Sterility Failure…Connecting the Dots

If you have never had to deal with the repercussions of a positive sterility result, it may interest you to know how complicated a simple open-drop-incubate test can become. This complexity originates with one simple question, “How did that organism get into the media?” Simple, right? Either the product was not sterile or the media was contaminated during testing. Picking one of these options is far from simple – it is nearly impossible to prove. Here is where I come in.

Surprisingly (for a microbiologist), I get paid to use my imagination. Imagine, for instance, that there was a tiny, invisible particle sitting on the floor of the cleanroom. This particle happened to host a bacterial cell. The analyst enters the cleanroom to perform the testing for the day, and the airflow suddenly changes, causing the particle to float and settle on some supplies. The analyst starts testing. Open packaging, drop sample into media. Open package, drop sample. Open, drop. The analyst reaches for a new lot of forceps and brushes her sleeve against the particle. As she starts testing again, her sleeve brushes the laminar flow hood surface. A media container slides along the hood, picking up the particle. The analyst removes that media container from the hood and grabs a fresh one. Her gloves are now contaminated. She pulls at the sealed packaging to expose the sterile device, docking the particle on the edge of the packaging. Forceps pinch the device, carefully pull it out of the package, just barely brushing against the edge, and the sample is dropped into the media container. That particle tags along into the media, and the organism slowly starts to grow.

Fourteen days later, I get a phone call saying that I have a new sterility failure to investigate. Can I scientifically and objectively re-construct this story from historical data, test videos, and interviews? The answer: Maybe. Investigating a sterility failure is essentially connecting the dots. These dots might not be numbered and some may be missing, but that is essentially what it is.

A microbe ended up in the media – connect the dots back to where it originated. What organisms have we been recovering lately from the room or analyst? How recently was the room cleaned? How does technique during testing look? Was the packaging adequately disinfected? Were the forceps sterile? Along with all these internally focused questions, confidence in the product’s sterility must also be assessed. How well did you validate the sterilization? Were there issues with sterilization this time? Are the organisms resistant? Have there been any manufacturing changes? Was the packaging an effective sterile barrier? The list of questions can go on and on. The art in all of this is knowing which dots are worth connecting.

Imagine that that particle mentioned earlier grew into a colony of Staphylococcus epidermidis. This is a common skin originating organism. The product was manufactured in an environment with S. epidermidis, but was then radiation sterilized. This organism is not known to be resistant to radiation. Bioburden testing shows that the devices, pre-sterilization, harbor some organisms. However, this has remained consistent over the past few years. Sterility testing since the validation has never shown any positives. Packaging was validated and no issues were noted. By this point, you may be thinking that it was the testing laboratory’s fault. According to most regulations and standards, that conclusion is far from justified. This is where the real work (fun) begins. Cleanroom data, such as differential pressures, microbial monitoring, videos, gowning records, cleaning records, maintenance records, sterilizer records, etc. are scrutinized to find evidence of contamination.

Every investigation I perform is unique. Variables combine and contradict in new, intriguing, and usually frustrating ways to paint a picture of contamination. Imagine that growth was observed in the sample as well as the instrument monitor. Did the instrument contaminate the sample, did the sample contaminate the instrument, or did both come into this situation independently? I liken this to the classic chicken or egg question. Which came first? Hard to think about. Even harder to prove.

Sterility testing (open-drop-incubate) can lead to stimulating debates, unanswerable questions, and the occasional headache, all in the name of science. So how did that organism get into the media? We may never know for sure… But I will do my best to find out!


Kevin R. Buckingham, RM(NRCM), Failure Investigator, Nelson Laboratories, Inc., Salt Lake City, UT; he earned his NRCM certification in 2010.


Copyright© National Registry of Certified Microbiologists. Reprinted from The Loop, 2011, Issue 3.

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