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Diagnostic Microbiology and Infectious Disease 107 (2023) 116025 Tanggal presentasi : Kamis, 28 Desember 2023

Contents lists available at ScienceDirect

Diagnostic Microbiology and Infectious Disease

jo ur na l h om ep ag e : w w w . el s e v ie r . c om /l oc at e / di ag m i c r o bi o

Comparative performance of microbiological methods for the detection

of tuberculous meningitis pathogens in cerebrospinal fluid
Yuling Lin, Weili Zhang, Ying Xiong, Yue Wang, Qiuju Yu, Ying Ma*, Yi Xie**
Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, China

A R T I C L E I N F O A B S T R A C T

Article history: The aim of this study was to comprehensively evaluate metagenomic next-generation sequencing (mNGS),
Received 2 May 2023 Acid-fast bacillus stain (AFB), MGIT960 culture, polymerase-chain-reaction (PCR), and Xpert MTB/RIF in the
Revised in revised form 11 June 2023 diagnosis of tuberculous meningitis (TBM). A cohort of 280 patients who presented with suspected TBM (ie,
Accepted 9 July 2023
headache or altered mental status with clinical signs of meningism) were analyzed. The sensitivities of the 5
Available online 15 July 2023
assays for the diagnosis of TBM ranged from 10.0% to 70.0%. The AFB had the lowest sensitivity of 10.0% (0.5-
45.9), while mNGS and PCR had the highest sensitivity, both at 70.0% (35.4-91.9). mNGS demonstrated a dis-
Keywords:
tinct advantage in identifying a wider array of pathogens, including viruses, in CSF samples. PCR was a cost-
Mycobacterium tuberculosis
Metagenomic Next-Generation Sequencing
effective option with excellent sensitivity and specificity. However, no single method was statistically signifi-
Tuberculosis meningitis cantly better than any other in the diagnosis of TBM. New diagnostic techniques are urgently needed for the
Cerebrospinal fluid independent, rapid and accurate detection of Mycobacterium tuberculosis to guide the diagnosis of TBM.
Xpert MTB/RIF © 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/)

1. Introduction
Tuberculosis (TB) is a serious infectious disease that has been the
leading cause of death from a single communicable agent until the
coronavirus (COVID-19) pandemic [1]. Among the various manifesta-
tions of TB, tuberculous meningitis (TBM) is one of the most devastat-
ing syndromes, which causes fatality or disability of nearly half of the
affected people [2]. Early diagnosis and timely treatment of TBM are
critical factors that significantly impact patient survival. However,
many patients are initially administered empirical antituberculous
therapy due to insensitive and time-consuming diagnostic methods
required for disease confirmation [3].
TBM is the most severe form of TB, affecting all ages, but young
children and people with untreated HIV are particularly vulnerable.
Although TBM accounts for about 1% of new TB cases, once
infected it causes death or disability in nearly half of all patients
[4]. The incidence of TBM is directly related to the prevalence of
pulmonary TB [2]. Therefore, early detection of M. tuberculosis is an
important tool in the control of disease progression. Conventional
clinical microbiological testing using cerebrospinal fluid (CSF) is the
primary approach, but it has limitations. M. tuberculosis culture is
considered the gold standard for diagnosing TBM, but has a pro-
longed turn-around-time (TAT) of at least 2 weeks and a low
* Corresponding authors. Tel.: 18980601863.
** Corresponding author. Tel.: 189 8060 5782.
E-mail addresses: majiying72@hotmail.com (Y. Ma), xie_yi_77@163.com (Y. Xie).
detection rate, making it less suitable for early diagnosis. Acid-fast
bacillus stain (AFB) of CSF is a cheap and widely available tech-
nique for rapid diagnosis of TBM, but is often insensitive [5]. In
recent years, emerging techniques have provided promising results
for the diagnosis of TBM. One such technique is Xpert MTB/RIF, a
rapid and sensitive nucleic acid amplification test (NAAT) that can
also detect rifampicin susceptibility [6]. By targeting the 81 bp
rifampicin resistance core region (RRDR) of the rpoB gene, Xpert
MTB/RIF can identify mutations associated with rifampicin resis-
tance, providing valuable information for both TB diagnosis and
drug resistance detection. A positive Xpert result is a strong indica-
tion that the patient had M. tuberculosis infection and was resistant
to rifampicin. A negative result does not rule out the possibility
that the patient was not infected and follow-up TB-DNA, imaging
and other methods are need to confirm the diagnosis [7]. Hence,
the limitations of current diagnostic methods, such as prolonged
TAT, low detection rates, and potential empirical antituberculous
therapy, which can lead to antibiotic resistance and increased
healthcare costs, necessitate the development of innovative diag-
nostic methods [3]. A method that can overcome the limitations of
current tests, accurately diagnose TBM and guide clinical manage-
ment is therefore urgently needed.
Metagenomic next-generation sequencing (mNGS) is a promising
diagnostic technology, which has been widely used in the detection
of infectious pathogens in recent years [8]. Compared with traditional
Sanger sequencing, which requires preamplification of a known
sequence and low-diversity sample, mNGS can identify a wide range

https://doi.org/10.1016/j.diagmicrobio.2023.116025
0732-8893/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
2 Y. Lin et al. / Diagnostic Microbiology and Infectious Disease 107 (2023) 116025
of pathogenic microorganisms in a single assay without bias [9]. This
breakthrough technology has revolutionized the field of infectious
disease diagnostics by enabling comprehensive and unbiased patho-
gen detection [10]. A recent large-scale, multicenter, prospective
study with CSF samples from infectious meningitis and encephalitis
evaluated the performance and efficacy of mNGS in comparison with
routine diagnostic testing. The results showed that conventional
microbiologic testing is often inadequate to detect all pathogens.
Hence, mNGS of CSF may represent a potential breakthrough in the
diagnosis of meningoencephalitis, which could guide earlier or
more targeted treatment of nerve-invasive infections and prompt
identification and treatment of non-infectious diseases [11]. Given its
capabilities, mNGS holds great promise in the rapid diagnosis of TBM
in CSF.
The current landscape of research comparing the performance of
clinical assays for TBM is dominated by small-scale studies, leaving a
critical gap in the understanding of their efficacy. To address this
limitation, we conducted a comprehensive retrospective study at
West China Hospital of Sichuan University between April 2020 and
April 2021, involving 280 adult patients with clinically suspected
TBM (headache for >3 days or altered mental status [Glasgow Coma
Scale <15] with clinical signs of meningism at examination—ie, neck
stiffness or Kernig’s sign) who were not infected with HIV [5]. The
aim of this study was to rigorously assess the diagnostic efficacy of
mNGS and other traditional microbiological methodologies, including
AFB, MGIT960 culture, PCR and Xpert MTB/RIF in the diagnosis of
TBM, using a standardized clinical case definition of TBM as the gold
standard for the study.
2. Materials and methods
2.1. Study design and participants
We conducted a retrospective study to evaluate the performances
of mNGS, AFB, MGIT960, PCR, and Xpert MTB/RIF for the diagnosis of
TBM. Patients aged 18 years or older with suspected TBM based on
clinical symptoms and signs were included in this study [12]. Patients
with contraindications to lumbar puncture, unsigned informed con-
sent, and HIV-coinfection were excluded. The study was approved by
the Ethics Committee of West China Hospital, Sichuan University
(approval number: 2022-270).
2.2. Diagnostic classification
Previous studies used different definitions of tuberculous men-
ingitis [13−19]. To make it easier to compare the results of differ-
ent studies and to optimize the use of existing data, Marais et al.
developed standardized diagnostic criteria for tuberculous menin-
gitis [12]. According to the uniform clinical case definition, TBM
was divided into 3 categories: definite, probable and possible,
based on the clinical, laboratory and radiological findings. The
diagnosis of TBM was considered to be definite when a patient
presenting with symptoms or signs consistent with meningitis ful-
fils 1 or more of the following criteria: AFB seen in the CSF, M.
tuberculosis cultured from CSF, or a CSF M. tuberculosis positive
commercial nucleic acid amplification test. If all the above criteria
were negative, probable or possible TBM was determined accord-
ing to a diagnostic scoring system in the uniform clinical case defi-
nition [12]. Specifically, the patient’s scores in each section (clinical
criteria, CSF criteria, cerebral imaging criteria, evidence of TB else-
where) were added, a patient may be classified as probable TBM if
imaging was available and the score was 12 or more, or if imaging
was not available and the score was 10 or more. A patient may be
classified as possible TBM if imaging was available and the score of
6 to 11, or if imaging was not available and the score of 6 to 9.
2.3. Acid-fast bacillus stain
Briefly, 2 to 4 mL of CSF was added to a disposable centrifuge tube,
the supernatant was discarded after centrifuging at 3500 rpm for 5
minutes, and the precipitate was taken for Acid-fast bacillus stain
(BA4015, BaSO, Guangdong, China) and smear microscopy.
2.4. MGIT960 culture
Briefly, 500 mL of CSF was added to MGIT culture tubes containing
800 mL of MGIT supplement. The tubes were then mixed and incu-
bated in the MGIT960 apparatus for 6 weeks. After the machine auto-
matically determined a positive result, the BACTEC MGIT SIRE kit
(Becton, Dickinson) was used to test all culture-positive strains for
susceptibility to isoniazid, rifampin, ethambutol, and streptomycin
according to the manufacturer’s instructions.
2.5. PCR
Briefly, 800 mL of CSF and 60 mL of proteinase K were added to
5 mL sample tubes. Then concert@HF 24 (CONCERT, Fujian, China)
was used to extract nucleic acid automatically. Finally, 100 mL of
nucleic acid was collected and frozen at -20°C for storage. The PCR
premix was prepared as follows: reaction solution 37.8 mL per well,
Taqase 0.2 mL and UNGase 0.06 mL. Subsequently, sample 2 mL was
added to each well. Amplification was performed using a LightCycler
480 with the following reaction conditions: 37°C 3 min 1 cycle; 94°C
1 min 1 cycle; then 95°C 5 s, 60°C 30 s, 40 cycles.
2.6. Xpert-MTB/RIF
Detection of M. tuberculosis was performed using the Xpert MTB/
RIF (Cepheid, CA) assay according to manufacturer’s recommenda-
tion. According to the original volume of the CSF, 1-2 mL of 4% NaOH
was added to the pretreatment tube, the sample was mixed thor-
oughly and allowed to liquefy for 20 minutes at room temperature.
After inverting the pretreatment tube 10-20 times, 2 mL of liquid was
added with a sterile pipette to the assay cartridge. The test cartridge
was loaded into the GeneXpertÒ System (Cepheid, CA) for testing and
the instrument automatically displayed the result after 2.5 hours.
Xpert MTB/RIF assay results were determined by measuring the
fluorescence signal and the built-in calculation algorithm of the
instrument (invalid [QC failure]; M. tuberculosis not detected; M.
tuberculosis detected and rifampicin resistance [rifampicin resistance
detected, not detected or indeterminate]). Based on the Ct value of M.
tuberculosis in the sample, the results of the semiquantitative quanti-
fication of M. tuberculosis was shown as high (Ct value <16), medium
(Ct value of 16-22), low (Ct value of 22-28) or very low (Ct value
>28).
2.7. mNGS
The MGISEQ-2000 (BGI, Guangdong, China) gene sequencer was
used for sequencing reactions, and the pathogen sequences were
compared with Mycobacterium TB (GenBank ID: GCF_001708265.1).
Three main steps were used: (1) Sample processing: 1.5 to 3 mL of
patient’s CSF was collected in accordance with standard sample col-
lection procedures. First, 7.2 mL Lyticase (RT410-TA, TIANGEN BIO-
TECH, Beijing, China) was added to 600 mL of sample for enzyme
breaking reaction, followed by 250 mL of 0.5 mm glass beads for
physical breaking, and finally the sample was mixed and shaken.
Then 300 mL of sample was taken for DNA extraction according to
the procedure of TIANamp Micro DNA kit (DP316, TIANGEN BIOTECH,
Beijing, China). (2) Library construction and sequencing: The
extracted nucleic acids were first subjected to enzymatic fragmenta-
tion, end-repair, adapter-ligation and PCR adapter-ligation for
 Y. Lin et al. / Diagnostic Microbiology and Infectious Disease 107 (2023) 116025
construction of DNA libraries. The DNA libraries were quantified by
Agilent 2100 Bioanalyzer and their concentrations were quantified
by Qubit dsDNA HS Assay kit (Thermo Fisher Scientific Inc). Finally,
the DNA libraries were sequenced using MGISEQ-2000 [20]. (3) Bio-
informatics analysis: Low-quality sequences were first removed to
generate high-quality sequencing data. Then, some of the high-qual-
ity data were excluded by comparison of Burrows−Wheeler Align-
ment [21] with human reference genome (hg19) matched. The
remaining data, consisting of bacteria, fungi, viruses, and parasites,
were classified by simultaneous alignment to the Pathogens Metage-
nomics Database (PMDB). The classification reference databases were
downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/). Posi-
tive results for the detection of M. tuberculosis complex matching
sequence (NC_00962.3).
3. Results
3.1. Flow chart of patient selection in this study
After considering the patient’s clinical features and relevant labo-
ratory tests, and applying a standardized case definition for TBM in
Fig. 1. Flow chart.
3
clinical research, a total of 82 cases were diagnosed as non-TBM,
while 198 cases were diagnosed as TBM. Among the TBM cases, 10
were classified as definite, 18 as probable, and 170 as possible,
according to the established rules (Fig. 1).
3.2. Patient characteristics
Between April 1, 2020, and April 1, 2021, a total of 605 HIV-unin-
fected individuals underwent screening. Among them, 325 did not
meet the criteria for enrollment, and 280 patients were finally
included and underwent testing. The average age of the 280 patients
was 48.4 years; 76 patients (27.1%) were over the age of 60 years;
63.5% of the patients were male (Table 1). A comparison of lateral
findings revealed that as the identification of TBM became more
accurate, the patients tended to be older and predominantly male
with suspected TBM. Furthermore, there was a gradual decrease in
the number of lymphocytes in the CSF, along with a lower CSF-blood
glucose ratio. We also performed a statistical analysis of 198 TBM and
82 non-TBM using the independent samples MannWhitney test,
which showed that CSF routine, cerebrospinal fluid biochemistry and
CSF-blood glucose ratio were significantly different from each other.
4 Y. Lin et al. / Diagnostic Microbiology and Infectious Disease 107 (2023) 116025

Table 1
Baseline characteristics.

Characteristic Value (280 participants) 198 TBM Value (82 non-TBM) P-Value (TBM vs non-TBM)

Value (170 possible) Value (18 probable) Value (10 definite)

Age
Mean - years 48.4 48.6 51.4 54.5 46.6 0.21
Distribution - no. (%)
19-25 years 26 (9.3) 17 (10.0) 2 (11.1) 1 (10.0) 6 (7.3) 0.42
26-40 years 84 (30.0) 46 (27.1) 5 (27.8) 1 (10.0) 32 (39.0) 0.49
41-60 years 94 (33.6) 62 (36.5) 3 (16.7) 5 (50.0) 24 (29.3) 0.00
>60 years 76 (27.1) 45 (26.5) 8 (44.4) 3 (30.0) 20 (24.4) 0.13
Male sex−no. (%) 178 (63.5) 111 (65.3) 11 (61.1) 7 (70.0) 49 (59.8) 0.58
CSF routine
CSF white cell count (per mL) 211.1 (108.1−314.1) 240.7 (92.0−389.4) 86.3 (54.4−118.3) 132.0 (28.6−235.4) 187.1 (13.9−360.3) 0.00
CSF lymphocytes 32.5 (27.6−37.3) 39.8 (33.3−46.4) 60.3 (42.4−78.2) 26.2 (2.9−49.5) 12.0 (5.7−18.2) 0.00
Cerebrospinal fluid biochemistry
CSF protein (g/L) 1.5 (1.0−2.0) 1.7 (0.9−2.6) 1.5 (1.2−1.9) 2.6 (1.5−2.8) 0.8 (0.6−1.0) 0.00
CSF glucose (mmol/L) 3.5 (3.3−3.7) 3.4 (3.1−3.6) 3.0 (2.3−3.8) 3.3 (1.2−5.5) 3.9 (3.5−4.2) 0.00
CSF Cl (mmol/L) 122.5 (121.6−123.5) 121.7 (120.5−122.9) 121.5 (116.7−126.3) 119.7 (107.6−131.8) 124.8 (123.5−126.0) 0.00
CSF-blood glucose ratio 0.6 (0.5−0.6) 0.5 (0.5−0.6) 0.4 (0.3−0.5) 0.3 (0.2−0.4) 0.6 (0.6−0.7) 0.00

3.3. Comparative performance of 5 microbiological methods for the positive by all methods, indicating the variability and complementary
diagnosis of tuberculous meningitis nature of these diagnostic techniques in identifying TBM.

The diagnostic sensitivities of 5 methods for detecting TBM in the
280 participants with a definitive diagnosis were as follows: 7.6% (95%
CI 4.5-12.4) for mNGS, 0.6% (95% CI 0.0-3.7) for AFB, 5.0% (95% CI 0.9-
18.2) for MGIT960, 6.1% (95% CI 2.7-12.6) for PCR and 8.8% (95% CI 3.9-
17.7) for Xpert MTB/RIF, when compared to the reference standard of
definite, probable and possible TBM. The specificity of all methods,
except mNGS, was 100% (Table 2). Although the sensitivity of mNGS
was relatively better than the other 4 methods when using definite and
probable TBM as a reference standard, it did not reach 100%. In cases of
definite TBM, both mNGS and PCR showed the same sensitivity.
3.4. Overlap of commonly used clinical methods
Among the CSF samples obtained from the included patients, a
total of 8 cases were diagnosed with TBM by both mNGS and routine
microbiologic testing methods, including AFB, MGIT960, PCR, and
Xpert MTB/RIF. Additionally, 12 cases were diagnosed with TBM
solely by mNGS. The observation of the allocation and overlap of pos-
itive CSF samples by mNGS, AFB, MGIT960, PCR, and Xpert MTB/RIF
revealed that out of the 20 cases that were positive by mNGS, 6 were
positive by Xpert MTB/RIF, 6 were also positive by PCR, 1 by
MGIT960, and 1 by AFB (Fig. 2). Notably, none of the cases were
3.5. Distribution of pathogenic microorganisms in CSF
A total of 250 pathogenic bacteria were found in CSF from 151
patients (Fig. 3A). Among them, the top 5 pathogens identified were
human herpesvirus type 4 (32 cases), M. tuberculosis (20 cases), human
herpesvirus type 1 (17 cases), Klebsiella pneumoniae (13 cases), and
Candida parapsilosis (12 cases). In contrast, conventional microbiolog-
ical methods such as culture and smear microscopy only identified 32
pathogenic strains in the same set of 280 CSF samples, with the high-
est detection rate of Cryptococcus neoformans in 16 cases (Fig. 3B).
Upon comparison of the 2 sets of data, the match rate was 12.8%,
highlighting the higher sensitivity and broader pathogen detection
capability of mNGS compared to traditional microbiological methods.
4. Discussion
In this study, no single test performed significantly better than the
others for the diagnosis of TBM in CSF. However, mNGS could detect a
larger number of potential pathogens compared to routine microbio-
logic testing. While CSF bacterial culture and smear microscopy
identified a total of 30 pathogens, mNGS identified a total of 85
pathogenic microorganisms. These findings highlight the enhanced

Table 2
Diagnostic performance of 5 commonly used methods against the uniform clinical case definition.

mNGS AFB MGIT960 PCR Xpert MTB/RIF

Reference standard: definite, probable, and possible tuberculous meningitis

Positive tests 15/198 1/173 2/40 7/115 7/80
Sensitivity 7.6% (4.5−12.4) 0.6% (0.0−3.7) 5.0% (0.9−18.2) 6.1% (2.7−12.6) 8.8% (3.9−17.7)
Specificity 93.9% (85.7−97.7) 100.0% (93.8−100.0) 100.0% (46.3−100.0) 100.0% (85.9−100.0) 100.0% (69.9−100.0)
PPV 75.0% (50.6−90.4) 100.0% (5.5−100.0) 100.0% (19.8−100.0) 100.0% (56.1−100.0) 100.0% (56.1−100.0)
NPV 25.0% (9.6−49.4) 29.8% (24.2−36.0) 11.6% (4.3−25.9) 21.7% (15.4−29.7) 14.1% (7.8−23.8)
Reference standard: definite, probable tuberculous meningitis
Positive tests 11/28 1/27 2/13 7/21 7/19
Sensitivity 39.3% (22.1−59.3) 3.7% (0.2−20.9) 13.3% (2.3−41.6) 33.3% (15.5−56.9) 36.8% (17.2−61.4)
Specificity 96.4% (93.1−98.2) 100.0% (97.9−100.0) 100.0% (86.7−100.0) 100.0% (96.3−100.0) 100.0% (93.8−100.0)
PPV 55.0% (32.0−76.2) 100.0% (5.5−100.0) 100.0% (19.8−100.0) 100.0% (56.1−100.0) 100.0% (56.1−100.0)
NPV 93.5% (89.6−96.0) 89.4% (84.7−92.8) 71.1% (55.5−83.2) 89.9% (83.3−94.1) 85.9% (76.2−92.2)
Reference standard: definite tuberculous meningitis
Positive tests 7/10 1/10 2/5 7/10 6/9
Sensitivity 70.0% (35.4−91.9) 10.0% (0.5−45.9) 40.0% (7.3−83.0) 70.0% (35.4−91.9) 66.7% (30.9−91.0)
Specificity 95.2% (91.7−97.3) 100.0% (98.0−100.0) 100.0% (89.1−100.0) 100.0% (96.6−100.0) 98.9% (92.5−99.9)
PPV 35.0% (16.3−59.1) 100.0% (5.5−100.0) 100.0% (19.8−100.0) 100.0% (56.1−100.0) 85.7% (42.0−99.2)
NPV 98.8% (96.4−99.7) 96.3% (92.9−98.2) 93.0% (79.9−98.2) 97.8% (93.3−99.4) 96.5% (89.3−99.1)
 Y. Lin et al. / Diagnostic Microbiology and Infectious Disease 107 (2023) 116025 5

Fig. 2. Venn diagram of positive diagnostic tests.

capability of mNGS in providing valuable clinical information beyond
traditional testing methods.
The sensitivity of mNGS in this study was found to be up to 70%,
with a specificity of 96.4%. Previous studies have reported varying
sensitivity (ranging from 27%-84%) and specificity (ranging from
96%-100%) for mNGS in the diagnosis of TBM [22]. Many of these
studies have concluded that mNGS is superior to traditional microbi-
ological methods for early diagnosis of TBM [11]. However, our study
results showed no significant superiority of mNGS compared to tradi-
tional laboratory testing methods (mNGS vs. AFB P = 0.03, vs.
MGIT960 P = 1, vs. PCR P = 1, vs. Xpert MTB/RIF P = 1), indicating sig-
nificant heterogeneity across studies for the same indicator. Several
factors could contribute to these differences. First, different sequenc-
ing methods were used in each study, resulting in variations in the
depth of sequencing and the number of genes detected. For example,
some studies used the fast-running model on the Illumina HiSeq

Fig. 3. mNGS versus other microbiological methods for the detection of pathogenic microorganisms.
6 Y. Lin et al. / Diagnostic Microbiology and Infectious Disease 107 (2023) 116025
instrument with a depth of 5-10 million single-end, 140-base-pair
reads per patient, while others used the BGISEQ-500/50 platform
with an average depth of 20 million reads per sample [11,23]. In the-
ory, increasing the sequencing depth can help improve the sensitivity
and specificity of the assay, but there is an upper limit to this cover-
age. Once the sequencing depth reaches a certain level, there are
enough reads that each base of the target gene to be detected has
been read at least once or even more times, and increasing the
sequencing depth at this point will not significantly improve sensitiv-
ity and specificity [24]. Due to the low total amount of DNA extracted
from CSF, good results can be obtained with different sequencing
platforms for the diagnosis of TBM when a sequencing depth of
100 million is achieved. Second, our study included a wider range of
patients compared to other studies, beyond Mycobacterium TB-con-
firmed patients only. While this may have made the results more
clinically relevant and instructive, stricter inclusion criteria for
patients tend to result in better assay performance. Lastly, sample
quality, particularly the low levels of pathogenic bacteria in CSF, can
also impact test results. While the sensitivity and specificity of mNGS
in our study were not as high as expected, several factors may have
contributed to these results, including differences in sequencing
methods, patient inclusion criteria, and sample quality. Further
research and optimization of mNGS protocols, including standardized
pretreatment steps for CSF samples, validation of specificity in HIV
coinfection cases, and comparison with other diagnostic methods,
are warranted to improve the laboratory testing of TBM.
Strengths of this study included its large sample size, retrospec-
tive study design and prior to the initiation of anti-TB treatment. A
graph evaluating the performance of mNGS with conventional meth-
ods indicates that the specificity of mNGS is not always 100%, mean-
ing that nondetection of the target pathogen does not necessarily
exclude disease. The sensitivity of all assays increased as the diagno-
sis became more accurate, with molecular assays (mNGS, PCR, Xpert
MTB/RIF) showing a greater increase in sensitivity compared to AFB
and MGIT960, with PCR demonstrating the greatest increase in sensi-
tivity. Therefore, PCR may be the best diagnostic method among the
5 methods for detecting TBM, considering sensitivity, specificity, and
cost of the test.
Although traditional microbiological methods may be more cost-
effective for detecting TBM, the application of mNGS is not limited to
TBM alone [25]. It has the ability to detect all pathogens in a sample
simultaneously, facilitating simultaneous diagnosis of multiple infec-
tious diseases [26]. A comparison of pathogenic distribution between
mNGS and conventional laboratory tests revealed that herpesvirus IV,
also known as EBV, was the most frequently detected pathogen in CSF.
This finding may be related to latent EBV infection or reactivation.
Additionally, mNGS showed significantly better detection rates for
fungi compared to traditional methods, with improved detection rates
for Candida parapsilosis and Aspergillus fumigatus, which can be valuable
in the diagnosis of fungal meningitis. Therefore, clinical selection of the
appropriate test should be based on the patient’s condition, and mNGS
may be considered as an additional diagnostic tool when necessary.
In this experiment, the detection performance of Xpert Ultra was
not assessed, and subsequent studies could address this research gap
by using CSF. Furthermore, based on the conclusions and experimen-
tal design of this study, 3 aspects could be optimized in future
research. First, due to the low bacterial content of CSF and the suscep-
tibility of mNGS results to various factors, standardized pretreatment
steps for CSF samples should be developed to minimize the occur-
rence of false positives and false negatives. Second, the specificity
estimates were primarily derived from HIV-uninfected individuals,
and further data are necessary to validate the specificity of mNGS for
HIV coinfection. Finally, this study only analyzed CSF, and subsequent
studies could add different categories of samples, such as sputum,
alveolar lavage fluid and puncture fluid, by testing them simulta-
neously for M. tuberculosis using AFB, MGIT960, Xpert MTB/RIF, PCR
and mNGS, and finally compare the sensitivity and specificity of the
same assay for different types of samples.
In summary, while 2 new technologies, mNGS and Xpert MTB/RIF,
show promising results, they do not completely solve the problem of
laboratory diagnosis of TBM. Currently, there is no single method to
independently diagnose TBM. Among the tested methods, PCR per-
formed best when using definite TBM as a reference standard. Both
mNGS and Xpert MTB/RIF have similar test performances, but a nega-
tive test does not exclude TBM and should be used in conjunction
with other conventional methods for accurate diagnosis. AFB staining
has limited value in diagnosing TBM. Although MGIT960 is time-con-
suming and moderately effective, it can provide subsequent drug sus-
ceptibility results if the culture is positive. Therefore, there is an
urgent need for developing new laboratory testing technologies for
TBM.
Authors’ contributions
Yuling Lin was involved in research design, performed the data anal-
ysis and interpretation, drafted and revised the manuscript. Weili Zhang
was responsible for collating data. Ying Xiong, Yue Wang and Qiuju Yu
were responsible for doing the test and recruiting patients. Yi Xie and
Ying Ma conceptualized the study, supervised the research design, data
analysis, and interpretation; and critically reviewed and revised the
manuscript. All authors read and approved the final manuscript.
Declaration of Competing Interest
The authors declare that they have no competing interests.
Funding
The work was supported by the Science and Technology Depart-
ment Project of Sichuan, China (2020YFS0555); and the Special Foun-
dation for National Science and Technology Basic Research Program
of China (2019FY101200).
Ethics declarations
Ethics approval and consent to participate. This prospective study
was approved by the Ethics Committee of West China Hospital, Sich-
uan University (approval number: 2022-270). Each participant or
their legal representatives gave written informed consent before
enrollment.
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