2. TB diagnostic tests--history
• Microscopy (1880s)
• Culture (1880s)
• Chest x-ray (1930s)
• Tuberculin skin test (Mantoux-1907; PPD-
1939))
• Nucleic acid amplification tests (1990s)
• Interferon release assays (2000

3. Direct demonstration of AFB in sample
Diagnosis of TB
The key to the diagnosis of tuberculosis is a high index of suspicion.
X-Ray
Skin Test
Direct demonstration of AFB in sample
• Growth of TB bacilli in culture

4. Role of Chest X-ray
No chest X-ray pattern is absolutely typical of TB.
10-15% of culture-positive TB patients not diagnosed by X-ray
40% of patients diagnosed as having TB on the basis of x-ray alone do not have active TB

5. Administering Tuberculin Skin Test
Purified protein derivative (PPD)
TU PPD tuberculin.
Read reaction hours
after injection
Measure only induration
Record reaction in millimet

6. Factors that affect the PPD Reaction
Type of Reaction Possible Cause
False-positive Nontuberculous mycobacteria
BCG vaccination
Anergy
False-negative Recent TB infection
Very young age (< 6 months old)
Live-virus vaccination
Overwhelming TB disease

7. AFB Smear Microscopy
Microscopy is a simple convenient test Requires minimal infrastructure and equipment
• Highly accurate, inexpensive and fast .
• Accessible to the majority of patients Prioritizes infectious cases

8. AFB Smear Microscopy
Fluorescence acid-fast staining is more expensive than conventional Ziehl–Neelsen staining but is associated with a higher rate of detection because the slides can be examined faster at lower magnifications.

9. M. Tuberculosis – the organism
Acid fast sputum
Acid fast tissue
Aurimine fluorescence
Scanning electron micrograph of Mycobacterium tuberculosis

10. Limitations of Microscopy Limitations of Microscopy
Can not distinguish between dead or
live bacteria .
• High bacterial load >3000–5000 AFB 5000 AFB/mL is required for detection
• Can not do species identification •
Can not perform Drug Sensitivity Test.

11. Culture Media main types
Egg = LJG, LJP, Stone brink, Ogawa
Agar = 7H10, 7H11, Blood
Liquid = Kirchner, 7H9, 7H12, Dubos
New Types= Bactec 460, MGIT, MB BacT BACTEC 9000 MB system
Septi-Chek AFB system (Becton Dickinson)

12. Radiometric Technology
The only well established rapid radiometric method for detecting mycobacteria in clinical specimens is the BACTEC 460TB system (Becton-Dickinson Diagnostic Instruments Systems, Maryland).
This system is based on the detection of radioactive carbon-dioxide produced by bacterial metabolism of palmitic acid labelled with carbon 14.
Growth of the mycobacteria can be detected within as few as 3 days, and the mean time to detect the M. tuberculosis complex is about 14 days (87-96%)

13. Radiometric Technology
BACTEC system, which employs a superscript 14C-labeled substrate medium that is almost specific for mycobacteria. Instrument Systems, Sparks, Md. has been reported to significantly decrease the time required for detection of mycobacterial TB
BACTEC method has provided more rapid growth (average, 9 -14days), specific identification of M. tuberculosis (5 days), and rapid drug susceptibility testing (6 days).

14. Non-Radiometric Technology
BACTEC 9000 MB system (Becton Dickinson).This system uses MYCO/F medium, a modified Middlebrook 7H9 broth.(8-13 days)
The system responds to changes in oxygen concentration. Each vial contains a silicon rubber disk, impregnated with a ruthenium metal complex, which serves as an oxygen-specific sensor. Oxygen quenches the fluorescent output of the sensor.
Oxygen consumption by microorganisms can be detected by the increase in fluorescence.

15. Non-Radiometric Technology
BACTEC 960 MGIT ,MGITstands for Mycobacteria Growth Indicator Tube, and 960 indicates the total number of culture tubes it can hold at any given time
Evaluation of mycobacteria recovery from the fluorometric BACTEC 960 and the radiometric BACTEC 460 TB system have shown that they are more sensitive in recovery of mycobacteria than the conventional L-J and smear microscopy.
There is no significant difference between the radiometric BACTEC 460 TB and the fluorometric BACTEC 960 with 91.9% positivity and 95.1% positivity respectively.
Results available in 7-14 days

16. Cytokine Release Assays
QuantiFERON-TB GOLD test .
Blood samples must be processed within 12 hours after collection while white blood cells are still viable.
followed by measurement of Interferon-gamma Assays released by sensitized lymphocytes in an enzyme-linked immunosorbent assay (ELISA).
At present, the QuantiFERON-Gold TB test is recommended for screening for latent tuberculosis infection .
After incubation of the blood with antigens for 16 to 24 hours, The white blood cells will release IFN-gamma in response to contact with the TB antigens ,the amount of interferon-gamma (IFN-gamma) is measured.

17. Cytokine Release Assays
QuantiFERON-TB GOLD test
Should not give false-positive result due to:
BCG vaccination
Nontuberculous mycobacteria.
The test’s performance may be enhanced by the use the Early Secreted Antigen Target -6 (ESAT-6 ) and Culture Filtrate Protien-10 (CPF-10).

18. The Xpert MTB/RIF TB Test
Mean time for Detection of MTB
GeneXpert = < day,
Microscopy = 1 day,
Liquid culture - MGIT = 17 days,
Solid Culture = > 30 days
Mean time for Detection of Rifampicin Resistance
GeneXpert = < 1day
Liquid DST = 30 days
Conventional DST ( Solid proportional Method) = 75 days

19. How does the test work?
Detects DNA sequences specific for Mycobacterium Tuberculosis and Rifampicin resistance by PCR
Based on Nucleic Acid Amplification Test (NAAT). The Xpert® MTB/RIF
purifies
concentrates
amplifies (by real-time PCR) and
identifies targeted nucleic acid sequences in the Mycobacterium tuberculosis genome,

20. The Xpert MTB/RIF TB Test
The Xpert MTB/RIF is a cartridge-based, automated diagnostic test that can identify Mycobacterium tuberculosis  (MTB) and resistance to rifampicin (RIF).
In December 2010 WHO endorsed the Xpert MTB/RIF technology and released a recommendation and guidance

21. The Xpert MTB/RIF TB Test
The Xpert MTB/RIF is a cartridge-based, automated diagnostic test that can identify Mycobacterium tuberculosis  (MTB) and resistance to rifampicin (RIF).
In December 2010 WHO endorsed the Xpert MTB/RIF technology and released a recommendation and guidance

22. Rapid tests immunochromatographic assays
lateral-flow tests or simply strip tests
1- Add 40 µl of serum or plasma sample to the T (Test) area of the test card add 1 drop of sample to the T (Test) area .
2- Follow sample addition with 2 drops of the diluent provided in the dropper
bottle by holding the bottle vertically over the T (Test) Area.
3- Results are then read in as little as 20 minutes.

23. Rapid tests immunochromatographic assays
1- Add 40 µl of serum or plasma sample to the T (Test) area of the test card add 1 drop of sample to the T (Test) area .
2- Follow sample addition with 2 drops of the diluent provided in the dropper
lateral-flow tests (strip tests)
Results are then read in as little as 20 minutes.

24. ESAT-6 and CFP10
Mycobacterium tuberculosis-specific antigens (ESAT-6 and CFP10) in experimental animals as well as during natural infection in humans and cattle.
combination of ESAT-6 and CFP10 was found to be highly sensitive and specific for both in vivo and in vitro diagnosis.
In humans, the combination had a high sensitivity (73%) and a much higher specificity (93%) for active tuberculosis than PPD (7%).

25. Enzyme-linked immunospot assay
T-cell–based interferon-γ release assay
The ELISpotPLUS assay incorporates a novel region of difference-1 encoded antigen, Rv3879c, alongside the ESAT-6 and CFP10.
ELISpotPLUS sensitivity is 89% higher than that of the standard ELISpot.
The combined sensitivity of ELISpotPLUS and tuberculin skin testing in confirmed and highly probable cases of TB was 99%.

26. Serologic Diagnosis of Tuberculosis
ELISA measurement of Ig antibody to mycobacterial antigens
Antigen 60 IgG measurement
Antigen 38kda IgG Antigen Kp90 IgA &measurement
Antigen 60 IgG  seemed to be superior to the others (i.e., the cutoff value was justified by both the sensitivity and the specificity .
Am. J. Respir. Crit. Care Med., Volume 156, Number 3, September 1997,

27. Serologic Diagnosis of Tuberculosis
Antituberculous glycolipid antigen TBGL.
The lipoarabinomannan (LAM) polysaccharide antigen.
Antigen 60 (A60), which is derived from purified protein derivatives.
The combination of LAM, A60, and TBGL appears to be the best choice of antigens for the serodiagnosis of TB

28. Chemical Detection of Biologic Compounds
Adenosine deaminase, a host enzyme produced by activated T cells and easily detected by a colorimetric procedure, was shown to increase in concentration during the active stages of tuberculous meningitis and to decrease to normal levels after effective antituberculosis therapy.
A more complicated technology detects the presence of tuberculostatic acid in the spinal fluid or serum of patients.

29. Gen-Probe AMPLIFIED TM
The MTT&MTD are chemical tests, the amplification is to produce sufficient nucleic acid, within a few hours, these tests can recognize MTC in an AFB-positive specimen, with nearly 96% sensitivity and 100% specificity.
The NAA result can be falsely negative if there are very few tubercle bacilli,
NAA test can amplify DNA from both viable and non-viable organisms.

30. Gen-Probe AMPLIFIED TM
Polymerase chain reaction (PCR)
Yield 95% of smear+ and only 50% of smear negatives.
main advantages: speed + sensitivity
sensitivity :
in principle able to pick up 1 TB bacillus
in practice : less sensitive than culture
Serve only the diagnosis not monitor the treatment outcome
Cannot replace culture
Not able to determine infectiousness.
Very expensive ($50-$100 per assay)

31. Real-time Polymerase Chain Reaction Techniques
Real-time PCR methods are based on hybridization of amplified nucleic acids with fluorescent-labelled probes spanning DNA regions of interest and monitored inside thermal cyclers.
The main advantage of real-time PCR methods is its speed in giving results, h after DNA extraction.

32. Non-molecular Techniques
The FastPlaque Tuberculosis Assay
The FastPlaque TB assay , relies on the ability of M. tuberculosis to support the growth of an infecting mycobacteriophage. The assay have shown a sensitivity of 50-65% in smear-negative specimens with specificity of 98% .
It is a rapid, manual test, easy to perform and has a higher sensitivity than microscopy, in newly diagnosed smear +ve pts.
Int J Tuberc Lung Dis 1998;2: 160