The Moldy Nightmare: Questions and Answers, Part 1

[Editor’s Note: The following questions were submitted during the Oct. 22 breakfast session, "Mold Contamination and Remediation—The Moldy Nightmare," of the 2020 PDA Pharmaceutical Microbiology Conference. The speaker, Ziva Abraham, an industry-leading mycologist, drafted answers to the questions. Questions were lightly edited for readability and organized into four broad categories: Mold Control, Disinfectants, Mold Identification, and Specific Molds. The Q&A for the last two categories will publish in Part 2 in a few weeks.]

Mold Control

Question

If a firm created a control plan specific to mold (a subset of the microbial control plan for the site), what unique controls would you expect to be implemented?

Answer

First, understand the type of product, its mode of administration, and the prevalence of infections via the mode of administration.

Second, identify the mold from all classes/grades to understand ingress and transport mechanisms for the mold.

Third, map out the biotic and abiotic factors. Biotic means the ingress path and abiotic means conditions in the cleanroom that will allow proliferation of the mold.

Fourth, adjust the disinfection and cleaning program based on mold type and predominance, and monitor the program effectiveness via trend analysis of mold recovered.

Question

If you see low-level recovery (1 or 2 CFU) on an airlock gowning bench every three months or so, with no other recoveries in the airlock, would you suspect inadequate cleaning practices?

Answer

Low recovery in gowning areas is a common phenomenon, as this is the entry point into the controlled areas from the uncontrolled areas. Having said that, it is important to have a science-based cleaning program to address all mold that can be tracked through foot-borne traffic. The cleaning and disinfection program should include use of a general-purpose disinfectant with a surfactant that helps break surface tension and aids in cleaning as well as periodic use of a sporicidal agent to address bacterial and fungal spores.

Question

Would you recommend that a firm create separate alert/action levels for mold, different from bacterial levels?

Answer

As mold does not have an infective dose like bacteria and infects by anchoring instead of endo or exotoxin production, it is beneficial to know the mold present in the cleanroom. More importantly, it is crucial to know if it is present proximal to open product. Setting alert and action limits may be beneficial, however, trending mold types recovered from all cleanroom classes/grades provides a better tool for understanding the mold type, its ingress path, and the effectiveness of the cleaning and disinfection program, as well as the possibility of the mold entering the product and causing harm to the patient. Often alert level excursions do not get much attention; thus, the risk may be missed.

Question

If mold is found only in air sampling (not on surface), what should be the action plan?

Answer

Mold recovery in air samples may be due to multiple reasons. Surface contaminants can become airborne, so the source could be foot- or wheel-borne contamination brought in. Inadequate storage of monitoring equipment and inadequate wipe-down procedures can be another reason. Monitoring equipment without a HEPA-filtered exhaust is known to be a contamination source. Growth of mold in walls after leaks, compromised HEPA filters, or mold growing on seals can cause airborne mold recovery. It is also important to track the mold to its source and map the transport into the area where it was recovered. Often dead spaces (e.g., in cleanrooms where the air is not cleared due to the location of HEPA filters), returns, and cleanroom and barrier system integration may allow the contaminants to linger for a long time.

If the mold is found in filling areas, especially RABs, the area between the HEPAs without a diffuser membrane is hard to clean and can hold contaminants that can become airborne.

Finally, depending upon the mold species recovered, especially the deuteromycotous fungi, which proliferate very fast, an abiotic factor such as moisture, carbon source, mineral oil, etc., should be looked at.

In summary, the investigation should be based on the genus recovered and the source, and the remedial action should be based on the above-mentioned points.

Once the source has been determined, increasing use of sporicidal agent, especially where the mold source is found, and trending monitoring data to assess the effectiveness of remedial measures is recommended. If a decision for using fogging is made, as the root cause is undetermined, the chemistry of the fog, the fog size, and the number of foggers to be used to cover the area in question should be considered. The smaller the fog particle size, the more buoyant, allowing it to stay in the air for longer periods of time.

Question

What are good preventative actions aging facilities can take to prevent mold?

Answer

For aging facilities, monitoring and trending provide valuable insight as to the health of the facility. Trending microorganisms provides insight into contamination types and sources. For example, leaks happen, surfaces become compromised. Once a leak happens, the building materials and moisture create an excellent breeding ground for mold. This mold stays dormant until the next leak. There have been facilities that have mold-infested walls, and mold contamination is transferred from one room to the walls of other adjacent rooms over time. Baseboards, if compromised, allow moisture to seep into the foundation, which has ample mold. Mold, being motile, can then start growing into the wall and flooring materials.

A routine audit for compromised structures, HEPA filters, seals, doors, etc., can help in incremental upgrades to the facility while keeping contamination under check.

Disinfectants

Question

What mold genus should be included in disinfection efficacy studies?

Answer

It is beneficial to use a USP-recommended Aspergillus strain along with the one or two predominant mold isolates recovered from the environment or product testing. Per the recent European standard, EN 13697, it is recommended to use the mature Aspergillus spores for disinfectant qualification.

Question

Why do some molds fail during disinfectant qualification studies?

Answer

Disinfectant label claim testing is performed using colorless deuteromycotous fungi Aspergillus and Trichophyton. Disinfectants with fungicidal claims, while using this testing method, may not be able to kill some colored Deuteromycota, most Ascomycota, and some Zygomycota.

It is recommended to understand the structures of the mold recovered in the cleanroom against those tested for fungicidal label claim. In some cases, increasing the contact time may help with the required kill, while in other cases prevention is the best strategy.

Question

Is rotation of cleaning agent essential? Is it true that microbes grow resistant to the cleaning agent?

Answer

There is no documented evidence of resistance, but to address all types of bacterial and mold contamination a rotation program utilizing a general-purpose disinfectant with surfactant, as well as a sporicidal agent, is effective at eliminating vegetative forms and spores.

Question

Do you recommend a routine rotation of a sporicide? Or whether, by exception, is it sufficient if we are not finding any systemic mold issue.

Answer

Using a sporicide with reasonable frequency is a good practice. Mold is mainly brought into the gowning room via foot- and wheel-borne traffic. If tacky mats are not used and changed adequately, the mold may also become airborne. The mold can subsequently be transported to more controlled areas via personnel.

Use of sporicidal agent helps reduce mold from being tracked into the controlled environment.

Question

Can you comment on the degree of disinfectant resistance between fungi and bacterial spores?

Answer

Among the bacteria, the spore formers are harder to eliminate. For example, Bacillus cereus has the least kill even with sporicidal agents. It is hard for the disinfectant to penetrate the spore structure. As for ascomycotous mold, whose sexual spores are protected by two layers as compared to one layer in bacterial spores, it is harder to eliminate these fungi. Hence, preventing the entry of hard-to-kill bacterial spores and ascomycotous fungi that are soil or cellulous material-borne respectively is a winning strategy.

Question

How do you recommend we clean or remove the residue from all of the cleaning agents on cleanroom surfaces?

Answer

Residue may be removed using quality water or residue strippers available on the market. Phenolic and quaternary ammonium compounds form most residues; hence the frequency of residue removal should depend on the use of residue-forming compounds.

Question

Do disinfectants make claims against the sexual stages of mold? Are they tested against this?

Answer

For fungicidal label claim, disinfectants are tested against Trichophyton and Aspergillus, which are both colorless deuteromycotous fungi. This testing is performed per AOAC methods in the U.S. and per EN methods in the EU for fungicidal label claim testing.

These tests do not require testing with colored deuteromycota, ascomycota, or zygomycota.