DST phenotypic

1. Introduction to Principles of Anti-
Tuberculosis Therapy
Content adopted from African Centre for Integrated Laboratory Training, 2019

2. Content outline
 Objectives and history of antituberculosis therapy
 First-line antituberculosis drugs
 Basic definitions of drug resistance
 Development and genetic basis of drug resistance
 Relationship of critical concentrations and proportion of resistant bacteria to
prediction of treatment failure
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3. Objectives of antituberculosis therapy
1. Quickly kill large numbers of rapidly growing bacilli in the
infected tissue
 Cure the patient and increase chances of survival
 Reduce the infectiousness of patient
2. Prevent the emergence of drug-resistant mutants due to:
 Treatment errors that select naturally occurring genetic mutations
which grow into resistant strains
3. Sterilization (elimination) of the dormant but still viable
bacilli from the infected tissue
 To avoid therapeutic failure and relapse
 To reduce the chance of transmission
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4. History of Anti-Tuberculosis Therapy
• Two drugs introduced for TB therapy in 1940’s
– Streptomycin (SM) and Para-Aminosalicylic Acid (PAS)
– Use of either SM or PAS in treatment of TB was initially found to
reduce deaths among treated patients
• Soon, it was found that single drug therapy
resulted in emergence of drug resistant strains in
approximately 70% of the patients
• Drug resistance seen when one drug used alone
• Multiple drug therapy introduced
– By combining SM + PAS the resistance rate was reduced to 9%
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5. Emergence of Drug Resistance
 Research confirmed selection of drug resistant
mutants from using one drug therapy
– One drug therapy - TB bacilli resistant to that drug
will grow
– Selection of drug resistant mutants
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MTBC resistant to S R and H
Strep resistant MTBC

6. WHO Classification of Resistance
 Drug resistance among new cases
 Newly identified cases with resistance to anti-tuberculosis
drugs
 No longer called primary resistance
 Drug resistance among previously treated patients
 Cases with a history of TB treatment and currently has drug
resistant TB
 No longer called acquired resistance
 Change in classification made
 Cannot tell if resistance occurred because of treatment failure
or if patient infected with new drug resistant strain unless
genotyping is done
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7. Resistance to more than one drug
 Polyresistance
 Strains of TB resistance to more than one
antituberculosis drug
 Multi-drug resistant TB (MDR-TB)
 Strains of TB resistance at least to INH and RMP
 Results in treatment failure and fatal outcomes more
often than with resistance to other drugs
 Extensively drug resistant TB (XDR-TB)
 MDR-TB also resistant to any of the fluoroquinolones
and to at least one injectable drug
 Often untreatable
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8. Estimated TB incidence rate 2017
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9. Percentage of new TB cases
with MDR TB
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10. Proportion of MDR among previously
treated TB cases
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11. Drug Resistance Profile in Ethiopia
2018 Global TB report
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12. Introduction of
More Effective Anti-TB drugs
• Isoniazid (INH), more potent
drug,1952
– Combination therapy – SM + PAS + INH
(1950’s) proved highly effective in
preventing emergence of resistance
– Still, 18-months of treatment required to
ensure adequate cure
• Pyrazinamide (PZA), Ethambutol
(EMB) and Rifampin (RMP)
– PZA (1952), EMB (1962) and RMP (1963)
effectively combined with INH for
combination therapy
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13. Current four drug TB therapy
(first-line, most effective drugs)
• Requirements for effective cure
– Treatment with multiple antibiotics
– Long therapy – 6 months
– High cure rate if drugs are taken exactly as prescribed and for
prescribed length of time
• Initial two months with RMP, INH and PZA and either
EMB or SM
• Another four months with RMP and INH
• This regimen
– Combines antibacterial activity
– Inhibits development of resistance
– Eliminates persisting organisms
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14. Compartmentalization of M. tuberculosis in
infected tissue
 Population A: Large
number of rapidly dividing
bacilli in pulmonary cavities
 Population B: Bacilli are
multiplying less rapidly due
to adverse local conditions
(usually acidic)
 Population C: Bacilli are
dormant and viable inside
granulomas
 Treatment plan: kill all
populations at the same time
Population C
Month 2 Month 6
Population A
Population B
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15. Bactericidal Activity
 Drugs that kill Pop. A are
considered to have rapid
Bactericidal Activity
 Bactericidal Activity is
measured by rapidity of
sputum and culture
conversion (from + to -)
 These drugs are most
effective in preventing the
emergence of drug
resistant cells that arise in
large populations
Population C
Month 2 Month 6
Population A
Population B
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16. Sterilizing effect
 Drugs that are more
effective against
Populations B and C are
regarded as Sterilizing
agents.
 The potency of Sterilizing
activity is reflected by a
high cure rate with limited
relapses in patients
completing therapy
Population C
Month 2 Month 6
Population A
Population B
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17. Why is chemotherapy so long?
 Antimicrobials are NOT effective against non-growing
bacteria
 Bacteria are in stationary phase
 Residual persistors or survivors not killed during antimicrobial
exposure
 Dormant bacteria
 Need continuous presence of antimicrobials to kill non-
growing TB when they become metabolically active
 RMP and PZA are good at killing persistors
 RMP is included in initial and continuous therapy phases
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18. TB Drugs Activity
 Isoniazid - bactericidal
 Inhibits fatty acid synthesis of mycolic acids
 Effective against actively growing TB
 Rifampicin - bactericidal
 Inhibits bacterial RNA polymerase
 Effective against actively growing TB and TB with spurts of
metabolism
 Pyrazinamide – bactericidal / bacteriostatic
 Inhibits fatty acid synthesis
 Effective against semi-dormant TB in acid environments
 Ethambutol - bacteriostatic
 Bactericidal at high doses – inhibits cell wall synthesis
 Effective against rapidly growing organisms
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19. TB First Line Drugs and
Mechanisms of Action
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20. Natural drug resistance in the MTB complex
 MTB complex members
 Most common - M. tuberculosis, M. bovis
 Vaccine strain - M. bovis BCG
 Rarely seen - M. africanum, M. canettii, M. microti,
M. caprae, M. pinnipedii
 Natural resistance to antibiotics:
 Hydrophobic cell envelope (permeability barrier),
 Drug efflux systems and drug modifying enzymes
 Efflux systems pump toxic substances out of cell
 Enzymes modify the drug configuration – can’t bind
 PZA resistance in M. canettii, M.bovis and BCG
(other members of MTBC are usually susceptible)
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21. Selection of Drug Resistant Mutants in TB
• Spontaneous mutations occur in the DNA of all cells
– Mutations can change the structure of a protein that is a drug target
– Protein still functions, but is no longer inactivated by the drug
– Thus, TB can grow in the presence of the drug
• Natural resistance is linked to large bacterial populations
– Mutants resistant to any drug naturally occur on average once in
every 100 million (108) cells
– Pulmonary TB - cavities often contain 107 – 109 organisms
– Non-cavitary lesions contain about 103 - 104 organisms
– By using two antibiotics, chances for both targets to be mutated and
naturally resistant to both drugs is extremely small (10-8 x 10-8 = 10-16)
– Monotherapy led to selection of drug resistant populations in cavitary
disease more often than in cases with non-cavitary lesions
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23. Other factors influencing the development of
drug resistance
 Metabolism of bacilli shifted to dormancy
 Impaired/ decreased drug uptake by M. tuberculosis cell
 Penetration of drugs to various body sites
 Suboptimal drug concentration at some body sites
 Less than therapeutic drug concentration
 Impaired drug absorption due to underlying host conditions such as
HIV/AIDS, diabetes, cancer, and other chronic diseases
 Treatment with inappropriate drugs, combinations or dosages
 Interruption or irregular treatments
 Incomplete treatments
 Duration – stop early because feel good
 required number of doses not taken (patient non-compliant)
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24. What does drug resistance mean to
the patient?
 A person infected with a drug resistant
TB strain would fail to respond to the
normal dosage of drug which would
cause a cured response in a person
infected with a drug susceptible TB strain
 Research determined that if 1% of the
TB population in a specimen is resistant
to the drug, then the person will not
respond to the normal drug dosage
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25. Critical concentrations of drugs used for
TB drug susceptibility testing
 Critical concentration is the
concentration of drug that inhibits the
growth of 95% of wild type TB strains
without appreciably affecting the growth of
resistant cells
 Wild type strains do not have any exposure to
drugs, therefore, not expecting resistance
 The critical concentrations of drugs
were found to vary dependent upon the
media used (solid vs. liquid)
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26. Determination of clinically significant
proportion of resistant TB bacteria
 TB isolate
 Isolate grown in media with drug
 Growth control media has 1% (1:100 dilution) of isolate
inoculum only – no drug is added
 Compare growth in growth control media to growth in test media
with isolate AND drug
 Clinically significant proportion
 Growth of resistant cells
 Growth control is growing – 1% of the inoculum
 Growth is present in the test media with the isolate and the critical
concentration of drug – isolate is growing in the presence of drug
 Why 1% of resistant cells?
 1% resistance seen in the organism population represents a significant
increase in proportion of drug resistant mutants where cure is less likely
 Laboratory assays are based on this assumption
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27. The proportion method for determining
drug resistance in M. tuberculosis
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Isolate in liquid
media
1:100 dilution
of isolate
inoculation
Undiluted
isolate plus
critical
concentration
drug
Diluted isolate
only -No drug
Growth Control

28. True and false exercise
1. TB treatment induces M. tuberculosis
resistance.
2. Monotherapy is recommended to treat TB.
3. The drug critical concentration is the amount of
drug in the medium that inhibits the growth of
susceptible organisms but not that of the
resistant mutants.
4. Drug resistance in a new case is defined as the
presence of drug-resistant organisms in a
previously untreated person.
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29. Why are YOU here for this course?
 Improve technical skills
 Improve work flow in the TB DST
laboratory
 Improve record keeping
 Patient records
 Quality control
 Equipment monitoring and maintenance
 Quality Assurance
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30. Take home messages I.
 Combined antituberculosis therapy is the cornerstone of effective
treatment and prevention of drug resistance.
 The first-line antituberculosis drugs are INH, RMP, PZA and
sometimes EMB and SM.
 M. tuberculosis may exhibit natural resistance to certain antibiotics
and may develop resistance to antituberculosis agents due to
spontaneous mutations in genes encoding drug targets or drug
activating enzymes.
 Anatomical, metabolic compartmentalization, mutation rates and
increase in the bacterial load in the cavity may also highly influence
the emergence of drug resistance.
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31. Take home messages II.
 Critical concentration is the amount of drug in the
medium that inhibits the growth of susceptible organisms
but not that of the resistant mutants
 The clinically significant proportion of resistant mutants
(determined to be greater than 1% of the population)
indicates the magnitude of drug resistant cells that either
predict or reflect therapeutic failure.
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