1. Bioaerosol Sampling John Scott Meschke 4225 Roosevelt Way NE, suite 2338 jmeschke@u.washington.edu 206-221-5470

2. Bioaerosols A collection of aerosolized biological particles (e.g. microbes, by-products of living organisms) capable of eliciting diseases that may be infectious, allergic, or toxigenic with the conditions being acute or chronic Size range 0.02–100 micrometers (typically 2-10 microns size range of most concern) Composition of the particles varies with source and environmental conditions Particles can contain varying amounts of water Some are colloidal particles of soil, vegetation, other material Viruses, bacteria and fungi (spores and hyphae) in aerosols due to small size Many protozoa too large to remain airborne

3. Examples: Agents of Respiratory Infections Viruses: influenza, measles (rubeola), chickenpox (herpes varicella ‑ zoster) and rhinoviruses (colds); Hantavirus (from a rodent; mouse) Bacteria: Legionella spp., tuberculosis and other mycobacteria ( Mycobacterium spp.), anthrax ( Bacillus anthracis ), and brucellosis (Brucella spp.). Fungi: diseases: histoplasmosis, cryptococcosis, blastomycosis, coccidiodomycosis, and aspergillosis Protozoans: Pneumocystis carinii pneumonia; prevalent in immunodeficient hosts such as AIDS patients. Acanthamoeba encephalitis; primary amebic meningoencephalitis (PAM)

6. Reservoirs and Amplifiers of Airborne Microbes Wide range, overall Depends on the microbe – humans, – animal, – soil – dust – water – air Amplifiers: Places where microorganisms multiply or proliferate. Most reservoirs are potential amplifiers.

7. Airborne Microbes and their Reservoirs Viruses: Mostly humans but some animals Some rodent viruses are significant: ex: Lassa Fever Virus and Hantavirus. Bacteria: Humans (TB & staphylococci), other animals (brucella and anthrax), water (Legionella ) soil (clostridia). Fungi: soil and birds ( Cryptococcus and Histoplasma ) dead plant material wet surfaces (wood and other building materials) indoor air (mycotic air pollution) stagnant water for the opportunistic fungi (e.g., Aspergillus sp.).

8. Disseminators Devices causing microbes to enter airborne state or be aerosolized; often the reservoir or amplifier. Any device able to produce droplets and aerosols: – Humans and other animals: coughs and sneezes, esp. – Mechanical ventilation systems – Nebulizers and vaporizers – Toilets (by flushing) – Showers, whirlpools baths, Jacuzzi, etc. – Wet or moist, colonized surfaces (wet walls and other structures in buildings) – Environments that are dry and from which small particles can become airborne by scouring or other mechanisms: Vacuuming or walking on carpets and rugs Excavation of contaminated soil Demolition of buildings

9. Bioaerosol Samplers Numerous sampler types Some adapted from dust or particle samplers Some designed specifically for microbes Few specifically for non-microbial bioaerosols (e.g. endotoxin), but generally thought samplers used for microbe collection are adaptable

10. Bioaerosol Samplers Gravitational samplers (e.g. settle plates) –No special equipment only coated microscope slide, agar plates, etc. –Passive (non-volumetric), relies on collection of particles by gravity settling –Oversamples for larger particles –Poor for collection in turbulent air; affected by turbulent deposition or shadowing

11. Inertial Bioaerosol Samplers Allow collection of particles by size selective sampling Includes impactors, sieves, stacked sieves Relies on particle tendency to deviate from air flow streamlines due to inertia Particles deposited to solid or semi-solid surface

12. Spore Traps E.g. Hirst, Burkhard, Air-o-cell, Allergenco Initially designed for fungal spore and pollen Sample at 10-20 Liters/minute Particles impacted on to coated glass slide or adhesive tape Advantages: non-selective, direct analysis after collection Disadvantages: may mask problem species, does not assess viability

13. Impactors Similar to spore trap, but collection on slide or agar plates Several designs tend to undersample smaller particles; particle bounce can also be an issue Used at air flows of 10-30 Liters/minute Types: –Single Stage or Multistage (e.g. Anderson) –Rotary arm samplers (e.g. Rotorod, Mesosystems BT550) –Slit to agar samplers –Sieve Samplers and Stacked Sieves (e.g. SAS)

14. Impactors

16. Impingers Air drawn through liquid (e.g. water, broth, mineral oil), particles removed by impingement Allows dilution Problems with pass through, particle bounce, bubbling, evaporation of liquid loss of viability Inlet efficiency decreased for particles above 10 microns Sampling rate 0.1-15 liters/minute (12.5 for AGI 30) Types: –AGI –Biosampler –Shipe –Multistage

18. Impingers

19. Cyclones or Centrifugal Samplers Creation of vortex creating sufficient inertia to trigger deposition of particles onto collection surface; recovered in liquid (cyclone) or semisolid medium (centrifugal) Allows dilution; high air sampling rates (up to 75- 1000 LPM for cyclones, 40-100 LPM for centrifugal samplers); small pressure drop Oversamples larger particles (can be used as trap); poor collection below 5 micron Can be used in series or paired with other samplers to overcome sampling bias (e.g. Innovatek)

21. Large Volume Aerosol Samplers Biocapture BT 550 (Mesosystems) –Rotary arm impactor, liquid collection –150L/min (~15 min) Bioguardian (Innovatek) –Wet-walled multi cyclone, w/centrifugal impactor for removal of large particles –100-1000L/min (1 min-12 hours) Spincon (Sceptor) –Centrifugal wet concentrator, w/cyclonic preseparation –450L/min (5 min-6 hours)

22. Aerosol Samplers

23. Non-Inertial Samplers E.g. Filtration, Electrostatic Precipitation, thermal precipitators, and Condensation traps Do not rely on inertia of particles for operation, thus less reliant on particle size (less particle size bias)

24. Filtration Simple equipment requirements Adaptable to personal sampling Less particle size bias (allows large and small particle collection; dependent on inlet size/shape) Continuous sampling over extended period Wide variety of sampling rates However, problems with desiccation leading to reduced viability and difficulties with particle recovery efficiencies

25. Filter Media Fiborous- mesh of material whose fibers are randomly oriented (creating nominal pore size); depth filter entrainment –Glass fiber (works for proteinaceous bioaerosols) Membrane- a gel with interconnected pores of uniform size (absolute pore size); depth filter entrainment –Cellulose esters (commonly used for water and other liquids for microbe concentration), PVC, PTFE, nylon, gelatin Flat disc or etched membranes- defined holes or pores (absolute pore size); surface collection –Silver, aluminum oxide, polycarbonate (most commonly filter media for collection of microbes from air)

26. Filters

27. Electrostatic Precipitators Particles removed from air stream by electrical rather than inertial forces Low pressure drop; low power; capable of large volume sampling and high rates Draws air across high voltage field or corona discharge inducing charge; surface collection Can be effective for very small particles, as well as larger ones Problem with ozone production; loss of viability Examples- –LVAS –LEAP

28. Thermal Precipitation and Condensation Traps Thermal precipitation –Not commonly used –Based on Thermophoretic motion –Air passed between two plates (one heated and one cooled); particles collected on cooler plate Condensation trap –Relies on manipulation of relative humidity –Bioaerosol used as condensation nuclei –Particles collected by settling

29. Recovery from Air Factors that will affect the recovery of microbes from air samples: –Sampling Rate –Environmental Factors may reduce sampling efficiency (e.g. Swirling winds) –Sampling Time –Organism Type and Distribution –Particle Size and Distribution – Target of detection method to be utilized –Sampler Choice Collection efficiency Recovery efficiency Particle Size Bias

30. Recovery from Air Factors that will affect the recovery of microbes from air samples: –Sampling Rate and Sampling Time (sampled volume) –Concentration factor –Environmental Factors may reduce sampling efficiency (e.g. Swirling winds) –Organism Type and Distribution (need for replication) – Target of detection method to be utilized –Sampler Choice Collection efficiency (d 50 ) Retention efficiency Recovery efficiency Particle Size Bias Loss of viability –Sampler Calibration

31. Collection Efficiency: Flowing Air

32. Sample Line Losses To minimize make as short as possible, minimize angles

33. Separation and Purification

34. Separation and Purification Methods Purification, separation and secondary concentration of target microbes in primary sample or sample concentrate –Separate target microbes from other particles and from solutes –Reduce sample size (further concentrate)

35. Separation/Purification Methods Variety of physical, chemical and immunochemical methods: –Sedimentation and flotation (primarily parasites) –Precipitation (viruses) –Filtration (all classes) –Immunomagnetic separation or IMS (all classes) –Flow cytometry (bacteria and parasites); an analysis, too

36. Secondary Concentration and Purification PEG (polyethylene glycol) Organic Flocculation IMS (Immunomagnetic separation) Ligand capture BEaDs (Biodetection Enabling Device) Capillary Electrophoresis Microfluidics Nucleic Acid Extraction Spin Column Chromatography Floatation Sedimentation Enrichment

37. Chemical Precipitation Methods Viruses: precipitate with polyethylene glycol or aluminum hydroxide –resuspend PEG precipitate in aqueous buffer –dissolve aluminum floc in dilute acid solution –both have been used as second-step concentration and purification methods Parasites: precipitate with calcium carbonate –dissolve precipitate in dilute sulfamic acid

38. Other Recovery and Concentration Methods Minerals, such as iron oxide and talc; used to adsorb viruses Synthetic resins: ion exchange and adsorbent Other granular media: glass beads and sand Less widely used; less reliable, cumbersome; uncertain elution, desorption, exchange efficiencies

39. Initial Recovery and Concentration of Pathogens Flotation centrifugation –Layer or suspend samples or microbes in medium of density greater than microbe density; centrifuge; microbes float to surface; recover them from top layer Isopycnic or buoyant density gradient centrifugation –Layer or suspend samples or microbes in a medium with varying density with depth but having a density = to the microbe at one depth. –Microbes migrate to the depth having their density (isopycnic) –Recover them from this specific layer Flotation: microbe density < medium density Isopycnic density gradient: microbe density = medium density at one depth

40. Immunomagnetic Separation Y Y Y Y Bead Antibody Microbe

41. Virus Capture Plus RT-PCR to Detect Infectious Viruses - The sCAR System The cell receptor gene for Coxsackieviruses and Adenoviruses has been cloned and expressed, producing a soluble protein receptor, sCAR Expressed, purified and bound sCAR to solid phases to capture infectious Coxsackieviruses from environmental samples – The nucleic acid of the sCAR-captured viruses is RT-PCR amplified for detection and quantitation

42. Application of sCAR with Para-Magnetic Beads for Virus Particle Capture and then RT-PCR : Virus Particle : sCAR Culture + media; :sCAR produced (RT-) PCR sCAR purification : Blocking protein Amine Terminated Support Magnetic Bead : BioSpheres(Biosource) Pre-coated to provide available amine groups for covalent coupling of proteins or other ligands by glutaraldehyde-mediated coupling method Covalent coupling to paramagnetic beads Blocking post-coupling Sample containing viruses NA extraction