Clinical Biochemistry 37 (2004) 450 – 455

Childhood tuberculosis and its early diagnosis

James W. Gray *
Department of Microbiology, Birmingham Children’s Hospital, Birmingham B4 6NH, UK

Received 7 November 2003; received in revised form 9 March 2004; accepted 9 March 2004

Available online 13 April 2004

Abstract

Traditional methods for laboratory diagnosis of tuberculosis are unsatisfactory, especially for children, in whose specimens mycobacteria
are usually sparse. Recent changes in tuberculosis epidemiology in developed countries, including a large increase in incidence in children
from certain ethnic minorities, have prompted interest in newer diagnostic methods. Liquid-based culture detection systems offer improved
sensitivity and speed of diagnosis, although the time taken for d etection of growth is still upwards of 1 week. Nucleic acid amplification
techniques offer more rapid results, but perform best on smear-positive samples; sensitivities may be as low as 50% in smear-negative
specimens. Although these newer techniques are widely used in some developed countries, in others, they are not perceived as offering
sufficient benefit to justify their routine use. The diagnostic accuracy of mycobacteriophage and serologic methods is insufficient to justify
their wide use even in developing countries. Despite recent developments, there is still no panacea for diagnosis of childhood tuberculosis.
D 2004 The Canadian Society of Clinical Chemists. All rights reserved.

Keywords: Childhood tuberculosis; Mycobacteriophage; Epidemiology

Introduction 66% of tuberculosis cases in the UK now occur in non-

Worldwide, there are an estimated 8 million new cases of
tuberculosis each year [1]. Not only is the global burden of
tuberculosis rising, but resistance to first-line antitubercu-
lous drugs is also increasing [2]. The highest incidences of
tuberculosis are in sub-Saharan Africa, much of Asia, and in
some Eastern European countries, where rates typically
exceed 100 per 100 000 population [2]. Developing coun-
tries account for over 90% of the tuberculosis burden.
Twenty-two of these countries, including India, Pakistan,
South Africa, and Brazil, account for 80% of all worldwide
cases each year, and are designated by the World Health
Organisation as high-burden countries [2]. In most high-
income developed countries, the overall incidence of tuber-
culosis is relatively static at 10 per 100 000 or less [2].
However there has been a significant change in the distri-
bution of disease in these countries, with ethnic minority
groups accounting for an increasing proportion of cases:
* Department of Microbiology, Birmingham Children’s Hospital,
Steelhouse Lane, Birmingham B4 6NH, UK. Fax: +44-121-333-9811.
E-mail address: jim.gray@bch.nhs.uk.
white ethnic groups [3].
Overall, children are believed to account for 5– 15% of the
total worldwide tuberculosis disease burden [4], but they may
account for a much higher proportion of cases in developing
countries [4]. The clinical presentation of childhood tuber-
culosis differs in developed and developing countries. In the
former, a substantial proportion of cases are asymptomatic
and are only diagnosed through contact investigation of
adults: in a recent Scottish study, only 60% of children
treated for tuberculosis had clinical disease [5]. In developing
countries, there is a greater incidence of symptomatic child-
hood tuberculosis [6]. The incidence and severity of child-
hood tuberculosis in some of these countries is compounded
by coexisting diseases, especially malnourishment and HIV
infection [7]. We need to be alert to the possibility that the
changing epidemiology of tuberculosis in developed coun-
tries will lead to an increase in the incidence of childhood
tuberculosis, and to a change in clinical presentation with a
greater number of symptomatic cases. In England and Wales,
there is as yet no evidence of a sustained overall increase in
childhood tuberculosis, with incidences in children aged
under 15 years of 3.3 and 3.6 per 100 000 in 1998 and
1998. However, these figures mask an increase in the

0009-9120/$ - see front matter D 2004 The Canadian Society of Clinical Chemists. All rights reserved.
doi:10.1016/j.clinbiochem.2004.03.003
 J.W. Gray / Clinical Biochemistry 37 (2004) 450–455
incidence of tuberculosis in children of black African ethnic
origin from 15 to 71 per 100 000 over the same period [8].
In all countries, the diagnosis of childhood tuberculosis is
often made without laboratory confirmation. In developing
countries, up to 90% of symptomatic cases are diagnosed
solely from clinical history and examination, and tuberculo-
sis score charts have been widely used to assist in this
process [9,10]. However, increasing numbers of malnour-
ished and HIV-positive children have compromised the value
of such scoring systems [9]. Non-laboratory investigations
have a limited role in the diagnosis of childhood tuberculo-
sis. Tuberculin skin testing (e.g., the Mantoux test) gives
negative results in up to 40% of children with tuberculosis,
and radiographic findings are often non-specific [11].
Reliable diagnosis of tuberculosis depends on laboratory
confirmation, but traditional diagnostic methods have long
been recognised to be less than optimal. In recent years,
several new diagnostic techniques have been developed to
improve the speed and reliability of tuberculosis diagnosis.
However, none of these has proven to be a diagnostic
panacea. Another consideration is that with the decline in
incidence of tuberculosis in the indigenous children of many
industrialised countries, clinicians’ expectations of tubercu-
losis testing are changing. Now, the need is often to exclude
tuberculosis from the differential diagnosis in low-risk
patients, so that drug therapy can confidently be withheld
or discontinued. A national audit found that only 3.3% of
primary samples submitted for mycobacterial diagnosis in
the UK produced a positive mycobacterial culture [12], and
in the author’s own laboratory, fewer than 1% of specimens
investigated for tuberculosis are positive.
The purpose of this article is to review the state-of-the-art
position on diagnosis of childhood tuberculosis.
Microbiological diagnosis of tuberculosis
The mainstays of diagnosis of tuberculosis remain mi-
croscopy, and culture on solid and liquid media. However,
neither microscopy nor culture offers good sensitivity. This
is a particular problem for pediatric microbiology laborato-
ries because the bacterial load in childhood tuberculosis is
substantially lower than that in post-primary tuberculosis in
adults. The problem of the relative paucity of mycobacteria
in pediatric samples is compounded by the difficulty in
obtaining good quality clinical samples of satisfactory vol-
ume, especially from younger children who are unable to
produce sputum [13]. Microscopy can provide same-day
preliminary information, but cannot distinguish between
Mycobacterium tuberculosis and other Mycobacterium spe-
cies. In adults, microscopy has the added advantage of
providing a measure of infectivity, but this observation
may be less important in pediatrics, where secondary cases
in contacts of children with smear-positive respiratory secre-
tions or gastric aspirates appear to be rare [14]. With
traditional culture methods, the time taken for isolation,
451
identification, and drug susceptibility testing of M. tubercu-
losis is typically 4 – 6 weeks, although cultures should be
maintained for up to 12 weeks before being reported as
negative [15]. However, the US Centers for Disease Control
have recommended that M. tuberculosis should be identified,
and first-line drug susceptibility testing completed, within 30
days of specimen collection [16]. Recent developments in
the laboratory diagnosis of tuberculosis have attempted, so
far with only limited success, to address the poor sensitivity
and slowness of traditional diagnostic methods.
Specimens
Almost all laboratory techniques for diagnosis of tuber-
culosis depend upon detection of mycobacteria. Therefore,
obtaining good quality specimens, containing the highest
possible numbers of mycobacteria, is central to the diagno-
sis of tuberculosis. Multiple samples will improve the
diagnostic yield, and specimen volumes should be as large
as possible, especially from sites such as CSF where myco-
bacteria are especially scanty.
Pulmonary tuberculosis
For childhood pulmonary tuberculosis, the best specimen
remains early morning gastric aspirates. However, the
sensitivity is still poor (<50% in most hands, and 33% in
one recent UK report) [5]. In theory, flexible fibreoptic
bronchoscopy might be expected to deliver good quality
specimens, however, in practice, it has proved inferior to
gastric aspirates [13]. However, bronchoscopy can be clin-
ically useful, especially in guiding the use of steroid therapy
for children whose chest radiographs are not suggestive of
bronchial involvement [17].
Pleurisy
In tuberculous pleurisy biochemical analysis of the
pleural fluid is indicative of a mild exudate, with a protein
concentration of 20 – 40 g/l. There are several thousand
leucocytes/mm3, with an early predominance of polymorphs
giving way to a later lymphocytosis. Smears are rarely
positive, because of the paucity of mycobacteria in the
fluid, and for the same reason, cultures are only positive
in 15 –70% of cases [18,19]. Although ZN-stained smears
of pleural biopsy material are positive in fewer than 10% of
cases, histopathological examination reveals granulomas
that are acid fast-positive in over 50% of cases, and cultures
are positive in over 90% [18].
Meningitis
The CSF leukocyte count is usually in the range of 10–
500/mm3 and lymphocytes usually predominate. The CSF
protein concentration is usually markedly elevated (4– 50 g/
l), the highest levels being associated with hydrocephalus
and spinal block. The CSF glucose concentration is reduced,
typically to 2.2 mmol/l or less [19]. The success of tests for
detection of M. tuberculosis depends on both the volume of
452 J.W. Gray / Clinical Biochemistry 37 (2004) 450–455
Table 1
Typical mean times to detection of mycobacterial growth using conven-
tional culture, the BACTEC 460TB system, and other liquid-based culture
detection systems
Type of infection Typical mean time (days) to detection of
mycobacterial growth
Conventional BACTEC Other
culture 460 systems
Smear-positive 20 – 25 9 – 13 9 – 20
tuberculosis
Smear-negative 21 – 33 15 – 22 18 – 36
tuberculosis
Non-tuberculous 22 – 40 13 – 17 12 – 25
mycobacterial
infections
CSF examined and on the techniques used. In one recent
series, microbiological confirmation was obtained in around
50% of cases, with liquid culture (positive in 34.5% of
cases) being the most sensitive method, followed by nucleic
acid amplification techniques (13.8%) and microscopy
(1.7%) [20]. Whilst comparative studies may give a fair
estimation of the overall success of microbiological diag-
nosis, they almost certainly underestimate the effectiveness
of individual techniques because smaller sample volumes
are inevitably used for individual tests.
Infection at other sites
Good-sized specimens of pus or tissue from the sus-
pected site of infection are the preferred specimens. Tissue
samples should also be referred for histopathological exam-
ination. Comprehensive information on specimen collection
from such sites has been produced by the American Tho-
racic Society and Centers for Disease Control [21].
Diagnostic techniques
Microscopy
Microscopy for mycobacteria has traditionally used the
Ziehl-Neelsen stain, which involves staining with carbol
fuchsin dye, which is retained by the mycolic acid-rich cell
wall of mycobacteria after washing with acid alcohol. The
time taken for microscopic examination can be reduced by
the use of fluorescent microscopy and staining with aur-
amine phenol. However, examination of smears stained by
either method is an insensitive technique that can only detect
5  103 AFBs/ml [15]. Another significant limitation of
these microscopic techniques is the inability to distinguish
between M. tuberculosis and other Mycobacterium species,
so that microscopy is unsuitable for examining specimens
such as urine that may be contaminated with non-pathogenic
mycobacteria. Pseudoepidemics of tuberculosis have also
been described where due to atypical mycobacteria being
present in the hospital water supply [22].
Peptide nucleic acids are DNA-like molecules in which
the sugar phosphate backbone is replaced by a peptide-like
structure. Because they are hydrophobic, they pass across
intact cell walls, and then bind to DNA or RNA in a
sequence-specific (and therefore species-specific) way. De-
spite reports of successful use of fluorescent-labelled pep-
tide nucleic acids to distinguish between species directly on
clinical material, this technology has never become widely
used in clinical practice [15].
Conventional culture
M. tuberculosis is a slow-growing and fastidious bacte-
rium that requires specialised culture media. Traditional
culture is performed using solid or liquid media, including
Lowenstein-Jensen, Kirchner, and the various Middlebrook
formulations [15]. The average time to detection of growth
on conventional culture media is 2 – 4 weeks, although
cultures are usually maintained for up to 12 weeks before
being reported as negative. Although liquid media may be
faster, manual handling of vials is cumbersome and time-
consuming. Primary isolates are then subcultured for species
identification and antibiotic susceptibility testing by pheno-
typic methods (see later), leading to a further delay of 2 to 3
weeks before a final result is available [15].
Liquid-based culture mycobacterial detection systems
It is now well established that these techniques offer
improved sensitivity and speed of detection of mycobac-
teria. The first of these was the radiometric BACTEC
460TB system (Becton Dickinson, USA), but this has
several limitations, including the need for manual loading,
potential for cross contamination related to invasive reading,
lack of computerised data management, and the accumula-
tion of low-level radioactive waste. Several non-radiometric
continuous monitoring culture systems have since been
introduced that circumvent mot or all of these drawbacks,
including the MB/BacT (Biomerieux, France), BACTEC
9000 (Becton Dickinson, USA) and the BACTEC MGIT
960 (Becton Dickinson, USA). The BACTEC MGIT 960 is
also available as a manual system for use in smaller
laboratories whose sample throughput does not justify the
high capital cost of monitoring equipment.
The relative performances of conventional culture, the
BACTEC 460 system, and other systems are outlined in
Tables 1 and 2 [15,23]. Although there are few specifically
pediatric-related data on these systems, it might be expected
Table 2
Typical recovery rates in mycobacterial infections using conventional
culture, the BACTEC 460TB system, and other liquid-based culture
detection systems
Type of infection Typical recovery rates (%) as a proportion of all
culture-positive cases
Conventional BACTEC Other
culture 460 systems
Tuberculosis 79 – 95 90 – 100 77 – 97
Non-tuberculous 33 – 60 76 – 85 58 – 80
mycobacterial
infections
 J.W. Gray / Clinical Biochemistry 37 (2004) 450–455
that they would be particularly useful in this setting because
of the need for a sensitive detection method for M. tuber-
culosis and other Mycobacterium species. Whilst the BAC-
TEC MGIT 960 system has been reported to offer
comparable sensitivity and speed of detection to the BAC-
TEC 460 in some studies [24], others have found the 460
system to be superior for detection of non-tuberculous
mycobacteria [23]. This is surprising, given the highly
enriched formulation of the MGIT medium that presumably
accounts for the high contamination rate that has been
consistently reported with the MGIT 960 [23 –25].
All of these systems are more expensive than conven-
tional culture, and even in developed countries, competition
for limited health care resources means that they have not
been adopted universally. Instead, in the UK at least, their
use has been largely limited to samples from individual
patients in whom there is a particular clinical need for a
rapid result, for example, in smear-negative clinically sus-
pected cases or where drug resistance is suspected.
Species identification and drug susceptibility testing
These final elements in the laboratory diagnosis of
tuberculosis are usually centralised at regional or national
reference laboratories. As such, detailed discussion of
developments in this area is outside the scope of this article.
Briefly, rapid genotypic methods are now widely used for
speciation, and rapid genotypic and phenotypic methods for
drug susceptibility testing, of Mycobacterium species [12].
Whilst molecular techniques are the most rapid means of
speciation, at present, their use in drug susceptibility testing
is largely restricted to screening for rifampicin resistance
(because most mutations conferring resistance to this drug
are confined to a short section of the gene encoding the h
subunit of bacterial RNA polymerase). The most widely used
phenotypic methods are based on the liquid-based culture
systems. Using these, identification of M. tuberculosis to
species level and drug susceptibility results are usually
available within 3 to 6 and 4 to 12 days, respectively [26].
Nucleic acid amplification techniques
The use of nucleic acid amplification techniques (NAATs)
is theoretically highly attractive for the diagnosis of tuber-
culosis because they offer potential for high sensitivity and
specificity as well as rapid results. Various NAATs are
commercially available, including PCR (Amplicor, Roche,
Switzerland); transcription-mediated amplification (Gen-
Probe, USA), strand displacement amplification (ProbeTec-
SDA, Becton Dickinson, USA), and ligase chain reaction
(LCx, Abbott Systems, USA). However, in practice, these
techniques have failed to fulfil some of their theoretical
promise. This compares with chlamydia diagnosis, where
NAATs rapidly established themselves as the diagnostic tests
of choice [27]. Since they were first developed, the specific-
ities of NAATs for detection of mycobacteria have improved
markedly, but remains at less than 100% [28]. However, the
most disappointing feature of NAATs has been their rela-
453
tively poor sensitivity, with positive results obtainable in
80 – 100% and 50– 95% of smear-positive and smear-nega-
tive culture-positive respiratory samples [29]. NAATs may
be even less sensitive with non-pulmonary samples [29].
DNA purification before amplification can improve sensi-
tivity, but probably at the expense of specificity [30,31]. A
nested PCR was recently reported to offer improved sensi-
tivity for detection of M. tuberculosis in children [32].
Another more straightforward means of improving sensitiv-
ity is simply to test multiple samples, presumably because
not all samples will contain detectable nucleic acid [15].
Whilst few studies have specifically evaluated the use of
NAATs in pediatrics, a metaanalysis of PCR for diagnosis of
smear-negative tuberculosis concluded that the sensitivity
and specificity of PCR compared less favourably in the six
studies that included gastric aspirates [31]. The same meta-
analysis concluded that NAATs are not consistently accurate
enough to be routinely recommended for the diagnosis of
smear-negative pulmonary tuberculosis, but that they might
be most useful for testing bronchial specimens (as opposed
to sputum or gastric aspirates) in highly suspicious TB cases
[31]. NAATs may also be sometimes useful to confirm M.
tuberculosis in smear-positive cases. Whenever and however
NAATs are used, it is important that requesting clinicians
have information on local experience of their sensitivities
and specificities to assist interpreting the significance of both
positive and negative results.
Mycobacteriophage
Many of the advances in tuberculosis diagnosis described
above are dependent on high capital cost equipment that is
inaccessible to most high-incidence countries. The FAST-
PlaqueTB (BIOTEC Laboratories, UK) is a commercial
technique that has been marketed to provide rapid diagnosis
of tuberculosis in settings where there is limited access to
diagnostic laboratories. It utilises a mycobacteriophage (a
virus that specifically infects mycobacteria) to reflect the
presence of M. tuberculosis in a specimen within 48 h.
Sensitivities of 58.3% to 87.5% in culture-positive samples
have been reported [33,34].
Serology
Despite the availability of purified and recombinant
antigens and monoclonal antibodies for competition assays,
serological testing has never been developed satisfactorily
for diagnosis of tuberculosis. A particular problem in
pediatrics is that children tend to have lower antibody titres,
making distinction from natural exposure antibodies more
difficult [35]. A number of protein and non-protein myco-
bacterial antigens have been investigated for use in antibody
detection assays. However, IgA, IgG, and/or IgM responses
to these appear to offer adequate sensitivity (29 – 75%) or
specificity (50 – 92%), either alone or in multiantigen assays,
to be useful as a confirmatory test for childhood tuberculosis
[36 –41]. Another problem is that sensitivity is markedly
lower in patients with HIV co-infection [40].
454 J.W. Gray / Clinical Biochemistry 37 (2004) 450–455
Provision of tuberculosis laboratory diagnostic services
In 1988, the World Health Organisation published defi-
nitions of peripheral, intermediate, and central tuberculosis
diagnostic laboratories [42]. The former are laboratories that
are capable of undertaking microscopy on sputum smears.
Intermediate laboratories have a greater repertoire of micro-
scopic techniques, undertake mycobacterial culture, and
undertake species identification of M. tuberculosis. Central
laboratories can identify all Mycobacterium species and
undertake antibiotic susceptibility testing. These definitions
were intended mainly for use in developing countries where
the lack of financial and infrastructure resources are major
limiting factors, and although they pre-dated the wide
commercial availability of newer diagnostic technologies,
they probably continue to represent a reasonable standard
for most developing countries to aim to attain.
In developed countries, where financial constraints are
less (but still) important, there is more scope to develop
diagnostic laboratory services according to local epidemio-
logical patterns and clinical need. For example, in areas
with a high incidence of HIV infection or multi-drug-
resistant M. tuberculosis, the need for rapid turnaround of
species identification and antimicrobial susceptibility results
may justify local testing rather than referral of isolates to a
distant reference laboratory. The US Centers for Disease
Control [43] and the American Thoracic Society [21] have
published recommendations for laboratories, which empha-
sise the timeliness of reporting, including the requirement
for a 7 days per week service. As a result, many US
laboratories have adopted the newer technologies to reduce
turnaround times for identification of M. tuberculosis and
other important species (e.g., Mycobacterium avium com-
plex) and for first-line drug susceptibility testing. In the UK,
there has been little central guidance on the provision of
mycobacterium diagnostic services, and it remains very
difficult to obtain funding to develop local diagnostic
services. Most hospital laboratories continue to rely on
microscopy and conventional culture, with investigation of
primary isolates centralised on five regional mycobacterium
reference centers [12]. Liquid-based mycobacterial detec-
tion and NAAT facilities are also mainly provided from
regional centers and are not as widely used as in the US
[12,15].
Conclusion
Despite recent developments, the laboratory diagnosis of
childhood tuberculosis remains unsatisfactory [44]. Newer
liquid-based detection systems such as the BACTEC and
MB/BacT systems offer improved sensitivity and more rapid
detection of growth than conventional culture, but the time
to reach a positive result is still measured in weeks rather
than days. NAATs can provide a same-day or next-day
result. However, the benefits of this need to be balanced
against the relatively low sensitivity (especially in speci-
mens that are negative by microscopy); the possibility of
false positives; the additional cost; and the fact that with
limited sample volumes (as is often the case in pediatrics),
use of multiple tests may actually decrease the overall
likelihood of obtaining a positive result.
Whilst newer diagnostic techniques for tuberculosis do
have some advantages over conventional methods, the
question that has to be addressed by laboratories consider-
ing their use is whether the benefits that they offer justify
the additional cost. In developed countries, where tubercu-
losis is confirmed in fewer than 5% of patients investigated,
the challenge is to determine evidence-based guidelines for
the clinical indications and specimen types at which the
newer tests should be targeted. The potential value of these
newer diagnostic techniques in developing countries with a
high incidence of symptomatic childhood tuberculosis is
clear, but these countries are unlikely to be able to provide
much of the necessary infrastructure for, or afford the
additional costs of, these tests in the foreseeable future.
Advances in the diagnosis of tuberculosis applicable to
children in high-burden countries remain frustratingly out
of reach.
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