- Email: [email protected]
To compare CSF adenosine deaminase levels and CSF-PCR for tuberculous meningitis
Clinical Neurology and Neurosurgery 112 (2010) 424–430
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To compare CSF adenosine deaminase levels and CSF-PCR for tuberculous meningitis S.V. Rana a,∗ , F. Chacko b , V. Lal b , S.K. Arora c , S. Parbhakar b , Sanjeev K. Sharma a , K. Singh a a b c
Department of Gastroenterology, Postgraduate Institute of Medical Education And Research, Chandigarh, India Department of Neurology, Postgraduate Institute of Medical Education And Research, Chandigarh, India Department of Immunopathology, Postgraduate Institute of Medical Education And Research, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 27 May 2009 Received in revised form 13 February 2010 Accepted 20 February 2010 Available online 29 March 2010 Keywords: Tuberculous meningitis Polymerase chain reaction Adenosine deaminase
a b s t r a c t This study was planned to compare the adenosine deaminase (ADA) levels and polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) as a rapid method to diagnose tuberculosis meningitis (TBM). Fifty-four adult patients with suspected TBM and 37 controls were included in this study. The median ADA level was 21 U/L of most likely TBM, 14 U/L of unconfirmed TBM and 5 U/L of controls. PCR for Mycobacterium tuberculosis was positive in 12 out of 27 most likely TBM cases, 5 out of 27 unconfirmed TBM cases and 3 out of 37 controls. Using a cut off level of >10 U/L, CSF-ADA had a sensitivity of 92.5% and specificity of 97% for the diagnosis of TBM. PCR for M. tuberculosis had a sensitivity of 44.5% and specificity 92% in the most likely TBM cases. This study shows that CSF-ADA is a more sensitive indicator than PCR for the diagnosis of M. tuberculosis. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis (TB) continues to be a major health problem world wide [1]. A variety of neurological complications were noted in military tuberculosis patients, tuberculous meningitis and cerebral tuberculomas being the most frequent complications [2]. The etiology, clinical features and outcome of patients with bacterial meningitis from an urban Chinese city over a 10-year period have been reported by Hui et al. [3]. Extra pulmonary disease accounts for nearly 20% of the cases in HIV seronegative people [4]. Tuberculous meningitis (TBM) is a serious complication of tuberculosis that is often diagnosed late due to its varied and insidious clinical presentation [5]. Early and accurate diagnosis helps in instituting proper treatment and better prognosis [6]. The conventional tests of demonstration of acid fast bacilli (AFB) in the cerebrospinal fluid (CSF) by staining and culture method are not very sensitive and are time consuming. There is often a delay in making the correct diagnosis and initiating the treatment in time [7]. Nucleic acid
Abbreviations: ADA, adenosine deaminase; AFB, acid fast bacilli; ATT, antitubercular treatment; CECT/MRI, contrast computed tomography/magnetic resonance imaging; CSF, cerebrospinal fluid; ESR, erythrocyte sedimentation rate; M, meningitis; OPD, out patient department; PCR, polymerase chain reaction; SSPE, sub acute scalloping pan encephalitis; TB, tuberculosis; TBM, tuberculosis meningitis; VDRL, Venereal Disease Research Lab. ∗ Corresponding author at: House # 137, Sector 15-A Chandigarh 160015, India. Tel.: +91 172 2716417; fax: +91 172 2744401. E-mail address: [email protected] (S.V. Rana). 0303-8467/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.clineuro.2010.02.012
amplification probes are expensive and do not add substantial benefit to the traditional techniques [8]. Therefore, there is a great need of other rapid diagnostic methods for diagnosis in patients with clinical suspicion of TBM. Adenosine deaminase, a polymorphic enzyme involved in purine metabolism is found to be elevated in CSF of TBM patients [9]. Estimation of ADA is easy, rapid inexpensive and can be carried out in routine. In bacterial meningitis with low cell counts, ADA level in CSF tends to be lower than that in TBM with a corresponding CSF cell counts [10]. Polymerase chain reaction (PCR) is a rapid, sensitive and specific method to identify Mycobacterium tuberculosis [10]. However, it cannot be used for initiating or concluding antituberculosis therapy in suspected cases of TBM [11,12]. The specificity and sensitivity of PCR can be further enhanced by modification in the methods directed to reduce contamination and by increasing sample volume. Polymerase chain reaction continues to be positive even after few weeks of starting ATT [11]. PCR has been successfully used for the detection of M. tuberculosis from other body fluids such as plural fluid [13]. This study is an endeavor to compare the ADA and PCR in CSF as rapid methods to diagnose TBM.
2. Materials and methods The number of total patients screened in this study was 105 attending Neurology and Emergency OPD of PGIMER, Chandigarh, India during a period of one year. Fourteen patients were excluded with traumatic lumber puncture and remaining 91 patients were
S.V. Rana et al. / Clinical Neurology and Neurosurgery 112 (2010) 424–430
included in the study. Out of 91 patients, 54 were of suspected TBM and 37 were of other neurological disorders. Selection of cases was made according to the criteria, which was based on history, clinical examination, CSF tests, radiology and response to treatment as done by Ahuja et al. [14] and Seth et al. [15]. All patients included in this study were HIV negative. Institute’s Ethical Clearance was obtained before starting this study. (I) Clinical symptoms and signs: • Mandatory – Fever and headache for more than two weeks • Optional – Vomiting, neck stiffness, altered sensorium, seizures or focal neurological deficit. (II) Supporting evidence
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2.1. ADA estimation ADA activity was measured by the method of Guisti [16]. The detection limit of this method for measurement of CSF-ADA levels is 0.5–300 U/L. The samples obtained were centrifuged at 2000 × g for 10 min and the supernatant stored at −20 ◦ C for few days. In short, 1 ml of 50 mM with 6.5 pH phosphate buffer, 1 ml of 21 mM adenosine in phosphate buffer, 0.5 ml CSF sample and 0.5 ml distilled water were added. Reaction mixture was incubated for 60 min in a water bath at 37 ◦ C. 3 ml of phenol solution and 3 ml alkaline hypochlorite solution were added and again incubated for 30 min at 37 ◦ C in a water bath. Absorbance was taken at 628 nm by spectrophotometer. 3. Procedure for TB-PCR
(A) CSF: Cells >20/cmm, lymphocytes >60%, proteins >100 mg%, sugar <60% of corresponding blood sugar, negative gram stain, India ink stain and VDRL (where relevant). (B) CECT/MRI showing one or more of: Exudates-basal cisterns/sylvian, fissures, gyral enhancement, hydrocephalus, infarcts, tuberculoma. (C) Active extra neural TB as evidenced by appropriate tests: Mycobacterium test, radiology and histopathological examination. (D) Clinical response to ATT and relief of symptoms The suspected TBM cases were classified as being: • Highly probable TBM (met clinical criteria and were positive for at least 3 of the 4 supporting evidences). • Probable TBM (met clinical criteria and were positive for 2 supporting tests). • Possible TBM (met clinical criteria and were positive for 1 supporting test). Cases were confirmed when AFB stain or culture of CSF was positive. The diagnosis of pyogenic meningitis was made on the basis of either positive CSF for bacterial culture or Gram stain in addition to the presence of >5 pus cells/mm3 , CSF with polymorph nuclear pleocytosis, CSF protein >40 mg/dl and CSF glucose < two-third of concomitantly determined blood glucose. - Detailed clinical history was recorded with main emphasis on presence of fever, headache, vomiting, altered sensorium, prior treatment and duration. - Clinical evaluation was done for signs of meningeal irritation, raised intracranial pressure, cranial nerves deficits, focal neurological deficits and other evidence of TB elsewhere in the body. - Routine blood tests (including ESR) and chest X-ray were done in all patients. CSF was tapped without blood staining and used for biochemistry (proteins and sugar), cytology (total and differential cell counts), AFB and gram stain. VDRL, India ink staining, cryptococcal latex agglutination test, estimation of ADA activity and PCR were also done. - Contrast enhanced CT scan/MRI was performed in all cases. ATT and steroids were started and response was evaluated. - Control samples were taken from 37 patients in whom TBM was not suspected and CSF examination was done for diagnostic purpose of other diseases like acute inflammatory demyelinating polyradiculoneuropathy, multiple sclerosis, polyneuropathy, and idiopathic intracranial hypertension, etc. Two ml CSF sample from all patients was collected in a sterile bottle. Fourteen patients, in whom the lumbar puncture was traumatic, were excluded from the study.
3.1. DNA extraction 0.4 ml CSF sample was taken in a micro centrifuge tube, centrifuged for 10 min at 10,000 rpm. Supernatant was decanted and the pellet, which contains cells, was obtained. 20 l of 0.1% Tritonx-100 was added to the cell pellet. The sample was incubated at 95 ◦ C for 15 min in a dry bath, centrifuged for 10 min at 10,000 rpm, the supernatant decanted and the pellet dissolved in Tris–EDTA buffer. Thus, the sample was ready for PCR. 3.2. Primers Primers as designed by Scarpellini et al. [17] and subsequently used by Arora et al. [13] were taken for amplification of a conserved repetitive insertion sequence 6110 (IS 6110) with 30 base pair in length. Primer Tb Pr-J Primer Tb Pr-K
5 CGC-GAC-CAC-CCG-CGG-CAA-AGC-CCG-CAG-CAG-3 5 -CAT-CGT-GGA-AGC-GAC-CCG-CCA-GCC-CAG-GAT-3
3.3. PCR The regents used for PCR, were dNTP, Taq polymerase enzyme, primers, MgCl2 , Taq buffer, distilled water and genomic DNA. 50–100 ng of genomic DNA was used in PCR reaction. In this method, 25 l reaction mixture contained 1 l DNA, 0.25 l Taq polymerase, 0.75 l each forward and reverse primers, 2.5 l of 10× Taq buffer, 1.25 l of 2.5 mM MgCl2 , 0.5 l dNTP and the volume made up with distilled water. PCR was carried out under the following conditions: 1. Pre Denaturation 2. Denaturation 3. Annealing 4. Extension 5. Final extension
- at 94 ◦ C for 7 min - at 94 ◦ C for 1 min - at 58 ◦ C for 1 min - at 72 ◦ C for 1 min - at 72 ◦ C for 10 min
35 cycles of PCR were carried out. 3.4. Statistical analysis Due to small study population for correlation calculation, the non-parametric Pearson correlation test was used. For all other statistical tests analysis also, non-parametric Mann–Whitney test was used. Values have been mentioned as mean ± SD. p Values <0.05 have been considered as significant. 3.5. Ethical considerations It was a prospective study and the CSF was obtained from the samples, which were routinely submitted to the laboratory for investigations. It involved no experimentation or undue trauma to the patients.
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S.V. Rana et al. / Clinical Neurology and Neurosurgery 112 (2010) 424–430 Table 1 CSF findings in TBM and non-TBM patients.
4. Results Fifty-four patients of TBM were included in the present study. Five of them were AFB culture positive (definite TBM). Remaining patients were classified as highly probable [22], probable [22] and possible [5] TBM based on the criteria mentioned above. CSF findings and neuro imaging studies of all patients were analyzed. Thirty seven control samples were taken from CSF of patients in the neurology ward, in whom TBM was not suspected and CSF was taken for other indications like demyelinating disorders, acute inflammatory demyelinating polyneuropathy, pyogenic meningitis, and polyneuropathy. Out of these 37 patients, 10 had demyelination, 5 idiopathic intracranial hypertension, 5 polyneuropathy, 3 acute inflammatory demyelinating polyneuropathy, 2 SSPE, 5 multiple sclerosis, and 7 neurocysticercosis.
Total cells per cumm Median (range)
TBM patients (n = 54)
Non-TBM patients (n = 37)
145** (20–1800)
0 (0–10)
Absolute lymphocyte count per cumm Median (range) 102** (4–497)
0 (0–10)
Proteins (mg%) Median (range)
40 (10–140)
158* (40–1500)
Ratio of CSF sugar to blood sugar Median (range) 0.32* (14–71) CSF-ADA levels (U/L) Median (range)
19 (8-74)
5 (2–14)
PCR positivity
17/54** (32%)
3/37 (8%)
** *
4.1. Age and sex Mean age of TBM patients was 30.2 ± 13.9 years with median of 26 years. Patients of pediatric age group were not included in this study. The male: female ratio of TBM patients was 22:32. While mean age of non-TBM patients was 32.4 ± 14.5 years with a median of 28 years, and the male:female ratio was 14:23.
0.62 (16–89)
p < 0.001 between TBM and non-TBM patients. p < 0.05 between TBM and non-TBM patients.
vestibulocohlear and glossopharyngeal nerves in one each. Eight of them had multiple cranial nerve palsies. Fever and headache were part of the inclusion criteria in the present study. Five patients had hemiparesis. All patients were started on ATT. Five patients developed drug induced hepatitis and were managed accordingly. Four patients expired while in the hospital and autopsy could not be performed on any of them.
4.2. Clinical characteristics
4.3. Chest X-ray
The mean duration of fever in TBM patients was 52.3 ± 73.1 days with a median of 30 days. Headache was present in all patients and 49 out of 54 (90.7%) patients had history of vomiting. Fortyseven out of 54 (87%) patients had alteration in sensorium with a mean duration of 6 ± 4 days. Seizures were present in 18 out of 54 (33%) patients with five of them having focal seizures and signs of meningeal irritation were present in 50 out of 54 (93%) patients. Papilledema was seen in 17 out of 54 (31%) patients, abducens nerve palsy in 17 (31%) patients and in 8 patients it was bilateral. Twentyone patients had ventriculomegaly and eleven of them required ventriculoperitoneal shunt insertion. Eighteen patients had other cranial nerve palsies. Oculomotor nerve was involved in 9 out of 54 (17%) patients, facial nerve in 7 (13%), vagus in 2 (4%), optic,
Fifteen out of 54 (28%) patients had an abnormal chest X-ray. Miliary mottling and cavitary changes were seen in 4 patients each while hilar nodes and pleural effusion in 3 patients each. 4.4. Cranial imaging Cranial imaging showed exudates in 22 out of 54 (41%) patients and hydrocephalus in 21(39%) patients. Seven out of 54 (13%) patients had evidence of infarcts and seven (13%) with tuberculomas. No abnormality was detected in the cranial imaging in seven patients.
Table 2 CSF findings in various subgroups of TBM patients. Definite (culture + ve) (n = 5)
Highly probable (n = 22)
Probable (n = 22)
Possible (n = 5)
300a
165a
145a
60a
Absolute lymphocyte count per cumm Median 100a
140a
82a
45a
Proteins (mg%) Median
125
160
92
80
Ratio of CSF sugar to blood sugar Median
0.33
0.48
Total cells per cumm Median
0.28
0.28
CSF-ADA levels (U/L) Median (range)
19 (19–74)
21 (13–54)
19 (11–40)
14 (8–33)
PCR positivity
4 (80%)
8 (36%)
5 (23%)
0 (0%)
Increase in ADA levels with increase in CSF absolute lymphocyte count was not statistically significant (p > 0.05) when student’s “t” test was used. a “r” value was 0.15 using Karl Pearson’s coefficient of correlation between CSF-ADA levels and absolute lymphocyte counts. Table 3 CSF-ADA levels and CSF-PCR positivity with Medical Research Council staging of TBM. Stage I (n = 6)
Stage II (n = 30)
Stage III (n = 18)
CSF-ADA (U/L) Median (range)
16.5 (8–30)
19 (10–45)
21.5 (22–74)
CSF-PCR positivity
2 (33%)
8 (27%)
7 (38%)
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Table 4 CSF-ADA levels and PCR positivity with the duration of illness.
4.5. CSF characteristics CSF was analyzed for cells, gram stain, AFB stain, India ink stain, proteins, sugars, AFB culture, bacterial culture, ADA levels and PCR for TBM and non-TBM. The results are shown in Table 1.Total cell counts, absolute lymphocyte counts and CSF proteins were significantly higher in TBM patients as compared to non-TBM patients. In contrast, the ratio of CSF sugar to blood sugar is significantly lower in TBM patients as compared to non-TBM patients. CSF-ADA levels were also significantly higher in TBM patients as compared to nonTBM patients. The PCR positivity was 32% in TBM patients and 8% in non-TBM patients.
Duration of illness (days) <30 (n = 26) CSF-ADA (U/L) Median (range) PCR positivity
30–59 (n = 13)
>60 (n = 15)
19(8–45)
22 (12–74)
19 (9–50)
7(27%)
6 (46%)
4 (27%)
5. Major CSF findings in various subgroups of TBM patients were as follows
5.3.1. CSF-ADA levels The median values of CSF-ADA with range in subgroups of TBM patients have been given in Table 2. CSF-ADA levels are higher in definite, highly probable and probable TBM patients as compared to possible TBM patients. Levels of CSF-ADA also showed a positive correlation with absolute lymphocyte counts and the ‘r’ value was 0.15 using Karl Pearson’s coefficient of correlation.
5.1. Pleocytosis
5.4. PCR results
Mean ± SD with median values of CSF total cells/cumm and absolute lymphocyte counts/cumm in various subgroups of TBM are given in Table 2. Total cells and absolute lymphocyte counts are higher in definite and highly probable TBM patients as compared to probable and possible TBM patients.
5.2. Biochemistry The mean ± SD with median values of protein (mg %) and the ratio of CSF glucose to blood glucose have been shown in Table 2. CSF proteins were higher in definite and highly probable TBM patients as compared to probable and possible TBM patients. While there is no change in the ratio of CSF sugar to glucose sugar in all the above-mentioned subgroups of TBM patients.
The PCR was also more often positive in definite subgroup of TBM patients as compared to highly probable, probable and possible subgroups of TBM patients as shown in Table 2. 5.5. Correlation with staging of the disease With increasing severity of the disease (according to the Medical Research Council staging), the CSF-ADA levels increased as shown in Table 3. 5.6. Correlation with duration of illness The CSF-ADA level was higher in those with illness duration between 1 and 2 months, as shown in Table 4. The PCR was also more often positive in this group, but the difference was not statistically significant.
5.3. AFB smear and culture
5.7. Correlation with prior ATT
AFB smear was positive only in 1 out of 54 (1.8%) patient and culture positivity in 5 (9%) patients.
The level of ADA tended to decrease with one week of ATT and further decreased with ATT for >1 week. The difference in the
Table 5 CSF-ADA levels and CSF-PCR positivity with ATT treatment. Group I
Group II
Group III
No prior ATT (n = 42)
ATT up to 1 week (n = 7)
>1 week of ATT (n = 5)
CSF-ADA (U/L) Median (range)
26 (20–74)
21 (13–55)
19* (8–35)
CSF-PCR positivity
14 (33%)
2 (29%)
1 (20%)
*
p < 0.02 between group I vs group III.
Table 6 CSF-ADA levels and PCR positivity in TBM and non-TBM patients: subgroups analysis. Groups
I II III IV # ## * ** ***
All TBM patients (n = 54) Most likely cases of TBM (definite and highly probable) (n = 27) Unconfirmed TBM (probable and possible) (n = 27) Non-TBM patients (n = 37) (control)
p < 0.01 between II and III. p < 0.001 between TBM (I, II, III) vs control (IV). p < 0.04 I vs IV. p < 0.008 II vs III. p < 0.0007 II vs IV.
CSF-ADA positivity with cut off level of ≥10 U/L
CSF-ADA positivity %
CSF-ADA activity (U/L) Mean ± SD
50 26 12 1
92.5%## 96.3%# 44.44% 2.7%
26.1 29 21 5.7
± ± ± ±
19## 24## 15## 3.9
PCR positivity
PCR positivity %
17 12 5 3
32* 44.5*** 19** 8 false positivity
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Table 7 Sensitivity and specificity of CSF-ADA levels in the diagnosis of TBM. Sensitivity
Specificity TBM vs controls
Cut off >8 (U/L) Cut off >10 (U/L) Cut off >15 (U/L)
100% (54/54) 92.5% (50/54) 72% (39/54)
TBM vs pyogenic meningitis
94% (35/37) 97% (36/37) 97% (36/37)
25% (1/4) 50% (2/4) 50% (2/4)
Table 8 Area under the curve, test result variable(s) of CSF-ADA levels. Area
SEa
Asymptotic Sig.b
0.992
0.008
0.000
Asymptotic 95% confidence interval Lower bound
Upper bound
0.977
1.007
The test result variable(s): CSF-ADA levels have at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased. a Under the non-parametric assumption. b Null hypothesis: true area = 0.5.
ADA levels of groups 1 (without prior ATT) and group III (ATT >1 week) was statistically significant (p < 0.02) as shown in Table 5.The PCR sensitivity also tended to decrease with ATT and duration of treatment, but one case was positive even after 3 months of ATT treatment. 5.8. Comparison of ADA levels CSF-ADA levels of subgroups i.e. TBM and non-TBM patients have been compared in Table 6. TBM group has been subdivided into “most likely (definite and highly probable)” patients of TBM [27], and unconfirmed (22 probable and 5 possible) TBM patients. The difference between CSF-ADA levels of TBM (group I) and non-TBM (group IV) patients was statistically significant (p < 0.001). However, the difference between most likely TBM (group II), and unconfirmed TBM (group III) patients was non-significant (p > 0.1). 5.9. Comparison of PCR results PCR for M. tuberculosis was positive in 44.4% of most likely cases of TBM (group II), and 18.5% of unconfirmed TBM patients (group III). This difference was significant (p < 0.008). Three samples from the non-TBM group were also positive by PCR for M. tuberculosis. This corresponds to a false positivity rate of 8% for PCR (Table 6). The difference of PCR positivity between TBM (group II) and non-TBM patients (group IV) was statistically significant (p < 0.0007). The sensitivity and specificity of CSF-ADA for the diagnosis of TBM (calculated by using a cut off value of CSF-ADA >10 U/L) were 92.5% and 97.3% respectively. By comparing TBM patients with pyogenic meningitis, the specificity was only 50%. By increasing the cut off level to >15 U/L (above the mean ADA of the pyogenic group), the specificity increased to 97% but the sensitivity falls to 72% as shown in Table 7. The sensitivity and specificity of PCR were 32% and 92% respectively in the diagnosis of TBM. The sensitivity was 44.5% for the most likely TBM cases. The positive predictive value for TBM patients was 85% and for most likely TBM patients 80%. The negative predictive value for TBM patients was 48% and for most likely TBM patients 69%. The false negative value in TBM patients was 68% and false positive value in non-TBM patients 8%. The comparison of sensitivity and specificity of CSF-ADA (by using cut off >10 U/L) and PCR to diagnose TBM has been mentioned in Table 8. This table shows that both the sensitivity and specificity to diagnose TBM using CSF-ADA is better than those of PCR.
The CSF-ADA levels showed a positive correlation with absolute lymphocyte counts and CSF proteins. The correlation was better with the proteins in CSF as reported in earlier studies [9,22]. The CSF-ADA levels were higher in those with severe disease, depending on the Medical Research Council staging (I–III), but the change was not statistically significant (Table 1). Among non-TBM patients, the patients with pyogenic meningitis had higher ADA levels (13.8 ± 6.7 U/L) and hence were analyzed separately. In pyogenic meningitis, the correlation of CSF-ADA levels with CSF absolute lymphocyte counts (r = 0.14) was less than that obtained in the TBM group (r = 0.15), even with comparable lymphocyte counts. Since the pyogenic meningitis group included only 4 patients, no specific conclusions can be drawn. It would require a larger series of patients with pyogenic meningitis to make any comparison (Table 9). 6. Discussion The incidence of fever (100%), headache (100%), vomiting (91%) and alteration in sensorium (85%) was higher in the present study than 78%, 37%, 45% and 53% respectively in a previous study (Thomas et al. [18]). The possible reasons may be absence of pediatric population and mandatory inclusion criteria of fever and headache in the present study. The incidence of seizures, alterations in sensorium and papilledema was comparable in both the studies. The CSF smear was positive for AFB only in 1 out of 54 (2%) patients in this study. Since the sensitivity is very low, CSF AFB smear cannot be relied upon to pick up cases of TBM. The CSF culture grew AFB in 5 patients (9%) in the present study. This is in comparison with the previous studies by Seth et al. (0%) [15], Shankar et al. (12%) [19], Miörner et al. (17%) [20] and Nguyen et al. (19%) [21]. The delay of 5–6 weeks to obtain positive culture coupled with low sensitivity (9%) makes this test less useful to the clinician for the diagnosis of TBM. The mean ADA in the TBM group in present study was 26.1 ± 18.8 U/L which is comparable with the mean ADA level of 26.2 U/L observed in TBM by Malan et al. [9]. They noted that the ADA level in TBM group was higher in adults (26.2 U/L) compared to children (14.5 U/L). Gambhir et al. [22], reported mean ADA of 9.6 ± 4.1 U/L in 36 patients with tubercular meningitis, which were significantly higher than cases of viral encephalitis and enteric encephalopathy. However, levels were comparable to pyogenic meningitis (7.92 + 0.95 U/L) and cerebral malaria. Using 8 U/L as cut off value for diagnosis of TBM, a sensitivity of 44% and specificity of 75% was observed while in the present study it was 100% and 94% respec-
S.V. Rana et al. / Clinical Neurology and Neurosurgery 112 (2010) 424–430 Table 9 Coordinates of the curve, test result variable(s) of CSF-ADA levels. Positive if greater than or equal toa
Sensitivity
1 − specificity
1.00 2.50 4.00 5.50 6.50 7.50 8.50 9.50 10.50 11.50 12.50 13.50 14.50 15.50 16.50 17.50 18.50 19.50 20.50 21.50 22.50 27.50 32.50 35.00 38.50 42.50 48.50 53.00 57.50 67.50 90.50 108.00
1.000 1.000 1.000 1.000 1.000 1.000 0.962 0.943 0.887 0.849 0.830 0.792 0.698 0.679 0.660 0.623 0.585 0.491 0.472 0.396 0.283 0.264 0.226 0.208 0.189 0.170 0.151 0.132 0.075 0.038 0.019 0.000
1.000 0.944 0.639 0.306 0.111 0.056 0.056 0.028 0.028 0.028 0.028 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
The test result variable(s): CSF-ADA levels has at least one tie between the positive actual state group and the negative actual state group. a The smallest cut off value is the minimum observed test value minus 1, and the largest cut off value is the maximum observed test value plus 1. All the other cut off values are the averages of two consecutive ordered observed test values.
tively for TBM vs controls and 100% sensitivity and 25% specificity for TBM vs pyogenic meningitis. Their study indicated that CSFADA is a non-specific method to diagnose TBM. The higher level of CSF-ADA in the present study may be due to the inclusion of only adults in the present study. CSF adenosine activity in 20 TBM, 10 non-TBM and 15 non-tubercular, non-meningitis patients were also measured by Gautam et al. [23]. They observed that the mean CSF-ADA activity (13.62 ± 8.45 U/L) in TBM was significantly higher as compared to non-TBM (6.51 ± 2.41 U/L) patients. The sensitivity and specificity of CSF-ADA for TBM was found to be 85% and 70% as compared to non-TBM and 85% and 100% non-TBM, non-meningitis patients respectively at a cut off value of 6.97 U/L to diagnose tubercular meningitis. Corral et al. [24] used ROC curve analysis and suggested a cut off value of 8.5 U/L for the diagnosis of TBM with 57% sensitivity and 87% specificity. ADA activity in CSF of 182 patients with meningitis has also been reported by Choi et al. [25]. In their study, (i) 36 cases were of TBM, (ii) 130 cases of viral or aseptic meningitis, (iii) 9 cases of bacterial meningitis, and (iv) 7 cases of cryptococcal meningitis. The mean ± SD of ADA activity was 12.76 ± 7.53 U/L in group (i), 2.58 ± 2.37 U/L in group (ii), 7.38 ± 3.27 U/L in group (iii), and 7.42 ± 4.38 U/L in group (iv). The sensitivity of the test for group (i) compared with group (ii) was 0.83 and specificity 0.95 when a cut off value of 7 U/L was used. When group (i) was compared with groups (iii) and (iv), the sensitivity was 0.58 and the specificity 0.89 and 0.71 respectively at a cut off value of >10 U/L. >15 U/L values were not observed in any of the non-TBM patients. They have also reported that ADA activity >15 U/L could be a strong indication of tuberculous meningitis. Thus, they concluded that determination of CSF-ADA can aid in the early differential diagnosis of TBM.
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In the present study using a cut off value of CSF-ADA at >8 U/L, the sensitivity and specificity were 100% and 94% respectively. However, by using the cut off level at >10 U/L, the sensitivity and specificity were 92.5% and 97% respectively. By raising the cut off level at >15 U/L (above the mean CSF-ADA of pyogenic meningitis group), the specificity was same (97%), but the sensitivity dropped to 72% from 92.5%. This indicates that with a cut off level at >10 U/L gives a good sensitivity and specificity. The present study also shows that CSF-ADA can reliably distinguish between TBM and non-TBM, but not between TBM and pyogenic meningitis. The overall results of present study are in comparison to previous studies [9,22]. In the present study, the sensitivity and specificity of PCR was 44.5% and 92% respectively in most probable TBM group. The PCR also had an overall sensitivity of 32% and specificity 92% in present study. The sensitivity of PCR is low when compared to previous studies by Shankar et al. [19] and Nguyen et al. [21]. Shankar et al. [19] reported a sensitivity of 65% and specificity of 89% for PCR in their study of 34 patients with TBM. The sensitivity was 75% in highly probable TBM group, 57% in probable TBM and 43% in possible TBM group. In a study of 33 patients by Miorner et al. [20], the sensitivity of PCR for M. Tuberculosis was 54% and specificity 94%. In a recent study of 40 cases of TBM by Bonington et al. [26], PCR for M. Tuberculosis was positive in 10 cases (25% sensitivity). Dora et al. [27] have also shown that the sensitivity and specificity of CSF-PCR for the diagnosis of TBM were 50% and 98.6% respectively with a concordance with CSF mycobacterial culture of 96%. Afroze et al. [28] have also reported 77.7% sensitivity and 100% specificity to diagnose TBM by using of MPB64-Target PCR. Recently, 86% sensitivity and 100% specificity to diagnose TBM by using rpoB nested PCR have also been reported by Huang et al. [29]. In earlier studies, PCR was evaluated in CSF from known cases and controls. An element of bias in the interpretation of PCR data cannot be completely excluded in those studies. In the present study, both TBM and non-TBM samples were marked as suspected TBM and thus were blinded for the processing of PCR. Previous studies tested a smaller number of patients and used a selected patient group or had a higher number of false positives in PCR. Out of the 5 culture positive cases, one was PCR negative in the present study. This discrepancy can be explained by the differences in the volume of samples tested. It would thus be advisable to send a large volume of CSF for PCR analysis. In the present study, three control CSF samples were positive by PCR. These were from a patient with polyneuropathy and one patient with fungal meningitis had positive cryptococcal latex agglutination test and improved with a course of amphotericin. Even though she had lymphocytic pleocytosis in CSF, co-infection with M. tuberculosis was unlikely because she improved completely with antifungal treatment. As other 2 patients had no cells in the CSF, silent tubercular lesions in the meningitis are unlikely in the absence of symptoms. These positive results may be attributed to possible cross contamination of samples during initial handling. In present study, the sensitivity (92.5%) and specificity (97%) of CSF-ADA in TBM was higher as compared to PCR overall sensitivity (32%) and specificity (92%). Thus it is evident that the CSF-ADA had an edge both in sensitivity and specificity over the CSF-PCR to diagnose tubercular meningitis.
7. Conclusion This study shows that ADA which is a simple, rapid and inexpensive test, can be used for the diagnosis of TBM with good sensitivity and specificity. PCR is also a rapid test for the diagnosis of TBM with a good specificity but low sensitivity. Although the CSF-ADA test is sensitive and specific and can suggest the diagnosis of TBM, other
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techniques like AFB smear, PCR for M. tuberculosis and rapid culture would be required for confirmation of the diagnosis. This pilot study remains to be confirmed in a large, prospective and preferably multi-centre study. References [1] Dye C, Watt CJ, Bleed DM, Hosseini SM, Raviglione MC. Evolution of tuberculosis control and prospects for reducing tuberculosis incidence, prevalence, and deaths globally. J Am Med Assoc 2005;293:2767–75. [2] Garg RK, Sharma R, Kar AM, Kushwaha RA, Singh MK, Shukla R, et al. Neurological complications of miliary tuberculosis. Clin Neurol Neurosurg 2010;112(March (3)):188–92. [3] Hui ACF, Ng KC, Tong PY, Mok V, Chow KM, Wu A, et al. Bacterial meningitis in Hong Kong: 10-years’ experience. Clin Neurol Neurosurg 2005;107:366–70. [4] Frieden TR, Sterling TR, Munsiff SS, Watt CJ, Dye C. Tuberculosis. Lancet 2003;362:887–99. [5] Molenaar RP, Jansen CL, Taphoorn MJ. Three patients with tuberculous meningitis: treatment started at tentative diagnosis. Ned Tijdschr Geneeskd 2008;152(11):593–8. [6] Thwaites GE, Lan NT, Dung NH. Effect of antituberculosis drug resistance on response to treatment and outcome in adults with tuberculous meningitis. J Infect Dis 2005;192:79–88. [7] Alarcon F, Duenas G, Cevallos N, Lees AJ. Movement disorders in 30 patients with tuberculous meningitis. Move Disord 2000;15:561–9. [8] Thwaites GE, Caws M, Chau TT. Comparison of conventional bacteriology with nucleic acid amplification (amplified mycobacterium direct test) for diagnosis of tuberculous meningitis before and after inception of antituberculosis chemotherapy. J Clin Microbiol 2004;42:996–1002. [9] Malan C, Donald PR, Marilyn G. ADA levels in CSF in the diagnosis of TBM. J Trop Med Hyg 1984;87:33–40. [10] Roberts MC, McMillan C, Coyle MB. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex. J Clin Microbiol 1987;25(July (7)):1239–43. [11] García JE, Losada JP, González VL. Reliability of the polymerase chain reaction in the diagnosis of mycobacterial infection. Chest 1996;110(July (1)):300–1. [12] Doucet-Populaire F, Lalande V, Carpentier E, Bourgoin A, Dailloux M, Bollet C, et al. A blind study of the polymerase chain reaction for the detection of Mycobacterium tuberculosis DNA. Azay Mycobacteria Study Group. Tuber Lung Dis 1996;77(August (4)):358–62. [13] Arora SK, Kumar B, Sehgal S. Development of a polymerase chain reaction dot-blotting system for detecting cutaneous tuberculosis. Br J Dermatol 2000;142(January (1)):72–6. [14] Ahuja GK, Mohan KK, Prasad K, Behari M. Diagnostic criteria for tuberculous meningitis and their validation. Tuber Lung Dis 1994;75(April (2)):149–52.
[15] Seth P, Ahuja GK, Bhanu NV, Behari M, Bhowmik S, Broor S, et al. Evaluation of polymerase chain reaction for rapid diagnosis of clinically suspected tuberculous meningitis. Tuber Lung Dis 1996;77(August (4)):353–7. [16] Guisti G. Adenosine deaminase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. New York Academic Press; 1974. p. 1092–6. [17] Scarpellini P, Racca S, Cinque S, Delfanti F, Gianotti N, Terreni MR, et al. Nested polymerase chain reaction for diagnosis and monitoring treatment response in AIDS patients with tuberculous meningitis. AIDS 1995;9(August (8)):895–900. [18] Thomas MD, Chopra JS, Wallia BNS. A clinical study of 232 patients. J Assoc Phys India 1977;25:633–9. [19] Shankar P, Manjunath N, Mohan KK, Prasad K, Behari M, Shriniwas, et al. Rapid diagnosis of tuberculous meningitis by polymerase chain reaction. Lancet 1991;337(January (8732)):5–7. [20] Miörner H, Sjöbring U, Nayak P, Chandramuki A. Diagnosis of tuberculous meningitis: a comparative analysis of 3 immunoassays, an immune complex assay and the polymerase chain reaction. Tuber Lung Dis 1995;76(October (5)):381–6. [21] Nguyen LN, Kox LF, Pham LD, Kuijper S, Kolk AH. The potential contribution of the polymerase chain reaction to the diagnosis of tuberculous meningitis. Arch Neurol 1996;53(August (8)):771–6. [22] Gambhir LS, Mehta M, Singh DS, Khanna HD. Evaluation of CSF-adenosine deaminase activity in tubercular meningitis. J Assoc Phys India 1999;47(February (2)):192–4. [23] Gautam N, Aryal M, Bhatta N, Bhattacharya SK, Baral N, Lamsal M. Comparative study of cerebrospinal fluid adenosine deaminase activity in patients with meningitis. Nepal Med Coll J 2007;9(June (2)):104–6. [24] Corral I, Quereda C, Navas E, Martín-Dávila P, Pérez-Elías MJ, Casado JL, et al. Adenosine deaminase activity in cerebrospinal fluid of HIV-infected patients: limited value for diagnosis of tuberculous meningitis. Eur J Clin Microbiol Infect Dis 2004;23(June (6)):471–6. [25] Choi SH, Kim YS, Bae IG, Chung JW, Lee MS, Kang JM, et al. The possible role of cerebrospinal fluid adenosine deaminase activity in the diagnosis of tuberculous meningitis in adults. Clin Neurol Neurosurg 2002;104(January (1)): 10–5. [26] Bonington A, Strang JI, Klapper PE, Hood SV, Rubombora W, Penny M, et al. Use of Roche AMPLICOR Mycobacterium tuberculosis PCR in early diagnosis of tuberculous meningitis. J Clin Microbiol 1998;36(May (5)):1251–4. [27] Dora JM, Geib G, Chakr R, Paris F, Mombach AB, Lutz L, et al. Polymerase chain reaction as a useful and simple tool for rapid diagnosis of tuberculous meningitis in a Brazilian tertiary care hospital. Braz J Infect Dis 2008;12(3): 245–7. [28] Afroze D, Mir AW, Kirmani A, Shakeel-ul-Rehman, Eachkoti R, Siddiqi MA. Improved diagnosis of central nervous system tuberculosis by MPB64-Target PCR. Braz J Microbiol 2008;39:209–13. [29] Huang HJ, Xiang DR, Sheng JF, Li J, Pan XP, Yu HY, et al. rpoB nested PCR and sequencing for the early diagnosis of tuberculous meningitis and rifampicin resistance. Int J Tuber Lung Dis 2009;13(June (6)):749–54.
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Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
To compare CSF adenosine deaminase levels and CSF-PCR for tuberculous meningitis S.V. Rana a,∗ , F. Chacko b , V. Lal b , S.K. Arora c , S. Parbhakar b , Sanjeev K. Sharma a , K. Singh a a b c
Department of Gastroenterology, Postgraduate Institute of Medical Education And Research, Chandigarh, India Department of Neurology, Postgraduate Institute of Medical Education And Research, Chandigarh, India Department of Immunopathology, Postgraduate Institute of Medical Education And Research, Chandigarh, India
a r t i c l e
i n f o
Article history: Received 27 May 2009 Received in revised form 13 February 2010 Accepted 20 February 2010 Available online 29 March 2010 Keywords: Tuberculous meningitis Polymerase chain reaction Adenosine deaminase
a b s t r a c t This study was planned to compare the adenosine deaminase (ADA) levels and polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) as a rapid method to diagnose tuberculosis meningitis (TBM). Fifty-four adult patients with suspected TBM and 37 controls were included in this study. The median ADA level was 21 U/L of most likely TBM, 14 U/L of unconfirmed TBM and 5 U/L of controls. PCR for Mycobacterium tuberculosis was positive in 12 out of 27 most likely TBM cases, 5 out of 27 unconfirmed TBM cases and 3 out of 37 controls. Using a cut off level of >10 U/L, CSF-ADA had a sensitivity of 92.5% and specificity of 97% for the diagnosis of TBM. PCR for M. tuberculosis had a sensitivity of 44.5% and specificity 92% in the most likely TBM cases. This study shows that CSF-ADA is a more sensitive indicator than PCR for the diagnosis of M. tuberculosis. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis (TB) continues to be a major health problem world wide [1]. A variety of neurological complications were noted in military tuberculosis patients, tuberculous meningitis and cerebral tuberculomas being the most frequent complications [2]. The etiology, clinical features and outcome of patients with bacterial meningitis from an urban Chinese city over a 10-year period have been reported by Hui et al. [3]. Extra pulmonary disease accounts for nearly 20% of the cases in HIV seronegative people [4]. Tuberculous meningitis (TBM) is a serious complication of tuberculosis that is often diagnosed late due to its varied and insidious clinical presentation [5]. Early and accurate diagnosis helps in instituting proper treatment and better prognosis [6]. The conventional tests of demonstration of acid fast bacilli (AFB) in the cerebrospinal fluid (CSF) by staining and culture method are not very sensitive and are time consuming. There is often a delay in making the correct diagnosis and initiating the treatment in time [7]. Nucleic acid
Abbreviations: ADA, adenosine deaminase; AFB, acid fast bacilli; ATT, antitubercular treatment; CECT/MRI, contrast computed tomography/magnetic resonance imaging; CSF, cerebrospinal fluid; ESR, erythrocyte sedimentation rate; M, meningitis; OPD, out patient department; PCR, polymerase chain reaction; SSPE, sub acute scalloping pan encephalitis; TB, tuberculosis; TBM, tuberculosis meningitis; VDRL, Venereal Disease Research Lab. ∗ Corresponding author at: House # 137, Sector 15-A Chandigarh 160015, India. Tel.: +91 172 2716417; fax: +91 172 2744401. E-mail address: [email protected] (S.V. Rana). 0303-8467/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.clineuro.2010.02.012
amplification probes are expensive and do not add substantial benefit to the traditional techniques [8]. Therefore, there is a great need of other rapid diagnostic methods for diagnosis in patients with clinical suspicion of TBM. Adenosine deaminase, a polymorphic enzyme involved in purine metabolism is found to be elevated in CSF of TBM patients [9]. Estimation of ADA is easy, rapid inexpensive and can be carried out in routine. In bacterial meningitis with low cell counts, ADA level in CSF tends to be lower than that in TBM with a corresponding CSF cell counts [10]. Polymerase chain reaction (PCR) is a rapid, sensitive and specific method to identify Mycobacterium tuberculosis [10]. However, it cannot be used for initiating or concluding antituberculosis therapy in suspected cases of TBM [11,12]. The specificity and sensitivity of PCR can be further enhanced by modification in the methods directed to reduce contamination and by increasing sample volume. Polymerase chain reaction continues to be positive even after few weeks of starting ATT [11]. PCR has been successfully used for the detection of M. tuberculosis from other body fluids such as plural fluid [13]. This study is an endeavor to compare the ADA and PCR in CSF as rapid methods to diagnose TBM.
2. Materials and methods The number of total patients screened in this study was 105 attending Neurology and Emergency OPD of PGIMER, Chandigarh, India during a period of one year. Fourteen patients were excluded with traumatic lumber puncture and remaining 91 patients were
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included in the study. Out of 91 patients, 54 were of suspected TBM and 37 were of other neurological disorders. Selection of cases was made according to the criteria, which was based on history, clinical examination, CSF tests, radiology and response to treatment as done by Ahuja et al. [14] and Seth et al. [15]. All patients included in this study were HIV negative. Institute’s Ethical Clearance was obtained before starting this study. (I) Clinical symptoms and signs: • Mandatory – Fever and headache for more than two weeks • Optional – Vomiting, neck stiffness, altered sensorium, seizures or focal neurological deficit. (II) Supporting evidence
425
2.1. ADA estimation ADA activity was measured by the method of Guisti [16]. The detection limit of this method for measurement of CSF-ADA levels is 0.5–300 U/L. The samples obtained were centrifuged at 2000 × g for 10 min and the supernatant stored at −20 ◦ C for few days. In short, 1 ml of 50 mM with 6.5 pH phosphate buffer, 1 ml of 21 mM adenosine in phosphate buffer, 0.5 ml CSF sample and 0.5 ml distilled water were added. Reaction mixture was incubated for 60 min in a water bath at 37 ◦ C. 3 ml of phenol solution and 3 ml alkaline hypochlorite solution were added and again incubated for 30 min at 37 ◦ C in a water bath. Absorbance was taken at 628 nm by spectrophotometer. 3. Procedure for TB-PCR
(A) CSF: Cells >20/cmm, lymphocytes >60%, proteins >100 mg%, sugar <60% of corresponding blood sugar, negative gram stain, India ink stain and VDRL (where relevant). (B) CECT/MRI showing one or more of: Exudates-basal cisterns/sylvian, fissures, gyral enhancement, hydrocephalus, infarcts, tuberculoma. (C) Active extra neural TB as evidenced by appropriate tests: Mycobacterium test, radiology and histopathological examination. (D) Clinical response to ATT and relief of symptoms The suspected TBM cases were classified as being: • Highly probable TBM (met clinical criteria and were positive for at least 3 of the 4 supporting evidences). • Probable TBM (met clinical criteria and were positive for 2 supporting tests). • Possible TBM (met clinical criteria and were positive for 1 supporting test). Cases were confirmed when AFB stain or culture of CSF was positive. The diagnosis of pyogenic meningitis was made on the basis of either positive CSF for bacterial culture or Gram stain in addition to the presence of >5 pus cells/mm3 , CSF with polymorph nuclear pleocytosis, CSF protein >40 mg/dl and CSF glucose < two-third of concomitantly determined blood glucose. - Detailed clinical history was recorded with main emphasis on presence of fever, headache, vomiting, altered sensorium, prior treatment and duration. - Clinical evaluation was done for signs of meningeal irritation, raised intracranial pressure, cranial nerves deficits, focal neurological deficits and other evidence of TB elsewhere in the body. - Routine blood tests (including ESR) and chest X-ray were done in all patients. CSF was tapped without blood staining and used for biochemistry (proteins and sugar), cytology (total and differential cell counts), AFB and gram stain. VDRL, India ink staining, cryptococcal latex agglutination test, estimation of ADA activity and PCR were also done. - Contrast enhanced CT scan/MRI was performed in all cases. ATT and steroids were started and response was evaluated. - Control samples were taken from 37 patients in whom TBM was not suspected and CSF examination was done for diagnostic purpose of other diseases like acute inflammatory demyelinating polyradiculoneuropathy, multiple sclerosis, polyneuropathy, and idiopathic intracranial hypertension, etc. Two ml CSF sample from all patients was collected in a sterile bottle. Fourteen patients, in whom the lumbar puncture was traumatic, were excluded from the study.
3.1. DNA extraction 0.4 ml CSF sample was taken in a micro centrifuge tube, centrifuged for 10 min at 10,000 rpm. Supernatant was decanted and the pellet, which contains cells, was obtained. 20 l of 0.1% Tritonx-100 was added to the cell pellet. The sample was incubated at 95 ◦ C for 15 min in a dry bath, centrifuged for 10 min at 10,000 rpm, the supernatant decanted and the pellet dissolved in Tris–EDTA buffer. Thus, the sample was ready for PCR. 3.2. Primers Primers as designed by Scarpellini et al. [17] and subsequently used by Arora et al. [13] were taken for amplification of a conserved repetitive insertion sequence 6110 (IS 6110) with 30 base pair in length. Primer Tb Pr-J Primer Tb Pr-K
5 CGC-GAC-CAC-CCG-CGG-CAA-AGC-CCG-CAG-CAG-3 5 -CAT-CGT-GGA-AGC-GAC-CCG-CCA-GCC-CAG-GAT-3
3.3. PCR The regents used for PCR, were dNTP, Taq polymerase enzyme, primers, MgCl2 , Taq buffer, distilled water and genomic DNA. 50–100 ng of genomic DNA was used in PCR reaction. In this method, 25 l reaction mixture contained 1 l DNA, 0.25 l Taq polymerase, 0.75 l each forward and reverse primers, 2.5 l of 10× Taq buffer, 1.25 l of 2.5 mM MgCl2 , 0.5 l dNTP and the volume made up with distilled water. PCR was carried out under the following conditions: 1. Pre Denaturation 2. Denaturation 3. Annealing 4. Extension 5. Final extension
- at 94 ◦ C for 7 min - at 94 ◦ C for 1 min - at 58 ◦ C for 1 min - at 72 ◦ C for 1 min - at 72 ◦ C for 10 min
35 cycles of PCR were carried out. 3.4. Statistical analysis Due to small study population for correlation calculation, the non-parametric Pearson correlation test was used. For all other statistical tests analysis also, non-parametric Mann–Whitney test was used. Values have been mentioned as mean ± SD. p Values <0.05 have been considered as significant. 3.5. Ethical considerations It was a prospective study and the CSF was obtained from the samples, which were routinely submitted to the laboratory for investigations. It involved no experimentation or undue trauma to the patients.
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S.V. Rana et al. / Clinical Neurology and Neurosurgery 112 (2010) 424–430 Table 1 CSF findings in TBM and non-TBM patients.
4. Results Fifty-four patients of TBM were included in the present study. Five of them were AFB culture positive (definite TBM). Remaining patients were classified as highly probable [22], probable [22] and possible [5] TBM based on the criteria mentioned above. CSF findings and neuro imaging studies of all patients were analyzed. Thirty seven control samples were taken from CSF of patients in the neurology ward, in whom TBM was not suspected and CSF was taken for other indications like demyelinating disorders, acute inflammatory demyelinating polyneuropathy, pyogenic meningitis, and polyneuropathy. Out of these 37 patients, 10 had demyelination, 5 idiopathic intracranial hypertension, 5 polyneuropathy, 3 acute inflammatory demyelinating polyneuropathy, 2 SSPE, 5 multiple sclerosis, and 7 neurocysticercosis.
Total cells per cumm Median (range)
TBM patients (n = 54)
Non-TBM patients (n = 37)
145** (20–1800)
0 (0–10)
Absolute lymphocyte count per cumm Median (range) 102** (4–497)
0 (0–10)
Proteins (mg%) Median (range)
40 (10–140)
158* (40–1500)
Ratio of CSF sugar to blood sugar Median (range) 0.32* (14–71) CSF-ADA levels (U/L) Median (range)
19 (8-74)
5 (2–14)
PCR positivity
17/54** (32%)
3/37 (8%)
** *
4.1. Age and sex Mean age of TBM patients was 30.2 ± 13.9 years with median of 26 years. Patients of pediatric age group were not included in this study. The male: female ratio of TBM patients was 22:32. While mean age of non-TBM patients was 32.4 ± 14.5 years with a median of 28 years, and the male:female ratio was 14:23.
0.62 (16–89)
p < 0.001 between TBM and non-TBM patients. p < 0.05 between TBM and non-TBM patients.
vestibulocohlear and glossopharyngeal nerves in one each. Eight of them had multiple cranial nerve palsies. Fever and headache were part of the inclusion criteria in the present study. Five patients had hemiparesis. All patients were started on ATT. Five patients developed drug induced hepatitis and were managed accordingly. Four patients expired while in the hospital and autopsy could not be performed on any of them.
4.2. Clinical characteristics
4.3. Chest X-ray
The mean duration of fever in TBM patients was 52.3 ± 73.1 days with a median of 30 days. Headache was present in all patients and 49 out of 54 (90.7%) patients had history of vomiting. Fortyseven out of 54 (87%) patients had alteration in sensorium with a mean duration of 6 ± 4 days. Seizures were present in 18 out of 54 (33%) patients with five of them having focal seizures and signs of meningeal irritation were present in 50 out of 54 (93%) patients. Papilledema was seen in 17 out of 54 (31%) patients, abducens nerve palsy in 17 (31%) patients and in 8 patients it was bilateral. Twentyone patients had ventriculomegaly and eleven of them required ventriculoperitoneal shunt insertion. Eighteen patients had other cranial nerve palsies. Oculomotor nerve was involved in 9 out of 54 (17%) patients, facial nerve in 7 (13%), vagus in 2 (4%), optic,
Fifteen out of 54 (28%) patients had an abnormal chest X-ray. Miliary mottling and cavitary changes were seen in 4 patients each while hilar nodes and pleural effusion in 3 patients each. 4.4. Cranial imaging Cranial imaging showed exudates in 22 out of 54 (41%) patients and hydrocephalus in 21(39%) patients. Seven out of 54 (13%) patients had evidence of infarcts and seven (13%) with tuberculomas. No abnormality was detected in the cranial imaging in seven patients.
Table 2 CSF findings in various subgroups of TBM patients. Definite (culture + ve) (n = 5)
Highly probable (n = 22)
Probable (n = 22)
Possible (n = 5)
300a
165a
145a
60a
Absolute lymphocyte count per cumm Median 100a
140a
82a
45a
Proteins (mg%) Median
125
160
92
80
Ratio of CSF sugar to blood sugar Median
0.33
0.48
Total cells per cumm Median
0.28
0.28
CSF-ADA levels (U/L) Median (range)
19 (19–74)
21 (13–54)
19 (11–40)
14 (8–33)
PCR positivity
4 (80%)
8 (36%)
5 (23%)
0 (0%)
Increase in ADA levels with increase in CSF absolute lymphocyte count was not statistically significant (p > 0.05) when student’s “t” test was used. a “r” value was 0.15 using Karl Pearson’s coefficient of correlation between CSF-ADA levels and absolute lymphocyte counts. Table 3 CSF-ADA levels and CSF-PCR positivity with Medical Research Council staging of TBM. Stage I (n = 6)
Stage II (n = 30)
Stage III (n = 18)
CSF-ADA (U/L) Median (range)
16.5 (8–30)
19 (10–45)
21.5 (22–74)
CSF-PCR positivity
2 (33%)
8 (27%)
7 (38%)
S.V. Rana et al. / Clinical Neurology and Neurosurgery 112 (2010) 424–430
427
Table 4 CSF-ADA levels and PCR positivity with the duration of illness.
4.5. CSF characteristics CSF was analyzed for cells, gram stain, AFB stain, India ink stain, proteins, sugars, AFB culture, bacterial culture, ADA levels and PCR for TBM and non-TBM. The results are shown in Table 1.Total cell counts, absolute lymphocyte counts and CSF proteins were significantly higher in TBM patients as compared to non-TBM patients. In contrast, the ratio of CSF sugar to blood sugar is significantly lower in TBM patients as compared to non-TBM patients. CSF-ADA levels were also significantly higher in TBM patients as compared to nonTBM patients. The PCR positivity was 32% in TBM patients and 8% in non-TBM patients.
Duration of illness (days) <30 (n = 26) CSF-ADA (U/L) Median (range) PCR positivity
30–59 (n = 13)
>60 (n = 15)
19(8–45)
22 (12–74)
19 (9–50)
7(27%)
6 (46%)
4 (27%)
5. Major CSF findings in various subgroups of TBM patients were as follows
5.3.1. CSF-ADA levels The median values of CSF-ADA with range in subgroups of TBM patients have been given in Table 2. CSF-ADA levels are higher in definite, highly probable and probable TBM patients as compared to possible TBM patients. Levels of CSF-ADA also showed a positive correlation with absolute lymphocyte counts and the ‘r’ value was 0.15 using Karl Pearson’s coefficient of correlation.
5.1. Pleocytosis
5.4. PCR results
Mean ± SD with median values of CSF total cells/cumm and absolute lymphocyte counts/cumm in various subgroups of TBM are given in Table 2. Total cells and absolute lymphocyte counts are higher in definite and highly probable TBM patients as compared to probable and possible TBM patients.
5.2. Biochemistry The mean ± SD with median values of protein (mg %) and the ratio of CSF glucose to blood glucose have been shown in Table 2. CSF proteins were higher in definite and highly probable TBM patients as compared to probable and possible TBM patients. While there is no change in the ratio of CSF sugar to glucose sugar in all the above-mentioned subgroups of TBM patients.
The PCR was also more often positive in definite subgroup of TBM patients as compared to highly probable, probable and possible subgroups of TBM patients as shown in Table 2. 5.5. Correlation with staging of the disease With increasing severity of the disease (according to the Medical Research Council staging), the CSF-ADA levels increased as shown in Table 3. 5.6. Correlation with duration of illness The CSF-ADA level was higher in those with illness duration between 1 and 2 months, as shown in Table 4. The PCR was also more often positive in this group, but the difference was not statistically significant.
5.3. AFB smear and culture
5.7. Correlation with prior ATT
AFB smear was positive only in 1 out of 54 (1.8%) patient and culture positivity in 5 (9%) patients.
The level of ADA tended to decrease with one week of ATT and further decreased with ATT for >1 week. The difference in the
Table 5 CSF-ADA levels and CSF-PCR positivity with ATT treatment. Group I
Group II
Group III
No prior ATT (n = 42)
ATT up to 1 week (n = 7)
>1 week of ATT (n = 5)
CSF-ADA (U/L) Median (range)
26 (20–74)
21 (13–55)
19* (8–35)
CSF-PCR positivity
14 (33%)
2 (29%)
1 (20%)
*
p < 0.02 between group I vs group III.
Table 6 CSF-ADA levels and PCR positivity in TBM and non-TBM patients: subgroups analysis. Groups
I II III IV # ## * ** ***
All TBM patients (n = 54) Most likely cases of TBM (definite and highly probable) (n = 27) Unconfirmed TBM (probable and possible) (n = 27) Non-TBM patients (n = 37) (control)
p < 0.01 between II and III. p < 0.001 between TBM (I, II, III) vs control (IV). p < 0.04 I vs IV. p < 0.008 II vs III. p < 0.0007 II vs IV.
CSF-ADA positivity with cut off level of ≥10 U/L
CSF-ADA positivity %
CSF-ADA activity (U/L) Mean ± SD
50 26 12 1
92.5%## 96.3%# 44.44% 2.7%
26.1 29 21 5.7
± ± ± ±
19## 24## 15## 3.9
PCR positivity
PCR positivity %
17 12 5 3
32* 44.5*** 19** 8 false positivity
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Table 7 Sensitivity and specificity of CSF-ADA levels in the diagnosis of TBM. Sensitivity
Specificity TBM vs controls
Cut off >8 (U/L) Cut off >10 (U/L) Cut off >15 (U/L)
100% (54/54) 92.5% (50/54) 72% (39/54)
TBM vs pyogenic meningitis
94% (35/37) 97% (36/37) 97% (36/37)
25% (1/4) 50% (2/4) 50% (2/4)
Table 8 Area under the curve, test result variable(s) of CSF-ADA levels. Area
SEa
Asymptotic Sig.b
0.992
0.008
0.000
Asymptotic 95% confidence interval Lower bound
Upper bound
0.977
1.007
The test result variable(s): CSF-ADA levels have at least one tie between the positive actual state group and the negative actual state group. Statistics may be biased. a Under the non-parametric assumption. b Null hypothesis: true area = 0.5.
ADA levels of groups 1 (without prior ATT) and group III (ATT >1 week) was statistically significant (p < 0.02) as shown in Table 5.The PCR sensitivity also tended to decrease with ATT and duration of treatment, but one case was positive even after 3 months of ATT treatment. 5.8. Comparison of ADA levels CSF-ADA levels of subgroups i.e. TBM and non-TBM patients have been compared in Table 6. TBM group has been subdivided into “most likely (definite and highly probable)” patients of TBM [27], and unconfirmed (22 probable and 5 possible) TBM patients. The difference between CSF-ADA levels of TBM (group I) and non-TBM (group IV) patients was statistically significant (p < 0.001). However, the difference between most likely TBM (group II), and unconfirmed TBM (group III) patients was non-significant (p > 0.1). 5.9. Comparison of PCR results PCR for M. tuberculosis was positive in 44.4% of most likely cases of TBM (group II), and 18.5% of unconfirmed TBM patients (group III). This difference was significant (p < 0.008). Three samples from the non-TBM group were also positive by PCR for M. tuberculosis. This corresponds to a false positivity rate of 8% for PCR (Table 6). The difference of PCR positivity between TBM (group II) and non-TBM patients (group IV) was statistically significant (p < 0.0007). The sensitivity and specificity of CSF-ADA for the diagnosis of TBM (calculated by using a cut off value of CSF-ADA >10 U/L) were 92.5% and 97.3% respectively. By comparing TBM patients with pyogenic meningitis, the specificity was only 50%. By increasing the cut off level to >15 U/L (above the mean ADA of the pyogenic group), the specificity increased to 97% but the sensitivity falls to 72% as shown in Table 7. The sensitivity and specificity of PCR were 32% and 92% respectively in the diagnosis of TBM. The sensitivity was 44.5% for the most likely TBM cases. The positive predictive value for TBM patients was 85% and for most likely TBM patients 80%. The negative predictive value for TBM patients was 48% and for most likely TBM patients 69%. The false negative value in TBM patients was 68% and false positive value in non-TBM patients 8%. The comparison of sensitivity and specificity of CSF-ADA (by using cut off >10 U/L) and PCR to diagnose TBM has been mentioned in Table 8. This table shows that both the sensitivity and specificity to diagnose TBM using CSF-ADA is better than those of PCR.
The CSF-ADA levels showed a positive correlation with absolute lymphocyte counts and CSF proteins. The correlation was better with the proteins in CSF as reported in earlier studies [9,22]. The CSF-ADA levels were higher in those with severe disease, depending on the Medical Research Council staging (I–III), but the change was not statistically significant (Table 1). Among non-TBM patients, the patients with pyogenic meningitis had higher ADA levels (13.8 ± 6.7 U/L) and hence were analyzed separately. In pyogenic meningitis, the correlation of CSF-ADA levels with CSF absolute lymphocyte counts (r = 0.14) was less than that obtained in the TBM group (r = 0.15), even with comparable lymphocyte counts. Since the pyogenic meningitis group included only 4 patients, no specific conclusions can be drawn. It would require a larger series of patients with pyogenic meningitis to make any comparison (Table 9). 6. Discussion The incidence of fever (100%), headache (100%), vomiting (91%) and alteration in sensorium (85%) was higher in the present study than 78%, 37%, 45% and 53% respectively in a previous study (Thomas et al. [18]). The possible reasons may be absence of pediatric population and mandatory inclusion criteria of fever and headache in the present study. The incidence of seizures, alterations in sensorium and papilledema was comparable in both the studies. The CSF smear was positive for AFB only in 1 out of 54 (2%) patients in this study. Since the sensitivity is very low, CSF AFB smear cannot be relied upon to pick up cases of TBM. The CSF culture grew AFB in 5 patients (9%) in the present study. This is in comparison with the previous studies by Seth et al. (0%) [15], Shankar et al. (12%) [19], Miörner et al. (17%) [20] and Nguyen et al. (19%) [21]. The delay of 5–6 weeks to obtain positive culture coupled with low sensitivity (9%) makes this test less useful to the clinician for the diagnosis of TBM. The mean ADA in the TBM group in present study was 26.1 ± 18.8 U/L which is comparable with the mean ADA level of 26.2 U/L observed in TBM by Malan et al. [9]. They noted that the ADA level in TBM group was higher in adults (26.2 U/L) compared to children (14.5 U/L). Gambhir et al. [22], reported mean ADA of 9.6 ± 4.1 U/L in 36 patients with tubercular meningitis, which were significantly higher than cases of viral encephalitis and enteric encephalopathy. However, levels were comparable to pyogenic meningitis (7.92 + 0.95 U/L) and cerebral malaria. Using 8 U/L as cut off value for diagnosis of TBM, a sensitivity of 44% and specificity of 75% was observed while in the present study it was 100% and 94% respec-
S.V. Rana et al. / Clinical Neurology and Neurosurgery 112 (2010) 424–430 Table 9 Coordinates of the curve, test result variable(s) of CSF-ADA levels. Positive if greater than or equal toa
Sensitivity
1 − specificity
1.00 2.50 4.00 5.50 6.50 7.50 8.50 9.50 10.50 11.50 12.50 13.50 14.50 15.50 16.50 17.50 18.50 19.50 20.50 21.50 22.50 27.50 32.50 35.00 38.50 42.50 48.50 53.00 57.50 67.50 90.50 108.00
1.000 1.000 1.000 1.000 1.000 1.000 0.962 0.943 0.887 0.849 0.830 0.792 0.698 0.679 0.660 0.623 0.585 0.491 0.472 0.396 0.283 0.264 0.226 0.208 0.189 0.170 0.151 0.132 0.075 0.038 0.019 0.000
1.000 0.944 0.639 0.306 0.111 0.056 0.056 0.028 0.028 0.028 0.028 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
The test result variable(s): CSF-ADA levels has at least one tie between the positive actual state group and the negative actual state group. a The smallest cut off value is the minimum observed test value minus 1, and the largest cut off value is the maximum observed test value plus 1. All the other cut off values are the averages of two consecutive ordered observed test values.
tively for TBM vs controls and 100% sensitivity and 25% specificity for TBM vs pyogenic meningitis. Their study indicated that CSFADA is a non-specific method to diagnose TBM. The higher level of CSF-ADA in the present study may be due to the inclusion of only adults in the present study. CSF adenosine activity in 20 TBM, 10 non-TBM and 15 non-tubercular, non-meningitis patients were also measured by Gautam et al. [23]. They observed that the mean CSF-ADA activity (13.62 ± 8.45 U/L) in TBM was significantly higher as compared to non-TBM (6.51 ± 2.41 U/L) patients. The sensitivity and specificity of CSF-ADA for TBM was found to be 85% and 70% as compared to non-TBM and 85% and 100% non-TBM, non-meningitis patients respectively at a cut off value of 6.97 U/L to diagnose tubercular meningitis. Corral et al. [24] used ROC curve analysis and suggested a cut off value of 8.5 U/L for the diagnosis of TBM with 57% sensitivity and 87% specificity. ADA activity in CSF of 182 patients with meningitis has also been reported by Choi et al. [25]. In their study, (i) 36 cases were of TBM, (ii) 130 cases of viral or aseptic meningitis, (iii) 9 cases of bacterial meningitis, and (iv) 7 cases of cryptococcal meningitis. The mean ± SD of ADA activity was 12.76 ± 7.53 U/L in group (i), 2.58 ± 2.37 U/L in group (ii), 7.38 ± 3.27 U/L in group (iii), and 7.42 ± 4.38 U/L in group (iv). The sensitivity of the test for group (i) compared with group (ii) was 0.83 and specificity 0.95 when a cut off value of 7 U/L was used. When group (i) was compared with groups (iii) and (iv), the sensitivity was 0.58 and the specificity 0.89 and 0.71 respectively at a cut off value of >10 U/L. >15 U/L values were not observed in any of the non-TBM patients. They have also reported that ADA activity >15 U/L could be a strong indication of tuberculous meningitis. Thus, they concluded that determination of CSF-ADA can aid in the early differential diagnosis of TBM.
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In the present study using a cut off value of CSF-ADA at >8 U/L, the sensitivity and specificity were 100% and 94% respectively. However, by using the cut off level at >10 U/L, the sensitivity and specificity were 92.5% and 97% respectively. By raising the cut off level at >15 U/L (above the mean CSF-ADA of pyogenic meningitis group), the specificity was same (97%), but the sensitivity dropped to 72% from 92.5%. This indicates that with a cut off level at >10 U/L gives a good sensitivity and specificity. The present study also shows that CSF-ADA can reliably distinguish between TBM and non-TBM, but not between TBM and pyogenic meningitis. The overall results of present study are in comparison to previous studies [9,22]. In the present study, the sensitivity and specificity of PCR was 44.5% and 92% respectively in most probable TBM group. The PCR also had an overall sensitivity of 32% and specificity 92% in present study. The sensitivity of PCR is low when compared to previous studies by Shankar et al. [19] and Nguyen et al. [21]. Shankar et al. [19] reported a sensitivity of 65% and specificity of 89% for PCR in their study of 34 patients with TBM. The sensitivity was 75% in highly probable TBM group, 57% in probable TBM and 43% in possible TBM group. In a study of 33 patients by Miorner et al. [20], the sensitivity of PCR for M. Tuberculosis was 54% and specificity 94%. In a recent study of 40 cases of TBM by Bonington et al. [26], PCR for M. Tuberculosis was positive in 10 cases (25% sensitivity). Dora et al. [27] have also shown that the sensitivity and specificity of CSF-PCR for the diagnosis of TBM were 50% and 98.6% respectively with a concordance with CSF mycobacterial culture of 96%. Afroze et al. [28] have also reported 77.7% sensitivity and 100% specificity to diagnose TBM by using of MPB64-Target PCR. Recently, 86% sensitivity and 100% specificity to diagnose TBM by using rpoB nested PCR have also been reported by Huang et al. [29]. In earlier studies, PCR was evaluated in CSF from known cases and controls. An element of bias in the interpretation of PCR data cannot be completely excluded in those studies. In the present study, both TBM and non-TBM samples were marked as suspected TBM and thus were blinded for the processing of PCR. Previous studies tested a smaller number of patients and used a selected patient group or had a higher number of false positives in PCR. Out of the 5 culture positive cases, one was PCR negative in the present study. This discrepancy can be explained by the differences in the volume of samples tested. It would thus be advisable to send a large volume of CSF for PCR analysis. In the present study, three control CSF samples were positive by PCR. These were from a patient with polyneuropathy and one patient with fungal meningitis had positive cryptococcal latex agglutination test and improved with a course of amphotericin. Even though she had lymphocytic pleocytosis in CSF, co-infection with M. tuberculosis was unlikely because she improved completely with antifungal treatment. As other 2 patients had no cells in the CSF, silent tubercular lesions in the meningitis are unlikely in the absence of symptoms. These positive results may be attributed to possible cross contamination of samples during initial handling. In present study, the sensitivity (92.5%) and specificity (97%) of CSF-ADA in TBM was higher as compared to PCR overall sensitivity (32%) and specificity (92%). Thus it is evident that the CSF-ADA had an edge both in sensitivity and specificity over the CSF-PCR to diagnose tubercular meningitis.
7. Conclusion This study shows that ADA which is a simple, rapid and inexpensive test, can be used for the diagnosis of TBM with good sensitivity and specificity. PCR is also a rapid test for the diagnosis of TBM with a good specificity but low sensitivity. Although the CSF-ADA test is sensitive and specific and can suggest the diagnosis of TBM, other
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