The document provides information on the molecular diagnosis of tuberculosis. It discusses the historical aspects of TB identification and increasing drug resistance. It notes that in 1993, WHO declared TB a global emergency, with one-third of the world's population infected. Current estimates from WHO in 2010 show over 8 million new TB cases annually. Molecular diagnostic methods like the AMTD and MTBDRplus tests can rapidly detect Mycobacterium tuberculosis complex and resistance patterns in days rather than the months needed for conventional culture. These new tests are especially useful for screening patients in high burden areas and for detecting drug resistant TB.

1. Molecular diagnosis of
Tuberculosis
Dr. Kanwal Deep Singh Lyall
M.D. Microbiology

2. Historical Aspects
• 24th Mar, 1882 : ROBERT KOCH identifies Mycobacterium
tuberculosis
• 1970s : 1st outbreak of Drug Resistant TB in the United States.
• 1993:
 WHO declares TB a GLOBAL EMERGENCY.
 1/3rd of the world’s population (2 billion people)
infected.
 7-8 million cases/yr.
 Dots under RNTCP launched in India.
 Only strategy proven effective in controlling TB on a
mass basis.
 India – Highest no. of TB cases in any one nation.

3. • Stop TB partnership: action plan to stop TB launched in 2006 by
WHO
 2015 – Decrease Prevalence & Death Rates by 50% compared
with 1990 levels.
 2050 – Eliminate TB as a global health problem (Global
rate<1/million population).
• 1995 : 1st recorded outbreak of MDR-TB at London hospital’s
HIV unit.
• 2000 : Green Light Committee (GLC) setup by WHO to support
countries in their fight against MDR-TB.
• Mar 2006 : MDR-TB subclass labeled as XDR- TB described
by WHO, IUALTD & CDC.

4. Burden of the Disease
• Each year: 9 million new cases with 2 million
deaths.
• India: Highest TB cases in any one nation; 1/5th of
the Global burden.
• >80% of TB patients : economically productive
age group (15-49 yrs)
• >1 million in HIV positive individuals.

5. WHO Global Tuberculosis
Control Report 2010

6. Global Tuberculosis Estimates (2010)
• 8.8 million incident cases
• Incidence rate has been ↓by 1.3% per year
since 2002
• Most cases were in SE Asia, African and
Western Pacific regions (35%, 30% & 20%,
respectively)
• MDR TB prevalence- 5.4%

7. TB Burden India (2010)
• Prevalence - 31,00,000
• Incidence - 23,00,000
• Incidence (HIV-positive) 1,10,000.

8. 2010/2011
Tuberculosis Global Facts- WHO

9. • 1.7 million people died from TB in 2009,
including 380 000 people with HIV
• Death rate has ↓by 35% since 1990
• 9.4 million new TB cases in 2009, including
1.1 million cases among people with HIV
• Since 1995, 41 million people have been
successfully treated
• Up to 6 million lives saved.

10. • 440 000 new MDR-TB cases in 2008 &
150 000 deaths from MDR-TB
• In 2009, 3.3% of new TB cases had MDR-TB
• The largest WHO MDR-TB survey (2010)
reported the highest rates ever of MDR-TB,
with peaks of up to 28% of new TB cases.

11. Mycobacteria
• Straight or slightly curved rods (≈ 3x 0.3µm)
• Slender, slightly curved or straight rod-shaped
organisms
• Non-motile
• Do not form spores
• Cell wall with extremely high lipid content
– Staining requires longer time or application of heat
– Once stained, resist decolorization with acid-alcohol
(acid-fast)

12. General Characteristics (cont’d)
• Strictly aerobic
• Grow more slowly than most bacteria
• Traditional characteristics used to identify Mycobacterium
– Rate of growth
– Colony morphology
– Pigment production
– Nutritional requirements
– Optimum incubation temperature
– Biochemical test results

14. MDR-TB:Resistant to at least Isoniazid & Rifampicin
XDR-TB : MDR-TB plus resistance to
• Any fluoroquinolone
• At least one of 2nd line injectable drugs
(Capreomycin, Kanamycin, Amikacin)

15. MOA of Anti-TB Drugs
ISONIAZID
Inhibits cell wall
synthesis
ETHAMBUTOL
Inhibits cell wall
synthesis
ETHIONAMIDE
Inhibits cell wall
synthesis
PYRAZINAMIDE
Inhibits cell wall synthesis
RIFAMPICIN
Inhibits RNA
synthesis
STREPTOMYCIN & OTHER
AMINOGLYCOSIDES
Inhibit protein synthesis
FLUROQUINOLONES
Inhibit DNA supercoiling
D-CYCLOSERINE
Inhibits cell wall
synthesis

16. Isoniazid: Inhibits Cell Wall Synthesis.
Activated INH acts on
enoyl-acyl carrier protein
(InhA) which is a
component of FAS-II
FAS-II synthesizes Long
Chain Mycolic Acids – an
essential component of cell
wall
Catalase Peroxidase
activates INH & is encoded
by the katG gene

17. Rifampicin: Inhibits RNA synthesis.
Rifampicin binds to ß-
subunit of DNA
dependent RNA
polymerase (rpoB) &
inhibits mRNA
transcription.

18. Diagnosis
Microscopy
• At least 10,000 bacilli /ml of sputum for them to be
readily demonstrable in direct smears.
Z N Stain Auramine

19. • Culture Methods
• Conventional (LJ Media)
• Rapid.
BACTEC 460 system
MGIT 960 system
MB/Bac T system
BACTEC MYCO/F Lytic system
ESP Culture System II.
• Serology ELISA, RIA, LA.
• Molecular Methods PCR, TMA, LiPA

20. Conventional Culture
• Lowenstein Jensen media
• NALC-NaOH decontaminated samples inoculated on
2 bottles of LJ media.
• Observed daily for the 1st week & then weekly for the
next 7 weeks.
• Suspected growth identified by Z N Staining & report
released as culture positive.
• Growth of MTB obtained after…. weeks of
incubation.
• Negative report after 8 weeks of incubation.

21. LJ Media
• At least 10-100 bacilli should be present per
ml of sputum for a culture to come positive.

22. Modern Diagnostic Methods Helps
In Quicker Diagnosis

23. • To increase the rate of detection
• To lessen the time of detection
• To be more specific in diagnosis
• Need of automated and molecular methods
• Added advantages.

24. BacT Alert 3D SYSTEM

25. BacT Alert 3D SYSTEM
• 10ml enhanced Middlebrook 7H9 broth with CO2,
N & O2 under vacuum.
• Antibiotics + growth factors + processed specimens.
• Gas permeable sensor at bottom of bottle → changes
from dark green to bright yellow with CO2 production
by metabolizing mycobact.
• Incubated bottles monitored cont. by reflected light

26. Advantages
• ↑ yield of positive culture.
• ↓ time of detection.
• broth based susceptibility testing can be performed.
• DST to 1st & 2nd line drugs
• Eliminates the need for handling & disposing of
radioisotopes.

27. Cost
• For rapid culture & sensitivity -
• For culture and DST to first line drugs-

28. GEN-PROBE®
AMPLIFIED™ Mycobacterium Tuberculosis
Direct (MTD) Test

29. Amplified Mycobacterium Tuberculosis Direct
Test (AMTD)
• In vitro diagnostic detection of MTBC rRNA
(signature genes)
• In AFB smear +ve & −ve conc sediments
• Prepared from pulm. & extra pulm. samples.
• Detects M. tuberculosis, M. bovis, M. bovis BCG,
M. africanum, M. microti, & M. canetti.

30. RNA versus DNA
DNA
• Very stable, present
also in dead
organisms
• Easily picked up by
PCR
RNA
• Present only in living
organisms
• Very labile
• Tell you Active
Infections

31. Advantages of rRNA targets
Sensitivity is Highly increased.
HOW????
Through «biological amplification

32. Sensitivity
10 000 copies
ribosomal RNA
C
A T
AT
A T
G C
G C
A T
T A
A T
C G
T A
G C
G C
G C
A T
A T
G C
T A
C G
T A
DNA
RNA Polymerase
Single Copy of DNA in Cell

33. rRNA Targeting Enhances Sampling Efficiency
• Suppose 5 ml sample contains one organism.
• If 0.1 ml aliquot is taken , odds of getting that
organism in that aliquot are1 in 50.

34. rRNA Targeting Enhances Sampling Efficiency
• If organism lysed prior to sampling
• There will be 2000 – 10,000 rRNA molecules in 5 ml
• On average there will be 40 −200 targets in each
0.1 ml aliquot.

35. Advantages of rRNA targets
• Target NA distributed through out sample
• ↓ false negatives d/t sampling error
• Absolute specificity by targeting unique sequences.
• No Cross Reactivity
• Positive only if active infection.
• Treat OR Not to Treat

36. • in vitro diagnostic detection of MTBC rRNA
• AFB smear +ve & −ve conc. sediments prepared
from specimens
• Very rapid, billion of copies of rRNA in <1hr
• Amplification & detection in single tube, reduces risk
of carryover contamination

37. Specimen Collection, Storage, Transport,
& Processing
• Specimen Collection and Storage:
• Specimens must be collected in sterile plastic
containers
• Stored at 2° to 8°C until transported or processed.

38. • Does not differentiate among members of the M.
tuberculosis complex, i.e., M. tuberculosis, M. bovis,
M. bovis BCG, M. africanum, M. microti, and M. canetti.
• Results may be affected by specimen collection, transport,
specimen sampling variability, laboratory procedural
errors, sample misidentification.

39. • Decontamination & Concentration by NALC-NaOH
method
• Lysis & Extraction by sonication
• Amplification → reverse transcriptase, RNA
polymerase & nucleoside triphosphate
• Hybridization with acridinium ester-labeled DNA
probes
• Selection by signal quenching of unlabeled probes
• Reading in Luminometer
• Results in Relative Light Units (RLU)

40. Principle of TMA & HPA

42. • Step1: promoter-primer binds to 3 end of RNA target;
• step 2: reverse transcriptase (RT) creates DNA copy
of RNA target;
• step 3: RNAse H activities of RT degrades the RNA;
• step 4: 5 ABL or BCR primer binds to the DNA;
• step 5: RT completes dsDNA template including the
RNA polymerase promoter sequence;
• step 6: RNA polymerase initiates transcription of
RNA from DNA template, producing 100–1000
copies of either BCR-ABL or ABL amplicon;

44. • step 6: RNA polymerase initiates transcription of RNA
from DNA template, producing 100–1000 copies of either
BCR-ABL or ABL amplicon;
• step 7: primer binds to the RNA amplicon;
• step 8: RTcreates an RNA:DNA heteroduplex;
• step 9: RNAse H degrades the RNA;
• Step 10: ABL promoter-primer binds to the newly
synthesised DNA;
• step 11: RTcreates double-stranded DNA. Products from
this step are then available as templates for step 6 in an
autocatalytic cycle
• amplicons serves as the template for a new round of
replication, leading to an exponential expansion of the
RNA target.

46. Results
• 500,000 RLU positive for M. tuberculosis complex rRNA
• < 30,000 RLU negative for M. tuberculosis complex rRNA
• 30,000 to 499,999 RLU probable M. tuberculosis complex
rRNA positive; repeat to verify results:
o Repeat 30,000 RLU positive for M. tuberculosis complex
rRNA
o Repeat < 30,000 RLU negative for M. tuberculosis complex
rRNA

47. MTBDRplus

48. GenoType MTBDRplus
• Based on DNA•STRIP technology
• Permits simultaneous molecular genetic identification
of
a. The M. tuberculosis complex
b. Resistance to rifampicin by detection of
mutations in rpoB gene
c. Resistance to isoniazid by detection of mutations
in katG gene and inhA gene
• From smear +ve pulmonary clinical specimens or
cultivated samples.
• Recommended by the WHO

49. Reverse Hybridization
• Probes immobilized on nitrocellulose strip
• Amplicon applied to strip
• Visible Lines form at site of amplicon-probe
hybridization

50. Indications for the Use of GenoType MTBDRplus
• Diagnosing patients after treatment failure and
relapse
• In high prevalence TB countries and high burden
MDR-TB regions
• For screening purposes to develop country-specific
TB action plans

51. Advantages of GenoType MTBDRplus
• Results are obtained in 4–5 hrs compared to
1 to 2 months with conventional methods.
• Allows early, appropriate treatment, which
reduces transmission and spread of MDR-TB.

52. Samples
• Cultivated material (solid/liquid)
• Pulmonary smear positive

53. Quality Control
• Each strip includes 5 control zones
• Conjugate control (CC)
• Amplification control (AC)
• Three locus control zones (rpoB, katG, inhA)
o rpoB for rifampicin
o katG for high level resistance to isoniazid
o inhA for low level resistance to isoniazid

54. • 95 C x 15 mnts
• Sonication x 15 mnts
• Centrifugation @
13000 rpm x 5 mnts

55. Reverse Hybridization

57. Evaluation & Interpretation of Results
CC & AC band present Test can be
evaluated
TUB band present M. tuberculosis
complex
detected
1. Absence of at least one Wild type probe or
2. Absence of at least one Wild type probe &
presence of at least one Mutation probe or
3. Presence of at least one mutation probe
Resistant
1. Presence of all the Wild type probes and
absence of all mutation probes
Sensitive

58. Example Results with the GenoType MTBDRplus

59. Advantages
• Fast: 4-5 hours only.
• Safe: internal controls & secure safe and
impeccable test procedures.
• Reliable: specific amplification and hybridization
• User-friendly
• High Sensitivity & Specificity.
• Cost-efficient
• Automation available.

60. Pulmonary Extra-Pulmonary
+ve -ve +ve -ve
AMTD
+ve -ve
MTBC
MTBC
Culture
MTBDRplus
Resistance
pattern
MTBDRplus
+ve -ve
May be MTBC or MOTT
Culture
Resistance
pattern
MAS/CM
MOTT
Culture
MTBDRsl
MAS/CMMTBDRsl
May be MTBC or MOTT
Smear

61. The GenoType® Mycobacterium
CM (Common Mycobacterium)

62. • Based on the DNA•STRlP® technology
• Identifies the following species;
• M. tuberculosis complex & 14 m/c NTM species
• M. avium, M. chelonae, M. abscessus, M. fortuitum,
M. gordonae, M. intracellulare, M. scrofulaceum,
M. interjectum, M. kansasii, M. malmoense,
M. peregrinum, M. marinum/ M. ulcerans, &
M. xenopi.

63. • Bacteria grown on culture plates or in liquid medium
• The test must not be used to detect mycobacteria
directly from patient material.

64. Example Results with the GenoType Mycobacterium CM

65. Thank You