Tropical Medicine and International Health doi:10.1111/tmi.

12099

volume 18 no 6 pp 783–793 june 2013

Review

Diagnosing tuberculous meningitis – have we made any

progress?
Jennifer Ho1, Ben J. Marais2,3, Gwendolyn L. Gilbert1,2 and Anna P. Ralph2,4,5

1 Centre for Infectious Diseases & Microbiology – Public Health, Westmead Hospital, Sydney, NSW, Australia
2 Sydney Emerging Infectious Diseases and Biosecurity Institute, Sydney, NSW, Australia
3 Division of Paediatrics and Child Health, Children’s Hospital at Westmead, Sydney, NSW, Australia
4 Global and Tropical Health Division, Menzies School of Health Research, Darwin, NT, Australia
5 Department of Medicine, Royal Darwin Hospital, Darwin, NT, Australia

Abstract Tuberculous meningitis (TBM) comprises a significant proportion of TB cases globally and causes
substantial morbidity and mortality, especially in children and HIV-infected patients. It is a
challenging condition to diagnose due to its non-specific clinical presentation and the limited
sensitivity of existing laboratory techniques. Smear microscopy and culture are the most widely
available diagnostic tools yet are negative in a significant proportion of TBM cases. Simplified and
more affordable nucleic acid amplification tests (NAATs) are increasing in use in resource-limited
settings but have not been optimised for cerebrospinal fluid (CSF) samples. Novel diagnostic methods
such as CSF interferon-gamma release assays and various biomarkers have been developed but
require further evaluation to establish their utility as diagnostic tools. There is an urgent need for
further research into optimal diagnostic strategies to decrease the morbidity and mortality as a result
of delayed or missed diagnosis of TBM. In this review, we discuss current and novel diagnostic tests
in TBM and areas where future research should be prioritised.

keywords tuberculosis, central nervous system, diagnosis, novel diagnostics, cerebrospinal fluid

predominance, increased protein content and low glucose

Introduction
Tuberculous meningitis (TBM) results in the highest rates
of morbidity and mortality out of all forms of tuberculosis
(WHO 2012). It is of particular concern in young chil-
dren, in whom it comprises up to 33% of all cases of TB
(Soeters et al. 2005). Outcomes of infection include death
in up to 50% of cases (Ruslami et al. 2013). Survivors can
suffer substantial neurological sequelae including develop-
mental delay in children, seizures, hydrocephalus, and cra-
nial nerve palsies (Thilothammal et al. 1994; Yarmis et al.
1998). Improved clinical outcomes are dependent on
timely diagnosis and initiation of appropriate treatment at
optimal doses (Kennedy & Fallon 1979; Garg 1999; Rus-
lami et al. 2013). Despite this, current diagnostic methods
are limited in sensitivity due to the paucibacillary nature
of this form of TB, and hence, the need for large volumes
of cerebrospinal fluid (CSF) that are impractical in most
cases especially in children. Smear microscopy and culture
are often negative in TBM, and a combination of consis-
tent CSF findings (moderate pleocytosis with a lymphocyte
concentration), clinical and radiological criteria are
required to optimise the diagnosis (Thwaites et al. 2002;
Marais et al. 2010). Atypical CSF findings have been
described in children (Yarmis et al. 1998; Stark 1999),
and the sensitivity and specificity of neurological symp-
toms and signs are particularly low in this vulnerable pop-
ulation (Kumar et al. 1999). A large proportion of TBM
cases remain undetected and probably die without access
to appropriate treatment (WHO 2011a). Molecular tech-
niques are routinely employed in developed countries and
increasingly in less wealthy settings. The sensitivity of
these assays is highly variable, however, and, in many
cases, offers little advantage over smear microscopy per-
formed by an experienced technician (Pai et al. 2003;
Thwaites et al. 2004a). The inadequacy of current labora-
tory diagnosis of TBM has encouraged a plethora of stud-
ies investigating novel diagnostic assays for TBM in the
past decade. While many are promising, studies evaluating
these assays are often small, and methods may be less suit-
able in resource-constrained, TB endemic settings.

© 2013 John Wiley & Sons Ltd 783

Tropical Medicine and International Health
J. Ho et al. Diagnosing tuberculous meningitis
Comparisons of TBM diagnostic studies are hindered by
the lack of a clear reference standard, low study subject
numbers and heterogeneity in both study design and spe-
cific diagnostic platforms. There is an urgent need for sim-
ple, affordable and effective techniques to enhance
detection and treatment of this debilitating disease. Our
objective in this review is to describe the utility and limita-
tions of current laboratory methods for TBM, discuss
recent advances, and propose best practice approaches
and future research priorities.
Search strategy and selection criteria
References were obtained using PubMed from the last
15 years using the search strategy ‘tuberculous meningi-
tis’ and ‘diagnosis’. References that included laboratory
diagnostic techniques were included. Relevant references
cited from these initial references were also reviewed.
Studies with low subject numbers (<10 cases) or unsuit-
able study design, such as no controls, were excluded.
Additional references concerning specific aspects of labo-
ratory diagnosis (microscopy, interferon-gamma release
assay, etc.) were further obtained by searching PubMed
using these subject headings specifically combined with
‘tuberculous meningitis’.
Microscopy
Detection of acid fast bacilli (AFB) in patient samples
using Ziehl–Neelsen (ZN) staining is the most widely
employed technique for diagnosing TB. It remains the only
laboratory test for TB diagnosis in many resource-limited
TB endemic regions and forms an integral part of the
WHO diagnosis and treatment algorithm (WHO 2010a).
Fluorescent microscopy using fluorochrome dye (aur-
amine-O or auramine-rhodamine) has improved the sensi-
tivity of microscopy over conventional ZN staining (by
approximately 10%) and significantly decreased the time
required to examine each slide (Steingart et al. 2006).
Cheaper light-emitting diode fluorescent microscopes have
enabled wider adoption of this technique (WHO 2011b;
Das & Selvakumar 2012). AFB microscopy is, however,
insensitive in extrapulmonary TB especially TBM, with
sensitivity rates of about 10–20% (Thwaites et al. 2000),
although this figure varies considerably (0–87%) (Kennedy
& Fallon 1979; Hosoglu et al. 2002; Bhigjee et al. 2007),
and is highly influenced by the clinical case definition used
for diagnosis, the volume of CSF sent and the skill of the
technician examining the slide. The sensitivity of smear
microscopy in TBM can be maximised by examination of
the spun deposit of large volume CSF samples (>6 ml),
several CSF specimens collected over a few days and
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volume 18 no 6 pp 783–793 june 2013
prolonged slide examination (30 min) (Thwaites et al.
2004b). However, these criteria are rarely achieved in
practice. Despite being the most practical and universally
adopted test for TB diagnosis, there have been few
advances towards improving smear microscopy. Recently
described fluorescent microscopy using small membrane
filters (Fennelly et al. 2012) and modified ZN stain using
cytospin and Triton (a detergent) processing of CSF sam-
ples (Chen et al. 2012) have provided promising avenues
for further developments in this field.
Culture
Culture of Mycobacterium tuberculosis (MTB) from the
CSF of TBM patients is slow and insufficiently sensitive to
be used as a ‘rule out’ diagnostic test, although it repre-
sents the most definitive ‘rule-in’ test – usually in retro-
spect (Tortoli et al. 1999; Venkataswamy et al. 2007).
Culture is essential for phenotypic drug susceptibility test-
ing (DST) and to confirm resistance detected by more
rapid molecular techniques (WHO 2011c). Reduced turn-
around times have been achieved using broth-based cul-
ture compared with solid media for the isolation of MTB
(13 days vs. 26 days) (Tortoli et al. 1999) and DST
(Tortoli et al. 2002). For CSF samples, both liquid and solid
culture media should be used for optimal detection and
should be incubated for longer periods (up to 8–10 weeks)
than routine culture (Venkataswamy et al. 2007). The
sensitivity of CSF culture for MTB varies (60–70% in
adults) (Hosoglu et al. 2002; Thwaites et al. 2004b) and
is considerably lower in children (Yarmis et al. 1998; Van
Well et al. 2009). As with microscopy, sensitivity can be
increased by culturing the deposit of relatively large
volumes of CSF (Thwaites et al. 2004b). Laboratories that
culture MTB and perform DST are required to meet spe-
cific biosafety level requirements beyond those of routine
microbiological testing to protect staff from aerosol trans-
mission and to prevent environmental contamination.
These include negative pressure ventilation, lockable doors
and restricted access, personnel protective equipment
including N95 masks, specific waste management and
emergency plans for spills and accidents. All specimens
that are processed for MTB culture and all culture manip-
ulation must be performed inside a certified and main-
tained biological safety cabinet. In addition, quality
assurance systems must be in place to ensure accurate and
reliable results (WHO 2004). In developing nations, these
considerable infrastructure requirements are generally
only met in national reference laboratories, further hinder-
ing timely identification and DST results.
In an attempt to overcome some barriers to conven-
tional culture, WHO has endorsed the microscopic
© 2013 John Wiley & Sons Ltd
Tropical Medicine and International Health
J. Ho et al. Diagnosing tuberculous meningitis
observation drug susceptibility (MODS) assay (WHO
2011d). This is a simple and inexpensive liquid culture
alternative for detection of MTB and DST in which
processed specimens are inoculated into broth media,
with and without antibiotics and examined frequently,
after 5–7 days incubation, for the presence of cording in
control tubes. In respiratory samples, MTB can be
detected by MODS in a median of 7–9 days (Moore
et al. 2006a; Ha et al. 2010; Shah et al. 2011) with sen-
sitivities and specificities of 85–87% and 93–97%,
respectively, against standard culture (Ha et al. 2010;
Shah et al. 2011). In TBM patients, MODS on CSF speci-
mens compares favourably (sensitivity 65% and specific-
ity 100% against clinical reference standard, with mean
CSF volume 4.6 ml) with conventional culture methods in
a significantly shorter detection time (median 6 days)
(Caws et al. 2007). In addition to more timely identifica-
tion and DST results, other advantages are cost, ease of
use and the ability to perform this technique without the
infrastructure required and with no greater risk of cross-
contamination than with conventional culture. (Moore
et al. 2006b; Ha et al. 2010; Shah et al. 2011) Further,
larger studies into the diagnostic utility of MODS in TBM
are required, including studies in children. Its performance
for DST in CSF has not been demonstrated and is likely to
be less successful than for sputum due to the paucibacil-
lary nature of this disease and the clumping of bacilli
within CSF (Thwaites et al. 2004a; Caws et al. 2007).
Nucleic acid amplification tests
The first NAATs for use on CSF specimens were
described over two decades ago (Kaneko et al. 1990;
Shankar et al. 1991), and there have been numerous in-
house polymerase chain reaction (PCR) assays developed
since. The design and performance of these are heteroge-
neous, however, which makes comparing them difficult.
This issue was highlighted by a meta-analysis of the diag-
nostic accuracy of commercial and in-house NAATs for
TBM diagnosis (Pai et al. 2003). Commercial assays were
found to be insensitive at detecting MTB in CSF samples
(sensitivity 56% and specificity 98%), whereas no useful
comparative information could be obtained for in-house
PCRs (Pai et al. 2003). Subsequently, a number of in-
house PCRs using a variety of targets (IS6110, INS, pro-
tein b, rpoB, MPB64) and testing platforms have
described, with improved overall performance, especially
with the use of multiplex PCRs, compared with commer-
cial assays (sensitivity 71–94% and specificity 88–100%)
(Kulkarni et al. 2005; Bhigjee et al. 2007; Rafi et al.
2007; Huang et al. 2009; Sharma et al. 2010; Kusum
et al. 2011; Chaidir et al. 2012). NAATs have a distinct
© 2013 John Wiley & Sons Ltd
volume 18 no 6 pp 783–793 june 2013
advantage over microscopy and culture, once antitubercu-
lous therapy has commenced with DNA remaining
detectable for up to a month after starting treatment
(Donald et al. 1993; Thwaites et al. 2004a).
Limitations of PCR tests for MTB detection include
cost, the need for laboratory infrastructure, trained tech-
nical staff and careful quality control to prevent cross-
contamination, detect inhibition and monitor assay per-
formance. In 2010, the WHO endorsed the use of Xpertâ
MTB/RIF (Cepheid, CA, USA), a rapid fully automated
NAAT for use on sputum specimens in resource-con-
strained TB endemic countries, at concessional pricing
(WHO 2011e). The main advantages of Xpertâ MTB/
RIF over pre-existing molecular assays are its simplicity
of use – it requires minimal technical expertise and bio-
safety requirements and the low rates of cross-contamina-
tion (Banada et al. 2010; Boehme et al. 2010; Van Rie
et al. 2010; Lawn & Nicol 2011). Another key advan-
tage is its ability to rapidly detect rifampicin resistance
and hence predict multidrug resistant disease (MDR). Of
note however, increasing rates of rifampicin monoresis-
tance have been reported in recent studies in more than
one-third of total rifampicin resistant cases (Anisimova
et al. 2012; Mukinda et al. 2012). Moreover, although
initial validation studies reported 100% specificity for the
detection of rifampicin resistance (Boehme et al. 2010),
more recent studies have shown specificity to be much
lower than this (Carriquiry et al. 2012), prompting modifi-
cations in product software (FIND 2011). So, while Xpertâ
MTB/RIF technology has allowed increased decentralisa-
tion of drug-resistance detection, all detected rifampicin
resistant isolates should ideally be confirmed with conven-
tional DST to detect false-positive results and in addition,
concomitant isoniazid testing is required to avoid potential
misassignment of MDR disease (WHO 2011c).
Xpertâ MTB/RIF has been extensively evaluated for
MTB detection in sputum specimens and performs well
on smear-positive samples (sensitivity 98% compared
with 68% in smear-negative samples; specificity 98%)
(Steingart et al. 2013). Preliminary studies on a range of
extrapulmonary TB samples have also been promising in
smear-positive samples (sensitivity 96–100% vs. 37–90%
in smear-negative specimens; specificity 98–100%)
(Armand et al. 2011; Causse et al. 2011; Hillemann et al.
2011; Vadwai et al. 2011; Tortoli et al. 2012). For CSF
samples specifically, further studies are required as the
few that have been performed have small subject num-
bers, variable results (sensitivity 27–86%, specificity
99–100%) (Vadwai et al. 2011; Tortoli et al. 2012) and
have not been performed in high TB-burden settings. The
use of a composite reference standard, combining micro-
biological, clinical, radiological and/or histological
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Tropical Medicine and International Health
J. Ho et al. Diagnosing tuberculous meningitis
findings may enable a more accurate interpretation of the
utility of Xpertâ MTB/RIF in diagnosing extrapulmonary
TB, where culture confirmation, the historical ‘gold stan-
dard’, may be absent in true TB disease (Vadwai et al.
2011). An important emerging challenge, given the wide
global rollout of Xpertâ MTB/RIF for use on pulmonary
specimens, is to demonstrate how access to this assay can
impact on patient outcomes, as the existing paradigm in
high TB-burden settings is to institute TB therapy for
cases of clinically suspected TBM even in the absence of
microbiological confirmation. Whether Xpertâ MTB/RIF
can, cost-effectively, improve time to initiation of appro-
priate treatment, patient outcomes, recognition of alter-
native diagnoses, resource allocation and epidemiological
understanding through improved case notification,
requires further investigation.
Another simple, rapid and cost-effective NAAT that
has been used for TB diagnosis is loop-mediated isother-
mal amplification (LAMP) (Eiken Chemical Co., Ltd,
Tokyo, Japan). This technology operates at isothermal
conditions without the need for sophisticated equipment
or skilled personnel, making it an attractive and feasible
option in resource-limited settings (Boehme et al. 2007).
It has been used for the diagnosis of a variety of infec-
tious diseases (Parida et al. 2008; Mori & Notomi
2009). In smear-positive sputum, samples LAMP has
good sensitivity and specificity (98% and 94–100%,
respectively), but it is less sensitive in smear-negative sam-
ples (49–56%) (Boehme et al. 2007; Mitari et al. 2011). A
small study evaluating the use of LAMP on CSF for TBM
diagnosis demonstrated good performance (sensitivity
88% and specificity 90%) with better sensitivity than
nested-PCR (Nagdev et al. 2011). Unlike PCR however,
LAMP cannot be multiplexed, hence simultaneous drug-
resistance detection is only possible with selected isother-
mal amplification methods. It is unclear how LAMP tech-
nology would be incorporated into TB testing algorithms
in laboratories that have existing Xpertâ MTB/RIF instru-
ments, but, given preliminary data suggesting superior sen-
sitivity to PCR in extrapulmonary samples (Nagdev et al.
2011; Yang et al. 2011), further larger studies will be
valuable to determine its role in this setting.
Adenosine deaminase and interferon-gamma activity
Adenosine deaminase (ADA) is an enzyme that is widely
distributed in tissues and body fluids and has been used
in the diagnosis of pleural, meningeal and pericardial TB
(Segura et al. 1989). Measurement of this enzyme is sim-
ple and affordable, but the evidence to support its utility
in TB diagnosis is inconclusive. Measurement of ADA
has not been standardised, and the cut-off level that
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volume 18 no 6 pp 783–793 june 2013
defines a positive result has not been determined (Tuon
et al. 2010; Xu et al. 2010). For TBM diagnosis, ADA
measurements on CSF may contribute to other supportive
diagnostic findings once other pathogens have been ruled
out and, by optimising the defined cut-off value used
(Tuon et al. 2010; Xu et al. 2010). However, in HIV
patients, this test is not useful for distinguishing TBM
from other clinically similar neurological illnesses (Corral
et al. 2004).
Interferon-gamma (IFN-c) plays a major role in the
immune response to MTB, stimulating macrophage activ-
ity and lymphocyte Th1 differentiation (Farrar & Schrei-
ber 1993). It has been used in a similar fashion to, and
often in conjunction with, ADA to aid in the diagnosis of
pleural (Jiang et al. 2007; Khan et al. 2013), pericardial
(Burgess et al. 2002; Reuter et al. 2006) and peritoneal
(Sharma et al. 2006) TB with good sensitivity and speci-
ficity. The quantification of IFN-c in CSF as a diagnostic
tool in TBM holds much promise, yet has only received
preliminary attention in recent years (Juan et al. 2006;
Patel et al. 2011). The measurement of unstimulated
IFN-c, on unprocessed CSF, when combined with crypto-
coccal antigen testing and gram stain, in a high HIV-
prevalence setting, has been shown to be 92% sensitive
and 100% specific in diagnosing TBM (Patel et al. 2011).
Further, larger studies in different settings are required to
validate these findings. IFN-c measurements share similar
accuracy and may have a complementary role to ADA in
diagnosing TB, however, may be limited in low-resource
settings due to technical and financial constraints (Reuter
et al. 2006; Sharma et al. 2006).
Interferon-gamma release assays (IGRAs)
The measurement of interferon-gamma release in whole
blood (QuantiFERON-TB© Gold In Tube [(QFT-IT);
Cellestis Limited Chadstone, Vic., Australia] or peripheral
blood mononuclear cells (T-SPOT.TB; Oxford Immuno-
tec, Abingdon, UK) in response to stimulation with spe-
cific MTB antigens, to diagnose latent TB, has been
incorporated into screening guidelines in many low-inci-
dence, high-income countries instead of, or together with,
tuberculin skin testing (TST) because of its high specific-
ity and need for only one healthcare visit (Denkinger
et al. 2011; National Institute for Health & Clinical
Excellence 2011). However, evidence to support the use
of IGRAs to diagnose active TB has been less compelling.
Meta-analyses have concluded that IGRAs cannot be
used to rule out active disease or to reliably distinguish
latent from active TB (Diel et al. 2010; Sester et al.
2011; Rangaka et al. 2012). The use of IGRAs directly
on CSF specimens has been evaluated for the diagnosis of
© 2013 John Wiley & Sons Ltd
 Table 1 Comparison of conventional and novel diagnostic tests for tuberculous meningitis performed on CSF specimens

Diagnostic test in Level of Sensitivity Specificity Reference

CSF specimens evidence* % % standard† Comments References

Conventional diagnostic tests

Ziehl–Neelsen A III-2 10–20 100 5 Highly dependent on volume of sample plus time Steingart et al. (2006);
(ZN) smear to examine slides and may be lower in clinical Thwaites et al. (2000)
microscopy practice than research settings

© 2013 John Wiley & Sons Ltd

Use of fluorescent microscopy increases sensitivity
Culture: liquid A III-2 60–70 100 6 Shorter time to detection with liquid media Thwaites et al. (2004b);
and solid media Increased sensitivity with larger volumes Hosoglu et al. (2002)
NAATs
In-house A III-2 71–94 88–100 1,2 or 4 Assays highly heterogeneous Rafi et al. (2007);
Sharma et al. (2010);
Tropical Medicine and International Health

Kusum et al. (2011);

Huang et al. (2009);
Kulkarni et al. (2005);
J. Ho et al. Diagnosing tuberculous meningitis

Bhigjee et al. (2007)

Commercial BI 56 98 1,2,3 or 4 Majority of commercial assays not licensed for Pai et al. (2003)
extrapulmonary samples
XpertâMTB/RIF C III-2 27–86 99–100 1 Requires further evaluation; very few studies, Tortoli et al. (2012);
small subject numbers Vadwai et al. (2011)
ADA CI 79–84‡ 84–91‡ 2 Variable results; possible additional Xu et al. (2010); Tuon
benefit to other diagnostic findings et al. (2010)
Newer and novel diagnostic tests
MODS B III-2 65 98–100 4 Requires further evaluation; shortens time to Caws et al. (2007)
achieve positive culture result
LAMP D III-2 88 90 2 Requires further evaluation Nagdev et al. (2011)
IGRAs D III-2 50–82§ 89–100§ 1 or 2 Requires further evaluation; may have a role Kim et al. (2008, 2010);
when combined with other rapid diagnostic Patel et al. (2010a,b);
tests, for example, gram stain and Juan et al. (2006)
cryptococcal antigen
Antigens and antibodies¶ D III-2 84–94 92–99 2 Requires further evaluation Kashyap et al. (2004);
Katti (2002); Mudaliar
et al. (2006);
Kashyap et al. (2005);
Katti (2001)
LAM D III-2 64 69 2 Requires further evaluation Patel et al. (2009,
2010a,b)

NAATs, Nucleic acid amplification tests; ADA, Adenosine deaminase; MODS, Microscopic observation drug susceptibility assay; LAMP, Loop-mediated isothermal
amplification; IGRAs: Interferon-gamma release assays; PBMC, Peripheral blood mononuclear cells; LAM, Lipoarabinomannan; CSF, cerebrospinal fluid.

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J. Ho et al. Diagnosing tuberculous meningitis

TBM, based on the premise that mononuclear cells local-

ised to infected sites produce more interferon than
peripheral blood mononuclear cells (PBMC) (Kim et al.
2008), which has also been demonstrated in pleural (Losi

†Reference standard: 1 = Culture, microscopy, clinical and radiological diagnosis; 2 = Culture, microscopy and clinical diagnosis; 3 = Culture and microscopy;
et al. 2007) and alveolar fluid (Jafari et al. 2006; Dheda
et al. 2009) in thoracic TB. There is some evidence that
non-consecutive persons with a defined clinical presentation
comparison of CSF vs. PBMC IGRA levels (Kim et al.
III-2 A comparison with reference standard that does not meet
2008), or combinations of CSF IGRA with other CSF
consecutive persons with a defined clinical presentation
A study of test accuracy with: an independent, blinded

III-1 A study of test accuracy with: an independent, blinded

tests such as ADA (Kim et al. 2010), gram stain and

comparison with a valid reference standard, among

comparison with a valid reference standard, among

cryptococcal antigen (Patel et al. 2010a) or with MTB

the criteria required for Level II and III-1evidence

Study of diagnostic yield (no reference standard) PCR (Juan et al. 2006) may improve the diagnosis of
TBM. Further studies are warranted to confirm these

¶Various Mycobacterium tuberculosis – specific antigens and antibodies including 65kD heat-shock protein and antigen 85 complex.
findings. It will be necessary to overcome barriers such as
A systematic review of level II studies

expense, availability and fastidious sample handling

Quality of evidence

requirements before widespread use of IGRAs becomes

feasible in most TB endemic countries. The value of other
III-3 Diagnostic case-control study

§Heterogeneous studies: IFN-c-based assays used alone or in combination with PBMC IFNc values or other CSF tests.

cytokine readouts (such as TNF alpha, IL-2 and IL-17,

etc.) is currently under investigation and may add value
compared with an isolated interferon-gamma read-out.

Novel biomarkers
The search for novel biomarkers for TB screening, diag-
*Level of evidence: strength of recommendation and the quality of evidence (Merlin et al. 2009).

4 = Clinical diagnosis; 5 = Culture and clinical diagnosis; 6 = Microscopy and clinical diagnosis.

nosis and treatment monitoring has come to the forefront

of attention in recent years (Parida & Kaufmann 2010;
‡Dependent on cut-off value used for positive result and method of ADA measurement.

McNerney et al. 2012). The deficiencies in our existing

IV
II

strategies for diagnosing TBM make research into diag-

I

nostic biomarkers for this condition appealing. Biomar-

Recommended but other alternatives may be acceptable

kers are defined as measurable characteristics that

indicate normal biological or pathogenic processes, or
Insufficient evidence to support a recommendation

pharmacological responses to a therapeutic intervention

(Biomarkers working group 2001), and encompass ADA,
IFN-c and IGRAs, as described above. Other biomarkers
Strength of recommendation

Weakly recommended: seek alternatives

such as MTB-specific antigen, antibody (Katti 2001,

2002; Kashyap et al. 2004, 2005; Mudaliar et al. 2006)
cytokine measurements (Kashyap et al. 2010; Misra et al.
2010), gene-expression profiles and metabolomics
(Kumar et al. 2012; Maertzdorf et al. 2012), in TBM
Strongly recommended

patient have been described, but their feasibility and

Never recommended

clinical utility as diagnostic tools are unknown.

The detection of lipoarabinomannan (LAM), a MTB
cell wall lipopolysaccharide antigen, in urine is useful in
the diagnosis of TB in HIV patients with advanced
immunodeficiency and has recently been developed as a
low-cost lateral flow assay for use as a point-of-care test
[(Determine TB-LAM) Alere, Waltham, MA, USA] (Min-
C
D
Table 1 (continued)

A
B

ion et al. 2011; Lawn 2012; Peter et al. 2012). Prelimin-

ary investigations measuring CSF LAM for TBM
diagnosis in immunosuppressed HIV-infected patients
have shown improved diagnostic value (sensitivity 64%
and specificity 69%) compared with smear microscopy
(Patel et al. 2009, 2010b).

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Tropical Medicine and International Health
J. Ho et al. Diagnosing tuberculous meningitis
Table 2 Best practice recommendations for laboratory diagnosis
of tuberculous meningitis
Diagnostic
test Best practice recommendation
Microscopy Collect an adequate volume of CSF
on CSF (>6 ml in adults)
Concentrate sample through cytospin technique
(or small membrane filter)
Use fluorescent microscopy
Ensure adequate time to examine each slide by
an experienced technician
Culture Collect an adequate volume of CSF
on CSF (>6 ml in adults)
Use liquid in addition to solid media culture
NAAT Use an in-house or commercial PCR which has
on CSF been validated for use in CSF
Non–CSF Test non-CSF specimens (e.g. sputum, gastric
specimens aspirate, urine) to aid diagnosis of disseminated
TB, or TBM with other sites of infection
CSF, cerebrospinal fluid; TBM, tuberculous meningitis.
A pragmatic approach
Table 1 summarises the accuracy and recommendations
for use of current and novel diagnostic techniques in
TBM, and a summary of the optimal use of existing tech-
niques is provided in Table 2. While optimising current
techniques is desirable and logical, in the majority of
regions most affected by TBM, clinicians are limited to
smear microscopy as the sole microbiological diagnostic
test, and clinical acumen governs empiric antimicrobial
treatment. The use of clinical prediction rules for differ-
entiating TBM from other forms of meningitis is sensitive
and specific (Kumar et al. 1999; Thwaites et al. 2002;
Hristea et al. 2012) and can significantly enhance the
utility of point-of-care diagnostic tests such as CSF LAM
(Patel et al. 2010a,b) and Xpert MTB/RIF (Vadwai et al.
2011). A standardised clinical case definition, which is
applicable irrespective of patient’s age, HIV status or
resource limitations, has been developed by a group of
experts in this field, and enables more accurate diagnosis
of TBM and consistency for study comparisons (Marais
et al. 2010). Components of this case definition are sum-
marised in Table 3. Sampling non-central nervous system
(CNS) sites such as sputum, lymph nodes, urine, bone
marrow and ascitic fluid can be particularly helpful in
diagnosing TBM (Hosoglu et al. 2002; Van Well et al.
2009), especially in children, where up to 68% of cases
in one study were MTB culture positive from gastric aspi-
rates (Doerr et al. 1995). Similarly, chest radiography
provides valuable additional information in TBM with up
© 2013 John Wiley & Sons Ltd
volume 18 no 6 pp 783–793 june 2013
Table 3 Components of a standardised clinical case definition
for tuberculous meningitis (Marais et al. 2010)
Component Features suggestive of tuberculous meningitis
Clinical criteria Duration of neurological symptoms >5 days
Systemic symptoms suggestive of TB
History of recent (within 1 year) close contact
with an individual with pulmonary TB
Focal neurological deficits or altered
consciousness
CSF findings Clear in appearance
Pleocytosis (10–500 cells/ll) with lymphocyte
predominance (>50%)
Raised protein concentration (>1 g/l)
Low glucose concentration (<2.2 mM or
CSF/plasma ratio <50%)
Cerebral Hydrocephalus, basal meningeal enhancement,
imaging tuberculoma, infarct or pre-contrast basal
(CT or MRI) hyperdensity)
Evidence of TB Chest radiograph suggestive of active
elsewhere tuberculosis
Other radiological findings suggestive of TB
outside the CNS
AFB identified or MTB cultured from another
source, e.g. sputum, lymph node, gastric
aspirate, urine, bone marrow or blood
Positive MTB NAAT from non-CNS site
Exclusion of Alternative diagnosis confirmed using gram
alternative stain, culture, NAAT, antigen test (e.g.
diagnosis cryptococcus), serology (e.g. syphilis) or
histopathology (e.g. lymphoma)
AFB, acid fast bacilli; TB, tuberculosis; MTB, Mycobacterium
tuberculosis; CT, computer tomography; MRI, magnetic reso-
nance imaging; CNS, central nervous system; NAAT, nucleic
acid amplification test; CSF, cerebrospinal fluid.
to 60% of cases demonstrating changes consistent with
active TB (Schutte 2001; Hosoglu et al. 2002; Van Well
et al. 2009). These adjuvant tests may also be diagnostic
in TBM cases where CSF cannot be obtained, for exam-
ple, when lumbar puncture is contraindicated, consent
for lumbar puncture is not provided, or there is technical
failure resulting in inability to obtain CSF.
Conclusion
While there have been encouraging developments in the
diagnosis of TB in general, none has translated directly
into significant improvements in TBM diagnosis. There
remains an important need for additional research to
identify optimal diagnostic strategies. In contrast to spu-
tum in pulmonary TB, CSF will invariably contain low
organism numbers and limit our current diagnostic
modalities. Concentrating bacilli in CSF specimens and/or
designing methods to enhance the sensitivity of culture
789
Tropical Medicine and International Health
J. Ho et al. Diagnosing tuberculous meningitis
and NAATs may overcome this.It seems logical that
research priorities be focused on improving the sensitivity
of XpertâMTB/RIF for use in TBM, given the expanding
distribution and utilisation of this simple to use technol-
ogy in TB endemic regions, many of which previously
relied solely on smear microscopy to guide patient man-
agement. Although the discovery of specific biomarkers
or gene-expressions profiles associated with TBM will
herald an innovative and exciting era, existing methods
for pathogen identification and DST will still be required
for optimal case management.
Tuberculosis is a challenging disease. It is entangled in
and highlights many of the complex factors contributing
to global socio-economic inequalities and barriers to
healthcare access. While the development of robust diag-
nostic tools for TBM is imperative, they will only be as
good as the healthcare systems in which they are imple-
mented. Concerted effort from all involved stakeholders
together with the necessary political commitment will be
fundamental to achieving global control over this age old
debilitating disease.
Acknowledgements
We would like to thank Peter Jelfs, head of the TB labo-
ratory at Centre for Infectious Diseases and Microbiology
Laboratory Services, Westmead Hospital, for his contri-
bution to several aspects of this paper.
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