Brazilian Journal of Microbiology (2008) 39:209-213
IMPROVED DIAGNOSIS OF CENTRAL NERVOUS SYSTEM TUBERCULOSIS BY
Dil-Afroze1; Abdul Waheed Mir1; Altaf Kirmani1; Shakeel-ul-Rehman2; Rafiqa Eachkoti2; Mushtaq A.Siddiqi2
Departments of Immunology & Molecular Medicine2, Neurosurgery1, Sher-I-Kashmir Institute of Medical Sciences, Soura,
Submitted: March 20, 2007; Returned to authors for corrections: September 22, 2007; Approved: April 25, 2008.
Central nervous system (CNS) tuberculosis is a serious clinical problem, the treatment of which is sometimes
hampered by delayed diagnosis. Clearly, prompt laboratory diagnosis is of vital importance as the spectrum
of disease is wide and abnormalities of the cerebrospinal fluid (CSF) are incredibly variable. Since delayed
hypersensitivity is the underlying immune response, bacterial load is very low. The conventional
bacteriological methods rarely detect Mycobacterium tuberculosis in CSF and are of limited use in diagnosis
of tuberculous meningitis (TBM). This double blind study was, therefore, directed to the molecular analysis
of CNS tuberculosis by an in-house-developed PCR targeted for amplification of a 240bp nucleotide sequence
coding for MPB64 protein specific for Mycobacterium tuberculosis. Based on the clinical criteria, 47 patients
with CNS tuberculosis and a control group of 10 patients having non-tubercular lesions of the CNS were
included in the study. Analyses were done in three groups; one group consisting of 27 patients of TBM, a
second group of 20 patients with intracranial tuberculomas and a third group of 10 patients having non-
tubercular lesions of the CNS acted as control. There were no false positive results by PCR and the specificity
worked out to be 100%. In the three study groups, routine CSF analysis (cells and chemistry), CSF for AFB
smear and culture were negative in all cases. PCR was positive for 21/27 patients (77.7% sensitivity) of the
first group of TBM patients, 6/20 patients (30% sensitivity) of the second group with intracranial tuberculomas
were positive by PCR and none was PCR-positive (100% specificity) in the third group. Thus, PCR was found
to be more sensitive than any other conventional method in the diagnosis of clinically suspected tubercular
Key-words: CNS tuberculosis, tuberculous meningitis (TBM), intracranial tuberculomas, PCR, Mycobacterium
*Corresponding Author. Mailing address: Department of Immunology & Molecular Medicine, Sher-I-Kashmir Institute of Medical Sciences, Soura,
J&K-190011 India. Phone No. +91-194-2401013 Ext. 2262 Fax No. +91-194-2403470. E-mail: email@example.com
Tuberculosis (TB) is a major global problem and a public
health issue of considerable magnitude. Approximately, eight
million new cases of TB and three million deaths are reported
annually (3). In recent times, there has been a resurgence of
tuberculosis in both developing and developed countries; the
incidence varies from 9 cases per 100,000 persons per year in
the US to 110-165 cases per 100,000 persons in the developing
countries of Asia and Africa (7,20,24). The attributing risk factors
include the increasing prevalence of HIV infection, over-
crowding in the urban population and in abnormal communities
(such as prisons, concentration camps and refugee colonies),
poor nutritional status, appearance of drug-resistant strains of
tuberculosis and ineffective tuberculosis control programmes.
TB is a chronic, systemic infectious disease caused by the
Mycobacterium tuberculosis primarily manifesting as pulmonary
Koch’s. The inhaled bacilli can localize in alternate sites, leading
to extrapulmonary TB (EPTB). Tuberculous involvement of the
central nervous system (CNS) is an important and serious type
Afroze, D. et al.
of extra-pulmonary involvement (26). It has been estimated that
approximately 10% of all patients with tuberculosis have CNS
involvement (27). Fatality rates in developing countries have
been reported to range from 44 to 69% (6,8,19). In fact, missed
diagnosis and delayed treatment often results in serious long-
term debilitating complications. Moreover, the clinical response
to antituberculosis therapy in all forms of neuro-TB is excellent,
provided the diagnosis is made early; before an irreversible
neurological defect occurs (delay in diagnosis is directly related
to neurologic sequelae in 20-25% of patients who do not receive
early treatment). Clearly, prompt laboratory diagnosis is of vital
importance. The great majority of patients with neuro-TB are
diagnosed on the basis of clinical criteria, radiographic findings
and laboratory investigation of the cerebrospinal fluid (CSF)
(11). Acid-fast staining of CSF sediment is the most rapid method
for detection of mycobacteria, but this method requires >104
cells ml–1 hence lacks sensitivity. Conventional methods like
microscopy and culture, although considered as gold standards,
are quite inadequate (12). The diagnostic reference standard,
isolation of Mycobacterium tuberculosis from CSF samples, is
insufficiently timely (it requires 2-6 weeks) to aid clinical judgment
with respect to treatment and because of the paucibacillary state
in the cerebrospinal fluid this method is insensitive if large
amounts of CSF are not tested. PCR and molecular analysis
techniques show promise as tools for rapid diagnosis of
pulmonary, EPTB and CNS tuberculosis (1,3,11,12,17,18,23).
However, the accuracy and reproducibility of these molecular
analysis techniques for the detection of M. tuberculosis in CSF
has not been clearly defined. Therefore, an in-house developed,
MPB64 gene targeted PCR was evaluated at our centre for rapid
and specific diagnosis of CNS tuberculosis.
MATERIAL AND METHODS
The subjects comprised patients and controls admitted in
the tertiary care referral centre in the department of Neurosurgery
Sher-i-Kashmir Institute of Medical Sciences, Srinagar, Kashmir,
India, between January 2003 and January 2005. The patients
presented history, clinical features and CT scan/MRI strongly
suggestive of tubercular lesions of the Central Nervous System
(CNS). The patients with the presumptive diagnosis of
tuberculomas without neuro-deficit were treated on out-patient
basis and those with TBM and tuberculomas with neuro-deficit
Based on the clinical criteria, 47 patients with CNS
tuberculosis and a control group of 10 patients having non-
tubercular lesions of the CNS were included in the study. Patients
were divided in three groups: Category I: 27 patients diagnosed
of tuberculous meningitis (TBM) based on clinical features of
fever, persistent headache, neck stiffness, vomiting, alteration
of sensorium or a focal deficit, mental changes and confusion,
lethargy and stupor for more than seven days with the
suggestive radiological and CT findings including basal exudate,
hydrocephalus, infarcts and gyral enlargement confirmed by
various diagnostic procedures like biochemical examination of
CSF showing the presence of >10 WBC/ml and >80%
lymphocytes, protein concentration of >40 mg/dl and sugar
concentration less than 60% of corresponding blood glucose;
Category II: 20 patients with intracranial tuberculomas and
Category III: 10 patients having non-tubercular lesions of the
The samples were received in ice and stored at -20ºC until
analysis. A bacteriological analysis of CSF was performed by
the Ziehl–Neelsen method and culture was done on
Lowenstein–Jensen slants for all samples. A double-blind study
was conducted; the samples were transferred to the laboratory
for PCR after coding.
PCR was performed in three different areas, physically
separated from each other as a precaution to avert cross-
contamination. DNA was extracted from CSF samples (23) by
centrifugation at 3000 rpm for 30 min, followed by treating 200
μl of the sediment with equal volume of lysis buffer (consisting
of 0.2M NaOH, 2M NaCl and 1% SDS) and 100μg proteinase-K
at 60ºC for 1hour followed by 95ºC for 15min to inactivate
proteinase K. The lysate was extracted successively with
chloroform. The aqueous phase was adjusted to 0.3M sodium
acetate (pH 5.2), precipitated with ethanol and dissolved in
double distilled water.
The target for the PCR assay was MPB64 gene (24) which
codes for an immunogenic protein specific to Mycobacterium
tuberculosis complex. The sequences of the two primers used
Forward primer (460-479) 5´-TCCGCTGCCAGTCGTCTTCC-3´
Reverse primer (700-681) 5´-GTCCTCGCGAGTCTAGGCCA-3´
DNA amplification by PCR was performed in the total reaction
volume of 25 μl with 5 μl of extracted DNA, 10mM Tris-Cl (pH
8.3), 1.5 mM MgCl2, 50 mM NaCl, gelatin 0.01% (w/v), 100 μM of
each dNTP (Genei-India), 0.5 μM of each primer & 0.5 Units of
Taq polymerase (Genei-India). Amplification was carried out on
a programmable MinicyclerTM (MJ Research, USA). Initial
denaturation at 94ºC for 5 min. was proceeded by 30 cycles
each of denaturation (94ºC for 30 sec.), annealing (60ºC for 1
min) and extension (72ºC for 2min) followed by a final extension
at 72ºC for 7min.
The amplified product was electrophoresed into 2% agarose
gels. The gels were stained with ethidium bromide and visualized
in a UV-transilluminator (Vilber Lourmat, France). The presence
of a 240 bp fragment indicated a positive test (Fig. 1).
RESULTS AND DISCUSSION
In this retrospective study, overall 57 patients were analyzed
by an in-house developed, MPB64 gene targeted PCR to
evaluate its diagnostic efficacy for rapid and specific diagnosis
Diagnosis of CNS Tuberculosis
of CNS tuberculosis. Based on the clinical criteria, 47 patients
with CNS tuberculosis and a control group of 10 patients having
non-tubercular lesions of the CNS included in the study were
analyzed for CNS tuberculosis. Microscopic examination by
ZN-staining indicated absence of AFB in all CSF samples (Table
1). The detection limit of microscopy is 104 mycobacteria per
milliliter whereas in view of the fact that delayed hypersensitivity
is the underlying immune response in CNS-TB, the pauci-
bacillary state could be accounted for these negative results.
These results are comparable to those reported in literature of
only 0% to 10% of Ziehl Neelsen positive results (5,9,11,13) in
TBM patients. CSF for AFB culture was also negative in all
cases. The chance of growing mycobacteria becomes higher
with the increase of sample volume. Only 2-3 ml of CSF per
patients were available, and part of this volume, had to be utilized
for ZN-staining and PCR, Thus negative culture results in all
the patients with CNS-TB could be attributed to sampling
Mycobacterium tuberculosis specific MPB64 targeted
sequence was detected (Fig. 1) in CSF samples from 21 out of 27
patients of the first group of TBM patients of Category I, whose
microscopy and culture results were negative (Table 1). Out of
20 cases of Category II, CSF analyses for AFB smear and culture
were negative while PCR was positive for six patients only,
while remaining cases were negative. There were no false
positive results by PCR (out of 10 control cases none tested
positive for PCR) and the specificity worked out to be 100%
(Table 2). The sensitivity of the in-house developed MPB64
targeted PCR as a diagnostic tool to detect CNS-TB worked out
to be 77.7% for TBM and 30% for intracranial tuberculomas.
Among the 47 cases considered either definitely TBM or
intracranial tuberculoma, on the basis of clinical and laboratory
findings, PCR proved to be a more sensitive method, detecting
21 out of 27 of TBM cases (77.7%), 6 out of 20 patients of
intracranial tuberculmas (30%) whereas none were positive either
by culture or AFB staining and only 10-11% of the CNS-TB
cases could be picked up on histopathological examination of
the biopsy samples (Table 2). Some studies report a relatively
higher sensitivity of PCR for the diagnosis of TBM, ranging
from 75- 90%, but some authors had tested very small number
of patients (22) or had used a selected patient group (21) or had
a considerable number of false positives (16). An explanation
for the lower sensitivity of PCR in our study could be attributed
to the small volume of CSF (mean volume 200 - 300 μl) available
for testing (after using for smear and culture) so that the sample
could not be concentrated. The volume of sample is of great
significance in PCR, especially in CNS-TB, due to frequent low
number of bacteria in the CSF. Culture of CSF also requires
larger volume and when both culture and PCR have to be done,
the minimum volume of CSF should be 2 ml. Another reason for
low sensitivity of PCR may be presence of PCR inhibitors in the
CSF as well as poor lysis of mycobacteria (15). False negatives
have occurred in two studies, in which the reported PCR
sensitivities (17,22) were 32% and 85%. These results suggest
that the PCR is more sensitive than other co-existing
conventional methods, but still not absolute to identify all cases
Table 1. Evaluation of various diagnostic methods for CNS-TB
27none none3 21
10 none nonenone none
AFB = Acid Fast Bacilli; HPE = Histo-pathological examination; PCR
= Polymerase chain reaction; TBM = Tuberculous Meningitis.
Figure 1. MPB64 gene-targeted PCR for detection of
Mycobacterium tuberculosis. Electrophoretic separation of the
amplicon into 2% agarose gel is documented across Lanes 1-7.
Lanes 1-3 represent the clinical cerebrospinal fluid samples,
Lanes 4 and 5 exemplify the colony-PCR from atypical
Mycobacterium (Mycobacterium bovis) as negative control and
lanes 6 and 7 stand for the colony-PCR from Mycobacterium
tuberculosis culture of the clinical samples (other than CSF) as
positive control. The presence of a 240 bp. amplicon in the
Lanes 1, 2, 3, 6 and 7 indicated the presence of the target.
Afroze, D. et al.
This study is the first of its kind from the Kashmir valley in
north India with such large number of samples to support the
credence that PCR deserves a place in the laboratory diagnosis
of central nervous system tuberculosis but careful adherence
to the test protocol is mandatory. Importantly, majority of
patients with tubercular meningitis and intracranial tuberculomas
can be managed non-operatively. Results of PCR are available
with speed comparable to microscopy; sensitivity is higher than
both microscopy and culture and the direct identification of the
organism, as belonging to the M.tuberculosis complex is
possible. To further enhance the sensitivity of PCR, alternative
procedures like double repetitive-element PCR (DRE-PCR) using
hot-Taq should be employed (2). However, over-reliance on
PCR should be avoided, as premature cessation of treatment
will have serious consequence in patients with TBM, in whom
PCR is negative. Hence, a combination of clinical criteria and
PCR is needed for the final outcome to address the disease.
Diagnóstico da tuberculose do sistema nervoso
central por MPB64-Target PCR
A tuberculose do sistema nervoso central (CNS) é um
problema clínico sério, cujo tratamento é dificultado pelo
diagnóstico tardio. O diagnóstico laboratorial rápido é de
importância vital considerando que o espectro da doença é amplo
e as anormalidades do liquor são muito variáveis. Considerando
que a hipersensibilidade tardia é a resposta imune fundamental,
a carga bacteriana é muito baixa. Os métodos bacteriológicos
convencionais raramente detectam Mycobacterium tuberculosis
no liquor e são de uso limitado para diagnóstico da meningite
tuberculosa (TBM). O presente estudo duplo-cego objetivou a
análise molecular da tuberculose do CNS através de um PCR
desenvolvido in-house direcionado para a amplificação de uma
seqüência de nucleotídios de 240pb que codificam a proteína
MPB64 especifica de Mycobacterium tuberculosis. Baseando-
se em critérios clínicos, selecionou-se 47 pacientes com
tuberculose do CNS e um grupo controle de 10 pacientes com
lesões não-tuberculosas no CNS. As análises foram divididas
em três grupos: um grupo de 27 pacientes com TBM, um segundo
grupo com 20 pacientes com tuberculomas intracraniais e um
terceiro grupo de 10 pacientes com lesões não-tuberculosas no
CNS (controles). O PCR não forneceu nenhum resultado falso-
positivo, com 100% de especificidade. Em todos os três grupos
de estudo, os resultados das análises de rotina do liquor por
histologia, química e baciloscopia e também cultura foram
negativos em todos os casos. No primeiro grupo de pacientes
com TBM, PCR foi positivo em 21/27 pacientes (sensibilidade
de 77,7%). No segundo grupo de pacientes com tuberculomas
intracraniais, 6/20 foram positivos (sensibilidade de 30%).
Nenhum dos pacientes do grupo controle foi positivo (100% de
especificidade). Dessa forma, o PCR mostrou-se mais sensível
que os métodos convencionais no diagnóstico de casos
suspeitos de meningite tuberculosa.
Palavras-chave: tuberculose do sistema nervoso central, me-
ningite tuberculosa, tuberculomas intracraniais, PCR,
1.Bonington, A.; Strang, G.J.I.; Klapper, P.E.; Hod, S.V.; Rubombora,
W.; Penny, M.; Willers, R.; Winkins, E.G.L. (1998). Use of the
Roche AMPLICOR Mycobacterium tuberculosis PCR in early
diagnosis of tuberculous meningitis. J. Clin. Microbiol., 36 (5), 1251-
Cavalcanti, H.R.; Marques, E.; Fonseca, L.S.; Saad, M.H.F. (2007).
Do DNA extraction methods and Taq polymerase quality improve
the double repetitive element (DRE) PCR typing method for
Mycobacterium tuberculosis strains? Braz. J. Microbiol., 38: 409-
Dil-Afroze; Sharma, D.; Dhobi, G.N.; Sonaullah Shah, S.; Eachkoti,
R.; Hussain, I.; Shah, Z.A.; Siddiqi, M.A. (2006). Evaluation of
polymerase chain reaction for rapid diagnosis of clinically suspected
tuberculous pleurisy. Ind. J. Clin. Biochem., 21 (2), 76-79.
Dolin, P.J.; Raviglione, M.C.; Kochi, A. (1994). Global tuberculosis
incidence and mortality during 1990-2000. Bull. World Health
Organ., 72, 213-220.
Table 2. Sensitivity and specificity of the methods for diagnosis of CNS-TB.
Number of positive
TBM Intracranial tuberculoma
Positive Sensitivity SpecificityPositiveSensitivity Specificity
AFB = Acid Fast Bacilli; HPE = Histo-pathological examination; PCR = Polymerase chain reaction; TBM = Tuberculous Meningitis; CSF =
Diagnosis of CNS Tuberculosis
5. Folgueira, L.; Delgado, R.; Palenque, E.; Noriega A.R. (1994).
Polymerase chain reaction in the diagnosis of tuberculous meningitis
in AIDS patients. Neurology, 44, 1336.
Girgis, N.I.; Sultan, Y.; Farid, Z.; Mansour, M.M.; Erian, M.W.;
Hanna, L.S.; Mateczun, A.J. (1998). Tuberculosis meningitis, Abbassia
Fever Hospital-Naval Medical Research Unit No. 3-Cairo, Egypt,
from 1976 to 1996. Am. J. Trop. Med. Hyg., 58 (1), 28-34.
Harries, A.D. (1990). Tuberculosis and human immunodeficiency
virus infection in developing countries. Lancet., 335 (8696), 387-
Hosoglu, S.; Ayaz, C.; Geyik, M.F.; Kokoglu, O.F.; Cerviz, A. (1998).
Tuberculous meningitis in adults: an eleven-year review. Int. J. Tuber.
Lung. Dis., 2 (7), 553-557.
Jatana, S.K.; Nair, M.N.; Lahiri, K.K.; Sarin, N.P. (2000). Polymerase
chain reaction in diagnosis of tuberculosis in children. Ind. Pediatrics.,
10. Kennedy, D.H.; Fallon, R.J. (1979). Tuberculous meningitis. JAMA.
241 (3), 264-268.
11. Kox, L.F.; Kuijper, S.; Kolk, A.H. (1995). Early diagnosis of
tuberculous meningitis by polymerase chain reaction. Neurology, 45
12. Kulkarni, S.P.; Jaleel, M.A.; Kadival, G.V. (2005). Evaluation of an
in-house-developed PCR for the diagnosis of tuberculous meningitis
in Indian children. J. Med. Microbiol., 54, 369-373.
13. Lin, J.J.; Harn, H.J.; Hsu, Y.D.; Tsao, W.L.; Lee, H.S.; Lee, W.H.
(1995). Rapid diagnosis of tuberculous meningitis by polymerase
chain reaction assay of cerebrospinal fluid. J. Neurol., 242 (3), 147-
14. Liu, P.Y.F.; Shi, Z.Y.; Lau, Y.J.; Hu, B.S. (1994) Rapid diagnosis of
tuberculous meningitis: a simplified nested amplification protocol.
Neurology, 44, 1161.
15. Melzer, M.; Brown, T.J.; Flood, J.; Lacey, S.; Bagg, L.R. (1999).
False negative polymerase chain reaction on cerebrospinal fluid
samples in tuberculous meningitis. J. Neura. Neurosurg. Psychiatry,
16. Miorner, H.; Sjorbring, U.; Nayak, P.; Chandramukhi, A. (1995).
Diagnosis of tuberculous meningitis: a comparative analysis of 3
immunoassays, an immuno-complex assay and polymerase chain
reaction. Tuber. Lung. Dis., 76: 38.
17. Nguyen, L.N.; Kox, L.F.; Lihn, D.P.; Sjoukje, K.; Kolk; A.H. (1996).
The potential contribution of the polymerase chain reaction to the
diagnosis of tuberculous meningitis. Arch. Neurol., 53 (8), 771-776.
18. Ogusku, M.M.; Sadahiro, A.; Hirata, M.H.; Hirata, D.C.R.; Zaitz, C.;
Salem, J.I.. (2003). PCR in the diagnosis of cutaneous tuberculosis.
Braz. J. Microbiol., 34 (2).
19. Pai, M.; Flores L.L.; Pai, N.; Hubbard, A.; Riley, L.W.; Colford,
J.M.Jr. (2003). Diagnostic accuracy of nucleic acid amplification
tests for tuberculous meningitis: a systematic review and meta-
analysis. Lancet Infect. Dis., 3 (10): 633-643.
20. Raviglione, M.C.; Snider, D.E.; Kochi, A. (1995). Global
epidemiology of tuberculosis. Morbidity and mortality of a world
wide epidemic. JAMA, 273, 220-226.
21. Scarpellini, P.S.; Racca, P.; Cinque, F.; Delfanti, N.; Gianotli, M.R.;
Terreni, L.V.; Lazzarin A. (1995). Nested polymerase chain reaction
for the diagnosis and monitoring of response in AIDS patients with
tuberculous meningitis. AIDS, 9 (8), 895-900.
22. Seth, P.; Ahuja, G.K.; Bhanu, N.V.; Behari, M.; Bhowmik, S.; Broor,
S.; Dar, L.; Chakraborty, M. (1996). Evaluation of polymerase chain
reaction for rapid diagnosis of clinically suspected tuberculous
meningitis. Tuber. Lung. Dis., 77, 353-357.
23. Shankar, P.; Manjunath, N.; Mohan, K.K. (1991). Rapid diagnosis of
tuberculous meningitis by polymerase chain reaction. Lancet., 337, 5-7.
24. Shinnick, T.M.; Jones, J. (1994). Molecular approaches to the
diagnosis of tuberculosis. In: Bloom BR, ed. Tuberculosis;
Pathogenesis, Protection and Control. Washington DC: Am. Soc.
25. Snider, D.E.Jr; Roper, W.L. (1992). The new tuberculosis. N. Engl.
J. Med., 226, 703-705.
26. Tandon, P.N. (1978). Tuberculous meningitis. In P.J. Vinken and
G.W. Bruyn (ed.), Handbook of clinical neurology. North Holland
Publishing Co., Amsterdam, The Netherlands, 195-262.
27. Wood, M.; Anderson, M. (1998). Chronic meningitis. Neurological
infections; major problems in Neurology, vol. 16. Philadelphia. WB