Clinical Microbiology and Infection 26 (2020) 213e219
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Clinical Microbiology and Infection
journal homepage: www.clinicalmicrobiologyandinfection.com
Original article
Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal
fluid for tuberculous meningitis in adult patients
Z. Li 1, y, L. Pan 1, y, L. Lyu 1, y, J. Li 2, H. Jia 1, B. Du 1, Q. Sun 1, Z. Zhang 1, *
1
Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic
Tumor Research Institute, Beijing, China
2
People's Liberation Army 263 Hospital, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 May 2019
Received in revised form
11 July 2019
Accepted 13 July 2019
Available online 20 July 2019
Objectives: Tuberculous meningitis (TBM) is difficult to diagnose. Digital PCR (dPCR) is a novel method
which can quantify trace nucleic acids. This study sought to evaluate the diagnostic accuracy of dPCR
analysis of cerebrospinal fluid (CSF) for TBM.
Methods: We collected CSF specimens from hospitalized TBM and non-TBM patients. Total CSF DNA was
purified and the concentrations of Mycobacterium tuberculosis insert sequence 6110 (IS6110) and gyrase
subunit B (gyrB) were quantified using droplet dPCR. The receiver operating characteristic curves of dPCR
were established and the diagnostic performances were obtained. We also compared the sensitivity of
dPCR with routine diagnostic tests.
Results: A total of 101 patients were recruited, 68 of whom suffered from TBM (26 definite, 34 probable
and eight possible TBM) and 33 from non-TBM. The sensitivity of IS6110-dPCR assay for total TBM was
higher than that of gyrB-dPCR assay (57.4% (44.8e69.3%) vs. 22.1% (12.9e33.8%)), and there was no
significant difference for specificity between them (97.0% (84.2e99.9%) vs. 100% (89.4e100.0%)). The
sensitivity of IS6110-dPCR in definite TBM was higher than that in probable and possible TBM (73.1% vs.
52.9% and 25.0%, respectively). IS6110-dPCR assay showed a higher sensitivity than smear microscopy
(53.3% vs. 6.7%), mycobacterial culture (50.0% vs. 12.5%), IS6110-quantitative PCR (53.1% vs. 21.9%) and
Xpert MTB/RIF (70.4% vs. 29.6%). Long anti-tuberculosis treatment time was found to be significantly
associated with negative dPCR results.
Conclusion: CSF IS6110-dPCR assay is a rapid and sensitive molecular test, which has the potential to be
used to enhance the diagnosis of TBM. Z. Li, Clin Microbiol Infect 2020;26:213
© 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All
rights reserved.
Editor: Franz Allerberger
Keywords:
Cerebrospinal fluid
diagnosis
digital PCR
tuberculosis
tuberculous meningitis
Introduction
Tuberculous meningitis (TBM) is the most lethal and disabling
form of tuberculosis (TB). The global burden of TBM is estimated to
be more than 100 000 new cases per year [1].
Rapid diagnosis and timely treatment have extraordinary significance in reducing the mortality and disability of TBM patients.
The early diagnosis of TBM remains difficult due to the nonspecific clinical features and the poor efficiency of diagnostic
* Corresponding author. Zongde Zhang, Beijing Key Laboratory for Drug Resistant
Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing
Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
E-mail address: zzd417@ccmu.edu.cn (Z. Zhang).
y
These authors contributed equally.
tests. The traditional cerebrospinal fluid (CSF) ZiehleNeelsen
staining and microscopy has a very low sensitivity (0e20%) [2,3].
Mycobacterial culture is more sensitive, but the long time to return
a result (10 days to 8 weeks) and requirement of a specialized
biosafety level 3 laboratory greatly limit its use. Xpert MTB/RIF
(Xpert; Cepheid, Sunnyvale, CA, USA), a cartridge-based nucleic
acid amplification test recommended by the World Health Organization (WHO), has an unsatisfactory sensitivity [4]. The secondgeneration Xpert MTB/RIF Ultra (Xpert Ultra) has a higher sensitivity, but the specificity decreases and the performance needs
further evaluation in larger cohorts [5]. Interferon-gamma release
assays (IGRAs), widely used to detect Mycobacterium tuberculosis
infection, only have moderate accuracy for TBM diagnosis [6].
Detection of M. tuberculosis antigens and changes in adenosine
deaminase (ADA) level in CSF also have low accuracy for TBM [7,8].
https://doi.org/10.1016/j.cmi.2019.07.015
1198-743X/© 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
214
Z. Li et al. / Clinical Microbiology and Infection 26 (2020) 213e219
Therefore, a new test with higher diagnostic efficiency for TBM is
urgently needed.
Recently, digital polymerase chain reaction (dPCR), the thirdgeneration PCR after end-point PCR and real-time PCR, has been
developed as a novel nucleic acid quantitative technique.
Compared with the most popular real-time quantitative PCR
(qPCR), dPCR is a quantitative method without a calibration curve
and has higher sensitivity and accuracy. Different dPCR platforms
have been developed according to various sample dispersion ways,
among which the water-in-oil droplet dPCR is the most frequently
used [9,10]. In recent years, dPCR has been utilized in many fields
such as clinical pathogen detection, prenatal diagnosis, DNA
methylation detection, circulating nucleic acid quantification and
gene mutation analysis [11e15]. It has also been reported to be
successful in detecting cell-free M. tuberculosis DNA in plasma of
pulmonary TB patients [16,17]. We aimed to assess the diagnostic
accuracy of dPCR analysis of CSF for TBM.
Methods
Study subjects
Eligible patients were aged 16e75 years and met the uniform
case definition of TBM and non-TBM [18]. Exclusion criteria
included immunosuppressive therapy, history of organ transplant,
pregnancy, lactation and human immunodeficiency virus (HIV)
infection. Patients were consecutively and prospectively enrolled
between January 2013 to March 2018 at Beijing Chest Hospital,
Beijing Tiantan Hospital and People's Liberation Army 263 Hospital.
Some of the following tests were performed to help make a final
diagnosis: computed tomography/magnetic resonance imaging
(CT/MRI) images; cytological, microbiological, biochemical and
histopathological examination of CSF; viral antibody; cryptococcal
antigen latex agglutination test (CLAT); CSF tests related to TB
including smear microscopy (for acid-fast bacilli), mycobacterial
culture (BACTEC MGIT 960; BD, Franklin Lakes, NJ, USA), Xpert
MTB/RIF, real-time IS6110-qPCR (DaAn Gene, Guangzhou, Guangdong, China), IGRA (T-SPOT.TB; Oxford Immunotec Ltd, Abingdon,
UK) and M. tuberculosis antibody detection (Huian, Shenzhen,
Guangdong, China); blood tests related to TB including IGRA and
M. tuberculosis antibody detection. Medical records were collected
on age, gender, time of anti-tuberculosis treatment, underlying
diseases and examination results. The study was conducted under
the approval of the Ethics Committee of Beijing Chest Hospital,
Capital Medical University.
Categorization of patients
Patients were divided into four groups according to the uniform
case definition [18]: (1) definite TBM: acid-fast bacilli were seen in
the CSF, M. tuberculosis was cultured from the CSF or the CSF
commercial nucleic acid amplification test (NAAT) was positive for
M. tuberculosis; (2) probable TBM: diagnostic score was 12 (cerebral imaging was available) or 10 (cerebral imaging was not
available), at least 2 points were from either cerebral imaging or
CSF, and alternative diagnoses were excluded; (3) possible TBM:
diagnostic score was 6e11 (cerebral imaging was available) or 6e9
(cerebral imaging was not available); (4) non-TBM: an alternative
diagnosis was made, without convincing signs of combining TBM.
CSF collection and DNA extraction
Cerebrospinal fluid samples were collected and centrifuged at
2000 g for 10 min at 4 C. The supernatants were stored at e80 C.
Total DNA from 400 mL of CSF was extracted in batches with DNeasy
Blood and Tissue Kits (Qiagen, Hilden, Germany) according to the
manufacturer's instructions, except for using a final elution volume
of 45 mL in order to increase DNA concentration. The DNA samples
were stored at e80 C until dPCR analysis in batches.
Droplet dPCR assay
The DNA sequences of insert sequence 6110 (IS6110) and gyrase
subunit B (gyrB) were both conserved in M. tuberculosis complex
and were used as detection targets in the study [17]. PCR primers
and the internal probes were designed according to the reference
and were synthesized by Sangon Biotech (Shanghai, China) [17].
Twenty microlitres of the reaction mixture contained 10 mL of
ddPCRTM supermix for probes (1863010, BioRad, Hercules, CA, US),
0.9 mM primers, 125 nM probe and 6.15 mL of extracted DNA without
dilution. The mixtures and droplet generation oil (1863005, BioRad) were added in cartridges and loaded into a QX200 droplet
generator (Bio-Rad) for droplet generation. The droplet emulsions
were transferred to a 96-well PCR plate and sealed with a foil heat
seal at 180 C for 10 s. PCR conditions were 95 C for 10 min, 40
cycles of 94 C for 30 sec and 54 C for 40 sec, and a final cycle of
98 C for 10 min. The temperature ramp rate was 2.0 C/s. The plate
was then loaded on a QX200 Droplet Reader (BioRad) to automatically measure the fluorescence signal of each droplet. The
quantification of target DNA was calculated using QuantaSoft
Version 1.7.4.0917 (BioRad) and presented as copy numbers per 20
mL of reaction mixture. No-template negative control and
M. tuberculosis H37Rv DNA positive control were adopted in all
dPCR assays. Tests for each sample were performed in duplicate.
Statistical analysis
The statistical analysis was performed using SPSS software
package Version 13.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were compared using the Student's t-test, Manne
Whitney U test or Wilcoxon test, while categorical variables were
tested using the chi-square test or McNemar test, as appropriate.
Accordance of repeated tests or different assays was analysed by
the Spearman correlation test. Risk factors were analysed using
univariate analysis. All tests were two-sided and p < 0.05 was
considered statistically significant.
Results
Characteristics of patients
Baseline characteristics of included patients are shown in
Table 1. A total of 101 patients were recruited, 68 of whom were
TBM patients (26 definite, 34 probable and eight possible TBM) and
33 were non-TBM patients. Among non-TBM patients, 16 were
diagnosed as viral meningitis, eight as tumours of central nervous
system, five as primary headache, one as Brucella meningitis, one as
arachnoidal cyst and syringomyelia, one as intractable epilepsy and
one as lung cancer with brain metastasis. Overall, 46 patients
(45.5%) were female and the median age was 30 years old (range
from 16 to 75).
Results of dPCR in detection of M. tuberculosis DNA in CSF
The target DNA concentrations are calculated based on the
Poisson distribution and some of the original results are shown in
Fig. S1. Results of two repeated tests for 42 randomly selected CSF
samples were highly correlated (IS6110, r ¼ 0.807, p < 0.001; gyrB,
r ¼ 0.726, p < 0.001, Fig. S2). IS6110- and gyrB-targeted tests were
correlated with each other (r ¼ 0.788, p < 0.001, Fig. 1A), and
Z. Li et al. / Clinical Microbiology and Infection 26 (2020) 213e219
215
Table 1
Basic demographic and clinical features of the included patients
Characteristics
Definite TBM
Patients number
26
Median age (age range)
25.5 (16e69)
Gender (female/male)
11/15
Anti-tuberculosis treatment time
1 month
14
>1 month
10
Not sure
2
Probable TBM
Possible TBM
Total TBM
Non-TBM
Total patients
34
32 (16e75)
20/14
8
43 (19e56)
3/5
68
29.5 (16e75)
34/34
33
30 (17e72)
12/21
101
30 (16e75)
46/55
21
11
2
5
1
2
40
22
6
e
e
e
e
e
e
TBM, tuberculous meningitis.
Fig. 1. Quantification of M. tuberculosis IS6110 and gyrB DNA in cerebrospinal fluid (CSF) samples by dPCR. (A,B) The correlation (Spearman correlation test) and the differences
(Wilcoxon test) in the number of copies detected between IS6110- and gyrB-dPCR, respectively. (C,D) IS6110 and gyrB copies detected in tuberculous meningitis (TBM) and non-TBM
group, respectively (ManneWhitney U test). (E,F) IS6110 and gyrB copies detected in definite, probable and possible TBM patients, respectively (ManneWhitney U test). All copy
numbers were obtained from the average of duplicated dPCR results. Results were considered significant when p < 0.05.
216
Z. Li et al. / Clinical Microbiology and Infection 26 (2020) 213e219
IS6110-dPCR detected more copies than gyrB-dPCR for the same
samples (p < 0.001, Fig. 1B). The number of copies detected in TBM
group was significantly higher than that in non-TBM group: median
(minimum, maximum), IS6110, 3.1 (0.0, 4260.0) vs. 0.0 (0.0, 4.6)
copies/20 mL of reaction mixture, p < 0.001; gyrB, 0.0 (0.0, 121.0) vs.
0.0 (0.0, 0.9) copies/20 mL reaction mixture, p ¼ 0.002 (Fig. 1C and
D). Moreover, the number of copies detected in definite TBM was
higher than that in probable and possible TBM (Fig. 1E and F).
sensitivity and specificity. The sensitivity of IS6110-dPCR assay for
total TBM was higher than that of gyrB-dPCR assay (57.4% vs. 22.1%,
p < 0.001), and there was no significant difference for specificity
between them (IS6110, 97.0%; gyrB, 100%). The sensitivity of dPCR
assay in definite TBM patients (IS6110, 73.1%; gyrB, 38.5%) was
higher than that in probable (IS6110, 52.9%; gyrB, 14.7%) and
possible TBM patients (IS6110, 25.0%; gyrB, 0.0%).
Evaluation of the diagnostic performance of dPCR for TBM
Sensitivity comparison of CSF IS6110-dPCR assay with routine
diagnostic tests
The ability to detect M. tuberculosis DNA in CSF of TBM patients
was assessed with a receiver operating characteristic (ROC) analysis. The overall area under ROC curve (AUC) of IS6110-dPCR (0.84,
95% confidence interval (CI) 0.76e0.91) was larger than that of
gyrB-dPCR (0.65, 95% CI 0.54e0.75) (p < 0.001, Fig. 2A). Further
analysis showed that the AUC of dPCR in patients with definite TBM
was larger than that in patients with probable TBM and possible
TBM (Fig. 2BeD).
The diagnostic performance of dPCR assay is presented in
Table 2. According to the ROC curves, we defined 2.0 (IS6110) and
1.0 (gyrB) copies per 20 mL of reaction mixture as the optimal cut-off
values to ensure high specificity and then the largest sum of
The positive detection rates of different tests in TBM patients are
listed in Table 3. The sensitivity of IS6110-dPCR was significantly
higher than that of smear microscopy (53.3% vs. 6.7%), IS6110-qPCR
(53.1% vs. 21.9%), Xpert (70.4% vs. 29.6%), TB antibody detection
(53.6% vs. 25.0%) and CSF ADA when cut-off value was 8 U/L (51.0%
vs. 16.3%) (all p < 0.05). It was significantly lower than that of peripheral blood IGRA (52.8% vs. 94.4%, p ¼ 0.001) and slightly lower
than CSF IGRA, TB antibody detection in peripheral blood and CSF
ADA when cut-off value was 3.8 U/L (all p > 0.05). The sensitivity of
IS6110-dPCR was also higher than that of mycobacterial culture
(50.0% vs. 12.5%), but the sample size was small (only eight cases)
and the difference was not significant (p ¼ 0.25).
Fig. 2. Diagnostic performances of cerebrospinal fluid (CSF) dPCR assays for TBM. (A, B, C and D) Receiver operating characteristic (ROC) curves in IS6110- and gyrB-dPCR for the
diagnosis of total, definite, probable and possible TBM, respectively. AUC, area under ROC curve; CI, confidence interval; TBM, tuberculous meningitis.
217
Risk factors for negative CSF IS6110-dPCR results
AUC, area under receiver operating characteristic (ROC) curve; LRþ, positive likelihood ratio; LR, negative likelihood ratio; PPV, positive predictive value; NPV, negative predictive value.
a
sensitivity comparison between IS6110- and gyrB- dPCR in total TBM patients (p < 0.001, McNemar test).
b
sensitivity comparison of IS6110-dPCR between definite TBM and probable group (p ¼ 0.112, Pearson chi-square test).
c
sensitivity comparison of IS6110-dPCR between definite TBM and possible group (p ¼ 0.033, Fisher's Exact Test).
d
sensitivity comparison of gyrB-dPCR between definite TBM and probable group (p ¼ 0.035, Pearson Chi-Square test).
e
sensitivity comparison of gyrB-dPCR between definite TBM and possible group (p ¼ 0.072, Fisher's Exact Test).
84.2 (68.7e94.0)
80.5 (65.1e91.2)
66.7 (4.0e99.8)
e
25.0 c (3.2e65.1)
0.0 e (0.0e36.9)
0.83 (0.67e0.99)
0.52 (0.29e0.75)
97.0 (84.2e99.9)
100.0 (89.4e100.0)
8.3 (0.8e80.1)
e
0.8 (0.5e1.2)
1.0 (1.0e1.0)
66.7 (51.6e79.6)
53.2 (40.1e66.0)
94.7 (74.0e99.9)
100.0 (47.8e100.0)
0.5 (0.3e0.7)
0.9 (0.7e1.0)
d
b
52.9
14.7
0.78 (0.67e0.90)
0.62 (0.49e0.76)
(35.1e70.2)
(5.0e31.1)
97.0 (84.2e99.9)
100.0 (89.4e100.0)
17.5 (2.5e123.5)
e
95.0 (75.1e99.9)
100.0 (69.2e100.0)
97.0 (84.2e99.9)
100.0 (89.4e100.0)
(52.2e88.4)
(20.2e59.4)
d, e
b, c
73.1
38.5
0.91 (0.82e0.99)
0.72 (0.58e0.86)
0.84
0.65
0.61
0.77
24.1 (3.5e168.5)
e
0.3 (0.1e0.5)
0.62 (0.5e0.8)
82.1 (66.5e92.5)
67.3 (52.5e80.1)
(39.3e65.4)
(28.1e49.5)
(28.1e49.5)
(39.3e65.4)
52.5
38.4
38.4
52.5
97.5 (86.8e99.9)
100.0 (78.2e100.0)
100.0 (78.2e100.0)
97.5 (86.8e99.9)
0.44 (0.3e0.6)
0.8 (0.7e0.9)
0.8 (0.7e0.9)
0.44 (0.3e0.6)
Cut point (copies/20mL reaction mixture)
Total TBM (n ¼ 68) compared with non-TBM (n ¼ 33)
IS6110-dPCR
2.0
gyrB-dPCR
1.0
IS6110- and gyrBedPCR positive e
IS6110- or gyrBedPCR positive
e
Definite TBM (n ¼ 26) compared with non-TBM (n ¼ 33)
IS6110-dPCR
2.0
gyrB-dPCR
1.0
Probable TBM (n ¼ 34) compared with non-TBM (n ¼ 33)
IS6110-dPCR
2.0
gyrB-dPCR
1.0
Possible TBM (n ¼ 8) compared with non-TBM (n ¼ 33)
IS6110-dPCR
2.0
gyrB-dPCR
1.0
Table 2
Diagnostic performances of CSF IS6110- and gyrB- dPCR assay for TBM
AUC
(0.76e0.91)
(0.54e0.75)
(0.51e0.71)
(0.68e0.85)
57.4 a (44.8e69.3)
22.1 a (12.9e33.8)
22.1 (12.9e33.8)
57.4 (44.8e69.3)
97.0 (84.2e99.9)
100.0 (89.4e100.0)
100.0 (89.4e100.0)
97.0 (84.2e99.9)
18.9 (2.7e131.8)
e
e
18.9 (2.7e131.8)
PPV (%)
Sensitivity (%)
Specificity (%)
LRþ
LR-
NPV (%)
Z. Li et al. / Clinical Microbiology and Infection 26 (2020) 213e219
Univariate analysis showed that long anti-tuberculosis treatment time (>1 month) was significantly associated with negative
IS6110-dPCR results in TBM group (p ¼ 0.001, Table S1). The
sensitivity of IS6110-dPCR in patients treated for no more than
1 month was significantly higher than that in those treated for
more than 1 month (72.5% (29/40) vs. 27.3% (6/22), p ¼ 0.001).
Moreover, the re-examination results of four patients after antituberculosis treatment (7 days to 32 days) also showed a significant reduction in the number of copies detected, two of which
changed from positive to negative (Table S2).
Discussion
Digital PCR is a novel method which can absolutely quantify
nucleic acids with low abundance [19]. Our study for the first time
reports the diagnostic accuracy of CSF dPCR for TBM. We selected
two M. tuberculosis-specific sequences as the amplification targets,
and IS6110-dPCR detected more copies and was more sensitive than
gyrB-dPCR. This is probably because IS6110 has more copies than
gyrB in the genome of most M. tuberculosis strains (e.g. 16 copies vs.
1 copy in H37Rv) [20,21]. Compared with IS6110-dPCR, joint
detection of gyrB and IS6110 did not significantly improve the
sensitivity and specificity (Table 2), so a single IS6110-dPCR is
enough for TBM diagnosis.
Tuberculous meningitis is usually classified into definite, probable and possible TBM, so it is better to evaluate the diagnostic
performance according to different types, which is also convenient
to compare the results with other studies. Our study showed the
sensitivity of IS6110-dPCR assay for total, definite, probable and
possible TBM was 57.4%, 73.1%, 52.9% and 25.0%, respectively,
indicating it was more sensitive than Xpert (57% for confirmed TBM
and 13% for suspected ones in a meta-analysis [4]) and Xpert Ultra
(44.19% for total TBM in a study [22]), two important tests recommended by WHO.
In this study, we also compared the sensitivity of IS6110-dPCR
with routine diagnostic tests conducted simultaneously or
earlier. We found IS6110-dPCR had higher sensitivity than CSF
smear (8.0-fold), mycobacterial culture (4.0-fold), IS6110-qPCR
(2.4-fold) and Xpert MTB/RIF (2.4-fold), suggesting that it was
more sensitive than current bacteriological and molecular tests
which can provide evidence of M. tuberculosis in CSF. Compared
with immunological tests, the sensitivity of CSF IS6110-dPCR for
TBM was only significantly lower than that of peripheral blood
IGRA (T-SPOT.TB) (52.8% vs. 94.4%, p ¼ 0.001). However, the
specificity of peripheral blood IGRA is very low (58% and 75.7% in
two studies, respectively) and it cannot be used to confirm TBM
[6,23]. Compared with CSF ADA, a biochemical index often
referred to in diagnosis, IS6110-dPCR was more sensitive than or
similar to CSF ADA when the cut-off value was high or low (two
thresholds based on previous studies), respectively [24,25].
Considering the poor specificity of ADA which often increases in
many infectious diseases, CSF IS6110-dPCR is far superior to CSF
ADA in TBM diagnosis.
The factors that may lead to negative dPCR results are also
noteworthy. We found that long anti-tuberculosis treatment time
was significantly associated with negative IS6110-dPCR results in
TBM patients. The overall sensitivity of dPCR increased significantly
in TBM patients treated with anti-tuberculosis drugs for no more
than a month. This is also evidenced by substantial reduction of
copies or even zero in re-testing after anti-tuberculosis treatment.
Therefore, these results remind us of the importance of early
detection, and also indicate that IS6110-dPCR might be used to
monitor the therapeutic effect of TBM.
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Z. Li et al. / Clinical Microbiology and Infection 26 (2020) 213e219
Table 3
Sensitivity comparisons between CSF IS6110-dPCR assay and routine tests in diagnosis of TBM
Tests
CSF
CSF
CSF
CSF
CSF
CSF
CSF
CSF
CSF
CSF
IS6110-dPCR
IS6110-dPCR
IS6110-dPCR
IS6110-dPCR
IS6110-dPCR
IS6110-dPCR
IS6110-dPCR
IS6110-dPCR
IS6110-dPCR
IS6110-dPCR
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
CSF smear microscopy
CSF mycobacterial culture
CSF IS6110-qPCR
CSF Xpert MTB/RIF
Peripheral blood IGRA
CSF IGRA
TB antibody detection in peripheral blood
TB antibody detection in CSF
CSF ADA (when cut-off value is 8.0 U/L)
CSF ADA (when cut-off value is 3.8 U/L)
Number of TBM patients
Sensitivity
30
8
32
27
36
13
36
28
49
49
53.3%
50.0%
53.1%
70.4%
52.8%
38.5%
52.8%
53.6%
51.0%
51.0%
(16/30)
(4/8)
(17/32)
(19/27)
(19/36)
(5/13)
(19/36)
(15/28)
(25/49)
(25/49)
p
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
vs.
6.7% (2/30)
12.5% (1/8)
21.9% (7/32)
29.6% (8/27)
94.4% (34/36)
46.2% (6/13)
55.6% (20/36)
25.0% (7/28)
16.3% (8/49)
57.1% (28/49)
0.000
0.250
0.006
0.001
0.001
1.000
1.000
0.021
0.001
0.678
qPCR, quantitative polymerase chain reaction; IGRA, interferon-gamma release assay; ADA, adenosine deaminase; TBM, tuberculous meningitis.
The cut-off value of CSF IS6110-dPCR assay is 2.0 copies/20mL reaction mixture. McNemar test was used to determine the significance between CSF IS6110-dPCR assay and
other tests.
Since Rich and McCordock [26] discovered M. tuberculosis
released into subarachnoid space from tuberculous granulomas
(now termed the Rich foci) in the 1930s, the two-step model of TBM
pathogenesis has remained unchallenged. We can make a definite
diagnosis of TBM by finding M. tuberculosis or detecting its nucleic
acid in CSF. However, some recent studies have reported the existence of cell-free DNA (such as fetal DNA and tumour DNA) in CSF
[27e29]. Cell-free M. tuberculosis DNA was also detected in tuberculous pleural effusion and plasma samples [16,17,30]. Therefore,
we speculate that the sequences detected in CSF may include
bacterial genomic DNA as well as cell-free DNA. But it still needs
further experimental confirmation.
Our study had some limitations. The number of enrolled patients was not large enough because of the low incidence of TBM.
Only two targets were selected and more multicopy targets or
combinations need to be assessed. Sensitivity comparisons between dPCR and other methods were not completely parallel due to
the limited volume of one CSF specimen, although all specimens
were collected at the early stage of admission. More parallel studies
using larger sample sizes, targeting more sequences or involving
different stages of disease, are needed to further clarify the diagnostic value of dPCR for TBM.
In conclusion, an IS6110-targeted droplet dPCR assay was shown
to be a rapid and accurate assay for TBM diagnosis. It is more
sensitive than aetiological tests currently used, and has considerable potential in improving the diagnosis of TBM.
Transparency declaration
The authors declare no conflict of interest. The study was supported by grants from Beijing Municipal Administration of Hospitals Incubating Program (PX2018055), National Natural Science
Foundation of China (81702097), National Science and Technology
Major Project of China (2015ZX10004801-003, 2017ZX10201301004 and 2018ZX10731301-003-003), Beijing Municipal Administration of Hospitals Ascent Plan (DFL20181601) and Beijing Natural
Science Foundation (7192038).
Acknowledgments
We thank the patients for their participation in this study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.cmi.2019.07.015.
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