One-third of the world's population has been infected with Mycobacterium tuberculosis, with new infections occurring in about 1% of the population each year. However 90–95% of infections remain asymptomatic. Thus early diagnosis of tuberculosis and drug resistance improves survival and helps to promote contact tracing, implementation of institutional cross-infection procedures, and other public-health actions. There have been many advances and modifications to the methodology for tuberculosis diagnosis some of which are very promising. But these advances have not kept pace with the explosion of tuberculosis or the outbreak of drug resistant tuberculosis. This review describes some of the newer advances in tuberculosis diagnostics and the challenges they face.

1.
Ne
ewer dia
agnostic
de
methods
etection
s in tuberculosis

2. Review Article
Newer diagnostic methods in tuberculosis
detection
Suneetha Narreddy a,*
, Sujit Muthukuru b
a
Infectious Diseases Consultant, Apollo Hospital, Jubilee Hills, Hyderabad, India
b
Research Assistant, Apollo Hospital, Jubilee Hills, Hyderabad, India
a r t i c l e i n f o
Article history:
Received 9 April 2014
Accepted 2 May 2014
Available online xxx
Keywords:
Tuberculosis
TB
Diagnostics
NAAT
Xpert
a b s t r a c t
One-third of the world's population has been infected with Mycobacterium tuberculosis, with
new infections occurring in about 1% of the population each year. However 90e95% of
infections remain asymptomatic. Thus early diagnosis of tuberculosis and drug resistance
improves survival and helps to promote contact tracing, implementation of institutional
cross-infection procedures, and other public-health actions. There have been many ad-
vances and modifications to the methodology for tuberculosis diagnosis some of which are
very promising. But these advances have not kept pace with the explosion of tuberculosis
or the outbreak of drug resistant tuberculosis. This review describes some of the newer
advances in tuberculosis diagnostics and the challenges they face.
Copyright © 2014, Indraprastha Medical Corporation Ltd. All rights reserved.
1. The history
Yaksma, Consumption, Romantic disease, White plague,
Scrofula, Phthisis, Pott's disease, chaky oncay; all these are
different terms used to refer to tuberculosis (TB) throughout
history. TB was discovered to have been prevalent as early as
9000 years ago1
but it was not until 1882 that the Tubercle
bacilli was first isolated by the German Physician Robert Koch
using staining techniques and forced the medical community
to accept TB as an infectious disease.2
Shortly after, a method
to culture the Tubercle bacilli was developed. Tragically the
development of TB diagnostics and their implementation
neither kept pace with the advances in medical technology
nor the calamitous explosion of TB in the wake of the Human
Immunodeficiency Virus (HIV) pandemic nor the outbreak of
multi-drug resistant tuberculosis (MDR TB). The older smear
microscopy and culture methods underwent slight modifica-
tions overtime and are still in use even today but they have
low sensitivity and more over drug susceptibility, which is the
need of the hour, is not known immediately.
2. The present diagnostic methods
Chest X-ray followed by acid-fast staining and microscopy of
the sputum sample along with culture are the most widely
used diagnostic methods today.
Chest X-ray is highly sensitive in the diagnosis of pulmo-
nary TB but is of low specificity, especially when co-existing
with HIV infection. Radiography is important in the diag-
nosis of several forms of extrapulmonary TB such as pleural,
vertebral and joint types.
* Corresponding author. Tel.: þ91 99660 22225.
E-mail address: suneethanarreddy@hotmail.com (S. Narreddy).
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.elsevier.com/locate/apme
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Please cite this article in press as: Narreddy S, Muthukuru S, Newer diagnostic methods in tuberculosis detection, Apollo
Medicine (2014), http://dx.doi.org/10.1016/j.apme.2014.05.008
http://dx.doi.org/10.1016/j.apme.2014.05.008
0976-0016/Copyright © 2014, Indraprastha Medical Corporation Ltd. All rights reserved.

3. The major drawback of sputum smear microscopy is that
it is laborious and has poor sensitivity, estimated to be
~70%.3
It's sensitivity further drops to around ~35% in some
settings with HIV co-infection and highly prevalent TB.4
This leads to increased workload, as more sputum tests
are needed per patient leading to diagnostic delay and pa-
tient loss to follow-up. The fluorescent AuramineeRhod-
amine stain is an additional armor apart from Ziehl Neelsen
acid-fast stain for smear microscopy, decreasing the time to
review the slide and improving the yield. Extrapulmonary
TB needs histopathological examination of the tissue spec-
imens; sensitivity and specificity depending on the ease of
sample collection; but the facilities and resources needed
for such methods are often unavailable in developing
countries.5
Culture can be performed using solid media, such as
LowensteineJensen (3e8 weeks), or liquid media, such as
Dubos' medium or Middlebrook 7H9 Broth etc. (10e14 days),
using the commercially available automated systems. Cul-
ture was also a prerequisite for phenotypic drug susceptibil-
ity testing until recent advances in molecular tests were
made. The longer waiting time for culture results makes it
difficult for clinicians to prove a diagnosis of TB in cases of
diagnostic doubt, especially in populations with low TB
incidence, and in the management of suspected drug-
resistant TB.
Tuberculin skin testing using purified protein derivative
(PPD) has poor sensitivity and specificity for active TB and is
used mainly to screen high risk population and diagnose
Latent TB.
3. Newer diagnostic tests e the need,
barriers and the impact they can have
With the limitation of the present day tests there is a need
for faster, user-friendly, low cost, highly specific and sen-
sitive diagnostic tests. New ways of performing “old” tests
(e.g. sputum smear microscopy) and completely innovative
tools (e.g. new technologies for molecular diagnosis) are
under investigation or have already been endorsed by
WHO.6
3.1. Optimizing smear
The strength of the smear test is its simplicity and low cost.
Thus approaches, which increase its sensitivity and reduce
the need for multiple visits will be beneficial.7,8
Practices,
which combine improved techniques with different ap-
proaches, have been endorsed by WHO to optimize yield of
microscopy.
Some of them include,
Collecting two supervised specimens in one visit (e.g. spot,
spot) instead of the age-old three early morning sputum
samples.9
Other practice which is not yet endorsed by WHO, but still
very promising, is the use of Light Emitting Diode (LED)
based microscopy as a replacement for conventional fluo-
rescent microscopy.10
3.2. Culture
The advances in culture methods are mostly in use already.
They employ the use of a liquid medium like Dubos' media,
Middlebrook 7H9 Broth, Sula's or Sauton's or Proskauer and
Beck's medium over the more traditional LowensteineJenson
medium. Not only is it more sensitive, it also reduces the delay
of drug sensitivity testing to about 10 days.
3.3. Antigen and antibody based tests
Antigen detecting test if developed into a point-of-care test
would allow for immediate diagnosis and initiation of treat-
ment. Urine samples and Breathalyzer are the most
commonly used. A urine specimen would be particularly
useful for children, who can have difficulty providing sputum.
In patients suspected of extrapulmonary TB, an antigen
detection test might prevent the use of more invasive tests.
The major and most promising antigens currently under
study are Lipoarabinomannan (LAM),11
a major glycolipid
component of the cell wall of Mycobacterium tuberculosis;
ESAT-6 (early secretory antigen target-6) and CFP-10 (culture
filtrate protein-10), both located in the RD1 (region of
difference-1) region that is lacking in BCG and in most Atypical
Mycobacteria.12
Antibodies to several antigens such as malate synthase,
TBF613,14
and cord factor are in use now, especially in the
developing countries. The accuracy of these tests has not been
found encouraging.15
There are still a large number of
commercially available serological tests in use, in developing
countries in spite of no International Guideline recommen-
dation. WHO issued a policy against these tests in 2011. The
Indian government banned antibody-based serological tests
in 2012.
3.4. Interferon-g release assays
These tests are currently used in many countries as a substi-
tute for Tuberculin test to diagnose Latent TB. These are based
on T-cell responses to antigens such as ESAT-6 and CFP-10,
which are more specific to M. tuberculosis than PPD. Presently
two such tests, in use in many countries, are the blood based
QuantiFERON-TB Gold In-Tube and T-SPOT.TB. They are
found to be more specific than the Tuberculin skin test16,17
in
diagnosing Latent TB and have good sensitivity but decreased
specificity to active TB.18
WHO has recommended against the
use of these tests for active TB diagnosis in high burden
countries, as these tests, like Mantoux, cannot separate latent
infection from active TB disease.
3.5. Molecular diagnostics
These tests use nucleic acid amplification techniques like
Polymerase Chain Reaction (PCR) for the diagnosis of TB and
drug susceptibility testing. The sensitivity of these tests has
been found to be 95% in sputum smear positive samples with
specificity around 90e100%,19
but in smear negative/culture
positive samples the sensitivities has been found to be
reduced. The main advantage of these tests, like GeneXpert, is
a p o l l o m e d i c i n e x x x ( 2 0 1 4 ) 1 e52
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4. Table 1 e Widely used and WHO endorsed tuberculosis diagnostic tests.29,30
Tests Intended use Advantages Drawbacks Comments
Chest X-ray Case detection of pulmonary TB Can diagnose other pulmonary
conditions too
Low specificity Widely in use
Acid-fast smear and
microscopy
Rapid, point-of-care test Rapid. Low cost. Minimal equipment
and training needed
Low sensitivity. Cannot detect drug
resistance and substantial quality
assurance is needed
Widely in use
Culture on solid media Confirmation of TB and drug
susceptibility testing
Good sensitivity Takes 8e12 weeks for results to
be available
Widely in use
Culture on liquid media Confirmation of TB and drug
susceptibility testing
Higher sensitivity than solid media,
takes lesser time (10e14 days); gold
standard for drug susceptibility testing
as of now
Time to detection is still slow.
Requires quality Lab environment
Endorsed by WHO
Tuberculin skin test Used to diagnose Latent TB Low cost and easy to administer Low sensitivity and specificity with
active TB, immunocompromised
host. Not recommended for active
TB. Low specificity for Latent TB
because of cross-reaction with
BCG vaccine and Nontuberculous
Mycobacteria
Widely in use
Interferon release assay Used to diagnose Latent TB More specific than Tuberculin skin
test and doesn't cross react with
BCG vaccination
Cannot differentiate active TB from
Latent TB, therefore not recommended
for active TB diagnosis. More expensive
than Tuberculin skin test
Widely in use
Serological tests Not recommended NA Highly inaccurate and expensive.
Used commercially in some developing
countries
In 2011 WHO issued a negative
policy against their use
Nucleic acid amplification
test (NAAT)
TB case detection High specificity but sensitivity lesser
than that of culture
Requires moderate training, increased
labor. Potential for cross contamination
Widely in use
Xpert MTB/RIF assay Detect TB and susceptibility
to Rifampin
Minimal processing time, high accuracy
even with some forms of extrapulmonary
TB like TB meningitis and lymphadenitis
Relatively expensive than other
conventional tests
WHO approved
Hain Genotype MTBDR
plus assay
Confirm smear positive
pulmonary TB and susceptibility
to Rifampin and Isoniazid
Minimal processing time if done singly
but usually done in batches. Highly
accurate
Poor sensitivity in extrapulmonary and
smear negative TB; expensive, requires
extensive training and stringent quality
control
WHO approved
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5. the simplicity of the fully automated machinery and that re-
sults can be obtained within a few hours.
Currently the most common commercially available NAAT
tests are the amplified Mycobacterium Tuberculosis Direct
Test (MTD, Gen-Probe) and COBAS®
TaqMan®
MTB Test
(Roche Diagnostics). Variations of the NAAT tests are
currently in use to detect drug resistance. Currently these
tests are only useful in identifying resistance to Rifampin and
Isoniazid and such tests for other anti-TB drugs are much less
developed and are currently being studied.20
Xpert MTB/RIF assay is a new test that is revolutionizing
the TB world. It detects the Tubercle bacilli and resistance to
Rifampin in less than 2 h21
compared to the weeks it takes by
culture method. It's accuracy and minimal processing time
help in selecting treatment regimens quickly. It is relatively
expensive, has a sensitivity of 98% in smear positive samples,
67% in smear negative samples and a specificity of 99%. Xpert
also detected 95% of rifampicin-resistant TB cases with
specificity of 98%.22
In 2013 Xpert MTB/RIF was endorsed by
WHO as the initial diagnostic test for pulmonary TB, MDR
pulmonary TB and HIV associated pulmonary TB in both
adults and children and as a follow-up test for smear negative
pulmonary TB in adults who are not at risk of MDR TB and HIV
associated TB. WHO also strongly recommended the use of
Xpert MTB/RIF for cerebrospinal fluid samples for rapid diag-
nosis of TB meningitis and conditionally recommended it for
diagnosis of other extrapulmonary TB samples.23
It's sensi-
tivity and specificity being 83% and 94% for lymph nodes, 81%
and 98% for cerebrospinal fluid and 46% and 99% for pleural
fluid samples.24
Another WHO endorsed molecular test is the Hain Geno-
type MTBDR plus assay. It is a rapid test that has been found to
be accurate only on smear positive pulmonary samples and
has been approved by WHO for that purpose.25
It can also
detect resistance to both Rifampin and Isoniazid unlike the
Xpert test; sensitivity and specificity being 98.1% and 98.7% for
Rifampin and 84.3% and 99.5% for Isoniazid.26
Though these are already a great improvement on the
widely used tests they can still be improved and investment is
needed.
To develop and implement new and better technologies for
DNA extraction and concentration methods.
Analyze the cost benefit aspects.
3.6. Adenosine deaminase
Adenosine deaminase testing, which has been in use in some
developing countries, has been abandoned now. It has been
found to have higher sensitivity than conventional tests when
performed on pleural fluid samples.16
3.7. Nose technology
It was documented that in ancient Greece, TB could be diag-
nosed by strong odors emitted from specimens. This was also
favored in Roman times and by traditional Chinese healers.
More recently there are reports suggesting that African pouch
rats can be trained to identify TB cases by smelling sputum.27
Instrument-based technologies using Electronic Noses28
have been developed and neural patterns were established
by exposure to a series of known TB-positive and TB-negative
samples and once such a pattern predictable of the disease is
established, it is programmed into the device. They have the
potential to be highly sensitive, portable and be able to screen
samples in a few minutes. The drawbacks being, it maybe
unstable over time, vulnerable to interference from water
vapor and background odors, and variation may occur be-
tween instruments requiring calibration.
Some of the widely used tests and those approved by WHO
are listed in Table 1.29,30
4. Conclusions
The future for TB diagnostics is bright with the tests like Xpert,
Hain and liquid cultures in use in many parts of the world and
many other advances in their late stages of evaluation. There
is the presence of large amount of funding and there is also a
huge challenge in translating these technological advances
into the public health settings. Initial studies show that these
advances are highly accurate in the normal setting but more
research is required to assess their performance in TB affected
children, MDR TB, HIV co-infection and with different types of
extrapulmonary TB.31
An ideal test, as also stated by the STOP
TB partnership, would have
~100% sensitivity and specificity
Be affordable
Have a quick turn around time of possibly 2 h
Be fully automatable with internal controls
Have a closed system to minimize contamination
Require minimal specimen processing
As of now existing tools like LED microscopy, Xpert, Hain
and liquid cultures should be scaled up as they are now more
affordable and accessible, even in India.
Conflicts of interest
All authors have none to declare.
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