Development of Disinfectant Efficacy Test Methods for Acanthamoeba

1
Development of Disinfectant Efficacy Test Methods
for Acanthamoeba

• Simon Kilvington, PhD
• Advanced Medical Optics
• University of Leicester

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Acanthamoeba
Cyst
Trophozoite

• Feeding dividing
• Asexual
• Cyst forming

• Response to adversity
• Dormant, resistant
• Double-walled with pores

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Need for standardisation of Acanthamoeba efficacy
testing

• Look to ISO 147292001 (bacteria and fungi)
• Test strain
• Method of culture and storage
• Assay method
• Presentation of results
• All apply equally to Acanthamoeba disinfectant
  testing

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Method 1 Qualitative 50 Challenge Inoculum

• 5 ml of test solution (x3 neutraliser control)
• Add 50 Acanthamoeba (50 µl)
• Incubate room temperature for 6 hr
• Neutralise (5 ml tween-lecithin E. coli)
• Centrifuge 1000 x g 10 min
• Culture pellet in well of NNA plate
• Incubate 28-32ºC 14 days

BD Falcon
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Method 1 50 Challenge Inoculum
3 days
5 days
7 days
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Method 1 Results
survival and growth of Acanthamoeba -
kill of Acanthamoeba NC disinfectant
neutraliser control
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Method 1

• Pros
• Relatively simple to perform
• Good for rapid screening for disinfectant and
  drug discovery
• Can assess experimental variables species or
  strain, inoculum size, exposure time, presence of
  contact lens, organic soil, etc.
• Cons
• Only qualitative, testing total kill (50 org or
  1.7 log)
• Does not measure rate of kill

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Method 2 QuantitativeTime Kill Assay

• 5 ml solution (x3 with neutraliser control)
  challenged to give 5 x104 /ml trophozoites or
  cysts
• Aliquots removed at 0, 1, 2, 4, 6 24 hr
• Neutralised and serial 10-fold dilutions made
  across rows of 96 well microtitre plate
• Live E. coli added and plates incubated 32ºC 14
  days
• Presence or absence of growth recorded and rate
  of kill or survival determined by Most Probable
  Number method using Spearman-Karber computation

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Acanthamoeba Challenge Testing
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2
1
Neutralise titrate

• T0 T24 neutralise 10-fold dilutions in ¼
  Ringers (1000 1)
• Add E. coli
• Incubate record excystment / trophozoite
  replication
• Spearman-Karber computation
• Delta-log plot

Acanthamoeba
Test solution
180 µl tween-lecethin neutraliser in outer rows
and ¼ Ringers in rest Take 4 x 20 µl of sample,
add to neutraliser row and then serial dilutions
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Acanthamoeba Challenge Testing
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A. castellanii (50370) Trophozoites
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Efficacy of Contact Lens Disinfectants Against A.
castellanii (50370) Trophozoites (6 hr)
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Trophozoite Strain (2000-2008)
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Effect of culture medium on trophozoite kill (6
hr)
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Cyst Testing

• Results dependent on strain and method of cyst
  production
• NNA-E. coli (poor yield, co-contamination with
  bacterium and clumping)
• NNA-Taurine (poor yield and significant clumping)
• Starvation in axenic medium (mixture of trophs,
  immature and mature cysts)
• Axenic medium with Mg2 (high yield but mixture
  and increased susceptibility to some MPS)
• Neffs constant pH encystment medium (high yield,
  gt99 mature form but increased susceptibility
  to some MPS?)

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Encystment Method
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A. castellanii (50370) Neff Cysts
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Method 2

• Pros
• Reliable and reproducible determination of
  Acanthamoeba trophozoite and cyst disinfection
  rate and extent
• Cons
• Although relatively simple to perform, requires a
  level of expertise and understanding beyond that
  of bacterial or fungal testing
• Requires large numbers of axenic trophozoites and
  cysts
• Results may be dependent on method used to
  produce cysts

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Acanthamoeba Regimen Testing

• Criteria as per ISO 147292001 for bacteria and
  fungi
• Inoculate Acanthamoeba (105) on to contact lens
• Regimen rub rinse, no rub but rinse or no
  rub or rinse (all with soaking step)
• Quantify remaining organism on lens and in
  soaking solution
• Expectation that Acanthamoeba trophozoites or
  cysts will be removed following manufacturers
  recommended regimen protocols

Kilvington, S. Lonnen, J. (2009). A Comparison
of Regimen Methods for the Removal and
Inactivation of Bacteria, Fungi and Acanthamoeba
from Two Types of Silicone Hydrogel Lenses.
Contact Lens Anterior Eye (in press).
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Recommendations Trophozoites

• Need for standardisation of Acanthamoeba
  disinfectant testing and should focus on
  trophozoites
• Two methods are presented that enable the
  efficacy of contact lens care solutions to be
  assessed uniformly
• Use A. castellanii ATCC 50370 (human eye
  infection, New York, NY, 1978 ) as reference
  strain
• Culture using Ac6 medium to log phase (75
  confluence in flask)
• Standardise tubes used for testing

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Recommendations Cysts

• Need for more work to achieve standardisation of
  Acanthamoeba cyst disinfectant testing
• Two methods presented that enable cysticidal
  efficacy of contact lens care solutions to be
  assessed
• Use A. castellanii (ATCC 50370) as reference
  strain
• Prepare cysts using Neffs Encystment Medium from
  late log phase trophozoites grown in Ac6 medium
• Need for an improved liquid encystment medium
  producing cysts with resistance comparable to
  those from NNA-E. coli or NNA-Taurine methods

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Conclusion

• Many contact lens disinfectant solutions on the
  market are not effective against Acanthamoeba
• No evidence to indicate this is a risk factor for
  acanthamoeba keratitis
• However, Industry should develop new solutions
  with efficacy against Acanthamoeba
• This will require standardised assay methods

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Recommendations

• Need for more work to achieve standardisation of
  Acanthamoeba disinfectant testing
• Work together towards developing standards
  acceptable to the Industry
• Establish workshops and reference centres for
  assay development and training
• e.g. University of Leicester (Simon Kilvington
  sk46_at_le.ac.uk or James Lonnen jdl3_at_le.ac.uk)
• Undertake quality assurance testing (similar to
  CLI Ring Test undertaken for bacteria and fungi)

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Acanthamoeba Physiological Response to Contact
Lens Solutions
Encystment
Aggregation
Multipurpose solution induced encystment and
aggregation of Acanthamoeba trophozoites
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Acknowledgements

• James Lonnen and Wayne Heaselgrave, University of
  Leicester, England
• John Lally and colleagues at Advanced Medical
  Optics, CA, USA
• The contact lens care industry that has supported
  my research over the years