Determining the performance of BSL-3 laboratory HEPA air filtration systems for reducing airborne contamination using model microorganisms

Transcription

1 Determining the performance of BSL-3 laboratory HEPA air filtration systems for reducing airborne contamination using model microorganisms ZhanBo Wen State key laboratory of pathogen and biosecurity Beijing Institute of Microbiology and Epidemiology

2 Main contents Introduction Materials and Methods Results Discussions Acknowledgements

3 Introduction (Laboratory biosafety manual, WHO, Third edition, 2004)

4 BSL-3 laboratory is designed and provided for work with risk group 3 microorganisms and with large volumes or high concentrations of risk group 2 that pose an increased risk of aerosol spread.

5 Introduction BSL-3 and BSL-4 laboratory are designed and provided for work with risk group 3 and group 4 microorganisms. (Kaiser, 2007, Science, Vol317, )

6 Introduction Emerging and re-emerging infectious diseases: SARS, H5N1 avian influenza, H1N1 influenza. China is a major contributor to the worldwide infectious disease burden because of its population size. Following the 2003 SARS epidemic, the concept of aerosol transmission was so well accepted. After that China has been built several BSL-3 laboratories.

7 Introduction High-efficiency particulate air (HEPA) filtration system of BSL-3 lab was to reduce airborne contamination to avoid microbial aerosol leak to the environment. The attempts to evaluate the respiratory protection equipments performance directly with biologic particles have been done using airborne bacteria and virus. (Brosseau1997, Reponen 1999, Balazy 2006, Eninger 2008, Wen 2010) But there is a lack of direct measurement data on the efficiency of BSL-3 laboratory HEPA filtration system against aerosolized biological particles.

8 Aim of this study To ensure there was no environmental leak risk of microbial aerosol through the BSL-3 laboratory air filtration system actually in a working laboratory using biological aerosol methodology.

9 Materials and Methods Test organism Serratia marcescens (0.7μm 1.0μm) one of the smallest bacteria was used as test organisms.

10 Materials and Methods BSL-3 laboratory air filtration system The BSL-3 laboratory HEPA filtration systems had two HEPA filters. First HEPA filters were in BSL-3 laboratory and distributed in each contaminated lab. The exhausted air through the first HEPA filters were aggregated together and filtrated through the second HEPA filter before exhausting into the out air. So the contamination air was ventilated out through two HEPA filter system to avoid the leak of the microbial aerosol.

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12 Materials and Methods Size of aerosol particles A multistage Andersen sampler was used to determine the size of S. marcescens aerosol particles. The size of particle collected was determined from the presence of growth on each of the plates used. Stage First Second Third Fourth Fifth Sixth Particles distribution (μm) >7 4.8~7 3.4~ ~ ~ ~1.0

13 Materials and Methods Aerosol challenge to test filters Inside lab The first HEPA filter Lab wall Outside lab Wind direction Nebulizer Wind chanel Upstream biosampler Downstream biosampler Figure 2 Test rig of BSL-3 lab HEPA air filtration system filtration efficiency against bacterial aerosol

14 Materials and Methods Aerosol challenge to test filters 1) DV40 nebulizer with S. marcescens suspension was aerosolized by applying compressed air to the nebulizer at 10L/min. 2) The bacterial aerosol was generated before the filter in the BSL-3 lab. At the upstream and downstream of the first HEPA filter and the second HEPA filter, air samplers were used to sample the air to detect the model bacterial aerosol concentration. 3) AGI-30 air sampler was used to collect the air before filtration, the flow rate was 12.6 L/min, 10min.

15 Materials and Methods 4) Merck100 air sampler was used to collect the air after filtration of the first HEPA and the exhaust of the second HEPA filter, the flow rate was 100L/min, 5min. 5) The collecting samples were incubated at 30 for 24h, counted the colonies on the agar plates and then transferred the result to the concentration of CFU (colony forming unit ) per liter. The filtration efficiency was determined by the aerosol concentration upstream and downstream of the tested HEPA filters.

16 Materials and Methods How to calculate Filtration efficiency The percentage efficiency of the HEPA filter was calculated using the following formula, where A was the concentration of bioaerosol challenging the HEPA filter and B was after filtration. S. marcescens was determined in terms of cfu/liter. Efficiency (%)=1-B/A 100%

17 Results Particle size distribution of S. marcescen aerosol 19% 1% 2% 8% 24% 46% >7μ m μ m μ m μ m μ m μ m Fi gur e 3 Par t i cl es si ze of S. mar cescens aer osol Count median diameter (CMD) of S. marcescen aerosol was 1.77μm

18 Results Table 2 Filtration efficiency of the first HEPA filter of BSL-3 filtration system HEPA filter Challenge concentration of bacterial aerosol (cfu/l) Concentration of bacterial aerosol after the first HEPA filters (cfu/l) Filtration efficiency of the first HEPA filter (%) 1# 4099 <0.002 > # 5207 <0.002 > # # 5324 <0.002 > # 2376 <0.002 > # # 3600 <0.002 > # 2280 <0.002 > Note: After replaced 3# filter and airproofed 6# filter fixed bracket no Serratia marcescens was detected in the filtration air, the filtration efficiency were > %.

19 Results Efficiency of second HEPA filter S. marcescens aerosol was not detected after filtrating through the second HEPA filter at each testing and filtration efficiency was > %.

20 Discussion Besides safe microbiological techniques, primary barriers (safety equipment and personal protective equipment) and secondary barriers (facility safeguards) are now regarded as vital elements of containment measures. Air filtration system with HEPA filters is one of the most important secondary barriers to prevent the escape of infectious agents from the working place of the BSL-3 lab to the environment.

21 Discussion There are no reports to evaluate the air filtration system of BSL-3 lab with the deliberate release of bacterial aerosol actually in a working laboratory setting. Due to aerosol safety issues involved with the generation of high bacterial aerosol concentrations, the method uses nonpathogenic microorganisms. In this study, a bacterial model (S. marcescens) was used to test the efficiency of air purification system of BSL-3 lab. S. marcescens has been used previously as model bacterial aerosol because of its aerosol stability. (Furuhashi 1981, Holton 1994, Heidelberg 1997, Barker 2005, Ko 2007, Patel 2008)

22 Discussion In our past study Merck 100 sampler collection efficiency was higher than AGI-30 when the concentration of the aerosol was at low level (Yang 2009). So Merck 100 air sampler was used to collect the air after filtration of the HEPA, the flow rate was 100L/min, 5min, so the testing limit was cfu/l(2 cfu/m3). In the BSL-3 lab the Serratia marcescens aerosol concentration was very high and mimicked the serious contaminated status and no Serratia marcescens was detected in the exhaust air.

23 Discussion This test method not only evaluated the HEPA filters but also the installation process such as: airproof of the filter fixed bracket, airproof glue, fixed screw and whether the HEPA filter was damaged during the installation. In the future research when evaluating BSL-3 lab doing viral research we will try to use viral model aerosol.

24 Acknowledgements The authors thank Dr Sun Zhenhai for technical assistance for this work. This work was supported by the National science and technology support program of China (No. 2008BAI62B05) and National importance infectious disease program of China (No. 2009ZX ).

25 Thank you for your attention!

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