
S. pneumoniae
S. suis
Neisseria
GCS at discharge
15
14
15
14
14
15
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
successful detections of Haemophilus influenzae in sputum and Campylobacter fetus
in culture materials by MinION sequencing of 16S rRNA have recently been reported.
8
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
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.
The clinical study
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).
MinION sequencing of 16S rRNA
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).
Declaration of Competing Interest
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).