determination of the transformair system’s efficacy ...labs+-+bioaerosols... · a flow diagram of...

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ARE Labs Inc. 2015 project # 10814.1 Transformair 1 of 24

Determination of the Transformair System’s Efficacy against Various Bioaerosols

Abstract

This in vitro study will characterize the Transformair System’s decontamination efficacy against

various aerosolized biologicals. The Transformair System is a prototype unit designed for use in

a room or air-conditioned environment to capture airborne bacteria, viruses, and fungal spores

photo catalytically deactivate the captured bioaerosols. The system consist of a pair of

proprietary photocatalytic coated filters with a long wavelength UV-A ultraviolet light source

housed in an enclosure with either an integrated blower or external blower system. This study

evaluated the efficacy of the system with an integrated blower against multiple species of

aerosolized bacteria, virus, and spores in a single-pass operating mode using a custom built

primary bioaerosol test system housed in a large secondary containment chamber.

The efficacy of the system was assessed for each of the six (6) following aerosolized biologicals:

Staphylococcus epidermidis, Escherichia coli, MS2 bacteriophage, Phi-X174 bacteriophage,

Aspergillus Niger spores, and Bacillus subtilis endospores. The study consisted of a total of

eighteen (18) separate trials conducted in triplicate for each of the six (6) aerosolized biologicals.

The triplicate single-pass bioaerosol trials showed that the Transformair System’s average log

reduction for S. epidermidis was 4.33 +/- 0.22 (average +/- standard deviation). The system’s

efficacy against Escherichia coli bioaerosol, was 4.91 +/- 0.24 log (Avg +/- STdev). The reduction

for viral bioaerosol concentrations were 4.19 +/ 0.23 logs and 4.19 +/- 0.51 logs (Avg +/- STdev)

for bacteriophage MS2 and PhiX174 respectively. A. niger fungal spore testing resulted in a

viable bioaerosol concentration reduction of 5.07 +/- 0.125 logs (Avg +/- STdev), and B. subtilis

endospores resulted in viable bioaerosol concentration reduction of 4.86 +/- 0.23 logs (Avg +/-

STdev). This study was conducted in compliance with FDA Good Laboratory Practices (GLP) as defined

in 40 CFR, Part 160.

Overview

This study was conducted to evaluate the ability

of a prototype Transformair disinfection unit

produced by Transformair Inc. (3802 Spectrum Blvd.

Suite 143 Tampa, FL 33612) to neutralize airborne

bioaerosols in a room environment. The unique

technology of the Transformair systems is the

employment of dual photo-catalytically coated high

efficiency air filters with a UV source sandwiched in-

between for decontamination captured bioaerosols.

This system could be employed in a variety of

various systems such as: various sized stand alone

units or clean room/hospital HVAC systems or other

air flow systems.

The Transformair unit tested in this study is

designed as a room air re-circulating stand-alone

portable air purification system with an integrated

blower system. Testing was conducted using a

primary bioaerosol test system for containment,

control, monitoring and collection of bioaerosol

challenges to the Transformair test system housed in

a secondary stainless steel aerosol chamber for

safety.

The Transformair effectiveness against the six

(6) separate Bio-safety level 1 (BSL1) organisms was

compared by simultaneously sampling viable

bioaerosols at upstream and downstream locations of

the Transformair unit in order to evaluate the

system’s effective log reduction of viable bioaerosols

from the flow stream. The test plan incorporated

challenging the test device in a closed single pass

flow through system with viable biological collection

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samplers located upstream and downstream of the

test unit.

Samplers were operated during the entirety of

each test for comparative measurements. Samples

were plated on nutrient agar and incubated for

enumeration of viable challenge and penetration

concentrations to determine the capture efficiency of

the Transformair unit. The system’s effectiveness

was evaluated against two vegetative bacteria, two

viruses, a fungal spore and a bacterial endospore as

simulants for a broad range of pathogenic organisms.

Testing was conducted to characterize a single

Transformair unit, approximately 12”x12”x18” in

size, against six separate and distinct organisms

(triplicate testing) in eighteen independent bioaerosol

tests to evaluate the capability of the Transformair

unit in single pass flow through operation.

Bioaerosol Test System

A large sealed secondary aerosol chamber was

used to house the bioaerosol test system and to

contain any potential release of aerosols into the

surrounding environment.

The aerosol test system was designed and

constructed as a closed system for bioaerosol

containment, controlled delivery and accurate

measurement of the bioaerosol challenge size

distribution and collection at both upstream and

downstream locations of the test unit. To accurately

reproduce operation of the test unit in a room

environment, the test system was operated in a push–

pull fashion with the Transformair test unit enclosed

and sealed within the aerosol test system. The test

system was operated to balance the bioaerosol

challenge flow rates and system exhaust flow rates at

equilibrium with the Transformair blower volumetric

flow rate to replicate an ambient test environment.

The bioaerosol test system was constructed of 10

inch diameter PVC piping and equipped with a pair

of internal blowers with independent flow rate

control at upstream and downstream locations of the

test system. The bioaerosol test system was equipped

with a Magenehelic differential pressure gauge with a

range of 0.0 +/- 0.5 inch H2O (Dwyer instruments,

Michigan City IN) to monitor the system differential

pressure and assure balanced and equilibrated flow

conditions upstream and downstream of the

Transformair unit. System pressures were

maintained at a slight negative pressure in relation to

the test chamber at - 0.05 to – 0.1 inches of water

during all testing to avoid any potential release of

aerosols outside of the test environment. Test

system flows were conditioned through HEPA

filtration units prior to introduction to the test system.

For all testing, bioaerosols were disseminated

using a Collison nebulizer (BGI Inc. Waltham MA)

driven by purified filtered house air supply. ¼ inch

diameter probes were located at upstream and

downstream locations of the Transform air test unit

for aerosol particle size monitoring via a TSI

Aerodynamic Particle Sizer (APS) model 3321. The

APS was equipped with a three-way valve and

manifold for selective sampling of the downstream

and upstream locations for comparative particle size

distribution and particle counts. A flow diagram of

the bioaerosol test system is shown in figure 2.

Figure 1: Transformair Unit in Chamber Test System.

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Blower

House Air

TransformAir® Bio-Aerosol TestingMonitors, Controls, Aerosol Generator, Decon System and Equipment

Primary Aerosol

Containment Chamber

TransformAir Unit

Ball

Valve

House Air

Bio-Aerosol Generator /

Dynamic Dilution System

Flow

Meters

Dilution Air

Generator Air

Regulator

Check

Valve

Three

way

Valve

HE

PA

Exhaust

Pump

Critical

orifice

Downstream

Sampling System

HE

PA

Exhaust

Pump

Critical

orifice

Upstream Sampling

System

APS 3321

Three-way Valve

Temperature

MonitorVaporous H2O2

Decontamination

System

Positive / Negative

Pressure Balance

HEPA

Challenge and Exhaust ducted directly to unit

Mixing Plate

Real-Time Aerosol Particle

Sizing Monitoring

Collison Nebulizer

Total System

Flow Rate

Monitor

HEPA HEPA

Blower

House Air

TransformAir® Bio-Aerosol TestingMonitors, Controls, Aerosol Generator, Decon System and Equipment

Primary Aerosol

Containment Chamber

TransformAir Unit

Ball

Valve

House Air

Bio-Aerosol Generator /

Dynamic Dilution System

Flow

Meters

Dilution Air

Generator Air

Regulator

Check

Valve

Three

way

Valve

HE

PA

Exhaust

Pump

Critical

orifice

Downstream

Sampling System

HE

PA

Exhaust

Pump

Critical

orifice

Upstream Sampling

System

APS 3321

Three-way Valve

Temperature

MonitorVaporous H2O2

Decontamination

System

Positive / Negative

Pressure Balance

HEPA

Challenge and Exhaust ducted directly to unit

Mixing Plate

Real-Time Aerosol Particle

Sizing Monitoring

Collison Nebulizer

Total System

Flow Rate

Monitor

HEPAHEPA HEPAHEPA

Blower

Figure 2: Bio-Aerosol Test System Flow Diagram.

Bioaerosol Generation System

Test bioaerosols were disseminated using a

Collison 24 jet nebulizer. A pressure regulator

allowed for control of disseminated particle size, use

rate and sheer force generated within the Collison

nebulizer.

Prior to testing, the Collison nebulizer flow rate

and use rate were characterized using an air supply

pressure from 28-50 psi (organism dependant), which

obtained an output volumetric flow rate of 50-80 lpm

with a fluid dissemination rate of approximately 0.8 –

1.5 ml/min. The Collison nebulizer was flow

characterized using a calibrated TSI model 4040

mass flow meter (TSI Inc, St Paul MN).

Bioaerosol Sampling and Monitoring System

For each test, a total of four (4) sterile AGI

impingers (Ace Glass Inc. Vineland NJ) with two

impingers located upstream, and two impingers

located downstream of the Transformair unit for

bioaerosol collection. Prior to testing, each impinger

was filled with 20 ml of sterile PBS + 0.05% tween

80 for sample collection. Impingers were operated

for simultaneous collection of challenge and

penetration bio-aerosols at both the upstream and

downstream for the entirety of each test. Following

each test, upstream and downstream impinger

samples were pooled respectively for plating and

enumeration to determine bioaerosol challenge and

penetration viable concentrations and calculation of

the Transformair log reduction in viable aerosols.

Impinger sample flow rates were operated at

critical vacuum and were controlled and monitored

using a valved Emerson 1/3 hp rotary vane vacuum

pump (Emerson Electric, St. Louis, MO) equipped

with a 0-30 inHg vacuum gauge (WIKA Instruments,

Lawrenceville, GA).

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The AGI-30 impinger vacuum source was

maintained at a negative pressure of 18 inches of Hg

during all characterization and test sampling to assure

critical flow conditions. The AGI-30 sample

impingers were flow characterized using a calibrated

TSI model 4040 mass flow meter. Sample flow rates

were maintained and monitored at 12.5 lpm for each

impinger using an in line calibrated TSI model 4040

mass flow meter.

Aerosol particle size distributions and count

concentrations were measured in real-time through

the duration of all tests using a model 3321

Aerodynamic Particle Sizer (APS) (TSI Inc, St Paul,

MN). The APS sampled for the entire duration of all

trials (10 minutes per trial) with 5 minute upstream,

and 5 minute downstream sampling intervals with 20

second sample times for each test.

Species Selection

Two vegetative bacteria were chosen for the

study as simulants for a broad range of pathogenic

bacteria both Gram-negative and Gram-positive

bacteria were selected. The first vegetative organism

used for this study was Staphylococcus epidermidis

(ATCC 12228). Staphylococcus epidermidis is a

Gram-positive bacterium and simulant for a wider

range of medically significant pathogens such as

Staphylococcus aureus.

Escherichia coli (ATCC 15997) is a gram

negative facultative anaerobic, rod-shaped bacterium

of the genus Escherichia. Most E. coli strains are

harmless, but some serotypes can cause serious food

poisoning in their hosts.

Two representative BSL1 viruses were chosen

to evaluate the Transformair unit’s performance

against both RNA and DNA based viruses. MS2

bacteriophage (ATCC 15597-B1) is positive-sense,

single-stranded RNA virus that infects the bacterium

Escherichia coli and other members of the

Enterobacteriaceae family. MS2 is routinely used as

a simulant for pathogenic RNA viruses, such as

influenza.

Phi-X174 (ATCC 13706-B1) bacteriophage is a

circular single stranded DNA based virus that infects

the bacterium Escherichia coli. Phi-X174 was

selected as a simulant for DNA based pathogenic

viruses, such as HIV.

Aspergillus niger (ATCC 16404) or A. niger is

one of the most common species of the genus

Aspergillus. A. niger is routinely defined as a

troublesome black mold and has been attributed to

many respiratory problems for infants, elderly and

immune compromised individuals. Purified A. niger

spores were obtained in bulk dry powder with an

approximate concentration of 1 x 109 cfu/gram.

Bacillus subtilis (also known as Bacillus

globigii) spores were used as the last representative

spore. Bacillus subtilis spores are routinely used as a

surrogate for Bacillus anthracis (Anthrax) for

bioterrorism/biowarfare research. Bacillus subtilis is

a Gram positive bacterium found in soil and the

gastrointestinal tract of ruminants and humans. B.

subtilis is rod-shaped, and can form a tough,

protective endospore, which allows it to tolerate

extreme environmental conditions.

Vegetative Cells Culture & Preparation

Pure strain seed stocks were purchased from

ATCC (American Type Culture Collection, Manassas

VA). Working stock cultures were prepared using

sterile techniques in a class 2 biological safety

cabinet and followed standard preparation

methodologies. Approximately 50mL of each

biological stock was prepared in tryptic soy liquid

broth media, and incubated for 24 – 48 hours at 37°C.

Biological stock concentrations were greater than 1 x

1010

cfu/ml for both Staphylococcus epidermidis and

Escherichia coli using this method.

Aliquots of these suspensions were enumerated

on tryptic soy agar plates (Hardy Diagnostics,

Cincinnati OH) for viable counts and stock

concentration calculation. For each organism, test

working stocks were grown in sufficient volume to

satisfy use quantities for all tests conducted using the

same culture stock material.

Viral Culture & Preparation

Pure strain viral seed stock and host bacterium were

obtained from ATCC. Host bacterium was grown in

a similar fashion to the vegetative cells in an

appropriate liquid media. The liquid media was

infected during the logarithmic growth cycle with the

specific bacteriophage. After an appropriate

incubation time the cells were lysed and the cellular

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debris discharged by centrifugation. MS2 stock

yields were greater than 1 x 1011

plaque forming units

per milliliter (pfu/ml) with a single amplification

procedure. Phi-X174, due to its much lower burst

size, required multiple amplification steps to produce

satisfactory viral yields. After amplification the cells

were lysed and the cellular debris separated from the

liquid media and discarded. Phi-X174 viral yields

were plated and enumerated and yielded viable

concentrations greater than 8 x 105 pfu /ml in the

stock used for aerosolization.

Fungal Spore Culture & Preparation

A. niger fungal spores were obtained in purified

bulk powder form at a concentration of 1 x 1010

cfu/g. To verify the bulk powder spore

concentration, an aliquot of weighed dry powder was

prepared in suspension in PBS + 0.05% Tween 80 at

a mass: volume ratio to obtain a concentration of 1 x

109 cfu/ml. The spore suspension was serial diluted,

plated on TSA plates and incubated at 30°C for 48-72

hours.

Plates were enumerated and bulk powder spore

concentration was verified to be in the range of 1 x

1010

cfu/g. Calculations were performed to obtain

mass use needed to generate aerosol test challenge

concentrations in the range of 5 x 104 cfu/L for

testing the Transformair system.

Bacillus Subtilis Spore Culture & Preparation

B. Subtilis spores were obtained in purified bulk

powder form. To verify the bulk powder spore

concentration, an aliquot of weighed dry powder was

prepared in suspension in PBS + 0.05% Tween 80 at

a mass: volume ratio to obtain a concentration of 2 x

109 cfu/ml.

The spore suspension was serial diluted, plated

on TSA plates and incubated at 30°C for 24 hours.

Plates were enumerated and bulk powder spore

concentration was verified to be in the range of 1 x

109cfu/g. Calculations were performed to obtain

mass use needed to generate aerosol test challenge

concentrations in the range of 4 x 104

cfu/L for

testing the Transformair system.

Plating and Enumeration

Impinger and stock biological cultures were

serially diluted and plated in triplicate (multiple serial

dilutions) using a standard spread plate assay

technique onto tryptic soy agar plates. The plated

cultures were incubated for 24 hours and enumerated

and recorded.

Bacteriophage samples and stock were plated

using the small drop plaque assay techniques outlined

by A. Mazzocco, T. Waddell, E Lingohr and R.

Johnson. The plates were then incubated 8-12 hours

and enumerated. All colonies and plaques counts

were manually enumerated and recorded.

Trial Run Species (description)

ATCC

Ref

Challenge

Suspension

Target

Mondispersed

Particle Size

Challenge

Conc. cfu(pfu)

/ ft3

Sampling Plating and Enumeration

1 Challenge Staphylococcus epidermidis

2 Challenge (Gram pos, vegetative)

3 Challenge

6 Challenge Escherichia coli

7 Challenge (Gram neg; vegetative)

8 Challenge

10 Challenge MS2 bacteriophage

11 Challenge RNA virus

12 Challenge (E. coli phage)

14 Challenge Phi-X174 phage

15 Challenge DNA virus

16 Challenge (E. coli phage)

17 Challenge A. niger

18 Challenge (endospores)

19 Challenge

20 Challenge Baccillus globigii

21 Challenge (endospore)

22 Challenge

APS, Upstream and

Downstream

Impingers

APS, Upstream and

Downstream

Impingers

APS, Upstream and

Downstream

Impingers

APS, Upstream and

Downstream

Impingers

ecoli ATCC 13706 for plaque, Tryptic

Soy Agar, All samples in triplicate or

greater

Tryptic Soy Agar, Spread Plate, All

samples in duplicate

APS, Upstream and

Downstream

Impingers

Tryptic Soy Agar, Spread Plate,

All samples in duplicate

APS, Upstream and

Downstream

Impingers



>106

>106

107-10

8

>105

>106

>106



ecoli ATCC 13706 for plaque,

Tryptic Soy Agar, All samples in

triplicate or greater

12228

15597

15597-B1

13706-B1

NA

NA

Broth 2.0-2.5 um

Broth 2.0-2.5 um

PBS <2.0um

PBS <2.0um

DI Water 3.0-4.0um

PBS 2.0um

Table 1: Test Matrices for all trials.

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Working stock concentrations of Aspergillus

spores were concentration verified prior to testing

using the small drop technique. Impinger samples

were plated using the small drop technique on TSA

agar plates. The plates were incubated at 30°C for 4-

728 hours and enumerated for concentration

measurement.

Test system Characterization

Calculations were performed to target biological

stock concentrations needed to obtain viable

bioaerosol concentrations to measure the efficacy of

the Transformair unit to obtain a 4 log reduction in

viability. The Transformair test unit was

characterized for volumetric flow rate to measure

total system aerosol dilution flow rates.

The unit was operated on low flow setting for all

bioaerosol tests, and prior to testing, a calibrated

Shortridge Instruments (Scottsdale, AZ) Air Data

Multimeter model ADM-870 anemometer was used

to verify the total flow rate throughput of the

Transform air blower and determine the total

bioaerosol dilution flow rate through the test system.

The system flow rate was verified and maintained at

85cfm for all biological testing.

The dilution flow rates, Collison nebulizer

dissemination rate, viable aerosol delivery and viable

bioaerosol collection efficiencies were used to

estimate test aerosol challenge duration and sampling

times to accurately assess the Transformair system

for capture and neutralization of viable bioaerosols in

a single pass test configuration. Viable bioaerosol

challenge concentrations were verified to be greater

than 1 x 104 cfu/L, or pfu/L of challenge air for each

biological.

Transformair Testing

Six challenge biological organisms:

Staphylococcus epidermidis (ATCC 12228),

Escherichia coli (ATCC 15997), MS2 bacteriophage

(ATCC 15597-B1), Phi-X174 bacteriophage (ATCC

13706-B1), Aspergillus niger spores and Bacillus

Subtilis spores were used for testing the viable

reduction capacity of the Transformair unit against

the broad spectrum bioaerosols. Transformair testing

was performed in triplicate for each biological

organism (18 total tests). The complete test matrix

for the study is shown in Table 1.

For each challenge test set, the Collison

nebulizer was filled with approximately 40 mL of the

applicable biological stock and disseminated for each

individual test. For testing, the impingers were filled

with 20 mL of sterilized PBS with the addition of

0.05% v/v Tween 80 for bioaerosol collection. The

addition of Tween 80 aids in increasing impinger

collection efficiency and reducing biological sample

agglomeration which can skew plating enumeration

results.

Preceding each test, the Transform air was set for

low blower speed operation and UV light operation

was verified. The aerosol test system is equipped

with internal blowers with independent flow rate

control at upstream and downstream locations of the

test system. For all testing, the blowers were

operated to balance the bioaerosol challenge flow

rates and system exhaust flow rates at equilibrium

with the Transformair blower volumetric flow rate to

replicate an ambient room test environment. A

magnehelic differential pressure gauge was used to

verify precise adjustment of the blowers to obtain

balanced system challenge and exhaust flow rates.

A set of Two (2) sterile AGI – 30 impingers

filled with 20 mL each of sterile PBS + 0.05% v/v

were connected to sampling ports at both the

upstream and downstream locations of the

Transformair test unit. Impingers sampling was

initiated, flow rates monitored and recorded, and the

Aerodynamic particle sizer (APS) sampling initiated

to take 20 second sequential samples during the

entirety of each test. At the initiation of testing, the

Collison nebulizer was turned on and the test time

recorded with a digital timer. At approximately 5

minutes into the test, the APS sampling was

redirected to the downstream location of the

Transformair unit to measure the particle size

distribution of aerosols penetrating the unit. At the

conclusion of each test, the Collison nebulizer,

impinger samplers and the APS sampling were turned

off.

Following each test, the two collected impinger

upstream samples were pooled, and the two collected

downstream impinger samples were pooled for viable

collected bioaerosol quantification. Decontamination

of the flow system used Hydrogen peroxide generator

to fully decontaminate the system between each trial.

For each of the eighteen tests, system

temperature and humidity, system pressures, sample

volumes, flow rates, and test start and stop times

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were recorded in a study test log. All samples were

plated in duplicate for vegetative cells and

endospores, and plated six times for viral samples on

growth media over a minimum of a 2 log dilution

range.

Plates were incubated for viable plaque forming

units (pfu) formation for the viral phase of the study,

and colony forming units (cfu) for fungal spore, and

bacterial endospore phases of the study. Plates were

incubated and enumerated for viable counts to

calculate aerosol challenge concentrations in the test

system and the reduction of viable microorganisms.

Post-Testing Decontamination and Prep

Following each test, the aerosol system was air

flow evacuated/purged for a minimum of twenty

minutes between tests and analyzed with the APS for

particle concentration decrease to baseline levels

between each test. The test system was

decontaminated at the conclusion of each test trial

with vaporous & aerosolized hydrogen peroxide to

eliminate any potential cross contamination between

organisms. The Collison nebulizer, and impingers

were cleaned at the conclusion of each test by

soaking in a 10% bleach bath for 20 minutes. The

nebulizer and impingers were then submerged in a DI

water bath, removed, and spray rinsed 10x with

filtered DI water until use.

Bioaerosol Particle Size Data

Aerosol particle size distributions were measured

with the APS. The APS has a dynamic measurement

range of 0.5 to 20µm and was programmed to take

consecutive real time 20 second samples throughout

the duration of each aerosol trial.

Vegetative Cells Native Particle Size DistributionsUpstream Sampling, Staphellococus Ep. & Escheria coli, Collison Nebulizer, APS 3321

0.0%

1.0%

2.0%

3.0%

4.0%

5.0%

6.0%

0 2 4 6 8 10 12 14 16 18 20

Aerodynamic Particle Size (um)

Ma

ss

Pe

rce

nt

(%)

Staph Ep E. coli Figure 3: Vegetative Aerosol Challenge Particle Size

Mass Distribution.

Data was logged in real time to an Acer laptop

computer, regressed, and plotted. Data shows the

upstream challenge bioaerosol particle size

distributions for each organism. The aerosol particle

size distributions for vegetative cells are shown in

Figure 3, virus particle size distributions in figure 4,

and fungal and endospore particle size distributions

are shown in figure 5.

Virus Native Particle Size DistributionsUpstream Sampling, MS2 & PhiX174 in PBS, Collison Nebulizer, APS 3321 Data

0.0%

1.0%

2.0%

3.0%

4.0%

5.0%

6.0%

7.0%

8.0%

9.0%

0 2 4 6 8 10 12 14 16 18 20

Particle Size (um)

Nu

mb

er

Pe

rce

nt

(%)

MS2 PhiX174

Figure 4: Viral Aerosol Challenge Particle Size

Number Distribution.

The particle size distributions for each bioaerosol

are shown to be within the respirable range for

alveolar region tract lung deposition and show a low

geometric standard deviation (GSD) indicating

monodispersed aerosol challenges were generated

into the test system for each biological. Figure 6

shows a summary of the MMAD and GSD for each

challenge organism.

Endospores Native Particle Size DistributionsUpstream Sampling, B. globigii & A. niger in PBS, Collison Nebulizer, APS 3321

0.0%

2.0%

4.0%

6.0%

8.0%

10.0%

12.0%

14.0%

0 2 4 6 8 10 12 14 16 18 20Aerodynamic Particle Size (um)

Mas

s P

erc

en

t (%

)

A. niger Spores B. globigii Spores

Figure 5: Fungal Spores and Endospore Aerosol

Challenge Particle Size Mass Distribution.

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MMAD and GSD of Disseminated OrganismsUpstream Sampling, Collison Nebulizer, APS 3321 Data

2.33 2.38

1.93

2.63

2.35

1.80 1.79 1.74 1.68

1.931.801.76

0.00

0.50

1.00

1.50

2.00

2.50

3.00

S. epidermitis E. coli MS2 Phage PhiX174

Phage

B. globigii

spores

A. Niger

Spores

Part

icle

Siz

e (

um

)

MMAD GSD

Figure 6: MMAD and GSD of Challenge Bioaerosols

Transformair Bioaerosol Results

Results from the bioaerosol test trials were graphed

and plotted to show the upstream and downstream

viable suspended bioaerosol after passing through the

Transformair unit for each biological organism. The

graphs are based on single pass collection testing of

the Transformair unit and show the averaged in

triplicate test results for each biological challenge.

Upstream and Downstream concentrations are plotted

showing the challenge and filtered viable aerosol

concentrations (Figures 7, 8 9, 10, 11 and 12).

Staphylococcus epidermidis: Upstream & Downstream Viable Concentrations

Upstream & Downstream, cfu/ft3

, TransformAir, Single Pass Efficiency, AGI-30 Impinger Enumeration

9.1E+01

1.8E+02

3.0E+062.5E+06

3.0E+06

3.5E+06

1.4E+021.6E+02

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

T1 T2 T3 Average

Trial Number

Via

ble

Co

nce

ntr

ati

on

cfu

/ft3

(lo

g S

cale

)

Upstream Concentration Downstream Concentrations

Figure 7: S. Epidermidis Transformair Upstream and Downstream Viable Concentration.

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Transformair Vegetative Bioaerosol Results

Bioaerosol challenge tests were conducted for each

organism in three (3) individual test trials. The

averaged test results for the net viable reduction of

airborne S. epidermidis was 4.33 +/ - 0.2 logs (Avg.

+/- STdev) above the challenge bioaerosol

concentration. Figure 7, shows the results of the

control and triplicate Staphylococcus Transformair

trial runs.

The averaged test results for the total viable

reduction of airborne E. coli was 4.91 +/ - 0.23 logs

(Avg. +/- STdev) above the challenge bioaerosol

concentration. Figure 8, shows the results of the

triplicate E. coli Transformair trial runs.

Escherichia coli: Upstream & Downstream Viable ConcentrationsUpstream & Downstream, cfu/ft

3, TransformAir, Single Pass Efficiency, AGI-30 Impinger Enumeration

1.6E+02

7.7E+06 9.7E+06 1.2E+07 9.8E+06

6.8E+011.6E+02 1.3E+02

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

T1 T2 T3 Average

Trial Number

Via

ble

Co

ncen

trati

on

cfu

/ft3

(lo

g S

ca

le)


Figure 8: E. Coli Transformair Upstream and Downstream Viable Concentration.

MS2 Bacteriophage: Upstream & Downstream Viable ConcentrationsUpstream & Downstream, pfu/ft

3, TransformAir, Single Pass Efficiency, AGI-30 Impinger Enumeration

2.1E+043.5E+04

5.5E+084.7E+086.9E+084.8E+08

3.7E+045.5E+04

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

T1 T2 T3 Average

Trial Number

Via

ble

Co

ncen

trati

on

pfu

/ft3

(lo

g S

cale

)


Figure 9: Bacteriophage MS2 Transformair Upstream and Downstream Viable Concentration.

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PhiX-174 Bacteriophage: Upstream & Downstream Viable Concentrations

Upstream & Downstream, p fu/ft3


6.0E+01

1.5E+01

6.9E+058.6E+05

5.1E+056.9E+05

9.1E+01 5.5E+01

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

T1 T2 T3 Average

Trial Number

Via

ble

Co

ncen

trati

on

pfu

/ft3

(lo

g S

ca

le)


Figure 10: Bacteriophage PhiX-174 Transformair Upstream and Downstream Viable Concentration.

Transformair Viral Bioaerosol Results


reduction of airborne MS2 bacteriophage were 4.19

+/ - 0.23 logs (Avg. +/- STdev) above the challenge

bioaerosol concentration. Figure 9, shows the results

of the triplicate E. coli Transformair trial runs.


reduction of airborne Phi-X174 were 4.19 +/ - 0.51

logs (Avg. +/- STdev) above the challenge bioaerosol

concentration. Figure 10 shows the results of the

triplicate Phi-X174 Transformair trial runs.

Aspergillus niger Endospores: Upstream & Downstream Viable Concentrations



2.3E+01 2.3E+01

3.4E+064.1E+06

3.6E+062.6E+06

3.0E+014.5E+01

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

T1 T2 T3 Average

Trial Number

Via

ble

Co

ncen

trati

on

cfu

/ft3

(lo

g S

ca

le)


Figure 11: Aspergillus niger spores Transformair Log Reduction in Viable Concentration.

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Bacillus globigii Endospores: Upstream & Downstream Viable Concentrations



2.3E+01 2.3E+01

2.8E+06

1.6E+06 2.0E+06

2.1E+06

4.5E+01 3.0E+01

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

T1 T2 T3 Average

Trial Number

Via

ble

Co

nce

ntr

ati

on

cfu

/ft3

(lo

g S

cale

)


Figure 12: B. Subtilis Transformair Log Reduction in Viable Concentration.

Transformair Aspergillus Spore Bioaerosol Results


reduction of airborne A. niger were 5.07 +/ - 0.13



triplicate A. niger Transformair trial runs.

Transformair Bg Endospore Bioaerosol Results


reduction of airborne B. subtilis were 4.86 +/ - 0.23



triplicate Bacillus globigii Transformair trial runs.

Avgerage Upstream Viable Concentrations (cfu or pfu/ft3)

S. epidermitis E. coli MS2 PhiX-174 A. niger B. globigii

Trial 1 3.46E+06 7.66E+06 2.63E+06 2.83E+06 4.83E+08 6.94E+05

Trial 2 2.95E+06 9.68E+06 3.62E+06 1.62E+06 6.88E+08 8.60E+05

Trial 3 2.47E+06 1.20E+07 4.08E+06 1.98E+06 4.68E+08 5.13E+05

Average 2.96E+06 9.80E+06 3.44E+06 2.14E+06 5.46E+08 6.89E+05

Avgerage Downstream Viable Concentrations (cfu or pfu/ft3)


Trial 1 91 158 23 23 20,768 60

Trial 2 181 68 23 23 35,469 15

Trial 3 158 158 45 45 54,940 91

Average 1.43E+02 1.28E+02 3.02E+01 3.02E+01 3.71E+04 5.53E+01

Table 3: Summary of Average Upstream & Downstream Viable Concentrations (cfu or pfu/ft3).

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4.58

4.21 4.19

4.68

5.15

4.88

4.29

3.934.06

3.75

5.065.20

4.955.10

4.85

4.644.33

4.91

4.37 4.19

4.76

4.19

5.07 4.86

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

T1 T2 T3 AverageTrial Number

Lo

g R

ed

ucti

on

fro

m U

pstr

ea

m C

on

cen

trati

on

Staph Ecoli MS2 PhiX174 A. niger spores Bg Spores

Net Viable Log Reduction of Bioaersols by TransformAir Unit(Single Pass Effeciency, S. epidermitis, E. coli, A. niger, Bg, MS2 & PhiX-174 , Impinger Collection)

Figure 13: Single-Pass Net Log Reduction of Viable Bioaerosols for Transformair unit.

Estimating Multi-Pass Efficiency

The Transformair unit was tested for single pass

efficiencies; however, the typical use of the device

(either stand alone unit or the HVAC insert) will be

in a re-circulatory mode. Thus it is interesting to

calculate the viable bioaerosol LOG reduction when

used in a typical room or enclosed environment with

the unit operating in a re-circulatory mode. If we

make the assumption of a well-mixed room then the

equation to show the reduction in total room

bioaerosols as a function of time is equal to:

AA C

dt

dCκ−= (1)

Where Ca = Room Concentration in the room at time t k = rate of removal of a by the Transformair Unit

The rate of removal by the Transformair Unit

can also be defined as defined by:

R

T

V

v•

=ε

κ (2)

Where:

ε = the average single pass efficiency of the Transformair Unit

•

ν = the volumetric flow rate of the

Transformair Unit VR = Total Room Volume

By solving the differential equation we obtain

the following solution to the equation.

R

T

V

tv

A

t

AA eCeCC

•

−

−==

ε

κ

00 (3)

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Theoretical Room Concentration vs. Number of Room TurnoversTransformAir Unit, Sealed Mixed Room, Average Single Pass Efficiency = 4.59 LOG reduction

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

0 2 4 6 8 10 12 14 16 18

Number of Room Turnovers

Ro

om

Co

nce

ntr

ati

on

4.0 LOG Reduction in Room

Viable Bioaerosol

Concentration with 9.21

Room Turnovers

6.0 LOG Reduction in Room

Viable Bioaerosol

Concentration with 13.81

Room Turnovers

Figure 14: Theoretical Multi-Pass Efficiency in a Sealed Room

The number of room turnovers is equal to the

Transformair flow rate multiplied by the time of

operation divided by the total room volume. This

reduces the equation eliminating the specific

volumetric flow and total room volume. This makes

it possible to solve for the concentration based on the

number of room turnovers.

R

TV

tvR

⋅=

•

(4)

Where: RT is the number of room turnovers

Substituting in for RT yields the equation used to

calculate the room concentration based on the

Transformair’s tested single-pass average bioaerosol

reduction efficiency. This equation follows an

exponential equation as expected.

TR

AA eCCε−

=0

(5)

The Average single pass efficiency (ε) for the

Transformair Unit was found to be 4.59 LOG

reduction (average of all six tested organisms), which

is equal to 99.99745% reduction in a single pass.

Setting the initial room concentration, CA0, arbitrarily

to unity (value of 1.0) we can graph the reduction in

concentration based on the total room volume

exchanges. Figure 14 shows the theoretical room

concentration as a function of total number of room

turnovers.

From the theoretical calculations (Eq 5) we can

see that it would take approximately 9.21 and 13.81

room exchanges respectively to achieve a 4 LOG and

6 LOG reduction in a multi-pass mode of operation.

To translate this to real world values, lets assume

a room that is 10’ x 10’ x 8’ in size. The total room

volume would be 800 ft3. The Transformair Unit has

a flow rate of approximately 80 ft3/min on the low

fan speed setting. Thus it takes 10 minutes for the

unit to achieve a single room turnover. To achieve

4.0 LOG reduction for the entire room would take

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9.21 room exchanges or 92.1 minutes; for 6.0 LOG

reduction over the entire room volume it would take

13.81 room exchanges or 138.2 minutes. If we

assume that the Transformair efficiency at 160 cfm

(high blower setting) is the same as the low blower

setting then the turnover rate would double for the

example room size given above which would yield

46.0 minutes and 69.1 minutes to achieve a 4 log and

6 log reduction in viable bioaerosols respectively.

Summary of Findings

Test results show that the Transformair system

was extremely effective at reducing viability of

bioaerosols in all conducted trials. The tested

Transformair system is designed for recirculation and

multiple pass purification of room air. The test

results reflect single pass operation results in viable

bioaerosols which showed and average collection and

disinfection result of over four logs of viability for

each organism. Based on these results, it would

infer that the Transformair system would have a

greater collection and reduction of biological aerosols

operating in a re-circulating multi-pass air

purification mode.

The Transformair System’s efficacy of reduction

of S. epidermidis viability = 4.33 +/- 0.22 logs

(average +/- standard deviation).

The Transformair System’s efficacy of reducing

E. coli bioaerosol viability = 4.91 +/- 0.23 log (Avg

+/- STdev).


MS2 bioaerosol viability, were 4.19 +/ 0.23 logs

(Avg +/- STdev)


PhiX174 viable aerosol were 4.19 +/- 0.51 logs (Avg

+/- STdev).


A. niger viable aerosols were 5.07 +/- 0.13 logs (Avg

+/- STdev).


B. Subtilis viable aerosols were 4.86 +/- 0.23 logs

(Avg +/- STdev).

In summary the Transformair unit showed 4 logs

or greater average reduction in viable bioaerosols for

all biological challenges operating in a single pass

mode for all tested organisms. Figure 13, on the

previous page, and Table 4, below, shows the net log

reduction during a single-pass through the

Transformair unit for all trials. Calculations show

that that when operating in multi-pass mode the

Transformair unit, theoretically, should reduce the

total viable bioaerosol concentration in the entire

room by 4.0 logs and 6.0 logs with 9.21 and 13.82

room turnovers respectfully.

Additional calculations for deriving the

Transformair test results are shown in Appendix A of

this report.

Net Log Reduction Summary Chart


Trial 1 4.58 4.68 4.37 4.06 5.06 5.10

Trial 2 4.21 5.15 4.29 4.76 5.20 4.85

Trial 3 4.19 4.88 3.93 3.75 4.95 4.64

Average +/- StDev 4.33 +/- 0.22 4.91 +/- 0.24 4.19 +/- 0.23 4.19 +/- 0.51 5.07 +/- 0.13 4.86 +/- 0.23

Table 4: Summary of Net Log Reduction Single-Pass Efficiencies (LOG reduction).

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References T. Reponen, K. Willeke, V. Ulevicius et al. Techniques of Dispersion of Microorganisms in Air. Aerosol Science

and Technology. 27: 1997. pp. 405-421.

Ding and Wing. Effects of Sampling Time on the Total Recovery rate of AGI-30 Impingers for E. coli. Aerosol and

Air Quality Research, Vol. 1, No. 1, 2001, pp. 31-36.

Flint et al. Principles of Virology. Principles of Virology (ASM). Chapter 2 Virological Methods. Vol. 2. 2008.

J.F. Heildelberg et al. Effects of Aerosolization on Culturabilty and Viability of Gram-Negative Bacteria. Applied

and Environmental Microbiology. Sept 1997, p 3585-3588.

A. Mazzocco et al. Enumeration of Bacteriophages Using the Small Drop Plaque Assay System. Bacteriophages:

Methods and Protocols, Vol. 1: Isolation, Characterization and Interactions. vol. 501. 2009. pp. 81-95.

P Hyman et al. Practical Methods for Determining Phage Growth Parameters. Bacteriophages: Methods and

Protocols, Vol. 1: Isolation, Characterization and Interactions. vol. 501. 2009. pp. 175-201.

A. Furiga, G. Pierre, et al. Effects of Ionic Strength on Bacteriophage MS2 Behavior and Their Implications of the

Assessment of Virus Retention. University of Toulouse. 2007.

Analytical Testing Facility

Aerosol Research and Engineering Labs, Inc.

15320 S. Cornice Street

Olathe, KS 66062

Project #

10814.1

Study Director

Jamie Balarashti

Aerosol Research and Engineering Laboratories

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GLP Statement

We, the undersigned, herby certify that the work described herein was conducted by

Aerosol Research and Engineering Laboratories in compliance with FDA Good Laboratory

Practices (GLP) as defined in 40 CFR, Part 160.

Study Director:

_________________________ __________

Jamie D. Balarashti Date

Study Director

ARE Labs, Inc.

Principal Investigator:

_________________________ __________

Richard S. Tuttle Date

Principal Investigator

Appendix A: Calculations

To evaluate the viable aerosol delivery efficiency and define operation parameters of the system,

calculations based on (theoretical) 100% efficacy of aerosol dissemination were derived using

the following steps:

• Plating and enumeration of the biological to derive the concentration of the stock

suspension (Cs) in pfu/mL or cfu/mL.

• Collison 24 jet nebulizer use rate (Rneb) (volume of liquid generated by the

nebulizer/time) at 35 psi air supply pressure = 1.0 ml/min.

• Collison 24 jet Generation time (t) = 10 minutes.

• Transformair Low setting blower flow rate = 85 cfm

Assuming 100% efficiency, the quantity of aerosolized viable particles (VP) per liter of

dilution air in the system for a given nebulizer stock concentration (Cs) is calculated as:

Nebulizer: tdil

RCV nebs

P

⋅=

11/20/2015

Date

11/20/2015

Date

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• Dilution flow rate (dil ) = 85 cfm or 2405 L/min

AGI – 30 impinger collection calculation:

• Viable aerosol concentration collection (Ca) = cfu or pfu/L of air.

• Viable Impinger concentration collection (CImp) = cfu or pfu/mL from enumeration of

impinger sample.

• Impinger sample collection volume (Ivol) = 20 mL collection fluid/impinger AGI–30

impinger sample flow rate (Qimp) = 12.5 L/min.

• AGI–30 impinger time (t) = 10 minutes.

For viable impinger aerosol concentration collection (Ca) = cfu or pfu/L of chamber air:

tQ

IC

imp

volImp ⋅=aC

The aerosol system viable delivery efficiency (expressed as %) is:

100 V

C

p

a⋅=Efficiency

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ARE Labs Inc. 2015 Project 10805.1 for HIG Industries Inc.. 18 of 24

Appendix B

Raw Data

Plating and Enumeration Tables

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S. epidermidis - Transformair Plating Results

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E. coli - Transformair Plating Results

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MS2 - Transformair Plating Results

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Phi-X174 - Transformair Plating Results

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A. niger spores - Transformair Plating Results

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B. Subtilis Endospores - Transformair Trial #1 Plating Results

determination of the transformair system’s efficacy ...labs+-+bioaerosols... · a flow diagram of...

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