1. DR.RAGHAVENDRA.H.GOBBUR
PROFESSOR OF PEDIATRICS
B.L.D.E.UNIVERSITY’S Shri.B.M.Patil MEDICAL COLLEGE ,BIJAPUR

2. Newer modalities in TB diagnosis
Guest lecture given at state PEDICON 2011
DR.RAGHAVENDRA.H.GOBBUR
PROFESSOR OF PEDIATRICS
B.L.D.E.UNIVERSITY’S
Shri.B.M.Patil MEDICAL COLLEGE ,BIJAPUR.

3. Newer modalities in TB diagnosis
IGRA assay Interferon (IFN)-γ Assay
Microscopy LED
Culture :Liquid Medias: BACTEC,
BAC T/ALERT 3D, MGIT
DNA NAAT: Real-time PCR, LIPA
Enzyme Assay: ADA

4. QUANTIFERON-TB GOLD in place of Mantoux test .
INTERFERON G ASSAY (IGRA)
IFN-γ

5. QUANTIFERON-TB GOLD . IN VITRO TEST
ESAT-6 ,CFP-10 synthetic peptides are used
(Absent in BCG and most NTM)
stimulate T-cells from infected people
releasing IFN-γ, from These T-cells
*early secretory antigenic target-6
**culture filtrate protein-10
LTBI V/S DISEASE ?
TST V/S IGRA

6. QUANTIFERON-TB GOLD . Objective , and controlled test
Reduces subjectivity in TB diagnosis
Simple diagnostic cut-off (> .35 IU/ml IFN-γ = + )
Straight forward positive/negative interpretation
Eliminates 2 step testing
No ‘booster’ effects in-vitro
Faster turn-around, results in hours
Results are electronic (computer generated reports)

7. INDIAN STUDY USING QUANTIFERONTB GOLD
Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold AL, Colford JM Jr, Riley LW, Pai M. Comparison of a
whole blood interferon-gamma assay with tuberculin skin testing
( J Infect 2007; 54:267–76.)
Compared QFT to the TST in 105 children ( suspected of TB, or had contact with an index case).
11 children (10.5%) were QFT positive, whereas the
TST was positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at
≥15mm .
Concordance of TST with QFT was high (95%) at the 10mm TST cut off
All subjects with≥15mm TST , were QFT positive.
There were
no indeterminate QFT results, despite 40% children being <4 years old , and 57% of them being malnourished.

8. SUMMARY of IGRA TESTING IGRAs are recommended for 1
SUMMARY of IGRA TESTING IGRAs are recommended for 1. Contacts of active TB Close contacts (HIGH RISK) TST OR IGRA if either is positive, treat for “L TB I” (latent infection) Casual contacts (LOW RISK) can have IGRA confirmation if TST is positive to verify infection v/s BCG or MOTT 2. Immune compromised “suspected child" TST first, if negative do IGRA and if IGRA positive treat as LTBI

9. M.TB. STAINING BY ZEIL-NEILSON STAIN
>1,000 Organism per ml sputum required for ordinary microscope.
Fluorescent, LED microscope detects even 100 M.TB. organism per ml

10. AFB + SPUTAM SMEAR

11. FIND and Carl Zeiss fluorescent LED microscope based on the proven Primo Star platform.( FIND/Zeiss microscope offers superior optics, reflected light illumination, easy switch from bright field to fluorescent light) 11

12. MYCOBACTERIAL CULTURE
Culture remains the gold standard for lab confirmation of TB
Advantages:
Increases number of case detection
Detects cases among smear negative patients
Establishes viability of organisms
Distinguishing between Mycobacterial species
Helps in performing DST (drug sensitivity test)
Helps in diagnosing cases of treatment failure
Limitations:
Expensive
Require enriched media
Require considerable expertise
Time consuming

13. Processing of sputum with CPC Method
If delay of more than 48 hours between collection and processing is anticipated, the sputum should be collected with 1%CPC and 2%NaCl2
CPC acts as homogenizing and decontaminating agent
It helps in retaining viability of Tubercle bacilli up to days
These specimens should not be treated with NaOH
( Petroff’s)

14. Culture: Extra-Pulmonary Samples
Aseptically collected samples
Body fluids:
Spinal ,Pleural, Pericardial, Synovial, ascitic, Blood, Pus & Bone marrow
Tissues:
Lymph node, Needle biopsies or Tissue biopsies
Specimens known to contain contaminating flora:
Gastric lavage, Bronchial washings & Urine

15. L J MEDIA

16. CORD LIKE GROWTH OF M.TB. IN MEDIA

17. NEWER CULTURE METHODS for M.TB.
Microscopic Observation of Broth Culture &
MODS Micro Colony Detection System (slide culture)
Septi-check AFB : Non radiometric, Non automated
MGIT : Automated. monitors every 60min O2 utilization, Intensification of O2 quenching fluorescent dye
MB/BAC T - ALERT : Non radiometric, colorimetric detection of CO2
BACTEC Radiometric

18. BACTEC 460 TB System(radio metric)
Developed in 1969 by Deland and Wagner.
Principle
BACTEC 12B vial , utilize 14C labeled substrate (fatty acid)
On inoculation, mycobacteria, grow & release 14CO2.
The BACTEC instrument measures quantitatively the radioactivity on a scale ranging from 0-999, as GI.(Growth Indicator)
The daily increase in GI is proportional to growth in the medium.
DST Drug Susceptibility Test
When ATT is introduced in the medium
reduced production of 14CO2 and decrease in GI.

19. MB BACT-ALERT 3D LIGHT EMITTING SENSORS

20. The MGIT 960 System
The MGIT 960 system is a non-radiometric automated system that uses the MGIT media & sensors to detect the fluorescence.
Advantages:
-The system holds 960 plastic tubes which are continuously monitored.
- Early detection with the machine monitoring & reading the tubes every hour.

21. II Mycobacteria Growth Indicator Tube (MGIT)
Tube contains modified Middle brook 7H9 broth base with OADC enrichment & PANTA antibiotic mixture.
All types of clinical specimens, pulmonary as well as extra-pulmonary ( except blood ) could be cultured on this type of media.

22. The OADC supplement O ----- Oleic acid ( Metabolic stimulant)
A Albumin ( to bind toxic free fatty acid )
D ---- Dextrose (Energy source )
C Catalase ( Destroy toxic peroxides that may be present in the medium )

23. The PANTA antibiotic mixture
P Polymyxin B
A ---- Amphotericin B
N ---- Nalidixic acid
T ---- Trimethoprim
A Azlocillin
+/- Vancomycin
The antibiotic mixture inhibits the growth of contaminating bacteria.

24. Principle of the procedure:(MGIT)
A fluorescent compound (which is sensitive to O2) is embedded in silicone on the bottom of the tube.
The actively respiring microorganisms consume the oxygen & allow the fluorescence to be observed using UV trans-illuminator lamp.

25. III Polymerase Chain Reaction (PCR) & Gene probe
Nucleic acid Amplification Tests polymerase enzymes amplify specific DNA sequences, using Nucleic acid probes, using DNA extracted from MTB in the sample.
Advantages:
- Rapid procedure ( 3 – 4 hours)
- High sensitivity (1-10 bacilli / ml sputum)
CDC recommends NAAT for all suspected TB cases

26. PCR ASSAY
The thermal cycling, DNA melting separates the strands of DNA double helix at 95°C Heat-stable DNA Taq polymerase, ( Bacteria Thermus aquaticus.) Enzymatically assembles new DNA strands(selectively amplify ) using DNA primers ( DNA oligonucleotides.) & template(each strand) at 55 °C The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

27. RR

28. REALTIME-PCR ASSAY
A TB specific primer and probe mix is provided and this can be detected through the FAM channel.
The primer and probe mix exploits the so-called TaqMan® principle.
During PCR amplification, forward and reverse primers hybridize to the TB DNA/Cdna
. A fluorogenic probe is included in the same reaction mixture , it consists of a DNA probe labelled with a
5`-dye (reporter) and a 3`-quencher. (5’3’DQ)
During PCR amplification, the probe is cleaved and the Reporter dye and Quencher are separated.
The resulting increase in fluorescence can be detected on a range of real-time PCR platforms

29. PCR ASSAY
The PrimerDesign™ genesig Kit for Mycobacterium Tuberculosis (TB) Genomes is designed
for the in vitro quantification of TB genomes.
The kit is designed to have the broadest detection profile possible whilst remaining specific to the TB genome.
The primers have 100% homology with all other reference sequences in the NCBI database.
Fig 1 Accession numbers for detected TB isolates.
CP , CP , AM , U , AE , BX , BX ,
BX , BX , U , U , AF , BX , BX ,

31. Disadvantages: - Very expensive.
- Require specialist training & equipment.
- False positive results.( CONTAMINATION)
Can not differentiate between living & dead bacilli. .
Sputum specimens (3%--7%) might contain inhibitors that prevent or reduce amplification and cause false-negative NAA results. 

32. RAPID RECOGNITION OF DRUG RESISTANCE
PCR PROBES ARE AVAILABLE
KAT-gene INH RESISTANCE
RPO gene RIFAMPICIN RESISTANCE
GYR –A FLUROQUINOLOE RESISTANCE
LIPA( Line Probe Assay ) amplified DNA is applied to strips with probe for M.TB. And Rif. resistance

33. MODS versus other culture methods*
Pos.each
Method(%)
Pos. by
atleast one cult.(%)
Sens. %
Median
detection
days
Auramine 0 76 98 78
MODS 89 97 92
9 (4-31)
MGIT 88 95 93
10 (3-39) LJ 73 96
24 ( 6-59)
Micro COL 7H11 75
14.5(4-28)
PCR 81 90
* Based on 172 samples
Caviedes.L. et al J..Clin.Microbiol. 2000, 38, 1203

34. Identification of M. tuberculosis from the growth
Growth temperature 35o-37oC only
No pigmentation
Niacin positive
Catalase negative at 68oC
No growth on LJ medium containing PNB
Positive reaction for nitrate reduction

35. Differentiation of Mycobacteria
M.tuberculosis
NTM
Colony morphology
Rough, eugonic
Mostly smooth
Growth at 37oC +
+/ -
Growth at 25oC -
Pigmentation
Niacin
PNB
Nitrate reduction
Catalase at 68oC

36. Can high drug dosage still have an effect on resistant strains?
Isoniazid
Mutants
katG – high MIC
inhA – low MIC
Early clinical trial
Guinea-pig study
Quinolones
Mainly in gyrA – low MIC

37. Evaluation of different methods of diagnosis
As regards the time:
MGIT shortest time to positivity at 13.3 days
BACTEC 460 system 14.8 days
& for L J medium 25.6 days .

38. As regards the no. of culture yield:
The best yield, was with BACTEC 460, followed by BACTEC MGIT 960 , & then with L J medium.
As regards contamination rate:
L J medium (17%) had the highest contamination rate (Tortoli E, Cichero P,Et al. 1999) then the MGIT 960 ( 10.0% )
Compared with radiometeric system (3.7%)

39. SUMMARY Useful newer modalities 3idiots?
QUANTIFERON-TB GOLD
MB BACT-ALERT 3D LIGHT EMITTING SENSORS
PCR PROBES for antibiotic resistance
INH,REF,ETH,FLORO Q

41. Tuberculosis (TB) Diagnostic Tests in Use, Recently Endorsed by the World Health Organization (WHO), and in Later Stages of Development.
Tuberculosis (TB) Diagnostic Tests in Use, Recently Endorsed by the World Health Organization (WHO), and in Later Stages of Development
Dorman S E Clin Infect Dis. 2010;50:S173-S177
© 2010 by the Infectious Diseases Society of America

42. Tuberculosis (TB) Diagnostic Tests in Use, Recently Endorsed by the World Health Organization (WHO), and in Later Stages of Development.

44. BACTEC Myco/F�Sputa Culture Medium, for use with the BACTEC 9000MB System to detect mycobacteria species in clinical samples.
BACTEC Myco/F Lytic.

45. Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold
AL, Colford JM Jr, Riley LW, Pai M. Comparison of a whole
blood interferon-gamma assay with tuberculin skin testing
for the detection of tuberculosis infection in hospitalized
children in rural India. J Infect 2007; 54:267–76.
An Indian study that compared QFT to the TST in 105 children who
were suspected of having TB, or had contact with an index case.
In this study 11 children (10.5%) were QFT positive, whereas the TST
was positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at
≥15mm. Concordance of TST with QFT was high (95%) at the 10mm
TST cut-off. All ≥15mm TST subjects were QFT positive.
There were no indeterminate QFT results, despite 40% of the children
being <4 years old and 57% of them being malnourished.

46. SUMMARY IGRAs are recommended for 1
SUMMARY IGRAs are recommended for 1. Contacts of active TB Close contacts (HIGH RISK) can get both TST and IGRA and if either is positive, be treated for LTBI Casual contacts (LOW RISK) can have IGRA confirmation if TST positive to verify infection vs BCG or MOTT 2. Immune compromised TST first, if negative do IGRA and if IGRA positive treat as LTBI 3. Low risk people who are TST positive Do an IGRA, if positive consider as LTBI

48. FIND and Carl Zeiss Micro Imaging GmbH have co- developed a fluorescent LED microscope based on the proven Primo Star platform. FIND/Zeiss microscope offers superior optics, reflected light illumination, easy switch from brightfield to fluorescent light 48

49. Components of the post-research-and-development process for promising new tuberculosis (TB) diagnostic technologies.
Components of the post-research-and-development process for promising new tuberculosis (TB) diagnostic technologies. QA, quality assurance.
Dorman S E Clin Infect Dis. 2010;50:S173-S177
© 2010 by the Infectious Diseases Society of America

53. Reporting of culture results
Reading
No Growth
1 – 19 colonies
colonies
>100 colonies
Confluent growth
Contaminated
Report
Negative
Positive ( No.of colonies)
Positive (1+)
Positive (2+)
Positive (3+)
Contaminated

55. INDIAN STUDY USING QUANTIFERONTB GOLD
Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold AL, Colford JM Jr, Riley LW, Pai M. Comparison of a
whole blood interferon-gamma assay with tuberculin skin testing
for the detection of tuberculosis infection in hospitalized
children in rural India. J Infect 2007; 54:267–76.
Compared QFT to the TST in 105 children ( suspected of TB, or had contact with an index case).
11 children (10.5%) were QFT positive, whereas the
TST was positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at
≥15mm.
Concordance of TST with QFT was high (95%) at the 10mm
TST cut-off. All ≥15mm TST subjects were QFT positive.
There were no indeterminate QFT results, despite 40% of the children
being <4 years old and 57% of them being malnourished.

56. Thank you