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Drug Resistant

Tuberculosis
A Great Human Concern
Dr.T.V.Rao MD
MDR TB –Great Human Concern
HISTORY of
Tuberculosis
Tuberculosis Is an
Ancient Disease
Identified as Spinal
Tuberculosis in
Egyptian Mummies
History dates to
1550 – 1080 BC
Identified
by PCR
A Tribute to Robert Koch
Discoverer of Mycobacterium
Tuberculosis
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.
USAID Report on Tuberculosis
in India
 India has more new tuberculosis (TB) cases
annually than any other country, ranking first
among the 22 high-burden TB countries
worldwide, according to the World Health
Organization’s (WHO’s) Global TB Report 2009.
TB remains one of the leading infectious causes
of mortality in India, causing more than 331,000
deaths in 2007. There were approximately 1.96
million new TB cases in India in 2007,
representing more than 21 percent of all TB
cases worldwide
Nobody is absolutely Immune to
Tuberculosis
Pharmacological discoveries
 1908-1920 (Calmette and Guerin)
 Vaccine (BCG)
 Attenuated strain Mycobacterium Bovis
 1943
 Streptomycin developed
 20th November 1944
 Critically ill TB patient injected dramatically recovered
Selman Abraham Waksman Nobel
Prize for his discovery in 1952.
Pharmacological discoveries
 1956-1960
 Combination therapy of INH and PZA cures TB
 1955 Cycloserine
 1962 Ethambutol
 1963 Rifampicin
 1970-1977
 Combination of Rifampicin and Isoniazid adopted as
International regime for treatment of TB
Introduction
Tuberculosis is an ancient disease & it
remains the leading cause of death of
human being.
 It is mainly caused by Mycobacterium
tuberculosis
Typical tubercle bacilli
 Human type M.tuberculosis.

 Bovine type M.bovis.

 Vole type M.microti.

 Human type M.africanum.


Multi Drug Resistant
Tuberculosis
MDR-TB
Definition
 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
MDR-TB & XDR-TB
THE 2008 REPORT
% of MDR-TB among new TB cases 1994-2007
Classification of Drugs
 3 Groups depending upon the degree of
effectiveness and potential side effects
 First Line: (Primary agents)
 are the most effective and have lowest toxicity. Isoniazid
Rifampin
 Second Line:
 Less effective and more toxic effects
 include (in no particular order): p-amino salicylic acid,
Streptomycin, Ethambutol
 Third Line
 are least effective and most toxic. Amikacin, Kanamycin,
Capreomycin, Viomycin, Kanamycin, Cycloserine
Several Drugs becoming
resistant
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)
What is multidrug-resistant
tuberculosis (MDR TB)?

 Multidrug-resistant TB (MDR TB) is TB


that is resistant to at least two of the best
anti-TB drugs, isoniazid and rifampicin.
These drugs are considered first-line
drugs and are used to treat all persons
with TB disease
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
What is extensively drug
resistant tuberculosis (XDR TB)?
 Extensively drug resistant TB (XDR TB) is
a relatively rare type of MDR TB. XDR TB
is defined as TB which is resistant to
isoniazid and rifampin, plus resistant to
any fluoroquinolone and at least one of
three injectable second-line drugs (i.e.,
amikacin, kanamycin, or capreomycin).
Why XDR - TB a grave concern

 Because XDR TB is resistant to first-line and


secondline drugs, patients are left with treatment
options that are much less effective.
 XDR TB is of special concern for persons with
HIV infection or other conditions that can
weaken the immune system. These persons are
more likely to develop TB disease once they are
infected, and also have a higher risk of death
once they develop TB.
Global Estimates
Classificati Estimated Number Estimated
on of Cases Number of
Deaths
All forms 8.8 million 1.6 million
TB
MDR TB 4,24,000 1,16,000,

XDR TB 27,000 16,000


Extensively Drug-Resistant
Mycobacterium tuberculosis, India

 The first XDR TB cases in India


and the emergence of XDR TB is
reported by Rajesh Mondal* and
Amita Jain*
*King George's Medical University,
Lucknow, India Volume 13, Number 9–
September 2007 in Emerging Infectious Diseases.
Global incidence of tuberculosis
Still rising as a result of the growing epidemic in
600 Africa
AFR high HIV
Incidence per 100,000 per year

500

400

300
AFR low HIV

200 Sth East Asia


World
West. Pacific
100 East. Medit.
East. Europe
Lat. America
Cent. Euro,
0 Est Market
1990 1995 2000 2005 2010 2015
Are we Returning to a Pre-
antibiotic Era

Drug
susceptible MDR-TB XDR-TB Total DR
TB*§ 1990§ 2006§ ?

*or limited Resistance Resistance Resistance to


resistance to H&R – to 2nd line all available
manageable drugs – drugs –
with 4 drug Treatable
Treatment
with 2nd
regimen -
line drugs
options No
DOTS seriously treatment
restricted
options
WHO Surveillance and Incidence of
MDR TB
Country % MDR TB of all new cases
Estonia 14.1
Latvia 9.0
China (non-DOTS) 7.7

China (DOTS) 2.8


Russia 6.0
India 3.4
Iran 5.8
Dominican 6.6
Ivory Cost 5.3

Dye et al. Global Burden of Multidrug-Resistant TB. JID 185(8), 2002


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.
Development of anti-tuberculosis drug resistance

Wild M. TB strain

Spontaneous mutation

Strains with genetic


drug resistance

Selection: inadequate treatment

Acquired drug
resistance

Transmission

Primary drug
resistance

Pablos-Mendez et al. WHO, 1997


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
Mechanism of resistance
 INH
 Chromosomally mediated
 Loss of catalase/peroxidase
 Mutation in mycolic acid synthesis
 Regulators of peroxide response
Mechanism of resistance
 Rifampin
 Reduced binding to RNA polymerase
 Clusters of mutations at “Rifampin Resistance
Determining Region” (RRDR)

 Reduced Cell wall permeability


Gene location associated
Drug-Resistant M.tuberculosis
Drug Gene
Isoniazid Kat G, Inh A, Kas A
Rifampicin rpo B
Ethambutol emb B
Streptomycin rps L
Pyrazinamide pnc A
Fluoroquinolones gyr A

Dubaniewicz A, et al. Molecular sub-type of the HLA-DR antigens


in pulmonary tuberculosis. Int J Infect Dis2000;4:129-33.
Drug Susceptibility
Testing
Susceptibility Testing

 � Direct and indirect testing


 � Primary Drugs testing
 � Isoniazid
 � Rifampicin
 � Ethambutol (*)
 � Pyrizinamide (*)
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 for this
Drug susceptibility Testing
 Assessment of grwoth inhibition on solid
media containing various dilutions of the
drug, in comparison with the test strains.
 As the method depend observation of
grwoth
Results are not available until several
weeks after isolation of the organism.
Other accredited Methods
 Radiometric and Non radiometric methods
 Nucleicacid technology – effective upto
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.
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.
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 ?
MODS
Microscopic Observation of
Drug Susceptibility Testing
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
microcolonies could be seen under the
microscope long before a colour change
occurred prompted the development of MODS.
Review Article in Indian Journal
of Medical Microbiology
 Caviedes L, Moore
DA. Introducing
mods: A low-cost,
low-tech tool for high-
performance
detection of
tuberculosis and
multidrug resistant
tuberculosis. Indian J
Med Microbial
2007;25:87-8
Observation of Grwoth 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
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
MODS more streamlined
 Recently completed operational field
studies have served to refine and
streamline the methodology further and
importantly validate MODS as a test for
TB detection and MDRTB detection
directly from sputum.
Inverted Microscope a minimal
need
 Characteristic “
tangles “ of
M.tuberculosis can
be visualised under
microscope long
before colonies to
the naked eye
MODS for detection of
MDR - TB

 The scientific observations have proved


that a single MODS culture of sputum
sample offers more rapid and sensitive
detection of tuberculosis and Multidrug-
resistant tuberculosis than the existing
gold standard methods used.
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.
Performing MODS Assay
 The MODS assay was performed as
described in standard protocols,
 Broth cultures were prepared in 24 well
tissue culture plates ( Becton Dickinson)
each containing decontaminant, 7H9 broth
(Becton Dickinson), oxalic acid, albumin,
dextrose, and catlase (OADC) (Becton
Dickinson) and Polymyxin, Amphotericin B,
Nalidixic acid,trimethoprim and azlocillin
(PANTA)
MODS Assay ( Contd)

 For each sample, 12 wells were used;


 Four in control wells, no drug was used
and each of the remaining 8 wells,
contained one of the four drugs at one of
the two concentrations tested.
 The cultures were examined under an inverted
light microscope at magnification of 40x every
day ( except weekends ) from 4 to day 15, on
alternative days from 16 today 25 and twice
weekly from 26 to 40day.
Sample layout on MODS plate
(2 samples per plate)
No plate contained 2 samples from the same
patient
Drug susceptibility Testing
In MODS
 Drug susceptibility testing was performed
with the use of MODS assay,
 Growth in the drug free control wells but
not in drug containing wells, indicates
susceptibility
 The drug concentration were as follows
 Isoniazid , 0.1 and 0.4 µg/milliliter
 Rifampicin 1 and 2 µg per millilitre
Differentiation from Typical and
Atypical Mycobacterium
 Nontuberculous
mycobacteria (NTM)
were recognized by
their lack of cording or
(in the case of
Mycobacterium
chelonae that uniquely
among NTM does form
cords) rapid overgrowth
of wells by day 5.
identified by cording on
Microscopy
MODS assay ( Contd)
 To minimize cross
contamination and
occupational
exposure, plates were
permanently sealed
inside plastic zip lock
bags after inoculation
and were
subsequently
examined with in the
bag
Observation of growth in
MODS

 Positive cultures
were identified by
cord formation,
characteristic of
M.tuberculosis
grwoth,in liquid
medium in drug
free control wells.
MODS in Atypical
Mycobacterium
 Non tuberculous
mycobacterium
were recognised by
their lack of cording
or, for M,chelonae (
which forms cords)
by rapid
overgrwoth by day
5.
Contamination in MODS Assay
Fungal or bacterial
contamination was
recognised by rapid
overgrowth and
clouding in wells.
If contamination was
detected, the original
samples was cultured
again after being
decontaminated once
more
Honduras study comparing the
LJ medium
 Per specimen, there was concordance
between MODS and LJ culture in 94.2%
MODS tests were also less prone to
contamination than LJ cultures. 62 [3.8%]
vs (95 [5.8%] of 1,639 samples,
respectively (P ≤0.01).
PCR: Molecular susceptibility
testing

Hain INNO-
Genotype LiPA
MTBDR Rif.TB
assay
RMP resistance

INH resistance
Confirming MODS results
 Spacer
Oligonucleotide typing
Spoligotyping,
polymerase chain
reaction with multiple
primers, or both were
applied to all isolates
from each of the three
types of cultures in
order to confirm the
presence of
M.tuberculosis.
MODS and MDR detection
• The drug sensitivity for Rifampicin and Isoniazid
can be tested and established the presence of
MDR.
• In view of being chronic disease it is highly
essential to establish MDR Tuberculosis at
centers serving DOTS under WHO guidelines
• Starting and establishing centers to identify MDR
at every district and Teaching Medical centers
leads to better control of Tuberculosis
Why MODS is a better
method for MDR TB
detection
 If a MODS culture was negative on day
15,there is 99.7% chance that the
sample is truly culture negative.
 The negative MODS cultures can be
discarded after 3 weeks
Biosafety concerns in MODS
technology
 Legitimate concerns about biosafety with
other liquid culture systems do not really
apply to MODS, indeed the converse is
the case. After inoculation with
decontaminated sample the MODS plates
are permanently sealed in ziplock
polythene bags through which the
microscopic examination is made, thus
spillage of the mycobacterial "soup"
cannot occur.
Lower Grade Biosafety is
adequate
N 95 mask protects from Biohazard
No transfer of Materials needed
in MODS
As no secondary sub-culture is needed
(because this is direct and not indirect
susceptibility testing) no further
manipulation is required - this zero potential
for aerosolisation or accident compares
favourably with the hazard associated with
preparation of a standardized inoculum for
indirect DST.
Computer pattern
recognition
of Mycobacterium
tuberculosis
in MODS culture
Automation in MODS
Day 6
M. tuberculosis in MODS x10 objective (sputum sample
inoculation)
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15

Day 16 Day 17
MODS can be used in Extra pulmonary
Tuberculosis
Draw backs of MODS
 One possible drawback however, could be
the inability of the laboratory technicians to
distinguish between TB and some NTM.
This could potentially have clinical impact
in settings where NTM prevalence is high
and not all mycobacteria respond to anti-
TB treatment.
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
WHO Controls the Tuberculosis
related work
 The laboratory methods for anti-
tuberculosis drug susceptibility testing
should be selected from among those that
are WHO-recommended, and all
laboratory processes should be quality-
assured in cooperation with a partner
Supranational Reference Laboratory
(SRL)
XDR-TB in South Africa
August 2006
 53 of 544 patients defined as XDR-TB
cases
 • 52 of the 53 patients died on average
within 25
 days, including those on antiretroviral
therapy
 • Further investigations being carried out
 • XDR-TB likely in bordering African
countries
Molecular Fingerprinting
 26 of 30 (87%) XDR TB isolates found to
be genetically similar
Majority of patients had no previous history
of TB treatment Suggestive of recent
infection with drug-resistant strain
Additional cases identified in 28 of the 68
hospitals in KZN KwaZulu Natal
CDC Updates Guidelines for Nucleic Acid
Amplification Techniques to Diagnose
Tuberculosis
 NAAT results should be interpreted in
conjunction with the AFB smear
results.
 NAAT and smear positive: start Rx
despite pending culture results. PPV
95%
 Smear negative, NAAT positive: use
clinical judgment to either treat or await
culture
Selection from automated systems for
molecular and
bacteriological rapid diagnostics
 PCR:
 Roche/COBAS®: Amplicor®
amplification kits
 Roche/COBAS® : LightCycler® (real-
time-PCR)
 Roche/COBAS® : TaqMan 48®
 (increases the specificity of real-time-
PCR)
Microscopy and Culturing still a
top priority
Is PCR methods a solution ?
 PCR can't yet
replace neither
microscopy,
culturing and
competent clinical
examination.
No testing method replaces
clinical assessment
Extreme Drug resistant
Tuberculosis (XDR-TB)
 Resistant to all first line drugs namely; Isoniazid and
Rifampin and
 Three or more second line drugs (SLD’S) that are
used to treat MDR-TB
 Thequinalones like Ofloaxin
Or
 Aminoglygocides like Capreomycin & Kanamycin
 No third-line drugs available to treat XDR-TB since
none has been developed in the last 40 years.

 Dr.T.V.Rao MD
XDR-TB: 8/23/06
“Rapidly Fatal in South Africa” Tugela
Ferry, KwaZulu-Natal

 10% isolates resistant to ALL 1st and


2nd line agents

 51/52 XDR dead in median 16 days


after first positive sputum

 67% AIDS deaths w/ MDR TB


Countries with confirmed XDR-
TB cases as of September 2007
Argentina Japan

border lines for which there may not yet be full agreement.  WHO 2005. All rights reserved
authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate
whatsoever on the part of the WHO concerning the legal status of any country, territory, city or area or of its
The boundaries and names shown and the designations used on this map do not imply the expression of any opinion
Armenia Latvia

Azerbaijan Lithuania

Australia Mexico

Bangladesh Mozambique

Brazil Netherlands

Canada Norway

Chile Peru

China, Hong Kong SAR

Czech Republic Poland

Ecuador Portugal

Estonia Republic of Korea

France Romania

Georgia Russian Federation

Germany Slovenia

Ireland South Africa

India Sweden

Islamic RepublicSpain
of Iran

Israel Thailand USA Based on information provided to WHO Stop TB Department


Italy UK Vietnam
13 September 2007
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
Health Care Workers and MDR
TB
 Recognised risk for health care workers
 Risk assessment
 High risk – Prolonged closed contact with
infectious patients
 Smear positive MDR TB patients
Medium risk –Primary health care centres
involved
Sputum collection on TB suspects
 Low risk –Health care support staff e.g.
cleaners
 Porters and admin staff
Dr.T.V.Rao MD
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*
Are there any solutions for
effective Diagnosis in TB ?
Many more powerful hands needed
to Control Tuberculosis
Contribute your Knowledge, Wisdom,
to prevent spread and control of
Tuberculosis
Created by Dr.T.V.Rao MD for
‘e’ learning Programme
Email
doctortvrao@gmail.com

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