Microbiologic Sampling of the Environment in Healthcare Facilities
Lynne M. Sehulster
Laura J. Rose
Judith Noble-Wang
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC). Any use or mention of trade names in this chapter is for identification purposes only and does not represent any endorsement by either the CDC or the U.S. Public Health Service.
In the world of medicine, three developments occurring in the second half of the 20th century have served to increase the complexity of infectious diseases epidemiology— opportunistic pathogens, sophisticated lifesaving medical therapies (e.g., solid organ transplants, bone marrow transplants), and the rapidly increasing diversity and magnitude of antibiotic resistance among bacteria. The experiences gained in dealing with each of these have heightened our awareness of man’s interaction with the environment, and the indoor environment in particular. Exposures to environmental pathogens can result in life-threatening infections among the most severely immunosuppressed patients. The identification of antibiotic-resistant bacteria in healthcare environments has drawn scrutiny to care-giving procedures as healthcare personnel move among patients from one area to another; the resistance pattern in one sense becomes a marker to help with the epidemiologic investigation to identify the source(s) of transmission. In the end, there is a renewed interest to understand how the indoor environment influences and/or facilitates transmission of infection. This necessitates the need to sample the environment in a way that is both practical and meaningful. Microbiologic sampling is the approach that most healthcare professionals often choose first when an epidemiologic investigation indicates some evaluation of the environment is needed, but this is not the only method, and it may not be the most appropriate method depending on the circumstances. Furthermore, environmental sampling methods are distinctly different from clinical microbiology methods, and clinical microbiology laboratories are often poorly equipped to carry out environmental sample analyses. This chapter addresses the basic principles and microbiologic methods of sampling indoor environmental surfaces and other environmental sources for microorganisms (1,2). Detailed methods for microbiologic sampling of the environment are included in this chapter since such sampling is frequently used during infectious disease outbreaks (2). Microbiologic sampling in response to a bioterrorism event is beyond the scope of this chapter; the reader is referred to other sources for more specific information about the unique sampling concerns for this endeavor (2).
GENERAL PRINCIPLES: MICROBIOLOGIC SAMPLING OF THE ENVIRONMENT
Before 1970, US hospitals conducted regularly scheduled culturing of the air and environmental surfaces (e.g., floors, walls, and table tops) (3). By 1970, the Centers for Disease Control and Prevention (CDC) and the American Hospital Association (AHA) were advocating the discontinuation of routine environmental culturing because rates of healthcare-associated infection had not been associated with levels of general microbial contamination of air or environmental surfaces, and because meaningful standards for permissible levels of microbial contamination of environmental surfaces or air did not exist (4, 5 and 6). During 1970 to 1975, 25% of US hospitals reduced the extent of such routine environmental culturing—a trend that has continued (7,8).
Random, undirected sampling (referred to as “routine” in previous guidelines) differs from the current practice of targeted sampling for defined purposes (5,9). Previous recommendations against routine sampling were not intended to discourage the use of sampling in which sample collection, culture, and interpretation are conducted in accordance with defined protocols (9). In this chapter, targeted microbiologic sampling connotes a monitoring process that includes (a) a written, defined, multidisciplinary protocol for sample collection and culturing; (b) analysis and interpretation of results using scientifically determined or anticipatory baseline values for comparison; and (c) expected actions based on the results obtained. Infection control, in conjunction with laboratorians, should assess the healthcare facility’s capability to conduct sampling and determine when expert consultation and/or services are needed.
Microbiologic sampling of air, water, and inanimate surfaces (i.e., environmental sampling) is an expensive and time-consuming process that is complicated by many variables in protocol, analysis, and interpretation. It is therefore indicated for only four situations (10). The first is to support an investigation of an outbreak of disease
or infections when environmental reservoirs or fomites are implicated epidemiologically in disease transmission (11, 12 and 13). It is important that such culturing be supported by epidemiologic data. Environmental sampling, as with all laboratory testing, should not be conducted if there is no plan for interpreting and acting on the results obtained (14, 15 and 16). Linking microorganisms from environmental samples with clinical isolates by molecular epidemiology is crucial whenever it is possible to do so.
or infections when environmental reservoirs or fomites are implicated epidemiologically in disease transmission (11, 12 and 13). It is important that such culturing be supported by epidemiologic data. Environmental sampling, as with all laboratory testing, should not be conducted if there is no plan for interpreting and acting on the results obtained (14, 15 and 16). Linking microorganisms from environmental samples with clinical isolates by molecular epidemiology is crucial whenever it is possible to do so.
The second situation for which environmental sampling may be warranted is in research. Well-designed and controlled experimental methods and approaches can provide new information about the spread of healthcareassociated diseases (17,18). A classic example is the study of environmental microbial contamination that compared healthcare-associated infection rates in an old hospital and a new facility before and shortly after occupancy (19).
The third indication for sampling is to monitor a potentially hazardous environmental condition, confirm the presence of a hazardous chemical or biological agent, and validate the successful abatement of the hazard. This type of sampling can be used to (a) detect bioaerosols released from the operation of healthcare equipment (e.g., an ultrasonic cleaner) and determine the success of repairs in containing the hazard (20); (b) detect the release of an agent of bioterrorism in an indoor environmental setting and determine its successful removal or inactivation; and (c) sample for industrial hygiene or safety purposes (e.g., monitoring a “sick building”).
The fourth indication is for quality assurance to evaluate the effects of a change in infection-control practice or to ensure that equipment or systems perform according to specifications and expected outcomes. Currently, much of the environmental assessment of practice in healthcare settings involves nonmicrobiologic methods such as covert visual inspection, use of ultraviolet (UV) fluorescent chemical markers, and adenosine triphosphate measurements of bioburden using relative light units recorded with a luminometer device (21,22). Nevertheless, any sampling for quality-assurance (QA) purposes (microbiologic sampling or any nonculture method) must follow sound sampling protocols. Microbiologic sampling in particular must address confounding factors through the use of properly selected controls. Results from a single environmental sample are difficult to interpret in the absence of a frame of reference or perspective. Evaluations of a change in infection-control practice are based on the assumption that the effect will be measured over a finite period, usually of short duration. Conducting QA microbiologic sampling on an extended basis, especially in the absence of an adverse outcome, is usually unjustified. A possible exception might be the use of air sampling during major construction periods to qualitatively detect breaks in environmental infection-control measures. In one study, which began as part of an investigation of an outbreak of healthcare-associated aspergillosis, airborne concentrations of Aspergillus spores were measured in efforts to evaluate the effectiveness of sealing hospital doors and windows during a period of construction of a nearby building (23). However, the only types of routine environmental microbiologic sampling recommended as part of a QA program are (a) the biological monitoring of sterilization processes by using bacterial spores (24) and (b) the monthly culturing of water used in hemodialysis applications and for the final dialysate use dilution (see Chapter 63 for more information on sampling in dialysis settings).
Microbiologic sampling of the environment involves selecting a representative sample of that environment and collecting microbial contaminants with appropriate sampling devices. The interpretation of results should be based on the understanding of the recovery efficiencies of the materials and the limitations of the processing method (2).
Air Sampling
Biological contaminants occur in the air as aerosols and may include bacteria, fungi, viruses, and pollens (25,26). Aerosols are characterized as solid or liquid particles suspended in air. Talking for 5 minutes and coughing each can produce 3,000 droplet nuclei; sneezing can generate approximately 40,000 droplets that then evaporate to particles in the size range of 0.5 to 12 µm (27,28). Particles in a biological aerosol usually vary in size from <1 to >50 µm. These particles may consist of a single, unattached microorganism or may occur in the form of clumps composed of a number of bacteria. Clumps can also include dust and dried organic or inorganic material. Vegetative forms of bacterial cells and viruses may be present in the air in a lesser number than bacterial spores or fungal spores. Factors that determine the survival of microorganisms within a bioaerosol include (a) the suspending medium; (b) temperature; (c) relative humidity; (d) oxygen sensitivity; and (e) exposure to UV or electromagnetic radiation (25). Many vegetative cells will not survive for lengthy periods of time in the air unless the relative humidity and other factors are favorable for survival and the microorganism is enclosed within some protective cover (e.g., dried organic or inorganic matter) (26). Pathogens that resist drying (e.g., Staphylococcus spp., Streptococcus spp., and fungal spores) can survive for long periods and can be carried considerable distances via air and still remain viable. They may also settle on surfaces and become airborne again as secondary aerosols during certain activities (e.g., sweeping and bed making) (26,29).
Microbiologic air sampling is used to determine the numbers and types of microorganisms, or particulates, in indoor air (30). Air sampling for quality control is, however, problematic because of lack of uniform air-quality standards. Although airborne spores of Aspergillus spp. can pose a risk for neutropenic patients, the critical number (i.e., action level) of these spores above which outbreaks of aspergillosis would be expected to occur has not been defined. Healthcare professionals considering the use of air sampling should keep in mind that the results represent indoor air quality at singular points in time, and these may be affected by a variety of factors including (a) indoor traffic; (b) visitors entering the facility; (c) temperature; (d) time of day or year; (e) relative humidity; (f) relative concentration of particles or microorganisms; and (g) the performance of the air-handling system components. To be meaningful, air-sampling results must be compared with those obtained from other defined areas, conditions, or time periods and outside air samples.
Several preliminary concerns must be addressed when designing a microbiologic air-sampling strategy (Box 72-1).
Because the amount of particulate material and bacteria retained in the respiratory system is largely dependent on the size of the inhaled particles, particle size should be determined when studying airborne microorganisms and their relation to respiratory infections. Particles >5 µm are efficiently trapped in the upper respiratory tract and are removed primarily by ciliary action (31). Particles <5 µm in diameter reach the lung, but the greatest retention in the alveoli is of particles 1 to 2 µm in diameter (32, 33 and 34).
Because the amount of particulate material and bacteria retained in the respiratory system is largely dependent on the size of the inhaled particles, particle size should be determined when studying airborne microorganisms and their relation to respiratory infections. Particles >5 µm are efficiently trapped in the upper respiratory tract and are removed primarily by ciliary action (31). Particles <5 µm in diameter reach the lung, but the greatest retention in the alveoli is of particles 1 to 2 µm in diameter (32, 33 and 34).
BOX 72-1 Preliminary Concerns for Conducting Air Sampling
Consider the possible characteristics and conditions of the aerosol, including size range of particles, relative amount of inert material, concentration of microorganisms, and environmental factors.
Determine the type of sampling instruments, sampling time, and duration of the sampling program.
Determine the number of samples to be taken.
Ensure that adequate equipment and supplies are available.
Determine the method of assay that will ensure optimal recovery of microorganisms.
Select a laboratory that will provide proper microbiologic support.
Ensure that samples can be refrigerated if they cannot be assayed in the laboratory promptly.
Bacteria, fungi, and particulates in air can be identified and quantified with the same methods and equipment (Table 72-1). The basic methods include (a) impingement in liquids; (b) impaction on solid surfaces; (c) sedimentation; (d) filtration; (e) centrifugation; (f) electrostatic precipitation; and (g) thermal precipitation (29). Of these, impingement in liquids, impaction on solid surfaces, and sedimentation (on settle plates) have been used for various air-sampling purposes in healthcare settings (30).
Several instruments are available for sampling airborne bacteria and fungi (Box 72-2). Some of the samplers are self-contained units requiring only a power supply and the appropriate collecting medium, but most require additional auxiliary equipment (e.g., a vacuum pump and an airflow measuring device [i.e., a flow meter or anemometer]). Sedimentation or depositional methods use settle plates (Petri plates with agar media) and therefore need no special instruments or equipment. Selection of an instrument for air sampling requires a clear understanding of the type of information desired and the particular determinations that must be made (Box 72-2). Information may be needed regarding: (a) one particular microorganism or all microorganisms that may be present in the air; (b) the concentration of viable particles or of viable microorganisms; (c) the change in concentration with time; and (d) the size distribution of the collected particles. Before sampling begins, decisions should be made regarding whether the results are to be qualitative or quantitative. Comparing quantities of airborne microorganisms to those of outdoor air is also standard operating procedure. Infection preventionists, healthcare epidemiologists, industrial hygienists, and laboratory supervisors, as part of a multidisciplinary team, should discuss the potential need for microbial air sampling to determine if the capacity and expertise to conduct such sampling exist within the facility and when it is appropriate to enlist the services of an environmental microbiologist consultant.
Liquid impinger and solid impactor samplers are the most practical for sampling bacteria, particles, and fungal spores, because they can sample large volumes of air in relatively short periods of time (30). Solid impactor units are available as either “slit” or “sieve” designs. Slit impactors use a rotating disc as support for the collecting surface, which allows determinations of concentration over time. Sieve impactors commonly use stages with calibrated holes of different diameters. Some impactor-type samplers use centrifugal force to impact particles onto agar surfaces. The interior of either device must be made sterile to avoid inadvertent contamination from the sampler. Results obtained from either sampling device can be expressed as microorganisms or particles per unit volume of air (CFU/m3).
Sampling for bacteria requires special attention, because bacteria may be present as individual microorganisms, as clumps, or mixed with or adhering to dust or covered with a protective coating of dried organic or inorganic substances. Reports of bacterial concentrations determined by air sampling therefore must indicate whether the results represent individual microorganisms or particles bearing multiple cells. Certain types of samplers (e.g., liquid impingers) will completely or partially disintegrate clumps and large particles; the sampling result will therefore reflect the total number of individual microorganisms present in the air.
The task of sizing a bioaerosol is simplified through the use of sieves or slit impactors, because these samplers will separate the particles and microorganisms into size ranges as the sample is collected. These samplers must, however, be calibrated first by sampling aerosols under similar use conditions (37).
The use of settle plates (i.e., the sedimentation or depositional method) is not recommended when sampling air for fungal spores, because single spores can remain suspended in air indefinitely (30). Settle plates have been used mainly to sample for particulates and bacteria either in research studies or during epidemiologic investigations (11,38, 39, 40 and 41). Results of sedimentation sampling are typically expressed as numbers of viable particles or viable bacteria per unit area per the duration of sampling time (i.e., CFU/area/time); this method cannot quantify the volume of air sampled. Because the survival of microorganisms during air sampling is inversely proportional to the velocity at which the air is taken into the sampler (25), one advantage of using a settle plate is its reliance on gravity to bring microorganisms and particles into contact with its surface, thus enhancing the potential for optimal survival of collected microorganisms. This process, however, takes several hours to complete and may be impractical for some situations.
Air samplers are designed to meet differing measurement requirements. Some samplers are better suited for one form of measurement than others. No one type of sampler and assay procedure can be used to collect and enumerate 100% of airborne microorganisms. The sampler
and/or sampling method chosen should, however, have an adequate sampling rate to collect a sufficient number of particles in a reasonable time period so that a representative sample of air is obtained for biological analysis. Newer analytical techniques for assaying air samples include polymerase chain reaction (PCR) methods and enzyme-linked immunosorbent assays.
and/or sampling method chosen should, however, have an adequate sampling rate to collect a sufficient number of particles in a reasonable time period so that a representative sample of air is obtained for biological analysis. Newer analytical techniques for assaying air samples include polymerase chain reaction (PCR) methods and enzyme-linked immunosorbent assays.
TABLE 72-1 Air-Sampling Methods and Examples of Equipment | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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BOX 72-2 Selecting an Air-Sampling Device
The following factors must be considered when choosing an air-sampling instrument:
Viability and type of the microorganism to be sampled
Compatibility with the selected method of analysis
Sensitivity of particles to sampling
Assumed concentrations and particle size
Whether airborne clumps must be broken (i.e., total viable microorganism count vs. particle count)
Volume of air to be sampled and length of time sampler is to be continuously operated
Background contamination
Ambient conditions
Sampler collection efficiency
Effort and skill required to operate sampler
Availability and cost of sampler, plus backup samplers in case of equipment malfunction
Availability of auxiliary equipment and utilities (e.g., vacuum pumps, electricity, and water)
(Data from Wolf HW, Skaliy P, Hall LB, et al. Sampling microbiological aerosols. Public Health Service publication No. 686. Washington, DC: Government Printing Office, 1964.)
Water Sampling
A detailed discussion of the principles and practices of water sampling has been published (42). Water sampling in healthcare settings is used to detect waterborne pathogens of clinical significance or to determine the quality of finished water in a facility’s distribution system. Routine testing of the water in a healthcare facility is usually not indicated, but sampling in support of outbreak investigations can help determine appropriate infection-control measures. Water-quality assessment in dialysis settings is another instance where routine microbiologic sampling of water is important and where standards have been established (see hemodialysis, Chapter 63).
Healthcare facilities that conduct water sampling should have their samples assayed in a laboratory that uses established methods and QA protocols. Water specimens are not “static specimens” at ambient temperature; potential changes in both numbers and types of microbial populations can occur during transport. Consequently, water samples should be sent to the testing laboratory cold (i.e., at ˜39.2°F [4°C]) and testing should be done as soon as practical after collection (preferably within 24 hours).
Because most water sampling in healthcare facilities involves the testing of finished water from the facility’s distribution system, a reducing agent (i.e., sodium thiosulfate [Na2S2O3]) needs to be added to neutralize residual chlorine or other halogen in the collected sample. If the water contains elevated levels of heavy metals, then a chelating agent should be added to the specimen. The minimum volume of water to be collected should be sufficient to complete any and all assays indicated; 100 mL is considered a suitable minimum volume. Sterile collection equipment should always be used.
Sampling of water from the distribution sytem from a tap requires flushing of the water line before sample collection. If the tap is a mixing faucet, attachments (e.g., screens and aerators) must be removed, and hot and then cold water must be run through the tap before collecting the sample (42). If the cleanliness of the tap is questionable, disinfection with 500 to 600 parts per million (ppm) sodium hypochlorite (1:100 v/v dilution of chlorine bleach) and flushing the tap should precede sample collection. If biofilm associated organisms are sought, samples are collected from inside the faucet head, screens and aerators with a non-cotton swab prior to flushing of the tap.
Microorganisms in finished or treated water often are physically damaged (“stressed”) to the point that growth is limited when assayed under standard conditions. Such situations lead to false-negative readings and misleading assessments of water quality. Appropriate neutralization of halogens and chelation of heavy metals are crucial to the recovery of these microorganisms. The choice of recovery media and incubation conditions will also affect the assay. Incubation temperatures should be closer to the ambient temperature of the water rather than at 98.6°F (37°C), optimum growth temperature of the specific microorganism sought, and recovery media should be formulated to provide appropriate concentrations of nutrients to support microorganisms exhibiting less than rigorous growth (42). High-nutrient content media (e.g., blood agar and tryptic soy agar [TSA]) may actually inhibit the growth of these damaged microorganisms. Reduced nutrient media (e.g., diluted peptone and R2A) are preferable for recovery of these microorganisms (42).
Use of aerobic, heterotrophic plate counts allows both a qualitative and quantitative measurement for water quality. If bacterial counts in water are expected to be high in number (e.g., during waterborne outbreak investigations), assaying small quantities using pour plates or spread plates is appropriate (42). Membrane filtration is used when low-count specimens are expected and larger sampling volumes are required (>100 mL). The sample is filtered through the 0.45 µm or 0.22 µm membrane, and the filter is applied directly face-up onto the surface of the agar plate and incubated.
Unlike the testing of potable water supplies for coliforms (which uses standardized test and specimen collection parameters and conditions), water sampling to support epidemiologic investigations of disease outbreaks may be subjected to modifications dictated by the circumstances present in the facility. Assay methods for waterborne pathogens may also not be standardized. Therefore, control or comparison samples should be included in the experimental design. Any departure from a standard method should be fully documented and should be considered when interpreting results and developing strategies. Assay methods specific for clinically significant waterborne pathogens (e.g., Legionella spp., Aeromonas spp., Pseudomonas spp., and Acinetobacter spp.) are more complicated and costly
compared with both methods used to detect coliforms and other standard indicators of water quality.
compared with both methods used to detect coliforms and other standard indicators of water quality.
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