my ophthalmology internship seminar..... 2013....

1. Newer investigations of TB
VISHNU AMBAREESH M S

2. • The conventional techniques
used in TB diagnosis like AFB
(acid fast bacilli) smear
microscopy lack sensitivity and
the gold standard, culture test
takes time.

3. AFB cons
• 6-8 weeks by culture
• not reliable for detection
• Elimination of this bacterium at the earliest level
not possible
• DOTS avails TB therapy to the patients with smear
positive tests only.
Sarbesh Dangol, B.Tech. BioTechnology © 2011

5. • The polymerase chain reaction (PCR) is a
biochemical technology in molecular
biology to amplify a single or a few copies of a
piece of DNA across several orders of
magnitude, generating thousands to millions
of copies of a particular DNA sequence
• 1983 by Kary Mullis

12. • PCR based assays offer high sensitivity by
amplification of small amount of DNA
• Many of the tests are based on amplification of
IS6110, an insertion element that is believed to be
restricted to members of the M. tuberculosis
complex
• The presence of multiple copies of this element in
the majority of M. tuberculosis strains undoubtedly
enhances the sensitivity of PCR.

13. • A test based on multiplex polymerase chain
reaction (PCR) targeting the 38 kDa gene and
IS6110 insertion sequence, specific to
Mycobacterium tuberculosis was developed to
further increase the sensitivity of a TB-PCR kit
targeting only 38 kDa gene developed earlier
in the same laboratory

14. Occasional strains from India lacking IS61107
targeting a house keeping gene of M. tuberculosis,
for 38 kDa protein(RV0934),involved in phosphate
transport
Was assembled in a kit form and was launched in
the market in August 2009 In collaboration with
Department of Atomic Energy’s commercial
department,BRIT(Board of Radiation & Isotope
Technology).

16. Results
• When the performance of various
tests was compared for diagnosis of TB
multiplex, PCR showed the highest
sensitivity as compared to AFB smear
microscopy and culture test(clinical
diagnosis as Gold standard) as also
observed in other studies evaluating in-house
PCR tests

17. • Because of high sensitivity observed
in paucibacillary smear negative
samples, this test may be used for
diagnosis of EPTB as well as PTB
which are difficult to diagnose with
available standard methods

18. Guidelines for nucleic acid amplification (NAA) tests
NAA testing has become a routine procedure in many
institutions
NAA tests can rapidly and reliably detect Mycobacterium
tuberculosis bacteria directly in a specimen one or more
weeks earlier than culture.
Earlier laboratory confirmation of TB can lead to earlier
treatment initiation, better patient care
and outcomes, greater opportunities to interrupt
transmission, and improved public health interventions.

19. Two NAA tests are approved for use in the United States FDA.
The Enhanced Amplified Mycobacterium Tuberculosis Direct
Test (E-MTD, Gen-Probe, San Diego, California) for detection
of M. tuberculosis complex bacteria in AFB smear-positive
and smear-negative respiratory specimens from patients
suspected of having TB.
The E-MTD test combines isothermal transcription-mediated
amplification of a portion of the 16S rRNA with a detection
method that uses a hybridization probe specific for M.
tuberculosis complex

20. • The MTD test displays a
sensitivity of >95% from AFB-smear
positive TB suspects
and 75% to 90% AFB-smear
negative TB suspects

21. The Amplicor Mycobacterium Tuberculosis Test
(Amplicor, Roche Diagnostics) in AFB smear-positive
respiratory specimens from patients
suspected of having TB.
uses the PCR to amplify a portion of the 16S
rRNA gene that contains a sequence that
hybridizes with an oligonucleotide probe
specific for M. tuberculosis complex bacteria.

22. • The Amplicor test displays a
sensitivity of >95% from AFB-smear
positive TB suspects and a
sensitivity of 60% to 70% from
AFB-smear negative TB suspects.

23. GeneProof Mycobacterium
tuberculosis PCR Kit
Real Time Polymerase Chain Reaction.
based on the amplification of a specific multicopy insertion
sequence IS 6110 and on measuring the amplification product
concentration in the course of the PCR proces by means of a
fluorescence marked probe.
specifically detects strains of the Mycobacterium
tuberculosis complex (M. tuberculosis, M. bovis, M.
africanum a M. microti) and also vaccination strains (e.g. BCG).

24. • Using the multicopy insertion sequence IS6110 for
mycobacteria detection enables 16 times higher
examination sensitivity in comparison to
conventional single copy genes detection methods,
which enables maximum sensitivity of mycobacteria
laboratory detection in clinical samples like sputum,
bronchoalveolar lavage or urine. The kit is designed
for in vitro diagnostics and provides qualitative
detection.

25. test procedure
steps: DNA extraction from decontaminated patient
specimens, amplification of mycobacterial DNA by PCR (M.
tuberculosis complex-specific target), hybridization of
amplicons with specific probes and detection of amplicon-probe-
complex on a lateral-flow-dipstick.
The duration of the test procedure takes only approx. 3 hours.

27. LIMTATIONS OF PCR

28. Limitations
The FDA-approved NAA tests for TB have
slightly less sensitivity than culture-isolation
methods, and the 15% to 20% of U.S. TB
cases that are reported with negative culture
results may also have negative NAA test
results. Thus, a negative NAA test result
does not exclude the diagnosis of TB.

29. • b. Sputum specimens (up to 20% in some
studies) may contain inhibitors that prevent
or reduce amplification to cause false-negative
NAA test results, although inhibitors
rarely cause false-negative NAA test results in
smear-positive specimens (<3% of samples).
• c. Several sporadic or systematic errors can
cause false-positive NAA test results

32. For MDR TB

33. FLOW CYTOMETRY

38. Confirmation of Mycobacterium tuberculosis
infection by flow cytometry after ex vivo incubation
of peripheral blood T cells with an ESAT‐6‐derived
peptide
The presence of a T-cell response to the early
secretory antigenic target-6 (ESAT-6) indicates
previous infection with or exposure to
Mycobacterium tuberculosis.Measuring this
response is useful for identifying individuals infected
It was also reported that the frequencies of ESAT-6-
specific T cells correlate with disease state..

39. Automation of fragment sizing
• Transgenomic WAVE
dHPLC
• - DNA fragment
sizing
• - No intermediary
sample manipulation
• - Based on novel
DNA separation
column
HPA North East Laboratory

40. Ex cyto test

41. • A kind of PCR
Registered by lucigen

42. instead of buying or making your own Taq, have
the bacteria make the Taq

43. • Lucigen’s high effciency E. cloni® 10G cells
engineered to constitutively express a
thermostable DNA polymerase for “ex cyto”
PCR directly from the cells.
• Cloning and amplifcation of the insert are
integrated, saving time, work and money

44. •
• ability to amplify DNA from different
templates, plasmids with different copy
numbers, and master mixes left on ice for up
to two hours.

45. • Ex cyto sequencing will eliminate the
overnight growth of bacterial cultures,
expensive template purification, and the
purchase of purified DNA polymerase

46. Seminar theernnu!!!!!!
ENJOY!!!!
Thank u….