Bioaerosol Sampling

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 (d50) • 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 Isopycnic density gradient: microbe density = medium density at one depth Flotation: microbe density < medium density

40. Immunomagnetic Separation Y Antibody Bead Y Y Y 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 sCAR purification Covalent coupling to paramagnetic beads Culture + media; :sCAR produced Blocking post-coupling (RT-) PCR : sCAR NA extraction Sample containing viruses : Virus Particle : 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