This document discusses the diagnostic steps for tuberculosis, including history and clinical examination, radiographic features, and bacteriological evaluation. It describes common symptoms and radiographic findings that suggest TB. Sputum smear microscopy and culture are important conventional diagnostic methods discussed in detail. Newer diagnostic tests like Xpert MTB/RIF provide rapid detection of M. tuberculosis and resistance to rifampin directly from sputum samples within 90 minutes. Overall, the document outlines the key diagnostic approaches and tests used to evaluate patients for possible pulmonary tuberculosis.

1. Diagnosis of Tuberculosis
part – 2
Presentation by : - Dr.Darshna Sarvaiya
1st year resident
Community Medicine
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2. Diagnostic Steps
History and clinical examination
Radiographic features
Bacteriological Evaluation
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3. History and clinical examination
 “The first rule of TB diagnosis : is to think of TB….”
 The physician includes TB in his differential diagnosis when history &
symptoms are consistent with TB diagnosis
He will recommended appropriate diagnostic tests to
prove the infection .
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Radiographic features

5.  Infiltrate or consolidation - Opacification of airspaces within the lung
parenchyma. Consolidation or infiltrate can be dense or patchy and might
have irregular, ill-defined, or hazy borders.
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6. Any cavitary lesion - Lucency (darkened area) within the lung parenchyma, with or without
irregular margins that might be surrounded by an area of airspace consolidation . The walls
surrounding the lucent area can be thick or thin. Calcification can exist around a cavity.
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7. Nodule with poorly defined margins - (tree-in-bud sign[3])
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8. Hilar lymphadenopathy (bihilar lymphadenopathy)
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9.  Milliary tuberculosis
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10. 10 Bacteriological Evaluation
Conventional diagnostic method
(smear , culture )
Immunological diagnosis
(Tuberculin test)
New diagnostic methods (NAAT
, BACTEC , MGIT ,CB-NAAT)

11.  Most confirmatory test for pulmonary
 Simple , appropriate technology which is relatively easy to perform and read
 Single direct smear  miss about 25 % of sputum positive cases
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Conventional diagnostic methods
( Smear , Culture )
Sputum examination :

12. Sputum collection
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Day 1 Sample 1 Patients provide an “ on –
the –spot” sample under
supervision when
presenting to the health
facility , give the patient a
sputum container to take
home for an early
morning sample the
following morning
Day 2 Sample 2 Patient brings an early
morning sample
Note : volume of sputum  3 ml – 6 ml ( minimum acceptable amount ; 2 ml )

13. Characteristic of a Good sputum specimen
 5 – 10 ml
 Thick and mucous , but may be fluid with pieces purulent material
 Colour varies from opaque white to green, but reddish to brown
when blood is present
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14. ZN ( ZIEHL NEELSEN ) staining
 Procedure :
• make the smear on a clean glass slide , the slide with the smear up about three
time slowly through a flame.
• Cover with Carbol fuchsin , kept for 5-7 min with stem gently over direct flame (
till fumes arise ) , wash with water
• Decolourize in 3.0 % acid alcohol ,until only a faint pink colour remains , wash with
water
• Counter stain for 1 minute with Loeffler’s methylene blue , wash with water
• Air dry and observe under oil immersion objectives of microscope
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15. Slide reporting
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Number of bacilli Result reported
No AFB per 100 oil immersion fields 0
1-9 AFB per 100 oil immersion fields Scanty
10-99 AFB per 100 oil immersion fields + ( 1 + )
1-10 AFB per oil immersion fields ++ ( 2 +)
> 10 AFB per oil immersion fields +++ ( 3 +)
Sputum smear microscopy for tubercle bacilli is positive when there are at least
10,000 organisms present per ml of sputum

16. 16 False positive result : sputum smear result is positive even through the
patient does not really have sputum smear positive PTB
Acid fast particle and other micro organisms
( nocardia , other mycobacteria )
False negative result : sputum smear result is negative even through the pt.
does have sputum smear positive PTB
Inadequate sputum collection , failure to select suitable sputum particle for
making smear , poor smear preparation and inadequate examination of
smear

17. Fluorescence microscopy
 The smear may be stained by
auramine – o dye . In this
method the TB bacilli are
stained yellow against dark
background & easily visualized
using florescent microscope
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18. Advantages
 Faster staining than ZN
 More sensitive than ZN
 Do not require oil –immersion
 No heat require for staining
 Fluorescence stain slide can be subjected to ZN stain
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19. Light emitted diode fluorescence microscopy ( LEDs)
 Less expensive
 In recent WHO evaluation, the diagnostic accuracy of LED microscopy was found
to be comparable to that of conventional fluorescence microscopy and superior
to that of conventional Z-N microscopy
 Much large area of the smear to be seen
 More rapid examination of the specimen and making it easier to count bacilli
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20. Limitations
 LED microscope require ( costly )
 -ve staining does not indicate specimen will be culture negative
 Auramine rhodamine  possible carcinogen
 KMNO4 / acid alcohol – strong irritant
 Stained slide should be examined within 24 hour (fluorescent
fading )
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21. Culture tests
 Lowenstein –Jensen ( egg and also contain high concentration of malachite green
to overcome contamination with other bacteria )
 Middlebrooks 7H10 & 7H11 are (contain defined vitamins , salts , catalase , glycerol
, oleic acid and albumin to neutralize toxic effect of fatty acids )
 Liquid media such as Middle brook 7H9 broth
 culture required ( 6-8 )weeks for weeks for isolation from media
 Drug sensitivity to Anti-TB require additional 4 weeks
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Mantoux test :
Dose : 1 TU of PPD in 0.1 ml intradermally
Area : flexor surface of the left forearm , mid way
between wrist & elbow
Result : read after 48-96 hours but 72 hour is ideal
Reaction : Erythema & Induration
>10 mm = positive
< 6 mm = negative
Immunological diagnosis
(Tuberculin test )

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25. Two step testing
 Use two – step testing for initial skin testing of adults who will be
retested within 1-3 weeks
- If first test (+) ,consider the person infected
- If first test (-) , give second test 1-3 weeks later
- If second test (+) , consider person infected
- If second test (- ) , consider person uninfected
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26. 26 Newer diagnostic method
 Radiometric BACTEC 460 TB method system
 MGIT 960 Mycobacteria detection system
 MB/ BACT System
 Nucleic acid amplification test (NAAT)
 Cartridge based nucleic acid amplification test
(CB-NAAT)

27. Radiometric BACTEC 460 TB method
 Where in C labelled palmitic acid in 7H12 medium is used
 Growth is ascertained by liberation of 14CO2 as metabolized by
mycobacteria measure by BACTEC system instrument
 Average time : 8 days
 Can also be used for drug susceptibility testing
 Drawbacks :
- High cost
- Disposal of radioactive waste
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28. MGIT( mycobacterial growth indicator tube) 960
 Rapid method
 Consist of round bottom tubes containing 4 ml of modified middlebrook
7H9 broth which has an oxygen sensitive fluorescent sensor at the
bottom
 When mycobacteria grow , they deplete the dissolved oxygen in the
broth & allow the indicator to fluoresce brightly in a 365 nm UV light
 Positive signal obtain in 10 – 12 days
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29. Advantages of MGIT
 All type of specimens
 Continuously monitored
 Positive signals over 10 -12 days
 Non-radiometric
 Cheaper than BACTEC
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30. MB/ BacT system
 Non radiometric continuous monitoring system
 Based on calorimetric detection of CO2
 Slightly longer time than BACTEC ( 14 days )
 Prone to contamination
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31. Detection and identification of mycobacteria directly clinical
sample
 Genotyping methods
- PCR ( polymerase chain reaction )
- LAMP ( loop mediated isothermal amplification )
- TMA ( transcription mediated amplification )
- Nucleic acid amplification ( NAA )
- Cartridge based nucleic acid amplification test ( CB- NAAT )
 Phenotypic methods
- FAST plaque TB method
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32. Nucleic acid amplification tests (NAAT)
 Amplifying M. tuberculosis nucleic acid sequence using a nucleic
acid probe
 Sensitivity at least 80 % in most studies
 Requires 10 bacilli / ml of given sample
 Specificity 98 % to 99%
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33. Xpert MTB / RIF
 Automated , cartridge – based nucleic acid amplification test ( CB-
NAAT )
 Detect DNA sequence specific for m. tuberculosis and rifampicin
resistance
 Results are obtain from unprocessed sputum sample in 90 min
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36. Advantages – Xpert MTB /RIF
 Simultaneously detect M.tuberculosis and rifampicin resistance in less
than 2 hours
 The sensitivity for detecting TB is similar to that of to liquid culture (
sensitivity 88 % ) ; the specificity is also high(99 %)
 The superior performance of Xpert MTB / RIF in detecting TB over that
of microscopy makes it a particular useful tool for case –finding among
people living with HIV
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37. Disadvantages
 A stable uninterrupted electrical supply is needed
 The ambient operating temperature of the instrument cannot
exceed 30degree Celsius , cartridge must be stored at less than 28
degree Celsius
 The shelf – life of the cartridge must be monitored to prevent them
from expiring before they are used
 Security measure must be put in place to prevent the theft of the
accompanying laptop or desktop computer
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38. LAMP ( loop mediated isothermal amplification )
 It is a novel nucleic acid amplification method in which reagents
react under isothermal conditions with high specificity , efficiency
and rapidity
 LAMP is used for detection of M.TB complex , M.avium , and M.
intracellular directly from sputum specimen as well as for detection
of culture isolate grown in a liquid medium (MGIT ) or on a solid
medium (Ogawa’s medium )
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39.  This method employs a DNA polymerase an a set of four specially
designed primers that recognize a total of six distinct sequence on the
target DNA
 Species – specific primers were designed by targeting the gyrB gene
 Simple procedure , starting with the mixing of all reagents in a single tube
, followed by an isothermal reaction during which the reaction mixture is
held at 63celsus
 60 min incubation time
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41. Thank you …!!!
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