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DIAGNOSIS
CLINICAL
LAB
LAB DIAGNOSIS
1.PUL TUBERCULOSIS
•SAMPLE COLLECTION
•MICROSCOPY
•CONCENTRATION METHODS
•CULTURE
•SENSITIVITY TESTS
•ANIMAL INOCULATION
•NUCLEIC ACID TECHNOLOGY
•IMMUNODIAGNOSIS
Sputum
•Samples collected---Laryngeal swabs
Bronchial washings
Gastric lavage
•Best collected-in morning before meal
•If scanty-24hr sample taken
MICROSCOPY
By Ziehl-Neelsen staining
ZN smear evaluation and AFB report
No. of AFB
0
1-2
Seen in
300 fields
300 F
1-9
1-9
1-9
10 or more
100 F
10 F
1F
1F
report
AFB not seen
Doubtful,
repeat smear
1+
2+
3+
4+
By fluorescent microscopy
Advantages
•Reliable method
•Rapid screening for epidemiological purpose
Disadvantages
•Has only 30-70% sensitivity
Concentration methods
For microscopy
For smear,culture &
animal inoculation
MYCOPROSAFE
CULTURE
Conc material into IUAT-LJ medium
Incubation at 37○C
Examine twice weekly
growth
No growth
_ve report after 8-12 wks
 Absolute conc method
In which no. of media containing serial conc of
drugs are inoculated(for MIC)
 Resistance ratio method
2 sets of media containing graded conc of drugs
are inoculated
 Proportion method
Indicates avg sensitivity of strain
Antibiotic sets for sensitivity testing.
Conc specimen
Inoculated IM into thigh of 2 healthy guinea pigs
12 weeks old
• Progressive loss of wt
• Infected animals show + tuberculin test
• 1 animal is killed after 4 weeks & autopsied
• On autopsy
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Caseous lesion at site of inoculation
Draining &internal LN are enlarged & caseous
Spleen enlarged with irregular necrotic areas
Tubercles seen in peritoneum

DISADVANTAGES:
• Cumbersome
• Costly
• Less sensitive than culture
 SAMPLES COLLECTED
• CSF
• Bone marrow &liver biopsy
• Blood
• Pus
• Pleural effusion--- exudate
• urine
•
CMI….manifests as
1. Delayed hypersensitivity
2. Resistance to infection
s/c injection given to a normal guinea pig
No immediate response
After 10 – 14 days
Nodule at site
Breaks up to form ulcer
Persists till animal dies of progressive TB
KOCH PHENOMENON
Guinea pig inj which has prior contact 4-6 wk before
After 1-2 days indurated lesions at site
Next day necrosis which forms shallow ulcer
Heals rapidly without involving draining LN
3 COMPONENTS----LOCAL REACTION,
FOCAL RESPONSE,
SYSTEMIC RESPONSE
ALLERGIC TESTS
• OT
•PPD - S
•1 TU= 0.01 ml of OT or 0.00002 mg of PPD-S
MANTOUX TEST
 METHOD
INTERPRETATION OF RESULT
HEAF TEST
•Multiple puncture testing
•For screening & surveys
Tine test
Disposable prongs carrying dried PPD is used
FALSE NEGATIVES
Miliary tuberculosis
Convalscents from viral infections like Measles
Lympho reticular malignancy
Immuno suppressive therapy
Inactive PPD preparation
Improper injection technique
FALSE POSITIVES
Infection or exposure to atypical mycobacteria
BCG vaccine
USES
Aids in diagnosing active infections in infants &
young children
To measure prevalence of infection in an area
To select susceptibles
As an indication of successful vaccination
Recent advances in detection of M.tb
1.Bactec 460 TB system
•Broth based growth system
•Medium contains 4ml of Middlebrook 7H12 broth
with carbon 14 labeled palmitic acid
•Clinical specimen inoculated with PANTA
•Use radiometric method for growth detection
BACTEC TB Instrument
ADVANTAGES
• rapid method
•Increase no. of +ve cultures
•Antibiotic sensitivity testing can be done
DRAWBACK—very expensive
2.Rapid evaluation of drug susceptibility by
bioluminescence assays
medium alone (+) or with INH
(H), rifampin (R), or ethambutol
(E). Erdman susceptible
reference strain (ERD), a CDC
INH-resistant strain (CDC-K),
and clinical monoresistant (Z)
and multidrug resistant (DS)
strains.
•Advantage---testing drug susceptibility
•Drawback---expensive & requires expertise
3.Serological tests
•To measure IgG antibody---ELISA
• Ag-capture ELISA test---for mycobacterial
lipoarabinomannan Ag
4.PCR-based tests
Detection of PCR
product of M.
tuberculosis gene on
polyacrylamide gel
electrophoresis. The
lane numbers are as
follows: lane M marker
of 50-bp ladder; lane
1,2,3, positive ; lane 4,
negative; and lane 5,
negative control.
Automated nucleic acid
amplification test
PCR-based.
For DNA extraction and
amplification and
detection of TB DNA and
rifampin-resistance
encoding mutations.
Restriction Fragment Length Polymorphism
(RFLP)
•Cutting by restriction enzymes
•Gel electrophoresis
Uses
•To know how many cases are due to reactivation of
latent infection &how many are recent transmission
•Epidemiological typing of strains
RFLP patterns.
Cluster I comprises
strains
9, 12, 16, 28, 38, 14, a
nd 40; cluster II,
strains 3, 7, 8, and
20; cluster III, strains
31 and 24; cluster IV,
strains 1 and 13; and
cluster V, strains
37 and
36. Mt, M. tuberculosi
s MT14323 reference
strain.