Diagnostic Accuracy of Quantitative PCR (Xpert MTB/RIF)
for Tuberculous Meningitis in a High Burden Setting: A
Prospective Study
Vinod B. Patel1*, Grant Theron2, Laura Lenders2, Brian Matinyena2, Cathy Connolly3, Ravesh Singh4,5,
Yacoob Coovadia6,7, Thumbi Ndung’u4,5, Keertan Dheda2,8*
1 Department of Neurology, University of KwaZulu-Natal, Durban, South Africa, 2 Lung Infection and Immunity Unit, Division of Pulmonology & UCT Lung Institute,
Department of Medicine, University of Cape Town, Cape Town, South Africa, 3 Biostatistics Unit, Medical Research Council, Durban, South Africa, 4 KwaZulu-Natal
Research Institute for Tuberculosis and HIV, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa, 5 HIV Pathogenesis Programme,
Doris Duke Medical Research Institute, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa, 6 Department of Medical Microbiology,
University of KwaZulu-Natal, Durban, South Africa, 7 National Health Laboratory Service, Durban, South Africa, 8 Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Cape Town, South Africa
Abstract
Background: Tuberculous meningitis (TBM) is difficult to diagnose promptly. The utility of the Xpert MTB/RIF test for the
diagnosis of TBM remains unclear, and the effect of host- and sample-related factors on test performance is unknown. This
study sought to evaluate the sensitivity and specificity of Xpert MTB/RIF for the diagnosis of TBM.
Methods and Findings: 235 South-African patients with a meningeal-like illness were categorised as having definite (culture
or Amplicor PCR positive), probable (anti-TBM treatment initiated but microbiological confirmation lacking), or non-TBM.
Xpert MTB/RIF accuracy was evaluated using 1 ml of uncentrifuged and, when available, 3 ml of centrifuged cerebrospinal
fluid (CSF). To evaluate the incremental value of MTB/RIF over a clinically based diagnosis, test accuracy was compared to a
clinical score (CS) derived using basic clinical and laboratory information. Of 204 evaluable patients (of whom 87% were
HIV-infected), 59 had definite TBM, 64 probable TBM, and 81 non-TBM. Overall sensitivity and specificity (95% CI) were 62%
(48%–75%) and 95% (87%–99%), respectively. The sensitivity of Xpert MTB/RIF was significantly better than that of smear
microscopy (62% versus 12%; p = 0.001) and significantly better than that of the CS (62% versus 30%; p = 0.001; C statistic
85% [79%–92%]). Xpert MTB/RIF sensitivity was higher when centrifuged versus uncentrifuged samples were used (82%
[62%–94%] versus 47% [31%–61%]; p = 0.004). The combination of CS and Xpert MTB/RIF (Xpert MTB/RIF performed if
CS,8) performed as well as Xpert MTB/RIF alone but with a ,10% reduction in test usage. This overall pattern of results
remained unchanged when the definite and probable TBM groups were combined. Xpert MTB/RIF was not useful in
identifying TBM among HIV-uninfected individuals, although the sample was small. There was no evidence of PCR
inhibition, and the limit of detection was ,80 colony forming units per millilitre. Study limitations included a predominantly
HIV-infected cohort and the limited number of culture-positive CSF samples.
Conclusions: Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals from a
tuberculosis-endemic setting, particularly when a centrifuged CSF pellet is used. Further studies are required to confirm
these findings in different settings.
Please see later in the article for the Editors’ Summary.
Citation: Patel VB, Theron G, Lenders L, Matinyena B, Connolly C, et al. (2013) Diagnostic Accuracy of Quantitative PCR (Xpert MTB/RIF) for Tuberculous
Meningitis in a High Burden Setting: A Prospective Study. PLoS Med 10(10): e1001536. doi:10.1371/journal.pmed.1001536
Academic Editor: David R. Boulware, University of Minnesota, United States of America
Received April 3, 2013; Accepted September 12, 2013; Published October 22, 2013
Copyright: ß 2013 Patel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Columbia University-Southern African Fogarty AIDS International Training and Research Program funded by the
Fogarty International Centre, National Institutes of Health (grant #D43TW00231; VP), a South African MRC grant (VP and KD), the EU FP7 programme (TB susgent;
VP and KD), the South African NRF Research Chairs Initiative (SARChI; TN and KD), a SA MRC Career Development Award (KD), and the EDCTP (TESA and TB-NEAT).
GT is supported by the Claude Leon Foundation, the SA National Research Foundation, and the Wellcome Trust. Open Access publication of this article has been
made possible through support from the Victor Daitz Information Gateway, an initiative of the Victor Daitz Foundation and the University of KwaZulu-Natal. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: Amplicor PCR, Amplicor Mycobacterium Tuberculosis PCR Test; CT, cycle threshold; CS, clinical score; CSF, cerebrospinal fluid; CT, computerised
tomography; IPC, internal positive control; IQR, interquartile range; M.tb., Mycobacterium tuberculosis; NPV, negative predictive value; PPV, positive predictive
value; TB, tuberculosis; TBM, tuberculous meningitis; TTP, time to culture positivity.
* E-mail: patelv@ukzn.ac.za (VBP); keertan.dheda@uct.ac.za (KD)
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Xpert for the Diagnosis of Tuberculous Meningitis
clinically assessed, underwent a computerised tomography (CT)
scan to exclude contraindications to a lumbar puncture, and had
blood drawn for routine tests including a serum fluorescent
treponemal antibody test, a venereal disease research laboratory
test, an HIV enzyme-linked immunosorbent assay, and a CD4
count. Approximately 15 ml of CSF, obtained by lumbar
puncture, was processed for the following tests: microscopy (Gram
stain and fluorescent staining for acid-fast bacilli [auramine]);
bacterial culture; Mycobacterium tuberculosis (M.tb.) culture (Bactec
MGIT 960; BD); fungal culture; cryptococcal latex agglutination
test; Roche Amplicor Mycobacterium Tuberculosis PCR Test
(Roche Diagnostic Systems) (Amplicor PCR); routine chemistry
(protein, glucose, chloride); viral PCR for cytomegalovirus,
varicella zoster virus, and herpes simplex; venereal disease
research laboratory test; fluorescent treponemal antibody test;
and test for cysticercus antibodies. An uncentrifuged specimen
and, volume permitting, a centrifuged sample of CSF was
biobanked for Xpert MTB/RIF analysis. The clinical information
recorded included demographic information, duration of symptoms, whether patients were being treated with anti-tuberculous or
steroid therapy, HIV status, past history of TB, and history of TB
contact.
Introduction
There are ,10 million new cases and 1.7 million deaths from
tuberculosis (TB) annually [1]. Although the incidence of TB is
decreasing worldwide, it remains a significant cause of morbidity
and mortality in sub-Saharan Africa, where, fuelled by the HIV
epidemic, it is out of control [1,2]. In this region, up to 80% of
patients infected with TB are co-infected with HIV. Up to 40% of
co-infected patients have extrapulmonary TB, and ,10% of those
with extrapulmonary TB have tuberculous meningitis (TBM)
[3,4]. These patients frequently require prolonged admission, they
have high morbidity rates due to neuro-pathology, and mortality is
substantial (,30%), particularly if the diagnosis and follow-on
therapy are delayed [5–8]. Consequently, TBM consumes a
disproportionate amount of health care resources. A rapid and
affordable confirmatory rule-in and rule-out test still eludes clinical
practice.
PCR (polymerase chain reaction) as a diagnostic test for TBM
has a sensitivity of ,50% and a specificity close to 100% [9].
Attempts at improving sensitivity using nested PCR and simultaneous testing using multiple target genes has generally not been
fruitful [10]. Given that PCR platforms are generally located in
reference laboratories, require technical expertise, are expensive,
and are prone to contamination, they are unsuited to resourcelimited settings. More recently, however, a closed PCR system,
Xpert MTB/RIF (Cepheid), has been developed. Xpert MTB/
RIF requires minimal training to operate, is potentially point-ofcare, has good accuracy in smear-negative pulmonary TB [11–
13], and costs ,US$10 per test, and it has become available as a
frontline diagnostic in several high burden countries including
South Africa [14]. However, its value has not previously been
rigorously evaluated for the diagnosis of TBM. Furthermore, the
impact of sample volume and how samples are processed
(centrifuged versus uncentrifuged), the limit of detection, the effect
of cerebrospinal fluid (CSF)–related PCR inhibition, and the
relationship between CSF bacterial load and Xpert MTB/RIF
cycle threshold (CT) values have not been determined. To address
these gaps in knowledge, we evaluated the accuracy of Xpert
MTB/RIF in an unselected cohort of patients with suspected
TBM. To meaningfully ascertain the clinical usefulness and
incremental value of the assay relative to pre-test probability (and
hence clinical impression), we compared test accuracy to a clinical
score (CS) derived from clinical, radiological, and basic laboratory
parameters.
Categorisation of Patients
Patients were categorised, based on standardised published
diagnostic criteria, as definite TBM if the CSF M.tb. culture and/
or Amplicor PCR was positive, probable TBM (treated empirically
with anti-TB drugs but not meeting the definite TBM criteria), or
non-TBM (alternate diagnosis confirmed and response to therapy
documented in the absence of anti-TB treatment) [16,17].
Amplicor PCR
197 samples were processed by an independent laboratory using
the Amplicor PCR kit, for the detection of M.tb. This procedure
was done as per manufacturer’s protocol. Briefly, DNA was
extracted from 0.5 ml of CSF using the Roche Magna Pure
automated DNA extraction system using the DNA high performance kit. Extracted DNA was then amplified using the
biotinylated primers KY18 and KY75 as described in the
Amplicor PCR kit protocol. PCR products were detected by the
Cobas Amplicor analyser according to the kit protocol.
Xpert MTB/RIF Assay and Related Bacterial Load Studies
Xpert MTB/RIF is an integrated automated sample-processing
and real-time PCR platform developed to simultaneously detect
M.tb. and rifampicin resistance in a single-use-cartridge hands-free
step [18–20]. The Xpert MTB/RIF assay consists of two main
components, namely, a Xpert MTB/RIF plastic cartridge
(containing the liquid sample processing and PCR buffers, and
lyophilized real-time PCR reagents with internal sample processing and PCR probe quality controls) and the automated Xpert
MTB/RIF machine (which controls the advanced automated
portion of the procedure involving the engagement of the fluidics
system within the cartridge, automated ultrasound lysis, and the
performance of the real-time PCR analysis) [21,22].
Batched, archived (270uC) uncentrifuged samples (n = 149) and
centrifuged and uncentrifuged samples (n = 59) were processed at
the Lung Infection and Immunity Unit (Department of Medicine,
Groote Schuur Hospital, University of Cape Town) for Xpert
MTB/RIF analysis. Samples were stored at 270uC, for ethical
reasons, for 4 to 6 wk prior to analysis. Both centrifuged and
uncentrifuged samples were archived only later in the study when
Methods
Ethics Statement
The study protocol was approved by the biomedical research
ethics committees of the University of KwaZulu-Natal and the
University of Cape Town.
Patient Selection
235 consecutive patients with suspected meningitis were
prospectively recruited between 1 January 2008 and 31 December
2011. Patients with a meningitic illness who were referred from
local district general hospitals were investigated at the tertiary
hospital, Inkosi Albert Luthuli Central Hospital, and included in
the study.
Written informed consent was obtained from the patient or a
close relative. If patients were unable to give consent and a lumbar
puncture was clinically indicated, the Head of the Department of
Neurology was approached for consent [15]. Patients were
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cut point with a high specificity, as we required a good rule-in test,
and so that the performance of the clinical assessment was directly
comparable to the performance of the diagnostic assays under
study. To determine whether this strategy could reduce test usage
in resource-limited settings, an analysis was also conducted to
determine the net reduction in test usage if the CS was combined
with Xpert MTB/RIF (i.e., Xpert MTB/RIF performed only if
the CS was below a rule-in threshold).
it was decided to test the effect of centrifugation on test accuracy.
The laboratory technicians performing the CSF culture, Xpert
MTB/RIF assay, and Amplicor PCR were blinded to all
participant details.
Samples were prepared for Xpert MTB/RIF according to the
manufacturer’s instructions [21]. Briefly, the frozen, unprocessed
samples were thawed and immediately processed. The CSF/
sample reagent mixture was shaken and incubated at room
temperature for a total of 15 min, with a second shake occurring at
10 min. 2 ml of the digested mixture was then transferred to the
Xpert MTB/RIF cartridge. The automated steps of the procedure
were initiated by placing the loaded assay cartridge into the Xpert
MTB/RIF instrument module and then selecting the ‘‘M.
tuberculosis-Rif’’ automated detection test option from the included
software. The test was started within 30 min of adding the sample
to the cartridge.
Results were interpreted using the automated software. The
data analysis algorithms report (1) ‘‘M.tb. detected’’ if the M.tb.
target DNA (rpoB) region was detected or (2) ‘‘M.tb. not detected’’
if the M.tb. target DNA (rpoB) region was not detected. If M.tb. was
detected, the results are further categorised into (1) ‘‘RIFresistance detected’’ (if a mutation in the rpoB gene was detected)
or (2) ‘‘RIF-resistance not detected’’ (if no mutation was detected
in the rpoB region). The detailed principle of the procedure, steps
of the automated assay protocol, and full details of the diagnostic
algorithms and threshold are described in the manufacturer’s
package insert [21]. In the initial period (up to 31 January 2011)
only 1 ml of uncentrifuged CSF was obtained for Xpert MTB/
RIF testing from 149 patients with suspected TBM. From 1
February 2011 onward, to evaluate the impact of centrifugation, a
,3-ml centrifuged pellet (3,000g for 15 min) was obtained from 59
patients with suspected TBM, and resuspended in 1 ml of
phosphate-buffered saline. In this latter period, if enough CSF
was obtained, both a 1-ml uncentrifuged and 3-ml centrifuged
sample were evaluated. Thus, either a 1-ml or 3-ml sample, or
both, was processed for Xpert MTB/RIF in 208 patients with
suspected TBM. Data were analysed according to HIV status.
Included in Text S1 is the detailed method of CSF processing by
Xpert MTB/RIF.
We further developed a CS and tested whether Xpert MTB/
RIF added diagnostic value above pre-test probabilities when
using basic clinical and laboratory values.
Preliminary experiments were performed to determine the
detection limit for Xpert MTB/RIF. Patients with motor neuron
disease provided written consent for CSF collection. The CSF
obtained was spiked with serial dilutions of M.tb. (H37Rv). CT
values were correlated with bacterial load (Bactec MGIT 960 time
to culture positivity [TTP]), and PCR inhibition was evaluated by
comparing the CT value of the internal positive control (IPC;
Bacillus globigii) in CSF to that in sputum samples obtained from a
previously described reference cohort [13]. In those with Xpert
MTB/RIF-positive samples, we also compared TTP between
those who had an uncentrifuged (1 ml) and those who had a
centrifuged (3 ml) Xpert MTB/RIF test performed.
Statistical Analysis
The demographic and clinical characteristics of subgroups
(definite TBM, probable TBM, or non-TBM patients, or by test
type) were compared using Fisher’s exact test for categorical
variables and Wilcoxon’s rank sum test (two groups; Tables 1 and
2) or the Kruskal-Wallis test (three groups; Table S1) for the
continuous variables. Wilcoxon’s rank sum test was also used to
compare TTP. Factors significantly associated with definite TBM,
p,0.05, were identified from a multiple logistic regression model.
Rounded regression coefficients were used to create an ordinal CS
for each patient. A cut-off point at the 90th percentile of the
frequency distribution of the scale was selected. Using this cut-off,
patients were classified into positive and negative, which forms the
dichotomous classification of the CS [23]. Patients negative on the
CS were then tested using Xpert MTB/RIF. Differences in
specificity and sensitivity between uncentrifuged Xpert MTB/RIF
and centrifuged Xpert MTB/RIF tests, differences between Xpert
MTB/RIF tests and the CS, and, finally, the incremental value of
adding the Xpert MTB/RIF test to the CS were determined using
McNemar’s Chi square test where tests were repeated on the same
sample. Culture and/or Amplicor PCR were used as the gold
standard. A two-sample Z test was used where the above two
sample groups being compared differed. Data were analysed using
Stata version 11 (Statacorp).
Results
Clinical and Laboratory Features
Of the 235 recruited patients, there were 31 exclusions (see
Figure 1 for details, including study overview). Of the remaining
204 patients, 59 had definite TBM (seven were smear positive; the
rest were culture and/or Amplicor PCR positive), 64 had probable
TBM, and 81 had a non-TBM disease. 179 (87%) of participants
were HIV-infected, and their median CD4 count was 141
(interquartile range [IQR]: 66–284) The non-TBM group
(n = 81) comprised the following breakdown of diagnoses (number):
cryptococcal meningitis (36), viral meningitis (25), mucormycosis
(1), acute bacterial meningitis (7), malignant meningitis (3),
cysticercal meningitis (1), neurosyphilis (2), parameningeal focus
(1), and other (5). There were 50 HIV-infected patients in the
definite-TBM group, 53 HIV-infected patients in the probableTBM group, and 70 HIV-infected patients in the non-TBM
group.
A detailed breakdown of CSF processing results is outlined in
Figure 1. Table 1 shows the demographic and laboratory data
when comparing the HIV-infected definite-TBM and non-TBM
groups. Both groups were comparable, except for the use of steroid
therapy and cryptococcal latex agglutination test negativity
(Table 1). The CD4 count was significantly higher in the nonTBM group (p = 0.02). Furthermore, all routine CSF parameters—including neutrophil and lymphocyte counts, protein level,
glucose level, and CSF:blood glucose ratios—were significantly
higher in the definite-TBM group. The same pattern of results was
seen in HIV-uninfected individuals, except for the lymphocyte:total cell ratio, for which the p-value was 0.9 (shown in Table 2). In
Development of the Clinical Score
By applying univariable and multivariable logistic regression
analysis to clinical and readily available imaging and basic
laboratory parameters, we developed a CS and further assessed
whether Xpert MTB/RIF had incremental value over the CS. To
develop the CS, factors significantly associated with definite TBM
(p,0.05) in HIV-infected individuals were identified from a
multiple logistic regression model, and scores proportionally
weighted to the level of significance were assigned. We chose a
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Xpert for the Diagnosis of Tuberculous Meningitis
Table 1. Clinical and cerebrospinal fluid data in HIV-infected patients with definite TBM (liquid culture or Amplicor PCR positive;
n = 55) and non-TBM disease (culture negative and no anti-TB treatment given; n = 70).
Definite TBM, n = 55
Non-TBM, n = 70
p-Valuea
Mean age in years (standard deviation)
33 (8.9)
34 (8.8)
0.8
Age ,36 y/$36 yb
37/18 (67/33)
44/26 (63/37)
0.6
Sex male/female
25/30 (45/55)
17/53 (24/76)
0.01
Ethnic group BA/M/E/Ic
55/0/0/0 (100/0/0/0)
70/0/0/0 (100/0/0/0)
na
HIV status positive/negative
55/0 (100/0)
70/0 (100/0)
na
Characteristic
Clinical characteristics
Previous TB yes/no/unknown
13/37/5 (23/67/9)
32/36/2 (46/51/3)
0.02
TB contact within last 2 y yes/no/unknown
15/35/5 (27/64/9)
19/48/3 (27/69/4)
0.5
Duration of illnessb ,6 d/$6 d/unknown
9/44/2 (16/80/4)
6/62/2 (9/89/3)
0.7
Steroid treatment yes/no
23/32 (42/58)
9/61 (13/87)
0.001
Cryptococcal latex agglutination test positive yes/no
4/49 (8/92)
32/38 (46/54)
0.001
CD4 cells/ml (IQR)
81.0 (43–140)
136 (54–253)
0.02
0.004
CSF parameters, median (IQR)
Lymphocytes (cells/ml)
89 (28–230)
36 (12–104)
Neutrophils (cells/ml)
65 (20–138)
8 (0–40)
,0.001
Protein (g/l)
1.9 (1.2–2.8)
1.0 (0.8–1.9)
,0.001
,0.001
CSF glucose (mmol/l)
1.2 (1.0–1.8)
2.2 (1.5–2.7)
CSF:serum glucose ratio
0.2 (0.2–0.3)
0.4 (0.2–0.5)
,0.001
Lymphocyte:total cell ratio
0.5 (0.3–0.8)
0.9 (0.5–1.00)
0.005
Values are number (percent) unless otherwise indicated.
a
Categorical variables were compared using Fisher’s exact test, and numeric variables using Wilcoxon’s rank sum test.
b
This cut point was chosen based on criteria derived by Thwaites et al. [35].
c
BA, Black African; M, mixed race; E, European; I, Indian.
na, not applicable.
doi:10.1371/journal.pmed.1001536.t001
the overall sensitivity and specificity (95% CI) of Xpert MTB/RIF
were 67% (53%–79%) and 94 (85%–98%), respectively (Table 4).
In HIV-infected individuals, the sensitivity of Xpert MTB/RIF
was significantly better than that of microscopy (p = 0.001), and the
sensitivity of centrifuged samples was significantly better than that
of uncentrifuged samples, p = 0.0003. When definite TBM
combined with probable TBM was compared with non-TBM,
regardless of whether the CSF was uncentrifuged (1 ml) or
centrifuged (3 ml), the sensitivity and specificity (95% CI) were
36% (27%–46%) and 94% (85%–98%), respectively (Table 4).
One patient, who had definite TBM, had an indeterminate Xpert
MTB/RIF result. This patient was excluded from the analysis.
The HIV-uninfected group had 0% sensitivity for all tests done
(Table 5).
the definite-TBM group, 73% of patients were being treated with
a fixed-dosed combination of first-line anti-TB treatment with a
median duration of treatment of 2.5 d (IQR: 0–4) at the time of
initial assessment. The non-TBM group had treatment appropriate to the diagnosis considered and were not on TB therapy. A
comparison of demographic data between patients who had
uncentrifuged, centrifuged, or both Xpert MTB/RIF tests
performed showed no differences (Table S1). Text S2 reflects
the percentage of TBM patients classified according the Medical
Research Council grading for severity. This article fulfils the
STARD requirements (Text S3).
Multivariable Regression Analysis Identifying Clinical and
CSF Parameters That Were Predictive of TBM in HIVInfected Patients
Xpert MTB/RIF Using Uncentrifuged CSF
Factors predictive for TBM are shown in Table 3. Applying
receiver operating characteristic curve analysis, a clinical prediction rule was developed where a rule-in score of CS$8 predicted
TBM with a sensitivity of 30% (95% CI 14%–50%) and a
specificity of 100% (95% CI 85%–100%) (C statistic 85% [95%
CI: 79%–92%]). When centrifuged Xpert MTB/RIF was added
to the CS, sensitivity improved to 89% (95% CI 71%–98%),
p = 0.001. Detailed accuracy data are shown in Table 4.
There were 149 samples processed for Xpert MTB/RIF
using 1 ml of uncentrifuged CSF. Details of the breakdown of
results are shown in Figure 1. The sensitivity and specificity
(95% CI) of uncentrifuged Xpert MTB/RIF were 51% (35%–
68%) and 94% (82%–99%), respectively (see Table 4). There
were two patients infected with TBM resistant to rifampicin.
Resistance was confirmed by drug sensitivity testing. Sensitivity in the uncentrifuged samples was 26% (17%–37%), and
specificity was 94% (82%–99%). Accuracy data for the
combined definite- and probable-TBM groups compared to
the non-TBM group are shown in Table 4. Data from HIVuninfected individuals (0% sensitivity for all tests) are shown
in Table 5.
Overall Accuracy of Xpert MTB/RIF Using Centrifuged or
Uncentrifuged Samples from HIV-Infected Individuals
When the definite-TBM (culture and/or Amplicor PCR
positive) and non-TBM groups were used to calculate accuracy,
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Table 2. Clinical and CSF data in HIV-uninfected patients with definite TBM (liquid culture or Amplicor PCR positive; n = 4) and
non-TBM disease (culture negative and no anti-TB treatment given; n = 11).
Definite TBM, n = 4
Non-TBM, n = 11
p-Valuea
Mean age in years (standard deviation)
30 (10.8)
28 (16.4)
0.9
Age ,36 y/$36 yb
3/1 (75/25)
8/3 (73/28)
0.9
Sex male/female
3/1 (75/25)
9/2 (82/18)
0.8
Ethnic group BA/M/E/Ic
3/1/0/0 (75/25/0/0)
9/0/0/2 (82/0/0/18)
0.3
HIV status positive/negative
0/4 (0/100)
0/11 (0/100)
na
Characteristic
Clinical characteristics
Previous TB yes/no/unknown
0/4/0 (0/100/0)
0/9/2 (0/82/18)
0.4
TB contact within last 2 y yes/no/unknown
0/4/0 (0/100/0)
1/9/1 (9/82/9)
0.7
Duration of illnessb ,6 d/$6 d/unknown
0/4/0 (0/100/0)
5/4/2 (45/36/18)
0.03
Steroid treatment yes/no
1/3 (25/75)
2/9 (18/82)
0.8
Cryptococcal latex agglutination test positive yes/no
0/4 (0/100)
0/11 (0/100)
na
CD4 cells/ml (IQR)
246 (120–426)
678 (344–735)
0.07
0.05
CSF parameters, median (IQR)
Lymphocytes (cells/ml)
128 (77–362)
14 (4–38)
Neutrophils (cells/ml)
73 (40–136)
8 (0–278)
0.3
Protein (g/l)
1.2 (1.2–1.4)
0.7 (0.5–1.4)
0.2
CSF glucose (mmol/l)
0.9 (0.6–1.0)
3.4 (2.5–4.1)
0.005
CSF:serum glucose ratio
0.2 (0.1–0.2)
0.6 (0.5–0.7)
0.005
Lymphocyte:total cell ratio
0.6 (0.4–0.8)
0.6 (0.02–1.0)
0.9
Values are number (percent) unless otherwise indicated.
a
Categorical variables were compared using Fisher’s exact test, and numeric variables using Wilcoxon’s rank sum test.
b
This cut point was chosen based on criteria derived by Thwaites et al. [35].
c
BA, Black African; M, mixed race; E, European; I, Indian.
na, not applicable.
doi:10.1371/journal.pmed.1001536.t002
patients. As the hospital prevalence of TBM is variable, we have
calculated the number of tests saved when assuming a hospital
prevalence varying from 10% to 59% (shown in Table 6). In an
alternative strategy using the 59% prevalence, CS (with CS$8
indicating TBM) would have categorised 17 patients as having
TBM. If centrifuged Xpert MTB/RIF were applied to the
remainder, a further 34 patients would have been detected. Thus,
the combined strategy would detect 51 (80%) patients. CS would
thus hypothetically have saved 17 tests, assuming that there were
no indeterminate results. Given the bias of selecting prevalence
based on definite-TBM and non-TBM cases only, and given that
the approximate prevalence in a hospital-based cohort is closer to
30% [24,25], this strategy would have detected nine patients by
CS and a further 17 patients in the remainder by centrifuged
Xpert MTB/RIF, i.e., a total of 26 (80%) patients (at a 30%
hospital-based prevalence of TBM). Centrifuged Xpert MTB/RIF
alone would have detected 24 patients (80%). Thus, CS would
have saved nine tests.
In summary, applying the CS reduced cartridge usage by 9%
with an in-hospital disease prevalence of 30%, and by 33% with a
prevalence of ,60% (outlined in Table 6).
Xpert MTB/RIF Using Centrifuged CSF
There were 59 samples prospectively tested via Xpert MTB/
RIF using 3 ml of centrifuged CSF. Details of the breakdown of
results are shown in Figure 1. In the HIV-infected group, the
sensitivity and specificity (95% CI) of centrifuged Xpert MTB/
RIF were 82% (62%–94%) and 95% (74%–100%), respectively
(see Table 4). When the combined definite- and probable-TBM
groups were compared with the non-TBM group, the sensitivity
and specificity (95% CI) of centrifuged Xpert MTB/RIF improved
relative to the uncentrifuged samples to 65% (47%–87%) and 96%
(78%–100%), respectively. Further accuracy data when the
combined definite- and probable-TBM groups were compared
to non-TBM is shown in Table 4. The HIV-uninfected group
showed a sensitivity of 0% across all tests (Table 5).
Influence of Prevalence and Impact of Test Usage
As positive predictive value (PPV) and negative predictive value
(NPV) are strongly influenced by prevalence of disease, we
included an analysis of how the CS, when hypothetically applied
to a cohort of 100 patients with suspected TBM, would perform,
and how many Xpert MTB/RIF tests would potentially be
unnecessary if the CS were applied prior to performing the
centrifuged Xpert MTB/RIF test. True hospital prevalence of
TBM in South Africa is not known. A hypothetical prevalence of
59% (based on the number of patients with definite plus probable
TBM in this cohort) was assumed for this calculation. When
applied to 100 patients with suspected TBM (thus potentially 59
TBM patients), 100 cartridges would have been used, and
centrifuged Xpert MTB/RIF would have detected 47 (79.6%)
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Testing of Paired Centrifuged and Uncentrifuged
Samples
Given limited available CSF volumes, there were only 12
culture-positive samples (note: one patient who was classified as
having probable TBM had both 1 ml and 3 ml tested but was
excluded from this analysis) that were tested concurrently via
Xpert MTB/RIF using both 1 ml of uncentrifuged and 3 ml of
5
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Xpert for the Diagnosis of Tuberculous Meningitis
Figure 1. Summary flow chart of patient recruitment and diagnostic testing performed. MGIT, Bactec MGIT 960; ND, not done; +ve,
positive; 2ve, negative. * These patients could not be clearly categorised as definite TBM, probable TBM, or non-TBM (e.g., reference negative and
lost to follow-up, and without initiation of TB treatment). { Note that the uncentrifuged and centrifuged Xpert MTB/RIF groups include 12 patients
who had both processes done, i.e., paired samples.
doi:10.1371/journal.pmed.1001536.g001
Table 3. Multivariable regression analysis identifying factors predictive for TBM and related derivation of the clinical score for HIVinfected patients.
Odds Ratio (95% CI)
p-Value
B-Coefficient
Score
Negative
17.6 (4.7–66.4)
,0.001
2.9
3
Positive
1
0
0
1.52
2
0
0
2.2
2
0
0
2.08
2
0
0
1.80
2
0
0
Parameter
Cryptococcal latex agglutination test
CSF:plasma glucose ratio
#0.2
4.6 (1.5–14.2)
.0.2
1
0.009
CD4 count
#200 cells/ml
8.6 (2.4–30.8)
.200 cells/ml
1
0.001
Lymphocyte count
.200 cells/ml
8.0 (1.9–34.0)
#200 cells/ml
1
0.005
Hydrocephalus
Yes
5.8 (1.7–19.2)
No
1
0.004
doi:10.1371/journal.pmed.1001536.t003
PLOS Medicine | www.plosmedicine.org
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Xpert for the Diagnosis of Tuberculous Meningitis
Table 4. Performance outcomes of Xpert MTB/RIF (overall, uncentrifuged, and centrifuged), smear microscopy, clinical score, and a
combination of Xpert MTB/RIF and clinical score.
Sensitivity
(95% CI) [n]
Specificity
(95% CI) [n]
PPV
(95% CI) [n]
NPV
(95% CI) [n]
Agreement
(95% CI) [n]
Smear microscopy
13%
(5–25)b
[7/55]
100%
(95–100)
[70/70]
100%
(59–100)
[7/7]
59%
(50–68)
[70/118]
62%
(53–70)
[77/125]
Xpert MTB/RIF (all samples, whether
uncentrifuged or centrifuged)
67%b
(53–79)
[36/54]
94%
(85–98)
[61/65]
90%
(76–97)
[36/40]
77%
(66–86)
[61/79]
82%
(73–88)
[97/119]
Uncentrifuged Xpert MTB/RIF
51%
(35–68)b,c
[20/39]
94%
(82–99)
[43/46]
87%
(66–97)
[20/23]
69%
(56–80)
[43/62]
74%
(64–83)
[63/85]
Centrifuged Xpert MTB/RIF
82%
(62–94)b,c
[22/27]
95%
(74–100)
[18/19]
96%
(78–100)
[22/23]
78%
(56–93)
[18/23]
87%
(74–95)
[40/46]
CS alone (score$8)
30%
(14–50)b,d
[8/27]
100%
(82–100)
[19/19]
100%
(63–100)
[8/8]
50%
(33–67)
[19/38]
59%
(43–73)
[27/46]
CS plus centrifuged Xpert MTB/RIF (only
done if CS,8)
89%
(71–98)d
[24/27]
95%
(74–100)
[18/19]
96%
(80–100)
[24/25]
86%
(64–97)
[18/25]
91%
(79–98)
[42/46]
Smear microscopy
6%
(3–13)
[7/108]
100%
(95–100)
[70/70]
100%
(59–100)
[7/7]
41%
(34–49)
[70/171]
43%
(36–51)
[77/178]
Xpert MTB/RIF (all samples, whether
uncentrifuged or centrifuged)
36%
(27–46)
[38/106]
94%
(85–98)
[61/65]
91%
(77–97)
[38/42]
47%
(38–56)
[61/129]
58%
(50–65)
[99/171]
Uncentrifuged Xpert MTB/RIF
26%f
(17–37)
[22/84]
94%
(82–99)
[43/46]
88%
(69–98)
[22/25]
41%
(32–51)
[43/105]
50%g
(41–59)
[65/130]
Centrifuged Xpert MTB/RIF
65%f
(47–80)
[22/34]
95%
(74–100]
[18/19]
96%
(78–100)
[22/23]
60%
(41–77)
[18/30]
76%g
(62–86)
[40/53]
Test Specifics
Definite TBM versus non-TBMa
Combined definite and probable TBM
versus non-TBMe
Sensitivity, specificity, PPV, NPV, and CS are expressed as percentages.
a
Performance outcomes when definite TBM is compared with non-TBM (liquid culture or Amplicor PCR positivity for M.tb. served as a reference standard).
b
Represents a comparison of sensitivity between microscopy and centrifuged Xpert MTB/RIF, p#0.001; microscopy and uncentrifuged Xpert MTB/RIF, p#0.001;
microscopy and CS alone, p = 0.8; microscopy and CS plus centrifuged Xpert MTB/RIF, p#0.001; microscopy with Xpert MTB/RIF (regardless of centrifugation or volume),
p#0.001.
c
Represents a comparison of Xpert MTB/RIF sensitivity between centrifuged and uncentrifuged samples, p = 0.004.
d
Represents a comparison of sensitivity between CS alone and CS combined with Xpert MTB/RIF when CS negative using centrifuged samples, p#0.001.
e
Performance outcomes when combined definite and probable TBM is compared to non-TBM (liquid culture or Amplicor PCR positivity for M.tb. and satisfaction of
probable TBM, as defined by Thwaites et al. [16,17], served as a reference standard).
f
Denotes a comparison of sensitivities between uncentrifuged and centrifuged Xpert MTB/RIF, p#0.001.
g
Denotes comparison for agreement between uncentrifuged and centrifuged Xpert MTB/RIF, p#0.006.
doi:10.1371/journal.pmed.1001536.t004
CSF changes. Details regarding these patients are shown in
Table 7.
centrifuged CSF. The sensitivity was not significantly different in
centrifuged versus uncentrifuged samples (58% versus 67%;
p = 0.6; see Table S2).
HIV-Uninfected Individuals
Patients Who Tested MTB Positive by Xpert MTB/RIF but
Negative by Culture
There were too few HIV-uninfected patients to generate reliable
performance outcome data. However, Xpert MTB/RIF was
negative in all four HIV-uninfected, TBM M.tb.–infected patients.
There were three patients who were classified in the non-TBM
group but were Xpert MTB/RIF positive (two had culture-proven
cryptococcal meningitis, and another had biopsy-proven leukemic
meningitis). Details are shown in Table 7.
There were seven patients who were classified only as probable
TBM even though they were Xpert MTB/RIF positive. All of
these patients were HIV-infected, with CD4 counts varying from
18 cells/ml to 350 cells/ml. A significant proportion had atypical
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Detection Threshold, Bacterial Load, and Inhibition
Studies
The detection threshold for Xpert MTB/RIF was 80 colony
forming units per millilitre (Figure 2). There was no correlation
between Xpert MTB/RIF CT values and time to positive culture
(TTP); (r2 = 0.02; p = 0.53; see Figure 3). Finally, we examined the
7
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Xpert for the Diagnosis of Tuberculous Meningitis
Table 5. Performance outcomes of Xpert MTB/RIF (overall, uncentrifuged, and centrifuged), smear microscopy, clinical score, and a
combination of Xpert MTB/RIF and clinical score.
Sensitivity (95% CI) [n]
Specificity (95% CI) [n]
PPV (95% CI) [n]
NPV (95% CI) [n]
Agreement (95% CI) [n]
Smear microscopy
0%
(0–60)
[0/4]
100%
(72–100)
[11/11]
0%
[0/0]
73%
(45–92)
[11/15]
73%
(45–92)
[11/15]
Xpert MTB/RIF (all samples,
whether uncentrifuged or
centrifuged)
0%
(0–60)
[0/4]
100%
(69–100)
[10/10]
0%
[0/0]
71%
(42–97)
[10/14]
71%
(42–92)
[10/14]
Uncentrifuged Xpert MTB/RIF
0%
(0–60)
[0/4]
100%
(54–100)
[6/6]
0%
[0/0]
60%
(26–88)
[6/10]
60%
(26–88)
[6/10]
Centrifuged Xpert MTB/RIF
0%
[0/0]
100%
(40–100)
[4/4]
0%
[0/0]
100%
(40–100)
[4/4]
100%
(40–100)
[4/4]
CS alone (score$8)
0%
[0/0]
100%
(40–100)
[4/4]
0%
[0/0]
100%
(40–100)
[4/4]
100%
(40–100)
[4/4]
CS plus centrifuged Xpert
MTB/RIF (only done if CS,8)
0%
[0/0]
100%
(40–100)
[4/4]
0%
[0/0]
100%
(40–100)
[4/4]
100%
(40–100)
[4/4]
Smear microscopy
0%
(0–22)
[0/15]
100%
(72–100)
[11/11]
0%
[0/0]
42%
(23–63)
[11/26]
42%
(23–63)
[11/26]
Xpert MTB/RIF (all samples,
whether uncentrifuged or
centrifuged)
0%
(0–23)
[0/14]
100%
(69–100)
[10/10]
0%
[0/0]
42%
(22–63)
[10/24]
42%
(22–63)
[10/24]
Uncentrifuged Xpert MTB/RIF
0%
(0–25)
[0/13]
100%
(54–100)
[6/6]
0%
[0/0]
32%
(13–57)
[6/19]
32%
(13–57)
[6/19]
Centrifuged Xpert MTB/RIF
0%
(0–84)
[0/2]
100%
(40–100]
[4/4]
0%
[0/0]
67%
(22–96)
[4/6]
67%
(22–96)
[4/6]
Test Specifics
Definite TBM versus non-TBMa
Combined definite and
probable TBM versus non-TBMb
a
Performance outcomes when definite TBM is compared with non-TBM (liquid culture or Amplicor PCR positivity for M.tb. served as a reference standard).
Performance outcomes when combined definite and probable TBM is compared to non-TBM (liquid culture or Amplicor PCR positivity for M.tb. and satisfaction of
probable TBM, as defined by Thwaites et al. [16,17], served as a reference standard).
doi:10.1371/journal.pmed.1001536.t005
b
Table 6. The number of cartridges potentially saved when using CS prior to centrifuged Xpert MTB/RIF testing in a hypothetical
cohort of 100 patients with suspected TBM.
Assumed Hospital
Prevalence of TBM
Cartridges Used
Number of
without CSa Being
Applied in 100 Patients Patients Identified
by CS Alone
with Suspected TBM
Number Identified
by Xpert MTB/RIF
Alone (No CS
Applied)
Number of Patients
Identified by CS
and Xpert MTB/RIF
(When CS,8)
Number of
Cartridges Saved
3/100 (3%)
10% (n = 10 TBM cases)
100
3/10 (30%)
8/10 (80%)
9/10 (90%)
20% (n = 20 TBM cases)
100
6/20 (30%)
16/20 (80%)
17/20 (85%)
6/100 (6%)
30% (n = 30 TBM cases)
100
9/30 (30%)
25/30 (83%)
26/30 (87%)
9/100 (9%)
40% (n = 40 TBM cases)
100
12/40 (30%)
33/40 (82%)
35/40 (88%)
12/100 (12%)
50% (n = 50 TBM cases)
100
15/50 (30%)
41/50 (82%)
44/50 (88%)
15/100 (15%)
59% (n = 59 TBM cases)
100
18/59 (30%)
48/59 (81%)
52/59 (88%)
18/100 (18%)
a
A CS was generated only to estimate the incremental value of Xpert MTB/RIF over clinical assessment using basic clinical and CSF parameters. The CS was not
independently validated.
doi:10.1371/journal.pmed.1001536.t006
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Table 7. Characteristics of patients classified as non-TBM and probable TBM who were Xpert MTB/RIF positive.
Neutrophils
Lymphocyte
Count (cells/ml)
CSF (Serum)
Glucose
Protein (g/l) (mmol/l)
Cryptococcal
Latex
Agglutination Test
132
0
10
3.52
2.0 (9.0)
+ve
305
138
20
0.89
+ve
350
66
92
2.26
HIV
Status
CD4 Count
(cells/ml)
1
+ve
2
3
Patient
Diagnosis
Outcome
+ve
Cryptococcal
meningitis
Died
0.6 (3.5)
2ve
B cellb lymphoma
Died
0.8 (6.5)
+ve
Cryptococcal
meningitis
Lost to follow-upc
CSF culture and microscopy
negative (categorised as
non-TBM)a
9
CSF culture negative
(categorised as probable TBM)d
1
+ve
84
0
2
0.79
0.8 (7.6)
2ve
TBM
Died
2
+ve
97
0
30
4.99
1.2 (9.3)
2ve
TBM
Improved
+ve
29
248
48
1.01
1.0 (5.1)
2ve
TBM
Improved
+ve
45
60
8
4.40
1.0 (7.5)
2ve
TBM
Died
5
+ve
177
0
18
0.93
3.1 (6.6)
2ve
TBM
Improved
6
+ve
14
286
50
2.93
1.0 (6.1)
2ve
TBM
Improved
7
+ve
426
38
292
2.19
0.7 (5.0)
2ve
TBM
Improved
a
Characteristics of patients who were CSF culture and microscopy negative but Xpert MTB/RIF positive (all the patients were classified as non-TBM).
Confirmed on histology of spinal cord lesion.
This patient improved in the short term (first 10 d) on anti-fungal treatment but was lost to follow-up after transfer to a peripheral health care facility. Thus, it is uncertain whether he had a dual infection (cryptococcal meningitis
and TBM).
d
Characteristics of patients who were CSF culture negative but Xpert MTB/RIF positive (all the patients were empirically treated for TB at presentation).
+ve, positive; 2ve, negative.
doi:10.1371/journal.pmed.1001536.t007
b
c
Xpert for the Diagnosis of Tuberculous Meningitis
October 2013 | Volume 10 | Issue 10 | e1001536
3
4
Xpert for the Diagnosis of Tuberculous Meningitis
Figure 2. Level of detection of CSF Xpert MTB/RIF for M.
tuberculosis using serial dilutions (500, 250, 100, 80, and 10
colony forming units per millilitre) of H37Rv. CFU, colony forming
units.
doi:10.1371/journal.pmed.1001536.g002
Figure 4. Comparison of PCR inhibition using the comparative
internal positive control CT values in CSF and sputum (all HIVinfected patients). CSF IPC: median (IQR) CT value is 27.2 (range:
27.83–35.4), n = 82. Sputa IPC: median (IQR) CT value is 29.85 (range:
31.9–40.5), n = 238. Comparison between CT values for CSF and sputum,
p#0.001.
doi:10.1371/journal.pmed.1001536.g004
degree of relative PCR inhibition by comparing the CSF-specific
IPC (B. globigii) CT values (n = 82) to those obtained from sputum
samples in a cohort of HIV-infected patients with suspected TB
(n = 238; CT but not IPC data previously published in Blakemore
et al. [26]). There was less inhibition using CSF compared to
previous published data from Cape Town using sputum
(p#0.0001; Figure 4) [13]. Using TTP as a surrogate marker,
we compared bacterial load between the centrifuged (n = 6) and
uncentrifuged (n = 9) groups. The median TTP was 19.0 (IQR:
18–24) and 20.0 (IQR: 18–21) d for the uncentrifuged and
centrifuged groups, respectively (p = 0.9). This may reflect a type 2
statistical error, as the numbers are small.
urgently required to guide appropriate and relevant usage of this
technology in biological fluids other than sputum. That Xpert
MTB/RIF performs poorly in fluids from some compartments,
e.g., the pleural space, highlights the need for such data [27]. The
key findings of this study were as follows: (1) Xpert MTB/RIF is
likely a good rule-in test for the diagnosis of TBM in HIV-infected
patients; (2) centrifugation of the sample improved sensitivity in
this context to almost 80%; (3) among HIV-infected patients,
Xpert MTB/RIF performed significantly better than the widely
available same-day alternative tests, i.e., smear microscopy, which
suggests that prompt diagnosis of TBM is potentially achievable in
the majority of patients in this setting; (4) the diagnostic value of
Xpert MTB/RIF for HIV-infected patients is clinically meaningful given that it performed significantly better than hypothetical
decision-making based on clinical characteristics and basic
laboratory data (the CS); and (5) when combined with the CS,
Xpert MTB/RIF test usage could be reduced by only a modest
,10% whilst retaining similar sensitivity and specificity compared
to using Xpert MTB/RIF alone. This last finding informs clinical
practice in resource-poor settings. Finally, we quantified the limit
of detection of the assay, its relationship to bacterial load, and the
impact of PCR inhibition. These data require reproduction in
HIV-uninfected and non-TB-endemic populations.
There are limited data about Xpert MTB/RIF performance in
TBM [28]. Published data include only small numbers of
microbiologically proven TBM cases (range of 0 to 23) [29–32],
often in a case-control design with a non-uniform reference
standard, and often CSF-associated data were published as part of
a laboratory-based evaluation of extrapulmonary TB samples,
usually including samples from countries with low TB prevalence.
Furthermore, there are no studies from high burden settings, and
technical performance evaluations, including bacterial load
studies, threshold level of detection, and impact of PCR inhibition,
have hitherto not been undertaken.
Xpert MTB/RIF sensitivity was as high as 80% when a
centrifuged CSF sample from an HIV-infected patient was used.
This suggests that Xpert MTB/RIF, at least in an HIV-endemic
environment, represents a possible new standard of care for the
Discussion
Although the utility profile and accuracy of Xpert MTB/RIF
has been well characterised in sputum samples, there are hardly
any data to guide its utility and implementation for TBM. This is
critical as the rollout of Xpert MTB/RIF means that quantitative
PCR is now available in many high burden settings, and data are
Figure 3. Correlation of CSF Xpert MTB/RIF cycle threshold (CT)
and Bactec MGIT 960 time to positive culture in all samples
(both centrifuged and uncentrifuged).
doi:10.1371/journal.pmed.1001536.g003
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Xpert for the Diagnosis of Tuberculous Meningitis
empiric treatment-based strategy given our study design and the
fact that management decisions were not based on Xpert MTB/
RIF results. However, this was because Xpert MTB/RIF had
not yet been endorsed by the World Health Organization when
the study commenced, had not been validated for use in CSF,
and had been used as a research tool only (thus, for ethical
reasons, study samples were evaluated only several weeks later).
Although the confidence intervals of some of our estimates are
wide (because of limited sample numbers), this is to our
knowledge the largest diagnostic study undertaken in TBM
(based on the number of microbiologically proven TBM cases;
n = 59). This reflects the challenge and difficulty in performing
such studies in resource-poor settings. It is possible that the
Xpert MTB/RIF performs much better in HIV-infected
individuals because of a possibly higher bacterial load, and
thus our findings need to be confirmed in other settings. Given
the small number of HIV-uninfected patients, we were unable to
meaningfully compare this sub-group. The CS was developed to
assess only incremental value above basic clinical and CSF
parameters. The CS and the combination of CS plus Xpert
MTB/RIF need prospective and independent validation. The
non-significant difference in sensitivity between the paired
centrifuged and non-centrifuged samples may reflect a type
two statistical error, as the number of culture-positive paired
samples was limited. Lastly, there were nine patients who could
not be categorised within our defined groups and were excluded
from the analysis.
In conclusion, Xpert MTB/RIF may be a good rule-in test
for the diagnosis of TBM in HIV-infected individuals in a
TB-endemic setting, particularly when a centrifuged CSF
pellet is used. A second Xpert MTB/RIF test had minimal
incremental benefit. Smear microscopy and the CS, when
combined with Xpert MTB/RIF, only modestly minimised
test usage in a resource-poor setting. Further studies are now
required in non-HIV-endemic settings, and using validated
scoring systems, to evaluate the impact of Xpert MTB/RIF
on diagnostic accuracy, and morbidity and mortality in
patients with TBM.
diagnosis of TBM. Sensitivity was considerably better than in
previous studies using commercially available or non-standardised PCR tools [9,32–34]. The ostensibly better performance is
likely related to a combination of centrifugation (and hence
concentration of bacilli) and technical aspects, including a more
efficient standardised extraction protocol, fractionation of
mycobacteria by a pre-sonication step, and a nested PCR
protocol, thus maximising amplification. However, possibly
higher bacterial loads in HIV-infected patients may have also
played a role. Our findings have practical relevance because
they imply that at least 3 ml of CSF should be set aside and
centrifuged, and re-suspended in phosphate-buffered saline,
before being run on the Xpert MTB/RIF. This high-sensitivity
and potentially rapid diagnosis in most cases is likely to benefit
HIV-infected patients suspected of having M.tb., as diagnostic
and treatment delay is associated with higher mortality [35–37].
Impact-related studies are now required to verify this hypothesis. It is noteworthy that a second sample improved sensitivity
minimally. These data suggest that, at least in an HIV-endemic
setting, using a second cartridge is unlikely to give further
benefit. However, larger studies are required to confirm this
possibility.
Similar to the findings when using sputum, the level of detection
of Xpert MTB/RIF was between 80 and 100 colony forming units
per millilitre. This explains the sub-optimal sensitivity of Xpert
MTB/RIF compared to culture, where the detection threshold is
as low as 1–10 organisms per millilitre [38]. We did not find a
correlation between TTP and Xpert MTB/RIF CT values, as has
been shown in sputum [39]. In contrast to previous PCR-based
studies [40,41], we found that CSF had a minimal inhibitory effect
on the PCR reaction when compared to sputum. This may be due
to the wash step incorporated into the assay that removes
extracellular debris. We did not find a difference in TTP between
the Xpert MTB/RIF–positive samples from centrifuged versus
uncentrifuged CSF. This may be due to a type two statistical error,
as the sample numbers were small.
There were three patients who were culture negative but Xpert
MTB/RIF positive, i.e., Xpert MTB/RIF positive in the non-TB
group. Our previous work has shown that such cases (Xpert
MTB/RIF positive but culture negative) are likely to be true TB
positives, and this is corroborated by high specificity obtained in
large sputum-based studies where a significant minority of the
patients had had previous TB [11]. If these culture-negative Xpert
MTB/RIF–positive individuals are hypothetically designated
definite-TB cases, then the overall case detection rate improves
by a further ,10%.
The proper and meaningful value of a test lies in its ability to
influence patient management through its incremental value over
pre-test probability, or to have an impact on decision-making
based on logical clinical judgement (based upon clinical features
and basic laboratory parameters). We therefore derived a CS,
hitherto unavailable for HIV-endemic settings, to evaluate Xpert
MTB/RIF utility in clinical practice. Xpert MTB/RIF had
significantly better performance outcomes than the clinical
prediction rule (using a rule-in cut point, so appropriate
comparisons could be made). Furthermore, hypothetically combining the CS with Xpert MTB/RIF resulted only in a modest
,10% reduction in test usage, but still maintained high sensitivity
and specificity. These data suggest that clinical algorithms or
scoring systems to limit test usage are unlikely to be significantly
useful in resource-poor settings.
There are several limitations of our study. We could not
determine the impact of Xpert MTB/RIF (time and proportion of
patients initiated on treatment) compared to a smear microscopy/
PLOS Medicine | www.plosmedicine.org
Supporting Information
Table S1 Comparison of demographic data amongst
the uncentrifuged, centrifuged, and both centrifuged
and uncentrifuged Xpert MTB/RIF test groups.
(DOCX)
Table S2 Diagnostic accuracy when comparing centrifuged and uncentrifuged Xpert MTB/RIF in the same
patients (all definite TBM, n = 12).
(DOCX)
Text S1 Method for processing CSF using Xpert MTB/
RIF.
(DOCX)
Text S2 Grading of TBM severity.
(DOCX)
Text S3 STARD document.
(DOC)
Acknowledgments
We are extremely grateful to the patients, registrars in the department of
neurology, and nurses for facilitating this study. The investigators are
grateful to the Province of KwaZulu-Natal and the TB programme for
facilitating the study.
11
October 2013 | Volume 10 | Issue 10 | e1001536
Xpert for the Diagnosis of Tuberculous Meningitis
Author Contributions
manuscript: GT KD. ICMJE criteria for authorship read and met: VBP
GT LL BM CC RS YC TN KD. Agree with manuscript results and
conclusions: VBP GT LL BM CC RS YC TN KD. Enrolled patients:
VBP. Supervised the study: TN KD. Statistician responsible for the
statistical analysis: CC.
Conceived and designed the experiments: VBP KD. Performed the
experiments: GT LL BM RS YC. Analyzed the data: VBP GT CC KD.
Contributed reagents/materials/analysis tools: YC TN KD. Wrote the
first draft of the manuscript: VBP. Contributed to the writing of the
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Xpert for the Diagnosis of Tuberculous Meningitis
Editors’ Summary
Standard diagnostic tests were used to categorize these
patients as either definitely having TB meningitis, possibly
having TB meningitis, or not having TB meningitis. Among
patients infected with HIV, the Xpert MTB/RIF correctly
identified 62% of those with TB meningitis and 95% of those
without TB meningitis.
The researchers also assessed whether it would be more
cost-effective to use the test only for cases where the
standard diagnostic procedure was uncertain, i.e., to avoid
testing in cases where TB meningitis was very likely following
the normal clinical assessment. Based on the researchers’
theoretical analysis, this would reduce test use by only about
10%.
Background.
Worldwide, tuberculosis (TB) is the leading cause of death
among people living with HIV. The risk of developing TB is
estimated to be 12–20 times greater in people with HIV than
in people without HIV. The World Health Organization
reported that, in 2011, there were 8.7 million new cases of
TB, of which 1.1 million were among people living with HIV.
TB infection in people living with HIV is a major problem in
sub-Saharan Africa, where up to 80% of individuals infected
with TB are also infected with HIV.
TB is caused by a bacterial infection spread through the air
from one person to another when the infected person
coughs or sneezes, for example. It usually affects the lungs,
but it can also affect other parts of the body including the
brain, where it leads to meningitis. People with meningitis
caused by TB are often seriously ill. Many may develop brain
damage, and 30% will die, particularly if they aren’t
diagnosed quickly and treatment is delayed. TB meningitis
is therefore a serious health concern in countries with high
rates of HIV and TB co-infection.
What Do These Findings Mean?
This study suggests that Xpert MTB/RIF is a useful diagnostic
test for TB meningitis in patients infected with HIV living in
areas where there are high levels of TB infection. It is not
known how well the test would perform in places where TB
levels are low, and the test did not perform well in
individuals without HIV, although there were very few of
these patients. The Xpert MTB/RIF test correctly identified
more positive cases than the other tests used to diagnose TB
meningitis, within 24 hours of first seeing a patient.
However, the test accuracy was best when the cerebrospinal
fluid sample was centrifuged (spinning the sample very fast
to concentrate the test material) to achieve the best results.
This means additional apparatus would be required, resulting in higher cost and requiring more training. The
researchers conclude that this test could still be useful in
settings where resources are limited.
There are also important questions that remain unanswered. This study shows only that the Xpert MTB/RIF test
is useful in determining that a patient has TB meningitis. It
is not useful in determining that a patient does not have
TB meningitis. Further research is needed to determine
whether the test will be effective in areas with lower rates
of TB, as well as whether its use will improve clinical
practice and ultimately lead to better outcomes for
patients. The hope is that the test will result in more
rapid diagnosis and faster treatment, reducing the number
of avoidable deaths from TB meningitis.
Why Was This Study Done?
There is currently no simple test to diagnose TB meningitis.
The tests that are available detect only about 50% of cases.
They are expensive and practical to use only in a high-tech
environment, and are therefore unsuitable for low-income
countries.
Recently, a new test has become available to detect TB,
known as Xpert MTB/RIF. The test is used to detect the DNA
(the molecular biological instructions for each organism) of
the bacteria that causes TB. It is accurate at detecting TB
lung infection, requires minimal training to operate, and is
relatively inexpensive. It is now being used to diagnose TB in
countries with high rates of the disease, including South
Africa. However, thus far its use has been limited to
detecting the TB bacterium in sputum samples (a mixture
of saliva and phlegm) from people with a lung infection. The
few studies that have assessed whether the test can be used
to detect TB meningitis have been small and inconclusive.
This study was carried out to determine whether Xpert MTB/
RIF could be used to detect TB bacteria in the cerebrospinal
fluid (the fluid that surrounds the brain and spinal cord) in
people with TB meningitis. The researchers wanted to find
out whether the test would be sensitive (correctly identifying
patients with TB meningitis) and specific (correctly identifying patients without TB meningitis). They also wanted to
address more practical questions such as how much
cerebrospinal fluid needs to be collected and how the
sample needs to be processed to ensure accurate results.
Additional Information.
Please access these websites via the online version of this
summary at
http://dx.doi.org/10.1371/journal.pmed.1001536.
N This study is further discussed in a PLOS Medicine
Perspective by David Boulware
N The US National Institutes of Health provides information
on TB meningitis
N The World Health Organization provides information on
tuberculosis and HIV
N The US Centers for Disease Control and Prevention has a
factsheet on HIV and TB
N AIDS.gov also provides information about HIV and TB
What Did the Researchers Do and Find?
The researchers used the Xpert MTB/RIF test to analyze
cerebrospinal fluid samples from 204 patients with suspected TB meningitis. These patients were recruited from
hospitals in South Africa between January 2008 and
December 2011.
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