NEW TECHNOLOGIES IN

DIAGNOSIS OF
TUBERCULOSIS
Dr.T.V.Rao MD

3/5/16

Dr.T.V.Rao MD

A Tribute to Robert Koch

The Mystery of Tuberculosis Continues?

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Dr.T.V.Rao MD

Samples required for

microbiological diagnosis
of TB

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Dr.T.V.Rao MD

Microscopy

Microscopy has
been a diagnostic
tool for TB for over
a century, and still
currently the most
rapid diagnostic
method. Standard
light microscopy
(LM) and
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Dr.T.V.Rao MD

3/5/16

The recent
development of
Advancing Microscopy
with
light emitting
Light Emitting diodes
Diodes
LED
(LED),
with
the appropriate
fluorescent light
output for FM and
low power
consumption, has
led to the
development of
simple, robust LED
FM microscopes,
requiring minimal
Dr.T.V.Rao MD

Microscopy and Culturing

Still feasible Alternatives
Microscopic
examination is
suitable for
laboratories at
peripheral and
higher levels and it
can be done safely
under minimal
biosafety
conditions.
Mycobacterial
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Dr.T.V.Rao MD

Antibiotic sensitivity testing by

Conventional methods getting out
dated

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Dr.T.V.Rao MD

WHO Recommending the

The WHO has
recommended
rolling it out as an
alternative to LMs in
resource-limited
settings, based on
studies that have
shown comparable
performance of LM
and standard FM
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Dr.T.V.Rao MD

SCIENCE IS MOVING
FROM BIOLOGY TO
MICROBIOLOGY

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Dr.T.V.Rao MD

Molecular Biology advances

with Nucleic acid based tests
NAATs are molecular
systems that can
detect small quantities
of genetic material
(DNA or RNA) from
microorganisms such
as Mycobacterium
tuberculosis. PCR is the
most common among
the variety of NAATs.
NAATs are available as
commercial kits and in3/5/16
Dr.T.V.Rao MD
house tests and are

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Molecular Biology advances with

Nucleic acid based tests
In-house PCR tests
are widely used in
the developing
countries because
these tests are less
expensive than
commercial kits. The
line probe assays
(LPA) are suitable
only for national or
regional level
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What are Molecular tests

In microbiology, the
term molecular test
is conventionally
used to describe
tests that detect
pathogen nucleic
acid (DNA or RNA).
All molecular
diagnostic tests
have three stages:
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12

Genotypic methods
Molecular techniques are aimed at the nucleic
acid of the mycobacterium as the analyte.
RibosomalrRNA is useful genetic target for the
identification of organisms, since it often
contains spesific sequences and is present in
the cells and media in high quantity due to the
growth of the mycobacteria. There are various
applications of molecular techniques for the
detection and identification of MTB.
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Extraction of Nucleic acid

The extraction process
should be carried out in
a containment level 3
laboratories and will
provide inactivation of
organisms since Hazard
Group 3 species may
be present
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Amplification
The nucleic acid in
the sample or
culture is amplified
so that it can be
detected.
Polymerase chain
reaction (PCR) is an
example of a
method for
amplification, but a
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Polymerase Chain Reaction

PCR is the common format
of nucleic acid
amplification tests (NAAT);
othemethodologiesinclude
ligase chain reaction, strain
displacement amplification,
loop-mediated isothermal
amplifi cation (LAMP) and
transcription mediated
amplification. More
recently, real-time
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Dr.T.V.Rao MD

16

RT PCR
RT PCR
technologies based
on fluorescentprobe detection or
melting-curve
analysis have been
developed
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17

 Methods of
detection of the
amplified nucleic
acid also vary and
include the use of
electrophoresis, line
probe assays or real
time detection.
Depending on the
format, some tests
may take two days
for results to be
available whereas
others may produce

Detection

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Different types of molecular

testing technology are
available for TB

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Rules of the Lab

Know before believe everything
No lab test is perfect
Do not order a lab test if you are
not ready to dealwith the result
Treat the patient, not the lab
testq For TBThere is a lot we still
need to learn about
DST and molecular detection of
drug resistance
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Dr.T.V.Rao MD

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NAAT for TB Diagnosis

Becoming standard of practice
CANNOT replace clinical judgment or be relied on as
the
ONLY guide for therapy or isolation practices (less
thanperfect sensitivity and specificity)
Sensitivity>95% for AFB smear-positive TB
patients5575% of AFB smear-negative, culturepositive TB Patients
Performance improves with increased clinical suspicion
ofTB
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Dr.T.V.Rao MD

Potential benefits impact on therapy, public

21

Molecular Methods helps

Detection Drug
Resistance
too
These molecular techniques also aimed

detecting resistance genes. Example

includes; DNA probe and DNA sequencing
of MTB gene such as catalase (katG) or
RNA polymerase(rpoB). Mutations in
these genes have been associated with
resistance to isoniazid and rifampicin
respectively.

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Dr.T.V.Rao MD

22

Molecular Tests are Rapid, but

expensive too
The using of molecular primers in real- time PCR
reaction can differentiate between the presence of
the wild- type sequence and mutated sequence
associated with drug resistance. Molecular tests
are rapid (within few hours), highly sensitive and
specific, but expensive, requires expertise and
may not differentiate active infection as DNA from
a dead organism during antibiotic treatment can
be detected and amplified by PCR
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Dr.T.V.Rao MD

23

Line-probe assays
Line-probe assays and
XPERT MTB/RIF: Line
probe assays (LPAs)
are actual molecular
tests. Three main LPAs
for the rapid diagnosis
of TB and/or rapid
detection of RMP
resistance and
MDR-/XDR-TB are
currently available on
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Dr.T.V.Rao MD

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Line probe assays (LPAs) for

tuberculosis were endorsed by the
WHO in 2008

Line probe assays

(LPAs) for tuberculosis
were endorsed by the
WHO in 2008 for
molecular detection of
drug resistance from
smear-positive
patients at risk of Multi
Drug ResistantTuberculosis (MDRTB)
Two commercial LPAs
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Dr.T.V.Rao MD

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Line Probe Assays Line

forProbe
tuberculosis
Assays for

3/5/16

tuberculosis uses
PCR/hybridization technique to
identify members of the
Mycobacterium Tuberculosis
while simultaneously
identifying drug-resistant
strains by detecting the most
common single nucleotide
polymorphorisms (SNPs)
associated with resistance.
Meta-analyses have shown
that LPAs are highly accurate
for the detection of first-line
drug resistance, especially in
Dr.T.V.Rao MD
26
smear-positive sputum

World Health Organization (WHO)

recommends the use of Xpert
MTB/RIF

World Health
Organization (WHO)
recommends the use
of Xpert MTB/RIF
assay for rapid
diagnosis of
tuberculosis (TB) and
detection of
rifampicin resistance.
This systematic
review was done to
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Dr.T.V.Rao MD

27

WHO Endorses New Testing

Method in 2011

In early 2011, WHO endorsed a novel, rapid, automated,

molecular diagnostic test, the Xpert MTB/RIF assay that can
simultaneously detect TB and rifampicin resistance. Xpert
MTB/RIF provides result within 2 h, thus the patients can be
started with proper treatment even on the same day.

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Dr.T.V.Rao MD

28

GenoTypeMTBDR/MTBDRplus and
Geno-Type

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GenoTypeMTBDR/MTBDRplus and Geno-Type

MTBDRsl (both HainLifescience, Germany). These
assays are based on the targeted amplification (PCR)
of specific fragments of the MTB genome, followed by
hybridisation of PCR products to oligonucleotide
probes immobilised on membranes

Dr.T.V.Rao MD

29

Newer Innovative Methods in

Diagnosis of Tuberculosis

INNO-LiPA Rif TB detects only RMP

resistance,
GenoType MTBDR/MTBDR plus detects both
RMP and INH resistance, and GenoType
MTBDRsldetects resistance to
fluoroquinolones, injectable second-line
drugs and ethambutol.
These tests are designed for detection the
MTB isolates in respiratory specimens.
Xpert
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Molecular Methods are not

routinely Used

Genotypic
methods are not
routinely used in
the
mycobacterium
laboratory; they
are essentially
for research

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Dr.T.V.Rao MD

31

Many Molecular Methods in offing

Multiple commercial tests
are available and new
tests are also being
developed. Some
laboratories have
developed local inhouse tests. The choice
of test depends on clinical
need and local laboratory
factors such as numbers
of specimens received by
the laboratory, access toDr.T.V.Rao MD
3/5/16
molecular platforms and

32

Line probe assays

3/5/16

Line probe assays and the Cepheid MTB/RIF test which

is run on the GeneXpert platform are commercial
systems that are recommended by the WHO, for
resource poor countries where financial support is
available for the cost of tests. In developed countries
this format of test is convenient but no more accurate
or reliable than other test systems and may be more
expensive.

Dr.T.V.Rao MD

33

Limitations and Considerations

on Molecular Methods
Sensitivity
Reduced for smear negative specimens and some
specimen types?
Do you want to rule in or rule out?
Platform dependent
q Specificity
Platform dependent
q Does not replace need for culture
Culture still needed for conventional DST, genotyping
q Amplicon cross contamination in open systems
q Cost and sustainability
Expense
can limit utilization
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Dr.T.V.Rao MD

34

Attention of Viewers
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and Hospital associated Infection wish to be your partner in educating many millions who
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Mob +91 7204113154

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

Benefit of Medical and Paramedical
Professionals in the Developing World
Created from World Wide Resources
Email
doctortvrao@gmail.com
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Dr.T.V.Rao MD

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