Download Mdr and xdr tuberculosis

MOLECULAR DIAGNOSIS OF
TUBERCULOSIS
MUHAMMAD MUNTAZIR SHAH
11-ARID-2287
Ph.D. Scholar (Biochemistry)
Tuberculosis
 Tuberculosis has been present in the human population since
antiquity
 1882 - Robert Koch discovered Mycobacterium tuberculosis
Tuberculosis in Humans
Treatment of Tuberculosis
 First Line Drugs
Rifampicin
Ethambutol
Pyrazinamide
Isoniazid
Streptomycin (injectable)
 Second Line Drugs
Flouroquinolones: Ciprofloxacin,Ofloxacin,Levofloxacin,Gatifloxacin
Aminoglycosides: Kanamycin ,Amikacin (Injectable)
Cyclic Peptide : Capreomycin (Injectable)
Ethionamide : Ethionamide and Protionamide
4
Mechanism of drug action
National Institute of Allergy And Infectious Disease
Drug-Resistant TB
Mono-resistant
Resistant to any one TB treatment drug
Poly-resistant
Resistant to at least any 2 TB drugs (but not both isoniazid and
rifampin)
Multidrug resistant
(MDR TB)
Resistant to at least isoniazid and rifampin, the 2 best first-line
TB treatment drugs
Extensively drug
resistant
(XDR TB)
MDR PLUS resistant to any fluoroquinolone AND at least 1 of
the 3 injectable second-line drugs (e.g., amikacin, kanamycin, or
capreomycin)
Mechanisms of resistance in M. tuberculosis
Zhang Y, et al. Int J Tuberc Lung Dis 2015; 19(11):1276-1289.
TB Diagnosis: WHO Recommended Technology
Xpert MTB/RIF Assay
Principle



Single-use disposable GeneXpert cartridges that hold the
PCR reagents and host the PCR process.
The Xpert MTB/RIF assay uses 3 specific primers and 5
unique molecular probes to ensure a high degree of
specificity.
Assay targets the rpoB gene, which is critical for
identifying
mutations
associated
with
rifampicin
resistance.
Molecular Beacon Technology
 Molecular beacon is a
short segment of
single-stranded DNA
composed of:

Target specific
sequence

Stem

Fluorescent dye

Quencher
Molecular Beacon Technology
Molecular Beacon
Target
Fluorescence
No Fluorescence
No Target = No Fluorescence
Internal Controls


Sample processing control (SPC) to control for
adequate processing of the target bacteria and
to monitor the presence of inhibitor(s) in the PCR
reaction.
The Probe Check Control (PCC) verifies reagent
rehydration, PCR tube filling in the cartridge,
probe integrity, and dye stability.
How GeneXpert Work
GenoType MTBDRsl Ver2.0
Loop Mediated Isothermal Amplification: LAMP
Notomi et al. 2000, developed LAMP-auto cycling
technique
Utilizes a polymerase with strand displacing property
Works under isothermal condition 60-65C
Results visualized directly, no post amplification procedure
needed
Less time-60 min.
10 to 100 times more than PCR
Enzyme – Heart of the LAMP
 Bst polymerase
Isolated from Bacillus stearothermophilus
Optimum temperature for activity 63 C
 Bsm polymerase
Isolated from Bacillus smithii
Optimum temperature for activity 60 C
 Phi29 DNA polymerase
Bacillus subtilis phage phi29
Primers
F3 also called Forward outer Primer major role during strand displacement – Strand
displacing primer
B3 also called backward outer Primer major role during strand displacement –
Strand displacing primer
FIP (Forward Inner Primer) function in loop formation
BIP (Backward Inner Primer) function in loop formation
LAMP Principle
Properties
PCR
LAMP
Denaturation
Required for separation of strands,
enabling primer binding
Denaturation step is not a mandate
Temperature
Usually employs 3 steps as
Works under a constant temperature
denaturation, annealing and extension, usually between 60-65 C
working at different temperature and
timing
Time required
Take 2-3 hour based on the different
parameters
60 min usually
Post amplification
Need agarose gel electrophoresis or
DNA binding dyes like SYBR green
or any material indicator like calcien
or HNB results can be interpreted
visually
Sensitivity
Can detect up to nanogram level of
DNA
Can detect up to femtogram level of
DNA
Instrument
Needs sophisticated instrument in
order to maintain different
temperature within a given time
Water bath can serve the purpose
Impurities
Impurities can hinder the PCR
reaction
Impurities won’t hinder the reaction
(Kaneko et al., 2007: Francois et al.,
2011)
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