Multi Drug Resistant

Tuberculosis
Basics, Concerns and Diagnosis
Dr.T.V.Rao MD
A Tribute to Robert Koch
Discoverer of Mycobacterium
Tuberculosis

Dr.T.V.Rao MD 2
 Nobody is absolutely
Immune to Tuberculosis

Dr.T.V.Rao MD 3
 Basic concepts – Keep facts
Primary (Initial) resistance
TB patient’s initial Mycobacterium tuberculosis population
resistant to drugs

Secondary (Acquired) resistance

Drug-resistant M. tuberculosis in initial population
selected by inappropriate drug use (inadequate
treatment or non-adherence)

Dr.T.V.Rao MD 4
 Changing Definition of
MDR TB
1950s-1970s:
M. tb resistant to INH,
streptomycin and/or PAS

1980s-current:

M. tb resistant to at least INH

and Rifampin
 Definition of MDR - TB
MDR-TB caused by strains of
Mycobacterium Tuberculosis resistant
both Rifampicin and Isoniazid with or
without resistance to other drugs.

Single Isoniazid or Rifampicin resistance

is not MDR - TB

MDR TB is a laboratory diagnosis, Not

a Clinical assumption
Dr.T.V.Rao MD 7
 Why INH and Rifampin
Most potent and bactericidal drugs for
Tuberculosis
Tb can be treated effectively with INH+Rif
alone
Mono-resistance to one of them can be
treated effectively with a regimen containing
the other agent with very low failure rate (2.5-
5%)
Failure rate when INH+Rif resistant is 44% in
non-HIV and 70% in HIV patients
Duration required for cure doubles to triples.
Dr.T.V.Rao MD 8
 MDR-TB & XDR-TB
THE 2008 REPORT
% of MDR-TB among new TB cases 1994-2007

Dr.T.V.Rao MD 11
Dr.T.V.Rao MD 12
Epidemiology of MDR TB
 Genesis of MDR TB
Resistance is a man-made amplification of a
natural phenomenon.

Inadequate drug delivery is main cause of

secondary drug resistance.

Secondary drug resistance is the main cause of

primary drug resistance due to transmission of
resistant strains.

MDR due to spontaneous mutations is not

possible as the genes encoding resistance for
anti TB are unlinked.
Dr.T.V.Rao MD 14
 When to suspect MDR
TB
Re-treatment patients who’s sputum smear
remains positive after three months’ of
intensive therapy
Treatment failure and interruption cases
Close contacts of MDR tuberculosis cases
Positive diagnoses with;
TB culture and susceptibility testing

Dr.T.V.Rao MD 16
 Factors Contributing to
Development
and Spread of MDR and XDR TB
Weak TB programs (DOTS)
Low completion/cure rates
Lack of treatment follow up and patient
support
Unreliable drug supply
Diagnostic delay
Absent or inadequate infection control measures
Uncontrolled use of 2nd line drugs
Dr.T.V.Rao MD 17
Mechanism of resistance
INH
Chromosomally
mediated

Loss of
catalase/peroxidase

Mutation in mycolic
acid synthesis

Regulators of
peroxide response

Dr.T.V.Rao MD 18
Mechanism of resistance
Rifampin
Reduced binding to RNA
polymerase

Clusters of mutations at
“Rifampin Resistance
Determining Region”
(RRDR)

Reduced Cell
wall permeability
Dr.T.V.Rao MD 19
LABORATORY DIAGNOSIS
OF MDR-TB AND XDR-TB
The laboratory is an essential component in TB
control programs, and broader access to DST is
a priority for most countries. Early choice of
appropriate treatment is an essential
determinant of favourable outcome, and rapid
determination of drug resistance can allow a
customized approach to treatment early in the
course of the disease and can potentially reduce
morbidity, mortality and infectiousness
Dr.T.V.Rao MD 20
CONVENTIONAL CULTURE-BASED
METHODS
Using standardized DST procedures with
conventional methods, eight to 12 weeks
are required to identify drug-resistant
microorganisms on solid media (ie,
Lowenstein-Jensen medium). In general,
such methods assess inhibition of M
tuberculosis growth in the presence of
antibiotics to distinguish between
susceptible and resistant strains
Dr.T.V.Rao MD 21
 Proportion method
The proportion method allows precise
determination of the proportion of resistant
mutants to a certain drug; the resistance ratio
method compares the resistance of an unknown
strain with that of a standard laboratory strain.
While relatively inexpensive and undemanding of
sophisticated equipment, results usually take
weeks and this is challenging; inappropriate
choice of treatment regimen may result in death
within weeks of initiation, such as in the case of
XDR-TB (especially in HIV-infected patients)
Dr.T.V.Rao MD 22
 Conventional Methods
are Outdated
In addition, delayed
identification of drug
resistance results in
inadequate treatment,
which may generate
additional drug
resistance and
continued
transmission in the
community.
 LIQUID CULTURE-BASED
METHODS
The BACTEC 460 TB radiometric system (Becton
Dickinson, USA) was considered to be a major
advancement when it was introduced, but has been
replaced by the Mycobacteria Growth Indicator Tube
system (Becton Dickinson, USA). Several published
studies have shown the excellent performance of the
Mycobacteria Growth Indicator Tube system for the
rapid detection of resistance to first- and second-line
anti-TB drugs . Detection of drug resistance can be
accomplished in days rather than weeks, although still
constrained by high cost (equipment and
consumables).
 Diagnosis of MDR-TB
and XDR-TB
The diagnosis of MDR-TB and XDR-TB is
hampered by the absence of effective and
affordable rapid diagnostic techniques for
drug sensitivity. Several approaches,
phenotypic and molecular, have been
explored to develop rapid, reliable and
accurate methods for the rapid detection of
drug resistance in M tuberculosis. These
methods should also be evaluated and
applied in high-incidence areas
Dr.T.V.Rao MD 25
 Susceptibility Testing
􀂄 Direct and indirect testing

􀂄 Primary Drugs testing

􀂄 Isoniazid

􀂄 Rifampicin

􀂄 Ethambutol (*)

􀂄 Pyrazinamide (*)

Dr.T.V.Rao MD 26
 Drug susceptibility testing
(DST)
DST is recommended for all new cases for all first line
drugs with specimens taken before initiating
treatment.?
Accuracy is more important than speed
DST results should come from a small number of well-
equipped, experienced laboratories who participate
and perform well in an international DST quality
control scheme.
The WHO Supranational Laboratory Quality
Control Network offers the greatest global
coverage.

Dr.T.V.Rao MD 27
Drug susceptibility Testing
Assessment of growth
inhibition on solid
media containing
various dilutions of the
drug, in comparison
with the test strains.
As the method depend
observation of growth
Results are not
available until several
weeks after isolation of
the organism.

Dr.T.V.Rao MD 28
 MODS
Microscopic Observation
of Drug Susceptibility
Testing

Dr.T.V.Rao MD 29
 MODS affordable
Technically Feasible
MODS arose during experiments conducted by
Luz Caviedes under the guidance of Professor
Robert Gilman at Universidad Peruana
Cayetano Heredia in Lima, Peru in the late
1990s in which a colorimetric test for TB growth
was being investigated. The observation that
micro colonies could be seen under the
microscope long before a colour change
occurred prompted the development of MODS.

Dr.T.V.Rao MD 30
 Observation of Growth in
liquid Media
MODS depends upon three key principles
(which have been known for decades): (1)
Mycobacterium tuberculosis grows faster in
liquid (broth) than on solid media, (2) in liquid
cultures M. tuberculosis grows in a visually
characteristic manner (tangles, cording) which
can be observed under the microscope long
before the naked eye could visualize colonies
on solid agar
Dr.T.V.Rao MD 31
Least time required for detection of
MDR
Incorporation of
anti-TB drugs
into broth
cultures at the
outset enables
direct
susceptibility
testing from
sputum samples
Dr.T.V.Rao MD 32
 Advantages of MODS
methodology in MDR detection
• All the chemical ingredients are available locally,
except few which can be acquired easily.
• Existing infrastructure in District and Teaching
hospital can be adopted for implementation of
MODS
• Risk to technician handling the specimens is
minimal, there is no absolute need to obtain
grade III safety cabinets,
Technology transfer is easier all the new
technical manpower can be trained easily.

Dr.T.V.Rao MD 33
 Other accredited
Methods
Radiometric and Non
radiometric methods
Nucleic acid
technology –
effective up to 95% in
mutations to
rifampicin resistance
to gene rpoB gene
 Drug susceptibility testing
(DST)
As a minimum,
laboratories
supplying DST data,
should correctly
identify resistance to
isoniazid and
rifampicin in over
90% of quality
control samples in
two out of the last
three quality control
rounds.
Dr.T.V.Rao MD 35
 Detection of Rifampicin Drug
susceptibility testing (DST) is more
important.
Early identification of mycobacterial growth as M.
tuberculosis complex and the identification of
rifampicin resistance should be the first priority as
rifampicin resistance invalidates standard 6 month
short-course chemotherapy and is a useful marker
in most countries for MDR-TB.

Laboratories should aim to identify isolates as M.

tuberculosis complex and perform rifampicin
resistance in 90% of isolates within 1-2 working
days. This is technologically feasible.

Dr.T.V.Rao MD 36
 Drug susceptibility testing
For DST laboratories,
modern molecular
techniques permit the
successful
identification of
isoniazid resistance in
at least 75% of
mycobacterial cultures
within 1-2 working
days and are useful
preliminary screens for
isoniazid resistance.
 Secondary Drugs testing:
[lack of standardized methods!]

Ofloxacin, quinolones

Ethionamide

Kanamycin

Capreomycin

! Ensure quality control and

quality assurance ?
 Other WHO-Endorsed
Tools
Liquid culture (e.g. MGIT, BacT/ALERT)

Capilia TB

Rapid strip test that detects a TB-specific antigen from

culture

Molecular line probe assays (e.g. Genotype

MTBDRplus, INNO-LiPA Rif.TB)

Strip test for detection of TB and drug-resistance

conferring mutations

Dr.T.V.Rao MD 39
 NOVEL, RAPID
MOLECULAR METHODS
The identification of specific mutations responsible for
drug resistance has facilitated the development of novel,
rapid molecular tools for DST. The detection of RIF
resistance is traditionally used as a predictor of MDR-TB
– its positive predictive value is a function of the
sensitivity and specificity of RIF resistance testing and
the prevalence of MDR and non-MDR RIF resistance,
which is highest among previously treated cases in
settings with high MDR prevalence and low non-MDR
RIF resistance.

Dr.T.V.Rao MD 40
Xpert MTB/RIF
The Xpert MTB/RIF
The Xpert MTB/RIF is a
cartridge-based,
automated diagnostic
test that can identify
Mycobacterium
tuberculosis (MTB)DNA
and resistance to
rifampicin (RIF)by
nucleic acid
amplification
technique(NAAT

Dr.T.V.Rao MD 42
WHO Endorses Xpert MTB/RIF

In December 2010,
the World Health
Organization (WHO)
endorsed the Xpert
MTB/RIF for use in
TB endemic
countries[2] and
declared it a major
milestone for global
TB diagnosis.
Dr.T.V.Rao MD 44
 Xpert MTB/RIF detects DNA
sequences
The Xpert MTB/RIF detects
DNA sequences specific
for Mycobacterium
tuberculosis and rifampicin
resistance by polymerase
chain reaction It is based
on the Cepheid GeneXpert
system, a platform for
rapid and simple-to-use
nucleic acid amplification
tests (NAAT).

Dr.T.V.Rao MD 45
 How Xpert® MTB/RIF
Works
The Xpert® MTB/RIF
purifies and
concentrates
Mycobacterium
tuberculosis bacilli from
sputum samples,
isolates genomic
material from the
captured bacteria by
sonication and
subsequently amplifies
the genomic DNA by
PCR.

Dr.T.V.Rao MD 46
 Xpert® MTB/RIF Helps in Faster
Diagnosis of Resistance to
Rifampicin
The process identifies all the clinically relevant
Rifampicin resistance inducing mutations in the
RNA polymerase beta (rpoB) gene in the
Mycobacterium tuberculosis genome in a real time
format using fluorescent probes called molecular
beacons. Results are obtained from unprocessed
sputum samples in 90 minutes, with minimal
biohazard and very little technical training required
to operate.This test was developed as an on-
demand near patient technology which could be
performed even in a doctor's office if necessary.
 Summary
Drug resistant TB
Drug-resistant TB poses a grave public health threat especially in
high HIV prevalence settings

XDR-TB strains have been found in all regions of the world

XDR-TB occurs as a result inadequate TB control programmes

XDR-TB, if identified early, can be treated and cured but

experience limited to low HIV prevalence settings

Infection control measures must be strengthened

XDR-TB underlines the need for investment in basic TB control

plus development of new TB diagnostics, treatments and vaccines

Dr.T.V.Rao MD 49
No testing method replaces clinical
assessment for Tuberculosis

Dr.T.V.Rao MD 50
Progress in diagnosing multidrug-
resistant tuberculosis
20 March 2014 |
Geneva - Almost half
a million people fell ill
with multidrug-
resistant tuberculosis
(MDR-TB) in 2012,
yet less than one in 4
of these people was
diagnosed, mainly
due to a lack of
access to quality
diagnostic services.
Dr.T.V.Rao MD 51
 Koch failed to conquer tuberculosis, which still
causes enormous health problems worldwide 100
years after his Nobel award.

The scientific academies

noted that the triumphant
discovery of 1882 was
followed by a succession
of failures: first of all, the
failed attempt to present
tuberculin as a remedy
against tuberculosis in
1890-91, which severely
damaged Koch's
reputation
Medical History, 2001, 45: 1-32
CHRISTOPH GRADMANN*

Dr.T.V.Rao MD 52
Tuberculosis is a Concern
for Everyone

Dr.T.V.Rao MD 53
MDR Tuberculosis is Global
Emergency

Dr.T.V.Rao MD 54
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Interest on Infectious Diseases

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Programme Created by Dr.T.V.Rao MD for
Medical and Health Professionals in the
Developing World

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