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Air sampling for Aspergillus
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Home Publications - Fungal infection - Fungi:toxic killers?
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Sampling of Aspergillus
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REVIEW Air sampling for Aspergillus spores Summary: Nosocomially-acquired aspergillosis typically occurs in the setting of treatment for leukaemia or other haematological malignancy.As Aspergillus species can be readily found in the environment, it has been widely believed that aspergillosis occurs as a consequnce of exogenous acquition of the fungus. Stringent environmental controls in transplant units have included high-efficiency air filtration, positive-pressure ventilation and frequent room air changes. Although there have been several well-documented examples of aspergillosis outbreaks as a result of hospital demolition and reconstruction, it has not always been possible to demonstrate elevated spore counts in clinical areas during building work. The sampling of air for Aspergillus is very problematic. Careful attention must be given to the choice of air sampler and sampling protocols, and the interpretation of air sampling data. This review outlines many of the physical and environmental parameters that influence meaningful air sampling and recommends a simple procedure that has been tried and tested in many aspergillosis outbreaks. Introduction Fungal infections in immunocompromised patients not only are difficult to treat, they are also common. Despite this they are often unsuspected at the time of death.1 Recent autopsy studies confirm this. For example, among bone marrow transplant patients, invasive yeast infection or pulmonary aspergillosis were present in about one half of the early deaths. Many haematologists who have treated such patients are acutely aware of the extremely high mortality of progressive fungal disease. An analysis of an individual patient’s risk factors for fungal infection, and the type of fungus to which they are most susceptible, indicates the preventative strategies that are likely to be successful. These include the prevention of endogenous fungal infections, such as candidosis, by means of prophylaxis and prevention of exogenous infections (mostly airborne and caused by filamentous fungi) through environmental protection and chemoprophylaxis.1, 2 Many studies have attempted to evaluate the importance of fungal aerobiology in relation to the acquition of invasive fungal infection by the compromised patient.3-8 The objectives of air sampling are stated in Table 1. Most authors recommend Aspergillus air counts of less than 5 colony forming units (CFU)/m3 in protective isolation suites, and counts of less than 0.1 CFU/m3 are desirable. However, there are few standards for performing and evaluating air counts in hospitals.9 The purpose of this review and protocol is to provide background material to the various parameters that can influence air sampling and to recommend a procedure that has been found useful in monitoring the level of airborne fungi in private dwellings and hospital enviroments. Sampling of biological aerosols: some general considerations The considerations relevant to sampling Aspergillus spp. spores in air are essentially the same considerations which apply to sampling any aerosol. Aerosols are made up of particles suspended in air with an upper size limit of approximately 25 mm. Because they may be derived from many sources, aerosol particles exhibit considerable compositional variation. They may, for example, be solid or liquid, comprise organic and/or inorganic material or contain living organisms. Inevitably, some aerosol particles are potentially toxic, allergenic or infective to man. However, all aerosols, whatever their composition, are governed by the same physical rules which determine their aerodynamic behaviour.10 Thus, many aspects of the behaviour of microbial particles in air might be predicted from a knowledge of their physical attributes but biological features can also be important as they influence the take off, aerial transport and landing in addition to survival and infectivity. Accordingly, successful isolation and subsequent identification of any microbial entity in air requires a clear understanding of the physical and biological properties of the species under investigation. The term aerobiology has been coined to describe the science of the aerial transport of microorganisms and other microscroscopic biological materials in air, their deposition and the ensuing consequences for life forms including the microscopic entities themselves.10 As direct microscopical examination of spores suspended in air is wholly impractical, detection and enumeration of spores in air requires that they be removed from the air to a surface where they can be examined under a microscope directly or after growth in culture.11 Sampler design and the human respiratory tract A number of factors are important when choosing the best sampling technique for any particular purpose but it is useful to remember that sampler design has traditionally been influenced by a desire to mirror the particle retention and deposition characteristics of the respiratory tract. Gregory (1973) described the respiratory tract as “a complicated instrument in which particles of different sizes are roughly sorted by a variety of deposition mechanisms in succession”.11 Deposition then is determined by the anatomy of the respiratory tract and by particle size, or more accurately aerodynamic diameter which is explained below. In all volumetric samplers, there is a pump which draws air at a constant rate yet, in contrast, air flow within the lungs is neither continuous nor uniform. Residual air (the volume that is not normally expelled from the lungs) oscillates between the alveoli and the bronchioles and bronchi where it mixes with the tidal air (the air inhaled in a single breath). For practical purposes it is useful to regard the respiratory tract as having three distinct regions.10 The first region comprises the nasopharynx and/or the mouth. Passage through this region ensures nearly 100% deposition of particles in the > 20 mm range although efficiency of capture falls off rapidly with decreasing particle size. Only the largest fungal spores will be deposited here.11 Mouth breathers, whether by habit, or due e.g. to rhinitis, will effectively by-pass the nose filter. The implication here is that in mouth breathers, larger particles will deposit lower down in the larynx, trachea and bronchi. The second principal region of the respiratory tract is the conducting passages of the larynx, trachea and bronchi. The larynx offers air resistance and thus permits some particle deposition. The air and the particles it entrains then pass through the trachea which at its lower end divides into two major bronchi. Some particle deposition occurs at this fork and at subsequent junctions as the bronchi repeatedly fork until the air enters the lobes of the lungs. For the nose breather, comparatively little particle deposition occurs in the trachea and bronchioles but where this does occur the particles involved are likely to range in size between 2mm and 20mm which at the lower end will include some of the smaller indoor fungal spores including Aspergillus spp. Gregory (1973) describes the reducing air velocity as air is drawn deeper into the lungs from a velocity of approximately 100 cm/s in the nose and larger bronchi to the order of tens of cm/s as the air passes into hundreds of secondary and tertiary bronchi.11 As the diameter of the passages in the bronchioles decreases to < 1 mm, the air speed further reduces to approximately 1 cm/s. The third region of the respiratory tract is that comprising the bronchioles which with successive branching are known as the “terminal respiratory bronchioles”. These in turn feed into the alveolar ducts and alveolar sacs, the latter being the oxygen exchange part of the lungs. This third region is commonly termed the “lower respiratory tract”, the first two regions being known as the “upper respiratory tract”. Particles of approximately 1-4mm are deposited in this third region mainly by sedimentation during the period of quiescence between breaths. The period is too short to permit sedimentation of the very smallest <1mm particles. The mouth breather is likely to experience greater particle deposition in the alveolar region but for both groups the optimal particle size for alveolar deposition is 2 mm.11 Hence the majority of the smallest particles which include Aspergillus spores reach and are optimally retained in the alveolar region. The boundaries for particle deposition reflected above are not as absolute as might be supposed which contrasts with the many air sampling instruments which endeavour to mirror respiratory deposition regimes. Volumetric samplers can produce very specific divisions in particle deposition because they have a constant flow rate and operate continuously throughout the sampling period. As stated above, the flow of air within the lungs however is neither continuous nor at a constant rate. Accordingly, at any point in the respiratory tract, there are changes in the speed and direction of air flow. Inevitably this makes the boundaries between areas of deposition less clear than in a sampler. An important function of the upper respiratory regions is to modify temperature and humidity of incoming air. Inhaled particles, including spores, are frequently hygroscopic and, on encountering the high humidity atmosphere of the respiratory tract, they will expand altering their site of deposition.11 Characteristics of the target species In some circumstances, the parameter of interest may be the total airspora whilst in others the aim may be to study a single species or a group of species, perhaps with certain shared characteristics. Knowledge of the physical and biological characteristics of the target organism or organisms will influence the choice of instrument. Biological characteristics including capacity for survival during aerial transport are important as are germination and growth requirements. In particular, biological characteristics influence the choice of culture medium used in any instruments which operate by trapping particles on an agar surface for subsequent culturing. Air sampler design imparts certain aerodynamic characteristics and thus sampling efficiency is never uniform for any instrument across the range of particle sizes. Hence a knowledge of particle size, or more specifically, aerodynamic diameter, is necessary to determine whether the instrument will efficiently capture the target species. Whilst the physical size of a particle is obviously an important factor in determining its aerodynamic behaviour, other characteristics such as particle shape, density and surface irregularities are also important. Prior to the development of the concept of aerodynamic diameter, linear size alone, as measured under an optical microscope, was used as the measure of particle size.12 Because particles come in many shapes, e.g spheres, cubes, flakes or fibres, it has proved useful to create a unit for particle size as deposited in the respiratory tract which takes cognisance of all the influences on deposition, and particularly on settlement and inertial impaction. This is known as the aerodynamic diameter (dae). This concept is derived from the way the particle behaves when airborne and considers gravitational and inertial forces acting on the particle proportional to its mass. It is defined as “the diameter of a sphere of density (ro = 1g/cm3) which settles through air with a velocity equal to that of the actual particle under consideration”.12 Appropriately then, dae reflects behaviour rather than linear diameter and is useful in understanding the behaviour of the non spherical irregular particles which exist in the practical circumstance. The aerodynamic diameter is less useful when particles are very small (<0.5mm) because diffusion becomes an important deposition mechanism and this is solely influenced by particle size as opposed to shape or density.13 Aerodynamic diameter also loses its utility in the larger size range (>15 mm) because of the very short residence time of larger particles in air. Nonetheless, because they exhibit significant variation in shape, dae is a an important measure of the capacity of fungal spores to lodge in the respiratory tract or be trapped in an air sampler.11 One complicating factor derives from the fact that particles absorb and lose water causing their size to change with changing atmospheric humidity.14, 15 It is more appropriate then, to think of aerodynamic diameter for a given species as falling within a range. In practice, aerodynamic diameters have been established by calculation from settling velocities. Lacey and Dutkiewity (1976), using this method, calculated the dae of Aspergillus fumigatus as 3.1 µm and of mixed Penicillium species as 3.2 µm.16 Pasanen et al (1991) reported aerodynamic diameters for viable fungal particles at different humidities, finding for example, a range of aerodynamic diameters for Penicillium spp of 2.2-3.9 µm depending on humidity.14 Madelin and Johnson (1992) conducted particle sizing of a number of common indoor fungal isolates in a relative humidity range of 40-98% using an aerodynamic particle sizer.15 These, they recorded as 1.9-2.2 µm for Aspergillus fumigatus, 2.6-3.0 µm for Penicillium chrysogenum and 2.3-2.5 µm for Cladosporium cladosporoides. Samson and van Reenen Hoekstra (1988) reported corresponding physical dimensions for these species of 2.5-3.0µm diameter (A.fumigatus), 3.0-4.0µm by 2.8-3.8µm (P.chrysogenum) and 3.0-7.0µm x 2.0-4.0µm (C.cladosporoides).17 A very important observation in relation to respiratory deposition was made by Lacey (1991) who noted that aggregated fungal particles occurring in chains behaved similarly, in aerodynamic terms, to single fungal particles.18 Sampling period In most circumstances air sampling is conducted to characterise the airborne microbial environment or some aspect of it. Often this is done in order to estimate human exposure as part of an epidemiological investigation. The requirement to present an integrated assessment of exposure implies that the sampling period should be long, perhaps hours or days. Yet sampling over long periods masks short term temporal or spatial variation in the airspora which may be important in the situation being investigated. In such cases a time discrimination of minutes or even seconds may be required. Many instruments, and particularly those which rely on culturing of viable organisms to permit enumeration and identification, are likely to become overloaded in contaminated environments and may only sample over short periods to provide a snapshot of conditions. Sample volume Quantification of airborne organisms requires that the volume of air from which the organisms were collected is known. This permits the numbers present to be expressed as a concentration per unit volume of air, perhaps as colony forming units per cubic metre of air (CFUs/m3) where viability is of interest, as it is e.g. to medical microbiologists or plant breeders. Where both viable and non viable organisms are of interest e.g. to those investigating allergic phenomena, the parameter of concern may be the total number of spores per cubic metre measurement. This requires a different type of instrument and different laboratory techniques which do not rely on spore viability. Some instruments incorporate a pump rated to deliver a particular flow rate consistent with the aerodynamic characteristics of the instrument. In others cases, a separate pump must be used and this must be calibrated. The volume of air sampled is altered by reducing or extending the sample period. Choice of sampling volume must be a compromise between the desire to obtain a sample sufficiently large to be representative and other practical considerations, most notably in the case of instruments providing viable spore counts, the requirement not to exceed the capacity of the instrument. Skill in selecting the appropriate sampling period will develop with increasing knowledge of the environment under investigation, but initially, and in the absence of any insight into expected airborne concentrations, appropriate sampling periods/volumes must be selected through trial and error. Practical and economic considerations With the exception of non-volumetric methods which rely on settlement under gravity to collect particles, most instruments require a power supply to operate the pump. Any instrument which requires a mains electricity supply is unlikely to be suitable in many outdoor situations. This has been addressed by some manufacturers who provide a rechargeable battery to power the equipment. Whilst this confers infinitely greater flexibility, true portability is also determined by the weight and dimensions of the instrument and its ancillaries. Quite apart from their requirement for a separate mains operated pump, large slit to agar samplers, for example, are considered by many as non portable due to their size and weight. Such considerations may be very important for extensive sampling programmes conducted at various sites such as might be the case in housing surveys. Capital and running costs are also important considerations as are robustness and reliability. Most important however is the observation that, subject to certain qualifications, operating a microbiological sampler is relatively straightforward. Enumeration, and particularly identification, of airborne organisms however, can be a skilled operation and, as Gregory (1973) has pointed out, it is uneconomic to use the time of a trained scientist with a good microscope to make determinations based on inefficient sampling methods.11 Sampling instruments for enumerating and identifying air spora Types of sampler It is convenient to consider microbiological samplers for collecting organisms in air as falling into several broad categories. The features of the different types of sampler are summarised in Table 2. Many popular microbiological air samplers use the principle of impaction to trap the organisms by impacting them directly onto agar or another surface. This group includes the Andersen Six-Stage Sampler (Andersen Instruments Incorporated, Atlanta, Georgia), the Casella Slit Sampler (Casella Ltd, Bedford, England) and the Surface Air Systems (SAS) Sampler (Cherwell Laboratories, Bicester, Oxon). A subset of impactors operate by using centrifugal acceleration to impact organisms onto agar as in the case of the Reuter Centrifugal Sampler (RCS) sampler (Biotest Folex, Birmingham, England) or onto liquid as for the Cyclone sampler (Aimer Ltd, London). A further distinct group are the impingers which operate by impinging organisms into liquid. This group includes the single-stage midget and micro impingers (SKC Ltd, Poole, Dorset), the multistage May liquid impinger (AW Dixon, Beckenham, Kent) and the Ace all glass impinger-30 Sampler (AGI-30) (Ace Glass, Vineland, New Jersey). Certain instruments use electrostatic precipitation to isolate particles from the air stream although the principle of electrostatic precipitation is more commonly seen in air cleaning applications. The large volume Litton-type sampler (Sci-Med, Inc, Eden Prairie, Madison) is an example of an air sampling device which relies on this principle but it is seldom used in indoor air sampling. Filtration methods also find application in microbiological air sampling. Such methods use either high or low volume pumps such as those produced by Casella Ltd (Kempston, Bedford, U.K.) to draw air through filters and in so doing, trap airborne organisms on the filters. Filters may be mounted in cassettes such as the Millipore cassette (Millipore, Watford, Herts, UK). Other filter options include cellulose-acetate and polycarbonate filters (Nuclepore, Sterilin, Houslow, Middlesex) or gelatin filters (Sartorius, Epsom, Surrey). The literature also contains many accounts of studies in which agar filled petri dishes were placed horizontally to isolate airborne particles through settlement prior to culturing.19 Impactors Samplers which operate by impacting particles from an airstream onto either an adhesive or agar surface are the most widely used in indoor air surveys. Because the air stream is drawn through by a pump, fan or aspirator whose flow can be regulated, these samplers are hence volumetric. Impaction onto agar surfaces is the more common method. Instruments are generally described as being single-stage or multi-stage. In single-stage instruments the air stream is directed towards the surface of a single agar filled plate. The air and the particles it carries accelerate on entering the instrument through a restricted inlet, a narrow slit in the case of a slit sampler such as the Casella Slit Sampler20 or a plate perforated with a number of uniform diameter holes as in the case of eg. the SAS sampler.21 The rapid change in the air direction as it approaches the collecting surface at right angles causes particles to be thrown from the stream to impact on the agar surface with an efficiency which depends on the velocity of the air and the size of the particles. In multi-stage or “cascade” impactors such as the Andersen Six-Stage Sampler the air is directed through a stack of perforated or sieve plates each with 400 uniform holes.22 As with a single stage impactor, particles are again deposited from the air stream onto an agar surface positioned below the perforated plate as the air stream rapidly changes direction. However, in a multi-stage impactor, the air stream (minus the particles deposited on the upper plate) proceeds to the next stage where it is drawn though a second plate, this time with smaller diameter holes, imparting a greater velocity to the air and causing smaller particles to be deposited from the airstream. The sequence is repeated for each plate in the stack. The total size range of the particles which can be collected over all the stages of an Andersen Six-Stage impactor is about 0.3 mm to 15 mm.19 This represents rather well the range of particle sizes which might present a hazard to the human respiratory tract. A capacity to separate particles according to size is important in the investigation of human health effects. In the case of the Andersen Six-Stage, sometimes termed the N6, there are six stages each capable of impacting particles in a different size range. Non-respirable particles are deposited on the top two plates and, the smaller respirable particles which would reach the alveoli are deposited on the lower plates.19 Most published data on the presence of fungal particles in non industrial environments has been obtained using Andersen samplers.23 The advantage of direct impaction onto an agar surface lies in the fact that the agar plates can be incubated without further treatment. This means that colonies grow directly from collected viable airborne particles. The statistical probability that a colony may be derived from more than a single colony forming unit passing through a hole is catered for by the application of probability tables supplied by the manufacturer. If, however, the environment is very heavily contaminated enumeration is impossible as the plate becomes overloaded. This problem can be overcome by drawing a smaller volume of air. Growth of colonies on an agar surface permits their identification using gross colony morphology and microscopic techniques. An enduring problem of impactors when sampling bacteria and the smaller fungal spores has been that the small size and resultant low inertia of the particles has demanded that that the air stream achieves a high velocity to permit impaction onto the capture surface. Pumps capable of doing this have tended to be bulky, noisy and require considerable power.24 Slit samplers and the Andersen Samplers have overcome this problem by separate mains operated pumps. Impactors introduced more recently have addressed this problem by using integral pumps operated from a rechargeable battery. An example in this latter category is the Surface Air Systems (SAS) sampler. Centrifugal acceleration The principle of centrifugal acceleration is used in certain types of instrument to remove particles from the sampled air. In the most commonly used instrument of this type, the Reuter Centrifugal (RCS) sampler, airborne particles are drawn in by an impeller and thereafter impacted onto an agar coated plastic strip lining the internal periphery of the impeller housing.24 Agar strips are subsequently removed for incubation. Whilst operating according to rather different aerodynamic principles, RCS samplers are impactors and share some of the advantages and disadvantages of other impactors described above. The results obtained using RCS instruments must however be regarded with caution. Macher and First (1983) found an instrument drawing 210 litres/min to be comparatively inefficient for particle sizes below 15 mm, a range of considerable importance in human respiratory deposition.25 Because the air stream enters and leaves the instrument through the same point, flow rate is difficult to evaluate making estimations of efficiency controversial. A second type of instrument relying on centrifugal acceleration collects the airborne particles in liquid. The sampler, known as the cyclone sampler, mixes the incoming air with a liquid supplied via a needle fed gravitationally or through a peristaltic pump.26 The mixture is drawn tangentially into an inverted cone. The air stream spirals down to the base of the sampler before being drawn up the centre of the instrument to the outlet.19 Particles are deposited on the internal wall of the sampler and wash to a collection point at the base of the instrument. The liquid containing micro-organisms can then be used as an inoculum. Several cyclones of differing design can be used to obtain particle size differentiation. Impingement into liquids These samplers work by drawing air through liquid causing the airborne particles to become suspended. As with impactors, impingers can be classified as single-stage and multi-stage. Single stage impingers differ from straightforward bubblers in that a small flask carries a wide inlet tube the inner end of which is fused to a piece of capillary tube which dips at least 5mm into the flask (in the original form) and terminates at least 4mm from the bottom of the flask. The capillary tube is a limiting orifice and thus controls the flow rate under suction from an attached pump.11 The multistage impinger (SKC Ltd, Poole, Dorset) confers advantages over the single stage device by having a gentler flow which is less damaging to particles and in its capacity to separate the retained particles into three particle size ranges corresponding to retention within (a) the upper respiratory tract, (b) the brochi and bronchioles and (3) the alveolar region of the human respiratory system. Sampled air passes through three liquid filled chambers at three different speeds (10 litres/min, 20 litres/min and 55litres/min). Particles collected in the first two chambers are impacted onto sintered glass discs washed by liquid. In the third stage particles are impinged tangentially into the liquid.19 Thus multi-stage impingers have the advantage of minimising damage to microbes and improving collection efficiency.11 Electrostatic precipitation
Movement of charged particle inelectrical fields has been used for dustextraction from airstreams. Berry (1941) is credited with recognising the value of applying this principle to microbiological sampling.27 Different designs for microbiological samplers have been described.11, 19 The Litton-type sampler draws 400 - 1000 litres of air/min. The charged particles are attracted to a rotating aluminium disc which carries the opposite charge. The disc is coated with a thin liquid film which moves centrifugally over the disk to a collection channel placed around the perimeter before being recirculated. The solution obtained can be used as an inoculum.
Filtration Passing air through a filter causes particles to be trapped on the filter medium. Micro-organisms collected can be resuspended in an aqueous solution and used as an inoculum. Polycarbonate membrane filters are particularly suitable due to the ease with which collected particles can be removed. The use of black polycarbonate filters and subsequent staining with acridine orange causes viable microbial cells to fluoresce orange allowing them to be counted directly under fluorescence microscopy.28 When collected on polycarbonate or cellulose acetate membrane filters, micro-organisms can be viewed directly under a microscope. Mounting and coating of polycarbonate filters allows examination under electron microscopy.19 Porous gelatin filters can be used to trap micro-organisms. The filters can then be dissolved and the trapped micro-organisms in solution used as an inoculum.29 Large volume filtration techniques can be used to trap airborne particles for biochemical or immunological assay.30
Gravity sedimentation methods Gravity sedimentation methods such as the open petri dish (OPD) method combine both gravitational and inertial processes. Burge and Soloman (1996) commented that because air is never still, settlement under gravity might play a relatively small part in particle collection.31 Indeed they went as far as to characterise a gravity slide or plate as “a continually changing inertial collector with a gravity component that varies inversely with wind speed and turbulance”. Gravity settlement methods, presumably for reasons of low cost, have been widely used but are defective in that they preferentially select larger particles. Results can also be misleading due to shadowing or turbulent deposition. Most importantly the method is not volumetric and provides qualitative rather than quantitative results. Verhoeff et al (1989) considered it to have some merit because its capacity to monitor over longer periods than most volumetric methods allow it to provide what they described as “an integrated assessment of exposure”.32 Contact plates Valuable information about the types of fungi in a particular environment can be obtained by sampling the accumulated dust on various surfaces such as tables, floors, horizontal blinds, fan blades and guards. Using, for example, the same contact plates of Czapek-Dox agar as described below for the SAS air sampler, a unit area of surface can be sampled, incubated and then the CFUs enumerated. Choosing a sampler for Aspergillus species Many factors require to be considered in choosing a sampling instrument for use in a particular situation. Some have been highlighted above and can now be applied in the context of Aspergillus spp spores. Inevitably other issues remain rather specific to the circumstances of the user and are less amenable to quantification. Amongst these are the resources available for purchase, the nature and scale of laboratory support and the environment under investigation. A further critical consideration is that, in practice, any sampler may require to be used in several applications. Finally, personal preference and experience of an instrument in use may greatly influence choice. It quickly emerges, even from the most cursory review of studies which characterise the indoor fungal environment that a wide range of samplers and sampling techniques are able to isolate Aspergillus spp spores from air simply because they have done so. This observation is of itself comparatively unhelpful as it gives no real clue as to comparative efficiency. Several investigators have sought to evaluate the efficiency of air sampling instruments either in isolation or comparatively applying performance criteria such as total yield (expressed as a concentration per unit volume of air), number of species isolated and the coefficient of variation between parallel or consecutive samples (as a measure of instrument precision).21, 32, 33 There is sufficient agreement within the literature to suggest that certain instruments consistently trap more organisms from the environment and are, in absolute terms, more efficient. Some instruments, such as the six stage Andersen N6 cascade sampler or the Casella slit samplers have been used over a long period of time and are regarded as efficient and robust. Indeed much of our knowledge of the aerobiology of occupational and other environments has accrued through the use of these instruments. Studies comparing the efficiency of different types of instrument in terms of their capacity to trap spores of a particular fungal species are less common and knowledge here often derives more from theoretical calculation based on information on the aerodynamic characteristics of the instrument and the target species or from experimental work using aerosols of known particle size. Accordingly, in selecting a sampler for isolating and enumerating Aspergillus spp. spores in air it is important to consider relevant biological and physical characteristics. Biological considerations Aspergillus spp. spores are intended for dissemination in air and share with most fungal spores a robustness and ability to resist impact damage and desiccation which means that sampling and enumeration techniques which might be unsuitable for more vulnerable particles are ostensibly suitable for Aspergillus spp. spores. Nonetheless, whilst biological considerations may be less relevant to the method employed to capture Aspergillus spores they are relevant to the selection of the medium used in impactors which rely on culturing to enumerate and identify viable spores from the catch. The minimum water activity for growth of is influenced by pH of the substrate and particularly by temperature. It varies too, according to species, but in the case of Aspergillus spp, the minimum Aw is accepted by most commentators to be around 0.78 with an optimum of around 0.97.34, 35, 36 Sui (1951) found that the range of relative humidity for spore germination on nutrient gelatin of seven different species of Aspergillus fell between 64% and 84%.37 Physical considerations Allowing for the fact that atmospheric humidity will influence aerodynamic diameter it is reasonable to express the aerodynamic diameter of Aspergillus spp spores as falling in the range 1.9-3.2 mm.14, 15, 16, 17 In addition to permitting the spores to penetrate the pulmonary defences and reach the alveolar spaces where they may germinate and form hyphae, their size also places them at the lower end of the aerosol particle size range and thus limits the choice of sampler. 38 The possibility of grouping of several spores in a single clump also merits consideration as this would clearly influence aerodynamic behaviour in sampling and site of deposition within the respiratory tract.19, 31 Gregory (1973) mentions that spore clumping is a recognised phenomenon with certain spores particularly with Cladosporium spp. and a few other types notably Ustilago.11 This, he took to denote failure of the spores to separate at the time of liberation rather than secondary aggregation during aerial transport, it being considered that many spores will be kept apart by like charges at the time of liberation. Lacey additionally observed that with Aspergillus fumigatus, the chains were so tightly bound together that they might break into clumps of chains with aerodynamic diameter determined by the size of the clump which would presumably vary. 18 Kozakiewicz (1989) commented on the significant variation in surface ornamentation within the same genera, highlighting Aspergillus spp as an example.39 Surface ornamentation is a primary determinant of aggregation. Thus, clumping inevitably occurs to some extent with Aspergillus spp. This has implications for particle capture within instruments. It can be appreciated that, irrespective of the physical principles on which they operate, instruments can be classified according to whether they are intended to measure only viable airborne propagules or both viable and non-viable particles. Filtration methods, some gravitational settlement techniques, liquid impingers and electostatic precipitators all gather both viable and non-viable particles although subsequent laboratory procedures can often permit enumeration of viable particles alone. A clear advantage is that, where the air is very contaminated, the large number of trapped spores will not exceed the capaity of the instrument and enumeration of the catch is possible using serial dilutions. A very pertinent consideration with regard to Aspergillus is that the focus of interest is normally the viable spores because they have the capacity to infect. Viable spore sampling techniques, i.e those which capture spores on an agar surface permit the catch of organisms to be cultured without further laboratory preparation. The development of colonies also facilitates enumeration and identification. Despite its widespread use, the open petri dish method of air sampling, relying, as it does on sedimentation is non-volumetric and inefficient for small particles. Whilst aggregations of Aspergillus spp spores will inevitably settle on horizontal surfaces or spores may impact when carried in rapidly moving air currents, open petri dish methods cannot be recommended for Aspergillus spores and will not be discussed further here. Cascade samplers, and in particular the Andersen Six-Stage samplers have been widely used and their efficiency in the sampling of smaller particles is recognised as an advantage particularly when allied to their capacity for particle size differentiation which can be important in many applications. Slit samplers too, have been evaluated and found to be efficient in their capacity to capture small particles. The larger slit samplers in particular the large model Casella (Casella Ltd, Bedford England) can draw some 700 litres of air per minute which may be an attractive feature in some hospital environments where there are low levels of airborne contamination. Unfortunately both these instruments have the disadvantage of a separate mains operated pump and the Casella in particular is bulky and heavy. Both instruments also require significant quantities of media. It is possible to operate the Andersen sampler using fewer stages and indeed the company have produced a two stage version intended to separate particles of respirable and non-respirable size. This reduces the quantity of media required but this does not overcome the inconvenience of an external pump which requires to be calibrated to align with the aerodynamic properties of the instrument. The supremacy of slit to agar samplers and the cascade samplers would suggest that, on the grounds of particle capture efficiency alone, these would be the natural choice for microbiological sampling. These instruments are however costly and the lack practicality for monitoring external or otherwise inaccessible areas. For sampling Aspergillus spp. spores, it is necessary at least to consider those instruments which capture viable particles and are powered by rechargeable batteries as these would appear to address some of the problems encountered with other impactors. This group is exemplified by the Reuter Centrifugal Sampler and the Surface Air Systems Microbiological sampler. Both instruments have been demonstrated to perform consistently below the level of the Casella slit sampler and the Andersen samplers in terms of the total number of organisms isolated.21, 32 This does not of itself rule these out as the instrument of choice but the findings of Clark and colleagues (1981) that collection efficiency of the RCS falls off rapidly for particles below 15mm and to approximately 50% for particles below 5mm suggests it is unsuitable for the capture of Aspergillus spp spores in air.24 The SAS sampler is a simple single stage impactor in which air is directed towards an agar filled contact plate of 50mm. diameter positioned behind the cover plate. The air is drawn into the instrument through a perforated cover plate which, in standard form, is perforated with 219 holes of 1mm. diameter. Particles leaving the air stream impact on the agar surface and thereafter the plate may be removed for subsequent incubation prior to enumeration and identification of colonies. The volume of air drawn into the instrument is regulated by pre-setting the period of operation using an integral timer. Capture efficiency in both quoted flow rates (180 l min-1 and 90 l min-1) falls to around 50% for particles of 2mm (approximating to the size of the smaller Aspergillus spp spores). The instrument is light, portable, robust and sufficiently versatile to sample in inaccessible areas or be pointed to detect a microbiological leak. The authors have found the 180 l min-1 SAS sampler to be practical and reliable when used in large epidemiological studies in the home environment and during outbreak investigations in hospital environments. The performance of the sampler and variations of it has been evaluated in comparison to other instruments.21,32 An important aerodynamic difference which affects capture efficiency is in the speed imparted to the incoming air with a markedly lower velocity in the case of the SAS than, say, the Six-Stage Andersen instrument. This is explained by the greater area of the combined orifices in the perforated cover plate of the SAS compared to the slit and Andersen samplers. A smaller inlet area offers greater resistance which can only be overcome using powerful pumps. Hence, low airspeed and reduced efficiency particularly in the impaction of smaller particles, may be an inevitable consequence of portability and convenience of the SAS sampler. The key question in this context is whether the lesser efficiency rules out the SAS as the instrument of choice for sampling Aspergillus spp. spores in the way it would seem to do for the RCS sampler. The performance of the SAS in standard 219 hole format (and a version with 260 holes) with a Bourdillon slit sampler have been compared.21 The version with 260 holes used a 90mm agar filled contact plate as opposed to the standard 50mm.contact plate. The most important characteristics of an air sampler have been defined as being the effective volume -rate of sampling and the range of particle sizes over which this is maintained.21 The tests showed that the effective sampling volume of both SAS instruments remained nearly constant over the particle sizes likely to be encountered during environmental sampling. But that collection efficiency fell off for particle sizes below 4mm and at 2mm reduced to 50%. Hence, efficiency of the SAS with a rated volume of 180 l min-1 is 50% for the smaller Aspergillus spp spores. In subsequent work conducted on behalf of the manufacturers, a version of the SAS sampler operating on the same principles but drawing 90 litres per minute was evaluated by the Centre for Aerobiological Research (CAMR) at Porton Down.40 Instead of using the 219, 1mm diameter hole cover plate, the test used a cover plate with 0.75mm holes. The study found that the reduction in the hole size resulted in an instrument which was over 100% effective for collecting particles in the size range of Aspergillus spp spores. Unfortunately this is a special adaptation and is not available as standard equipment. Whilst on first appraisal it might appear that the simple substitution of a new coverplate would create the ideal instrument, certainly for Aspergillus spp spores, it must be remembered that the greater air velocities created by the smaller diameter holes would reduce the volume sampling rate whilst the increased air velocity a would render the instrument less suitable for general microbiological air sampling where less robust organisms are of interest. The writers’ experience of the standard 180 l min-1 SAS instrument has shown that it regularly captures Aspergillus spp. spores. We nonetheless recognise that it may appear illogical to recommend an instrument which may be only 50% efficient for particles of the size of the target organism. However, the greatest advantage conferred by use of the SAS, particularly in 180 l min-1 form is its high flow rate which is particularly suited to environments where there may be low concentrations of the target organism.21 A further justification must be that the instrument is commonly used permitting comparison of monitoring conducted in different locations. The purchase price too is comparatively low, yet in the authors’ experience it has proved robust and reliable The accepted efficiency deficit in the critical 2mm range requires, in our view, to be set against the significant advantages offered in terms of practicality. The efficiency gained by using a modified SAS has been highlighted above yet this is not a practical option as the instrument is not available as standard in this form. Accordingly, the method set out below is recommended for sampling Aspergillus spp. spores in air. A recommended method for the sampling of Aspergillus species spores in air Sampling to isolate and enumerate Aspergillus spp spores in air should be conducted as follows: Material and equipment Sampling Instrument: Surface Air Systems microbiological air sampler. Manufacturer: Pool Bioanalyse Italian (PBI), Milan Italy U.K. Supplier: Cherwell Laboratories, Churchill Rd., Bicester, Oxon, U.K. Collection Rate: 180 l min-1 Medium: Czapek Dox Agar (Oxoid) Method Sampling Procedure (general) The air sample is aspirated through the instrument at a nominal rate of 180 liters per second for a preselected period of between 20 seconds and 6 minutes giving a volume range between 60 litres and 1080 litres. The airflow is directed towards the agar surface of a 50 mm diameter contact plate which contains 12.5 ml. of agar so that particles whose aerodynamic diameter causes them to leave the airstream are deposited on the agar surface. The plate is then removed for incubation.
Sampling location Sampling location is dependent on circumstances but should accord broadly with the breathing zone of potentially affected personnel. The choice of a sampling height of 1.2 meters for room hygiene. With other samples taken for exploratory purposes near suspected or potential sources of contamination. Multiple samples are preferable to single samples as they will highlight temporal and spatial variation in spore levels within any environment.
Selection of sampling time Selection of an appropriate sampling period is vital to the success of the sampling operation. If the sampling time selected is long in a heavily contaminated environment then the colonies, once cultivated, can only be expressed as exceeding a particular number. Where confluent growth occurs the colonies may even be uncountable. A chart is supplied with the instrument to assist in selection of the sampling time which, on the basis of an assumption regarding the level of contamination in the environment permits the number of sampling units (periods of 20 ) to be estimated. In practice, whilst some knowledge of the levels of contamination may build up over time, certainly initially the selection of the sampling period must largely be done by trial and error.
Sampling steps 1. Unscrew the top cover plate avoiding contact with the inner or outer surfaces of the drilled area. The cover plate should be cleaned after each use. 2. Insert a contact plate with Czapek Dox agar with the lid still in place, remove the contact plate lid and replace the instrument cover plate. 3. Set the digital selector on the instrument to zero units. 4. Switch on the battery pack. 5. Turn the timer to the desired setting. 6. Press the instrument start button. 7. Following completion of the sampling the instrument will switch off. The cover plate can then be removed and the exposed contact plate agar surface immediately covered by replacing the contact plate lid. 8. The contact plate should then be removed for incubation.
Laboratory procedure 1. On receipt of the contact plates, these are placed in a pre-heated incubator ot 28oC for 48 hrs to permit germination and colony formation. 2. The plates are then microscopically examined at x100 magnification to enumerate colonies growing on the plate. 3. Identification of fungal colonies is based on colony characteristics and micromorphological characteristics ascertained through microscopic examination ot x400 magnification. 4. Specimens for examination should be prepared using a wet needle mount using Lactophenol with Cotton Blue Stain (0.75%). 5. A colour key is available for the specific identification of different Aspergillus species grown on Czapek Dox agar or broth.
N.B. Czapek Dox (Oxoid Code ) is recognised as a suitable medium for isolation and culturing of Aspergillus species whilst permitting growth of the majority of airborne fungal species. This permits general levels of fungal contamination to be simultaneously appraised as an indicator of building hygiene.
Enumerating the colony forming units There is a possibility that any colony which grows on the contact plate derives from more than one colony forming unit passing through a single hole in the cover plate. This possibility increases with the number of colonies on the plate and for higher counts a correction factor is applied according to the following formula:-
Pr = n(1/n+ 1/(n-1) + 1/(n-2) +………….1/(n-r+1))
Where
Pr is the probable statistical total N is the number of holes in the sampling head r is the number of colonies counted
Tables have been prepared by the manufacturer which can be read off to give a value for P once r has been established.
The number of colony forming units calculated from the above formula is normally expressed as CFUs/m3. This is calculated using the formula:-
X=Adjusted colony count on plate x 1000 Volume of air drawn into sampler (litres)
[accepted thresholds here for different types of hospital enviroments]
The interpretation of air sampling data and recommendations for intervention are given in Table 3.
Conclusions
In published outbreaks of invasive aspergillosis, local fungal contamination of ducts, grids, and filters may release spores intermittently as may nearby demolition and building works. Although in some outbreaks of aspergillosis the patient’s isolates may have been different from those in the environment, some degree of control is more important than measuring air counts, because these are taken only at one points in time within the ward or clinic, and if an outbreak of infection has occurred, it reflects an episode of air contamination that happened some days or weeks before. “Normal” air counts are therefore not a reason for complacency, and a search for a specific source of air contamination is required, together with continued vigilance against the introduction of large numbers of spores into the patient’s room.
References
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40 Whittard
A. Personal Communication, 1999. Table 1 : Objectives of air sampling
- To correlate outbreaks of invasive aspergillosis with hospital construction/demolition - To identify potential sources of nosocomial aspergillosis, eg. potting soil, damp ceiling voids, damp fire proofing material, carpeting, etc. - To predict environmental spore contamination from outside sources - To identify defects/breakdown in hospital ventilation/filtration systemsa - To monitor cleaning procedures that may release bursts of airborne Aspergillus conidia - To determine the efficacy of HEPA filters in laminar flow facilities - To monitor efficacy of procedures to contain hospital building work from hospital wards and other areas where high-risk patients are managed - To determine level of contamination prior to initial occupancy of special controlled environments
a regular engineering maintenance of the air supply system (whether HEPA-filtered or not) is more important than regular air sampling Table 2: Air samplers for quantitation of viable fungal spores
Sampler type Principle Flow rate Cut-off diameter (litres/min) (d50)(um)
Sieve impactor impaction 28.3 0.65-7.0 (Anderson) onto agar plate
Slit sampler Impaction 30-700 ~0.5 (e.g. Casella) onto rotating agar plate
Centrifugal Impaction 40 4.0 Impactor due to (RCS) centrifugal acceleration
Impingess Impingement 12.5 0.3 (e.g. AGI) into liquid
P.B.I. SAS Impaction 90/180 2.0 Sampler onto agar (Single stage plate impaction)
Settle plates Gravity Non- N/A volumetric
Contact plates Surface Non- N/A Sampling volumetric
Table 3: Interpretation of air sampling data and recommendations Ř Levels of fungal spores vary by several orders of magnitude during the course of a day due to: Ř Activity levels in any one particular area Ř Fluctuations in temperature Ř Fluctuations in humidity Ř Fluctuations in air flow Ř Changes in light level Ř A single air sample will often underestimate the fungal contamination in the air: multiple air sampling has to be performed Ř No strict numerical guidelines are available which are appropriate for assessing whether the contamination in a particular location is acceptable or not but the following threshold levels have been recorded: Ř Outdoor air: total fungal count: 103 to 105 CFU/m3 Ř Aspergillus: 0.2 – 3.5 conidia/ m3 Ř Note: seasonal variation recognised Ř HEPA filtered air (>95% efficiency and >10 air changes per hour): <0.1 CFU/m3 Ř No air filtration: 5.0 conidia/ m3 Ř Construction/defective ventilation: 2.3 – 5.9 conidia/ m3 Ř If total fungal count exceeds 1.0 CFU/m3 on several occasions the air systems or procedural practice in patient areas requires intensive evaluation. Ř Further investigation of sources of contamination is warranted in the following circumstances: Ř Total indoor counts are greater than outdoor counts Ř Comparison of indoor and outdoor levels of fungal organisms show one of the following: Ř Organisms are present in the indoor sample and not in the outdoor sample Ř The predominant organisms found in the indoor sample is different from the predominant organism in the outdoor sample Ř A monoculture of an organism is found in the indoor sample. It may be absent from samples taken in other areas of the building Ř Persistently high counts Ř If persistently high counts are recorded, or nosocomial invasive aspergillosis suspected or confirmed: identify source of contamination by sampling: Ř dust Ř fabrics Ř ventilation ducts/screens/fans Ř ceiling voids Ř kitchen areas Ř excreta of roosting birds in close proximity of windows [Table 3 continued] Ř Restrict food stuffs known to carry Aspergillus conidia, eg. black pepper Ř Consider painting contaminated surfaces with copper-8-quinolinolate Ř Start appropriate antifungal prophylaxis or pre-emptive therapy if not already used Ř Perform an intensive retrospective review of microbiological, histopathological and post-mortem records for other cases. Ř Alert clinicians caring for high risk patients to the possibility of infection Ř Establish a system for prospective surveillance of patients and their environment for additional cases Ř If further cases arise in the absence of a nosocomial source consider monitoring home enviroments of patients pre admission.
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