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The proper design and execution of an environmental sampling plan is a central component of USP <797>. A good environmental sampling program will not only allow a pharmaceutical compounding laboratory to know whether or not it is within the recommended action levels of USP <797>, but will also provide valuable information for determining sources of potential contamination and counteracting them.

Testing Requirements and Methodology
Successful implementation of a USP <797> environmental sampling program starts with an understanding of the different tests required by USP <797>. The most basic division between the required tests is nonviable particle sampling and viable particle sampling. Nonviable airborne particle testing seeks to measure the density of airborne particles based strictly on their size (0.5 µm in diameter or larger) without regard for the nature of the particles themselves. Viable particle sampling only measures those particles that are living organisms (typically bacterial and fungal spores). USP <797> breaks viable particle testing into several different categories, each of which is designed to test a separate aspect of pharmaceutical compounding for potential contamination. These tests are viable airborne particle testing, viable surface particle testing, gloved fingertip sampling and media-fill testing (also called aseptic manipulation testing).

Nonviable airborne particle sampling must be performed by a qualified operator using anelectronic particle counter. Testing needs to occur once every six months at a minimum. Additional testing is required any time that the primary engineering controls (laminar flow hoods, isolators, etc.) are moved or altered, or in the event of major changes to the facilities surrounding the primary engineering controls. At a minimum, each separate ISO class 5, 7 or 8 area needs to be tested (1). A thorough testing plan will also investigate areas and items that could be potential sources of nonviable particulates. Potential sources of nonviable particles include, but are not limited to, potential leaks in a clean room's containment (possibly at windows, doors and pass-through cabinets), mechanical and electrical equipment (refrigerators, computer printers, etc.) and areas with high personnel traffic during compounding operations. A map of the laboratory being sampled can be very useful for pinpointing critical areas for sampling. If a map or blueprint of the compounding laboratory cannot be obtained from the laboratory manager, a hand-sketched map can prove to be useful. All data collected must be thoroughly documented. Documentation should include the specific locations sampled, the time of day sampling took place, copies of the calibration certificate for the particle counter used to collect the data, and training certificates for the individual(s) who performed the sampling.

Airborne bacteria and fungi can pose a significant threat of contamination during the manufacture of compounded sterile preparations. Viable airborne particle testing is performed to monitor this threat and to ascertain that physical and procedural controls in place are keeping the airborne microbial load of a facility's air to an acceptable level. The frequency of testing for airborne viable particulates is identical to that for airborne nonviable particulates (2). It is recommended that sampling take place during ongoing compounding operations. If it is not possible to take viable air samples while compounding is taking place, then sampling should take place immediately after compounding has ended for the day (3). Volumetric sampling devices must be used and 400 to 1000 liters of air must be tested for each sample (4). Gravimetric sampling may be used to supplement volumetric sampling, but gravimetric sampling is not sufficient for meeting the requirements of USP <797>. Air samples taken for the purpose of measuring bacterial load utilize TSA (Tryptic Soy Agar) or soybean casein digest as growth media for collected organisms, while air samples taken to examine airborne fungal load are collected using MEA (Maltose Extract Agar) or Sabouraud Dextrose Agar. It is important to note that testing for fungi is only required for compounding categorized as high risk (5).

Viable surface sampling is required by USP <797> in order to assess the success of the laboratory's cleaning program in keeping surfaces free of microbial contamination. While USP <797> mandates periodic surface sampling, it does not specify a definitive period or time frame for sampling. A feasible option is for surface sampling to take place at the same time as air sampling. Sampling surfaces in parallel with air sampling allows for a more thorough investigation of any detected contamination and maximizes convenience for personnel performing the sampling. At least one surface sample must be taken from each ISO 5, 7 and 8 area after compounding has concluded (6). Additional samples can be taken from any surface that personnel, materials or produced compounds are exposed to regularly. Work surfaces, storage surfaces, door handles and equipment are all good targets for surface sampling. Surface samples may be collected using TSA contact plates with added lecithin and Polysorbate 80 (TWEEN 80) or swabs. Contact plates are generally the preferred method for surface testing because of their ease of use, but sample collection with swabs is perfectly acceptable under USP <797>. Swabs are also the only means of sampling from curved and oddly shaped surfaces, such as door handles or sink faucets.

Gloved fingertip sampling is performed to evaluate the efficacy of compounding personnel's hand washing and garbing techniques. Workers wash their hands and don all of their protective equipment while being observed by their supervisor, in order to make certain that their technique is in accordance with the laboratory's standard operating procedures. Once they are completely garbed, the worker presses four fingers and a thumb to a TSA plate (one for each hand) with lecithin and Polysorbate 80 (7). It should be noted that only TSA plates with lecithin and Polysorbate 80 are utilized for this test. There are no requirements for a separate gloved fingertip test specifically to check for fungal contamination. Gloved fingertip tests must be performed prior to any new personnel beginning work at a compounding facility, annually for established personnel performing low and medium risk compounding, and once every six months for personnel performing high risk compounding.

Media-fill testing (also called aseptic manipulation testing) is performed to measure the ability of procedures, personnel and equipment to successfully produce a sterile end product. This test is a simulation of the actual compounding taking place in a facility. During media-fill testing, personnel perform all of the steps for a given compounding procedure using the same equipment and facilities as they would when compounding, except the typical medications and diluents normally used in the procedure, are replaced with soybean casein digest media. It is important for the laboratory manager or head pharmacist of a facility to participate extensively in the development and execution of this particular test. This is because extensive knowledge of the laboratory's compounding procedures is necessary to properly set up a media-fill test that closely resembles the compounding taking place in their lab or facility. A media-fill test is considered successful if no growth is seen in the final containers prepared by the test after incubation. Media-fill tests must be performed by all personnel prior to being allowed to begin compounding at a given facility, annually for personnel working in low and medium risk compounding facilities, and once every six months for personnel engaging in high risk compounding (8).

Quality Control
While performing viable or nonviable sampling it is important to be aware of quality control. Copies of calibration certificates should be provided to the laboratory manager for all equipment used in sampling. Likewise, a copy of the sampling personnel's training qualifications should also be made available. All media used for viable sampling should have an accompanying certificate of analysis that also should be provided to the laboratory manager. It is generally considered good practice to utilize positive and negative controls for viable testing, however USP <797> does not require these controls. Positive controls require that a viable sample plate be exposed to a known quantity of microorganisms and incubated alongside samples taken in a compounding facility. In practice, it is very difficult for personnel working in the field to obtain and maintain stock cultures of organisms with known concentrations. In the field, personnel seeking to make a positive control, sometimes sample from an area that is not ISO rated. Sampling air outside of an ISO classified area is likely to detect microorganisms of an unknown concentration. This type of "positive control" will demonstrate the media's ability to support microbial growth if microorganisms are in the area sampled, but without any specific data available for microbial concentrations in a specific area, it is impossible to be certain how well the media performs. As a result, this type of sample represents a compromise between the precision of a true positive control and the ambiguity of having no positive control at all. Negative controls are much easier for field personnel to obtain. A negative control is provided by sealing an unopened media plate taken from the same batch as the sample plates, then sending it to a laboratory for incubation and analysis without ever exposing the plate to an air sample. For media plates that are received sterile, the negative controls should show no growth after incubation.

Data Interpretation
Data interpretation begins by comparing the results of nonviable and viable air particle testing and viable surface particle testing to the recommended action levels in USP <797>. These recommended action levels have been reproduced in Table 1 (9). If the number of particles detected by nonviable air particle testing, viable air particle testing or viable surface particle testing, exceeds the levels given in table 1, then it is necessary to begin a full root cause investigation of the source of contamination, followed by whatever steps are necessary to bring the particulate levels detected to within acceptable limits.

Table 1: Recommended Action Levels. (9)

For example, if a viable airborne particle sample from an ISO 7 clean room yielded a total of 11 cfu/m³, it would be higher than the recommended action level of greater than 10 cfu/m³and a full investigation into the source of contamination would be necessary. Alternatively, if the same sample had only 9 cfu/m³, the recommended action level would not have been exceeded and a full investigation would not need to be initiated. It is also necessary to implement an investigation and remediation in the event that there are particulates identified above historically detected levels for a given facility.
There is also a special requirement for viable air particle samples. If any cfu's are detected on a viable airborne particle test plate from an ISO 5, 7 or 8 area, then USP <797> requires that the colonies growing on that plate be identified to at least the genus level, even if the number of colonies is below the recommended action level (10). The reasoning behind this requirement for genus identification is that there are some organisms that cannot be tolerated in a clinical environment at any concentration.
For example, Methicillin-resistant Staphylococcus aureas (MRSA) is a very dangerous organism in hospitals, and has been discussed extensively in the popular media. If even one cfu of MRSA is detected in the air of a pharmaceutical compounding laboratory, then extensive investigation and remediation would be warranted. Full speciation of viable colonies is highly recommended in order to avoid expending extensive resources for remediation of non-dangerous organisms in the same genus as highly dangerous ones.
To return to our previous example, Staphylococcus epidermidis would not warrant the same response as Staphylococcus aureas. However, a genus identification for these two organisms would yield the same result, Staphyloccoccus. A laboratory manager would not be able to distinguish which of the two organisms were present in the laboratory and would have to respond as if the more dangerous organism was present. Species level identification allows for a more precise response by laboratory personnel to the actual threat posed by any given organism.
Interpretation of data from gloved fingertip and media-fill tests is less complex than interpretation of air and surface plates. For gloved fingertip plates, a new worker must have three successful tests with 0 cfu's, prior to beginning work in a compounding laboratory or facility. For experienced workers, the recommended action level is >3 cfu's per test total, whereby the counts from both the left and right hand plates are added together. Media-fill tests are either positive or negative for growth. Any positive media-fill tests require a root cause investigation and the implementation of remediation to fix the problem (11).

Investigation and Remediation
Investigation of viable particle concentrations above action levels, or away from historical baseline levels of contamination, must take into account both physical and personnel factors. Physical factors include items such as making certain that all equipment is working properly. The maintenance records of all equipment should be reviewed to be certain that everything in the lab has been properly maintained. Regularly scheduled cleaning of the lab itself is also critical. Cleaning logs should be reviewed and cleaning equipment inspected to be certain that it is still capable of cleaning properly. Cleaning equipment should be non-shedding and dedicated for use strictly in the controlled environment. Cleaning agents need to be rotated on a regular basis. Not all cleaning agents are equally effective against all microorganisms. For example, 70% ethanol is a cleaning agent that is highly effective against bacteria, but has very little effect on bacterial endospores, some fungal spores and certain viruses (12, 13). Rotating through a series of different cleaning agents over time increases the likelihood that all organisms in a laboratory are eventually exposed to a susceptible substance. Cleaning agents must also be stored and used according to their labels. Changes in the weather or in the building containing the compounding facility also need to be investigated. A rapid climate change can cause previously quiescent organisms to begin growing. Changes to the physical structure of the building could affect the ability of the compounding laboratory to prevent environmental incursions. Personnel factors are the ability of compounding personnel to successfully implement the laboratory's standard operating procedures to prevent contamination. Direct observation of personnel during ongoing compounding operations should be conducted to verify proper implementation of all laboratory standard operating procedures. The procedures themselves should be reviewed to make certain they are adequate to the task of preventing contamination.

Documentation
At all stages of USP <797> implementation it is very important to remember to document every action taken and every test result. The historical record is the only means that a laboratory manager has for recognizing trends and spotting potential problems before they become severe. Ideally, observing how the contamination levels change over time in a laboratory will allow potential contamination issues to be isolated and solved early, before they have a chance to cause injury or illness in pharmacy patients or laboratory personnel. In the event that a patient illness leads to an investigation by legal authorities, these records are the only proof that a laboratory has been following the regulations established by USP <797>. Without thorough documentation that the laboratory has been meeting the requirements of USP <797>, they will likely be subject to legal consequences.
Design and execution of a USP <797> environmental sampling plan is a task that requires attention to detail, extensive knowledge of the tests required and precision in reporting data. At EMLab P&K we are dedicated to providing the technical expertise and laboratory capabilities necessary to meet the challenge of implementing a rigorous USP <797> program. We are prepared to provide the commitment to quality that you have come to expect from EMLab P&K. Contact us for USP <797> services.

References:
1. The United States Pharmacopeial Convention. <797> Pharmaceutical Compounding - Sterile Preparations.Revision Bulletin. 2008, p. 1-61.
2. Ibid., Revision Bulletin, p. 25.
3. The United States Pharmacopeial Convention. <1116> Microbiological Evaluation of Clean Rooms and Other Controlled Environments. National Formulary. 2000, p. 2099-2106.
4. Ibid., Revision Bulletin, p. 25.
5. Ibid., Revision Bulletin, p. 25.
6. Ibid., Revision Bulletin, p. 33.
7. Ibid., Revision Bulletin, p. 31-32.
8. Ibid., Revision Bulletin, p. 30.
9. Ibid., Revision Bulletin, p. 2, 26 and 34.
10. Ibid., Revision Bulletin, p. 26.
11. Ibid., Revision Bulletin, p. 31-32.
12. Allen, L.V., Jr. and Okeke, C.C. 2007. Basics of Compounding: Considerations for Implementing United States Pharmacopeia Chapter <797> Pharmaceutical Compounding - Sterile Preparations, Part 4: Considerations in Selection and Use of Disinfectants and Antiseptics. International Journal of Pharmaceutical Compounding. 11(6): 492-496.
13. Utama, I.M.S., Wills, R.B.H., Ben-yehoshua, S. and Kuek, C. 2002. In Vitro Efficacy of Plant Volatiles for Inhibiting the Growth of Fruit and Vegetable Decay Microorganisms. Journal of Agricultural Food Chemistry. 50(22): 6371-6377.

They say a picture is worth a thousand words; well then a video is worth a hundred thousand. Utilizing online videos is one of the fastest trends in marketing today and the good news is it's easier and cheaper to accomplish than ever before.

As internet speeds and compression technology have advanced the dominant trend on the internet has been to harness the power of videos. Online visitors want quick access to information and properly constructed videos provide just that. They also allow visitors the chance to see and hear directly from the company.

It's no wonder that one of the most highly ranked websites in the world is YouTube. It consistently ranks in the top 5 websites globally and close to 700,000 websites currently link to YouTube. Chad Hurley, a co-founder and Chief Executive Officer of YouTube, claimed last October that the website has "well over a billion views a day." There was indeed a good reason Google paid $1.65 billion for YouTube in 2006 (the company was only founded in 2005).

Companies of all types are now posting video content to YouTube and many are also directly linking that content to their own company websites (which is very easy to accomplish).  IAQ videos can also be posted at no charge to the just introduced IAQ Video Network.  Company videos consist of commercials, infomercials, press releases, instructional videos, news and some that are mostly for entertainment. Many savvy companies also link their videos to their Twitter, Facebook and other social media pages.

One of the great features of utilizing YouTube to post video content for your indoor air quality or environmental business is that it's completely free to do and people do not need to be registered with YouTube to watch your company videos. Paid promotions for videos is an option for registered users, but is not at all required.

To get started you only need a digital camcorder (which cost as little as a hundred dollars - although ones that record in high definition are preferred and these can be found for $250+) or digital camera and a computer (for editing and uploading) to get started.

When creating video content be sure to consider who your audience is and tailor your video to them. The great thing about YouTube, or loading videos directly to your existing website, is you can make individual videos targeted to as many specific groups as you want. Another thing to consider is keeping the videos short, 1 to 10 minutes is ideal for most circumstances (YouTube limits videos to 10 minutes in length). Viewers will lose interest and close out of long videos more often than not.

Once you have your digital camcorder shoot your video. Long individual video clips should usually be avoided and instead include individual clips of 10 to 30 seconds. A tripod ($25) is strongly recommended to prevent the video from appearing shaky. Most camcorders also allow still photography and a mixture of video and rolling still photographs is often the easiest type of video to produce. Once you have your content, download it onto your computer and use one of the many free video editing programs that are currently available (Windows Live Movie Maker is a good one to start with).

Video editing software will allow you to edit video clips, add photos, transitions, titles, text, music and voice narration. For voice narration there is a great free program called Audacity that allows recording and editing of any narration directly on your computer. To use the software simply download the program and purchase a digital microphone (ideally one with a headset - $50).

To see YouTube's video and audio specifications please click here. The page is full of all the information you will need and these same specifications can generally be considered even if you are making a video to simply upload directly your own website.

Cochrane & Associates, LLC, the environmental, mold and indoor air quality industries' only dedicated marketing, public relations and business development consulting firm works with clients to develop ways to create and utilize online videos for customers. We hope you consider making online videos a prominent part of your company's business development strategy. We also invite you to visit our newest venture, the IAQ Video Network www.IAQTV.com.  Companies can post their videos or links to videos from our industry at no charge on the website.

Spring has sprung, the snow is subsiding and the flowers are beginning to bloom coast to coast. With the change of seasons, many Americans have spring cleaning on their minds. According to a recent study done by the Soap and Detergent Association (SDA), nearly two-thirds of Americans participate in the annual tradition known as spring cleaning.[1] After being cooped up in the home all winter long, spring cleaning is a great way to rid the house of dirt and dust. The study also revealed that the most commonly cleaned areas of the home are the kitchen, living room, bathroom, and master bedroom. While taking the time to clean is a necessary task, many people forget that the quality of air in the home is just as important in maintaining a clean, healthy home environment.

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“E●Z Breathe is excited to be able offer an effortless solution for improving the quality of air within the home this spring,” states Erika Lacroix, President of E●Z Breathe. “This revolutionary system allows people all over the nation to live healthier lives just by being in their homes.”

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The online video channel and website is available to all mold, IAQ, HVAC, industrial hygiene and environmental professionals.

The indoor air quality (IAQ) industry’s only dedicated marketing and public relations firm, Cochrane & Associates, recently announced the debut of the IAQ Video Network. The online video network brings news and video content to industry professionals and can be found at www.IAQTV.com.

Cochrane & Associates launched the online video service to provide online educational content that highlights industry events, conferences, companies, equipment and other news that is affecting the IAQ industry. The first news video posted takes place at the 2010 Indoor Air Expo presented by the Indoor Air Quality Association (IAQA) and the Air Conditioning Contractors of America (ACCA). Another conference video will be placed on April 1st following the upcoming Environmental Information Association’s (EIA) conference in Austin, Texas.

“We wanted to develop an online video source for news and events for the IAQ industry,” reported Paul Cochrane, President of Cochrane and Associates who are developing the IAQ Video Network and IAQTV.com. “We hope to make the website a popular video portal that other companies in our industry will utilize to post video content and links to their videos. The website was just launched last week so we have a lot of development work to do to get the website up to its final configuration which we hope to reach in about a month.”

In addition to the online videos, Cochrane & Associates also continues to publish the ever popular Environmental Marketer newsletter. The monthly electronic publication brings marketing ideas and concepts to the industry that illustrate the many ways that companies in the IAQ, HVAC and environmental industries can stand out from the crowd without the expenditure of a lot of resources. The complimentary publication is received by thousands each month and an automated signup form is located on the company website atwww.cochraneassoc.com.

Opportunistic fungi attack people with severely compromised immune systems either resulting from medical treatment or disease. Infections with these pathogens are increasing due to the increased use of immunosuppressive medications and diseases such as AIDS that reduce immune competence. Industrial hygienists, hospital infection control personnel, mold investigators and remediators are more and more frequently being called upon to discover the sources of outbreaks of diseases caused by these fungi. Unfortunately, while in other types of environments concentrations of fungi must be relatively high to cause problems, in hospital settings where immunosuppressed patients are housed, airborne concentrations of pathogenic fungi must be maintained at near zero levels. Thus, even very limited growth may be important, and unusual sources must be investigated.

The Fungi Involved
The most common opportunist that is also the most frequent cause of disease is Aspergillus fumigatus, a thermotolerant fungus that not only causes infections, but also colonizes the lungs of asthmatics and people with cystic fibrosis. Other species of Aspergillus that are frequently reported as pathogens are A. terreus, A. niger, and A. flavus. Aspergillus ustusless frequently causes infection (Saracli et al., 2007). Fungi from other genera have also been reported to cause opportunistic disseminated infections, including Scedosporium prolificans (Alvarez et al., 1995), Geotrichum candidum (Scora et al., 2009), Mucor(Sochaj et al., 2009), Pseudallescheria boydii (Thornton 2009), Blastoschizomyces capitatus (Celik et al., 2009), Trichoderma longibrachiatum and closely related Hypocrea orientalis (Druzhinina et al., 2008), and Absidia corymbifera (Parra-Ruiz et al., 2008).Candida albicans, Pneumocystis carinii and other species of Pneumocystis, andCryptococcus neoformans are also important opportunistic pathogens. Candida is a human commensal that attacks the host when immunosuppression occurs. However, the organism can be transferred between patients and health care workers (Marco et al., 1999).Pneumocystis probably does not grow in the hospital environment, although the fact that it is not culturable may contribute to this perception. It can be readily detected through PCR methods and has been recovered from air (Bartlett et al., 1997). Cryptococcus neoformansis extremely common in the environment, and many people either have or have had subclinical infections. It may become invasive and cause central nervous infections in immunosuppressed patients. This is extremely common in AIDS patients.

The Array of Sources
Person to person transmission. Most fungal opportunists are not transmitted from one person to another. Candida albicans is an exception as is Pneumocystis jerovecii, and probably P. carinii as well. Candida is probably transferred between people by direct contact (Buffington et al., 1994). On the other hand, Pneumocystis is airborne (Yazaki et al., 2009).

Construction. Outbreaks of aspergillosis (invasive Aspergillus infection) related to hospital construction and remodeling have been reported repeatedly (e.g., Alvarez et al., 1995; Ansorg et al., 1996; Lai 2001). As discussed below, outbreaks of infection can be avoided if proper attention is paid to containment during construction activities.

Ventilation Systems. Arnow (1978) was one of the first to note the growth of Aspergillus fumigatus in ventilation systems and the relationship of this growth to aspergillosis. His group conducted environmental monitoring in a new hospital, culturing for Aspergillus and conducting surveillance for aspergillosis cases (Arnow et al., 1991). After airborne concentrations of Aspergillus flavus and A. fumigatus increased to average levels >1 colony forming unit per cubic meter (cfu/m³) and the incidence of aspergillosis increased, filters in the ventilation system were found to be heavily colonized with Aspergillus fumigatus, andA. flavus was found within the hospital rooms. Remediation reduced both airborne concentrations of these fungi and the incidence of aspergillosis. Lutz et al. (2003) reported a similar situation, attributing the contamination to deterioration of insulating material in variable air volume units.
As is the case in all buildings, cellulosic filters that get wet are inevitably colonized with fungi. While Cladosporium is often the dominant type in these situations, Penicillium andAspergillus species have been recovered as well (Price et al., 2005).

Cleaning Activities. Cleaning activities clearly can raise Aspergillus concentrations in hospitals as well as other sites. In one study, geometric mean concentrations before cleaning were 5.5 cfu/m³ compared to 18.9 one hour after cleaning (P=0.0047) (Lee et al., 2007). In another case, a vacuum cleaner used to clean the floor in a pediatric oncology/hematology ward was found to be the source of Aspergillus fumigatus that led to an outbreak of infection. During vacuum cleaner operation, Aspergillus fumigatus recoveries were 65 cfu/m³ compared to less than 6 cfu/m³ in rooms where this vacuum cleaner was not used (Anderson et al., 1996).

Waste Containers. Hospital waste containers are an obvious potential source for all sorts of infectious agents. Blenkharn (2006) found many organisms including Aspergillus species in these reservoirs.

Plants. Controversy over potted plants as a source for fungal aerosols in hospitals has existed for many years. Thompson et al. (1994) found Aspergillus in 80.5% of his samples of potted plant soil. The most common species were Aspergillus fumigatus and A. niger(Thompson et al., 1994).

Water Systems. Water can contain fungi, and water systems may become colonized.Aspergillus species have been recovered from water taps, patient showers, and ice making machines (Anaissie 2001). Anaissie et al. (2002) found significantly higher concentrations of airborne Aspergillus propagules in bathrooms, where water use was highest (2.95 cfu/m³). In a comparison of different kinds of water sources in hospitals, Kauffmann-Lacroix et al. (2008) found that 52% of the cold water samples contained fungi while only 4% of the hot-water samples had positive cultures. In two hospitals there was generalized growth in the water pipes; one with Exophiala species and the other with Fusarium species. Otherwise, colonization was usually minimal. Nucci et al. (2002) traced the source of infections withExophiala jeanselmei to deionized water in the hospital pharmacy as well as a water tank and a sink. Genetic comparisons revealed that the cause of the outbreak of infection was the deionized water in the pharmacy. In another interesting study, filamentous fungi were found in more that 94% of all water samples taken in a hospital and all of the samples collected from the intake reservoir. Eighty five percent of the intake reservoir samples contained Aspergillus fumigatus suggesting that this was the source for the fungus in the hospital water. In a second study, this group found that Aspergillus fumigatus strains from air were different genetically from those in water, and that patients had been infected from both sources (Warris et al., 2003).

Matching Sources To Infection Outbreaks
Most of us have our own protocols for building investigations. Hospitals differ primarily in the risk associated with exposure, and the need to document very low concentrations of specific organisms in air. Anderson et al. (1996) describe the steps he took in one investigation where the source turned out to be a vacuum cleaner.

1. Identify all sources of air intake.
2. Map dispersal routes of air throughout the hospital.
3. Visit the site of the outbreak (i.e., specific rooms) on several days to evaluate activities that might be relevant.
4. Discuss recent building records with building services managers and engineers.
5. Consult external ventilation contractors regarding the overall function of the building ventilation.
6. Collect samples in triplicate including at the intakes, inside, and exhausts of each of the mechanical ventilation units and in each ward. Anderson's group sampled in 15 sites in the suspect ward including the ceiling void, soft toys (encouraged to release aerosol by firm handling), and the exhausts of the ward vacuum cleaners.

Sampling is most often done using culture (since the organisms must be alive to cause infection). Having found a possible source one can remove or remediate the source, then continue sampling to document that the problem has been solved. However, to be sure that the actual source for the ongoing infections has been identified, it is important to match environmental strains of the fungi to those recovered from the patients. This is done using DNA fingerprinting. Unfortunately, this is more complicated than it appears, at least forAspergillus fumigatus. It appears that this species is extremely diverse genetically (Symoens et al., 2002). Bart-Delabesse et al. (1999) analyzed 62 environmental isolates to reveal 43 genotypes represented only once. Likewise, isolates from patients were diverse. Chazelet et al. (1998) fingerprinted more than 700 clinical and environmental isolates ofAspergillus fumigatus and found that 85% of the isolates recovered from air represented different strains. To qualify as nosocomial (hospital acquired) patients must be infected with an isolate found in the environment, or multiple patients at the same site must be infected with the same genotype (Bart-Delabesse et al., 1999; Chazelet et al., 1998). Chazelet et al. (1998) considers that the frequent lack of common strains among patients involved in an outbreak is due to the extreme genetic diversity of Aspergillus fumigatus. It is also true that the same strain can appear throughout a hospital, and can persist for many months (Girardin et al., 1994) and that the same patient can be infected with more than one strain (Menotti et al., 2005).

Aspergillus flavus is less common than A. fumigatus, and infections in patients appear to be more likely to match environmental isolates. Ao et al. (2007) matched two patient isolates to two environmental strains of Aspergillus flavus. Three other patients had strains that differed from those in the environment, but two of these patients had the same strain. Thus, four out of five of these patients were assumed to have nosocomially acquired infections. In another Aspergillus flavus case, Buffington et al. (1994) found environmental strains in a patient and a health care worker, but different strains in two other patients. Heinemann et al. (2004) investigated an outbreak of surgical site infections with Aspergillus flavus. He found a single clone of the organism throughout the surgical suite and in the patients. On the other hand, Leenders et al. (1996) found distinctly different strains ofAspergillus flavus in a group of patients arguing against a hospital source for the infections.

Outbreaks related to Aspergillus terreus have also been investigated. Lass-Flori et al. (2000) was able to match Aspergillus terreus strains from potted plant soil to infections in four patients.

Monitoring
Some people strongly recommend monitoring of the hospital environment in order to detect the beginning of an episode of contamination. The problem is, how often and how extensive should monitoring protocols be? Alberti et al. (2001) recommend monitoring for changes in the entire fungal population (not just Aspergillus species) as this indicates the potential for conditions that could lead to growth of opportunists. Falvey & Streifel (2007) found spikes of Aspergillus associated with infection during a monitoring period and considered this data useful in the search for sources. Monitoring protocols should include notation of activities going on before or during the monitoring period. Some transient activities as well as conditions that become chronic clearly affect aerosolization of fungi and the presence of these conditions and activities help to focus on the source of spikes. On the other hand, monitoring may reveal spikes not related to any apparent activity and may indicate the need for more extensive investigations.

Prevention
Prevention of hospital contamination and patient infection is a multifaceted task. The outdoor air must be filtered, the systems supplying outdoor air to the hospital environment must be maintained so that they are water free, activities within the hospital that raise dust must be minimized or isolated, and patients may have to be protected directly through medications and/or masking. All of these precautions apply especially to immunocompromised patients.
HEPA filtration of the outdoor air is almost universally recommended (Araujo 2008; Benet et al., 2007; Brenier-Pinchart et al., 2009; Falvey & Streifel 2007). Comparing outdoor air, HEPA-filtered air and other hospital locations, Falvey & Streifel (2007) found Aspergillusspecies in 95% of outdoor air samples, 33% of HEPA-filtered locations, and 50% of other hospital areas.
Laminar air flow is also sometimes used to protect immunocompromised patients, but it involves high air exchange rates, is expensive, and causes noise and drafts (Humpfreys 2004).
Air purifiers and mobile units have been marketed to protect immunosuppressed patients either routinely or under especially hazardous conditions. Poirot et al. (2007) tested a mobile unit that provided an environment with no detectable airborne fungi regardless of levels outside of the unit's influence. Bergeron et al. (2007) tested a mobile nonthermal plasma air treatment unit that significantly reduced airborne spore concentrations. However, portable air filtration units were not capable of significantly reducing air concentrations in rooms (Englehart et al., 2003). Of course the amount of air processed per unit time and the amount of disturbance of dust during unit operation would contribute to these results. On the other hand, compliance was poor due to noise and thermal discomfort.
Protection from activities within the hospital that may produce fungal aerosols is extremely important. Many hospitals have installed anterooms next to rooms housing at-risk patients in part so that gowning and hand washing activities can take place outside of the patient's environment (Araujo 2008; Brenier-Pinchart et al., 2009). Activity with rooms without anterooms and other restrictions on aerosol production led to a direct correlation between activity and fungal spore levels. On the other hand, aerosols in rooms with control measures were related to outdoor concentrations, emphasizing the need for high quality filtration.
One activity in hospitals that is well known to produce fungal aerosols is construction. Many approaches have been used to prevent construction-related outbreaks of aspergillosis in hospitals. Most follow the same principles of containment that should be used for all occupied spaces. Given the greatly enhanced susceptibility of immunosuppressed patients, additional efforts may need to be made. Cornet et al. (1999) used laminar flow and HEPA filtration in rooms adjacent to the construction and no Aspergillus was found in any subsequent air sample in these protected spaces. In addition to HEPA filtration, Loo et al. (1996) used biocide containing paint and non-perforated ceiling tiles, sealed all windows, replaced horizontal blinds with roller shades, and called for systematic and regular cleaning of surfaces. With these efforts he was able to reduce the incidence of aspergillosis to levels below pre-construction levels, and far below those that had pertained during the initial phase of construction. Use of water to reduce airborne dust concentrations in construction areas has been used, but the risk of mold growth resulting from damp conditions must be considered (Berthelot et al., 2006). Daily particle count measurements in high risk areas can encourage compliance with infection control measures during construction (Prezant et al., 2005).
Finally, one can act directly at the patient level to prevent infection. Removing patients from high risk areas and the use of well fitted masks are two approaches that are commonly used (Maschmeyer et al., 2009; Chang et al., 2008; Berthelot et al., 2006; Raad et al., 2002). In addition, some physicians use prophylactic anti-fungal agents in their patients to reduce the risk of fungal infections (Chang et al., 2008).

Guidelines
There are currently no specific guidelines for acceptable concentrations of any individual opportunistic fungus in hospitals. It appears that any guideline would have to be in a range less than 1 cfu/m³ to be effective for immunocompromised patients. Alberti et al. (2001) reviewed the literature on "safe" levels of Aspergillus. He found opinions on decreased levels that ranged from 0.009 - <0.2 cfu/m³ (total Aspergillus spores). On the other hand, risk appears to start increasing near 1 cfu/m³.

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