MOLECULAR METHODS IN

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

Discovery of staining technique

that identified Tuberculosis
bacillus
Definite diagnosis made possible
and thus treatment could begin

1890 (Robert Koch)

Tuberculin discovered
Diagnostic use when injected into
skin

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

CHALLENGES WITH TUBERCULOSIS

INFECTION
Re-emerging problem
in industrialized
countries
Infections in immunocompromised patients
Multi-drug resistant
strains (MDR-TB)

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
3
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

EFFECTIVE LABORATORY DIAGNOSIS

Sputum smear
examinations rapid
classification of
species (atypical
mycobacteria common
in AIDS)
Culture examinations
Rapid drug sensitivity testing

Emerging Molecular
Methods are trend
setters in rapid
Diagnosis of TB

TUBERCULOSIS CONTINUES TO BE GLOBAL

HEALTH PROBLEM

Tuberculosis continues to be, as

it has been for centuries, one of
the most prevalent infectious
diseases of humans and is the
leading cause of mortality from a
single infectious disease
worldwide . Laboratory methods
play a crucial role in establishing
the diagnosis, monitoring
therapy, and preventing
transmission of tuberculosis.

DR.T.V.RAO MD

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TUBERCULOSIS A DISEASE OF GROWING

IMPORTANCE
In addition, the importance
of the mycobacteriologist
has grown, in view of a
changing epidemiology
(e.g. social factors, the
acquired immune deficiency
syndrome (AIDS pandemic)
and an increasing
resistance of M.
tuberculosis to drugs

DR.T.V.RAO MD

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MOLECULAR METHODS IN
DIAGNOSIS OF
TUBERCULOSIS
Several methods are available, mainly used as
Research tools

DR.T.V.RAO MD

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MEDICAL PROGRESS: EVOLUTION OR REVOLUTION?

Historic Drivers of Medical Progress
Clinical expertise

Genetics

Differential diagnosis
Risk assessment - prevention

Classical epidemiology
More differentiated, molecular understanding of pathology and drug action
Clinical Disease Definition
Clinical Diagnosis

Molecular Disease Definition

Molecular Diagnosis
in-vitro Diagnostics
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MOLECULAR DIAGNOSTICS
WHY?

Detection and Diagnosis

uncultivable or difficult to
culture

need for rapid answer

inadequacy of phenotypic methods

(biochemical)

Prognosis and management

need for quantitative information

(viral load)

susceptibility testing (drug

resistance) without culture

Molecular resistance testing

DR.T.V.RAO MD

15

MOLECULAR TESTS FOR DETECTION OF

NUCLEIC ACIDS
The majority of molecular tests have been focused on detection
of nucleic acids, both DNA and RNA, that are specific to
Mycobacterium tuberculosis, by amplification techniques such as
polymerase chain reaction (PCR); and detection of mutations in
the genes that are associated with resistance to ant tuberculosis
drugs by sequencing or nucleic acid hybridization. Recent
developments in direct and rapid detection of mycobacteria, with
emphasis on M. tuberculosis species identification by 16S rRNA
gene sequence analysis or oligohybridization and strain typing,
as well as detection of drug susceptibility patterns, all contribute
to these advance

DR.T.V.RAO MD

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BEGINNING OF GENE
AMPLIFICATION METHODS

Gene amplification can achieve

the goal of reducing the
generation time of
microorganisms to minutes, and
of replacing biological growth on
artificial media by enzymatic
reproduction of nucleic acids in
vitro . The importance of nucleic
acid amplification methods lies in
their wide applicability in the life
sciences, and their potential to
revolutionize the practice of
medicine. Examples are nucleic
acid sequence analysis and
genetic fingerprinting

DR.T.V.RAO MD

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DR.T.V.RAO MD

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MOLECULAR DIAGNOSIS OF TUBERCULOSIS .

Rapid and sensitive tools for the diagnosis of tuberculosis are
needed, due to the increased incidence of tuberculosis
epidemics and the length of time required by classical diagnostic
tests, especially among human immunodeficiency virus (HIV)infected patients. In this context, the recent advances in cloning
and characterization of M. tuberculosis genes has allowed the
application of basic molecular biology techniques to the
examination of clinical samples, such as sputum and
bronchoalveolar lavage (BAL), for the molecular diagnosis of
tuberculous infection. By using the polymerase chain reaction
(PCR) for the amplification of mycobacterial nucleic acids and
nonradiometric revelation techniques, the time required for the
identification of mycobacteria has been considerably shortened
(24-48 h), in comparison to the time required by microbiological
tests
DR.T.V.RAO
MD
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BREAKTHROUGH WITH MOLECULAR

TECHNOLOGIES

The rapid development and

availability of a variety of new
molecular genetic
technologies present the
clinician with an array of
options for the accurate
diagnosis of infectious
diseases. This is particularly
the case for tuberculosis,
since molecular methods have
been rapidly introduced into
all working areas of the
mycobacteriology laboratory.

DR.T.V.RAO MD

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MYCOBACTERIUM TUBERCULOSIS
GENOME

DR.T.V.RAO MD

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NUCLEIC ACID AMPLIFICATION

ASSAYS
NAA assays amplify M. tuberculosis-specific nucleic acid
sequences using a nucleic acid probe.
The sensitivity of the NAA assays currently in commercial
use is at least 80% in most studies
Require as few as IO bacilli from a given sample
NAA assays are also quite specific for M. tuberculosis,
with specificity in the range of 98% to 99%.

Official statement of ATS and CDC, July 1999

DR.T.V.RAO MD

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NAAS- VARIOUS TYPES

AMPLICOR M. TUBERCULOSIS assay
Amplified M.tuberculosis Direct (AMTD2) assay
LCx MTB assay, ABBOTT LCx probe system
BD ProbeTec energy transfer (ET) system (DTB)
INNO-LiPA RIF.TB assay
DR.T.V.RAO MD

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NAAS- VARIOUS TYPES

DR.T.V.RAO MD

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AMPLICOR M. TUBERCULOSIS
ASSAY

DR.T.V.RAO MD

Cohen, R. A., 1998. Am. J. Respir. Crit. Care Med. 156:156161.

Bonington, A., 1998. J. Clin. Microbiol. 36:12511254.
Al Zahrani, 2000. Am. J. Respir. Crit. Care Med. 162:13231329.

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DNA AMPLIFICATION ASSAY

Amplification of specific
DNA sequences
(eg. Polymerase Chain
Reaction PCR)
Provide rapid diagnosis
High sensitivity &
specificity
Possible to use crude
DNA e.g. boiling

DR.T.V.RAO MD

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DIRECT DETECTION OF M. TUBERCULOSIS IN

SPUTUM
BY DNA AMPLIFICATION

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

DR.T.V.RAO MD

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TARGET AMPLIFICATION

Target amplification

PCR Thermal cyclic synthesis of dsDNA by hybridization of IS6110 Roche Molecular

Systems

specific oligonucleotides to ssDNA target, extension to 65 kDa protein gene (16S rRNA,
Amplicor

dsDNA by a thermostable polymerase and denaturation 16S rDNA gene Testkit).

of ssDNA, which serves as a new target for the next MPB64 gene Colorimetric,
automated

cycle [1923]. 35 kDa protein gene sandwich-hybridization

assay using horseradish

peroxidase (Cobas Analyzer).

DR.T.V.RAO MD

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REAL TIME PCR REPLACING

OLDER METHODS

DR.T.V.RAO MD

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DNA FINGERPRINTING OF M. TUBERCULOSIS

Subtyping M. tuberculosis strains

used to rely mainly on testing for
one or several phenotypic
markers, notably unusual drug
susceptibility patterns, and on
phage typing.
These markers have been
replaced by more powerful DNAtyping methods, since the
discovery and characterization of
repetitive DNA in M. tuberculosis,
such as direct repeat (DR)
sequences and insertion
sequences (IS6110 and IS1081),
in the early 1990s

DR.T.V.RAO MD

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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

DR.T.V.RAO MD

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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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ALARMING RISE OF RESISTANT TUBERCULOSIS

WHO Report on Anti-TB Drug
Resistance

490,000 new cases of MDR-TB each year, with

>110,000 deaths1

Accounts for 5% of 9 million new cases of TB 2

MDR-TB rates higher than ever (up to 22.3%),

particularly in former Soviet Union countries

XDR-TB reported by as many as 49 countries

(by June

2008)3

Recent WHO/IUATLD Global Surveillance report

indicated 7.5% (301/4012) of MDR TB to be XDR 4

Around 40,000 XDR-TB cases emerge every year 1

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)

DR.T.V.RAO MD

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UNDERREPORTED THREAT OF MULTIDRUGRESISTANT TUBERCULOSIS IN AFRICA

DR.T.V.RAO MD

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MOLECULAR METHODS FOR DRUG

RESISTANCE

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%

DR.T.V.RAO MD

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EMERGING MOLECULAR METHODS

MADE MORE AFFORDABLE

Xpert MTB/RIF
DR.T.V.RAO MD

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AFFORDABLE PCR TB DIAGNOSTIC TOOL

DEVELOPED

Researchers have developed an

automated PCR diagnostic test
that can detect the presence of
Mycobacterium tuberculosis
(MTB) and the resistance to
rifampin (RIF), an antibiotic used
to treat it. The test, which was
described in paper published in
the New England Journal of
Medicine, promises to help the
public health sectors of lowincome countries, where the
occurrence of multidrug-resistant
pulmonary tuberculosis (TB) is
high.

DR.T.V.RAO MD

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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.
DR.T.V.RAO MD

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XPERT MTB/RIF

Dual PCR reactions sampleprocessing PCR is followed by

hemi-processing PCR increase
the tests sensitivity and
specificity: according to the
results published in NEJM, the
PCR test was 98.2% sensitive in
patients with smear-positive,
culture-positive TB. And,
because it is automated, there is
little technical training needed to
administer the tes t.

DR.T.V.RAO MD

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RAPID TB TEST GETS NOD OF APPROVAL FROM WHO

The World Health Organization

(WHO) announced Wednesday
its support of the Xpert MTB/RIF
for rapid diagnosis of
tuberculosis, multidrug resistant
TB (MDR-TB) and TB in HIVinfected individuals. The results
of the test demonstration study
were announced in September of
this year in the New England
Journal of Medicine, producing
results for many patients in
approximately 100 minutes .

DR.T.V.RAO MD

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WITHOUT INFORMATION,
THE DOCTOR CANNOT ACT.

With information,
he cannot but
act.
DR.T.V.RAO MD

42

HL MENCKENS LAW
Every complex problem
has a simple solution.

And it is always wrong in

Tuberculosis
DR.T.V.RAO MD

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 Created by Dr.T.V.Rao MD for e

learning resources for Medical
Microbiologists in the Developing World
Email
doctortvrao@gmail.com

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