- Email: [email protected]
Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients
Clinical Microbiology and Infection xxx (xxxx) xxx
Contents lists available at ScienceDirect
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 xxx
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 2019;▪:1 © 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: [email protected] (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.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
2
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
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
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
3
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.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
4
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
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.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
5
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 xxx (xxxx) xxx
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.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
6
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
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.
References [1] Wilkinson RJ, Rohlwink U, Misra UK, van Crevel R, Mai NTH, Dooley KE, et al. Tuberculous meningitis. Nat Rev Neurol 2017;13:581e98. [2] Rufai SB, Singh A, Singh J, Kumar P, Sankar MM, Singh S. Diagnostic usefulness of Xpert MTB/RIF assay for detection of tuberculous meningitis using cerebrospinal fluid. J Infect 2017;75:125e31. [3] Thakur R, Goyal R, Sarma S. Laboratory diagnosis of tuberculous meningitis - is there a scope for further improvement? J Lab Physicians 2010;2:21e4. [4] Gupta R, Talwar P, Khurana S, Kushwaha S, Jalan N, Thakur R. Diagnostic accuracy of nucleic acid amplification based assays for tuberculous meningitis: a meta-analysis. J Infect 2018;77:302e13. [5] Bahr NC, Nuwagira E, Evans EE, Cresswell FV, Bystrom PV, Byamukama A, et al. Diagnostic accuracy of Xpert MTB/RIF Ultra for tuberculous meningitis in HIVinfected adults: a prospective cohort study. Lancet Infect Dis 2018;18:68e75. [6] Pan L, Liu F, Zhang J, Yang X, Zheng S, Li J, et al. Interferon-gamma release assay performance of cerebrospinal fluid and peripheral blood in tuberculous meningitis in China. Biomed Res Int 2017;2017:8198505. [7] Bahr NC, Tugume L, Boulware DR. A word of caution in considering the use of the lipoarabinomannan lateral flow assay on cerebrospinal fluid for detection of tuberculous meningitis. J Clin Microbiol 2016;54:241e2. [8] Talwar P, Gupta R, Kushwaha S. Diagnostic test accuracy of adenosine deaminase for tuberculous meningitis: a systematic review and meta-analysis - an update. J Infect 2018;77:249e57. [9] Tong Y, Shen S, Jiang H, Chen Z. Application of digital PCR in detecting human diseases associated gene mutation. Cell Physiol Biochem 2017;43:1718e30. [10] Cao L, Cui X, Hu J, Li Z, Choi JR, Yang Q, et al. Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications. Biosens Bioelectron 2017;90:459e74. [11] Malatinkova E, Kiselinova M, Bonczkowski P, Trypsteen W, Messiaen P, Vermeire J, et al. Accurate quantification of episomal HIV-1 two-long terminal repeat circles by use of optimized DNA isolation and droplet digital PCR. J Clin Microbiol 2015;53:699e701. [12] Zhu G, Ye X, Dong Z, Lu YC, Sun Y, Liu Y, et al. Highly sensitive droplet digital PCR method for detection of EGFR-activating mutations in plasma cell-free DNA from patients with advanced non-small cell lung cancer. J Mol Diagn 2015;17:265e72. [13] Vidal-Folch N, Gavrilov D, Raymond K, Rinaldo P, Tortorelli S, Matern D, et al. Multiplex droplet digital PCR method applicable to newborn screening, carrier status, and assessment of spinal muscular atrophy. Clin Chem 2018;64: 1753e61. [14] Cui X, Cao L, Huang Y, Bai D, Huang S, Lin M, et al. In vitro diagnosis of DNA methylation biomarkers with digital PCR in breast tumors. Analyst 2018;143: 3011e20. [15] Tian H, Sun Y, Liu C, Duan X, Tang W, Li Z. Precise quantitation of microRNA in a single cell with droplet digital PCR based on ligation reaction. Anal Chem 2016;88:11384e9. [16] Yang J, Han X, Liu A, Bai X, Xu C, Bao F, et al. Use of digital droplet PCR to detect Mycobacterium tuberculosis DNA in whole blood-derived DNA samples from patients with pulmonary and extrapulmonary tuberculosis. Front Cell Infect Microbiol 2017;7:369. [17] Ushio R, Yamamoto M, Nakashima K, Watanabe H, Nagai K, Shibata Y, et al. Digital PCR assay detection of circulating Mycobacterium tuberculosis DNA in pulmonary tuberculosis patient plasma. Tuberculosis (Edinb) 2016;99:47e53. [18] Marais S, Thwaites G, Schoeman JF, Torok ME, Misra UK, Prasad K, et al. Tuberculous meningitis: a uniform case definition for use in clinical research. Lancet Infect Dis 2010;10:803e12. [19] Huggett JF, Cowen S, Foy CA. Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 2015;61:79e88. [20] Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537e44.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx [21] Cave MD, Eisenach KD, McDermott PF, Bates JH, Crawford JT. IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting. Mol Cell Probes 1991;5:73e80. [22] Wang G, Wang S, Jiang G, Yang X, Huang M, Huo F, et al. Xpert MTB/RIF Ultra improved the diagnosis of paucibacillary tuberculosis: a prospective cohort study. J Infect 2019;78:311e6. [23] Park KH, Cho OH, Lee EM, Lee SO, Choi SH, Kim YS, et al. T-cell-based assays on cerebrospinal fluid and PBMCs for rapid diagnosis of TB meningitis in non-HIV patients. Eur Respir J 2012;39:768e70. [24] Tuon FF, Higashino HR, Lopes MI, Litvoc MN, Atomiya AN, Antonangelo L, et al. Adenosine deaminase and tuberculous meningitis–a systematic review with meta-analysis. Scand J Infect Dis 2010;42:198e207. [25] Li XL, Ma LP, Chen HM, Zhang LQ, Wu XG, Gao MQ. Cerebrospinal fluid adenosine deaminase and its dynamic changes in tuberculous meningitis. Zhonghua Jie He He Hu Xi Za Zhi 2017;40:339e42.
7
[26] Rich AR, McCordock H. The pathogenesis of tuberculous meningitis. Bull Johns Hopkins Hosp 1933;52:5e37. [27] Angert RM, Leshane ES, Yarnell RW, Johnson KL, Bianchi DW. Cell-free fetal DNA in the cerebrospinal fluid of women during the peripartum period. Am J Obstet Gynecol 2004;190:1087e90. [28] Mouliere F, Mair R, Chandrananda D, Marass F, Smith CG, Su J, et al. Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients. EMBO Mol Med 2018;10:e9323. [29] Seoane J, De Mattos-Arruda L, Le Rhun E, Bardelli A, Weller M. Cerebrospinal fluid cell-free tumour DNA as a liquid biopsy for primary brain tumours and central nervous system metastases. Ann Oncol 2019;30:211e8. [30] Che N, Yang X, Liu Z, Li K, Chen X. Rapid detection of cell-free Mycobacterium tuberculosis DNA in tuberculous pleural effusion. J Clin Microbiol 2017;55: 1526e32.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
Contents lists available at ScienceDirect
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 xxx
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 2019;▪:1 © 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: [email protected] (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.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
2
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
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
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
3
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.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
4
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
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.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
5
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 xxx (xxxx) xxx
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.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
6
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx
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.
References [1] Wilkinson RJ, Rohlwink U, Misra UK, van Crevel R, Mai NTH, Dooley KE, et al. Tuberculous meningitis. Nat Rev Neurol 2017;13:581e98. [2] Rufai SB, Singh A, Singh J, Kumar P, Sankar MM, Singh S. Diagnostic usefulness of Xpert MTB/RIF assay for detection of tuberculous meningitis using cerebrospinal fluid. J Infect 2017;75:125e31. [3] Thakur R, Goyal R, Sarma S. Laboratory diagnosis of tuberculous meningitis - is there a scope for further improvement? J Lab Physicians 2010;2:21e4. [4] Gupta R, Talwar P, Khurana S, Kushwaha S, Jalan N, Thakur R. Diagnostic accuracy of nucleic acid amplification based assays for tuberculous meningitis: a meta-analysis. J Infect 2018;77:302e13. [5] Bahr NC, Nuwagira E, Evans EE, Cresswell FV, Bystrom PV, Byamukama A, et al. Diagnostic accuracy of Xpert MTB/RIF Ultra for tuberculous meningitis in HIVinfected adults: a prospective cohort study. Lancet Infect Dis 2018;18:68e75. [6] Pan L, Liu F, Zhang J, Yang X, Zheng S, Li J, et al. Interferon-gamma release assay performance of cerebrospinal fluid and peripheral blood in tuberculous meningitis in China. Biomed Res Int 2017;2017:8198505. [7] Bahr NC, Tugume L, Boulware DR. A word of caution in considering the use of the lipoarabinomannan lateral flow assay on cerebrospinal fluid for detection of tuberculous meningitis. J Clin Microbiol 2016;54:241e2. [8] Talwar P, Gupta R, Kushwaha S. Diagnostic test accuracy of adenosine deaminase for tuberculous meningitis: a systematic review and meta-analysis - an update. J Infect 2018;77:249e57. [9] Tong Y, Shen S, Jiang H, Chen Z. Application of digital PCR in detecting human diseases associated gene mutation. Cell Physiol Biochem 2017;43:1718e30. [10] Cao L, Cui X, Hu J, Li Z, Choi JR, Yang Q, et al. Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications. Biosens Bioelectron 2017;90:459e74. [11] Malatinkova E, Kiselinova M, Bonczkowski P, Trypsteen W, Messiaen P, Vermeire J, et al. Accurate quantification of episomal HIV-1 two-long terminal repeat circles by use of optimized DNA isolation and droplet digital PCR. J Clin Microbiol 2015;53:699e701. [12] Zhu G, Ye X, Dong Z, Lu YC, Sun Y, Liu Y, et al. Highly sensitive droplet digital PCR method for detection of EGFR-activating mutations in plasma cell-free DNA from patients with advanced non-small cell lung cancer. J Mol Diagn 2015;17:265e72. [13] Vidal-Folch N, Gavrilov D, Raymond K, Rinaldo P, Tortorelli S, Matern D, et al. Multiplex droplet digital PCR method applicable to newborn screening, carrier status, and assessment of spinal muscular atrophy. Clin Chem 2018;64: 1753e61. [14] Cui X, Cao L, Huang Y, Bai D, Huang S, Lin M, et al. In vitro diagnosis of DNA methylation biomarkers with digital PCR in breast tumors. Analyst 2018;143: 3011e20. [15] Tian H, Sun Y, Liu C, Duan X, Tang W, Li Z. Precise quantitation of microRNA in a single cell with droplet digital PCR based on ligation reaction. Anal Chem 2016;88:11384e9. [16] Yang J, Han X, Liu A, Bai X, Xu C, Bao F, et al. Use of digital droplet PCR to detect Mycobacterium tuberculosis DNA in whole blood-derived DNA samples from patients with pulmonary and extrapulmonary tuberculosis. Front Cell Infect Microbiol 2017;7:369. [17] Ushio R, Yamamoto M, Nakashima K, Watanabe H, Nagai K, Shibata Y, et al. Digital PCR assay detection of circulating Mycobacterium tuberculosis DNA in pulmonary tuberculosis patient plasma. Tuberculosis (Edinb) 2016;99:47e53. [18] Marais S, Thwaites G, Schoeman JF, Torok ME, Misra UK, Prasad K, et al. Tuberculous meningitis: a uniform case definition for use in clinical research. Lancet Infect Dis 2010;10:803e12. [19] Huggett JF, Cowen S, Foy CA. Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 2015;61:79e88. [20] Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998;393:537e44.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015
Z. Li et al. / Clinical Microbiology and Infection xxx (xxxx) xxx [21] Cave MD, Eisenach KD, McDermott PF, Bates JH, Crawford JT. IS6110: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting. Mol Cell Probes 1991;5:73e80. [22] Wang G, Wang S, Jiang G, Yang X, Huang M, Huo F, et al. Xpert MTB/RIF Ultra improved the diagnosis of paucibacillary tuberculosis: a prospective cohort study. J Infect 2019;78:311e6. [23] Park KH, Cho OH, Lee EM, Lee SO, Choi SH, Kim YS, et al. T-cell-based assays on cerebrospinal fluid and PBMCs for rapid diagnosis of TB meningitis in non-HIV patients. Eur Respir J 2012;39:768e70. [24] Tuon FF, Higashino HR, Lopes MI, Litvoc MN, Atomiya AN, Antonangelo L, et al. Adenosine deaminase and tuberculous meningitis–a systematic review with meta-analysis. Scand J Infect Dis 2010;42:198e207. [25] Li XL, Ma LP, Chen HM, Zhang LQ, Wu XG, Gao MQ. Cerebrospinal fluid adenosine deaminase and its dynamic changes in tuberculous meningitis. Zhonghua Jie He He Hu Xi Za Zhi 2017;40:339e42.
7
[26] Rich AR, McCordock H. The pathogenesis of tuberculous meningitis. Bull Johns Hopkins Hosp 1933;52:5e37. [27] Angert RM, Leshane ES, Yarnell RW, Johnson KL, Bianchi DW. Cell-free fetal DNA in the cerebrospinal fluid of women during the peripartum period. Am J Obstet Gynecol 2004;190:1087e90. [28] Mouliere F, Mair R, Chandrananda D, Marass F, Smith CG, Su J, et al. Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients. EMBO Mol Med 2018;10:e9323. [29] Seoane J, De Mattos-Arruda L, Le Rhun E, Bardelli A, Weller M. Cerebrospinal fluid cell-free tumour DNA as a liquid biopsy for primary brain tumours and central nervous system metastases. Ann Oncol 2019;30:211e8. [30] Che N, Yang X, Liu Z, Li K, Chen X. Rapid detection of cell-free Mycobacterium tuberculosis DNA in tuberculous pleural effusion. J Clin Microbiol 2017;55: 1526e32.
Please cite this article as: Li Z et al., Diagnostic accuracy of droplet digital PCR analysis of cerebrospinal fluid for tuberculous meningitis in adult patients, Clinical Microbiology and Infection, https://doi.org/10.1016/j.cmi.2019.07.015