Aerosol Generation, Ventilation and Risk

Assessment

CDC Biosafety Symposium 2014

Contents • Laboratory Acquired Infection and Aerosols (1910-79). Where the

regulations came from

• How many Pascals? The Laboratory Protection Factor

• An introduction to Microbial Aerosols

• Aerosol Generation in the Modern Microbiology Laboratory

• Gloves and laboratory discipline

2 BEDA Business plan

Laboratory Acquired Infections (1910-50) -

Aerosols & Outbreaks • A Sharples centrifuge used for centrifuging live Brucella organisms leading to

45 clinical cases and one death was located in the hallway of the basement.

• 20 lab workers infected with VEE when 9 freeze dried ampoules dropped

• Numerous Q fever outbreaks associated with buildings centrifuging and

blending infected eggs

• 3 died of glanders following centrifuge accident

• 11 cases of typhus due to intranasal infection of mice

• Many of these associated with biological weapons research. High titre, high

aerosol risk.

Laboratory Acquired Infections (Pike 1976)

CAUSE

Accident 17.9

Animal or

ectoparasite

16.8

Clinical Specimen 7.3

Glassware 1.2

Autopsy 1.9

Intentional

Infection

0.5

Aerosols (known) 13.3

Work with agent 21.1

Others, unknown 20.0

AGENT

Brucellosis 426 (5)

Q fever 280 (1)

Hepatitis 268 (3)

Typhoid fever 256(20)

Tularemia 225 (2)

Tuberculosis 194 (4)

Typhus 124

Psittacosis 116 (10)

Leptospirosis 78

Streptococci 87

Smallpox Lab 1978

Aerosols, Aerosols, Aerosols

The focus on lab safety was now firmly on prevention of aerosol transmission as:

The report concluded an aerosol route of infection

The distance this aerosol had to travel

The lab contained a malfunctioning cabinet

The tragic outcome

Control measures can be monitored

Therefore as regulations were developed it was focussed on preventing a recurrence

Decency prevented focussing on the human factors behind the outbreak

Overwork

Poor training

Lack of separation between safety and management

Rumours of a relationship between the professor and the victim

Negative Pressure – How many

Pascals?

7 Detailed Design

Introduction • How many Pascals?

• Do I need an anteroom?

8 Detailed Design

How Many Pascals Do I Need?

• In a UK survey a range of 10-150Pa was

encountered

• Imploding laboratories

• UK Guidance (ACDP) -40Pa, -70Pa for Cat 4

• Calibration

• Testing of aerosol release during entry and exit

from laboratories was undertaken using the

Potassium Iodide method

Detailed Design 9

When a Laboratory Is At Negative Pressure To The

Rest of The Building And The Doors Are Closed No

Microbial Aerosol Will Be Released Into The Building

Aerosol Release Will Only Occur When The Lab Doors

Are Opened

Laboratory Protection Factor

(LPF)

LPF = Laboratory aerosol Released aerosol

LPF v DP for Lab Entry

Maximimum Pressure Differential (Pa)

20 40 60 80 100 120 140

Labora

tory

Pro

tecti

on F

acto

r

104

105Anteroom LPF

was less

than 4.4 x

102

LPF for lab

without

anteroom

1.5 x 103

Detailed Design

Volumetric Inflow (m3sec-1)

0 2 4 6 8 10 12 14 16 18

Lab

ora

tory

Pro

tecti

on

Facto

r

104

105Operational Laboratory

Experimental Laboratory

LPF v Inflow for Lab Entry

Detailed Design

LPF v Inflow for Anteroom Entry

Laboratory Inflow (m3min-1)

0 2 4 6 8 10 12 14 16 18

Lab

ora

tory

Pro

tect

ion F

acto

r

103

104

105

106

Anteroom, Cat 3 Lab

Corridor, Cat 3 Labs

Anteroom, Experimental Room

Corridor, Experimental Room

Detailed Design

Conclusions of Study Laboratory Protection Factor is a useful measure of containment

LPF is proportional to inflow velocity and not pressure differentials

Anterooms increase containment 10 -100 fold

An inflow of 10m3/min through a standard laboratory door (0.17m3/sec) gives

LPF of ca105

Detailed Design 15

Aerosol Generation • The modern BSL3 laboratory is designed to control workers being exposed

to aerosols and aerosols leaving the facility.

• This governs requirements for

• Filtration

• Negative pressure

• Safety cabinets

• While we can measure the protection afforded by these measures to ensure

they are adequate we need to know something about aerosols generated

within the microbiology laboratory

16 Detailed Design

Introduction to Aerobiology –

Deposition & Exposure Deposition Velocity (u) = dp

2g

18

The deposition velocity is directly proportional to the particle diameter squared

Particle size also influences deposition in the respiratory tract

V=4/3πr3

The cube rule. The number of micro-organisms in a 10 micron particle will be 1000 times those in a 1 micron particle

10 micron particle has volume 1.3x 10-9 mls

Aerodynamic Particle Diameter (microns)

0.1 1.0 10.0 100.0

Depositio

n V

elo

city (

cm

/sec)

0.0001

0.001

0.01

0.1

1

10

100

Accident

(109 spore/ml

suspension)

Aerosol

Generated

(cfu/l of air)

Centrifuge Rotor

Leak*

23.0

Flask Break in

Shaking Incubator*

1.15

Dropping Large 2l

Bottle*

13.7

15ml Spill from 1m* 2.07

•A direct relationship

was found between

titre and aerosol

concentration. The

lower the titre the

less likely is that

significant aerosol

exposure will occur

•50% of aerosol

particles were less

than 3 microns

Where do aerosols come from ?

Bennett & Parks (2006)

Detailed Design 18

Accident

(109 spore/ml

suspension)

Aerosol

Generated

(cfu/l of air)

CONTROL

Centrifuge Rotor Leak* 23.0 SEALED ROTOR

Flask Break in Shaking

Incubator*

1.15 USE

PLASTICWARE

Dropping Large 2l

Bottle*

13.7 USE

PLASTICWARE

15ml Spill from 1m* 2.07 WORK IN

CABINET

Where do aerosols come from

and how do we prevent them

Bennett & Parks (2006)

Detailed Design 19

Participant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

To

tal A

vera

ge

CF

U

0

10

20

30

40

50

60

Ave

rag

e T

ime

Ta

ken

(S

eco

nd

s)

0

100

200

300

400

500

Experienced

Inexperienced

Average Time Taken

Aerosol Generation from Serial Diluting a Spore Solution

Aerosol Generation from Pipetting

Detailed Design 20

Visual Contamination for Serial Diluting Procedure

Participant Surface Gloves

Experienced 1 N.D. N.D.

2 2 N.D.

3 N.D. N.D.

4 N.D. N.D.

5 N.D. N.D.

6 N.D. N.D.

7 N.D. N.D.

Inexperienced 1 >10 N.D.

2 N.D. N.D.

3 N.D. N.D.

4 N.D. N.D.

5 1 N.D.

Detailed Design 21

Aerosols from Plating Out

Participant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Tot

al A

vera

ge C

FU

0

20

40

60

80

100

120

140

160

Ave

rage

Tim

e T

aken

(S

econ

ds)

0

100

200

300

400

500

600

700

800

Experienced

Inexperienced

Average Time Taken

Aerosol Generation from Plating Out and Spreading a Spore Solution

Detailed Design 22

Visual Contamination for Plating Out Procedure

Participant Surface Gloves

Experienced 1 9 N.D.

2 >10 N.D.

3 >10 3

4 5 N.D.

5 7 N.D.

6 7 N.D.

7 9 1

Inexperienced 1 >10 N.D.

2 8 N.D.

3 2 N.D.

4 5 2

5 >10 N.D.

Detailed Design 23

Participant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

To

tal A

vera

ge

CF

U

0

10

20

30

40

50

60

Experienced

Inexperienced

Aerosol Generation from Serial Diluting a Spore Solution

Containment Level 3 Trained Non Containment Level 3 Trained

Detailed Design 24

Conclusions • In most cases aerosols should not be generated in a laboratory

• Any aerosols should be contained in primary containment

• To create a significant aerosol you need a high titre agents

• Unrecognised surface contamination occurs

• Training can reduce the extent of aerosol and surface contamination

25 BEDA Business plan

Gloves and Laboratory Acquired

Infection

26 Detailed Design

Causes of Laboratory Acquired Infections

1995-2010 Davies review to be published

Cause Number %

Needlestick/accidental inoculation 18 27

Open Bench working (no clear

exposure)

12 18

Aerosol exposure suspected 8 12

Contact/Ingestion/mouth pipetting 18 27

Unknown/no information given 7 10

Improper Inactivation 3 5

Detailed Design 27

Clinical cases of hep B/100 000 persons and year ((health care staff, upper line, and general population 16-64 years, lower line.

0

20

40

60

80

100

120

140

160

1969 71 73 75 77 79 81 83 85 87 89 91 93

health care staff

vaccination

Ng et al 2011 Bacterial contamination of hands and the environment in a microbiology laboratory

Journal of Hospital Infection Volume 78, Issue 3 231-233

Six episodes of MRSA

contamination were detected from

20 hand-washing episodes where

the technician did not use latex

gloves, as compared to one MRSA-

positive culture from 51 hand-

washing episodes where he or she

used latex gloves (risk ratio 15.3,

P < 0.05).

No potential pathogens were

cultured from any hand episodes

following hand washing.

Gloves Prevent Direct Contact

Infection

Detailed Design 28

Laboratory Discipline & Biosafety Compliance –

Someone is watching you (BSL2)

Compliance with Glove Use

Only 46% of staff removed

gloves on leaving BSL2 lab

Hand hygiene compliance

before exiting laboratory was

10.3% (0-85%) in labs.

Training was not predictive

Face Touching 72% touched face during study

3.4 contacts per hour

Wide variation between labs

Nose> Forehead> Cheek>

Mouth>Eye

Face touching linked to vaccinia and meningitidis lab infections

Detailed Design

James Johnston, University of Utah, ABSA conference

2010. 29

Outbreak of Salmonella Typhimurium Infections

Associated with Exposure to Clinical & Teaching

Microbiology Laboratories CDC April 28th 2011

Illnesses identified among students in teaching laboratories and employees in clinical microbiology laboratories.

Ill persons (60%) were significantly more likely than control persons (2%) to report exposure to a microbiology laboratory in the week before the illness began.

multiple ill persons reported working specifically with Salmonella bacteria in microbiology laboratories.

The outbreak strain was indistinguishable from a commercially available Salmonella Typhimurium strain used in several laboratories associated with people infected with the outbreak strain.

Several children who live in households with a person who works or studies in a microbiology laboratory have become ill with the outbreak strain.

Detailed Design 30

CONCLUSIONS • Aerosols are not the only source of infection. Designing facilities to prevent

aerosol transmission is very expensive

• Since the regulations were written aerosol control methods have become

widely available

• We still have problems in the developed world with compliance to simple

cost effective protective measures

• We need to educate, train and ensure compliance in good microbiological

and biosafety practise

• Aerosols can also be avoided by good practise

Detailed Design 31

Questions?

32