ESPID Reports and Reviews

CONTENTS
Diagnostic Tests for Childhood TB
Molecular Fungal Diagnostics
EDITORIAL BOARD
Co-Editors: Delane Shingadia and Nicole Ritz
Board Members
David Burgner (Melbourne, Cristiana Nascimento-Carvalho George Syrogiannopoulos
Australia) (Bahia, Brazil) (Larissa, Greece)
Kow-Tong Chen (Tainan,Taiwan) Ville Peltola (Turku, Finland) Tobias Tenenbaum (Mannhein, Germany)
Luisa Galli (Florence, Italy) Emmanuel Roilides (Thessaloniki, Marc Tebruegge (Southampton, UK)
Steve Graham (Melbourne, Greece) Marceline van Furth (Amsterdam,
Australia) Ira Shah (Mumbai, India) The Netherlands)

Diagnostic Tests for Childhood Tuberculosis

Past Imperfect, Present Tense and Future Perfect?
Marc Tebruegge, DTM&H, DLSHTM, MRCPCH, MSc, FHEA, MD, PhD,* Nicole Ritz, MD, PhD,
Nigel Curtis, DCH, DTM&H, MRCP, FRCPCH, PhD,** and Delane Shingadia, DTM&H, MPH, MRCP, FRCPCH

O ver the past decade, there has been

significant progress in developing new
diagnostic tools for childhood tuberculosis
(TB).1 However, there are still many unan-
Accepted for publication June 10, 2015.
From the *Academic Unit of Clinical & Experimen-
tal Sciences, Faculty of Medicine, University of
Southampton, Southampton, United Kingdom;
Department of Paediatric Infectious Diseases &
Immunology, University Hospital Southampton
NHS Foundation Trust, Southampton, United
Kingdom; Institute for Life Sciences, University
of Southampton, Southampton, United Kingdom;
NIHR Respiratory Biomedical Research Unit,
University Hospital Southampton NHS Foun-
dation Trust, Southampton, United Kingdom;
Department of Paediatrics, The University of
Melbourne, Parkville, Victoria, Australia; Uni-
versity Childrens Hospital Basel, Paediatric
Infectious Diseases and Pharmacology, University
Basel, Basel, Switzerland; **Murdoch Childrens
Research Institute, Parkville, Victoria, Australia;
Infectious Diseases Unit, Royal Childrens Hos-
pital Melbourne, Parkville, Victoria, Australia;
Department of Paediatric Infectious Diseases,
Great Ormond Street Hospital, London, United
Kingdom; and University College London Insti-
tute of Child Health, London, United Kingdom.
The authors have no funding or conflicts of interest to
disclose.
Address for correspondence: Marc Tebruegge, MD, PhD,
Wellcome Trust Clinical Research Facility (Mailpoint
218), University Hospital Southampton NHS Founda-
tion Trust, Tremona Road, Southampton, SO16 6YD,
United Kingdom. E-mail: m.tebruegge@soton.ac.uk.
Supplemental digital content is available for this article.
Direct URL citations appear in the printed text and
are provided in the HTML and PDF versions of this
article on the journals website (www.pidj.com).
Copyright 2015 Wolters Kluwer Health, Inc. All
rights reserved.
ISSN: 0891-3668/15/3409-1014
DOI: 10.1097/INF.0000000000000796
swered questions, and the search for accurate
diagnostic tests is far from over. This review
provides an overview of existing immuno-
logical and microbiological tests for TB, with
particular focus on their strengths and limita-
tions, and discusses novel methods and direc-
tions for future research.
THE EPIDEMIOLOGY OF
CHILDHOOD TUBERCULOSIS
The true global incidence and preva-
lence of childhood TB remain uncertain,2
largely because microbiological confirma-
tion of active TB (also called TB disease) is
not obtained in the majority of children for
a number of reasons. First, children typically
have paucibacillary disease, which hampers
detection of Mycobacterium tuberculosis.
Second, respiratory samples are difficult to
obtain in young children and few healthcare
facilities are set up to obtain induced spu-
tum samples in routine practice, which have
been shown to increase detection yields.3
Consequently, worldwide the majority of
children with active TB are started on anti-
mycobacterial treatment based on history,
symptoms and clinical signswith or with-
out supporting radiological findingsalone.
Until recently, the World Health Organiza-
tion (WHO) and other public health agen-
cies made little effort to capture children
with non-microbiologically confirmed active
TB, as official figures for TB incidence were
simply based on the number of smear- and
culture-confirmed cases. The WHO Global
Tuberculosis Report in 2012 was the first to
include estimates for childhood TB. The lat-
est edition (2014) includes estimates for new
TB cases in children (an estimated 550,000
cases) and TB-related deaths in HIV-negative
children (an estimated 80,000 cases).4 Nota-
bly, the report does not include an estimate
for TB-related deaths in HIV-positive chil-
dren, which is likely to eclipse the figure in
HIV-negative children. However, the WHO
estimates are based on detection rates in
adults, and therefore likely considerably
underestimate the burden of TB in children. It
is therefore not surprising that a recent study
based on mathematical modeling arrived at
substantially higher figures, estimating the
annual global incidence of active TB in chil-
dren to be greater than 650,000 cases.5
ACTIVE TUBERCULOSIS VERSUS
LATENT TUBERCULOSIS
INFECTION
Traditionally, infections with
M.tuberculosis have been categorized into
active TB and latent TB infection (LTBI).
Patients with active TB typically have symp-
toms and/or signs, which depend on the site
of the infection, and the infection may be
confirmed by conventional (ie, microscopy
or culture) or molecular (eg, PCR) micro-
biological methods (depending on the infec-
tion site, adequacy of the sample and the
methods used). In contrast, classical dogma
suggests that patients with LTBI are asymp-
tomatic, and that their immune system is

The ESPID Reports and Reviews of Pediatric Infectious Diseases series topics, authors and contents are chosen and approved
independently by the Editorial Board of ESPID.

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The Pediatric Infectious Disease Journal Volume 34, Number 9, September 2015 Diagnostic Tests for Childhood Tuberculosis
containing M. tuberculosis. Consequently,
current microbiological methods are unable
to detect the mycobacteria, and the diagnosis
of LTBI is therefore solely based on immune-
based tests that detect memory T cells (and
potentially other immune cells) induced by
exposure to antigens expressed by M. tuber-
culosis [ie, either tuberculin skin test (TST)
or interferon-gamma release assay (IGRA)].
The segregation between active TB and LTBI
remains useful from a clinical as well as a
programmatic perspective as this distinction
currently determines the treatment approach
(ie, treatment with 1 or 2 antimycobacterial
drugs for LTBI vs. 3 or more drugs for active
TB). However, there is increasing evidence
that active TB and LTBI are not discrete
infection states, but rather opposite ends of
a continuum. Historical data from before the
advent of antimycobacterial drugs illustrate
that some patients with active TB survive
without treatment; more recent data show
that these asymptomatic survivors maintain
TB-specific immunological memory for
decades.6 Thus, if tested with immune-based
tests (TST or IGRA) after recovery, such
individuals would currently have to be clas-
sified as LTBI.7 Furthermore, recent stud-
ies in both nonhuman primates and humans
using newer radiological methods, such as
positron emission tomography, show that
disease activity can be detected in a signifi-
cant proportion of individuals with a positive
immune-based test who are asymptomatic,
and therefore would be classified as LTBI
according to current criteria.8,9 It is uncertain
whether those individuals would convert to
overt active TB if left untreated. This ques-
tion will likely remain unanswered given that
withholding anti-mycobacterial treatment
under those circumstances would be unethi-
cal. In addition, several recent publications
have described patients with subclinical TB
disease, who are clinically asymptomatic,
but have positive sputum cultures (with or
without smear-positivity).10,11 Despite these
issues, for clarity we will continue to use the
terms active TB and LTBI in this review. to source PPD from the Statens Serum Insti-
IMMUNE-BASED TESTS AND
THEIR LIMITATIONS
Tuberculin Skin Test
Since the early 20th century, purified
protein derivative (PPD), a heterogeneous
mixture of mycobacterial peptides, has been
used as the test substance for the TST (also
called Mantoux test). The TST is commonly
used to support the presumptive diagnosis of
active TB, and was the only available test for
the detection of LTBI, until IGRA became
commercially available in 2002.
The key limitation of the TST lies in its
limited specificity. False-positive results can
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occur as a result of prior BCG immunization
or infection with nontuberculous mycobacte-
ria (NTM), as both BCG and NTM express
peptides that are present in PPD. False-
negative results can also occur as a result
of immunodeficiency, immunosuppression,
malnutrition and errors in test administration
or reading. The test requires reading after 48
to 72 hours, which is inconvenient for both
healthcare providers and patients; if reading
is not performed within this time window, the
validity of the test result becomes question-
able. Despite those limitations, the latest rec-
ommendations of the American Academy of
Pediatrics Committee on Infectious Diseases
state that for LTBI screening in children less
than 5 years of age TST should be used in
preference of IGRA.12 However, the recom-
mendations also highlight that the combined
use of TST and IGRA results in an increase in
diagnostic sensitivity.
An additional major limitation of the
TST remains the subjectivity in the read-
ing of the resulting induration, the extent of
which determines the test result. The current
standard technique for readingthe ball-
point techniqueinvolves palpating for the
outer edges of the induration, marking these
with a ballpoint pen and measuring the diam-
eter of the induration using a flexible ruler or
a caliper. This technique is prone to consid-
erable inaccuracy with both intra- and inter-
observer variability, particularly when the
induration is not circular.13 Optimization and
standardization of the reading technique to
achieve greater accuracy and reproducibility
have recently been investigated using a vari-
ety of novel approaches, including measure-
ment by Doppler imaging, spectrophotom-
etry and ultrasound.14
Another significant problem with the
TST are recurrent shortages of PPD. World-
wide, only a small number of manufactur-
ers produce PPD for clinical use, so supply
shortages at 1 manufacturer can affect the
global market. Following production prob-
lems at Evans Vaccines in 2003, the UK had
treatment is overall poor (the extent of which
tut (SSI) in Denmark as an unlicensed medi-
cine.15 In 2013, there was a national short-
age of PPD in the US caused by production
problems at Sanofi Pasteur (Tubersol) leaving
only 1 FDA-licensed product (Aplisol; JHP
Pharmaceuticals).16 Later that year, 29 of 52
US jurisdictions reported a shortage of at
least 1 of the 2 PPD products to the extent
that routine activities were being threatened
or had been curtailed.17 Canada also experi-
enced a less well-publicized PPD shortage in
late 2012.18 A further, Europe-wide shortage
of PPD was highlighted by a recent survey
of TB experts based in 23 different European
countries.19 Sixty percent of these (from 14
different countries) reported a PPD shortage
at the time the survey was conducted (June to
July 2014). The majority of those reporting a
PPD shortage were using RT23 (SSI; 81.0%);
fewer reported shortages of Tubertest (Sanofi
Pasteur; 9.5%), PPD Tuberculin (BulBio;
4.8%), and PPD Tuberculin (St. Petersburg
Institute of Vaccines and Sera; 4.8%).
Interferon-gamma Release Assays
Currently, 2 commercial IGRA are
available for clinical use, the QuantiFERON-
TB Gold In-Tube (QFT-GIT; Cellestis/Qia-
gen, Carnegie, Australia) and the T-SPOT.TB
assay (Oxford Immunotec, Abingdon, UK).
Although IGRA are solely cleared for the
diagnosis of LTBI, in clinical practice they
are commonly used to support a presumptive
diagnosis of active TB. Both assays rely on
the detection of interferon-gamma secreted
by memory T cells following stimulation with
mycobacterial antigens. Both assays incorpo-
rate the relatively M. tuberculosis-specific
RD1 peptides antigens early secretory anti-
genic target 6 (ESAT-6) and 10kDa cul-
ture filtrate protein (CFP-10); the QFT-GIT
incorporates and additional antigen, TB7.7.
Based on the test principle, IGRAs likely
have greater specificity than the TST and are
not confounded by prior BCG immuniza-
tion, as all 3 stimulatory antigens are absent
from all BCG vaccine strains in current use.
Also, the impact of current or previous NTM
infection and/or exposure on test specificity
is relatively limited, since only a small num-
ber of NTM express ESAT-6 and CFP-10.20
However, (false) positive IGRA results
have repeatedly been reported in individu-
als infected with Mycobacterium kansasii,
Mycobacterium marinum and Mycobacte-
rium szulgai.21,22
Although initially heralded as a tool
that has the potential to revolutionize the
diagnosis of TB, there are now legions of
publications highlighting the limitations of
IGRA.12,2325 First, similar to the TST, the per-
formance of IGRA in patients with immuno-
deficiencies or receiving immunosuppressive
depends on the type of the immunodeficiency
and the degree of immunosuppression), and
false-negative assay results remain a sig-
nificant problem in these patient groups.26,27
This problem is further compounded by
those patients also being at greatest risk of
progression from LTBI to active TB, making
the accurate identification of latent infection
in those individuals crucially important.12,25
As there is no gold standard for the diagno-
sis of LTBI (with the TST having previously
been regarded as the gold standard) the true
sensitivity of IGRA for the detection of LTBI
cannot be determined. Nevertheless, several
robust meta-analyses on the performance of
IGRA as a supportive tool for the diagnosis
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Tebruegge et al The Pediatric Infectious Disease Journal Volume 34, Number 9, September 2015
of active TB have shown that IGRA perform
no better than the TST in this setting, report-
ing a pooled sensitivity of approximately
6080% in immunocompetent individuals,
and even lower pooled estimates in HIV-
infected patients.28,29
There is now a considerable body
of evidence that the performance of IGRA
is worse in young children compared with
adults.12 Although the precise underlying
mechanisms remain uncertain, it is likely that
incomplete immune maturation plays a sig-
nificant role.30 Indeterminate IGRA results
(resulting from failed negative or positive
control samples), which convey no informa-
tion regarding the TB infection status of the
patient, are significantly more common in
young children than in adults.26,30,31 One pedi-
atric study reported that indeterminate QFT-
GIT results occurred in 83 (35%) of the 237
study participants.26 However, in that study,
a large number of children were immunode-
ficient or were receiving immunosuppressive
medication. The majority of pediatric studies
in countries with low HIV prevalence have
reported lower rates of indeterminate IGRA
results, generally ranging between 5% and
20%.3234 In addition to young age and immu-
nodeficiency/immunosuppression, further
factors that have been found to be associated
with indeterminate IGRA results include
malnutrition, chronic renal disease, autoim-
mune conditions, malaria and co-existing
helminth infections.35,36 Preanalytical sources
of assay variability that can result in indeter-
minate assay results include delays in sample
incubation and inadequate shaking/mixing of
QFT-GIT tubes.37,38
Further limitations of IGRA include
their relatively high cost and the need for ade-
quate laboratory facilities, which precludes
their use in many resource-limited, high TB
prevalence settings. In addition, several studies
have shown that the reproducibility of IGRA
is suboptimal when serial testing is performed
(ie, with unexplained conversions from posi-
tive to negative and vice versa).12 Furthermore,
while there is a large amount of longitudinal
data related to the TST and its predictive value
for the development of active TB, those data in
relation to IGRA, particularly in children, still
remain relatively limited.25
Discordance Between TST and
IGRA Results
Many pediatric TB experts use the TST
and IGRA in parallel with the aim of increas-
ing sensitivity, but this can be complicated by
contradictory results. The underlying mecha-
nisms of this discordance remain uncertain. In
most pediatric studies, the number of children
with a TST+/IGRA result constellation is far
greater than that with a TST/IGRA+ con-
stellation. Some authors contend that TST+/
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IGRA discordance primarily results from
prior BCG immunization or immune sensiti-
zation induced by exposure to NTM, and that
it should thus be interpreted as a false-positive
TST result (assuming the IGRA is true-neg-
ative as a result of greater specificity). How-
ever, the evidence to support this conjecture
is limited. There is no doubt that BCG immu-
nization can produce (false) positive TST
results. However, an analysis of published
data that included more than 240,000 subjects
vaccinated with BCG in infancy found that,
overall, only 8.5% had a positive TST result
(here defined as 10mm induration) that was
attributable to the vaccine, while only 1%
were TST-positive when tested 10 years or
more after vaccination with BCG.39 Therefore,
BCG immunization alone cannot account for
the large proportion of children with TST+/
IGRA discordance reported by the majority
of pediatric studies, which typically ranges
between 10% and 40% of the study popula-
tion.26,3234 There is also no doubt that NTM
disease produces positive TST results in a
substantial proportion of patients.40 Neverthe-
less, NTM disease is rare with most publica-
tions estimating the incidence in children to be
less than 10 per 100,000 children per year.41,42
Therefore, NTM disease can also not account
for the substantial proportion of individuals
with TST+/IGRA discordance in pediatric
studies. The concept that environmental NTM
exposure alone can induce memory T cells and
thereby produce false-positive TST results is
unproven, and the experimental data to sup-
port this theory are unconvincing.24 NTM are
ubiquitous and the rate of isolation of NTM
from environmental sources is identical world-
wide.43 Despite this, the vast majority of indi-
viduals living in low TB prevalence countries
are TST-negative,44,45 strongly suggesting that
NTM exposure alone has limited impact on
TST results.
The alternative explanation for TST+/
IGRA discordance is that the TST result
is true-positive, while the IGRA result is
false-negative (as a result of the latter hav-
ing inferior sensitivity). Notably, recent data
provide strong evidence that a significant
proportion of individuals with TST+/IGRA
discordance are in fact latently infected with
M. tuberculosis.46 Using whole blood assays
with ESAT-6 and CFP-10 as stimulatory anti-
gens, pro-inflammatory cytokine responses
(including interferon-gamma, IP-10, TNF-
alpha, IL-2, IL-12(p40) and IL-13 responses)
were found to be significantly higher in a
group of children with TST+/IGRA dis-
cordance compared with a group of TB-
uninfected children. This observation can not
be explained by confounding caused by prior
BCG vaccination, since neither ESAT-6 nor
CFP-10 are expressed by any of the currently
used BCG vaccine strains.
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In the light of these uncertainties, in
a child with a TST+/IGRA result constel-
lation, the possibility of latent TB cannot be
reliably ruled out. Consequently, we, as well
as other TB experts, strongly recommend that
children with risk factors for TB infection
(including known TB exposure or birth in a
high TB prevalence country) with this dis-
cordant result constellation should be offered
preventive treatment until such time that bet-
ter immunodiagnostic assays that can reliably
rule out latent infection with M. tuberculosis
become available.47,48 This recommenda-
tion takes into account that children are at
far greater risk of progression from LTBI
to active TB over their lifetime compared
with adults, and the fact that serious adverse
events with standard preventive treatment
(ie, isoniazid alone or in combination with
rifampicin) are very rare in children.
Molecular Assays for Tuberculosis
Currently, there are a considerable
number of commercial polymerase chain
reaction (PCR)-based assays available for
the detection of M. tuberculosis, some of
which incorporate testing for the presence
of drug resistance genes, including the
COBAS TaqMan MTB (Roche Diagnostics,
Basel, Switzerland), the ProbeTec ET Direct
TB (Becton Dickinson, Franklin Lake, NJ),
the FluoroType MTB (Hain Lifescience,
Nehren, Germany), the m2000 RealTime
MTB (Abbott Laboratories, North Chicago,
IL) and the Xpert MTB/RIF assay (Cep-
heid, Sunny Vale, CA). Following the official
endorsement by the WHO in 2010, attention
has focused on the latter assay. By September
2014, a total of 3553 Xpert instruments and
almost 9 million Xpert MTB/RIF cartridges
had been procured by countries eligible for
concessional pricing (ie, low- and medium-
resource countries; see http://who.int/tb/labo-
ratory/GeneXpert_rollout_large.jpg).
The Xpert MTB/RIF assay is based
on a qualitative, nested real-time PCR that
allows M. tuberculosis complex to be directly
detected in clinical samples, and can simul-
taneously detect mutations in the rpoB gene
associated with rifampicin resistance. The
assay has a number of advantages: it can be
operated by personnel with minimal training,
the sample preparation required is minimal,
and the test result is available within 2 hours
once the assay is initiated.49 However, disad-
vantages include the cost of the cartridges
(currently $9.98 at concessional pricing),
the need for a laboratory infrastructure with
reliable power supply and air conditioning,
and the need for regular maintenance of the
analytical instruments, all of which pose con-
siderable challenges in low-resource settings.
Also, the assay cannot distinguish between
live and dead M. tuberculosis, and can
The Pediatric Infectious Disease Journal Volume 34, Number 9, September 2015 Diagnostic Tests for Childhood Tuberculosis
therefore not be used to confirm treatment
success or failure, or for the identification of
relapse. The costeffectiveness of the assay
remains uncertain, but it appears obvious that
its implementation and scale-up in countries
with an annual health expenditure of $1020
per capita represents a major challenge.49
A meta-analysis of early studies
assessing the performance of the Xpert MTB/
RIF assay reported that in pulmonary TB, the
overall pooled sensitivity of the assay was
90.4% (95% confidence interval: 89.291.4),
with a pooled specificity of 98.4% (98.0
98.7).50 However, disappointingly, in smear-
negative pulmonary TB (which applies to the
majority of children) the pooled sensitivity
was only 75.0%. It is therefore not surprising
that studies in children with pulmonary TB
have generally reported sensitivities ranging
between 65% and 76%, although universally
with specificities above 98%.49,5153 While this
yield represents a 2- to 3-fold improvement
over sputum smear microscopy, these data
also highlight that a quarter to one-third of
pediatric cases would elude microbiological
confirmation if the Xpert MTB/RIF assay
was used as a replacement for mycobacte-
rial cultures, which currently remain the gold
standard in active TB.
Availability and Current Use of
Immunological and Molecular
Assays
Data on the availability and use of
immunological and molecular assays in the
routine clinical setting are surprisingly lim-
ited. A recent survey provides some insight
into the current European landscape.54 The
vast majority (93.6%) of participating Euro-
pean TB experts (based in 31 different coun-
tries) had access to PCR-based TB assays.
Approximately two-thirds had access to com-
mercial and the remaining one-third to non-
commercial (in-house) PCR-based assays.
Interestingly, the survey found that a large
proportion of participants had used the Xpert
MTB/RIF assay for the analysis of a variety
of nonrespiratory samples (including pleu-
ral fluid, gastric aspirate fluid, cerebrospinal
fluid, stool samples and blood/serum) despite
the assay having been optimized and solely
being licensed for the analysis of sputum
samples.49 Although increasing data suggest
that the Xpert MTB/RIF assay has relatively
high sensitivity with cerebrospinal fluid and
biopsy samples, recent reports highlight the
assays suboptimal performance with stool
samples, and pleural, peritoneal and joint
fluids.54,55 Furthermore, the survey of Euro-
pean TB experts found that IGRA were also
widely available across Europe, and that
far more had access to the QFT-GIT than
to the T-SPOT.TB assay (84.7% vs. 52.2%,
respectively). This may reflect the practical
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Copyright 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
difficulties of integrating an ELISPOT-based
assay, such as the T-SPOT.TB assay, into the
routine diagnostic laboratory setting.
Novel Methods for the Diagnosis
of Tuberculosis
A detailed account of the current stage
of development of novel TB diagnostics is
outside the scope of this review, but can be
found in a recent UNITAID report.56 Table,
Supplemental Digital Content 1, http://links.
lww.com/INF/C183 provides an overview of
existing tests for TB, tests that are currently
in development for commercial use, and
diagnostic methods that are currently only
available in the research setting. The require-
ments for new diagnostic tools for TB and
the pathways of progression from the proof-
of-principle stage to adoption in the clinical
setting and in public health programs are out-
lined elsewhere.57
The SSI is currently developing a new
skin test, the C-Tb test, which is based on
recombinant ESAT-6 and CFP-10 and there-
fore likely to achieve greater specificity than
the TST. A phase I trial (TESEC-01) shows
that the test is well tolerated and that adverse
events are rare.58 Data from the phase I dose
finding trial (adults with confirmed and prob-
able active TB; n = 38) and the phase II trial
(healthy, BCG-vaccinated adults; n = 151)
suggest that the C-Tb test has greater speci-
ficity (>95%) than the TST, but only limited
sensitivity in active TB (17/24 individuals
tested with a dose of 0.1 g of C-Tb posi-
tive at the 5mm induration cut-off; 71%).59
The results of a follow-on study (TESEC-
04), which included 251 participants, 100
of which were HIV co-infected, will soon be
available (personal communication; Dr Sren
Tetens Hoff, SSI). Further studies (TESEC-
05/-06/-07) in more than 3000 participants
(including 722 children) have concluded
recently (personal communication; STH).
These preliminary results indicate that the
C-Tb test will likely have greater specificity
than the TST, but will have similar issues in
relation to the subjectivity of the test result
reading and interpretation. Nevertheless, the
key advantages of the C-Tb test are that it can
be performed at the primary care/community
level without the need for laboratory infra-
structure, that healthcare professionals are
familiar with the test method (as it is identi-
cal to the TST), and that the anticipated costs
are lower than those of IGRA-based testing.
The manufacturer of the QFT-GIT
assay (Cellestis/Qiagen) has recently released
the fourth generation of the assay, called
QuantiFERON-TB Gold Plus, in some Euro-
pean countries, with releases in further coun-
tries planned for the second half of 2015. As
of March 2015, the FDA approval for the
assay is still pending. The assay comprises 4
tubes (2 antigen-stimulated tubes, 1 positive
and 1 negative control tube). According to the
package insert, the antigen-stimulated tubes
contain only ESAT-6 and CFP-10, while TB
7.7which forms part of the current ver-
sion of the assayhas been removed. The
manufacturer claims that the performance
of the first antigen-stimulated tube will be
similar to the existing assay, while the sec-
ond tube will primarily determine CD8+ T
cell responses, with the aim of improving
assay sensitivity in patients with active TB.
Currently, there are no peer-reviewed publi-
cations on this new assay. Data included in
the package insert suggest that the increase
in assay sensitivity resulting from inclusion
of the second antigen-stimulated tube will
be marginal; in the evaluation studies, which
included 174 individuals with culture-con-
firmed TB, the overall increase in assay sen-
sitivity (compared with use of the first tube
alone) resulting from the inclusion of the sec-
ond tube was only 1.7%. The package insert
and product presentations indicate that the
assay has not been validated in children. An
additional challenge for use of the assay in
the pediatric setting is that the assay requires
a larger blood volume (4mL, instead of 3mL
for the QFT-GIT).
The current commercially available
TB antibody-based (serological) tests have
highly variable sensitivity and specificity,
and the WHO therefore advises against their
use.60 Notably, the WHO has encouraged
the development of more robust antibody-
based assays, and promising new multiplexed
assays are currently in development.
Recent studies using a variety of flow
cytometric methods for the detection of M.
tuberculosis-specific T cells have also shown
considerable promise. Data from 1 study in
adults suggest that analysis of polyfunctional
T cells (ie, T cells producing more than 1
cytokine) may allow the distinction between
LTBI and active TB based on a blood test
alone.61 A more recent study in children high-
lights the diagnostic potential of T cell acti-
vation marker analysis (based on CD27).62
However, the existing data for both assays
are currently limited, and translation of these
methods into a routine diagnostic setting in
the near future appears challenging consid-
ering their current cost and complexity. In
contrast, assays based on the detection of
cytokine response signatures in supernatants
following stimulation with M. tuberculosis-
specific antigens (ie, based on similar princi-
ples as IGRA) are more likely to be translated
into the routine diagnostic setting, as this can
be achieved with much simpler and more
robust methods. A small number of stud-
ies provide promising data suggesting that
these assays may also enable the distinction
between LTBI and active TB.46,6365
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Tebruegge et al The Pediatric Infectious Disease Journal Volume 34, Number 9, September 2015
A recent high-profile publication
highlights the potential of using host RNA
expression for the diagnosis of TB.66 The
investigators identified a 51-transcript signa-
ture that achieved a sensitivity of 83% and
specificity of 84%. However, this approach
currently requires complex technology and
it is uncertain whether this method can be
downscaled for use in a routine diagnostic
setting.
Assays based on the detection of
lipoarabinomannan (LAM) in sputum or
in urine have been extensively evaluated in
the research setting. Recently a commercial
test, the Determine TB LAM Ag (Alere,
Waltham, MA), has become available. LAM-
based urine tests generally have insufficient
sensitivity (5060%) to be used as rule-
out tests, but perform marginally better in
HIV-infected patients with low CD4 T cell
counts.67 Adenosine deaminase-based assays
can only be used for the diagnosis of certain
forms of extrapulmonary TB (TB meningitis,
pleuritis, pericarditis and ascites), and have
been shown to have limited sensitivity (typi-
cally 5070%).68
The recent detection of characteris-
tic volatile organic compounds in the breath
of patients with pulmonary TB has opened
promising new avenues.69 Its noninvasiveness
makes this approach attractive, but it is likely
that this method will require relatively expen-
sive instruments and perform worse in indi-
viduals with paucibacillary disease (includ-
ing the majority of children). Also, volatile
organic compound-based tests will not have
the ability to detect extrapulmonary TB.
DIRECTIONS FOR FUTURE
RESEARCH
This review highlights that despite
considerable advances in recent years, the
search for TB tests with better performance
characteristics must continue. Existing
diagnostic tests have suboptimal sensitiv-
ity, and generally perform worse in children
compared with adults. Novel tests should
particularly aim to achieve greater sensitiv-
ity, and would ideally be breath-, blood- or
urine-based, as these samples can be obtained
relatively easily from children. To be useful
in settings in which childhood TB is most
prevalent, tests should ideally be point-of-
care assays, without the need for an exist-
ing laboratory infrastructure.70 Furthermore,
a test that can reliably distinguish between
LTBI and active TB is highly desirable, as
this would help to guide management deci-
sions. Future tests for LTBI should also have
a better predictive value for the develop-
ment of active TB, given that the predictive
values of both TST and IGRA are compara-
tively low. As new tests are developed, it is
crucially important that their performance
1018|www.pidj.com
Copyright 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
characteristics are determined in sufficiently
large studies in children, in light of the fact
that existing tests almost universally perform
worse in this patient group. Finally, further
research is needed to determine how existing
diagnostic approaches, in particular the use
of the Xpert MTB/RIF assay in the routine
clinical setting, impact on patient care and
ultimately outcomes.
REFERENCES
1. Shingadia D. The diagnosis of tuberculosis.
Pediatr Infect Dis J. 2012;31:302305.
2. Seddon JA, Shingadia D. Epidemiology and dis-
ease burden of tuberculosis in children: a global
perspective. Infect Drug Resist. 2014;7:153165.
3. Zar HJ, Hanslo D, Apolles P, et al. Induced spu-
tum versus gastric lavage for microbiological
confirmation of pulmonary tuberculosis in infants
and young children: a prospective study. Lancet.
2005;365:130134.
4. World Health Organization. Global Tuberculosis
Report 2014. Available at: http://apps.who.int/
iris/bitstream/10665/137094/1/9789241564809_
eng.pdf Accessed March 5, 2015.
5. Dodd PJ, Gardiner E, Coghlan R, et al. Burden of
childhood tuberculosis in 22 high-burden coun-
tries: a mathematical modelling study. Lancet
Glob Health. 2014;2:e453e459.
6. Millington KA, Gooding S, Hinks TS, et al.
Mycobacterium tuberculosis-specific cellular
immune profiles suggest bacillary persistence
decades after spontaneous cure in untreated
tuberculosis. J Infect Dis. 2010;202:16851689.
7. Ritz N, Curtis N. Novel concepts in the epide-
miology, diagnosis and prevention of childhood
tuberculosis. Swiss Med Wkly. 2014;144:w14000.
8. Kim IJ, Lee JS, Kim SJ, et al. Double-phase
18F-FDG PET-CT for determination of pulmo-
nary tuberculoma activity. Eur J Nucl Med Mol
Imaging. 2008;35:808814.
9. Heysell SK, Thomas TA, Sifri CD, et al.
18-Fluorodeoxyglucose positron emission tomog-
raphy for tuberculosis diagnosis and management:
a case series. BMC Pulm Med. 2013;13:14.
10. Oni T, Burke R, Tsekela R, et al. High preva-
lence of subclinical tuberculosis in HIV-1-
infected persons without advanced immunode-
ficiency: implications for TB screening. Thorax.
2011;66:669673.
11. Robertson BD, Altmann D, Barry C, et al.
Detection and treatment of subclinical tuberculo-
sis. Tuberculosis (Edinb). 2012;92:447452.
12. Starke JR; Committee on Infectious Diseases.
Interferon- release assays for diagnosis of
tuberculosis infection and disease in children.
Pediatrics. 2014;134:e1763e1773.
13. Erdtmann FJ, Dixon KE, Llewellyn CH. Skin test-
ing for tuberculosis. Antigen and observer vari-
ability. JAMA. 1974;228:479481.
14. Yang H, Kruh-Garcia NA, Dobos KM. Purified
protein derivatives of tuberculinpast, present,
and future. FEMS Immunol Med Microbiol.
2012;66:273280.
15. Scottish Executive Health Department. Supplies
of tuberculin PPD for Mantoux testing.
Available at: http://www.sehd.scot.nhs.uk/cmo/
CMO(2005)6.pdf. Accessed March 5, 2015.
16. Centers for Disease Control and Prevention.
National shortage of purified-protein derivative
tuberculin products. MMWR Morb Mortal Wkly
Rep. 2013;62:312.
2015 Wolters Kluwer Health, Inc. All rights reserved.
17. Centers for Disease Control and Prevention.
Extent and effects of recurrent shortages of puri-
fied-protein derivative tuberculin skin test antigen
solutions - United States, 2013. MMWR Morb
Mortal Wkly Rep. 2013;62:10141015.
18. Manitoba Health Communicable Disease Control.
Current shortage of tubersol for administering the
tuberculin skin test. Available at: www.gov.mb.ca/
health/publichealth/cdc/docs/hcp/2012/121101.
pdf. Accessed February 15, 2015.
19. Tebruegge M, Bogyi M, Soriano-Arandes A, et al;
Paediatric Tuberculosis Network European Trials
Group. Shortage of purified protein derivative for
tuberculosis testing. Lancet. 2014;384:2026.
20. Andersen P, Munk ME, Pollock JM, et al. Specific
immune-based diagnosis of tuberculosis. Lancet.
2000;356:10991104.
21. Tebruegge M, Connell T, Ritz N, et al.
Mycobacterium marinum infection following
kayaking injury. Int J Infect Dis. 2010;14(Suppl
3):e305e306.
22. Kobashi Y, Mouri K, Yagi S, et al. Clinical evalu-
ation of the QuantiFERON-TB Gold test in
patients with non-tuberculous mycobacterial dis-
ease. Int J Tuberc Lung Dis. 2009;13:14221426.
23. Tebruegge M, Connell T, Curtis N. Tuberculosis
in children. N Engl J Med. 2012;367:1568.
24. Tebruegge M, Connell T, Ritz N, et al. Discordance
between TSTs and IFN-gamma release assays:
the role of NTM and the relevance of mycobacte-
rial sensitins. Eur Respir J. 2010;36:214215.
25. Pai M, Denkinger CM, Kik SV, et al. Gamma
interferon release assays for detection of
Mycobacterium tuberculosis infection. Clin
Microbiol Rev. 2014;27:320.
26. Haustein T, Ridout DA, Hartley JC, et al. The
likelihood of an indeterminate test result from a
whole-blood interferon-gamma release assay for
the diagnosis of Mycobacterium tuberculosis infec-
tion in children correlates with age and immune
status. Pediatr Infect Dis J. 2009;28:669673.
27. Clifford V, Zufferey C, Germano S, et al.
The impact of anti-tuberculous antibiotics
and corticosteroids on cytokine production
in QuantiFERON-TB Gold In Tube assays.
Tuberculosis (Edinb). 2015;95:343349.
28. Sester M, Sotgiu G, Lange C, et al. Interferon-
release assays for the diagnosis of active tubercu-
losis: a systematic review and meta-analysis. Eur
Respir J. 2011;37:100111.
29. Metcalfe JZ, Everett CK, Steingart KR, et al.
Interferon- release assays for active pulmo-
nary tuberculosis diagnosis in adults in low- and
middle-income countries: systematic review
and meta-analysis. J Infect Dis. 2011;204(Suppl
4):S1120S1129.
30. Tebruegge M, de Graaf H, Sukhtankar P, et al.
Extremes of age are associated with indetermi-
nate QuantiFERON-TB gold assay results. J Clin
Microbiol. 2014;52:26942697.
31. Connell TG, Tebruegge M, Ritz N, et al. Indeterminate
interferon-gamma release assay results in children.
Pediatr Infect Dis J. 2010;29:285286.
32. Connell TG, Ritz N, Paxton GA, et al. A three-
way comparison of tuberculin skin testing,
QuantiFERON-TB gold and T-SPOT.TB in chil-
dren. PLoS One. 2008;3:e2624.
33. Connell TG, Curtis N, Ranganathan SC, et
al. Performance of a whole blood interferon
gamma assay for detecting latent infection with
Mycobacterium tuberculosis in children. Thorax.
2006;61:616620.
34. Cruz AT, Geltemeyer AM, Starke JR, et
al. Comparing the tuberculin skin test and
The Pediatric Infectious Disease Journal Volume 34, Number 9, September 2015 Diagnostic Tests for Childhood Tuberculosis
T-SPOT.TB blood test in children. Pediatrics.
2011;127:e31e38.
35. Jeong SJ, Han SH, Kim CO, et al. Predictive fac-
tors for indeterminate result on the QuantiFERON
test in an intermediate tuberculosis-burden coun-
try. J Infect. 2011;62:347354.
36. Thomas TA, Mondal D, Noor Z, et al. Malnutrition
and helminth infection affect performance of
an interferon gamma-release assay. Pediatrics.
2010;126:e1522e1529.
37. Herrera V, Yeh E, Murphy K, et al. Immediate
incubation reduces indeterminate results for
QuantiFERON-TB Gold in-tube assay. J Clin
Microbiol. 2010;48:26722676.
38. Gaur RL, Pai M, Banaei N. Impact of blood vol-
ume, tube shaking, and incubation time on repro-
ducibility of QuantiFERON-TB gold in-tube
assay. J Clin Microbiol. 2013;51:35213526.
39. Farhat M, Greenaway C, Pai M, et al. False-
positive tuberculin skin tests: what is the absolute
effect of BCG and non-tuberculous mycobacte-
ria? Int J Tuberc Lung Dis. 2006;10:11921204.
40. Tebruegge M, Curtis N. Mycobacterium spe-
cies non-tuberculosis. In: Long S, Pickering
LK, Prober CG, eds. Principles and Practice
of Pediatric Infectious Diseases. 4th ed.
Philadelphia, PA: Saunders; 2012.
41. Winje BA, Oftung F, Korsvold GE, et al. School
based screening for tuberculosis infection in
Norway: comparison of positive tuberculin skin
test with interferon-gamma release assay. BMC
Infect Dis. 2008;8:140.
42. Blyth CC, Best EJ, Jones CA, et al.
Nontuberculous mycobacterial infection in chil-
dren: a prospective national study. Pediatr Infect
Dis J. 2009;28:801805.
43. Falkinham JO 3rd. Nontuberculous myco-
bacteria in the environment. Clin Chest Med.
2002;23:529551.
44. Larsson LO, Bentzon MW, Lind A, et al. Sensitivity
to sensitins and tuberculin in Swedish children.
Part 5: a study of school children in an inland rural
area. Tuber Lung Dis. 1993;74:371376.
45. Hansen KN, Heltberg I, Hjelt K. Sensitivity to
tuberculin and sensitins from atypical mycobac-
teria (M. chelonae subsp. abscessus, M. avium,
M. intracellulare, M. scrofulaceum) in 100 Danish
school children. Dan Med Bull. 1989;36:399401.
46. Tebruegge M, Dutta B, Forbes B, et al.
Mycobacteria-specific cytokine responses detect
TB infection and distinguish latent from active
TB. Am J Respir Crit Care Med. [published
2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
online ahead of print June 1, 2015]. doi:10.1164/
rccm.201501-0059OC.
47. Connell T, Tebruegge M, Ritz N, et al. Interferon-
gamma release assays for the diagnosis of tuber-
culosis. Pediatr Infect Dis J. 2009;28:758759.
48. Connell TG, Tebruegge M, Ritz N, et al. The
potential danger of a solely interferon-gamma
release assay-based approach to testing for latent
Mycobacterium tuberculosis infection in chil-
dren. Thorax. 2011;66:263264.
49. Lawn SD, Mwaba P, Bates M, et al. Advances
in tuberculosis diagnostics: the Xpert MTB/RIF
assay and future prospects for a point-of-care test.
Lancet Infect Dis. 2013;13:349361.
50. Chang K, Lu W, Wang J, et al. Rapid and effective
diagnosis of tuberculosis and rifampicin resist-
ance with Xpert MTB/RIF assay: a meta-analysis.
J Infect. 2012;64:580588.
51. Nicol MP, Workman L, Isaacs W, et al. Accuracy
of the Xpert MTB/RIF test for the diagnosis of
pulmonary tuberculosis in children admitted to
hospital in Cape Town, South Africa: a descrip-
tive study. Lancet Infect Dis. 2011;11:819824.
52. Rachow A, Clowes P, Saathoff E, et al. Increased
and expedited case detection by Xpert MTB/
RIF assay in childhood tuberculosis: a prospec-
tive cohort study. Clin Infect Dis. 2012;54:
13881396.
53. Zar HJ, Workman L, Isaacs W, et al. Rapid molec-
ular diagnosis of pulmonary tuberculosis in chil-
dren using nasopharyngeal specimens. Clin Infect
Dis. 2012;55:10881095.
54. Tebruegge M, Ritz N, Koetz K, et al. Availability
and use of molecular microbiological and immu-
nological tests for the diagnosis of tuberculosis in
europe. PLoS One. 2014;9:e99129.
55. Tortoli E, Russo C, Piersimoni C, et al. Clinical
validation of Xpert MTB/RIF for the diagnosis
of extrapulmonary tuberculosis. Eur Respir J.
2012;40:442447.
56. UNITAID. Tuberculosis diagnostics technol-
ogy and market landscape (3rd edition, 2014).
Available at: http://www.unitaid.eu/images/
marketdynamics/publications/UNITAID_
TB_Diagnostics_Landscape_3rd-edition.pdf.
Accessed March 5, 2015.
57. Pai M, Minion J, Steingart K, et al. New and
improved tuberculosis diagnostics: evidence, pol-
icy, practice, and impact. Curr Opin Pulm Med.
2010;16:271284.
58. Bergstedt W, Tingskov PN, Thierry-Carstensen B,
et al. First-in-man open clinical trial of a combined
rdESAT-6 and rCFP-10 tuberculosis specific skin
test reagent. PLoS One. 2010;5:e11277.
59. Aggerbeck H, Giemza R, Joshi P, et al.
Randomised clinical trial investigating the
specificity of a novel skin test (C-Tb) for diag-
nosis of M. tuberculosis infection. PLoS One.
2013;8:e64215.
60. Steingart KR, Henry M, Laal S, et al. Commercial
serological antibody detection tests for the diag-
nosis of pulmonary tuberculosis: a systematic
review. PLoS Med. 2007;4:e202.
61. Harari A, Rozot V, Bellutti Enders F, et al.
Dominant TNF-+ Mycobacterium tuberculo-
sis-specific CD4+ T cell responses discriminate
between latent infection and active disease. Nat
Med. 2011;17:372376.
62. Portevin D, Moukambi F, Clowes P, et al.
Assessment of the novel T-cell activation marker-
tuberculosis assay for diagnosis of active tubercu-
losis in children: a prospective proof-of-concept
study. Lancet Infect Dis. 2014;14:931938.
63. Frahm M, Goswami ND, Owzar K, et al.
Discriminating between latent and active tuber-
culosis with multiple biomarker responses.
Tuberculosis (Edinb). 2011;91:250256.
64. Sutherland JS, de Jong BC, Jeffries DJ, et al.
Production of TNF-alpha, IL-12(p40) and IL-17
can discriminate between active TB disease and
latent infection in a West African cohort. PLoS
One. 2010;5:e12365.
65. Clifford V, Zufferey C, Street A, et al. Cytokine
biomarkers for monitoring anti-tuberculous ther-
apy: a systematic review. Tuberculosis (Edinb).
2015;95:217228.
66. Anderson ST, Kaforou M, Brent AJ, et al; ILULU
Consortium; KIDS TB Study Group. Diagnosis of
childhood tuberculosis and host RNA expression
in Africa. N Engl J Med. 2014;370:17121723.
67. Minion J, Leung E, Talbot E, et al. Diagnosing
tuberculosis with urine lipoarabinomannan: sys-
tematic review and meta-analysis. Eur Respir J.
2011;38:13981405.
68. Gui X, Xiao H. Diagnosis of tuberculosis pleurisy
with adenosine deaminase (ADA): a systematic
review and meta-analysis. Int J Clin Exp Med.
2014;7:31263135.
69. Syhre M, Chambers ST. The scent of
Mycobacterium tuberculosis. Tuberculosis
(Edinb). 2008;88:317323.
70. Pai NP, Pai M. Point-of-care diagnostics for HIV
and tuberculosis: landscape, pipeline, and unmet
needs. Discov Med. 2012;13:3545.
www.pidj.com|1019