Presented by : Victor BANDALY

EHESP– Rennes & IMT- Atlantique– Nantes

Dr. Aurélie JOUBERT - Dr. Pierre LE CANN - Pr. Yves ANDRES

1. INTRODUCTION

2. MATERIALS AND METHODS

3. RESULTS

4. DISCUSSION

5. CONCLUSION

SUMMARY

INTRODUCTION

3

Air quality is one of the most important topics for public health

Air + bioaerosols

InsideOutside

Micro-organisms- Bacteria-Fungi- Viruses

Respiratory virus :

- High infectivity

- Major public health problem

- Easily transmitted (direct contact, droplets, aerosolization)

-Responsible for absenteeism, loss of working time and high socio-economic cost

INTRODUCTION

4

90% of the time spent is in enclosed spaces (homes, workplaces , gym and transport)

Estimated costs for the French system of care for major respiratory diseases and hospitalizations attributable to air pollution is between 0.9 and 1.8 billion euro/year

(Rafenberg, 2015)

+ -Outdoor Air

Rejeted air

Recovered air

Air conditioned

RecycledAir

Vitiated air Outdoor air

Insufflation of airAir extraction

Ventilator Heatexchanger

Spread and transport of viral particles within buildings

Ventilation System Air Handling Units (AHU)

Filter

5

Understand the fate and persistence of respiratory viruses on filters used in

air handling units (AHU)

MATERIALS ET METHODS : VIRUS AND AEROSOLIZATION

7

Choice of a model of RNA viruses :- Mengovirus (RNA virus / 30 nm ) similar to Rhinovirus

Cellular support of virus replication: Mengovirus BGM cells (Monkey African Green kidney cell)

Detection of virus multiplication Cytopathic effect (BGM cells)

BGM CELL After 30 min After 30 h

BGM cell Infected by Mengovirus(Cytopathic effect)

MATERIALS ET METHODS : EXPERIMENT SET-UP

6

Air pump (generator Omron C29 – CompAir pro) + Suspension of Aerosolized Viruses

(Flow rate 4.5L/min)

Methyl polyethacrylate column

Filter analyzed

No filter

4 cm

BioSampler SKC Flow rate 13 L/min

Fiberglass filter F7 (EN 779-2002)

Laminar Flow Hood

MATERIALS ET METHODS : EXAMPLE OF AN AEROSOLIZATION PROCESS

8

Filter F7Camfil TM7 60+

Aerosolization time = 25 min

Laminar Flow Hood

Quantification and detection

Quantification of persistent viruses on filters and those passing

through filters

BioSampler Filter

Detection of infectious viruses(Cell Culture TCID50 )

Quantification of viruses(qPCR)

Quantification of viruses on filters and those passing through filters

9

F B

Virus culture

TCID : Tissue culture Infective Dose

MATERIALS ET METHODS : QUANTIFICATION AND DETECTION OF

AEROSOLIZED VIRUSES

MATERIALS ET METHODS : AEROSOLS CHARACTERIZATION

Scanner Mobility Particle Size

(SMPSTM, TSI)

Electrical Low Pressure Impact (ELPITM, DEKATI)

Aerodynamic size

Electric mobility size

10

10 nm – 1 µm

7 nm – 4 µm

Validation by fluorescein (CAS: 518-47-8, Merck KGaA) : Mass balance = 98,72%

Validation by qPCR of Mengovirus : Efficiency = 81%(Initial solution aerosolized of 5,24 1010 GU and 4,24 1010 GU of viral particles recovered)

Evaluation of the effectiveness of the system

Initial sol = 0.795 mg

Rest not aerosolized = 0.064 mg

BioSampler recovered = 0.143 mgFilters recovered = 0.454 mg

Not recovered = 0.13 mg(column + pipes)

(Bailly et al., 2001)The filter F7 shows efficiency between 83,8 and 98,5 %.

Filter 1 Filter 2 Filter 3

Exp.1 efficiency 76,41 98,49 87,81

Exp.2 efficiency 94,73 96,07 91,19

Exp.3 efficiency 92,13 99,70 89,96

Efficiency of filtration = qty collected / qty up-stream

12

Evaluation of the filtration efficiency

RESULTS

T°C constante and RH% (min 40 % - max 89%) in the system

13

Aerosols characterization of the system

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

10 100 1000 10000

ELPI_BGM_blanc

Mengo

d(N)/d[log(dp)]

d. Aerodynamic (nm)

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

10 100 1000

SMPS_BGM_Blanc

Mengo

d(N)/d[log(dp)]

d. Electric mobility (nm)

d50 ELPI_blanc 52,6 nm

d50 SMPS_blanc 87,15 nm

d50 ELPI_Mengo 80 nm

d50 SMPS_Mengo 76,52 nm

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

7 28.4 56 92.8 154 261 382 612 947 1600 2390 3950

ELPI Mengo (L-1 air)

ElLPI Blanc

qPCR (UG. L-1 air)

(nm)

- Virus can not be aerosolized as single particle

- Aerosolized as groups of viruses (more than 3 viruses) - Associated to proteins or cells debris present in the medium

Mengovirus size = 30 nm

RESULTS

Limit detection

14

Viral quantification (qPCR) /

1 log virus lost in the system According to the fluorescein experiment:It is assumed that the loss is on the column and pipes

GU : Genomic unit

Quantity (UG.L-1) Quantity (UG.cm-2)Initial 6,68.107

Fltr.1 3,48.106

Fltr.2 3,02.106

Fltr.3 2,71.106

Bios.1 4,61.105

Bios.2 2,43.105

Bios.3 4,90.105

Bios.4 5,33.106

RESULTS

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

Initialsol.

Fltr.1 Fltr.2 Fltr.3 Bios.1 Bios.2 Bios.3 Bios.4

Quantity (UG.cm-2)

Quantity (UG.L-1 air)

14

Evaluation of viral infectivity (TCID)

TCID : Tissue culture Infective Dose

Quantity (TCID.L-1) Quantity (TCID.cm-2)Initial 4,00.108

Fltr.1 3,26.102

Fltr.2 4,23.102

Fltr.3 1,59.102

Bios.1 3,77.101

Bios.2 2,13.101

Bios.3 3,91.101

Bios.4 8,48.102 1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

Initialsol.

Fltr.1 Fltr.2 Fltr.3 Bios.1 Bios.2 Bios.3 Bios.4

Infectivity (TCID.cm-2)

Infectivity (TCID.L-1 air)

1,0.102 TCID.L-1 of infectious mengovirus pass through the filter (BioSamplers)

1,0. 103 TCID. cm-2 of infectious mengovirus are recovered on the filter

Lost of ≈ 5,6 log of viruses infectivity between initial solution and BioSampler

Remaining viruses are infectious upstream and downstream of the system

Vir

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(q

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Ev

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f vi

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(TC

ID)

RESULTS

Comparison : Quantification (qPCR) / Infectiosity (TCID)

On the filter : 106 GU.cm-2 of Mengovirus was detected whose 102 PFU. cm-2 are infectious

In the BioSampler: 105 GU.L-1 of Mengoviruswas detected whose 101 PFU.L-1 are infectious

Loss of 4 log of infectivity1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

Fltr.1 Fltr.2 Fltr.3 Bios.1 Bios.2 Bios.3 Bios.4

Infectivity (PFU.cm-2)

Infectivity (PFU.L-1 air)

Quantity (GU.cm-2)

Quantity (GU.L-1 air)

Persistence of Mengovirus on filters as a function of time

Aerosolization time : 25 min - 24h(25mn-50mn-75mn-6h-10h-24h )

Mengovirus remains infectious on the filter until 10 hours after aerosolization

Continuous air flow in the system

Average Initial 25 50 75 360 600 1440Infectivity (PFU.L-1) 4,43.108 3,43.102 6,27.103 2,11.103 1,63.103 1,82.101 0

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

Initial 25 50 75 360 600 1440

Infectivity (PFU.cm-2)

15

16

Understanding the behavior and persistence of adenovirus on F7 filter in air handling units

The system was validated by fluorescein and is ready to study the aerosolization of viruses

Effect of aerosolization on virus infectivity: loss of 4 log compared to initial solution

A model of DNA respiratory virus, the adenovirus, was tested with the same process

Work in progress :

Viruses pass through filters and remain infectious

Viruses stopped by the filter remain infectious

Effect of time: No infectious virus after 10h of aerosolization on the filter

The virus can not be aerosolized as single particle, it’s associated to other viruses or cell debris or protein

CONCLUSION

The Labrotory team works on bioaerosols

(Bacteria, viruses and fungal)

Pr. Yves ANDRES (co-director): yves.andres@imt-atlantique.fr

Victor BANDALY (PhD student): victor.bandaly@imt-atlantique.fr

Dr. Aurélie JOUBERT : aurelie.joubert@imt-atlantique.fr

Dr. Pierre LE CANN (co-director): pierre.lecann@ehesp.fr