2. Part 2 Performing drug susceptibility testing on solid media Module 10 1

3. 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

4. Content outline Drug susceptibility test methods: – –direct – –Indirect Proportion method: technical protocol and interpretation criteria Recording and reporting DST results Storage of strains 3

5. 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

6. 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, Argentina 5

7. Direct drug susceptibility method: advantages Bacillary populations more representative of those existing in vivo. Takes 3–4 weeks less than indirect testing. 6

8. 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

9. Indirect drug susceptibility methods Organisms isolated in culture. A homogeneous suspension of growth is inoculated onto control and drug- containing egg-based solid media. 8

10. Indirect drug susceptibility methods 9

11. 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

12. 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. 11

13. Proportion method: critical parameters Use of LJ medium and a critical proportion of 1% of growth for all 4 drugs Critical drug concentration DrugCritical conc. (μg/ml)Critical % Streptomycin4 1 Isoniazid0.2 1 Rifampicin40 1 Ethambutol2 1 12

14. TOUCH ALL COLONIES! Procedure 5 ml Sterile distilled water BEADS Vortex Media should be from the same batch Sediment for 15 min and transfer the supernatant to another tube 13

15. 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 SN 1 MacFarland suspension 14

16. Inoculation Loop (10 μl, diameter 3 mm) Calibrated pipettes 15

17. Procedure – inoculum with loop 0.01 ml 2 ml distilled water twice 10 –2 GC S I R E S I R E 16 BEADS + inoculum of 2 GC with suspension 10 –4 MacFarland 1

18. Procedure – inoculum with loop../.. 2 ml distilled water 10 –1 GC S I R E S I R E 2 ml distilled water 10 –3 17 + inoculum of 2 GC with suspension 10 –5 twice

19. 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 18 BEADS 10 –4 10 –5 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

20. 10 –3 GC S I R E S I R E 0.1 ml 19 0.1 ml GC 10 –5 Inoculum with pipette

21. 10 –4 GC S I R E S I R E 0.1 ml 20 GC 10 –6 Inoculum with pipette../

22. Incubation Incubate at 35–37 ºC in slanted position, loose caps. At 48 hours: tighten caps check for contaminants move to upright position 21

23. 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

24. Growth on control tubes (>20 colonies on GC) and no growth on drug tubes Interpretation SENSITIVE TO ALL DRUGS 23

25. 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 24

26. 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 25

27. 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 26

28. 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 27

29. 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 28

30. 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 29

31. 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 30

32. 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 31

33. 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) 32

34. No growth on control tubes (or <20 colonies on GC) and no growth on drug tubes Interpretation INVALID RESULT – REPEAT 33

35. 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 34

36. 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

37. 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

38. Recording and reporting form 37 Date: _____/______/20_____Signature:________________

39. 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

40. 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

41. Storage of strains Liquid media Skim milk 40

42. True and false exercise 1. 1. DST is considered to be a procedure that risks generating aerosols. 2. 2. Misidentification of a strain can lead to clinically irrelevant DST results. 3. 3. Modified proportion method on LJ is currently considered as the reference method for DST of first-line drugs 4. 4. Results should be reported only if quality assurance criteria are satisfied. 41

43. 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

44. 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