Tuberculosis (TB) is one of the most serious diseases which affect millions of people annually. It is caused by Mycobacterium tuberculosis (MTB), specifically causing tuberculosis in humans. Tuberculosis infection spreads mainly through inhalation of the aerosolized bacteria present in the form of droplet nuclei. These infective droplets are coughed or sneezed into the environment by the patients suffering from pulmonary tuberculosis. The bacteria multiply within the alveolar macrophages and might spread to other parts of the body. Thus tuberculosis should be treated effectively by providing the patients with proper medication or else the strain develops resistance against the drugs. MDR - TB has become prevalent mainly due to late diagnosis and inadequate treatment regimens. Tuberculosis is detected by culturing the specimens on the Lowenstein and Jensen (LJ) medium. MTB, being a slow growing strain, takes about 6-8 weeks of time to show demonstrable colonies on the medium. Biochemical testing for speciation was time consuming and gives varied results for species which were genetically similar. Thus there was a need for rapid detection of tuberculosis infection which would help in adequate treatment of the patients without allowing the progression of the disease. Liquid culture was introduced in the year 1980’s which reduced the turnaround time to 2-3 weeks. Drug sensitivity testing by the conventional solid or liquid methods took an additional 4-6 weeks and 2 weeks respectively. Many molecular methodologies have been adopted for rapid detection of tuberculosis. Genotype MTBDRplus assay (Hain Life science) is a Line Probe Assay which amplifies the mycobacterial DNA and detects the sensitivity or resistivity of the strain against first - line anti-tubercular drugs (Rifampicin and Isoniazid). GeneXpert (Cepheid) is an advanced technology in detecting tuberculosis and it’s susceptibility to Rifampicin only. It is a semi quantitative Nested PCR and is based on the principle of Cartridge Based Nucleic Acid Amplification Testing (CB - NAAT). It gives the result within 2 hours. Thus we discuss the exact procedure of these techniques which can detect tuberculosis rapidly and can help to treat the patients adequately.

1. Rapid Detection of Multi-Drug
Resistant Tuberculosis (MDR-TB)
Dissertation carried out under the guidance
of Dr. Ameeta Joshi, In-Charge, Department
of Mycobacteriology at Sir J.J group of
Hospitals, Byculla, Mumbai.
MAYUR D. CHAUHAN
M.Sc. BIOTECHNOLOGY
1

2. INTRODUCTION
Tuberculosis & Types
Causative Agent
Morphology
2

3. Cell Wall of MTB
3

4. Unique Characteristics imparted by
Mycolic Acid of the Cell wall
Resistance to detergents
Unusual resistance to killing by acids and alkalies
Resistance to commonly used antibiotics
Slow growth by delaying permeation of nutrients
Acid Fastness (AFB)
4

5. Mycobacterium tuberculosis Complex
• M. bovis
• M. cannettii
• M.
tuberculosis
• M. microti
Bacillus-
Calmette
Guerin
M. caprae
M.
africannum
M.
pinnipedii
5

6. Transmission of
Tuberculosis
• Person to Person
• Present in Air-borne
particles termed as
Droplet Nuclei.
• 1-5 µm in size
• Generated when
infected patients cough
or sneeze
• Bacteria may remain
viable in the
environment uptill 8
months.
6

7. Pathogenesis of Tuberculosis
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8. 8

9. Treatment of Tuberculosis
First-Line Anti-tubercular
Drugs
• Streptomycin
• Isoniazid
• Rifampicin
• Ethambutol
• Pyrazinamide
Second-Line Anti-tubercular
Drugs
• Ciprofloxacin
• Cycloserine
• Ethionamide
• Ofloxacin
• Kanamycin
• Levofloxacin
• Capreomycin
9

10. MDR, XDR & TDR
MDR is resistance to RIF & INH
Caused by poor treatment regimens
XDR is MDR patient resistant to
quinolones and aminoglycosides
TDR is resistant to all the anti-
tubercular drugs
10

11. Molecular Mechanism of Drug Action
A] Rifampicin
rpoB
ß-subunit of
RNA Pol
Transcription
11

12. rpoB
ß-subunit of
RNA Pol
Transcription
is
Inhibited
RIF
12

13. rpoB
Mutated
ß-subunit of
RNA Pol
Transcription
RIF
13

14. B] Isoniazid
katG
Catalase-
Peroxidase
inhA
NADH dep.
Enoyl ACP
reductase
Mycolic acid
synthesis
(Lipid
Synthesis)
14

15. katG
Catalase-
Peroxidase
inhA
NADH dep.
Enoyl ACP
reductase
Mycolic acid
synthesis is
inhibited
INH
15

16. katG mutated
Catalase-
Peroxidase
inhA mutated
NADH dep.
Enoyl ACP
reductase
Mycolic acid
synthesis takes
place
INH
16

17. Conventional Methods of Detection
Culture on LJ medium
DST by solid culture
Species detection by Biochemical
testing
17

18. Disadvantages of Conventional
Methods
Growth of MTB takes 2-8 weeks
DST takes further 3-6 weeks
Delay in Treatment
18

19. AIM
To Carry out the rapid Detection of
Tuberculosis by using various
Molecular Methods (Line Probe
Assay (LPA) and GeneXpert)
19

20. MATERIALS & METHODS
Laboratory
Equipment
Instruments
• Situated at a Distance
• Proper Ventilation
• BSL-2 and BSL-3
• N95 respirators
• PPE
• Latex Gloves
• Headgear
• Shoe covers
• BSC
• Hot air oven
• Thermal Cycler
• GT Blot-48 or Twincubator
20

21. Procedures to follow for LPA
Sample collection and Decontamination
by NALC-NaOH
Smears preparation, ZN staining and
Smear Grading
Positive samples go for LPA
21

22. Line Probe Assay
DNA
extraction
Master Mix
preparation
Amplification
&
Hybridization
22

23. A] Sample Collection
23

24. B] Decontamination by NALC-NaOH
24

25. C] Ziehl- Neelsen Staining and Grading
25

26. 26

27. 27

28. D] Line Probe Assay for Positive
Samples
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29. 29

30. 30

31. 31

32. 32

33. 33

34. Results for LPA
34

35. GeneXpert
Catridge Based Nucleic Acid
Amplification Testing
Semi-Quantitative Nested real time
PCR
Gives result in less than 2 hours
35

36. 36

37. 37

38. RESULTS
38

39. 39

40. 40

41. Discussions and Conclusions
Sensitivity of the Tests were 99%
Specificity was 100%
Superior to Conventional Methods
41

42. Future Work
Assay for Second Line Drugs
Novel Drugs
Awareness among people
42

43. Major References
Marinus Barnard, Linda Parsons, Paolo Miotto, Daniella Cirillo, Knut Feldmann,
Cristina Gutierrez, Akos Somoskovi, 2012, Molecular Detection of Drug-Resistant
Tuberculosis By Line Probe Assay, Laboratory Manual for Resource-Limited Settings,
FIND, Unit Aid and Expand-TB project
Soundiram Indira, Cepheid, 2012, GeneXpert
Technology.
Subhash Chandra Parija, 2012, Textbook of
Microbiology and Immunology, Second edition,
published by Elsevier
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