Module 10
Part 2
Performing drug
susceptibility testing
on solid media
1
Learning objectives
At the end of this module you will be able to:
 explain the different methods for
performing DST;
 perform and interpret the proportion
method;
 record and report DST results;
 store strains.
2
Content outline
• Drug susceptibility test methods:
– direct
– Indirect
• Proportion method: technical protocol and
interpretation criteria
• Recording and reporting DST results
• Storage of strains
3
Direct drug susceptibility method:
proportion method
• Sputa with smear grading of 2+ or more can be
used as pure cultures after decontamination using
standard procedure.
• The neutralized suspension is used for inoculation
directly onto drug-containing and drug-free media,
according to the proportion method (see below).
• Inoculum size is adjusted according to the number
of AFB observed during smear microscopy of
concentrated sputum.
• Identification should be performed before results are
released.
4
Example of a susceptibility test, proportion method,
inoculated directly after decontamination from the sample
(4 AFB/field)
Courtesy of Dr Isabel N. de Kantor, Buenos Aires, Argentina
5
Direct drug susceptibility
method: advantages
• Bacillary populations more representative
of those existing in vivo.
• Takes 3–4 weeks less than indirect
testing.
6
Direct drug susceptibility
method: disadvantages
• Only for highly positive smear.
• NOT possible in liquid culture.
• High contamination rate.
• Difficult to calibrate (live + dead bacilli).
• Failure rate: 10–15%
7
Indirect drug susceptibility methods
• Organisms isolated in culture.
• A homogeneous suspension of growth is
inoculated onto control and drugcontaining egg-based solid media.
8
Indirect drug susceptibility methods
9
Indirect drug susceptibility method:
proportion method
• The proportion method is the most widely used
method for DST and is currently used as a
reference for testing new methods.
• The proportion method determines the
percentage of growth (number of colonies) of a
defined inoculum on a drug-free control medium
vs. growth on culture media containing the
critical concentration of an anti-TB drug.
• The critical drug concentration, as well as the
critical proportion of resistant colonies, has been
evaluated from clinical data.
10
Indirect proportion method
• A pure culture of tubercle bacilli (test strain) in
the active phase of growth (2–4 weeks). In the
absence of sufficient growth (<20 colonies, 1+),
DST should not be performed unless the culture
comes from a patient who has completed antiTB treatment.
From cultures with scarce growth, the bacillary
population used for performing DST may not be
representative of those existing in vivo.
11
Proportion method: critical parameters
Use of LJ medium and a critical proportion
of 1% of growth for all 4 drugs
Critical drug concentration
Drug
Critical conc. (μg/ml)
Streptomycin
4
Isoniazid
0.2
Rifampicin
40
Ethambutol
2
Critical %
1
1
1
1
12
Procedure
Sediment for 15 min
and transfer the
supernatant to
another tube
TOUCH ALL COLONIES!
5 ml
Sterile distilled
water
BEADS
Media should be from the
same batch
Vortex
13
Calibration of the bacterial
suspension
Calibrate the inoculum to 1 MacFarland by
comparing with standard 1 MacFarland
suspension on a dark background.
SN
1 MacFarland suspension
Clumps and
beads
14
Inoculation
• Loop (10 μl,
diameter 3 mm)
• Calibrated
pipettes
15
Procedure – inoculum with loop
GC
GC
S
S
I
I
R
R
E
E
16
0.01 ml
twice
2 ml
distilled
water
BEADS
MacFarland 1
10–2
+ inoculum of 2 GC with suspension 10–4
Procedure – inoculum with loop ../..
GC
GC
S
S
I
I
R
R
E
E
17
twice
2 ml
distilled
water
10–1
2 ml
distilled
water
10–3
+ inoculum of 2 GC with suspension 10–5
Procedure – inoculum with pipette
0.5 ml
0.5 ml
BEADS
4.5 ml
distilled
water
10–1
0.5 ml
0.5 ml
0.5 ml
0.5 ml
4.5 ml4.5 ml 4.5 ml 4.5 ml4.5 ml
4.5 ml
distilled distilled distilleddistilled
distilled
distilled
water water
waterwater
water
water
10–2
10–3
10–4
0.5 ml
4.5 ml
4.5 ml
4.5
ml
distilled
distilled
distilled
water
water
water
10–5
4.54.5
mlml
distilled
distilled
water
water
10–6
18
Inoculum with pipette
GC
GC
GC
GC
0.1 ml
0.1 ml
S
S
I
I
R
R
10–3
10–5
E
E
19
Inoculum with pipette ../
GC
GC
GC
GC
0.1 ml
0.1 ml
S
S
I
I
R
R
10–4
10–6
E
E
20
Incubation
• Incubate at 35–37 ºC in slanted position,
loose caps.
• At 48 hours:
• tighten caps
• check for contaminants
• move to upright position
21
Reading results
• Read after 4 weeks as a provisional result and after 6 weeks for the
definitive interpretation of result.
• The growth on GC tube 1/100 should allow easy counting of 30–100
colonies.
• If fewer than 20 colonies have grown on this control, report only if resistant
(repeat if susceptible).
• A strain is resistant if the medium containing the critical concentration of
the corresponding drug shows more colonies than the GC with the 1%
inoculum .
• “Borderline cases” (about 1% growth on drug-containing medium) should
be reported as resistant and retested.
• If the criteria described above are met and quality control of the batch
meets the standards, the result is interpreted and reported as “susceptible”
or “resistant” using the report form sheet.
22
Interpretation
Growth on control tubes (>20 colonies on GC)
and no growth on drug tubes
SENSITIVE TO ALL DRUGS
23
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on isoniazid tube
≥ number of colonies on control tube 1/100
RESISTANT TO ISONIAZID
24
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on isoniazid tube
< number of colonies on control tube 1/100
SENSITIVE TO ISONIAZID
25
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on rifampicin tube
≥ number of colonies on control tube 1/100
RESISTANT TO RIFAMPICIN
26
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on rifampicin tube
< number of colonies on control tube 1/100
SENSITIVE TO RIFAMPICIN
27
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on streptomycin tube
≥ number of colonies on control tube 1/100
RESISTANT TO STREPTOMYCIN
28
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on streptomycin tube
< number of colonies on control tube 1/100
SENSITIVE TO STREPTOMYCIN
29
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on ethambutol tube
≥ number of colonies on control tube 1/100
RESISTANT TO ETHAMBUTOL
30
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on iethambutol tube
< number of colonies on control tube 1/100
SENSITIVE TO ETHAMBUTOL
31
Interpretation
Growth on control tubes (>20 colonies)
and number of colonies on isoniazid tube and
on rifampicin
≥ number of colonies on control tube 1/100
MULTIDRUG-RESISTANT (MDR)
32
Interpretation
No growth on control tubes (or <20
colonies on GC) and no growth on
drug tubes
INVALID RESULT – REPEAT
33
Proportion method ‒
advantages and disadvantages
Advantages:
Disadvantages:
• Standardization of
• Influenced by degree
the size inoculum is
not critical
• Simple
of dispersion of the
inoculum suspension
34
Possible errors
• Related to drug concentrations and stability:
– preparation of drug solutions and drug-containing media;
– excessive or prolonged heat during inspissation – steaming of media;
– improper storage of drug solutions and drug-containing media.
Related to media quality:
– excessive moisture on the surface of the medium;
– excessive dehydration of the medium.
Related to the inoculum:
– use of an inoculum not representative of the bacterial population;
– use of too little inoculum;
– presence of large aggregates of bacteria.
Related to NTM contamination:
– failure to recognize the simultaneous presence of MTB complex and
other mycobacteria. Use only pure M. tuberculosis cultures.
35
Quality assurance issues
• Every new batch of drug-containing media
prepared for DST must be quality-controlled.
• For each drug, a slant of the critical
concentration and media with lower drug
concentrations are tested with H37Rv strain.
• Compare results with the MIC of H37Rv.
• External quality control should be organized and
supervised annually by the national research
laboratory, using a panel of 20 test strains
provided by the SRL network.
36
Recording and reporting form
Request and reporting form for TB culture and Drug Susceptibility Test (DST)
Patient identification (ID):
TB register number:____________ Previous TB register number:____________ MDR register number:_____________
Surname and first name of patient:________________________________
Address: _________________________________
1 week
Date reported
Identification
ID test 2**
ID test 1**
PNB
History:  new (never treated before for ?1 month)
 relapse
 failure
Cat.1
 return after default
Cat.2
 chronic excretor
Drug
in µg/ml*
Cat.4concentrations
(second-line drugs)
 MDR contact
Other _________________
 uncertain
Final results
ID test 3**
Ethambutol-4
Ethambutol-2
Rifampicin-4
Previous treatment: 



Rifampicin-2
Isoniazid-4
Isoniazid-2
Control -4
Control -2
Reading time
TB Disease type and treatment history
Site:  pulmonary
 extrapulmonary (specify):_______________
Streptomycin-4
Streptomycin-2
Quantified
results of growth / reactions
*HIV-status: Pos / Neg
/ Unknown
_________________________________
Resistance profile
Ward / Department: __________________
Age (yrs):_____ Sex:____
Origin of request:
4 weeks
6 weeks
Region
ID:_______________
Date:
_____/______/20_____
Date specimen was collected:
1 week
Local laboratory:
4 weeks
6 weeks
District ID:_______________
Local laboratory ID:________________
Signature:________________
____/____/20____
st
nd
Specimen ID number:_______________
rd
smear result: 1 ____ 2 ____ 3 ____ specimen
microscopy technique used:
 hot Ziehl-Neelsen
 cold staining
 fluorescence
 direct smear
 concentrated smear
Request for testing at the reference laboratory:
Legend: S = susceptible; R = resistant; C = contaminated; ND = not done
ID # ____________
Reason:  diagnosis
Specimen:  sputum
 follow-up
at …. monthsEthambutol
during treatment Streptomycin sputum
in preservative, type
…………… Kanamycin
INH
Rifampicin
Pyrazinamide
Ofloxacin
µg/ml
result
 follow-up at …. months after treatment
Requested tests:
 microscopy (type _______ )
 other specify):__________________
 culture
Person requesting examination: Name:_________________________
* Information that can be disclosed optionally
Date: _____/______/20_____
ID = identification number or code
 DST (first / second line)
Position:________________
Signature:________________
37
Storage of strains
• As part of good laboratory practice,
cultures should be stored in appropriate
conditions:
– to preserve viability;
– for safety reasons.
• Viability will decline over time if bacteria
are stored at room temperature or at 4 ºC.
(Check national policy on strain storage.)
38
Storage of strains
• Short-term storage:
– 4 ºC (1 year solid)
– 20 ºC (2 weeks)
– liquid cultures – no more than 1 month.
Long-term storage of patient cultures:
– –20 ºC.
Long-term storage of reference cultures:
– –20 ºC.
39
Storage of strains
•
•
Liquid media
Skim milk
40
True and false exercise
1. DST is considered to be a procedure that
risks generating aerosols.
2. Misidentification of a strain can lead to
clinically irrelevant DST results.
3. Modified proportion method on LJ is
currently considered as the reference
method for DST of first-line drugs
4. Results should be reported only if quality
assurance criteria are satisfied.
41
Module review: take-home messages
 The proportion method is valid for susceptibility testing of M.
tuberculosis complex with anti-TB drugs.
 The proportion method is considered to be a reference standard,
against which other routine methods should be assessed.
 The proportion method determines the percentage of growth of a
defined inoculum on a drug-free control medium vs. growth on
culture media containing the critical concentration of an anti-TB
drug.
 Slants must be read after 4 weeks of incubation for a provisional
result and after 6 weeks of incubation for the definitive
interpretation of results.
 The critical concentration to define a resistant strain is 1% of the
inoculum.
 Test with invalid growth control should be repeated.
42
Self-assessment
• Explain the differences between direct and
indirect DST methods.
• List the possible errors in performing the
proportion method.
• List the consequences of a false resistance.
• List the consequences of a false sensitivity.
43