Microbiology count test failure

[hoanght2]

Hello everyone,

We are performing a sterilization validation for our Ethylene Oxide sterilization process on one of our product. Right now, we are still on the cycle development phase in which we are running multiple fractional cycles with different gas exposure time to obtain evidences proving that the resistances of product < iPCD < ePCD. In these fractional runs, we are performing STERILITY test for product, and MICROBIOLOGY COUNT test for iPCD and ePCD. Our expected results are the product is sterile and the quantity of CFU of the iPCD is less than that of the ePCD. For the microbiology count test, we follow exactly to the guidance in USP 41 - chapter 55 ( Biological indicators - Resistance performance tests). However, for the first couple fractional runs, the result shows no growth at all for the iPCDs and the ePCDs, we couldn't find/count even a single CFU on them. This result doesn't make senses to us because the sterility test's result on those same iPCDs and ePCDs were positive in cycles that have longer gas exposure time. Our iPCDs and ePCDS are constructed using the Self-Contained-Biological-Indicators (SCBI). We already successfully conducted method validation for our Microbiology count test. But we used the SCBIs that were not exposed to the sterilization cycle, bascially we used brand new SCBIs for the method validation. I think one of the reasons for the failure to detect any viable spore in the fractional cycles's iPCDs & ePCDs is that the spore is somehow injured and thus does not response well to our current recovery technique. But i'm not really savvy in the microbiology field, so I'm still clueless why our microbiological count test failed. Can somebody help me how to investigate this issue ? What potential root causes should I look into ?

Thanks a lot.

 

[planB]

Some input for thought:

1. Try to reduce the complexity /unknowns / variables in your test set-up, such as focusing on one specific single fractional cycle time and not comparing fractional cycles of different duration. Maybe also drop product and iPCD for the moment (with the additional effect to safe materials and test cost) and focus on ePCDs only, until you make progress in your investigation.
2. ou may want to benchmark /re-confirm your test results against the microbiological analysis of an accredited lab by repeating one fractional cycle with a duplicate set of products, iPCDs and ePCDs; send off 1 set of product and PCDs to an external lab for the very same microbiological tests that you perform inhouse.
3. A sterility test has typically a higher sensitivity than a (spore) count test; so if you are on the brink of a fractional cycle becoming actually a half cycle, you might encounter a test result that you reported, namely non-sterility, but still no actual spore count.
4. You may want to talk back to manufacturer of the SCBI and ask for their thoughts on your suspicion about your recovery technique. For completeness sake, also re-confirm with the manufacturer that the used SCBI lot has no issues know to them
5. You may want to confirm that you indeed have nothing changed in your EO sterilization process parameters, the presentation of your load and PCDs to the cycle, that could impact lethality - e.g. does you half cycle still work, and deliver inactivated PCDs only?
6. And maybe unrelated to the product under investigation, but maybe relevant for other, already marketed similar product: could your current issue have an impact other product's SAL (sterility assurance level) or residuals that you sterilise with the same/similar cycle?

HTH,