EUCAST: Mycobacteria FAQ

Mycobacteria FAQ

Frequently asked questions (FAQ) about the EUCAST reference method for MIC testing of the Mycobacterium tuberculosis complex and the SOP for calibrating surrogate methods.

Reference method

Is the intention for the media for it to be prepped from scratch, or can it be bought premade from a manufacturer? You refer to it as "media from base"

The media for the reference method should be prepared from the powder base which is supplied by the manufacturer.

The reference protocol for MTBC MIC determination with 7H9 sec. 1.3 stipulates 7H9 must be prepared freshly from powder; is fresh preparation of 7H9 required to be done the day of plate inoculation? We understand that nothing can be added to the plates until the day of testing, but can 7H9 be prepared and stored refrigerated beforehand?

As stated in the reference method, 7H9 needs to be prepared according to the manufacturers’ instructions and should thus also be stored according to the instructions of the manufacturer. It is not an additional requirement to use fresh 7H9 as long as the media is handled according to the instructions of the manufacturer.

If 7H9 can be prepared from powder before the day of testing, can OADC be added to it before the day of testing?

The OADC should be added according to the instructions from respective manufacturer.

Can you confirm that OADC may be purchased pre-made and does not need to be prepared from scratch?

OADC can be purchased pre-made. 7H9 or OADC does not need to be produced from each component but may be purchased ready-made and then prepared, used and stored according to the instructions of the manufacturer.

Can multiple inoculum preps be tested on a given micro-well plate (thinking of H37Rv in particular)?

As long as the reference method description is adhered to, it is possible to use several inoculum preparations from the same isolate on the same plate.

Is it permitted to test more than one isolate on a given 96-well plate? If not, can multiple drugs for the same isolate be tested on the same plate?

As long as the plate outline is adhered to (Appendix 1), several drugs can be tested but as there has to be an inoculum control for each isolate (GC1% and GC100%) it may not be suitable to use more than one isolate per plate.

Calibration SOP

Can media variability be done internally with 2 operators to mimic 2 sites?

As outlined in the SOP, there has to be two separate laboratories.

Does media variable need to be run for every drug?

As outlined in the SOP, there is a need to check for media variability for each drug as the potential media-drug variability is specific to each drug.

For the media distributor variability portion (stage 1b)- is it 3 separate McFarland preps or 3 dilutions from the same MF?

It should be 3 separate McF preparations (individual inocula).

For stage 3 in the calibration protocol it says that two different media should be covered by the four labs- is this correct? Shouldn’t we have already addressed media variability in stage 1? What does this do to the results interpretation?

It is correct according to the calibration SOP. Stage 1 is to ensure that there is no technical issue in the drug-media combination which would make the following testing problematic and needs to be solved first. In stage 3, two different media are needed to include any media specific variability when the ECOFF is determined.

The reference protocol for MTBC MIC determination with 7H9 sec. 1.1 stipulates testing at least 8 drug concentrations. The largest MGIT carrier set holds 8 tubes, which is only 7 drug concentrations because the leftmost tube must be a growth control. Will MGIT calibration with only 7 drug concentrations vs. the reference be acceptable? If not, then only labs with TBeXiST can run the calibration

The rule about testing 8 drug concentrations applies to the reference method. As long as there is no truncation, it is possible to use 7 tubes for the comparator method. If needed, two ranges of MIC values for the same drug may be used (low and high).

BD will provide lyophilised drugs for MGIT testing. Can the same drug be used for the reference method or should the drug from an independent manufacturer (e.g. Sigma) be used instead for the reference method?

The same drug as used in the comparator method in the calibration SOP should not be used (ie lyophilised drugs for MGIT tubes produced by BD).

Preferably one should use the same inoculum/isolate for both methods; the reference method initially grows the inocula on 7H10 plates and then subculture them in 7H9 medium whereas we normally prepare the inocula from freezer stock in 2 MGIT tubes in such a way that the tests are ready for interpretation within 6 to 9 days in the MGIT.

It is important that the inoculum for the reference method is prepared according to the protocol. The preparation of the inoculum in the comparator method is up to the manufacturer to decide. However, it must be prepared in a reproducible way.

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Mycobacteria FAQ

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