Fungal infections in immunocompromised patients are not only difficult to treat, but are common and often unsuspected at the time of death. Recent autopsy studies confirm this, for example, among bone marrow transplant patients, invasive yeast infection or pulmonary aspergillosis was 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 by means of prophylaxis and prevention of exogenous infections (mostly airborne and caused by filamentous fungi) through environmental protection and chemoprophylaxis.

Many studies have attempted to evaluate the importance of fungal aerobiology in relation to invasive fungal infection in the compromised patient. 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 (refs: Humpreys J Med Micro 37: 81-82, 1992; ASM Clinical Microbiology Procedures Handbook, Isenberg). 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 environments. This article does not discuss in detail methods of sampling surfaces for Aspergillus spores although in order to fully assess fungal contamination, surface sampling is also recommended.

 

Sampling Biological Aerosols: Some General Considerations

The considerations relevant to sampling Aspergillus spp. spores in air are essentially the same as those that apply to sampling any aerosol. Aerosols are made up of particles suspended in air with an upper size limit of approximately 25 µm.  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 (Cox, 1989).  Thus, many aspects of the behaviour of microbial particles in air might be predicted from knowledge of their physical attributes. Biological features, however can also be important as they influence the take off, aerial transport and landing of microbial particles in addition to their 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 (Cox, 1989).

As direct microscopic 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 (Gregory, 1973).

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".  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 that 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 (Cox, 1989).  The first region comprises the nasopharynx and/or the mouth. Passage through this region ensures nearly 100 % deposition of particles in the > 20 µm range although efficiency of capture falls off rapidly with decreasing particle size. Only the largest fungal spores will be deposited here (Gregory, 1973). Mouth breathers, whether by habit or due, for example, 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 contains then pass through the trachea that divides at its lower end 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 2 µm and 20 µm.  At the lower end this 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.  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 that 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 to 4 µm 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 <1 µm 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 µm. 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.  This contrasts with the many air sampling instruments that 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 (Gregory, 1973).

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  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 (Knutson and Lioy, 1989).   Because particles come in many shapes such as, spheres, cubes, flakes or fibres, it has proved useful to create a unit for particle size as deposited in the respiratory tract which takes account of all the influences on deposition, and particularly on settlement and inertial impaction.  This is known as the aerodynamic diameter (dae).  The 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 (µo = 1g/cm3) which settles through air with a velocity equal to that of the actual particle under consideration" (Knutson and Lioy, 1989).

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.  Sem (1984) points out that aerodynamic diameter is less useful when particles are very small (<0.5 µm) because diffusion becomes an important deposition mechanism and this is solely influenced by particle size as opposed to shape or density.  Aerodynamic diameter also loses its utility in the larger size range (>15 µm) because of the very short residence time of larger particles in air.  Nonetheless, because they exhibit significant variation in shape, dae is an important measure of the capacity of fungal spores to lodge in the respiratory tract or be trapped in an air sampler (Gregory, 1973).

One complicating factor derives from the fact that particles absorb and lose water causing their size to change with changing atmospheric humidity (Pasenen, et al., 1991; Madeline and Johnson, 1992). 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.  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 to 3.9 µm depending on humidity.  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.  These, they recorded as 1.9 to 2.2 µm for Aspergillus fumigatus, 2.6-3.0 µm for Penicillium chrysogenum and 2.3 to 2.5 µm for Cladosporium chadosporoides.  Samson and van Reenen Hoekstra (1988) reported similar physical diameters for these species of 2.5 to 3 µm (A.fumigatus), 3 to 7 µm (P.chrysogenum) and 2 to 4 (C.cladosporoides).  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.

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 the measurement of the sample air volume.  This permits the numbers present to be expressed as a concentration per unit volume of air, or  where viability is of interest (for example, medical microbiologists or plant breeders) as colony forming units per per cubic metre of air (CFUs/m3).  Where both viable and non viable organisms are of interest for example, to those investigating allergic phenomena, the parameter of concern may be the total number of spores per cubic metre.  This requires a different type of instrument and different laboratory techniques which do not rely on spore viability.  Some instruments incorporate a pump set 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.  Reducing or extending the sample period alters the volume of air sampled.  Choice of sample 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/volume 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 unsuitable in many outdoor situations.  This has been addressed by some manufacturers who provide a rechargable battery to power the equipment.  Whilst this confers infinitely greater flexibility, true portability is also determined by the weight and dimensions of the instrument.  Quite apart from their requirement for a separate mains operated pump, large slit samplers for example, are construed 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 on inefficient sampling methods.

Sampling Instruments for Enumerating and Identifying Air Spora

Types of Sampler

It is convenient to consider microbiological samplers for collecting organisms in air falling in several broad categories.

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 Cascade Sampler, the Casella Slit Sampler (Casella Ltd, Bedford, England) and the Surface Air Systems (SAS) Sampler (Cherwell Laboratories,  Bicester, Oxon).

Some instruments  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). Despite the quite different design characteristics these instruments are also impactors.

A further distinct group is the impingers which operate by impinging organisms into liquid.  This group includes the single-stage midget and micro impingers (SKC Ltd, Poole, Dorset) and the multistage May liquid impinger (AW Dixon, Beckenham, Kent).

Certain instruments use electrostatic precipitation to isolate particles from the air stream, although the principle of electostatic 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 of 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 (Crook and Lacey, 1989).

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 these samplers can be calibrated and 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.  This restricted inlet is a narrow slit in the case of a slit sampler such as the Casella Slit Sampler (Bourdillon, 1985) or a plate perforated with a number of uniform diameter holes in the case of for example, the SAS sampler (Lach, 1985).  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 Cascade Sampler (Andersen, 1958) the air is directed through a stack of perforated or sieve plates each with 400 uniform holes.  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.  This imparts a greater velocity to the air and causes smaller particles to be deposited from the airstream.  The sequence is repeated for each plate in the stack and the total size range of the particles, which can be collected over all the stages of a cascade impactor, is between 0.3 µm to 15 µm.  This represents rather well the range of particle sizes that might present a hazard to the human respiratory tract. The capacity to separate particles according to size is important in the context of human health.  In the case of the Andersen 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 (Crook and Lacey, 1989).  Most published data on the presence of fungal particles in non-industrial environments has been obtained using Andersen samplers (IEH, 1996).  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.  However, if the environment is very heavily contaminated, then 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 the airstream 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 (Clark et al., 1981).  Slit samplers and the Andersen Cascade Samplers have traditionally used separate mains operated pumps.  Impactors introduced more recently have addressed this problem by using integral pumps operated from a rechargeable battery.

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 the RCS sampler (Clark et al., 1981), 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.

Agar strips are subsequently removed for incubation.  Whilst operating according to rather different aerodynamic principles, these 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 µm, a range of considerable importance in human respiratory deposition.

A second type of instrument relying on centrifugal acceleration collects the airborne particles in liquid.  The sampler, known as the cyclone sampler (Errington and Powell, 1969) mixes the incoming air with a liquid supplied via a needle fed gravitationally or through a peristaltic pump.  The mixture is drawn tangentially into an inverted cone.  The airstream spirals down to the base of the sampler before being drawn up the centre of the instrument to the outlet (Crooks and Lacey, 1989).  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 the flask is fused to a piece of capillary tube that dips at least 5 mm into the flask (in the original form) and terminates at least 4 mm 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 (Gregory, 1973).
 
The multistage impinger (SKC Ltd, Poole, Dorset) confers advantages over the single stage device as it has a gentler flow which is less damaging to particles and by 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 (c) 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 55 litres/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 (Crook and Lacey, 1989).

Multi-stage impingers also have the advantage of minimising damage to microbes and improving collection efficiency (Gregory, 1973).

Electrostatic Precipitation

 Movement of charged particles in electrical fields has been used for dust extraction from airstreams.  Berry (1941) is credited with recognising the value of applying this principle to microbiological sampling.  Different designs for microbiological samplers have been described (Gregory, 1973; Crook and Lacey, 1989).  The Litton-type sampler draws 400 - 1000 litres of air/min.  The charged particles are attracted to a rotating aluminium disc that carries the opposite charge.  The disc is coated with a thin liquid film that 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.  Microorganisms 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 (Rolmyren, 1986).  Alternatively, 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 (Palmgren et al., 1986).  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 (Crook and Lacey, 1989).

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 (Rotter et al., 1973).

Large volume filtration techniques can be used to trap airborne particles for biochemical or immunological assay (Swanson et al., 1985).

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.  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 this method 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".

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, horizonatal blinds, fan blades and guards. Using 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 spp.

Many factors need to be considered when 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.  These include whether the sampler is required for one specific task or for varying tasks, purchase price, the nature and scale of laboratory support, the environment under investigation, and not least, personal preference.
 
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 species from air simply because they have done so.  This observation is unhelpful, as it gives no real clue as to comparative efficiency of the samplers. Several commentators (e.g. Lach, 1985; Verhoeff et al., 1989; Smid et al., 1989) have sought to evaluate the efficiency of air sampling instruments, either in isolation or comparatively, by 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).

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 been 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.  Information here often derives from theoretical calculation, based on knowledge of 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 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 in theory suitable for Aspergillus 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 enumerate and identify viable spores from the catch. The minimum water activity for growth is influenced by the 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 (Al Doory & Domson, 1984;  Summerbell  et al., 1994; Samson et al.,1994). 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 %.
 

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 2 to 3 µm.  In addition to permitting them to penetrate the pulmonary defences and reach the alveolar spaces where they may germinate and form hyphae (Walsh and Dixon, 1989) their size also places them at the lower end of the aerosol particle size range and thus limits the choice of sampler.  The possibility of spore clumping also merits consideration as this would clearly influence aerodynamic behaviour in sampling and site of deposition within the respiratory tract (Lacey & Crooke, 1988; Burge, 1989).  Gregory (1973) mentions that spore clumping is a recognised phenomenon with certain spores particularly Cladosporium spp. and a few other types notably Ustilago.  He took this to denote failure of the spores to separate at the time of liberation rather than secondary aggregation during aerial transport, since it is considered that many spores will be kept apart by like charges at the time of liberation.  Whilst clumping cannot be categorically ruled out, for practical purposes it is reasonable to assume Aspergillus spores will occur as separate entities in air and their aerodynamic behaviour will be determined by aerodynamic particle diameter.

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 capacity 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 - 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.  Therefore it cannot be recommended for Aspergillus spores and will not be discussed further here.

 
Cascade samplers, and in particular the Andersen N6 samplers have been widely used.  Their efficiency in the sampling of smaller particles is recognised as an advantage particularly when allied to their capacity for particle size differentiation that can be important in many applications.  Slit samplers have also 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 only those stages suitable for capture of smaller airborne particles thus reducing the quantity of media required, but this does not overcome the inconvenience of an external pump which requires calibration to align with the aerodynamic properties of the instrument. Thus, for reasons of cost and practicality, it has been decided that neither the Slit samplers or  the Andersen N6 qualify for recommendation as the instrument of choice for universal use in sampling Aspergillus spp. spores.  It is logical then to consider those instruments that capture viable particles and are powered by rechargable batteries as these would appear to address some of the problems encountered with other impactors.    The Reuter Centrifugal Sampler and the Surface Air Systems Microbiological sampler exemplify this group.  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 (Lach, 1985; Clarke et al., 1981; Verhoeff et al., 1988)  This does not rule these out as the instrument of choice.  However, Clark et al., 1981 found that collection efficiency of the RCS falls off rapidly for particles below 15 µm and falls to approximately 50 % for particles below 5 µm.  These data suggest it is unsuitable for the capture of Aspergillus spp spores in air.

It is necessary then to consider the suitability of the SAS sampler.  The Surface Air Systems sampler is available with quoted flow rates of 180 l min-1 and 90 l min-1.  Air is drawn into the instrument through a perforated cover plate which, in standard form, is perforated with 220 holes of 1 mm diameter.   Air is directed towards  an agar filled contact plate of 50 mm diameter positioned behind the cover plate.  Particles leaving the airstream  impact on the agar surface and thereafter the plate may be removed for subsequent culturing prior to enumeration and identification. Pre-setting the period of operation using an integral timer regulates the volume of air drawn into the instrument.  The instrument is light, portable and 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 have been evaluated in comparison to other instruments ( Lach, 1985; Verhoeff et al., 1988; Verhoeff et al., 1989).  As stated above, the efficiency of both the RCS and the SAS samplers is less than the Andersen N6 and the Slit samplers.  Lach (1985) found that the velocity imparted to the airstream was 4.36 m s-1 compared to 24.46 m s-1  for the Andersen N6 sampler.  Other features of the instrument serve to reduce the velocity in the SAS to 2.17 m s-1.   This marked difference in air velocity is explained in large part 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 such as the mains, operated separate pumps used with the N6 and Casella slit samplers.  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.

Lach (1985) compared the performance of the SAS in standard 220 hole format and a version with 260 holes with a Bourdillon slit sampler. The version with 260 holes used a 90 mm agar filled contact plate as opposed to the standard 50 mm contact plate.   Lach defined the most important characteristics of  an air sampler as being the effective volume rate of sampling and the range of particle sizes over which this is maintained.  The tests showed that the effective sampling volume of both instruments  remained nearly constant over the particle sizes likely to be encountered during environmental sampling. However, that collection efficiency fell off for particle sizes below 4 µm and at 2 µm 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.  Lach also observed that the high air flows of the samplers made them particularly suited to the measurement of low levels of micro-organisms.

In subsequent work conducted on behalf of the manufacturers, a later version of the SAS sampler operating on the same principles, but drawing 90 litres per minute was evaluated by the Centre for Aerobiological Research at (CAMR) at Porton Down (Whittard, 1999).  Instead of using the 220, 1 mm diameter hole cover plate, The test used a cover plate with 0.75 mm 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.

In our opinion the standard 180 l min-1 SAS instrument has shown that it regularly captures Aspergillus spores.  The instrument can also sample large volumes of air that is a significant advantage in areas where there is little contamination. We nonetheless recognise that it may appear illogical to recommend an instrument, which may capture as little as 50 % of the target organism.  The justification must be that the instrument is commonly used, is comparatively low cost and, in our own experience is robust and reliable.  The advantages conferred by using the modified instrument with 0.75 µm holes are obviously significant in terms of absolute efficiency in the particle size range, but because the instrument is not available as standard it must be discounted.  Accordingly, this method is recommended for sampling Aspergillus spp. spores.
 

References

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Richardson, M.D. (1998) Aspergillus and Penicillium species. In: Topley and Wilson's Microbioogy and Microbial Infections, Volume 4: Medical Mycology.  Ajello, L. and Hay, R.J. (eds) London: Edward Arnold.

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Goodley, J.M., Clayton, Y.M. & Hay, R.J. (1994) Environmental sampling for aspergilli during building construction on a hospital site, J. Hosp. Infect., 26: 27-35.

Pennington, J.E. (1993) Aspergillus. In:Sarosi, G.A., Davies,  S.F. (eds) Fungal Diseases of the Lung. 2nd edn. New York: Raven Press. pp133-48.

Rossi, G., Tortorana, A.M., Viviani, M.A., Pagona, A., Colledan, M. & Fassati, L.R. (1989) Aspergillus fumigatus infections in liver transplant patients. Transplant Proc, 21: 2268-70.

Smid,T., Schokkin, E., Boley, J.S.M., & Heederik, D.E. (1989) enumeration of viable fungi in occupational environments: a comparison of samplers and media. Am. Ind. Hyg. Assoc, J. , 50(5): 235-39.
 

Staib, F., Folkens, U., Tompak, B., Abel, T. and Thiel, D. (1978) a comparative study of antigens of Aspergillus fumigatus from patients and soil of ornamental plants  in the immunodiffusion test, Zbl. Bakt. Hyg. I. Abt. Orig. A. 242: 93-99 cited in Walsh, T.J. & Dixon, D.M. (1989) Nosocomial Aspergillosis: Environmental microbiology, hospital epidemiology, diagnosis and treatment.  Eur. J. Epidemiol., 5: 131-142.

Walsh, T.J. & Dixon, D.M. (1989) Nosocomial Aspergillosis: Environmental microbiology, hospital epidemiology, diagnosis and treatment.  Eur. J. Epidemiol., 5: 131-142.

Seeliger, H.P.R. & Tintelnot, K. (1988) Epidemiology of aspergillosis.  In: Vanden Bossche., H., Mackenzie, D.W.R. and  Cauwenburgh, G., (eds). Aspergillus and aspergillosis. New York. Plenum.

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Gustafson, T.L., Schaffner, W., Lavely, G.B., Stratton, C.W., Johnson, H.K. & Hutcheson Jr, R.H. (1983) Invasive aspergillosis in renal transplant patients: correlation with corticosteroid therapy. J. Infect. Dis., 147:230-8.

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[additional references]

Richardson MD, Kokki M (1999). Aspergillus and aspergillosis. In: Clincial Mycology (Eds. Aniassie, Pfaller, McGinnis). Lippincott Williams and Wilkins (in press).

Richardson MD, Kokki M (1999). Diagnosis and prevention of fungal infection in the immunocompromised patient. Blood Reviews 12: 241-254.

Richardson MD, et al (1999). Fungal surveillance of an open haematology ward. Journal of Hospital Infection (submitted for publication).

Cole EC, Cook CE (1998). Characterisation of infectious aerosols in health care facilities: an aid to effective engineering controls and preventative strategies. Am J Infect Control 26: 453-464.

Hospenthal DR, Kwon-Chung KJ, Bennett JE. (1998). Concentrations of airborne Aspergillus compared tot he incidence of invasive aspergillosis: lack of correlation. Medical Mycology 36: 165-168. (lots of debate about this paper on the HIS WWW site).

Loo VG et al. (1996). Control of construction-associated nosocomila aspergillosis in an antiquated hematology unit. Infect Control Hosp Epidemiol 17: 360-364.

Philpott-Howard J (1996). Prevention of fungal infections in haematology patients. Infection Control and Hospital Epidemiology 17: 545-551.

Reponen T. (1995). Aerodynamic diameters and respiratory deposition estimates of viable fungal particles in mold problem dwellings. Aerosol Science and Technology 22: 11-23.

Health Implications of Fungi in Indoor Environments (Eds. Sampson et al.). Elsevier, 1994, pp. 622.

Iwen PC et al. (1994). Airborne fungal spore monitoring in a protective environment during hospital construction, and correlation with an outbreak of invasive aspergillosis. Infection Control and Hospital Epidemiology 15: 303-306.