Journal of Medical Microbiology (2009), 58, 616–624 DOI 10.1099/jmm.0.

006015-0

Efficient diagnosis of tuberculous meningitis by

detection of Mycobacterium tuberculosis DNA in
cerebrospinal fluid filtrates using PCR
Sagarika Haldar,1 Neera Sharma,2 V. K. Gupta2
and Jaya Sivaswami Tyagi1
Correspondence 1
Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi
Jaya Sivaswami Tyagi 110029, India
jstyagi@aiims.ac.in 2
Department of Biochemistry and Department of Pediatrics, Dr Ram Manohar Lohia Hospital, New
Delhi 110001, India

Tuberculous meningitis (TBM) is the most devastating form of meningitis and prompt diagnosis
holds the key to its management. Conventional microbiology has limited utility and nucleic acid-
based methods have not been widely accepted for various reasons. In view of the paucibacillary
nature of cerebrospinal fluid (CSF) and the recent demonstration of free Mycobacterium
tuberculosis DNA in clinical specimens, the present study was designed to evaluate the utility of
CSF ‘filtrates’ for the diagnosis of TBM using PCR. One hundred and sixty-seven CSF samples
were analysed from patients with ‘suspected’ TBM (n581) and a control group including other
cases of meningitis or neurological disorders (n586). CSF ‘sediments’ and ‘filtrates’ were
analysed individually for M. tuberculosis DNA by quantitative real-time PCR (qRT-PCR) and
conventional PCR. Receiver-operating characteristic curves were generated from qRT-PCR data
and cut-off values of 84 and 30 were selected for calling a ‘filtrate’ or ‘sediment’ sample positive,
respectively. Based on these, TBM was diagnosed with 87.6 % and 53.1 % sensitivity (P ,0.001)
in ‘filtrates’ and ‘sediments’, respectively, and with 92 % specificity each. Conventional devR and
IS6110 PCR were also significantly more sensitive in ‘filtrates’ versus ‘sediments’ (sensitivity of
87.6 % and 85.2 % vs 31 % and 39.5 %, respectively; P ,0.001). The qRT-PCR test yielded a
positive likelihood ratio of 11 and 6.6 by analysing ‘filtrate’ and ‘sediment’ fractions, respectively,
which establishes the superiority of the ‘filtrate’-based assay over the ‘sediment’ assay. PCR
findings were separately verified in 10 confirmed cases of TBM, where M. tuberculosis DNA was
detected using devR PCR assays in ‘sediment’ and ‘filtrate’ fractions of all samples. From this
study, we conclude that (i) CSF ‘filtrates’ contain a substantial amount of M. tuberculosis DNA
Received 20 August 2008 and (ii) ‘filtrates’ and not ‘sediments’ are likely to reliably provide a PCR-based diagnosis in
Accepted 19 January 2009 ‘suspected’ TBM patients.

INTRODUCTION most severe form of disease, namely tuberculous meningitis

(TBM). Prompt diagnosis is crucial for successful disease
Tuberculosis (TB) is a devastating disease with approxi-
management; the case fatality rate for untreated TBM is
mately 8.8 million new cases and 1.6 million deaths each
almost 100 %, and delay in treatment often leads to
year worldwide (WHO, 2006). While pulmonary disease is
permanent neurological damage (Bonington et al., 2000;
the most common manifestation of TB, the involvement of
Katrak et al., 2000). In developing countries, TBM
the central nervous system (CNS) is associated with the
primarily afflicts children; the highest incidence is noted
in the first 3 years of life (Tandon, 1978). It is the most
Abbreviations: CIIMS, Central India Institute of Medical Sciences; CNS, commonly dreaded complication and cause of death in
central nervous system; CSF, cerebrospinal fluid; NIND, non-infectious childhood tuberculosis and 2–4 % of all hospital paediatric
neurological disorders; NTIM, non-tuberculous infectious meningitis; admissions in India were diagnosed as TBM (Udani et al.,
OIND, other infectious neurological disorders; qRT-PCR, quantitative
1971). In a study of 292 autopsy-proven cases of TB, 200
real-time PCR; RML, Dr Ram Manohar Lohia Hospital; ROC, receiver-
operating characteristic; TB, tuberculosis; TBM, tuberculous meningitis; were in the paediatric age group (,15 years), and CNS
USP, universal sample processing. involvement was observed in 60 % of children compared to
Supplementary tables are available with the online version of this paper.
31.5 % of adults (Vithalani & Udani, 1982).

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 Detection of M. tuberculosis DNA in CSF ‘filtrates’

The prompt and accurate diagnosis of TBM is a daunting After analysis of 167 samples (RML, New Delhi), the performance of
challenge especially in paediatric subjects (Kumar et al., the PCR tests in ‘filtrates’ and ‘sediments’ was validated in CSF from
10 known culture-positive cases of TBM (‘definite’ TBM) from the
1999; Jatana et al., 2000; Narayanan et al., 2001) because of
Central India Institute of Medical Sciences (CIIMS), Nagpur.
the difficulty in obtaining a precise history and collecting
an adequate volume of cerebrospinal fluid (CSF) for Specimen processing and DNA isolation. CSF specimens were
laboratory investigation. Definitive diagnosis requires processed by universal sample processing (USP) methodology as
detection of tubercle bacilli in CSF. Smear microscopy is outlined in Fig. 1. The utility of USP methodology to efficiently detect
inexpensive and rapid but insensitive (0–20 %), while M. tuberculosis DNA by PCR in multibacillary and paucibacillary
culture techniques are unacceptably slow and insensitive. samples of pulmonary and extrapulmonary origin has been previously
established (Chakravorty & Tyagi, 2005; Chakravorty et al., 2005a, b,
Nucleic acid-based amplification (NAA) tests have 2006; Haldar et al., 2005, 2007; Pathak et al., 2007). Briefly, CSF was
emerged as potentially important tools for diagnosing filtered through a 0.22 mm membrane filter to yield ‘filtrate’ and
TBM though no commercial test is licensed for use in non- ‘sediment’ fractions. Filtration concentrated and entrapped intact
respiratory specimens. A meta-analysis of NAA tests used mycobacteria on the membrane (Kumar et al., 2008). The membrane
in the diagnosis of TBM concluded that commercial tests (‘sediment’ fraction) was divided into two halves: one half was
yielded results with high specificity but low sensitivity suspended in 200 ml 0.05 % Tween 80 and inoculated on Löwenstein–
while heterogeneity and low diagnostic accuracy were a Jensen medium while the other half was placed in 500 ml USP
solution and subjected to processing as described by Chakravorty &
concern with in-house PCR tests (Pai et al., 2003). The Tyagi (2005). Briefly, after incubation with USP solution for 10–
overall test sensitivities ranged from ~2 to 100 % and 15 min, the contents were centrifuged at 20 000 g for 20 min. The
specificities ranged from 75 to 100 % (Pai et al., 2003; sediment was rinsed twice with water and resuspended in 0.1 %
Hooker et al., 2003; Jonsson & Ridell, 2003; Kulkarni et al., Triton X-100. Smears were prepared and microscopically examined
2005; Bhigjee et al., 2007; Rafi et al., 2007a, b), highlighting after Ziehl–Neelsen staining. The remaining sediment was incubated
the need for an improved NAA-based test for diagnosing at 90 uC for 40 min for lysis of mycobacteria and DNA isolation. The
lysate was centrifuged at 15 000 g for 10 min and an aliquot was used
TBM.
in PCR. ‘Filtrate’ fractions were extracted with an equal volume of
We recently demonstrated the presence of both phenol/chloroform (1 : 1, v/v) and chloroform and the DNA was
Mycobacterium tuberculosis DNA and intact bacilli in precipitated overnight with ethanol. The DNA pellet was suspended
in sterile water and an aliquot was used for PCR. Two different
freshly collected sputum samples and suggested that
methods were employed to isolate DNA since ‘filtrates’ contain free
amplification of free DNA may improve diagnostic DNA and ‘sediments’ contain intact bacteria. DNA from ‘filtrate’
accuracy in paucibacillary specimens (Pathak et al., requires purification, which was done using the conventional phenol/
2007). This hypothesis was tested for the PCR-based chloroform extraction method. Purified DNA from ‘sediments’ was
detection of M. tuberculosis DNA in CSF taken predomi- obtained after bacterial lysis using USP methodology described above.
nantly from paediatric subjects. DNA-based diagnosis was
PCR. DNA from ‘sediment’ and ‘filtrate’ fractions of all CSF samples
compared with the currently used TBM diagnostic criteria.
was subjected to devR quantitative real-time PCR (qRT-PCR), gel-based
devR and IS6110 PCR assays. The qRT-PCR assay amplified a 144 bp
region of devR (Haldar et al., 2007). Fifty microlitre reactions contained
METHODS 0.5 mM each primer, PCR buffer, 3 mM MgCl2, 0.2 mM dNTPs, 0.56
SYBR Green (Amresco), 2.5 U AmpliTaq Gold DNA polymerase
Patients and samples. The approval of the Institutional Ethics
(Applied Biosystems) and specimen DNA in an I-Cycler (Bio-Rad). The
Committee and informed consent of the patients was obtained for
tubes were incubated for 10 min at 94 uC and 40 cycles each of 1 min at
this diagnostic study, which included 167 CSF samples from 167
94 uC, 1 min at 52 uC and 30 s at 72 uC. A standard curve was
patients, predominantly from the paediatric age group, which were
generated under identical amplification conditions by plotting
collected from Dr Ram Manohar Lohia Hospital (RML), New Delhi.
increasing dilutions of input M. tuberculosis DNA (ranging from 1.4
CSF was subjected to biochemical and cytological analysis and the
to 1.46107 genome equivalents) versus threshold cycle (Ct) and was
remaining sample aliquots were stored at 4 uC and subjected to PCR
used to determine the bacterial DNA load in the sample volume
testing within the next 6–36 h without knowledge of the clinical
analysed. The Ct was determined from SYBR Green fluorescence
diagnosis (Fig. 1). All CSF samples were collected prior to the
measured at 72 uC in the extension stage of each thermal cycle.
administration of antitubercular treatment. The diagnosis of TBM
was established on the basis of CSF culture, biochemistry and The gel-based devR PCR assay amplified a 162 bp sequence with
cytology, clinical findings, neuroimaging findings from CT/MRI scans primers devRf3 (59-ATCTGTTGTCCCGCATGCC-39) and devRr3
and the occurrence of extraneural TB as described in Supplementary (59-GTCCAGCGCCCACATCTTT-39). The assay contained 0.5 mM
Table S1 in JMM Online. Neuroimaging was performed only of each primer, PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 U Taq
selected subjects under clinical suspicion of TBM. After PCR analysis, DNA polymerase and specimen DNA. The tubes were subjected to
patients were classified into ‘highly probable’ TBM (n52), ‘probable’ 10 min at 94 uC, 45 cycles each of 1 min at 94 uC, 1 min at 52 uC and
TBM (n510), ‘possible’ TBM (n569) and non-TBM (n586). No 30 s at 72 uC, and finally 7 min at 72 uC. Volumes of ‘sediment’ and
culture-positive definite TBM was diagnosed. The ‘highly probable’, ‘filtrate’ fractions added to PCR were normalized; since only half of
‘probable’ and ‘possible’ TBM groups were grouped together as the membrane filter was processed for ‘sediment’ PCR 10 ml
‘suspected’ TBM (n581) for analysis. The diagnoses for non-TBM ‘sediment’ DNA was used versus 5 ml ‘filtrate’ DNA. IS6110 gel-
control cases (n586) and their classification into non-tuberculous based PCR was performed as described by Haldar et al. (2007). A
infectious meningitis (NTIM), other infectious neurological disorders positive control, having M. tuberculosis DNA, and two negative
(OIND) and non-infectious neurological disorders (NIND) groups control reactions, lacking DNA, were always included. The amp-
were based on clinical and laboratory findings compatible with the lification products in gel-based assays were detected by ethidium
respective disease conditions for the individual groups (Table 1). bromide staining and visualization under UV light after electrophor-
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S. Haldar and others

Fig. 1. Flow chart of CSF sample processing.

esis on a 2.3 % agarose gel. The absence of PCR inhibitors (residual subjects were grouped as ‘suspected’ TBM (n581) and
amounts of guanidinium or phenol from the sample preparation control subjects having NTIM (n522), OIND (n521) and
step) in ‘sediment’ and ‘filtrate’ samples was established by inhibitor NIND (n543). Approximately 91 % of the subjects
check reactions that were spiked with M. tuberculosis DNA.
belonged to the paediatric age group (0–16 years), of
Statistical analysis. The diagnostic performance of PCR was which ~36 % were ¡3 years of age.
evaluated against that of clinical diagnosis as gold standard. The
various performance characteristics were calculated as described by
Altman (1991). The diagnostic accuracy was calculated as [Tp+Tn/ Conventional CSF examination is of limited utility
N]6100, where N5167 (Tp and Tn represent true positives and true
negatives). Significance of differences between various PCR assays was There were no significant clinical differences between
calculated by the chi-squared test. Receiver-operating characteristic patients from the various groups except that seizures were
(ROC) curves were plotted using the STATA 9.0 software (College associated more frequently with the NIND group and
Station, Texas, USA) and cut-off values of DNA genome equivalents positive CT/MRI findings were generally associated with
were selected that conferred a high sensitivity (~ 88 %) without ‘suspected’ TBM patients. Seven paediatric subjects in the
compromising on specificity (92 %). The cut-off values were used to ‘suspected’ TBM category had associated pulmonary and
call a sample positive or negative by qRT-PCR.
abdominal tuberculosis (Table 1). All patients that were
clinically diagnosed with ‘suspected’ TBM (n581) were
administered antitubercular treatment and were included
RESULTS AND DISCUSSION in the analysis. Of these, 61 subjects responded and
This study evaluated 167 CSF samples for the diagnosis of information on 20 subjects was not available as they were
TBM using PCR. After the PCR assays were completed, the lost to follow up.
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 Detection of M. tuberculosis DNA in CSF ‘filtrates’

Table 1. Clinical features in TBM and non-TBM patients

Suspected TBM* NTIM* OIND* NIND* Definite TBMD

(n581) (n522) (n521) (n543) (n510)

Clinical findings
Fever 48 19 17 29 5
Headache 18 4 3 3 3
Vomiting 22 13 8 6 1
Altered sensorium 9 2 2 1 1
Seizures 17 8 4 29 2
Focal signs 1 0 0 0 0
Neck stiffness 20 8 0 4 0
Nausea 2 0 0 0 0
Convulsions 7 4 2 5 0
Head injury 3 1 0 2 1
Nerve palsy 10 0 0 0 0
History of contact 7 0 3 1 0
CT/MRI
Basal exudates 4 0 0 0 0
Hydrocephalus 30 1 0 3 3
Ring enhancing lesions 5 0 0 0 1
Infarcts 4 0 0 0 1
Tuberculoma 3 0 0 0 0
Pulmonary TB 5 0 0 0 0
Abdominal TB 2 0 0 0 0

*From RML, New Delhi (n5167), ‘suspected’ TBM category includes ‘highly probable’ (n52), ‘probable’ (n510) and
‘possible’ TBM (n569) cases as defined in Supplementary Table S1; the NTIM group (n522) includes nine cases of bacterial
meningitis and 13 cases of viral meningitis; the OIND group (n521) includes four cases of cerebral malaria, three cases each of
bronchopneumonia, chicken pox and viral fever, and two cases each of acute gastroenteritis, sepsis, pneumonitis and enteric
fever; the NIND group (n543) includes 29 cases of febrile seizures, three cases of head injury, two cases each of Guillain–Barré
syndrome, metabolic encephalopathy, intracranial space occupying lesion and brain stem glioma, and one case each of CNS
lupus, CNS malignant lymphoma and hypocalcaemic seizures.
DTen culture-positive samples of definite TBM from CIIMS, Nagpur.

All the specimens were subjected to biochemical analysis, difficult to obtain CSF from paediatric subjects in volumes
cytology examination, USP smear microscopy, culture and reported to give reasonably good culture yield.
PCR. Approximately 41 % of the ‘suspected’ TBM patients
had protein levels .100 mg%. Only 6.7 % of the non-TBM
devR qRT-PCR
group had an elevated protein level of .100 mg%, most of
whom were of the NTIM category. With reference to CSF A standard curve was generated in eight independent
glucose levels, ~55 % of the ‘suspected’ TBM patients had experiments for real-time detection of amplified M.
levels ,60 mg% as compared to 46.4 % of the non-TBM tuberculosis DNA over an 8-logarithmic range of genome
patients. A clear-cut difference in the biochemical char- equivalents based on Ct data (Fig. 2a). The variation between
acteristics of CSF samples between the various patient the standard curves was marginal and demonstrated the
groups was not apparent (Supplementary Table S2). reproducibility of the qRT-PCR assay (Fig. 2a). M.
However, the CSF lymphocytes versus polymorphs tuberculosis DNA was quantified in 167 CSF samples and
response was greater in the case of ‘suspected’ TBM ROC curves were generated to establish cut-offs that
patients, ~62 % of whom had .60 % lymphocytes distinguished between TBM and non-TBM samples. The
compared to only 15 % of non-TBM patients, most of DNA load in each sample was quantified by extrapolation
whom were placed in the NTIM group. None of the from the standard curve generated for the same batch of
samples were positive by microscopy in spite of using assayed samples. To construct ROC curves, DNA loads from
concentrated smears. Culture yield was also nil. Enhanced ‘suspected’ TBM were considered true-positive values and
culture positivity could be obtained by using sediments those from control subjects as true-negative values. A
from larger volumes of CSF (Thwaites et al., 2004) and combined ROC curve for ‘filtrate’ and ‘sediment’ samples
using liquid culture media versus Löwenstein–Jensen was first constructed. However, a common threshold value
slopes. However, it is to be noted that it is extremely that provided reasonably favourable specificity and sensiti-
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S. Haldar and others

Fig. 2. (a) A qRT-PCR standard curve was generated using M. tuberculosis DNA over a range of 1.4”1.4¾107 genome
equivalents. The standard curve data from eight independent experiments (mean±SD) are shown. Ct (threshold cycle) is plotted
versus logarithm of the number of M. tuberculosis genome equivalents added to each tube at the start of the reaction. (b) ROC
curves were generated from bacterial DNA load quantified in 167 ‘filtrate’ and ‘sediment’ fractions. (c) Scatter plot showing
bacterial DNA load in individual CSF ‘filtrate’ and ‘sediment’ fractions of (i) 167 samples from RML, New Delhi, and (ii) 10
definite TBM samples from CIIMS, Nagpur. The encircled value was excluded for determination of mean DNA load. The
horizontal bar drawn across each dataset denotes the median value. DNA negative values are not depicted in the scatter plot.
The horizontal line across the ‘filtrate’ and ‘sediment’ plots denotes the cut-off points determined by ROC curve analysis.

vity (.~85 %) was not attainable. Therefore, two separate respectively, were obtained (Fig. 2b). A 0.92 value for
ROC curves were constructed and values of 0.92 and 0.68 for ‘filtrate’ implies that a randomly selected DNA value from
areas under the ROC curves for ‘filtrate’ and ‘sediment’, the TBM group has a test value larger than that from a
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 Detection of M. tuberculosis DNA in CSF ‘filtrates’

Fig. 3. Summary of PCR results obtained in various patient groups. F and S denote ‘filtrate’ and ‘sediment’, respectively. devR
and IS6110 are gel-based assays. Values in parentheses are % positivity.

randomly chosen individual from the control group 92 % of (n5167), the ‘filtrate’ assay sensitivity was ~88 % while that
the time. Likewise, a 0.68 value for ‘sediment’ indicates that of ‘sediment’ PCR was ~53 % (Figs 2c, 3).
test values were larger than that from a randomly chosen
individual of the control group only 68 % of the time (Zweig
& Campbell, 1993). Thus to attain a specificity of 92 %, a Conventional gel-based PCR assays
cut-off value of 84 genome equivalents was selected for devR and IS6110 gel-based assays were performed on all
calling a CSF ‘filtrate’ TBM-positive. To call a ‘sediment’ ‘filtrate’ and ‘sediment’ fractions. PCR results are summar-
TBM-positive at equivalent specificity, a threshold of 30 was ized in Fig. 3 and Table 2. Sensitivities of 87.6 % and 85.2 %
selected. On applying these cut-offs to the study samples were obtained with ‘filtrate’ in contrast to 31 % and 39.5 %

Table 2. Performance of the various PCR assays

Based on analysis of 167 CSF specimens from RML, New Delhi.

PCR assay Sensitivity* (%) Specificity (%) Diagnostic accuracy (%) LR+ LR”

Filtrate
qRT-PCR 87.6 92.0 90.0 11 0.14
devRD 87.6 87.2 87.4 6.8 0.14
IS6110D 85.2 83.7 84.4 5.2 0.18
Sediment
qRT-PCR 53.1 92.0 73.1 6.6 0.51
devRD 31.0 94.2 63.5 5.3 0.73
IS6110D 39.5 93.0 67.1 5.6 0.65

*P ,0.001 for all ‘filtrate’ versus ‘sediment’ assays.

DConventional gel-based assays.

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S. Haldar and others

with the ‘sediment’ fraction by devR and IS6110 assays, further improved by use of the dUTP-uracil glycosylase
respectively. Thus PCR positivity was ~2 to 3 times more in system. In the present study, qRT-PCR gave the highest
the ‘filtrate’ fraction versus ‘sediment’ fraction. specificity (92 %) and was associated with the lowest false-
positivity rate (7 samples were false-positive compared to
the gel-based assays which detected 11 and 14 samples as
Performance of the various PCR assays
false-positives; Fig. 3), which is consistent with the
‘Filtrate’-based qRT-PCR was adjudged the single best suggestion that cross-over contamination can be mini-
assay with sensitivity and specificity of 87.6 % and 92 %, mized in real-time PCR that employs a closed tube system.
respectively. PCR sensitivity was enhanced by 6–11 % when
the results of any two ‘filtrate’-based assays were con-
sidered; however, it was compromised by a 7–10 % CSF ‘filtrates’ are superior to ‘sediments’ for PCR
decrease in specificity. In contrast, all the ‘sediment’-based The highlight of this study was the demonstration of a
assays performed at unacceptable levels of sensitivity (~31– substantial quantity of M. tuberculosis DNA in CSF
53.1 %; Table 2) and a large number of false-negatives were ‘filtrates’ from ‘suspected’ TBM subjects (n581, mean
obtained (Fig. 3). It may be argued that USP treatment load 18 066 genome equivalents; Fig. 2c). The detection of
lowered DNA yields in ‘sediments’. However, this pos- a large quantity of DNA in ‘filtrate’ argues against the
sibility is excluded as DNA recovery was not adversely commonly held view that CSF is paucibacillary per se.
affected by USP treatment (Chakravorty & Tyagi, 2005). Several studies have reported the use of real-time PCR for
We conclude that restricting PCR analysis to CSF M. tuberculosis detection (Wada et al., 2004; Aldous et al.,
‘sediments’ may be a contributory factor for poor test 2005; Takahashi & Nakayama, 2006; Takahashi et al., 2007;
sensitivity in addition to other previously cited reasons Bhigjee et al., 2007) but its quantification in CSF has been
such as low bacterial load, insufficient sample volume, documented in a limited number of samples (Takahashi &
inefficient DNA extraction and incomplete removal of Nakayama, 2006; Takahashi et al., 2007). The DNA content
inhibitory substances (Gascoyne-Binzi & Hawkey, 1999). estimated in the present study broadly agreed with that of
Most studies have used IS6110 as a target for PCR-based recent reports (Takahashi & Nakayama, 2006; Takahashi et
diagnosis of TBM with varying degrees of success (Miörner al., 2007) but substantially exceeded an earlier estimation
et al., 1995; Kox et al., 1995; Nguyen et al., 1996; Jatana et of ,100 organisms ml21 (Davis et al., 1993). By contrast,
al., 2000; Narayanan et al., 2001; Bhigjee et al., 2007; Rafi et lower amounts of DNA were recovered from ‘sediments’ of
al., 2007a, b). However, this insertion element is absent in a the same samples (mean load 1303 genome equivalents;
proportion of M. tuberculosis isolates from India Fig. 2c). Not a single sample that was positive by ‘sediment’
(Narayanan et al., 2001; Radhakrishnan et al., 2001), PCR was negative by ‘filtrate’ PCR. ‘Sediment’-based PCR
which argues against its utility as a sole target for gene was significantly less sensitive than the ‘filtrate’ assays (P
amplification. In contrast, devR sequences (Dasgupta et al., ,0.001, Table 2).
2000) were universally detected in M. tuberculosis strains The outcome of microbiological tests in CSF is reported to
and PCR tests based on this target have been useful for be volume-dependent (Thwaites et al., 2004). In the
pulmonary and extrapulmonary tuberculosis diagnosis present study, relatively small volumes of CSF (500 ml–
(Chakravorty & Tyagi, 2005; Chakravorty et al., 2005a, b, 1.5 ml) were adequate to achieve a sensitivity of ~88 %
2006; Haldar et al., 2005, 2007; Pathak et al., 2007). Only using ‘filtrate’ PCR. PCR positivity of CSF ‘filtrates’
few studies have assessed amplification of multiple gene indicates the occurrence of bacterial lysis, which may be
targets from the same CSF sample (Bhigjee et al., 2007; Rafi an outcome of cell-mediated immune response in TBM
et al., 2007b). An increased sensitivity was noted when all (Thwaites et al., 2000) or a consequence of antitubercular
three assays were analysed together: the sensitivity of treatment (Kox et al., 1995). However, the latter possibility
‘filtrate’ assays increased to ~99 % and that of ‘sediment’ is excluded since the samples were collected before
assays increased to ~74 %. However, in another study, an treatment was initiated. An additional advantage of
increase in diagnostic yield was not noted on adding a ‘filtrate’ PCR is that the entire ‘sediment’ fraction can be
second or a third target (Bhigjee et al., 2007). A ~22 % used for conventional microbiology.
increase in sensitivity was observed with qRT-PCR versus
conventional PCR in ‘sediment’ assays. This is consistent
with a recent report of ~16 % improvement in sensitivity PCR analysis of CSF from ‘definite’ TBM subjects
with real-time PCR compared to conventional PCR tests All 10 culture-positive CSF samples were also positive by
when applied to CSF deposits (Bhigjee et al., 2007).
qRT-PCR in both ‘filtrate’ and the ‘sediment’ fractions.
A limited number of false-positive results were obtained in Higher DNA loads were detected in individual ‘sediment’
spite of physical separation of areas used for sample versus ‘filtrate’ fractions, which was consistent with culture
processing, PCR-setup and analysis of amplification positivity of these samples; 9/10 samples had higher DNA
products, meticulous handling procedures and unambigu- load in ‘sediments’ (mean load, 21 299 genome equivalents;
ous negative results with control PCRs lacking M. Fig. 2c) as compared to the corresponding ‘filtrates’ (mean
tuberculosis DNA. We believe that assay specificity can be load, 2343 genome equivalents; Fig. 2c). Amongst the
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 Detection of M. tuberculosis DNA in CSF ‘filtrates’

conventional assays, PCR was positive in both fractions commercial tests (92 % vs 98 %; Pai et al., 2003) while the
with both targets in all CSF samples, except for an IS6110 sensitivity was substantially higher (~88 % vs 56 %; Pai et al.,
PCR-negative result in three ‘filtrates’. 2003). Note that no commercial test is licensed for diagnosis
of TBM or any other form of extrapulmonary TB. Therefore,
rapid tests are required with better diagnostic accuracy, and
Comparison of PCR results with conventional
the present report is a step in this direction.
diagnostic parameters
The devR gel-based assay (87 %) was equally sensitive as
Since culture yield was nil in the present study, we
qRT-PCR (87.6 %) and would be a useful tool for clinicians
compared individual TBM-associated clinical and labor-
in settings that do not have access to real-time PCR
atory criteria proposed by Ahuja et al. (1994) to PCR
instrumentation. In summary, an excellent diagnostic
positivity. The commonly observed clinical symptoms were
accuracy of 90 % was achieved using limited volumes of
not discriminatory enough for TBM diagnosis. Pleocytosis
CSF in ‘filtrate’-based devR PCR. These tests hold promise
appeared to be the most informative laboratory parameter
as valuable aids in the rapid diagnosis of TBM, particularly
for ruling in cases of infectious meningitis that included
in the paediatric age group. Lastly, the presence of lysed
‘suspected’ TBM and NTIM groups. Approximately 62 %
bacteria in CSF is likely to have implications for detecting
(50/81) of ‘suspected’ TBM patients had a lymphocytic
predominance versus only ~23 % (5/22) of NTIM patients. DNA of other CNS pathogens which are often difficult to
Therefore, by corollary, a CSF sample with predominantly culture.
neutrophils was unlikely to indicate TBM. Amongst 50
subjects of the ‘suspected’ TBM category with a lympho- ACKNOWLEDGEMENTS
cytic predominance, 46 were positive by two or more PCR
assays; by contrast, amongst five subjects of the NTIM S. H. is thankful to the Department of Biotechnology, Government of
group with a lymphocytic predominance, only one CSF India (DBT), for a Junior Research Fellowship, and N. S. is thankful
sample was positive by one PCR test. However, Kumar et to the World Health Organization for an In-country fellowship. Dr S.
N. Dwivedi and Ms M. Kalaivani are gratefully acknowledged for
al. (1999) reported that 36 % of paediatric subjects with expert advice and assistance with statistical analysis. The technical
TBM had a predominant polymorphonuclear type of CSF assistance of Sanjay, Jitendra Singh and Sunil Kumar is acknowledged.
pleocytosis. These results point to limitations in the use of The authors sincerely thank Dr Hatim Daginawala and Dr Rajpal
CSF cytology in an individual subject. Imaging techniques Kashyap, Central India Institute of Medical Sciences (CIIMS),
such as CT/MRI are being increasingly used in conjunction Nagpur, for providing 10 CSF samples for the study. Financial
with conventional tests to diagnose TBM. In our study, assistance to J. S. T. from the Department of Biotechnology,
CT/MRI analysis was performed on 44/81 ‘suspected’ TBM Government of India, is gratefully acknowledged.
patients and ~91 % (40/44) were positive by two or more
PCR assays. The evidence of extraneural TB is also a
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