Diagnosis of tuberculosis by direct demonstration of the pathogen or by indirect demonstration of cell mediated immunity through activation of CD 4 and / or CD 8 T lymphocytes.

1. Recent Advances
in
Laboratory Diagnosis of Tuberculosis
Prof. Ashok Rattan,
MD, MAMS, Common Wealth Fellow, SEARO TA, INSA DFG, WHO Lab Director (CAREC/PAHO)
Advisor: Pathkind Labs Knowledge Forum, R & D and Quality,

2. A disease of great antiquity
• Fossile bones 9000 BC show evidence
• Egyptian mummies: Spinal caries 2400 BC
• 1500 BC mention of consumption in India
• Hippocrates 460 – 370 BC recognized phthisis (felt it was heredity)
Tuberculosis, Phthisis

3. Since its discovery 137 years ago TB has claimed more
than 1 billion lives
• Which is more than those claimed by (all of the below)
• Malaria
• Small Pox
• HIV/AIDS
• Cholera
• Plague
• Influenza

6. Commonest method for diagnosis of TB
1885
2019
A change in mind set is required
Mark Twain said, “ You cannot keep doing
the same thing and expect different
results”

7. Can you submit sample
likely to contain Mtb ?
Yes
Gene xpert combo
Mtb + or –
RIF R or S
Culture
In Smear positive cases
LPA 1 or LPA 2
MDR & XDR
No, submit 5 ml blood
in lithium heparin vial
Normal
Latent TB [CD4]
Recent exposure /
active TB [CD8]
Tuberculosis Clinically suspected ? Pulmonary or Extra Pulmonary ?

8. Improving detection
• CO2 production
• O2 consumption
– Radiometric
– Fluorescent
– Colourimetric
– Pressure sensor
– Decrease TAT by half
•14 C Palmitate  14 CO2

10. Speed Of Growth Of M.tuberculosis (MGIT 960 vs L J Media)
1st wk 2nd wk 3rd wk 4th wk 5th wk 6th wk
166
0
132
23
55
46
23
63
13
48
2
24
391 204
Data generated in 2007 – 08 by Dr Bansidhar Tarai under guidance of
Dr Ashok Rattan during their tenure in Religare SRL Reference Lab, Gurgaon, unpublished
Time-to-Detection in Culture Predicts Risk of
Mycobacterium tuberculosis Transmission:
Matthew K. O’Shea et al. [UK] Clinical Infectious Diseases 2014;59(2):177–85
TTD < 9 days identifies patients at high risk of transmitting tuberculosis and is superior to
sputum smear.

11. Molecular Biology Detection on clinical samples
• In the past: Polymerase Chain Reaction (PCR)
• Lab made tests
• Variable performance
• High cross contamination
• Inhibitors in clinical samples
• Real Time PCR [Cepheid]
• Closed system
• Simple sample processing
• Bacteria are made inactivated, no chance of infection
• Rapid results [2 hours]
• Detection of M tb and Rif Resistance [surrogate marker for MDR]
• Extensively field tested in different parts of the world
• 30 million cartridges used
• Cost subsidized for developing countries by Bill Clinton Foundation

12. Gene Xpert MTB/RIF
• WHO endorsed Cartridge based, closed RT PCR for
• Detection of MTB &
• RIF Resistance (as surrogate for MDR TB)
• Contains Internal quality controls for
• Sample Processing (SPC)
• Probe Check (PCC)
• Amplifies a 192 bp region of MTB rpoB gene
• RIF resistance is detected by 5 overlapping molecular beacon probes
that are complementary to entire 81 bp region of RRDR of rpoB gene

13. Gene Xpert Report
Gene Xpert MTB/RIF is Positive / Negative
Mycobacterium tuberculosis DNA Detected / Not Detected
Quantitation High / Medium / Low / Very Low
Rifampicin Resistance Present / Absent
Result:
MTB Quantitation Result Ct Range
High < 16
Medium 16 – 22
Low 22 – 28
Very Low  28
Rifampicin result result types, when MTB is DETECTED
 Rifampicin resistance Detected: a mutation in the rpoB gene has been detected that falls within the valid delta Ct setting.
 Rifampicin resistance Not detected: no mutation in the rpoB has been detected
 Rifampicin resistance Indeterminate: The MTB concentration was very low and resistance couldnot be detected.

14. The First Choice Diagnostic Laboratory

17. Antibodies tests
• From Agglutination to SAFA all techniques have been tried
• Cannot differentiate between infection and disease
• Positive even in healthy individuals
• WHO and GOI have banned the use of Antibody tests

18. In Vitro Diagnostic Tests : Cell Mediated Immunity
Antigen
presenting
cell
Memory
T-cell
Presentation of
mycobacterial antigens
IFN-
IFN-
IL-8, etc.
IL-8, etc.
TNF-
TNF-
Andersen P, et al. Lancet 2000;356:1099

20. Early Secreted Antigenic Target 6 & Culture Filterate Protein 10

21. 3rd Generation
Quantiferon TB Gold IT (in Tube method)
• Grey cap tube (Nil control)
• Red cap tube (TB Ag)
• Purple cap tube (Mitogen control)
• Collect 1 ml blood into each tube and mix well
• Transfer to Lab ASAP
• For detection of Latent TB, has negative predictive value

23. Attribute Tuberculin Skin Test
(Mantoux Test)
IGRA
Type In vivo In vitro
Sample Injected into skin (intradermal) 5 mL of Blood in Lithium Heparin tube
Visits 2 1
Antigen used PPD ESAT 6 & CFT 10 from Mtb RD1 region
Cross reaction with BCG Yes No
Use For diagnosis of LTBI Latent TB & now recent exposure of
contacts or active TB
Results Read after 48 or 72 hours ELISA test after 24 hours of exposure
Reading Subjective Objective
Booster effect Yes No
Cost Low High

24. CD 8
CD 8+ cells assessed in adults with LTBI or active TB disease by FACS
CD 8+ cells found more frequently in PTB (67%), ETB (37%) than LTBI (15%)
CD 8+ cells found in high frequency in Smear + than Smear - patients

27. Cellular Immunity plays an important role in containing Tuberculosis

29. CD8 T-cells
• Suppress Mycobacteria tuberculosis growth
• Kill infected cells
• Directly lyse intracellular Mycobacteria
• TB specific CD8 T cells that produce IFN-Ύ have been
• More frequently detected in those with active TB disease vs latent infection
• Associated with recent exposure
• Detectable in active TB subjects with HIV co-infection & young children

31. We are calling this test TB Sure to avoid any confusion
TB SURE

34. ©
In conclusion, QFT-Plus and QFT-GIT assays showed a substantial agreement
and similar accuracy for active TB detection. Interestingly, a higher proportion of the
LTBI subjects responded concomitantly to TB1 and TB2 compared to those with active
TB, whereas a selective TB2 response associated with active TB.

36. HIV + HIV --

37. 108 TB patients; 73% HIV +ve;
90 were TB Sure +ve; 11 Negative & 7 indeterminate at initiation
Sensitivity 83%

41. TB SURETB Sure®

42. Caution
• A NEGATIVE TB Sure® result doesnot preclude the possibility of
Mycobacterium tuberculosis infection or disease
• 1. Sample may have been collected before development of CMI response
• 2. Hetrophil antibodies or non specific IFN-Ύ production from other
inflammatory conditions may mask specific response to ESAT 6 or CFP 10.
• A POSITIVE TB Sure® shouldnot be the sole or definitive basis for
determining infection or disease with M tuberculosis. It should be
accompanied by addition clinical and laboratory investigations.

44. TB SURETB Sure

47. Greater Specificity for M tuberculosis exposure with TB Sure®

50. Quantiferon TB Gold Plus [TB Sure®]
• Is a blood test, provides indirect evidence of infection
• Can detect LTBI as well as recent exposure & active infection
• Can be used for monitoring of response to treatment [0, 3 & 6 Month]
• Not effected by HIV status [till CD4 count is < 100 /mm3
• Doesnot detect Drug resistance
• Gene Xpert & MGIT culture [Gene xpert combo] is preferred if bacteria
containing sample can be submitted e.g. sputum, pus, fluid, biopsy, csf

54. Can you submit sample
likely to contain Mtb ?
Yes
Gene xpert combo
Mtb + or –
RIF R or S
Culture
In Smear positive cases
LPA 1 or LPA 2
MDR & XDR
No, submit 5 ml blood
in lithium heparin vial
Normal
Latent TB [CD4]
Recent exposure /
active TB [CD8]
Tuberculosis Clinically suspected ? Pulmonary or Extra Pulmonary ?

56. TB Sure® Reports
Possible
Negative Mitogen TB 1- N TB 2- N TB 2 – TB 1 Report
IU / mL
< 8 > 0.5 < 0.35 < 0.35 < 0.5 Reference Range
0.05 4.08 0.10 0.12 0.02 Infection with M tuberculosis unlikely
0.05 7.61 4.14 3.16 -- 0.92 LTBI
0.20 1.10 4.26 9.34 5.09 Active disease / Recent Infection
0.2 0.2 0.2 0.2 0 Indeterminate
8.2 > 10 > 10 > 10 ? Indeterminate