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Research and Reviews: A Journal of Life Sciences
ISSN: 2249–8656
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Standard Diagnostic Procedure for
Tuberculosis: A Review
Suresh Jaiswal*, Jay Prakash Sah, Bhoopendra Sharma
Department of Medical Lab Science, School of Health and Allied Sciences, Pokhara University,
Lekhnath, Kaski, Nepal
Abstract
Despite continuous effort in monitoring and treatment of tuberculosis, the disease
remains a major public health issue. Rapid diagnosis and appropriate therapies become
the first priorities in controlling the growing epidemics. The bedside decision on the
initiation of anti-tuberculous drug therapy are based on epidemiologic, clinical,
radiolographic, and/or histological findings, which can generally be supported by a
rapid microbiologic test, commonly a positive acid-fast bacilli (AFB) smear result.
However, AFB smear is positive in only half of patients with subsequently culture positive
for Mycobacterium tuberculosis. Although the sensitivity of the smear is improved by
fluorescent staining, the test fails to distinguish between tuberculous and nontuberculous
mycobacteria. Diagnosis of Tuberculosis (TB) is mainly based on the culture method and
non-culture methods. Molecular techniques are becoming more advanced and
confirmatory diagnostic procedure of TB. Recent surveys also reveal that drug-resistant
tuberculosis is still ubiquitous and alarmingly high in several countries. The situation is
further complicated by the emergence of multi-drug resistant tuberculosis (MDR-TB).
MDR-TB results from improper administration of antibiotics in chemotherapies of TB
patients and is recognised as Mycobacterium tuberculosis resistant to at least isoniazid
(INH) and rifampin (RIF), the two most common first-line antituberculosis drugs.
Keywords: Tuberculosis, AFB smear, Multi Drug Resistant (MDR), LJ medium
*Author for Correspondence: E-mail [email protected]
INTRODUCTION
Tuberculosis (TB) remains the leading cause
of death from a curable infectious disease,
despite the availability of short-course therapy
that can be both inexpensive and effective.
Clinical management of cases in developing
countries is hampered by the lack of a simple
and effective diagnostic test [1].
Active tuberculosis (TB) is diagnosed by
detecting
Mycobacterium
tuberculosis
complex bacilli in specimens from the
respiratory tract (pulmonary TB) or in
specimens from other bodily sites (extra
pulmonary TB). Although many new
(molecular) diagnostic methods have been
developed, acid fast bacilli (AFB) smear
microscopy and culture on Lowenstein-Jensen
medium are still the “gold standards” for the
diagnosis of active TB and, especially in lowresource countries, the only methods available
for confirming TB in patients with a clinical
presumption of active disease. AFB smear
microscopy is rapid and inexpensive and thus
is a very useful method to identify highly
contagious patients. Culture is used to detect
cases with low mycobacterial loads and is also
requested in cases at risk of drug-resistant TB
for drug susceptibility testing, or in cases
where disease due to another member of the
Mycobacterium genus is suspected. AFB
smear microscopy and culture can also be used
to monitor the effectiveness of treatment and
can help to determine when a patient is less
likely to be infectious. Two manuals are
recommended for the laboratory diagnosis of
TB [2, 3].
Correct diagnosis of TB is needed to improve
treatment, reduce transmission, and control
development of drug resistance. In patients
with active pulmonary TB, only an estimated
45% of infections are detected by sputum
microscopy. This test, first developed in the
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Standard Diagnostic Procedure for Tuberculosis
Jaiswal et al.
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1880s and basically unchanged today, has the
advantage of being simple, but is hampered by
very low sensitivity: it may only detect half of
all cases with active infection. It is also very
dependent on the skill of the technician, and a
single technician can only process a relatively
small number of slides per day [3].
Furthermore, a staggering three million people
who present annually with suspected TB may
not be properly diagnosed, because their
infection (so-called smear-negative disease)
cannot be detected by sputum microscopy [4].
There are specific epidemiological factors that
present additional challenges to TB diagnosis.
HIV infection is thought to be a major
contributor to the increase in TB incidence
across the world [2]. An estimated 9% of
adults globally with newly diagnosed TB are
HIV positive.
HIV co-infection with TB presents challenges
to effective diagnosis of TB and diagnosis can
also be more difficult in children. The rapid
rise of drug-resistant (DR) TB has further
complicated TB diagnosis [6]. Tests that
measure drug susceptibility are essential to
monitor the spread of resistant TB strains, and
ensure that patients are given effective
treatment.
New diagnostic tests that are simple and robust
enough to be used in the field, accurate enough
to diagnose all infected individuals, and able
to identify drug resistance are desperately
needed, and represent an essential complement
to new drug development efforts and to
effective control and treatment programmes.
An individual who is suspected of having TB
disease requires a complete medical
evaluation, including the following [4]:
1. Medical history, including exposure,
symptoms, previous treatment for TB, and
risk factors.
2. Human immunodeficiency virus (HIV)
screening.
3. Physical examination.
4. Tuberculin skin test (TST) or interferon
gamma release assay (IGRA).
5. Chest radiography.
6. Bacteriologic examination.
MEDICAL HISTORY
Clinicians should ask about the patient’s
history of TB exposure, infection, or disease.
It is also important to consider demographic
factors, (e.g., country of origin, age, ethnic or
racial group, occupation) that may increase the
patient’s risk for exposure to TB or to drugresistant TB. Also, clinicians should determine
whether the patient has medical conditions,
especially HIV infection, that increases the
risk of latent TB infection progressing to TB
disease [5].
HUMAN IMMUNODEFICIENCY
VIRUS SCREENING
Voluntary counseling and testing for HIV is
recommended for all patients with TB. HIV
counseling and testing has also been
recommended for contacts of persons with TB
[6].
The Centers for Disease Control and
Prevention (CDC) recommends the following:
 Routine HIV screening for all patients
ages 13–64 seeking health care for any
reason, without regard to any patient’s
known risks for HIV infection.
 Annual HIV screening of patients known
to be at high risk [7].
PHYSICAL EXAMINATION
A physical exam can provide valuable
information about the patient’s overall
condition and other factors that may affect
how TB is treated, such as HIV infection or
other illnesses.
TEST FOR TB INFECTION
The Mantoux tuberculin skin test (TST) or the
special TB blood test can be used to test for M.
tuberculosis infection. Additional tests are
required to confirm TB disease.
The Mantoux tuberculin skin test is performed
by injecting a small amount of fluid called
tuberculin into the skin in the lower part of the
arm. The test is read within 48 to 72 h by a
trained health care worker, who looks for a
reaction (induration) on the arm (Figure 1).
The special TB blood test measures the
patient’s immune system reaction to M. tuberc
ulosis.
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Fig. 1: Mauntax Tuberculin Test and its Reading in 48–72 h.
CHEST RADIOGRAPH
A posterior-anterior chest radiograph is used
to detect chest abnormalities (Figure 2).
Lesions may appear anywhere in the lungs and
may differ in size, shape, density, and
cavitation. These abnormalities may suggest
TB, but cannot be used to definitively
diagnose TB. However, a chest radiograph
may be used to rule out the possibility of
pulmonary TB in a person who has had a
positive reaction to a TST or special TB blood
test and no symptoms of disease [8].
Fig. 2: Showing Chest Radiograph with Lesions and Chest Abnormalities.
DIAGNOSTIC MICROBIOLOGY
The presence of acid-fast-bacilli (AFB) on a
sputum smear or other specimen often
indicates TB disease. Acid-fast microscopy is
easy and quick, but it does not confirm a
diagnosis of TB because some acid-fast-bacilli
are not M. tuberculosis. Therefore, a culture is
done on all initial samples to confirm the
diagnosis. (However, a positive culture is not
always necessary to begin or continue
treatment for TB). A positive culture for M.
tuberculosis confirms the diagnosis of TB
disease. Culture examinations should be
completed on all specimens, regardless of
AFB smear results. Laboratories should report
positive results on smears and cultures within
24 h by telephone or fax to the primary health
care provider and to the state or local TB
control program, as required by law.
MATERIALS AND METHODS
Specimens
The successful isolation of the pathogen
requires that the best specimen be properly
collected, promptly transported and carefully
processed. Many different types of clinical
specimens may be obtained for the
microbiological diagnosis. If pulmonary TB is
suspected, specimens originating from the
respiratory tract should be collected, i.e.,
sputum, induced sputum, broncho alveolar
lavage or a lung biopsy. For the diagnosis of
pulmonary TB, three first-morning sputum
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Standard Diagnostic Procedure for Tuberculosis
Jaiswal et al.
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specimens (not saliva) obtained after a deep,
productive cough on non-consecutive days are
usually recommended. Several studies have
shown, however, that the value of the third
sputum is negligible for the diagnosis of TB,
as virtually all cases are identified from the
first and/or the second specimen (Yassin 2003,
Nelson 1998, Dorronsoro 2000, Finch 1997).
Specimens to be collected for the diagnosis of
extrapulmonary disease depend on the site of
the disease. The most common specimens
received in the laboratory are biopsies,
aspirates, pus, urine, and normally sterile body
fluids, including cerebrospinal fluid, synovial,
pleural, pericardial, and peritoneal liquid.
Stool can be collected when intestinal TB is
suspected and also in the case of suspected
Mycobacterium avium infection in AIDS
patients.
AFB Smear
AFB smear microscopy plays an important
role in the early diagnosis of mycobacterial
infections because most mycobacteria grow
slowly and culture results become available
only after weeks of incubation (Table 1). In
addition, AFB smear microscopy is often the
only available diagnostic method in
developing countries. Smear staining is based
on the high lipid content of the cell wall of
mycobacteria which makes them resistant to
decolorization by acid-alcohol after the
primary staining. To determine that a clinical
specimen contains AFB, the specimen is
spread onto a microscope slide, heat-fixed,
stained with a primary staining, decolorized
with acid-alcohol solution and counterstained
with a contrasting dye in order to obtain better
differentiation between the microorganism and
the background. The slide is observed under
the microscope for the detection of AFB
(Figure 3). Several methods can be used for
determining the acid-fast nature of an
organism.
Fig. 3: A and B showing Myctobacterim Bacilli in Sputum and Slide Smear.
Table 1: Showing the Count of Mycobacterium Bacilli in per Fields of 100x.
Count on Ziehl-Neelsen/Kinyoun stain (1000x)
0
1–9/100 fields
10–99/100 fields
1–10/field
> 10/field
Diagnostic methods in detection of
Mycobacterium Tuberculosis are as follows:
1. Culture based methods
2. Non culture based methods.
Culture Based Methods
Culture of Mycobacterium tuberculosis
remains the gold standard for both diagnosis
Report
Non AFB observed
Exact count
1+
2+
3+
and drug sensitivity testing. This section
reviews culture tests currently in use, and
newly developed techniques. Conventional
culture methods using Lowenstein-Jensen (LJ)
or 7H11 medium, while cheap and simple,
have the major disadvantage of being very
slow. LJ cultures take 20–56 days for
diagnosis and four to six weeks after initial
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__________________________________________________________________________________________
culture for drug sensitivity testing. 7H11
medium slightly accelerates the process, but
requires antibiotics in the medium to prevent
contamination and a CO2 incubator. Diagnosis
with 7H11 medium takes 17–21 days,
Daylight Saving Time (DST) information is
available three to six weeks later. Some more
rapid culture methods have been developed
and are commercially available, most of which
are difficult to implement in the field due to
the complexity of the technique or the required
equipment. There are also some emerging
simplified culture techniques that can reduce
time to diagnosis or DST that seem more
appropriate for use in resource-limited
settings.
The sensitivity of culture is limited by the
need to have bacilli present in the sample to be
cultured. HIV positive patients and children
have difficulty in producing sputum and
sputum culture will not detect extrapulmonary
(EP) forms of TB. EP TB is very common in
HIV positive patients and is rapidly fatal. Even
in patients with active pulmonary TB, the
bacilli may be protected in lung cavities or not
present in a particular sputum sample, or may
be lost in the decontamination treatment
required to process sputum for mycobacterial
culture. All these factors limit the usefulness
of the technique.
New Rapid Commercial Methods for
Diagnosis and DST
A number of commercial systems are available
for culture and DST, some of which may have
slight advantages in certain settings. However,
none of the tests are easily adaptable to the
realities of field projects, given the difficulties
in setting up and running culture laboratories.
Phage-based Tests
Phage-based tests require limited culture
facilities and promise rapid results (~2 days).
However, MSF field evaluations shown that it
is very hard to implement in non-culture
facilities in the field, even in relatively wellsupported urban settings. Dedicated areas are
required, careful control of access to the rooms
is needed to reduce contamination, and even
seemingly simple requirements, like a stable
power supply and a functioning biosafety
hood, are very difficult and often enormously
expensive to ensure. Metanalyses comparing
phage based tests to culture in field settings
have shown that in most cases they are no
more informative that smear microscopy.
Non-Culture Methods
A number of strategies to detect and report the
presence of M. Tuberculosis have been
developed. Serology (detection of antibodies)
has not produced any reliable, informative
tests despite decades of work. Detection of
antigens is a more promising approach, as it
detects the presence of the organism and thus
may be able to diagnose active infection. The
use of nucleic acid amplification (NAA) tests
in non-specialised laboratories is technically
challenging. These tests have been shown to
be highly specific, but sensitive if starting
from patient samples, low and highly variable
and is difficult to assess 28. These tests can
also be used from primary culture. Although
this improves the sensitivity, the technique is
then very slow. For this reason, we have
decided to include NAA tests in the nonculture section of this report, in order to focus
on the tests’ use on direct clinical samples.
Here we also look at some polymerase chain
reaction (PCR) based techniques which are
being validated for use on patient samples for
rapid detection of rifampicin/isoniazid
resistance. There are also some tests being
developed
that
detect
immunological
responses (interferon gamma assays). These
tests are rather expensive and complicated to
perform, and still need to be validated in
endemic areas, and their interpretation is not
clear.
Techniques using Antibody Detection
In 2005, WHO/TDR performed an evaluation
of commercially available rapid diagnostic
tests (RDTs). Twenty-seven manufacturers
were invited to submit their products for
evaluation, but of the 19 who agreed, only six
provided information on the antigen used. All
tests detect antibodies in serum. Test samples
came from the TDR specimen bank.
The WHO study found that TB rapid
diagnostic tests currently available on the
market vary widely in performance, with some
products showing a high lot-to-lot and readerto-reader variability. At less than 80%, the
specificity was poor in the majority of
products when tested in TB suspected cases
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Standard Diagnostic Procedure for Tuberculosis
Jaiswal et al.
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from endemic settings. Those tests with a
better specificity (over 90%) had poor
sensitivity, detecting fewer than 40% of TB
patients. The tests performed even worse in
HIV co-infected samples. The conclusion of
the study was that none of the assays perform
well enough even to replace microscopy [9].
Based on this and other information, it seems
that antibody detection is unlikely to be a good
strategy for the development of a reliable
diagnostic test for TB. It is important to note
that the tests named in the above table are only
those that agreed to participate in the study:
absence from the list does not imply that the
test works. We found no convincing evidence
supporting the use of any existing antibody
detection tests [10].
with the variability and lack of sensitivity in
sputum negative and extra pulmonary TB do
not support their use [11, 12].
Techniques using Antigen Detection
Several tests using antigen detection are
currently
commercialised
or
under
development.
CONCLUSION AND
RECOMMENDATION
Nucleic Acid Amplification (NAA)
NAA techniques require strong laboratory
capacities, good quality control procedures,
and remain relatively expensive. In recent
years, some improvements have been made,
such as isothermal amplification steps, the
inclusion of internal amplification controls to
ensure that inhibitors (resulting in false
negatives) are not present, the design of
single-tube reactions to reduce contamination
and the development of detection by emitted
light or by dipstick. The use of NAA
techniques remains technically challenging.
Despite being usually highly specific, NAA
tests have lower (and greatly variable)
sensitivity. A positive NAA test is considered
good evidence of infection but a negative
result is not informative enough. Use of NAA
tests has not been recommended for sputum
negative patients. As these tests cannot
distinguish live from dead bacteria, they
cannot be used for patients receiving
treatment. One study considered that current
NAA tests cannot replace microscopy or
culture, and should be used only in
conjunction with these tests. While some NAA
assays reported seem to work quite well
(sometimes sensitivities near 90% were
reported), there is very wide variability, even
from very resource rich laboratories, making
their use in the field uncertain. Their difficulty
of use and quality control issues combined
DRUG RESISTANCE
For all patients, the initial M. tuberculosis
isolate should be tested for drug resistance. It
is crucial to identify drug resistance as early as
possible to ensure effective treatment. Drug
susceptibility patterns should be repeated for
patients who do not respond adequately to
treatment or who have positive culture results
despite three months of therapy. Susceptibility
results from laboratories should be promptly
reported to the primary health care provider
and the state or local TB control program [9,
13].
This overview and development pipeline for
tuberculosis diagnostics and drug sensitivity
testing gives a mixed picture. On the one hand,
the pipeline is unquestionably active, with a
number of different tracks being explored,
culture or otherwise, and some improvements
have been made. However, if it is now
possible to increase throughput, or obtain
results faster than can be achieved with
traditional
culture
methods,
these
improvements often come at the expense of
sensitivity and simplicity. In part this is due to
the complexity of the problem for TB
diagnostics today.
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