Annex 10.1
Proposed protocol: Drug susceptibility testing, proportion method
RESISTANCE OF M. TUBERCULOSIS COMPLEX STRAINS
Resistance of an M. tuberculosis strain determined by the proportion method differs from the
usual concept of resistance in clinical microbiology. The proportion method determines the
proportion of resistant bacilli present in a strain: below a certain proportion, the strain is
classified as susceptible; above that proportion the strain is classified as resistant. The strain is
presumed to be resistant when growth of more than a certain proportion of the inoculum (critical
proportion) occurs on culture media with a defined concentration (critical concentration) of the
drug.
Critical concentration
Critical concentration is the lowest concentration of an anti-TB drug in the culture medium at
which growth (equal to or larger than the critical proportion) of tubercle bacilli indicates
resistance of clinical significance.
Critical proportion
Critical proportion is the percentage of tubercle bacilli of the inoculum whose growth on culture
media containing the critical concentration of an anti-TB drug signifies clinical ineffectiveness.
Growth control
Growth control is the culture after inoculation of tubercle bacilli on a culture medium without any
test drug in order to exhibit unrestricted growth.
PROCEDURE
The proportion method (Canetti et al., 1969, modified) determines the percentage of growth
(number of colonies) of a defined inoculum on a drug-free control medium vs. growth on the
culture medium 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. While Canetti et al. originally based their method on three
concentrations per drug, they proposed later versions based on one or two critical
concentrations only (see table 1). A single critical concentration is most commonly used today
‒ and that is the procedure described here.
Table 1. Critical concentrations of first-line drugs
Drug
INH
RMP
EMB
DSM
Critical concentration (µg/ml)
0.2
40
2
4
Samples
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), DST cannot be safely interpreted.
A culture of M. tuberculosis strain H37Rv as control strain, freshly subcultured.
Sputa with smear grading of 2+ or more can be used as pure cultures. They are
decontaminated using a standard procedure but not centrifuged. The neutralized suspension is
used for inoculation.
Equipment and materials
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BSC, with exhaust air ducted or vented to the outside.
Safety Bunsen burner, with device to light on demand or micro-incinerator.
Rack for glass tubes.
Glass tubes for dilutions (sterile culture tubes).
Glass beads, diameter 3 mm.
Closed loop, internal diameter 3 mm, calibrated to 10 µl, and/or short sterile pipettes for
exact 0.1-ml portions.
Pipettes, graduated, for 0.1 ml, 0.2 ml and 1.0 ml.
Pipetting aids.
Autoclave.
Buckets, stainless steel or polypropylene.
Autoclavable bags.
Vortex mixer.
Sterile NaCl solution, 0.9%.
McFarland standard No. 1.
Disinfectants.
Separate waste containers for pipettes and disposals (autoclavable).
Incubator.
Refrigerator.
Drug-containing media.
Plain culture media.
Reagents and solutions
Drug-containing media, all from the same batch. For practical reasons, use a colour code to
identify different batches or indicate the date of preparation on each tube.
Bacterial suspension
1. With a loop, scrape colonies from all over the culture strain (try to pick up portions from all
colonies).
2. Use a sterile, small, thick-walled, screw-capped glass tube containing 5‒ 7 clean glass
beads.
3. Gently shake the loop over the beads.
4. Repeat step 1.
5. Add 2 drops of sterile saline or distilled water, shake, add 2 additional drops, and shake.
Note: Avoid using any detergent; do not use an open system such as a mortar or
mechanical homogenizer
6. Let stand for 15–30 minutes to allow the larger aggregates of bacteria to settle. The
homogenous upper part of the supernatant should be aseptically transferred to another tube
with similar dimensions to the McFarland tube for comparative purposes. The bacterial
suspension is adjusted with sterile distilled water to a turbidity matching a McFarland
standard No 1.
Dilution steps
Starting from the adjusted bacterial suspension (equivalent to McFarland No. 1), factor 10 serial
dilutions are prepared down to 10‒ 4 (for an inoculum of 10 µl) and 10‒ 5 (for an inoculum of 0.1
ml).
Note: The inoculum of 10 μl can be done with a closed platinum/iridium loop of diameter 3 mm;
some skill is needed but, in areas with high humidity, a small inoculum volume is highly
recommended to avoid water accumulation in the tube. Moreover, the loop may be used for
preparing 10‒ 2 and 10‒ 4 dilutions by adding one loopful of suspension to 1 ml of water (more
exactly, 0.99 ml) in two steps, starting from the one adjusted to McFarland 1) Strictly speaking,
only these two dilutions – one a hundred-fold more dilute than the other – are needed for the
proportion method.
Inoculation of test media with patients’ strains
1. Mark all sets of culture media properly with patient’s identification (at least laboratory
number).
2. Discard condensed moisture from the slants before inoculation.
3. The inoculation may be performed with pipettes (0.1 ml) or a calibrated loop (10 µl).
The suspensions must be adjusted to the inoculum size. The objective of the inoculum
technique is to achieve a growth of 30–100 colonies on the growth control (drug-free) medium
using S3 (see below) for inoculation.
Care must be taken to distribute the inoculum evenly over the lower 80% of the culture medium;
avoid inoculating the upper (thin) part of the slant). The tube cap should allow a little gas
exchange but also prevent drying out; screw-caps have proved suitable.
Method 1: inoculum 10 µl, calibrated loop (table 2)
Dilutions 10‒ 2 (suspension 1, S1), [10‒ 3 (S2)], 10‒ 4 (S3) are required for inoculum, i.e. the 2nd,
3rd and 4th tubes of the serial dilution.
For growth control (GC), drug-free culture media from the same batch are inoculated with S1,
[S2], S3 and designated GC1, [GC2], GC3.
Test media ‒ containing isoniazid, rifampicin, dihydrostreptomycin, ethambutol ‒ are
inoculated with S1.
Note: This is the absolute minimal procedure. It may be advisable for beginners, who usually
have difficulty in obtaining the correct suspension, to perform duplicate inoculations as a control
for accurate (reproducible) results.
Table 2. Modified configuration, extended concentration range without identification
Suspension
S1, 10‒
S2, 10‒
S3, 10‒
S4, 10‒
2
3
4
5
x = inoculation, ‒
Growth
control
No.
Isoniazid
0.2 µg/ml
Rifampicin
40 µg/ml
GC1
GC2
GC3
GC4
1x
1x
2x
2x
x
x
‒
‒
x
x
‒
‒
Dihydrostreptomycin sulfate
4 µg/ml
x
x
‒
‒
Ethambutol
2 µg/ml
= no inoculation
This configuration needs 14 slants and yields two sets for reading (7 slants each)
x
x
‒
‒
Inexperienced staff are recommended to inoculate all dilutions indicated by x in Table 3a;
experienced staff need use only those dilutions marked with a bold x.
Method 2: inoculum 0.1 ml, short graduated pipette or single channel microlitre pipette with
sterile tips (table 3)
The procedure described above for Method 1 is followed, except bacterial suspensions
one order of magnitude lower are used for inoculation. The dilution may be extended to
10‒ 6 for beginners.
Table 3.
Suspension
S2, 10‒
S3, 10‒
S4, 10‒
S5, 10‒
3
4
5
6
x = inoculation, ‒
Growth
control
No.
Isoniazid
0.2 µg/ml
Rifampicin
40 µg/ml
Ethambutol
2 µg/ml
x
x
Dihydrostreptomycin sulfate
4 µg/ml
x
x
GC1
GC2
GC3
GC4
1x
1x
2x
2x
x
x
‒
‒
‒
‒
‒
‒
‒
‒
x
x
= no inoculation
Inexperienced staff are recommended to inoculate all dilutions indicated by x in Table 3b;
experienced staff need use only those dilutions marked with a bold x.
Method 3: direct test using a sputum of at least 2+ after decontamination/liquefaction and
neutralization (table 4 and 5)
Table 4
Suspension
from 2+
sputum
undiluted
GC1
1x
x
x
Dihydrostreptomycin sulfate
4 µg/ml
x
‒ 2
GC2
2x
x
x
x
x
‒ 3
GC3
2x
‒
‒
‒
‒
Ethambutol
2 µg/ml
10
10
x = inoculation, ‒
Growth
control
No.
Isoniazid
0.2 µg/ml
Rifampicin
40 µg/ml
Ethambutol
2 µg/ml
x
= no inoculation
Table 5
Suspension
from 3+
sputum
10‒ 1
GC1
1x
x
x
Dihydrostreptomycin sulfate
4 µg/ml
x
‒ 3
GC2
2x
x
x
x
x
‒ 4
GC3
2x
‒
‒
‒
‒
10
10
x = inoculation, ‒
Growth
control
No.
Isoniazid
0.2 µg/ml
Rifampicin
40 µg/ml
= no inoculation
Incubation
The incubation temperature shall be 36 ± 1 °C.
x
The seeded media are examined for contamination after 1 week of incubation. The first reading
of drug susceptibility test results is done after 4 weeks of incubation, when all strains showing
drug resistance can be reported as drug-resistant.
Because some (especially MDR) strains grow very slowly, a further 2 weeks of incubation are
needed before final interpretation (reporting susceptibility).
If there is no growth on the drug-free media after 6 weeks, no evaluation is possible.
Reading, interpretation, recording and reporting
General remarks
Slants must be read after 4 weeks of incubation for a provisional result and after 6 weeks of
incubation for the definitive result.
The growth on GC3 tube (1% inoculum of the strain suspension to be tested according to
Tables 2 and 3 should allow easy counting of 30‒ 100 isolated colonies. If fewer than 20
colonies have grown on this control, a reliable interpretation is possible only for resistant strains;
for susceptible strains the result should be reported as preliminary. The test should be repeated.
If the MIC of the control strain (H37Rv) indicates drug activity in the test medium that is much
too high, the result “susceptible” cannot be evaluated with sufficient confidence.
If the MIC of the control strain indicates an exceedingly low activity of the drug in the test
medium, the result “resistant” cannot be evaluated with sufficient confidence.
If growth on GC3 exceeds the upper limit for counting isolated colonies, GC4 may be used for
reading against inoculum S2 (Table 2) or S3 (Table 3).
Reading, interpretation
Growth on culture media shall be recorded in accordance with the following schema:
No growth
<50 colonies
50‒ 100 colonies
100‒ 200 colonies, light bacterial lawn
200‒ 500 colonies, almost confluent
> 500 colonies, confluent growth
0
Actual count
+
++
+++
++++
For all four drugs of this proposed protocol, the critical proportion is 1% and the critical
concentrations are listed in Table 1. The bacterial growth on the culture medium with the
respective critical concentration inoculated with S1 is therefore compared with GC3. If the
modified inoculum schema (Table 2 is used, a second set of slants, S2 vs. GC4, can be
evaluated. One or both combinations should always fulfil the criteria to show countable colonies
on one of the two GCs. For Table 3 the corresponding schema is applicable.
A strain is considered to be susceptible if no growth or considerably less than 1% growth is
detected on the test medium containing the critical concentration of the corresponding drug
compared with GC with 1% inoculum (GC3 or GC4).
A strain is considered to be resistant if the growth on the culture medium containing the critical
concentration of the corresponding drug shows more growth than the GC with the 1% inoculum
(GC3 or GC4).
“Borderline” cases ( about 1% growth on drug-containing medium) should be retested before
reporting.
If the criteria described above are met and the quality control of the lot meets the required
standards, the result is interpreted and reported as “susceptible” or “resistant” using the report
form sheet shown below (Figure 1).
RELATED DOCUMENTS
1. Canetti G et al. Advances in techniques of testing mycobacterial drug sensitivity, and the
use of sensitivity tests in tuberculosis control programmes. Bulletin of the World Health
Organization, 1969, 41:21‒ 43.
2. Collins CH, Grange JM, Yates MD. Tuberculosis bacteriology: organization and practice.
Oxford, Butterworth-Heinemann, 1997.
3. Kent PT, Kubica GP. Public health mycobacteriology: a guide to the level III laboratory.
Atlanta, GA, Centers for Disease Control, 1985.
4. Abdel Aziz M. Guidelines for surveillance of drug resistance in tuberculosis. Geneva, World
Health Organization, 2003 (WHO/TB/2003.320).
5. http://www.who.int/tb/dots/r_and_r_forms/en/index.html
Figure 1. DST quality control form sheet
Drug
INH
RMP
EMB
DSM
INH
RMP
EMB
DSM
Date
Inoculation Reading
Critical conc.
Concentrations for MIC
Low
Middle
High
Validation
Remarks
Sign