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DIAGNOSIS OF
TUBERCULOSIS
EMERGING TRENDS
Dr.T.V.Rao MD
DR.T.V.RAO MD
HISTORICAL BACKGROUND
Neolithic Time
2400 BC - Egyptian mummies spinal columns
460 BC
Hippocrates, Greece
First clinical description: Phthisis / Consumption
(I am wasting away)
500-1500 AD
Roman occupation of Europe it spread to Britain
1650-1900 AD
White plague of Europe, causing one in five deaths
HISTORICAL BACKGROUND
1800-1900
Industrial revolution (Europe)
50 mil. Infected & 7 mil. Dying annually
1844
Half of Englands population infected with TB
1900s
Approximately all of Europes adult population infected with TB
1850-1952
Sanatorium Movement ( Brehmer and Trudeau)
Emphasis on rest, good nutrition, and fresh mountainous air
Isolation led to decrease in transmission
DIAGNOSTIC DISCOVERIES
24th March 1882 (Robert Koch)
TB Day
1895 (Roentgen)
Discovery of X-rays
Early diagnosis of pulmonary
disease
GLOBAL STATUS
Nine million people suffer
from tuberculosis
Two million people die each
year.
Tuberculosis accounts for
one-third of Aids deaths
world wide every year.
Globally, there have been
just 347 identified cases of
XDR-TB, mainly in the
former USSR and in Asia
DR.T.V.RAO MD
MYCOBACTERIUM TUBERCULOSISCHARACTERISTICS
GRAM POSITIVE
OBLIGATE AEROBE
NON-SPORE-FORMING
NON-MOTILE ROD
MESOPHILE
0.2 TO 0.6 X 2-4UM 1
SLOW GENERATION TIME: 15-20 HOURS
May contribute to virulence1
LIPID RICH CELL WALL CONTAINS MYCOLIC ACID50% OF CELL WALL DRY WEIGHT 1
Responsible for many of this bacteriums characteristic properties
Acid fastretains acidic stains
Confers resistance to detergents, antibacterial
DR.T.V.RAO MD
Diagnostics of Mycobacterium
Initial screening:
-TB skin test (Purified Protein Derivative).
Drawbacks: BCG injected subjects are
positive, 3 days delay for result
- QFT-G test (measures INF- response to TB
specific antigen)
TB tests Active, depending on the
suspected location of bacterium:
-3-5 samples of sputum
- multiple gastric aspirate
- urine (UTI)
- CSF (meningeal)2
Cultures
Samples are processed for fast acid stain
(FAS smear positive indicates
Mycobacterium) and cultured after alkali
decontamination (30s in 1-2% NaOH)
Molecular methods use species-specific
genes, including light and heave ribosomal
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RNADR.T.V.RAO
MD
Clinical
specimen/
decontamination
cultur
e
Direct detection:
- Microscopy
- PCR
- MTB rifampin
resistance
Species identification:
- 16S rRNA hybridization (MTB
and MAC)
-16S rRNA gene PCR sequencing
(NTM)
- restriction fragment length
polymorphism
Susceptibility testing
Rifampin resistance
(PCR oligohybridization
sequencing)
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SYMPTOMS
What are the symptoms of TB?
Fever
Fatigue
Weakness
Weight loss
Night sweats
Symptoms of pulmonary TB include:
Coughing
Pleurisy (pain when taking deep breaths)
Coughing up blood4.
DR.T.V.RAO MD
Emerging Molecular
Methods are trend
setters in rapid
Diagnosis of TB
DR.T.V.RAO MD
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MOLECULAR METHODS IN
DIAGNOSIS OF
TUBERCULOSIS
Several methods are available, mainly used as
Research tools
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Genetics
Differential diagnosis
Risk assessment - prevention
Classical epidemiology
More differentiated, molecular understanding of pathology and drug action
Clinical Disease Definition
Clinical Diagnosis
MOLECULAR DIAGNOSTICS
WHY?
uncultivable or difficult to
culture
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BEGINNING OF GENE
AMPLIFICATION METHODS
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MYCOBACTERIUM TUBERCULOSIS
GENOME
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AMPLICOR M. TUBERCULOSIS
ASSAY
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Automatic system
Roche (Cobas Amplicor) : PCR for 16S rRNA gene
Abbott (LCx) : PCR/LCR for PAB gene
Becton Dickenson (BD ProbeTec) : SDA
Gen-Probe : Transcription-Mediated Amplification
(TMA) for rRNA
Manual method
QMH-single tube nested PCR for IS6110 gene
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TARGET AMPLIFICATION
Target amplification
specific oligonucleotides to ssDNA target, extension to 65 kDa protein gene (16S rRNA,
Amplicor
of ssDNA, which serves as a new target for the next MPB64 gene Colorimetric,
automated
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NAA- SUMMARY
Useful technology for rapid diagnosis of smear negative cases
of active TB
Able to identify 50-60% of smear -ve culture +ve cases
Distinguish M.tb from NTM in smear +ve cases
Should not be used to replace sputum microscopy as an initial
screen & culture remains the gold standard
Very high degree of quality control required
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NAA- LIMITATIONS
They are able to detect nucleic acids from both living
and dead organisms so in pts on ATT, PCR should not
be used as an indicator of infectivity as this assay
remains positive for a greater time than do cultures
A major limitation of NAA tests is that they give no
drug-susceptibility information.
NAA should always be performed in conjunction with
microscopy and culture
DR.T.V.RAO MD
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(by June
2008)3
Tuberculosis: MDR-TB & XDR-TBThe 2008 Report. The Stop TB Department, WHO.
2
Hargreaves S. http://infection.thelancet.com, Vol 8, April 2008, p.220
3
Raviglione MC. NEJM 2008;359:636-8.
4
Anti-TB Drug Resistance in the World: Report No. 4. The WHO/IUATLD Global Project on Anti-Tuberculosis Drug
Resistance Surveillance 2002-2007. World Health Organization, 2008 (WHO/HTM/TB2008.394).
MOLECULAR METHODS
DRUG RESISTANCE
Reverse
hybridization
Line probe assays
RNase Cleavage
Diagnostic
Sequencing
(Genotyping)
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Rifampin (RIF)
Binds to subunit of RNA polymerase (rpoB)
96% of resistant Mtb isolates have mutations in 81-bp
region . well-studied
Four (4) mutations . 75% of resistant clinical isolates
Isoniazid (INH) . two genes
katG and inhA . 75-85%
Pyrazinamide . pncA . 70%
Streptomycin . rpsL . 65-75%
Ethambutol .embB . 70%
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Xpert MTB/RIF
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XPERT MTB/RIF
The new PCR-based TB
diagnostic testcalled
Xpert MTB/RIFis fast,
sensitive, and automated.
An accurate diagnosis can
be obtained in less than 2
hours by adding a reagent
to a sputum sample and, 15
minutes later, pipetting it
into a cartridge that is
inserted into the diagnostic
instrument for 12 minutes.
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XPERT MTB/RIF
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WITHOUT INFORMATION,
THE DOCTOR CANNOT ACT.
With information,
he cannot but
act.
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HL MENCKENS LAW
Every complex problem
has a simple solution.
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