Download An Update on the Laboratory Diagnosis of Tuberculosis

Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47
Review Article
An Update on the Laboratory Diagnosis of
Tuberculosis
1
Khurshid Ahmad1, Farhat Khurshid2
Consultant Microbiologist, Islamabad Diagnostic Centre; Scientist Emeritus, National Institute of Health, Islamabad
2
Department of Microbiology, Islamabad Diagnostic Centre, Islamabad
fundamental epidemiological attributes, mycobacterial
species are divided into two major groups:
 Mycobacterium
tuberculosis
complex
(MTB
Complex). Comprises 3 major species:
o Mycobacterium tuberculosis
o Mycobacterium bovis (including M. bovis
BCG)
o Mycobacterium africanum
 The Non-tuberculous Mycobacteria (NTM) – also
called Atypical Mycobacteria : All mycobacterial
species other than MTB complex have been included in
this group.6
Species belonging to Mycobacterium tuberculosis complex
occur in humans (and animals); although all three are
capable of causing disease, Mycobacterium tuberculosis is
the predominant species responsible for tuberculosis in
humans. Differentiation of these three species for routine
microbiological diagnosis is not
only difficult and
complicated but also of little or no medical importance in
the diagnosis and clinical management of the disease except
when required for epidemiological and public health
purposes.7
Tuberculosis primarily affects the lungs. Infection occurs by
inhalation of infectious airborne droplet nuclei containing
Mycobacterium tuberculosis exhaled during coughing and
sneezing (even speaking and singing) by “open” cases of TB
lungs. Therefore pulmonary tuberculosis is by far the
commonest form of disease produced by these organisms;
also it has probably the smallest infective dose of any
infectious disease – inhalation of a single viable organism of
MTB complex has been shown to lead to infection.7 In a
small percentage of infected patients, extra-pulmonary TB
occurs which may affect the lymph nodes, gastrointestinal
tract, central nervous system (meningitis), bones and joints,
peritoneum, genitor-urinary tract, pericardium etc.
Outcome of primary infection with MTB complex is
variable; a) in the majority the cellular immune system of
the host is able to contain the organisms; bulk of the
organisms is killed; those that survive are surrounded by T
cells and macrophages to form granulomas which limit their
multiplication and spread. After a variable period of time
15-20% of these people develop clinical tuberculosis. b) In
others the mycobacteria do not produce disease but lie
within the granulomas in a dormant state for years and
decades. Such people have a latent TB infection (LTBI)
Introduction
Tuberculosis is one of the most serious diseases afflicting
mankind since times immemorial; it has been present in all
ages and climates.1 Using the most modern DNA
techniques, presence of Mycobacterium tuberculosis has
been confirmed in the lesions from the lungs of a 1000-year
old mummy in Peru.2 Recent decades have witnessed
resurgence in tuberculosis, both in the developing as well as
in developed countries – more people are currently dying of
TB than at any other time in human history. According to
WHO estimates, one third of the entire human population is
presently infected with TB, most of it in developing
countries; a person is newly infected every second. Up to 8
million individuals develop active TB infection, with 2.9
million deaths, annually.
Among the 22 countries that have the highest burden of TB
cases in the world Pakistan is listed in the top eight.3
Prevalence of pulmonary tuberculosis in Pakistan among
adults over the age of 15 years stands confirmed at 295 per
100,000 populations.4 At least 400,000 new TB cases occur
every year including an alarming 15000 cases of multi-drug
resistant tuberculosis. These disturbing statistics call for a
redoubling of efforts to properly diagnose and treat all those
who have been infected with tuberculosis.
Mycobacteria
Microorganisms belonging to the genus Mycobacterium are
thin, non-motile, non-sporing and non-capsulated rods
measuring 0.2-0.4 x 2-10um. Three characteristic features
differentiate these organisms from other pathogenic bacteria:
a) they cannot be stained by the classical Gram stain, b) they
are acid-fast – once stained, they resist decolourisation by
acid - hence the name Acid Fast Bacilli or AFB; both these
characteristics owe their occurrence to the high lipid content
of the cell wall, and c) they have extremely slow growth in
culture – 2-8 weeks as opposed to 18-24 hours for other
common pathogens. Currently the genus Mycobacterium
comprises more than 70 recognized species.5 Based on their
Corresponding Author:
Dr Khurshid Ahmad
Email: [email protected]
association with human or animal disease and on their
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Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47
but not clinical tuberculosis. c) A variable percentage of
primary infections (up to 5%) develop directly into
progressive primary tuberculosis.
Thus tuberculosis can be active which is symptomatic and
highly infectious or it can be latent and inactive without
any symptoms. Individuals with latent TB infection are at
the risk of developing active disease whenever their immune
status changes (e.g. due to immunosuppressive therapy or
HIV infection) or clinical circumstances are altered. Each
person having a LTBI has about 10% chance of progressing
to active disease.
successive days are usually recommended for detection of
AFB by smear microscopy; at least one, if not all three, must
be early morning sample. Other pulmonary specimens may
include broncho-alveolar lavage, broncho-alveolar brushings
and trans-tracheal aspirate.
Extra-pulmonary Tuberculosis
Most common specimens from cases of extra-pulmonary
tuberculosis consist of body fluids (including pleural,
peritoneal, pericardial, synovial and CSF), urine and pus.
Others include lymph nodes, bone marrow, gastric aspirate
and tissues. From cases of renal tuberculosis, mid-stream
urine is collected on three successive days. Specimens are
transported to the laboratory immediately, at most within 2
hours of collection. In case of delay, they should be
refrigerated.
Tuberculosis and HIV/AIDS
HIV/AIDS infection has played a crucial role in the
resurgence of tuberculosis particularly in Africa and South
Asia. TB and HIV/AIDS are fatally synergistic. Immunity to
MTB complex is primarily cell-mediated. Because of
suppressive effect of HIV on cell mediated immune
function, co-infection with HIV greatly increases the risk of
developing active tuberculosis with rapidly developing
primary disease, instead of latent infection, and markedly
accelerates its progress.8 WHO estimates that up to 40% of
AIDS patients in Africa and South Asia die of tuberculosis. 3
Diagnostic Modalities Currently
Available
The clinical microbiology laboratory is faced with the
challenge of providing a rapid, reliable and cost effective
diagnosis of both active and latent tuberculosis. Techniques
presently available include the following:
Safety Precautions
Organisms of MTB complex carry a very high risk of
laboratory acquired infection (Hazard Group 3). Even the
simplest procedures like making AFB smears should be
undertaken using disposable gloves and a face mask.
Extreme care should be taken when sterilizing the
inoculating loop after making smear. Any amount of sputum
remaining in the loop spurts in the flame and may create
aerosols. This maneuver should be performed using a
hooded flaming device. It is safer to use a tooth pick instead
of wire loop which can be discarded in disinfectant without
flaming. Beyond this, all procedures on TB specimens
should be carried out in a bio-safety level 2 facility (Class II
safety cabinet). WHO further advises that all laboratories
handling TB cultures, must have Biosafety level 3 (BSL-3)
facilities.
Conventional techniques
1 AFB Smear Microscopy
2. Tuberculin Skin Test (TST) – (Mantoux test)
3. AFB Culture – Lowenstein-Jensen (LJ) solid
medium
New technologies
1. BACTEC MGIT 960 Culture System (Becton
Dickinson, USA)
2. Interferon Gamma Release Assays – IGRAs
a. Quantiferon TB Gold
b. T-SPOT.TB
3. Nucleic acid Amplification Tests (NATs) - PCR
4. Bacteriophage-based Tests – FASTPlaqueTB
Serological tests
1. Rapid Immunochromatographic assays (ICTs)
2. Enzyme-linked Immunosorbant Assays (ELISA)
Except for the basic procedures of smear microscopy and
tuberculin skin test, most of the advanced technologies,
including culture, conventional as well as MGIT, described
herein belong to the domain of specialized tuberculosis
laboratories.
Collection and Transport of Clinical Specimens
Successful detection and isolation of MTB complex depends
on the type and quality of the specimen obtained from the
patient which in turn is determined by the clinical
presentation of the case.
Pulmonary tuberculosis
Spontaneously produced early morning sputum from a deep
and vigorous cough is the specimen of choice. It should be
collected in a clean (not necessarily sterile), dry, widemouthed, leak-proof container and should be free from
saliva. Patients should be asked to cover their mouths
carefully during sputum collection or they should be asked
to collect it in a bath room. Up to three specimens on three
AFB Smear Microscopy
In Pakistan and other resource poor countries with a high
TB prevalence, smear microscopy continues to be the
mainstay of TB diagnosis. Because of its simplicity and
minimum requirements in terms of equipment and supplies,
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Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47
it is by far the most widely used procedure in TB diagnostics
not only in detecting TB cases but also in establishing their
infectiousness.
AFB smear microscopy may be performed directly or after
concentration.
If positive, result should be reported only as “Acid Fast
Bacilli seen” with quantification/grading as follows (WHO /
CDC):
> 9 AFB / oil field
(++++)
1 – 9 AFB / oil field
(+++)
1 – 9 AFB / 10 oil fields
(++)
1 – 9 AFB / 100 oil fields
(+)
1 – 2 AFB in the entire smear
Doubtful. Repeat
on fresh specimen
Direct smears
A loopful from a thick purulent portion of the sputum is
spread evenly on a slide to make a smear taking care that it
should neither be too thin nor too thick. A slide which has
already been used for sputum smear and washed should
never be reused for making a fresh smear. The smear is airdried and fixed by gently heating the slide on the flame.
Sensitivity and specificity of AFB smear microscopy
An AFB stained smear from clinical specimens requires at
least 5000-10000 bacilli per ml for detection. In skilled and
experience hands, overall sensitivity and specificity of
smear microscopy is excellent – up to 80% and 100%
respectively.11,12 There have been suggestions that sputum
smear microscopy may be more reliable for evidence-based
diagnosis of pulmonary tuberculosis than X-ray chest which
may often result in over-diagnosis.
Use of direct ZN microscopy for spinal, pleural and other
body fluids for detection of AFB is an unrewarding exercise;
some experts consider it a wasteful use of time and energy
as these samples are rarely positive. Many laboratories have
stopped reporting direct AFB smears on CSF and other
effusions.7 However a highly specialized test, adenosine
deaminase level, is now available for the reliable diagnosis
of tuberculous effusions particularly the pleural and ascetic
effusions.13
Concentration method: AFB can be concentrated by
centrifugation after treating the sputum with an equal
volume of 5% sodium hypochlorite (commercial bleach) for
10-15 minutes which also renders the specimen nonConcentration
technique
significantly
infectious.9,10
increases the sensitivity of smear microscopy (by up to
50%) for the detection of AFB in sputum .
Staining
Smears are stained by the standard Zeihl-Neelsen technique
or by Auramine-O stain (if facilities for fluorescent
microscopy are available). ZN staining is a hot stain
procedure as it requires heating the slide for better
penetration of stain into the mycobacterial cell wall.
AFB Fluorescent microscopy
Fluorescent staining using auramine O stain is more
sensitive than conventional ZN staining but this advantage is
more than offset by the high cost of fluorescent microscope
and reagents as well as the requirement of highly trained
and experienced staff for interpretation. It has been strongly
recommended that all positive results of auramine staining
must be confirmed either by a second trained observer or by
staining the same smear with ZN stain.7,12,14
Examination, interpretation and reporting of AFB
smears
ZN stained smears are examined under oil immersion. While
applying oil on the smear the slide should never be touched
with oil applicator or the nozzle of oil-containing bottle. A
small oil droplet should be dropped on the smear without
touching it. Examination of smears should be carried out by
scanning at least 300 oil immersion fields (roughly
equivalent to 3 horizontal sweeps over an area 2 cm x 1 cm)
before giving a negative result.7 Oil should be wiped off
from the objective before examining the next smear,
especially in case of a positive smear; AFBs from a positive
smear may come off the slide and float in the oil remaining
on the objective and may give a false positive result while
examining the next smear. In a well stained smear, typical
acid fast bacilli, when present, appear as red or purple,
slightly curved rods, 2-8 um long, against a crisp blue
background of mucous threads and debris. Bacilli of MTB
complex often appear beaded or banded (Fig.1).
Tuberculin Skin Test (TST) – Mantoux test 15
Next to smear microscopy, tuberculin skin test, in spite of its
limitations, is probably still the most commonly performed
test for detection of tuberculosis. Introduced by Robert Koch
nearly 100 years ago, it is still used as a standard screening
test in the United State for latent TB infection. Reaction is
based on the premise that infection with MTB complex
elicits delayed type hypersensitivity based on cell mediated
immune response to antigenic components of MTB
complex. In the standard Mantoux skin test, 0.1 ml of
purified protein derivative (PPD) from tubercle bacilli
containing 5 tuberculin units is injected intradermally on the
forearm. After 72 hours, the site is examined for induration
(area of firmness as a result of influx of immune cells). The
diameter of induration (not erythema which is usually
present with or without induration) is measured. Result is
Reporting
The following information should be included in the final
report: Specimen quality (Thick, mucoid, muco-purulent,
muco-salivary, blood stained etc), staining method used (ZN
stain), quantification or grading of result.
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Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47
reported according to the interpretative criteria given in
table 1. A positive Mantoux skin test establishes exposure to
tuberculosis but does not distinguish between active and
latent
infection.
Infection
with
non-tuberculous
mycobacteria and BCG vaccination may yield a false
positive tuberculin skin test; BCG effect may last for several
years. On the other hand a negative skin test does not rule
out the presence of tuberculosis.
instrument monitors fluorescence every 60 minutes and
gives a signal when detected.
Cultural methods
Specimen processing: Sputum and other specimens from
sites containing resident flora require decontamination and
digestion before culture. 4% NaOH or a mixture of NaOH
and N-acetyl l-cystein – NALC (a mucolytic agent) not only
decontaminates but also digests and liquefies the mucoid
material and organic debris which may wall off AFBs from
nutrients in the medium.16,17 Specimens from sterile sites do
not require decontamination. They should only be
concentrated by centrifugation.
AFB smear
microscopy
Table 2: Summary of diagnostic procedures for
tuberculosis
Test
Culture
Medium
0 – 4 mm
5 – 9 mm
= / > 10 mm
All individuals
- Healthy individuals
- Close contacts of
active TB cases
- HIV positive
individuals
- Injecting drug users
All individuals
4-8
weeks
11-13
days
48-72
hours
Comments
Mainstay of TB
diagnosis; rapid,
simple, cost-effective
Gold standard for TB
diagnosis
Does not distinguish
between active & latent
TB; mainly used to
evaluate a person who
has symptoms of TB or
for detecting LTBI in
asymptomatic persons;
false negative & false
positives (after BCG
and NTM infection)
may occur
As above but highly
IFN-γ release assays 12-24
hours
sensitive & specific;
(IGRAs) –
most reliable test for
T-SPOT.TB,
LTBI, not affected by
Quantiferon TB
BCG and most NTMs;
but costly & labour
intensive
Technically demanding,
Bacteriophage based 2 days
assay -FASTplaque
can only be used for
TB
sputum specimens.
Few
Highly sensitive and
NA amplification
hours
specific but technically
tests – PCR
complex and expensive
20 - 30
Inconsistent results –
Rapid Serological
tests- ICTs
minutes not endorsed by WHO
Conventional ELISA 2-3
Insufficient evidence of
hours
diagnostic accuracy
A schematic representation of the processing of sputum by
smear microscopy and AFB culture is given in Figure 2.
Footnote: ICT: Immuno-chromatographic Test, IGRA:
Interferon Gamma Release Assay, LJ: Lowenstein-Jensen
Medium, LTBI: Latent Tuberculosis Infection, MGIT:
Mycobacterial Growth Indicator Tube, NTM:: Nontuberculous Mycobacteria,
PCR: Polymerase Chain
Reaction, TST: Tuberculin Skin Test.
BACTEC MGIT (Mycobacterial Growth Indicator Tube)
consists of modified Middlebrook 7H9 broth medium which
is known to yield better recovery and faster growth of MTB
complex.18
Table 1: Interpretation of Mantoux skin test (5
tuberculin units)
Person tested
LJ
Bactec
MGIT
Tuberculin Skin Test
(TST) –
Mantoux test
Conventional AFB Culture
A definitive diagnosis of tuberculosis requires culture which
is still considered to be the gold standard for laboratory
diagnosis of tuberculous disease. It is much more sensitive
than microscopy, being able to detect as low as 10 viable
tubercle bacilli per ml of specimen. However, the
conventional culture on egg-based solid LJ medium has a
long detection time, taking anywhere from 4 to 8 weeks for
the growth to appear.
Area of
induration
Turnaround
time
<2
hours
Result
Negative
Borderline
positive
Positive
Positive
Positive
Positive
A fluorescent compound embedded in silicon sensor at the
base of the tube monitors oxygen consumption by growing
AFB if present in the specimen. Growth of AFB and
resulting O2 depletion is detected as an increase in
fluorescence. Fluorescence can be detected visually in UV
light, manually by Mini-MGIT or automatically by the
MGIT 960 system (Beckton Dickensen, USA) in which the
Mean detection time in the MGIT system is 11 – 13 days.
Growth is confirmed by ZN staining of a smear from the
45
Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47
culture tube which will show AFBs in groups or cords
(cording).
Interferon Gamma Release Assays – IGRAs
Interferon-gamma release assays (IGRAs) are based on the
principle that the T-cells of individuals who have acquired
TB infection respond to re-stimulation with M. tuberculosis
by secreting interferon gamma (IFN-γ). These assays
employ specific antigens of M. tuberculosis (ESAT-6 &
CFP-10) which are shared neither by BCG strains nor (with
rare exceptions) by non tuberculous mycobacteria, making
these assays virtually specific for MTB complex. The
QuantiFERON-TB Gold (Cellestis U.K. - [email protected])
measures the IFN-γ produced in response to these M.
tuberculosis antigens in whole blood.19,20 In contrast, the TSPOT.TB assay (Oxford Immunotec UK) detects the
number of “spot forming effector T cells” in peripheral
blood which produce IFN-γ in response to the above
antigens (Fig 3). Like Mantoux skin test, IGRAs do not
distinguish between active and latent TB infection. However
unlike skin test, these assays are not affected by BCG and
by most non-tuberculous mycobacteria. 21-24
Nucleic acid Amplification Tests (NATs) - PCR
NATs have been successfully used for the direct detection
of MTB in sputum and blood.25 In spite of being rapid,
highly sensitive and specific, these tests suffer from a
number of limitations: they are too expensive to be used in
countries where they are most needed, require special
expertise and may not be specific for active infection as
DNA from a dead organism can be amplified and detected
by PCR with equal efficiency. Assays from Roche and GenProbe have been approved by FDA for use on AFB-smearpositive specimens only because of less than optimum
sensitivity on smear-negative specimens.
Recently WHO has endorsed a NA assay for TB control
programmes in developing countries by the name of
GeneXpert MTB/RIF.3. It is an automated, cartridge-based
NA amplification assay specifically for the simultaneous
detection of MTB complex as well as rifampicin resistance
directly from sputum samples in less than two hours.
Fig. 1: Sputum smear strongly positive for AFB – ZN
stain
Sputum
Direct
Concentration
Decontaminate & liquify
4% NaOH (or NaOH + NAC)
Neutralise (1N-HCl)
AFB smear Microscopy
(ZN stain)
Centrifuged deposit
Inoculate
Bactec MGIT
Incubate
37oC – 42d
LJ Slope
Incubate
37oC 6-8 wks
If there is growth confirm by AFB smear microscopy
Fig. 2: Suggested flow diagram for processing of sputum
samples for AFB
Bacteriophage-based assay – FASTplaque TB
FASTPlaque TB (Biotec UK) utilizes the ability of a
specific mycobacteriophage (Actiphage) to lyse and destroy
viable MTB complex in decontaminated sputum. The test
takes 48 hours to complete but is technically demanding and
can only be used for sputum specimens.10
Serological tests
Antibody-based serological tests for the detection of
antibodies
to
MTB
complex,
including
immunochromatographic assays and ELISA, are simple to
use, comparatively inexpensive and easily available. Despite
widely being used in routine laboratories, these tests have
important limitations as clearly stated in the following WHO
recommendations.26
 “It is strongly recommended that these commercial
tests not be used for the diagnosis of pulmonary and
extra-pulmonary diagnosis.
Fig. 3: T-SPOT.TB test showing “spot forming effector T
cells”. Each spot represents an IFN-γ-producing T cell
in response to TB antigens
46
Journal of Islamabad Medical & Dental College (JIMDC); 2015:4(1):42-47

Currently available commercial sero-diagnostic tests
(also referred to as serological tests) provide
inconsistent and imprecise results.
 There is no evidence that existing commercial
serological assays improve patient outcomes, and high
proportions of false positive and false negative results
may have an adverse impact on the health of the
patients.”
A summary of diagnostic procedures for tuberculosis and
their clinical correlations is given in table 2.
12. Lipsky BJ, Gates J, Tenover FC. Factors affecting the clinical
value of microscopy for acid fast bacilli. Rev Infect dis
1984;6:214
13. Banales JL , Pineda PR, Fizgerald JM. Adenosine deaminase in
the diagnosis of tuberculous pleural effusions: a report of
218 patients and review of literature. Chest 1991; 99:355
14. Steingart K, Henry M, Ng V, et al. Fluorescence versus
conventional sputum smear microscopy for tuberculosis: A
systematic review. Lancet Infectious Diseases 2006;
6(9):570-81.
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Clin Infect Dis 1993; 17:968
16. Sommers HM, Good RC. Mycobacterium. In Lennette EH,
Balows A, Hausler WJ, Shadomy HJ editors: Manual of
Clinical Microbiology, 4th ed. 1985. Washington DC;
American Society of Microbiology: pp 216 – 248.
17. Yajko DM, Nassos PS, Sanders CA et al. Comparison of four
decontamination methods for recovery of Mycobacteria. J
Clin Microbiol 1993;31:302
18. Anargyros P, Astill DS, Lim IS. Comparison of improved
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1990; 142: 725.
19. Centres for Disease Control and Prevention. Guidelines for
using
the
QuantiFERON-TB
test
for
detecting
States.
Mycobacterium tuberculosis infection, United
MMWR Morb Mortal Wkly Rep 2005; 54 (RR-15):4955.
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Lancet 2001; 357 : 2017-21.
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countries: policy statement. 2011; ISBN 978 92 4 150267 2
(NLM classification: WF 220): Geneva, World Health
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26 World Health Organization. Laboratory Based Evaluation of
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Acknowledgments
We are grateful to Dr. Aftab Ahmad for the use of his
excellent camera microscope for taking picture of AFB
smear (Fig. 1) and to Mr. Najam Farooq for providing
picture of a positive T-Spot.TB test performed at Islamabad
Diagnostic Centre (Fig. 3)
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