AN APPROACH TO

M.TUBERCULOSIS

DR.ASWINI
ESTIMATED INCIDENCE OF ACTIVE TB

WHO, Global TB Report, 2011 2

Notification of new TB cases in
Malaysia 2005 - 2011
 Causes
• Mycobacterium tuberculosis
– Bacilli, highly aerobic
– Infects lungs
– Divides every 15-20 hours
– Unable to be digested by microphages
– Very resistant to many disinfectants, acid, alkali, drying, etc.
• Contagious, spread through air by inhalation of airborne
bacteria from infected
• Easier to contract with weak immune system
• Some will acquire the infection.

• But, many of those infected will develop

adequate immunity to keep the infection at
bay.

• Hence, only a small number will eventually

develop active disease.

• Those infected but remain asymptomatic are

said to have Latent TB Infection (LTBI).
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 Symptoms
• Cough (with bloody sputum), loss of appetite, fever,
loss of energy/weight, night sweats, SOB
• Could spread to other parts of the body causing
other symptoms
 LTBI

LTBI is diagnosed when an individual:

– shows positive reaction to the TST/IGRA

but

– does not have any clinical, bacteriological (if

done), or radiographic evidence of active TB

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TUBERCULIN SKIN TEST (TST)

Robert Koch 1843 -1910

Pioneered the
Tuberculin Skin Test

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 PRINCIPLE OF TST
– TST is based on the principle of delayed-type
hypersensitivity response to intradermal
inoculation of PPD, or tuberculin.

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 THE SHORTCOMINGS OF TST

Tuberculin contains
>200 proteins, widely
shared among the TB
mycobacteria.

BCG NTM

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 NEW TESTS - IGRA
• Recently, with the invention of 2 new tests,
collectively known as IGRA
•QuantiFERON-Gold-In-Tube (QFT-GIT) test
•Elispot test (T-SPOT)
 the prospect of differentiating LTBI from BCG
vaccination/NTM infection has becoming promising.

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PRINCIPLE OF IGRA

TB

BCG NTM

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 Specimen Collection
Procedure
• Obtain 3 sputum specimens for smear
examination and culture

•Spot, first morning, spot

• Follow infection control precautions during
specimen collection
-If possible, specimens should be obtained in an
airborne infection isolation (AII) room or other isolated,
well-ventilated area (e.g., outdoors)
 SPECIMENS COLLECTED
• Pulmonary secretions –
** spontaneously produced or induced sputum
** gastric lavage
** transtracheal aspiration
** bronchoscopy
** laryngeal swabbing

• Gastric lavage specimens.

• Urine samples.
• Faecal specimens.
• Tissue & Body Fluid specimens.
• Blood specimens
• Wound ,skin lesion aspirates.
• Cervical swab.
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 STORAGE AND TRANSPORTATION
• Refrigerate if delayed examination ( non-freezing)
• Collection of specimen to processing in the lab <4
days
• Pack each specimen container in a plastic bag,
kept vertically.
• The transport box must have separators to
prevent the containers to knock into each other.
• The transport box must be kept as cool as
possible.
 SPUTUM EXAMINED BY :
• DIRECT METHOD
# Z.N.Staining.
# Fluorescent Auramine Rhodamine staining

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Biosafety Cabinet
 Procedure for ZN Staining
1. Place slides on staining rack so they are at least 1 cm apart( to prevent
contamination), and flood with carbol fuchsin.
2. Heat the slide to steaming with the flame from a Bunsen burner. An
electric heating block may also be used. Apply only enough additional
heat to keep the slide steaming for 5 minutes. Do not let the stain boil
or dry. Add additional stain if necessary. Leave the warm stain on the
slides for 5-10min.
3. Wash off the stain with distilled water.
4. Flood slides with 3% hydrochloric acid-ethyl alcohol
5. Let stand for 2-3 min (more acid-alcohol should be used if the smear is
heavily stained).
6. Wash off the acid-alcohol with distilled water and tilt the slides to drain.
7. Flood the slides with 0.3% methylene blue and let stand for 1-2 minute.
8. Wash off the methylene blue with distilled water.
9. Tilt the slides to drain.
10. Allow slides to air dry in the slide rack.
Acid-fast bacilli

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 GRADING OF SMEAR
No. of AFB Interpretation Report
0 No AFB in 2x3cm 0 AFB/300VF
length
1-9/100 visual 1 to 9 AFB seen in 1-9 AFB/100VF
field 100 visual field
Report as 1+ 10-99 AFB seen in 10-99 AFB/100VF
100 visual fields
Report as 2+ 1 to 10 AFB seen 1-10 AFB/VF in
in each of 50 50VF
visual field
Report as 3+ >10 AFB in each of >10/VF in 20VF
20 visual field 24
 Auramine or Auramine/Rhodamine
Procedure
1. Place slides on staining rack so they are at least 1 cm apart, and flood with
auramine or auramine/rhodamine stain and let stand for 20 min.
2. Rinse the stain away with distilled water and tilt slide to drain. Water must
be chlorine free.
3. Flood the slide with 0.5% acid alcohol and let stand for 2 min.
4. Wash off the acid alcohol with distilled water.
5. Flood slides with 0.5% potassium permanganate for 1-2 min. Do not allow
potassium permanganate to act over 2 min, or it might quench the
fluorescence of acid-fast bacilli.
6. Wash off the stain with distilled water.
7. Allow slides to air dry in the slide rack.
8. Protect smears from light and examine immediately using the fluorescent
microscope. If unable to read right away, place slides in covered box.
Grading Scale for Fluorescent Stain
What you see (200x) What you see (400x) What to report*

No AFB in one length length No AFB in one No AFB observed

length
1-4 AFB in one length 1-2 AFB in one length Confirmation required**

5-49 AFB in one length 3-24 AFB in one length Scanty

3-24 AFB in one field 1-6 AFB in one field 1+

25-250 AFB in one field 7-60 AFB in one field 2+

> 250 AFB in one field > 60 AFB in one field 3+

 Indications for Culture
• Failures of re-treatment cases
• Seriously ill cases;
– extra-pulmonary cases
– smear negative cases
– childhood TB & HIV-TB
• For DRS

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 MYCOBACTERIAL CULTURE
Advantages:

 Increases number of cases found ( 30-50%)

 Detects cases among smear negative patients
 Establishes viability of organisms
 Distinguishing between Mycobacterial species
 Helps in performing DST
 Helps in diagnosing cases of failure

Limitations:

 Expensive
 Require enriched media
 Require considerable expertise
 Time consuming 30
 Screen by AFB smear & inoculate media (one liquid & one solid)

Liquid Medium Solid Media

LJ
MGIT BACTEC SEPTI- CMS
CHEK L J with RNA
Incubate Incubate Incubate LJ with
Incubate
At 37ºC At 37ºC inverting Pyruvic
At 37ºC
For 6 wks For 6 wks At 37ºC acid
For 6 wks
For 8 wks
Incubate
Growth At 37ºC
Fluoresc- Colonies Growth
-ence
Index For 8 wks
detected or detected
>10 turbidity
If growth
Confirm on
Reinoculate AFB smear
Confirm by AFB smear
on 31
Colonies growing on media

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Characteristics of Tubercle bacilli
Growth of Primary culture takes 2 – 4 weeks to obtain
visible colonies
Colonies are buff colored and rough, having the
appearance of bread crumbs or cauliflower
Not easily emulsified but give a granular suspension
Microscopically frequently arranged in serpentine cords of
varying length or show linear clumping

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 Direct Detection of M. tuberculosis in Clinical
Specimen Using Nucleic Acid Amplification
NAA tests are used to amplify DNA and RNA segments to
rapidly identify the microorganisms in a specimen. NAA
testing can reliably detect M. tuberculosis bacteria in
specimens in hours as compared to 1 week or more for
culture Possible benefits of using NAA tests include
• Earlier laboratory confirmation of TB disease;
• Earlier treatment initiation;
• Improved patient outcomes;
• Interruption of transmission by early diagnosis, respiratory
isolation and appropriate treatment;
• Earlier, more efficient use of respiratory isolation;
• Earlier initiation of contact investigation; and
• More effective public health interventions.
 MOLECULAR TECHNIQUES TO DETECT
M.TUBERCULOSIS

• Amplicor Test

** Detects the presence of the

mycobacterial 16S ribosomal ( rRNA)
gene by the PCR amplification
followed by ELISA reaction.

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Mycobacteria (AFB) sample

AFB
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Extract 16S RNA

16S
RNA
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Probe for specific 16S RNA

Probe Probe

16S
RNA
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Detector binds to probe

******* *******
Probe

16S
RNA
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Detector identifies 16S RNA

*******
Probe

16S
RNA
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 Molecular diagnostic methods for drug
resistant M.tuberculosis

• DNA Sequencing : reliable & accurate.

• Line Probe Assay (LiPA) : to identify mutations in the rpoB core
gene.
• DNA microarrays : based on principle of hybridization
• Molecular beacons :they are hair-pin shaped probes to detect
presence of specific nucleic acids.
• Single strand conformation polymorphism :determines
presence of mutations in specific DNA regions
• FRET ( Fluorescent resonance energy transfer) probes: used to
detect presence of mutations in real time PCR.

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 REFERENCE
• CPG Management of Tuberculosis ( 3rd edition)
• http://www.who.int/tb/laboratory/mycobacte
riology-laboratory-manual.pdf
• Laboratory Technical Manual ( Direct Sputum
Smear Microscopy for Acid Fast Bacilli-MOH
• http://microbeonline.com/auramine-
rhodamine-fluorochrome-staining-principle-
procedure-results-limitations/