TB Diagnosis

1. Diagnosis of
Tuberculosis,
Tuberculin skin test,
New techniques
Dr. Ajaypal Singh
Junior Resident
Pulmonary medicine
Gmc patiala

2. Definition of presumptive TB
• Presumptive pulmonary TB : Refers to person with
any of the symptoms and signs suggestive of TB including:
1) Persistence Fever > 2 Weeks ,
2) Weight loss
3) Night sweats
4) Cough for > 2 weeks
Any pulmonary abnormality in chest radiograph
• Presumptive Extra pulmonary TB – Refer to as organ
specific symptoms and signs like Swellings of lymph node,
Pain and Swelling Joint, Neck stiffness, Disorientation etc

3. METHODS OF DIAGNOSIS OF
PULMONARY TUBERCULOSIS
1)DIRECT METHODS: Detects mycobacteria
and its products
2)INDIRECT METHODS : Antigen and
Antibody Detection
3)RADIO-DIAGNOSIS :CXR,CT AND MRI

4. DIRECT METHODS
1)Direct Microscopy -ZN stain ,Flourence
microscopy.
2)Culture -Traditional, Rapid methods.
3) molecular methods – CBNAAT ,LPA

5. • INDIRECT METHODS
• 1)Antibody detection : Banned by GOI
 TB STAT-PAK
 ELISA
 Insta test TB
• 2) Antigen detection : Banned by GOI
 TB MPB 64 patch test.
 Quantiferon-GOLD test.
• 3) Biochemical Assays :
 ADA

6. Antibody Detection Test : Banned by GOI in june 2012
• A60 is the antigen used for both pulmonary and extra
pulmonary, adult and childhood TB.
• sensitivity of these test has ranged between 30 to 100 per
cent.
• presence of antibody does not indicate current disease or
past infection.
• presence of antigen may be a better indicator of the disease
than the antibody
• However, antigen quantity in circulation is usually very limited
and masked by the antibody and hence difficult to detect

7. Antigen Detection Test
1) Lipoarabinomannan Urine Test : Lipoarabinomannan
is a component of the TB bacterial cell wall.
• The test exists in ELISA and simplified “tube” format.
2) Flow-through Filter Tests : detection of
Mycobacterium tuberculosis in sputum or body fluids
with a polyclonal antibody, using a flow-through device.

8. Sputum smear microscopy
• Use of microscopy in diagnosis of TB is of
paramount importance, as culture takes a long
time before the results are ready.
• 10,000 AFB /ml required.
• -Sensitivity = 50%
• The tubercle bacilli are Gram positive but they do
not take the stain readily.
• Mycobacteria retain the primary stain even after
decolourization with acid alcohol; hence the
term as “acid-fast”.

9. • A counter-stain is employed to highlight
the stained organisms for easier
recognition.
• In the carbol-fuchsin [Ziehl-Neelsen] ,
acid-fast organisms appear red against a
blue background.
• Acid fastness is based on the integrity of
the cell wall.
• Cell wall of Myc. Tuberculosis is made
up of MYCOLIC ACID which connect
outer Arabinoglactan and inner
peptydoglycan.
• These form a dense pallisade, arranged
in rope-like structure, which gives the
cell wall its thickness and is largely
responsible for acid fastness

13. Fluorescent Staining
• Ziehl-Neelsen staining is a time
consuming process for staining
as well as examination.
• Fluorescence microscopy is
recommended where > 50
smears are examined per day,
and if electricity is
continuously available.
• Fluorescence staining utilizes
basically the same approach as
Z-N staining, but carbol fuchsin
is replaced by a fluorescent
dye .

15. Grading for FM
1 length =40 fields
Negative Zero AFB/1 length
scanty 1 – 19 AFB / 1 length
1+ 20 -199 AFB /1 length
2+ 5 – 50 AFB /1 field
3+ > 50 AFB/1 field

16. Value of Smear Examination in Extra-pulmonary
Specimens
 Benefit of microscopy in these specimens is limited because of their pauci
bacillary nature and it is, therefore, recommended that the extra-
pulmonary specimens be referred for culture and other molecular
techniques.
 Various extra pulmonary specimen includes :
1 ) Gastric Washings
2) Laryngeal Swabs
3) Pus and Thick Aspirates
4) Pleural and Pericardial Fluid
5) Cerebrospinal Fluid
6) Urine
7) Ascitic fluid
8) lymph node FNAC
9) Blood

17. ISOLATION OF MYCOBACTERIA BY
CULTURE
• Provide definitive diagnosis by establishing the viability and identity of
the organisms
• Distinguish between different mycobacterial species as well as to
perform drug susceptibility tests.
• M. tuberculosis proliferates extremely slowly [generation time 18 to 24
hrs ].
• Detect as few as 10 to 100 bacilli per ml of sputum.
• M. C used medium is L-J medium. It contains eggs, asparagine, glycerol
and some mineral acids
Cultural Characters : Growth appears in about two weeks
but may be delayed up to six to eight weeks.
• Optimum temperature for growth is 37 °C.
• Increased [CO2] tension [5% to 10%] enhances growth

18. Culture Media

19. Culture Colony Characteristics
• tubercle bacilli give rise to discrete, raised, irregular, dry and wrinkled
colonies which may be white in colour in begning to buff colour .
• Virulent strains form long serpentine cords in the liquid media while
avirulent strains grow in a more dispersed fashion.
• The clinical specimen after concentration, is inoculated onto two bottles
of L-J medium and incubated at 37 °C.
• Cultures are examined initially after 7 days to rule out the presence of
rapid growing mycobacteria.

20. • Negative result is given, if no growth appears after eight weeks.
• Z-N stained smear made from the same is examined and routine
biochemical tests put up.
• All cultures should be examined 3 – 4 days after inoculation to
detect gross contaminants.
• Thereafter cultures are examined weekly.
• With doubtful cultures, the acid-fastness should be confirmed by Z-
N staining.
• Diagnosis for sputum positive material is obtained within a week
while for negative results incubation has to continue for six weeks.
• culture system has also been used for drug susceptibility testing for
the first-line drugs.
• DST is set up after identification tests
• Takes 7-9 weeks for DST.

23. Rapid Culture Methods
BACTEC radiometric system : measure radioactive CO2 liberated
during decarboxylation of 14C labelled substrates.
• BACTEC 12B vial is inoculated, mycobacteria, utilize the 14C labelled
substrate [palmitic acid] and release 14CO2.
• BACTEC instrument measures quantitatively the radioactivity in terms of
numbers on a scale ranging from 0 to 999, designated as the growth index
[GI].
• The daily increase in GI is directly proportional to the rate and amount of
growth in the medium.
• By adding inhibition agents inhibition of metabolism is indicated by
reduced production of 14CO2, which in turn is indicated by decrease in GI.
• This basic principle is utilized in isolation of mycobacteria and drug
susceptibility testing in this method.
• Not used due radioactive hazards.

25. Rapid Liquid Tuberculosis Culture
• Also known as Mycobacteria
Growth Indicator Tube [MGIT].
• culture is done using manual or
automated systems in which tubes
contain enriched Middlebrook 7H9
broth and an oxygen sensitive
fluorescent sensor embedded in
silicone on the bottom of the tube.
• The presence of oxygen dissolved in
the broth quenches emissions from
the compound.
• As the actively growing and
respiring mycobacteria consume
the dissolved oxygen, the sensor
glows indicating mycobacterial
growth.
• The fully automated version can
incubate up to 960 samples.

26. • Higher recovery rates especially in smear negative
specimens.
• DST –4-14 days
• Negative –42 days
• Can be made to read out negative by 28 days
• Manual version also available.
• MPT64 protein detection-based immunochomatographic
test is applied for confirmation of myco. Tuberculosis.
• If culture became positive and myco. Tuberculosis
detected on MPT64 then result given as culture positive
and MTB detectected.
• If culture became positive and MTB not detected on MPT64
then result given as culture positive and MTB not detected.

27. MOLECULAR DIAGNOSIS OF
TUBERCULOSIS
• Rapid and sensitive tools for the diagnosis of
tuberculosis are needed, due to the increased
incidence of tuberculosis epidemics and the length
of time required by classical diagnostic tests,
especially among human immunodeficiency virus
(HIV)-infected patients.
• In this context, the recent advances in cloning and
characterization of M. tuberculosis genes has
allowed the application of basic molecular biology
techniques to the examination of clinical samples,
such as sputum and bronchoalveolar lavage (BAL),
for the molecular diagnosis of tuberculous infection.

28. Polymerase Chain Reaction
Sequencing
• The most widely used method for defining genetic resistance
for drug sensitivity testing.
• commonly used for characterising mutations in the rpoB gene
in rifampicin resistant strains and to detect mutations
responsible for other antituberculosis drugs.
• This process is repeated sequentially 25-40 times, thereby
creating millions of copies of target sequence. The amplified
sequence can then be detected by agarose gel electrophoresis

29. Ingredients of Polymerase Chain
Reaction
• Primers: µM 0.1-0.5
• Deoxy-nucleotides triphosphate (dNTPs): µM 200-
250nucleotides
• Co-factors:
1. Cations: MgCl2 mM 1.5-6
2. Buffer pH 8.3-8.8
• DNA polymerase: 0.5-2.5 U
• Target DNA:  1 µg

30. Three steps of PCR: Denaturation,
annealing and extension

31. Role of PCR in pulmonary TB
• Useful technology for rapid diagnosis of smear –ve cases
of active TB.
• Able to identify 50-60% of smear -ve cases; this would
reduce the need for more invasive approaches to smear -
ve cases
• Distinguish M.tb from NTM in smear +ve cases as IS6110
sequence is not found in NTM.
• Should not be used to replace sputum microscopy.
• Sensitivity, specificity, & for PCR is 83.5%, 99% &
respectively.

32. Disadvantages
• Very high degree of quality control required.
• Variation from lab to lab remain significant.
• In pts. on ATT, PCR should not be used as an
indicator of infectivity as this assay remains
+ve for a greater time than do cultures
• High false +ve results in patients previously
treated with ATT in contacts of sputum +ve
active cases.As it can detect dead bacteria.
• High Cost

33. Geno Type Assays
1. Two Geno Type assays :For TB diagnosis
[Geno Type Mycobacteria Assay] CBNAAT
2. For detection of rifampicin and isoniazid
resistance [Geno Type MTB DR Assay].LPA

34. Xpert MTB/RIF assay (CBNAAT-
cartridge based NAA test
• Automated PCR diagnostic test that can
detect presence of M.tb & resistance to
RIFampicin(by detecting mutation in 81bp
region)
• It is automatic , fast & sensitive
• Accurate diagnosis is obtained in 1hr 45
mins by adding a reagent to the sputum
sample & 15mins later its pippeted into a
cartridge that is inserted into the
diagnostic instrument.
• Sensitivity for PTB in adults= 88%
• (98% for smear positive TB, 68% for smear
negative TB).

35. CBNAA- Limitations
• They are able to detect nucleic acids from
both living and dead organisms so in pts on
ATT, CBNAAT should not be used as an
indicator of infectivity as this assay remains
positive for a greater time, than do cultures
• NAA should always be performed in
conjunction with microscopy and culture .
• Used only for diagnosis and not for follow ups.

37. LPA(Line Probe Assay)
• It is a genotypic method use PCR and reverse
hybridization with specific oligonucleotide probes
fixed to nitrocellulose strips in parallel lines.
• Resistance targets are
1. rpoB for RIF
2. Kat G and inhA for INH.
• higher sensitivity for H resistance
Both H&R
• Sensitivity for R is >95%, H is 70-80 %
• Specificity for R & H is 98%
• Results within 48 hours

38. Differences types
Hain GenotType
MTBDRplus
It targets 23s rRNA
gene space region.
It detects both R and
H resistance
• InnoLiPA Rif
• It targets 16s - 23s
rRNA gene space
region.
• It detects only R
resistance

40. CONCLUSION:
1) LPA is applicable on sputum+ve specimens only.
2) Gene Xpert can’t monitor progress of treatment.
3) HENCE PHENOTYPIC CULTURE REMAINS THE GOLD
STANDARD

42. Interferon-gamma release assays for
latent tuberculosis infection
• Banned by GOI in 2012
• markers of Myc. TB infection.
• Indicate a cellular immune response to M.
tuberculosis.
• Cannot distinguish between latent infection and
active TB disease.
• should not be used for diagnosis of active TB.
• A positive result not necessarily indicate active
TB
• a negative IGRA result may not rule out active
TB.

43. Blood Assays for M. tuberculosis
• 1) QuantiFERON®-TB Gold In-Tube (Banned by GOI)
 Measures Interferon-gamma (IFN-y)
• 2) T-SPOT.TB
 Measures peripheral blood mononuclear cells that
produce IFN-γ

44. Loop-mediated isothermal
amplification[ LAMP]
• LAMP is used for detection of M.tb complex,
M.avium, and M.intracellulare directly from
sputum specimens as well as for detection of
culture isolates grown in a liquid medium
(MGIT).
• Simple procedure, starting with the mixing of
all reagents in a single tube, followed by an
isothermal reaction during which the reaction
mixture.

46. ADVANTAGES:
Due to its easy operation without
sophisticated equipment, it will be simple enough
to use in:
• Small-scale hospitals,
• Primary care facilities
• Clinical laboratorie in developing countries.
Difficulties :
• Sample preparation
• Nucleic acid extraction
• Cross-contamination

47. Microscopic-Observation Drug-
Susceptibility Assay[MODS]
• facilitates the detection of Mycobacterium tuberculosis,
directly from the sputum.
• This innovative technique utilizes a liquid medium which
facilitates faster growth of the TB bacillus and thereby aids in
the early microscopic visualization of characteristic cord
formation.
• incorporation of drugs allows rapid and direct drug-
susceptibility testing concomitantly with the detection of
bacterial growth.

48. IMMUNOLOGICAL MAREKER
Adenosine deaminase : enzyme catalyzing the deamination
reaction from adenosine to inosine.
• 2 isoforms of ADA.
• ADA-1 - many tissues including red blood cells.
• ADA-2 - only in macrophages and monocytes.
• ADA acts in proliferation and differentiation of lymphocyte,
especially T lymphocyte.
• If the pleural fluid ADA level is berween 40 and 70 U/L and the
patient has a lymphocyte-to-neutrophil ratio of more than
0.75, a presumptive diagnosis of TB can be made.
• If the patient's pleural fluid ADA level is below 40, the
diagnosis of TB is unlikely.

49. Interferon-γ :
• produced by the CD4 lymphocytes from
patients with tuberculous pleuritis
• cutoff level of 3.7 IU/mL yielded a sensitivity
of 0.98 and a specificity of 0.98
• measurement of IFN-γ levels in pleural
effusions is also likely to be a useful tool for
diagnosis of TB pleurisy.

50. Tuberculin Skin Test
• Tuberculin skin test [TST] is mainly used to detect infection
with tubercle bacilli.
• Test is based on the fact that infection with Mycobacterium
tuberculosis produces sensitivity to certain components,
which are contained in culture extracts called tuberculins.
• However, has a limited value for the diagnosis of TB disease
among adults living in areas where TB is highly endemic
• The interpretation of TST is complicated by cross-sensitivity to
tuberculin induced by infection with environmental
mycobacteria or by bacille Calmette-Guerin [BCG] vaccination

51. HISTORICAL BACKGROUND
• Sir Robert Koch : produced a filtrate prepared from heat
sterilized concentrated broth cultures of human tubercle bacilli, in
the late 19th century.
• This product named as old tuberculin [OT].
• OT was gradually replaced by a low dose, intradermal test using a
purified protein derivative [PPD] that was well tolerated.
• Clement Von Pirquet observed in 1907 that a tiny scratch
with a little quantity of tuberculin resulted in a local reaction at the
test site.
• Moro in 1908 announced his patch test, where tuberculin was
incorporated into an ointment that was smeared onto the skin, with
a piece of gauze over it

52. • Charles Mantoux developed the intradermal test to be
administered by injection as a measured volume
[Mantoux test].
• Heaf test which used a simple instrument, that caused six
spring loaded needles to pierce the skin with a drop of
undiluted OT.
• Tine test was developed as a disposable multiple
puncture test where the tuberculin was introduced into
the skin by puncture with four tines coated with dried
tuberculin.
• most of these tests have become obsolete and only the
Mantoux technique which allowed quantitative
measurement has stood the test of time and is now the
standard method of administration of the TST.

53. IMMUNOLOGICAL BASIS OF
TUBERCULIN TEST
• Individuals infected with Mycobacterium tuberculosis respond with
delayed type hypersensitivity [DTH] to the TST.
• The sensitization is induced by natural mycobacterial infection or by
vaccination with BCG, a live attenuated mycobacterial strain derived
from Mycobacterium bovis.
• Clinically, it is a manifestation of previous infection with tubercle
bacilli or a variety of nontuberculous mycobacteria [NTM].
• Injection of the tuberculin antigen leads to migration and
proliferation of the sensitized T-cell lymphocytes to the test site.
These T-cells release cytokines and chemokines, which further
attract other lymphocytes and monocytes.
• These reactions along with increased permeability of the local
blood capillaries lead to an induration at the test site

54. SKIN CHANGES IN TUBERCULIN SKIN
TEST
• characteristic features : include a delayed course reaching a
peak more than 24 hours after injection and an induration
with occasional vesiculation and necrosis..
• DTH reaction peaks at 48 to 96 hours with an area of
erythmatous induration, which resolves in a week’s time.
• size of this induration is, thus, maximal between 48 to 96
hours after the test
• Product and Dosage :Standard test employs a single batch
tuberculin, i.e., PPD RT-23
• However, 2 TU of PPD RT-23 is now recommended as the
standard dose.

56. Administration of Test
• given on the mid-volar aspect of the forearm since this area
is usually hair free.
• The skin is lightly stretched and the needle point is inserted
with its bevel facing upward into the superficial layers of the
skin and 0.1 ml of tuberculin is injected slowly.
• hold the syringe only by the barrel and not to touch the
plunger before the needlepoint has been satisfactorily
inserted into the skin.
• A satisfactory test should raise a flat pale pea-sized wheal
with clear pits of hair follicles and a well demarcated border
and without leakage of tuberculin.

58. Reading of the Test
• Result is read between 48 to 96 hours after the test in good day
light.
• keeping the forearm flexed, by carefully palpating the site of
injection using one finger
• induration may be easily recognized as a firm well circumscribed
density.
• Small indurations may be missed if not sought carefully with a light
touch.
• Marked with the ballpoint pen and the maximum transverse
diameter is measured in ‘millimetres’ [mm] with a transparent ruler.
• test result should never be recorded as ‘positive’ or ‘negative’ and
must always be recorded in ‘mm of size’.
• Record should also be made of vesicles, bullae, lymphangitis,
ulceration and necrosis at the test site. B

62. False positive reaction
• Infection with non tubercular mycobacterium.
• Previous BCG vaccination
• Incorrect method of TST
• Incorrect interpretation of reaction
• Incorrect bottle of antigen used
False negative reaction
• Cutaneaus anergy (inability to react to antigen due to
weak immune respone)
• Recent TB infection
• Very old TB reaction
• Very young age(<6 months)
• Recent live virus vaccination
• Overwheling TB disease
• Incorrect method of TST
• Incorrect interpretation

64. New techqnies
• URINE-BASED SCREENING [stamp trial] Rapid
urine-based Screening for Tuberculosis to
reduce AIDS-related Mortality .
• February 2015 to January 2018.
• The overarching aim of this work is to
determine whether use of a fundamentally
new approach to rapid diagnostic screening
for HIV-associated tuberculosis ..

65. Trunat tb test
• The TrueNat TB test is a new molecular test
that can diagnosis TB in one hour as well as
testing for resistance to the drug rifampicin.
• This test for TB uses a sputum sample taken
from each patient. Only about 0.5 ml of the
sample is required compared with about 1 ml
needed for the American GeneXpert machine

66. Urease activity of M. tuberculosis
• M. tuberculosis urease is a likely bacterial
virulence factor, which may subvert the host
acidification of the phagosome
microenvironment.
• It may used as suportive diagnostic technique.

67. Diaskintest
• Diaskintest is an innovative skin test for the mass
screening of tuberculosis.
• Diaskintest belongs to the IGRA tests, which are
the most promising line in the diagnostics
of infectious diseases. Diaskintest is highly
specific, and does not show false-
positive reactions in BCG-vaccinated persons.
Diaskintest allows the identification of only
a group of individuals with active tuberculosis
infection or those at high risk of TB, minimizing
the rate of false-positiveresults.