3. • History
• Drug resistance type
• Mechanism of drug resistance
• Detection methods for drug resistance
• TB and HIV
• PMDT
• New Anti TB Drug
• Vaccines
4. Problem of TB in India (WHO SEARO
REPORT 2015-2016)
• Estimated incidence
• -2.8 MILLION CASES
o 167 (156-179) per 1 lac population annually
o 75 new smear positive PTB cases/1lakh population per year
• Estimated prevalence of TB disease
o 195 (131-271) per 1 lac population annually
• Estimated mortality
o 480,000 deaths due to TB each year
o Over 1000 deaths a day
o 2 deaths every 3 minutes
Gujarat and Maharashtra has indicated multi drug resistance levels
of 3% among new TB cases and 12-17% among previously treated
TB patients.
5. HISTORY
• MDR-TB was first seen in the early 90’s
• First case of XDR –TB in India was diagnosed in P. D.
Hinduja National Hospital and Medical Research Centre,
Mumbai in 2006
• In 2007 (May) – First report of two documented cases of
extremely drug resistant (XXDR) TB) in Italy
6. XXDR TB in India
• In January 2012 it was reported that twelve cases of TB had
been diagnosed in Mumbai which were referred to as totally
drug resistant (TDR) TB/XXDR
• NTI Bangalore confirms 8 strains to be resistance to all known
first and second line TB drugs and 2 from Mumbai were
sensitive to second line drug
• There have also been six cases in Bangalore and a further two
in New Delhi⁵
“We have little to offer these patients except for drastic surgery
and medication for some relief,”
Dr Zarir Udwadia4
7. Categories of Antituberculosis Drugs:
WHO
Group 1 – First-line drugs: Isoniazid, rifampicin,
ethambutol, pyrazinamide
Group 2 - Injectable agents: Kanamycin, amikacin,
capreomycin, streptomycin
Group 3 - Fluoroquinolones: Levofloxacin,
moxifloxacin, ofloxacin
Group 4 - Oral bacteriostatic agents: Ethionamide,
cycloserine, para-aminosalicylic acid (PAS),
prothionamide, terizadone
Group 5 – Unclear role: Clofazamine, linezolid,
amoxicillin/clavulanate, Imipenem/cilastatin,
thioacetazone, high-dose isoniazid, clarithromycin,
8. Drug resistance - types
• defined as presence of drug resistance to
one or more antiTB drugs in TB patient
who has never received prior antiTB
chemotherapy
PRIMARY
DRUG
RESISTANCE
• defined as resistance to one or more
anti TB drugs, which arises during the
course of t/t, usually as a result of non-
adherence to the recommended
regimen or faulty prescribing.
ACQUIRED
DRUG
RESISTANCE
9. 9
Definitions of drug resistant TB
TB condition Definition
Monoresistant TB: resistant to a single drug
Polyresistant TB: resistant to at least two drugs, but not involving
Isoniazid and Rifampicin simultaneously.
Multidrug resistant (MDR) Resistance to:
+ Isoniazid
+ Rifampicin
Extensively drug
resistant (XDR)
Resistance to:
+ Isoniazid
+ Rifampicin
+ Any fluoroquinolone
+ At least one of the three injectable drugs:
capreomycin, kanamycin, amikacin
Extremely drug resistant
(XXDR)
Resistance to:
+ All first-line anti-TB drugs
+ All second-line anti-TB drugs
10. 10
Random spontaneous mutations creating
wild TB bacteria drug-resistant
Selection of drug-resistant TB bacteria by
inadequate treatment (resulting in
monotherapy) due to:
inadequate prescription ,supply and
intake of drugs
NATURAL
DRUG RESISTANCE
ACQUIRED
DRUG RESISTANCE
MDR /XDR/XXDR
MDR-TB can be amplified
into XDR-TB OR XXDR-TB
by:
Inadequate/interrupted
treatment with second
line anti-TB drugs
Indiscriminate use of
second-line drugs
Non-adherence to
national and/or
international guidelines
Development of anti-tuberculosis drug resistance
PRIMARY
DRUG RESISTANCE
11. Antimycobacterial
agents
Genes involved in
resistance
Mechanism of resistance
Isoniazid i) katG (catalase
peroxidase)
i) mutations in KatG result in failure to
generate an active intermediate of INH.
ii) inh A (enoyl –ACP
reductase synthesis)
ii) overexpression of inhA allows
continuation of mycolic acid
iii) ahp C (alkyl
hydroperoxide reductase)
iii) ahpC mutations as a marker for lesions
in KatG
Rifampicin rpoB (β subunit of RNA
polymerase)
Mutations in rpoB prevent interaction with
rifampicin
Streptomycin rpsl (ribosomal protein
S12)
Mutation prevents interaction with
Streptomycin
Ethambutol EmbAB (arabinosyl
transferase)
Overexpression or mutation of embAB
allows continuation of arabinan
biosynthesis.
Pyrazinamide PncA Loss of pyrazinamidase activity results in
decreased conversion of pyrazinamide to
pyrazinoic acid, the putative active moiety
Fluoroquinolones GYra (DNA gyrase
subunit A)
Mutations in gyrA prevent interaction with
fluoroquinolones.
Mechanism of resistance of Anti-tubercular drugs
12. Spontaneous mutations
develop as bacilli
proliferate to >108
Drug Mutation Rate
Rifampisin 10-8
Isoniazid 10-6
Pyrazinamide 10-6
13. Drug resistance in XXDR
TB ³
Atomic force microscopes confirmed morphological variation
in XXDR-TB isolates . bacilli were round , oval or even
multiple branching forms
In addition, various type of cell division i.e., symmetrical,
asymmetrical and budding were found.
The cell wall thicker MDR-TB isolates
Pilli like structure that protruded from the head, tail or side
poles of the bacilli were also detected
14. Does HIV infection favor
TB drug resistance?
Documented evidence:
Nosocomial transmission (HIV facilities, prisons, etc.)
Poor adherence to TB treatment by HIV-positive patients
Malabsorption of TB drugs (advanced
immunosuppression, chronic diarrhea)
Acquired rifampicin resistance (diarrhea, antifungal
treatment, antiretroviral treatment)
Poor TB programme performance (overload of TB cases
due to HIV epidemic, unknown association of HIV)
14
16. AUTOMATED LIQUID BASED CULTURE
BACTEC 460-TB
• Based on microbacterial metabolism
• Faster than solid culture
MB/Bact T® system
• Based on detection of CO2
• DST 8 to 12 days
BACTEC Mycobacteria Growth indicator Tube (MGIT)960
• Consumption of oxygen
• DST 8 to 12 days
17. ESP culture system II
Based on the detection of pressure changes
within the headspace,due to gas production
or gas consumption by microbial growth.
.
Phage Assay Methodology
• Expose strains to drug (RMP)
• Infect with D29 phage
• Grow with M.smegmatis
• Resistance correlated with plaque
formation
18. Microscopic Observations Drug
Susceptibility (MODS) Assay
It is rapid drug susceptibility testing method for M.
tuberculosis (Rapid detection of MDR-TB)
Due to faster growth of M. tuberculosis in liquid culture
characteristic cord formation observed early
Incorporation of drugs permit concomitant DST
19. MOLECULAR METHODS
DNA sequencing
The Line Probe assay (LiPA) WHO
recommended
GenoType MTDBRplus
Strips (Hain Lifescience
• Most accurate and reliable method
• Detect both previously recognized
and unrecognized mutations
• For rifampicin only
• Detect rpoB mutations of rifampicin
• Can be used directly on sputum
specimens,
• Results within 1-2 days
21. PCR SSCP
• Based on property of single stranded DNA to fold into a
tertiary structure whose shape depends on it’s sequence
• Single strands of DNA differing by only one or few bases
will fold into different conformations with different
mobility's on a gel ,producing single strand conformation
polymorphism
• Detect -RIF , INH ,SM and ciprofloxacin resistance.
22. Microarrays
Gryadunov et al : developed a biochip for detection of
rifampicin-resistant and isoniazid-resistant strains of M.
tuberculosis .
The newest generation of TB-biochips identifies mutations
responsible for the emerging resistance of M. tuberculosis
so the highly effective second-line fluoroquinolone
antibiotics can be administered
23. Secondary Drugs testing: [lack of
standardized methods!]
XXDR Diagnostic
technology
CHOICE
Molecular DST (LPA
DST)
FIRST
Liquid culture isolation
and LPA DST
SECOND
Solid culture isolation
and LPA DST
THIRD
Liquid culture isolation
and Liquid DST
FOURTH
Solid culture isolation
and DST
FIFTH
24. New Anti TB drugs
• Bedaquiline
• Delamanid
• Pretomanid
• NC-002, NC-003
• Sutezolid
• SQ 109
• Benzothiazinones
Repurposed Anti TB drugs
• Linezolid
• Clofazamine
• Imipenem/ Meropenem
• Amoxicillin – Clavulanate
• Thioacetazone
• Clarithromycin
(Mendell, et al.Principle and Practice of INFECTIOUS DISEASES;2005:2852-
2886.)
Bedaquiline
2016 – RNTCP has
introducing BDQ
through
conditional access
programme at 6
sites in India
25. Programmatic Management of Drug
Resistant TB (PMDT) in India
Efficient and timely identification of patients who require DST
Quality-assured laboratory capacity (Smear, Culture-DST,
rapid molecular test)
Efficient drug procurement and supply chain management
Adherence to difficult-to-take regimens for long periods
Prompt identification and management of side-effects
Recording and reporting and Human and financial resources
27. REFERENCES
• http://tbcindia.nic.in Central Tuberculosis Division, Govt Of India.
• WHO website http://www.who.int/tb/en/
• Velayati AA, et al. New insight into extremely drug-resistant
tuberculosis: using atomic force microscopy. Eur Respir J 2010;36:
1490-3.
• Udwadia, Zarir “Emergence of New Forms of Totally Drug-Resistant
Tuberculosis Bacilli”, Chest, April 6
2009 www.ncbi.nlm.nih.gov/pubmed/
• Anand, G “India in Race to Contain Untreatable Tuberculosis”, The
Wall Street Journal, 2012 //online.wsj.com/article/Mandeep Jassal,
William R Bishai. Extensively drug-resistant tuberculosis . Lancet
Infect Dis 2009; 9: 19–30
• RNTCP Briefing and Response to Dual Challenges of MDR and
XDR TB
• Topley and Wilsons Microbiology and Microbial infections
Bacteriology Vol-2,10th edition
