Newer investigations of TB

VISHNU AMBAREESH M S

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

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

• 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

• 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.

• 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

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).

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

• 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

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.

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

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

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.

• 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.

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).

• 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.

test proceduresteps: 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.

LIMTATIONS OF PCR

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.

• 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

For MDR TB

FLOW CYTOMETRY

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..

Automation of fragment sizing

• Transgenomic WAVE dHPLC

• - DNA fragment sizing• - No intermediary

sample manipulation• - Based on novel DNA

separation column

HPA North East Laboratory

Ex cyto test

• A kind of PCR

Registered by lucigen

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

• 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

• • ability to amplify DNA from different

templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours.

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

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