Part 2

Performing drug susceptibility testing

on solid media

Module 10

11

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.

22

Content outline

• Drug susceptibility test methods:– direct– Indirect

• Proportion method: technical protocol and interpretation criteria

• Recording and reporting DST results• Storage of strains

33

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.

44

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, Courtesy of Dr Isabel N. de Kantor, Buenos Aires, Argentina55

Direct drug susceptibility method: advantages

• Bacillary populations more representative of those existing in vivo.

• Takes 3–4 weeks less than indirect testing.

66

Direct drug susceptibilitymethod: disadvantages

• Only for highly positive smear.• NOT possible in liquid culture.• High contamination rate.• Difficult to calibrate (live + dead bacilli).• Failure rate: 10–15%

77

Indirect drug susceptibility methods

• Organisms isolated in culture.

• A homogeneous suspension of growth is inoculated onto control and drug-containing egg-based solid media.

88

Indirect drug susceptibility methods

99

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.

1010

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 anti-TB treatment. From cultures with scarce growth, the bacillary population used for performing DST may not be representative of those existing in vivo.

1111

Proportion method: critical parameters

Use of LJ medium and a critical proportion of 1% of growth for all 4 drugs

Critical drug concentrationDrug Critical conc. (μg/ml) Critical %

Streptomycin 4 1

Isoniazid 0.2 1

Rifampicin 40 1

Ethambutol 2 1

1212

TOUCH ALL COLONIES!

Procedure

5 ml Sterile distilled

water

BEADS

VortexMedia should be from the same batch

Sediment for 15 min and transfer the supernatant to another tube

1313

Calibration of the bacterial suspension

Calibrate the inoculum to 1 MacFarland by comparing with standard 1 MacFarland suspension on a dark background.

Clumps and beads

SN1 MacFarland suspension

1414

Inoculation

• Loop (10 μl, diameter 3 mm)

• Calibrated pipettes

1515

Procedure – inoculum with loop

0.01 ml

2 ml distilled

water

twice

10–2

GC

S

I

R

E

GC

S

I

R

E1616

BEADS

+ inoculum of 2 GC with suspension 10–4

MacFarland 1

Procedure – inoculum with loop ../..

2 ml distilled

water

10–1

GC

S

I

R

E

GC

S

I

R

E

2 ml distilled

water

10–3

1717+ inoculum of 2 GC with suspension 10–5

twice

Procedure – inoculum with pipette

4.5 ml distilled

water

10–1

0.5 ml

4.5 ml distilled

water

10–2

4.5 ml distilled

water

10–3

0.5 ml 0.5 ml

1818

BEADS

10–4 10–5

0.5 ml 0.5 ml 0.5 ml

4.5 ml distilled

water

4.5 ml distilled

water

4.5 ml distilled

water

4.5 ml distilled

water

4.5 ml distilled

water

4.5 ml distilled

water

4.5 ml distilled

water

4.5 ml distilled

water

4.5 ml distilled

water

10–6

0.5 ml

10–3

GC

S

I

R

E

GC

S

I

R

E

0.1 ml

1919

0.1 ml

GC GC

10–5

Inoculum with pipette

10–4

GC

S

I

R

E

GC

S

I

R

E

0.1 ml

2020

0.1 ml

GC GC

10–6

Inoculum with pipette ../

Incubation

• Incubate at 35–37 ºC in slanted position, loose caps.

• At 48 hours: • tighten caps• check for contaminants• move to upright position

2121

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.

2222

Growth on control tubes (>20 colonies on GC) and no growth on drug tubes

Interpretation

SENSITIVE TO ALL DRUGS2323

Growth on control tubes (>20 colonies)

and number of colonies on isoniazid tube

≥ number of colonies on control tube 1/100

Interpretation

RESISTANT TO ISONIAZID

2424

Growth on control tubes (>20 colonies) and number of colonies on isoniazid tube < number of colonies on control tube 1/100

Interpretation

SENSITIVE TO ISONIAZID

2525

Growth on control tubes (>20 colonies) and number of colonies on rifampicin tube ≥ number of colonies on control tube 1/100

Interpretation

RESISTANT TO RIFAMPICIN

2626

Growth on control tubes (>20 colonies) and number of colonies on rifampicin tube < number of colonies on control tube 1/100

Interpretation

SENSITIVE TO RIFAMPICIN

2727

Growth on control tubes (>20 colonies) and number of colonies on streptomycin tube ≥ number of colonies on control tube 1/100

Interpretation

RESISTANT TO STREPTOMYCIN

2828

Growth on control tubes (>20 colonies) and number of colonies on streptomycin tube < number of colonies on control tube 1/100

Interpretation

SENSITIVE TO STREPTOMYCIN

2929

Growth on control tubes (>20 colonies) and number of colonies on ethambutol tube ≥ number of colonies on control tube 1/100

Interpretation

RESISTANT TO ETHAMBUTOL

3030

Growth on control tubes (>20 colonies) and number of colonies on iethambutol tube < number of colonies on control tube 1/100

Interpretation

SENSITIVE TO ETHAMBUTOL

3131

Growth on control tubes (>20 colonies) and number of colonies on isoniazid tube and on rifampicin≥ number of colonies on control tube 1/100

Interpretation

MULTIDRUG-RESISTANT (MDR)

3232

No growth on control tubes (or <20 colonies on GC) and no growth on

drug tubes

Interpretation

INVALID RESULT – REPEAT

3333

Proportion method ‒ advantages and disadvantages

Advantages: • Standardization of

the size inoculum is not critical

• Simple

Disadvantages:• Influenced by degree

of dispersion of the inoculum suspension

3434

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.3535

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.

3636

Recording and reporting form

3737

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:________________________________ Age (yrs):_____ Sex:____

Ward / Department: __________________ Address: _________________________________

*HIV-status: Pos / Neg / Unknown _________________________________ TB Disease type and treatment history Site: pulmonary History: new (never treated before for ?1 month) extrapulmonary (specify):_______________ relapse failure Previous treatment: Cat.1 return after default Cat.2 chronic excretor Cat.4 (second-line drugs) MDR contact Other _________________ uncertain Origin of request:

Region ID:_______________ District ID:_______________ Local laboratory ID:________________

Date specimen was collected: ____/____/20____ Specimen ID number:_______________

Local laboratory: smear result: 1st ____ 2nd ____ 3rd ____ specimen microscopy technique used: hot Ziehl-Neelsen direct smear cold staining concentrated smear fluorescence

Request for testing at the reference laboratory: Reason: diagnosis Specimen: sputum follow-up at …. months during treatment sputum in preservative, type …………… follow-up at …. months after treatment other specify):__________________

Requested tests: microscopy (type _______ ) culture DST (first / second line)

Person requesting examination: Name:_________________________ Position:________________

* Information that can be disclosed optionally ID = identification number or code

Date: _____/______/20_____ Signature:________________

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Quantified results of growth / reactions

Drug concentrations in µg/ml*

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Final results

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ID # ____________ Legend: S = susceptible; R = resistant; C = contaminated; ND = not done

INH Rifampicin Ethambutol Streptomycin Pyrazinamide Ofloxacin Kanamycin

µg/ml

result

Date: _____/______/20_____ Signature:________________

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.)

3838

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.

3939

Storage of strains

• Liquid media • Skim milk

4040

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.

4141

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.4242

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.

4343