|A 55 year old man
cough, fever, night sweats, loss of weight,
anorexia, malaise and weakness since 3 months
presents himself to the hospital.
Chest X-ray suggested upper lobe consolidation.
Hematological examination revealed mild leucocytosis.
What is your diagnosis?
It could be a case of pulmonary tuberculosis,
however differential diagnosis includes
aspergillosis, actinomycosis, bronchiectasis,
histoplasmosis, lung abscess, blastomycosis etc.
What is the etiological agent of tuberculosis?
Typical pulmonary tuberculosis is caused by
Mycobacterium tuberculosis, however other species of
Mycobacteria such as M. bovis,
M.avium-intercellulare complex too can cause
What is the specimen collected?
sputum specimen is the preferred specimen
in the laboratory diagnosis of pulmonary
tuberculosis. According to the revised guidelines of
National Tuberculosis Control Programme (India), two
sputa samples (spot-morning) must be collected from
the patient. When the patient approaches the center
to collect the container, spot specimen is
collected. Patient expectorates the second sample on
the next morning in the given container and submits
it to the laboratory. After rinsing the mouth,
patient should take deep breath, cough and expel the
sputum into the container that is held close to the
mouth. In patients without spontaneous sputum
production, sputum induction may be induced using
Which are the other specimen that can be
obtained from patients?
In case of children (who tend to swallow sputum), a
morning gastric lavage may be collected.
Alternatively laryngeal swabs may also be collected.
Other invasive techniques that can be employed
include fiberoptic bronchoscopy with transbronchial
biopsy, bronchial brushings, and transtracheal
How is the specimen processed?
A gram stained smear may be made from the thick
part of the sputum to exclude other bacterial
Acid fast staining of the smear is
considered the gold standard in the laboratory
diagnosis. After assessing the suitability of the
sample, the smear is stained with any of the acid
fast staining techniques (Ziehl Neelsen, Kinyoun,
Gabbett). Hundred fields must be observed in each
smear before giving a negative report. If acid fast
bacilli are seen, they should be counted and the
smear is graded.
What are your observations?
Pink coloured, slightly curved bacilli in singles or
clumps, with occasional branching and beaded
appearance are seen against a blue background
consisting of many pus cells and few epithelial
cells. The given smear contains
acid fast bacilli.
How is sputum graded?
According to the RNTCP, sputum sample should be
graded in the following way:
>10 AFB/oil immersion field in at least 20 fields:
1-10 AFB/oil immersion field in at least 50 fields:
10-99 AFB/100 oil immersion field: 1+
1-9 AFB/100 oil immersion field: record exact number
What is the significance of grading the sputum
The initial count gives a picture of the extent of
disease. It has a very important role in monitoring
the progress of treatment, as the counts decrease
with successful treatment.
What is the sensitivity of sputum smears?
AFB smear is not a very sensitive technique, if
numbers of bacilli are less than 1000/ml of sputum,
they may be missed. The sensitivity of the smear can
be increased by subjecting the sample to
concentration techniques such as Petroff's method,
Cetyl pyridinium chloride, Zepharin chloride method,
or NALC method etc. The sensitivity of microscopic
examination of sputum can be increased by using
fluorescent dyes such as
How are Mycobacteria cultured?
Direct sputum sample (or sputum concentrate) is
usually cultured on
Lowenstein Jensen Medium and incubated at 37oC
for 4-8 weeks. Cultures are not routinely performed,
but may be done to identify the species or for drug
susceptibility testing. Mycobacterium tuberculosis
produces rough, buff and tough
colonies on LJ medium. The
acid fast smear of these colonies show acid
fast bacilli. Conventional culture media used for
Mycobacterial isolation are most often
Lowenstein-Jensen (LJ), or Middlebrook 7H9, 7H10 or
7H11.Growth in these media is observed in a range of
3 – 56 days, depending on the species isolated and
concentration of viable bacteria. The
SEPTI-CHEK AFB system is intended for use as
an integrated in-vitro diagnostic system for
the detection and isolation of Mycobacteria from
various clinical specimens, which provides a
presumptive identification as well as the ability to
perform susceptibility testing. In miliary
tuberculosis, Mycobacteria can be recovered from
BACTEC-460 TB system; a system employing
radiometric technology providing rapid and accurate
detection in as little as 4-8 days and
susceptibility testing in as little as 4-12 days.
Other system includes Mycobacterial growth indicator
tube (MGIT) system. Culture using animal models are
no longer employed for routine diagnosis.
How is Mycobacterium tuberculosis identified?
Conventional method of identification are
aryl sulfatase test,
thermocatalase test etc. High pressure
liquid chromatography for detection of mycolic acid,
nucleic acid hybridization, PCR have replaced the
conventional system in developed countries.
Which are the other investigation techniques
employed in the laboratory diagnosis of
Serological methods detecting mycobacterial antigens
or IgA, IgG or IgM antibodies against Mycobacterium
using ELISA have been employed but have not met with
Skin testing (tuberculin test such as PPD,
Mantoux) have been employed to test for sensitivity
Reading the test has been prone to error and
are subjected to many false positive and negative
What is tuberculin test?
Please read this
What is the pathogenesis of tuberculosis?
Tuberculosis (TB) is spread from person to person
through the air by droplet nuclei that contain M.
tuberculosis. Droplet nuclei are produced when
persons with pulmonary tuberculosis cough, sneeze,
or speak.Infectious dose is less and few bacilli can
cause infection. Droplet nuclei are small enough to
reach the alveoli within the lungs. Alveolar
macrophages ingest the bacilli and enclose them in
phagosomes. If these macrophages are activated, the
mycobacteria containing phagosomes fuse with
lysosomes, and the bacteria are killed. If, on the
other hand, the alveolar macrophages are not
activated, the bacilli survive and multiply within
the phagosomes. The macrophages lyse and the
mycobacteria are released into the surrounding lung
tissue, where they are phagocytized by tissue
macrophages. Again, if the macrophages are
activated, the bacteria are killed. However, if
these tissue macrophages are not activated, the
mycobacteria continue to multiply within the
phagosomes and, upon release, are phagocytized by
additional tissue macrophages and the infection
spreads. As this process continues, a primary lesion
forms. As the primary lesion enlarges, some
mycobacteria are transported to the regional
draining lymph nodes and the lymph nodes enlarge as
the bacilli multiply intracellularly. Extension from
the lung parenchyma or the lymph nodes lead to
progressive primary tuberculosis. They may also
become dormant and remain asymptomatic, or may
proliferate after a latency period (reactivation
disease). The main determinant of the pathogenicity
of tuberculosis is its ability to escape host
defense mechanisms, including macrophages. Among the
several virulence factors in the mycobacterial cell
wall are the cord factor, lipoarabinomannan, and a
65-kd heat shock protein. Progression of the primary
complex may lead to enlargement of hilar and
mediastinal nodes. Lymphohematogenous dissemination
of the mycobacteria to other body parts and their
multiplication results in miliary or disseminated
tuberculosis. Tubercular meningitis may also result
from hematogenous dissemination. Bacilli may remain
dormant in the apical posterior areas of the lung
for several months or years, which may later
progress resulting in the development of
How is the diagnosis made using sputum smears?
The following is the recommendation of RNTCP: If one
or two samples of sputa are smear positive it may be
considered as sputum smear positive tuberculosis and
the patient is put on anti-tuberculosis treatment.
If only one smear is positive, but X-ray findings is
suggestive of TB, the patient is considered smear
positive and put on treatment. If the x-ray is not
suggestive, then TB is ruled out. If all the smears
turn out to be smear negative but X-ray finding
suggest tuberculosis, the patient is considered to
be sputum smear negative tuberculosis and is put on
treatment. Tuberculosis is ruled out if all the
smears are negative and x-ray finding too is not
How is tuberculosis treated?
Directly observed short-course chemotherapy for
newly diagnosed cases and sputum smear negative but
seriously ill with tuberculosis are subjected to
intensive phase treatment regimen comprising of
isoniazid, rifampicin, pyrazinamide and ethambutol
that is administered three times a week for two
months. When the patient has completed the initial
intensive phage of two months and the sputum smear
becomes negative, the continuation phase is begun.
If the sputum remains positive despite the intensive
phase, then the four drugs of intensive phase is
continued for another month. After this period,
continuation phase is begun irrespective of smear
status. The continuation phase consists of isoniazid
and rifampicin given three times a week for four
Why are anti-tubercular drugs given in
Spontaneous mutation can result in development of
resistant strains even during the course of
treatment. Inclusion of more than one drug ensures
that strains resistant to one drug are killed by the
What is MDR-TB?
Please read this
Are there any rapid commercial methods of diagnosis?
Interferon-Gamma Release Assays (IGRAs) measure a person’s immune reactivity to M. tuberculosis. Freshly drawn patient's blood samples are mixed with purified M. tuberculosis antigen (synthetic peptides). White blood cells of the patient that are reactive to M.tuberculosis will release interferon-gamma, which is quantitatively measured. Results of this test can be available within 24 hours. However, this test does not help in differentiating latent tuberculosis infection from tuberculosis disease. Prior vaccination with BCG does not cause a false-positive result.
How can antibiotic resistance be detected in short time?
Line probe assay is a PCR and DNA hybridization based molecular tool that can detect resistance to rifampin and isoniazid. This test can be performed directly on smear-positive sputum samples and results would be available in five hours.