Moldy Stories: Tales from the Frontlines

Master mycologist Ziva Abraham shares her experiences remediating mold contamination events.  

Welcome to Part 3 and the conclusion of this mold contamination series! In Part 1, we highlighted how easy it is for mold to get into your product – It can ride the coattails of your raw materials! In Part 2, we outlined how this mold contamination can have a serious impact on the final product. 

In this finale, Ziva Abraham and I sat down to discuss her experiences with manufacturing sites that had similar mold contamination concerns. Here are the highlights of our conversation.

Jon: Let us rehash the first two parts of this series to clarify what happened at that site. The site used a raw material that could be thought of as sawdust. It was processed wood pulp dumped directly into product manufacturing vessels. However, the site had no tolerance for mold in the process stream. What I am most interested in discussing here- have you come across those types of situations where companies have trapped themselves, claiming “no mold!,” but inevitably mold pops up? How do they deal with that?

Ziva: Oh, yes! Multiple times. With a limited understanding of the sources of mold, companies out of fear of regulatory implications claim mold free cleanrooms; most of the time that is not achievable. Such a commitment to a mold free facility leads to setting unsustainable limits for mold in all Class/Grade rooms resulting in excursions and investigations. Often the root cause is not established, hence excessive cleaning and multiple fogging cycles are implemented to no avail. 

The lack of knowledge about the various patterns of mold sporulation, some easy to kill and others hard to kill even with the sporicidal agents, leads to compromising cleanroom surfaces due to excessive exposure to harsh disinfectants. This leads to mold growth in the compromised walls, gaskets, silicone, and other cleanroom surfaces which the mold can use as a carbon source. Ironically, agents used to kill mold ultimately encourage mold to grow more due to excessive moisture and compromised surfaces.

Mold should not be allowed in the critical areas and there is no way that any facility, no matter how well built, will be able avoid mold in the support areas. The key is to understand the mold, know the correct identification, analyze the sporulation patterns, create heat maps to map out the transport of the mold into the support areas and from the support areas to the critical areas. The last step is to ensure that mold is not feeding in the critical areas that allows it to proliferate. The materials coming into the cleanroom, material flows, airflows, personnel flows all play an important role in transporting mold from outside to the support areas and subsequently into the critical areas.

Jon: I would love to hear some examples like that, and how you have dealt with the remediation response in the past.

Ziva: I had the opportunity to audit a facility whose contamination control strategy claimed to have a mold free facility; the interesting part is that the process had ample wet operations in the support areas which greatly accelerated mold growth. The audit discovered external sources of mold which were transferred into the support areas where the mold had the opportunity to grow due to the presence of moisture. The mold sources were pallets and the main cold room outside the controlled area that was never cleaned. This external cold room had exposed drywall; mold was everywhere. The piping had mold growth hanging which looked like spider webs. Most of the bulk containers were stored in this mold ridden cold room - an easy transport of mold spores as these containers were transferred into the processing areas. The cold rooms within the manufacturing facility also had visible growth. The labels on the materials stored within these cold rooms had mold growth all over. 

This company conducted many GEMBA walks in the clean rooms, but the lack of knowledge on mold transfer made them miss sources outside the clean rooms. The cold rooms were not a part of the GEMBA walks so the main source was missed. Their claim of a mold free facility was not based on understanding their own facility and processes.

There was major remediation work involved in reducing mold in the support areas. It included construction of a new cold room, cleanup of other cold rooms, establishing informational monitoring of all cold rooms and re-evaluation of the cleaning and disinfection program to discontinue excessive use of harsh chemicals and avoid soaking of cleanroom surfaces. Gaskets which had started growing mold, especially in the autoclaves due to the excessive cleaning, were changed and all other sources that mold could use as food were identified and removed or changed. 

The remediation efforts we at Microrite implemented, though extensive, resulted in substantial control of mold. It was a wakeup call to the site management. Even with all the remedial efforts their facility could never be mold free. Again, the goal is to avoid mold in Grade A and B and keep it under control in Grades C and D.

Jon: Yeah, great point. If a site allows for, and sometimes sees, low counts of bacteria at certain sample points, at some point they can expect to see mold show up as well. The goal is to keep a state of control. In the case study from the previous posts, the site investigated the environment where mold could be coming from. It took a while to make the connection that, well, it was the raw material contaminating the environment, not the environment contaminating the product.

Ziva: Yes, exactly! So, you have to look for the source and that cannot be done without a valid ID. Then you can look at the transport, proliferation, and the clinical implication of the mold. I must tell you honestly not many understand the clinical implication of mold. I can give another case study with your permission.

Jon: Please go on!

Ziva: A nasal spray manufacturer was failing batches with Aspergillus fumigatus. They were producing for 3 years and had no such issues. Some consultant told them that people breathe 23 to 25 spores with every breath they take so it should not be a concern. What the consultant did not realize - all the CFUs recovered were A. fumigatus. The airborne fungus A. fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. It is a major cause of chronic pulmonary aspergillosis (CPA), invasive aspergillosis, aspergillomas, as well as fungal sinusitis. Our investigation found that a biological safety cabinet from the warehouse was pulled into the cleanroom to temporarily hold partially sealed vials. 

So, you see why you also have to look at non-sterile products seriously. We have seen multiple recalls of both sterile and non-sterile products with mold contamination. Additionally, the list of 19 most objectionable fungi released by the WHO in December of 2022, is a sign that mold is more resilient, and it is evolving at becoming more pathogenic as the humans become more immunocompromised.

Companies take nonsterile products very lightly; they just look at the microbial limits test result and say, oh, it is OK. There is more to the test results.
It is also concerning that inhalation products are taken lightly with lax limits for mold. Immunocompromised patients and those who have survived COVID are prone to fungal pneumonia. The compromised soft tissue is the target for fungi as seen during the mucormycosis incidents in India. The correct identification is so key! Not only for investigative purposes, but also to determine the objectionability of the mold via mode of administration. I have another one for you Jon.

Jon: Ziva you are on a roll. Give me another example.

Ziva: A company wrongly identified the mold isolate in-house. They had limited mycology skills or understanding of the morphological or microscopic features of fungi. They used Google images to identify and came up with the organism Trichophyton mentagrophytes. That is a foot fungus! As you can guess, they came up with an inappropriate CAPA. They ended up soaking the operators’ feet in peracetic acid while the contamination prevailed in the cleanrooms and product. You do a wrong ID, you don't have the right path to investigation. The entire premise for the revision of Annex 1 is inadequate investigations and ineffective CAPAs. This is a perfect example of inadequate investigation and ineffective 

Jon: Oh my gosh Ziva, that give me flashbacks of a contamination event with C. acnes. Our management team was seriously considering swabbing the acne on operators to look for a matching strain. It felt like high school peer pressure all over again. But given some of the comments from the auditor in our last story, I would like to know a little bit of your thoughts on mycotoxins. Do you have anything to share there?

Ziva: Most mycotoxin data come from agriculture and the food industry. With the few CFUs recovered in cleanrooms, there is lack of literature that has tried to tease out mycotoxin concentrations of the basis of single conidia or single CFU. The recovery of a few CFUs from a batch of product will most likely yield undetectable levels of any and all mycotoxins that might be associated with the particular taxon. The same is likely true of allergens and other antigens. 

We have talked about so much today. What conclusions can we make?
Mold investigations are difficult, considering that many in the industry do not have good understanding of mold sources, their nutritional requirements, and their sporulation patterns. Unless you have a valid identification, it becomes difficult to gather the required information about the mold species and perform an appropriate investigation and implement effective CAPAs. Often, excessive cleaning, use of harsh chemicals, fogging, and performing disinfectant efficacy studies is what the industry engages in, in hope that the mold will go away. However, more often than not the mold reappears. Inappropriate investigations and ineffective CAPAs were some of the reasons that prompted the revision of EU Annex 1.

Understanding the mold, its sporulation patterns, and identifying the source are the first steps. Then, how it is transported into the cleanroom and how it moves about the cleanroom through airflows, material flows and personnel flows needs to be understood. In case of major contamination events with in-process samples and finished products, how the mold is being fed is important to understand. In your case study Jon, sawdust, which mold loves to eat, was the source as well as the point of proliferation.

Jon: Understanding. That is such a key word. It is hard to know the root cause, impact, and proper corrective actions if you don’t understand your process, facility, and the mold involved. You cannot look at a mold investigation, see it is not on an objectionable organism list, and conclude it’s acceptable in your product. You also need to understand how the mold is getting into your facility, otherwise efforts to remove it will be futile. It was so great talking with you today. Thank you again for your time. I look forward to your presentation on Biofilms in Water systems at the 2024 QC Micro Summit! We have both run into our struggles with those.