5/2/2014 Understanding, Preventing, and Remediating Mold in Cleanrooms

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Understanding, Preventing, and Remediating Mold in Cleanrooms
By Ziva Abraham Dec 17, 2013 1:21 pm PST
Peer Reviewed: Microbiology
Introduction
Mold contamination has been considered a hazard in residential and commercial buildings for many years. In
the past few years, however, mold contamination issues in pharmaceutical products have caught the attention of
regulators. In the wake of mold findings and patient deaths, regulators have honed in on compounding
pharmacies that are alleged to be deficient in their contamination control practices (1).
Even though there is a lot of buzz about this topic, there is very little understanding with regard to the nature of
mold, the reason it is found in cleanrooms, and how it happily proliferates in the cleanroom environment. All
mold cannot be placed in the same basket as mold is capricious. To explain this point, let us consider the
mold found in the now so famous New England Compounding Center (NECC) pharmacy. Exserohilum
rostratum, Aspergillus fumigatus,and the common cleanroom mold Cladosporium were the isolates recovered
from the sterile products aseptically compounded in an ill-managed pharmacy. All over the Internet, it was an
interesting read when Exerohilum was referred as a black mold and Aspergillus as a common mold; as there
are many other black and common molds, such generalization of molds can lead to misconceptions about their
characteristics. This is what had occurred, as important characteristics of Exserohilum rostratum and
Aspergillus fumigatus were not presented in the publications that describe the NECC incident.
Aspergillus fumigatus is a fungus whose spores are ubiquitous in the atmosphere; it is also an opportunistic
human pathogen in immuno-compromised individuals, causing potentially lethal invasive infections. The
species is known to reproduce by asexual means (A.fumigatus-asexual reproduction). However, this mold also
possesses a fully functional sexual reproductive cycle that leads to the production of cleistothecia and
ascospores, which is the teleomorphic/sexual stage called Neosartorya fumigata. It is this understanding of
phase specific structures that allows for successful mold remediation. The asexual phase of a mold may be
killed by many disinfectants while the sexual phase may not be killed even by sporicidal agents if the correct
contact time and dilution are not used during disinfection. This is also the very reason behind disinfectant
qualification studies.
Similarly Exserohilum rostratum is the asexual stage (anamorph) while Setosphaeria rostrata is the sexual stage
(teleomorph) of the same mold. Microbiologists in the quality control (QC) laboratory are often not
microbiologists by profession. Many biochemists or others have learned the art of microbial testing. Lack of
understanding of mold, their origin, their prevalence, as well as modes of proliferation and mode of infection is
what leads to mold-related contaminants in products, thereby creating undesired consequences.
Nature of Fungi
Fungi are neither plants nor animals but have some characteristics of both. They cannot move about like an
animal, but do consume organic matter, have no chlorophyll as plants do, and cannot manufacture their own
energy. They have a true nucleus in their cells and are able to sexually reproduce by combining like strains of
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nuclei. They can also reproduce by spore formation like some early plants such as ferns and mosses. Current
molecular methods have shown that fungi are more closely related to animals than to plants.
Most structures of fungi isolated from cleanrooms are microscopic and not visible to the naked eye. Some are
unicellular like yeast, but most coalesce their cells into long, thread-like filaments called hyphae. Most
filamentous fungi produce an extensive system of hyphal strands, which may be visible when growing thickly in a
mass called mycelium, commonly referred to as mold. Mycelium can be of any size, from tiny bundles or clusters
to substantial structures, which effectively form the feeding and growing body of the fungus. The lower fungi, or
micro fungi as they are often called, form asexual spores on their surface simply by budding off from the tips of
their aerial hyphae and do not produce any structures that are visible. The vast majority of fungi found in
cleanrooms, such as Penicillium or Aspergillus, are of this type. In the case of higher fungi or macro fungi, when
they are ready to reproduce, the hyphae from two different parents fuse and form into a solid structure called the
sporocarp, ascocarp in case of Ascomycetes, and basidiocarp in Basidiomycetes. Mushrooms are
basidiocarps. The sexual spores are formed within or on the sporocarp after a fusion of nuclei from the different
parents (2).
Classification
Since the 18th century, fungi have been classified according to their morphology, for example: characteristics
such as color, spore shape and size or special microscopic features or physiology (3). DNA analysis may soon
be incorporated into taxonomy; however, this will pose a major challenge to the historical groupings based on
morphology and other traits that are used to date and to which mycologists are accustomed. Fungi can also be
classified according to their clinical importance.
Without going into the details of classification, such as phyla and family, a simple classification scheme put forth
in this paper helps in separating each class using distinctive morphological and microscopic characteristics.
The key features of each group are presented below:
Zgomycetes
This group has hyphae that are aseptic and exhibit both sexual and asexual reproduction patterns, producing
sexual spores known as zygospores as well as asexual sporangiospores.
Bread mold Rhizopus stolonifer belongs to this class. Cunnighamella, Syncephalastrum, Absidia, Rhizopus,
and Mucor are some of the common cleanroom isolates belonging to this class of fungi.
Aseptate Hyphae Asexual reproductive structure in Zygomycetes (Sporangium)
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Ascomycetes
This group has septate hyphae and only has a sexual reproduction phase; the sexual spores are formed within
a sac called an ascus; hence they are also known assac fungi. The asci are formed within a structure called the
ascocarp (4).
Some of the common cleanroom isolates belonging to this class are Eurotium, Chaetomium, and Microascus.
Septate Hyphae Ascocarps in Ascomycetes (Courtesy of APS Press)
Basidiomycetes
Members of the class are also called club fungi (5). After the sexual cells have united, they undergo division and
produce a club shaped structure called a basidium. Sexually produced basidiospores form at the tips of the
basidia. Basidia are often found on huge, visible, fruiting bodies called basidiocarps. The typical mushroom is a
basidiocarp. One should not see basidiocarps in the clean environments.
Deuteromycetes
These fungi have septate hyphae, lack a known sexual cycle of reproduction, and are said to be imperfect,
hence the name Fungi Imperfecti (3). When its sexual cycle is discovered, a fungus from this class is usually
reclassified to one of the other classes. A common example of Deuteromycetes that has a sexual stage is
Eurotium; its asexual stage is Aspergillus glaucus.
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A.glaucus group (anamorph) and Eurotium (teleomorph) in same culture.
Two types of hyphae are depicted in this class. Those where the septate hyphae have no color, Hyaline, and
hence are easily stained blue by lactophenol aniline blue stain during examination, and those that are
dematiaceous. The term "dematiaceous" refers to the characteristic dark appearance of this group of fungi as it
grows on agar. Colonies are commonly dark gray, brown, or black and, importantly, have a dark colored
underside when the reverse of the agar plate is examined. This distinguishes the dematiaceous
Deuteromycetes from hyaline Deuteromycetes (4). Hyaline Deuteromycetes may have dark spores, but the
hyphae are colorless and hence pale on reverse of the agar plate. Aspergillus, Penicillium, Fusarium, and
Verticillium are some of the examples of Hyaline Deuteromycetes, while Alternaria, Ulocladium, Cladosporium
and Epicoccum are examples of pigmented or dematiaceous Deuteromycetes.
Hyaline Deuteromycetes Dematiaceous Deuteromycetes
In residential and commercial buildings, not excluding cleanrooms, construction is very complex and contains
many components and systems for various purposes. These components and systems may be microbiological
reservoirs; for example, heating, ventilating, and air conditioning (HVAC) systems have been known to contain
mold growth, requiring regular maintenance and remediation. Materials used for building cleanrooms; such as
gypsum board, paper, wood, glue etc.; can pose a problem because of their nutritional value and ability to absorb
moisture. Wood and paper products are notoriously susceptible to moisture, leading to problems of fungal
infestation and decay (7). Below are a few molds that thrive on cellulose based materials: Cladosporium,
Eurotium, Exophiala, Fusarium, Paecilomyces, Acremonium, as well as Aspergillus, Aureobasidium,
Chaetomium, Penicillium, Stachybotrys, Trichoderma, Ulocladium. Many ofthese molds thrive on paint, glues,
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and plastics; some also grow on cement and mineral insulation. Thus, cleanroom management is critical as the
nutrition for mold is present in the building materials of the cleanrooms themselves.
It is well established that fungi and bacteria have health effects in humans. Many species of fungi can cause
infections. They also produce a wide range of chemical biproducts, called mycotoxins, which are mostly
associated with spores and have numerous health effects, primarily in ingestion exposures (7). Fungi also
include allergens and triggers of asthma. Among the well-studied effects of fungal spores causing asthma,
Alternaria alternata, a common indoor and cleanroom isolate, has often been cited. Fungal glucans are
inflammatory in the lungs and have been reported to be associated with headaches. In cleanrooms, the causes
of occasional mold could be attributed to improper gowning procedures and inadequate personnel and material
flows. However, it is not uncommon to find major mold contamination of the cleanroom environment and product
due to one or more causes such as:
Mold growth in unclean cold rooms or refrigerators where materials are stored
Inadequately cleaned incubators
Leaks in cleanroom structures
Unmaintained HVAC systems and high-efficiency particulate absorption (HEPA) filters
Inadequate pressure differentials between controlled areas
Seldom-cleaned warehouses
Cardboard boxes, wooden pallets, and more.
In cleanrooms with unattended moisture, issues mold may proliferate within the walls, which provides food
source for sustenance.
Medically Important Fungi
Fungal isolates recovered from product testing may be of clinical importance and may be objectionable
depending upon the mode of administration and patient population; hence it is beneficial to understand the
clinical importance of the common fungi recovered in cleanrooms and product (9). Fungal infections are broadly
categorized as superficial, systemic, and opportunistic (2).
Prevention of Mold
Fungi pose a multitude of problems in the pharmaceutical, medical device, diagnostic, food, and cosmetic
industries; from structural damage of cleanroom facilities due to poor maintenance to contaminating organic and
inorganic raw materials and spoilage of product by circumventing the effectiveness of preservatives. Fungal
contaminations lead to many issues such as product recalls, economic loss, regulatory issues, patient safety
issues, and public relation damage. There have been a significant number of recalls of products in each
category and additionally for supplies and consumables such as cleanroom(s).
To say there will be no mold in the cleanroom, at all, is wishful thinking. However, mold can be prevented and its
prevalence controlled. Mold can be prevented by:
Restricting its entry into the clean area
Not providing a food source for it to proliferate in the cleanroom.
Cleanroom construction materials may be prone to mold contamination for many reasons. Drywall may become
damaged when exposed to water, especially if the drywall remains exposed to the water for an extended period
of time. Often, during cleanroom construction, unintended introduction of water occurs if the water comes in
contact with the drywall at the base of the wall where the drywall touches the ground; wicking will occur. Capillary
action may introduce moisture anywhere from several inches to several feet above the floor depending upon the
length of time the wall is exposed to water. Water that enters a room from overhead may cause ceiling drywall
tape to separate from the ceiling as a result of the grooves immediately behind the tape where the drywall pieces
meet becoming saturated. Excessive cleaning of cleanroom surfaces, especially when the construction is older,
may allow water to seep into the cleanroom walls and floors.
Inadequately maintained HVAC systems may pose a risk of mold related contamination, either due to damaged
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filters or leaks. Filter media is made up of borosilicate fibers; however, there are many other components such
as the laminated plywood, particle board, gaskets, and sealants that can provide breeding grounds for mold if
not maintained. Moreover, if the filter media is physically compromised, particles along with mold can enter into
the cleanroom.
Warehouses are not controlled, and most of the materials are stored in cardboard boxes or on pallets. Mold
breaks cellulose into simple sugars, which then attracts other simpler mold forms, yeast, and bacteria. Both
these materials are a food source for mold; hence materials entering from the warehouse into the cleanrooms
have to be diligently wiped down. Also personnel and material flows, if not adequate, can allow mold to hitchhike
on materials or personnel and gain entry into the clean areas. Mold, like bacteria, is abundant in soil; gowning
procedures, especially tacky mats, have to be used as designed and changed frequently to prevent soil along
with mold spores from entering the clean areas. Often cleanroom personnel step on tacky mats after donning
disposable shoe covers. The function of the tacky mats is to remove dirt from the soles of street shoes. Stepping
on tacky mats after donning shoe covers compromises the shoe covers, thereby making the soles of the shoe
covers like sieves that allow soil to seep out while walking in the cleanroom.
Having cardboard in the vicinity of, or in, the cleanrooms increases the chances of mold related bioburden.
Pallets pose a similar issue. Often, gowns are stored in cardboard boxes in an uncontrolled warehouse. Though
the gowns may be double-bagged and terminally sterilized, mold spores from the cardboard boxes may fall onto
packaged gowns; this gives them easy access to the gown areas and subsequently into the clean areas.
Occasionally a small, invisible, and consistent leak can allow mold to grow unchecked in the walls. Watching for
change in color or appearance of cleanroom surfaces provides clues to mold proliferation in walls and under
flooring. Compromised baseboards are another common cause for the spread of mold into cleanroom walls
and floors. During cleaning of cleanroom walls, water seeps through the wall and the baseboard creating an
environment for mold to grow under flooring and in walls. Puddles of water around buildings are known to trigger
mold contamination of clean facilities.
In fact, for any place where even a little organic matter and moisture is available, mold will appear. Cold-rooms
and refrigerators are good examples. Product, media, and other materials stored in refrigerated units are often
carriers of mold into production areas or laboratories. Cardboard boxes stored in cold rooms and refrigerators
are a recipe for mold contamination in process or samples. Incubators are also well documented as a source
for mold contamination. Mold grows on the walls and floor of the incubators and may go undetected. Often
laboratory results with mold growth may be due to mold-ridden incubators and not due to product contamination.
Water baths, if not cleaned and dried, also promote mold growth. The same is true for any process equipment
that is cleaned and not thoroughly dried.
Cleaning is not the only way to prevent mold; as seen above, it is crucial to have systems in place to stop mold
from entering and proliferating in cleanrooms. Cleaning and disinfection are good means for both preventing
and remediating mold; although, without understanding how commonly used disinfectants work and what their
label claims mean, the process of cleanroom cleaning and disinfection may not yield the desired results.
Cleaning and Disinfection
The most commonly used sanitizers, disinfectants, and decontaminating and cleaning agents used in the
pharmaceutical industry include: phenolic compounds, quaternary ammonium compounds, Peroxygens, Bleach
(sodium hypochlorite), and, one favored by all, alcohol. What are these chemistries, and what is their function in
protecting our process, product, and ultimately the patient? Phenolic and quaternary ammonium compounds are
general-purpose disinfectants that are not necessarily sporicidal or fungicidal in nature (10). They act as good
cleaning agents due to the presence of a surfactant that overcomes surface tension when applied to hard
surfaces and, hence, allows even coverage of cleanroom surfaces. Sporicidal agents such as bleach and PAA
(peracetic acid chemistries) are sporicidal and fungicidal in nature; however, they do not contain surfactants and
thus are not suited as cleaning agents. Alcohol is a sanitizer and may not kill some bacteria. Gram negative
bacteria and mold, in fact, may use alcohol as a food source.
Whether one is choosing a cleanroom disinfection program for the first time or planning a disinfectant
qualification study; understanding how disinfectants work, what the label claims are for the disinfectant in use,
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and what type of mold are most prevalent in the facility is crucial (11). The effect of such microorganisms,
whether due to their numbers or species, on the processes or the product also need to be understood. As
explained above, mold belongs to four distinct groups. Though sporicidal agents may kill mold spores, the
exposure time, strength of the disinfectant, and the severity of the contamination should be considered. Among
the mold types discussed above, the hyaline Deuteromycetes are the easiest to kill. Disinfectant manufacturers
are required to perform fungicidal activity testing using AOAC methods for label claims (8, 12). Both the
organisms tested in United States and European Union (Trichophyton mentagrophytes and Aspergillus
brasiliensis) belong to this hyaline Deuteromycetes group. This is the very reason why regulators expect
companies to qualify disinfectants against their in-house flora. Companies do not like using sporicides due the
pungent or repulsive smell both bleach and PAA chemistries have, thus increasing their chances for mold
related contamination.
Disinfectant qualification studies are expensive, time consuming, and also very misunderstood. These studies
are prone to many errors if performed by personnel who have little or no understanding of:
The chemistries involved
Nature of hard surfaces
The method chosen to neutralize the disinfectant
The method chosen to recover the organisms from the hard surfaces
The viability of microorganism chosen for testing.
Also, there are no standard methods to perform these studies in the pharmaceutical industry, though USP
Chapter <1072> provides some general guidelines (4). The most important consideration when planning a
study is understanding that this study cannot prove efficacy of cleaning procedures. Cleaning procedures
translate to chemical kill by disinfectant activity and physical removal by mops and wipes. This study should be
undertaken to prove the chemical kill of disinfectants on various surfaces for a battery of organisms at the
manufacturer-recommended or other contact time-points. The chemical kill of microorganisms in the cleanroom
occurs by choosing the right dilution of disinfectant, a rotation between non-sporicidal disinfectants/cleaning
agents, and sporicidal disinfectants (11). Physical removal of microorganisms from cleanroom surfaces is
attained by:
Adequate coverage of the surfaces cleaned by the disinfectant/cleaning agent
Using good quality cleaning mops/wipes that can physically remove the microorganisms.
Additionally, a successful disinfection program includes implementation of the disinfectant effectiveness study
findings into routine disinfection practices. Hence, a poorly executed disinfectant qualification study may have an
overall bearing on the bioburden in the cleanroom.
Cleaning supplies play a very important role in both mold prevention and remediation. String mops hold dirt and
contaminants between the strings near the handle, are hard to dry, and do not allow full coverage of the surfaces
during cleaning. Because they cannot be dried easily, they provide breeding grounds for mold and bacteria. If the
disinfectants used in the cleanroom are not sporicidal in nature, these mops actually apply contamination
instead of removing contamination. Cotton string mops are even worse as they actively promote mold. Mops
used for cleanroom cleaning should:
Allow even application of disinfectants without soaking
Reach behind heat generating equipment to clean walls
Physically and efficiently remove dirt and debris
Not promote mold growth
Not generate particulates.
Fogging
Application of chemical disinfectants to clean areas with disinfectant fogs or mists is a method gaining
popularity. The purpose is to create and disperse a disinfectant aerosol to reduce the numbers of mold spores
in the air and on surfaces that may be difficult to reach during cleaning. However, one must understand the
nature of the mold contaminant before implementing fogging. As mentioned earlier, some mold is easily killed
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while others are not. For some mold, one fogging cycle may be adequate; for other species, multiple cycles may
be necessary. Often fogging companies use biological indicators to prove effectiveness of fogging. There are
mold species that can be more resilient than Bacillus, hence a 5 to 6 log kill of Bacillus does not equate to the
same level of reduction of the mold. It is advisable to use the same mold contaminant as the indicator of
effectiveness.
During fogging, the equipment supersaturates the atmosphere with a disinfectant fog; the area covered will vary
depending on the application system being used. Under typical conditions, fogging is carried out for a minimum
of 1530 minutes to enable the fog to disperse and the chemical action to occur. After fogging, an additional
period of 4560 minute is required to allow the droplets to settle out of the air and onto the surfaces. Electrostatic
charging of chemical fogs during aerosolization can improve the application as the droplets will be attracted to
surfaces that are electrically charged. The fogging technologies commonly used in the pharmaceutical industry
are:
Vaporized hydrogen peroxide (VHP), which is widely used for disinfection of the pharmaceutical
environment, including cleanrooms and production filling lines
Peracetic acid and hydrogen peroxide, which is used for disinfection of cleanrooms, cold rooms,
biological safety cabinets, incubators, etc.
Generally, decontamination cycles consist of steps such as humidification/dehumidification (depending upon
the technology), conditioning, vaporization, and aeration to remove the residual disinfectant
Conclusion
To prevent, control, and remediate mold, understanding the nature of the mold in question is crucial. One cannot
prevent mold from entering the cleanroom unless the source is known. Knowing the type of mold contaminant
leads to the source, which then can be corrected. There are many ways mold can enter into a cleanroom; having
proper procedures in place can reduce mold in clean environments. The selection of disinfectants used to
control mold contamination in a cleanroom is a complex decision as there is no single disinfectant that is
effective against all mold and possesses cleaning capability with surfactant activity. Choosing the best
disinfection program involves knowing the predominant mold types present in the facility. It is not just a choice of
the chemical agents, but also practical considerations such application methods and type of supplies, that are
important for mold control. Cleanroom cleaning procedures should address both the chemical kill by effective
disinfectants and adequate physical removal by appropriate cleaning supplies such as cleanroom compatible
non-linting mops. Physical removal of contamination is equally important; hence, supplies should not promote
microbial growth, should reach hard to clean surfaces, and should be able to hold and deliver the disinfectant.
Fogging may be used for remediating mold contamination, but the effectiveness of the fogging technique should
be evaluated against the mold being remediated.
References
1. R. Vijayakumar, T. Sandle, and C. Manoharan, (2012). A Review of Fungal Contamination in
Pharmaceutical Products and Phenotypic Identification of Contaminants by Conventional Methods,
European Journal of Parenteral and Pharmaceutical Sciences 17 (1), 4-19, 2013.
2. D. Sutton, A. Fothergill, and M. Rinaldi, Guide to Clinically Significant Fungi, Williams & Wilkins, Maryland,
US, 1998.
3. M. Griffin and D. Reber, Microbial Identification: The Keys to a Successful Program, PDA, Bethesda, MD,
DHI Publishing, LLC, River grove IL, USA.
4. St-Germain G, Summerbell R, Identifying Filamentous Fungi, A Clinical Laboratory Handbook, Star
Publishing Company, Inc., Belmont, California, USA, 2012.
5. T. Watanabe, Pictorial Atlas of Soil and Seed Fungi, Morphologies of Cultured Fungi and Keys to Species,
CRC Press, Florida, US, 2002.
6. H.L. Barnett and B. Hunter, Illustrated Genera of Imperfect Fungi, APS Press, St. Pail, Minnesota, US,
1998.
7. B. Andersen, J. Frisvad, S. Rasmussen, and L. Larsen , Associations between Fungal Species and
Water-Damaged Building Materials, Environmental Microbiology 77 (12), 4180-4188, 2011.
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8. EN 1650, Quantitative Suspension Test for the Evaluation of Fungicidal Activity of Chemical Disinfectants.
9. T. Sandle, Dimorph and Filamentous Fungi, in Mascellino, M. T. (Ed.) Bacterial and Mycotic Infections in
Immunocompromised Hosts: Clinical and Microbiological Aspects, (OMICS Group Inc.: Henderson, NV,
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