|A 55 year old man with
persistent cough, fever, night sweats, loss of weight, anorexia,
malaise and weakness since 3 months presents himself to the
hospital. Chest X-ray suggested
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 tuberculosis.
What is the specimen collected?
is the preferred specimen in the laboratory diagnosis of
tuberculosis. According to the revised guidelines of National
Tuberculosis Control Programme (India), three sputa samples
(spot-morning-spot) must be collected from the patient. When the
patient approaches the center to collect the container, first spot
specimen is collected. Patient expectorates the second sample on
the next morning in the given container and the third spot
specimen is collected when the patient arrives at the center to
submit the second specimen. In hospitalized patients, sputum may
be collected every 8 hours. In patients without spontaneous sputum
production, sputum induction may be induced using hypertonic
Which are the other specimen that can be obtained from
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 aspirate.
How is the specimen processed?
A gram stained smear may be made from the thick part of the
sputum to exclude other bacterial infection. 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
>10 AFB/oil immersion field in at least 20 fields: 3+
1-10 AFB/oil immersion field in at least 50 fields: 2+
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 smear?
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
or NALC method etc. The sensitivity of microscopic examination of
sputum can be increased by using fluorescent dyes such as Auramine
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 blood using
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,
niacin test, nitrate reduction, 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 tuberculosis?
Serological methods detecting mycobacterial antigens or IgA, IgG
or IgM antibodies against Mycobacterium using ELISA have been
employed but have not met with great success. Skin testing
(tuberculin test such as PPD, Mantoux) have been employed to test
for sensitivity to Mycobacteria. Reading the test has been prone
to error and are subjected to many false positive and negative
What is tuberculin test?
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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 reactivation-type tuberculosis.
How is the diagnosis made using sputum smears?
The following is the recommendation of RNTCP: If two or three
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 out of three samples is
smear 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 three
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
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 months.
Why are anti-tubercular drugs given in combination?
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 other drug.
What is MDR-TB?
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