Académique Documents
Professionnel Documents
Culture Documents
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.
500
400
300
AFR low HIV
Drug
susceptible MDR-TB XDR-TB Total DR
TB*§ 1990§ 2006§ ?
Wild M. TB strain
Spontaneous mutation
Acquired drug
resistance
Transmission
Primary drug
resistance
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
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
Dr.T.V.Rao MD
XDR-TB: 8/23/06
“Rapidly Fatal in South Africa” Tugela
Ferry, KwaZulu-Natal
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
Ecuador Portugal
France Romania
Germany Slovenia
India Sweden
Islamic RepublicSpain
of Iran