B. anthracis

Dead or alive on your slide?

Dr Suzanna Hawkey, Senior Microbiology Trainer Novel and Dangerous Pathogens Training, PHE Porton

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Overview

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- UK handling requirements

- Suspicion of anthrax in animals

- Tasked to produce control and proficiency slides

- Biosafety considerations

- Validation

- Summary

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UK handling requirements

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- Categorisation

- UK Animal Health Regulations

- Anti-Terrorism, Crime & Security

(ACTSA)

- Biocontainment

B. anthracis - RG3

Intact pathogen

GMO & attenuated pathogen

Nucleic acid

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2006 2008 2009 – 2013 2015

Anthrax in the UK

Inhalational anthrax

Injectional anthrax

1970s -

2006

Sporadic

animals

cases

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NADP Training

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B. anthracis microscopy

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‘

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Viability of B. anthracis material on microscopy slides following treatments

Blood culture microscopy

Heat fixation 70˚ C

Heat fixation

(70˚ C) minimum

2 minutes

Heat fixation

(85˚ C) minimum

2 minutes

Alcohol fixation

(1 minute 95%

methanol)

100% (9/9) 11.1% (1/9) 0% (0/9 )

B. endophyticus B. anthracis

Gram 11.1% (1/9 ) 0% (0/9)

M’Fadyean 100% (9/9 ) 100% (9/9)

Methodology informed by Blackwood et al . 2005

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Westbury cases

Veterinary procedures:

Sudden death of cow

Blood swabs and blood films taken

Suspicious stained blood film

Premises placed under restriction

Incineration of carcase

Referral of samples to Rare and

Imported Pathogens Laboratory

(RIPL):

Unstained and stained slides

Swabs extracted for PCR and cultured

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Control and proficiency slides

Large scale production of blood films representing:

- low and high concentrations of B. anthracis (x600)

- Low B. anthracis mixed with Cl. perfringens (x200)

- Negative slides (x650)

Cl. septicum, Cl. novyi, Cl. perfringens

- Blood no organisms present (x500)

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Rendered safe?

Sterilisation – complete destruction or elimination of microbial viability

including spores

Previous methods involved:

- formaldehyde fumigation followed by

- dry heat 2 hours 160˚ C

Surrogates do not always predict the behaviour of target organisms

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Procedure

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Blood culture Bacteria on gel plug Re-suspended in formalin

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Formalin fixation Re-suspend in blood Slide production

Formalin

overnight and

centrifugation to

remove formalin

Adjust for low

and high

concentrations

Large scale and

addition of Cl.

perfringens for

mixed slides

BSC III BSC III BSC III

BSC III BSC I BSC I

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Biosafety - principles

Eliminate Biological, Chemical, Thermal, Ergonomic, Sharps

Reduce Volume / titre, delivery of chemical, time

Isolate Primary containment (BSC III & I), centrifugation

Control Equipment, procedure, staff

“The application of knowledge, techniques and equipment

to prevent personal, laboratory and environmental

exposure to potentially infectious agents or biohazards’.

Validation

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Confirmation of method suitability:

- Formalin fixed material provided capsule visualisation

Testing inactivation method:

- 1ml 108 CFU ml-1 B. anthracis

- Resuscitate formalin fixed concentrated bacteria in broth overnight

- Culture to plates and monitored for 1 week

- Residual formalin? (wash, tube transfer, dilution and broth dilution)

- Spiked formalin fixed material

Confidence in inactivation – 100% of material sterility tested

then 50% and now 10% on every batch

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Summary

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Biosafety principles used to re-examine methods

- Reduce risk of spore formation

- Inactivate concentrated pathogen prior to slide production

Successful production of control and proficiency slides

- Growth and capsule production from low inoculum blood culture

produced shorter chains

Validated procedure for distribution

- Reproducible method, validation agreed for distribution

- Control of subsequent handling

Acknowledgements

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References and images

Blackwood, K. S., Burdz, T. V., Turenne, C. Y., Sharma, M. K., Kabani, A. M., & Wolfe, J. N. (2005).

Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a

containment level-III laboratory as part of a Laboratory Risk Assessment Program. Bio Med Central

Infectious Diseases, 5, (4).

B. anthracis images – NADP Training, PHE Porton

Cow blood smears – RIPL, PHE Porton

PHE Biosafety Programme Lead

• Heather Sheeley

NADP Training

• Prof. Nigel Silman

• Dr Jane Shallcross

• Amber Lansley

• Ben Gannon

• Dr Christopher Logue

• Clare Shieber

• Sara Fraser

Biosafety

• Allan Bennett

• Simon Parks

Rare & Imported Pathogens Laboratory

• Dr Andy Simpson

• Jason Busuttil

• Daniel Carter

Diagnostic Support

• Angela Sweed

• Anthony Crook