Physiological Response of Acanthamoeba to Contact Lens Disinfectants

1
Physiological Response of Acanthamoeba to
Contact Lens Disinfectants

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

2
Acanthamoeba
Cyst
Trophozoite

• Feeding dividing
• Asexual
• Cyst forming

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

3
Robustness Studies Organic Soil and Contact
Lenses
1 lens per 3 ml test solution for 4 days NC
Neutraliser control survival and growth of
Acanthamoeba
4
Incidence of AK in CLW
Cases per million lens wearers USA (1989)
1.36 Stehr-Green et al., 1989. The epidemiology
of Acanthamoeba keratitis in the United States.
American Journal of Ophthalmology 107331-6. USA
(2000) 10 (based on non-culture
methods) Mathers, et al.,. 2000. Confirmation of
confocal microscopy diagnosis of Acanthamoeba
keratitis using polymerase chain reaction
analysis. Arch Ophthalmology 118178-183. Holland
(1997) 3.06 Morlet, et al., 1997. Incidence of
acanthamoeba keratitis associated with contact
lens wear. Lancet 350414-416. UK (2002) 17.53
-21.14 (70 cases / annum) but may be 33
higher Radford, et al., 2002. Acanthamoeba
keratitis in England and Wales incidence,
outcome, and risk factors. British Journal of
Ophthalmology 86536-42. Seal et al.,
Acanthamoeba keratitis. Br J Ophthalmol.
200387516-7.
5
Acanthamoeba keratitis and Complete Moisture Plus
Acanthamoeba keratitis is not a notifiable
disease and incidence is unknown

• USA Salt tablets and deionised water in late
  1980s
• Stehr-Green et al., 1989. The epidemiology of
  Acanthamoeba keratitis in the United States.
  American Journal of Ophthalmology 107331-6.
• USA 29/30 cases linked to regional flooding,
  Iowa 1993-94
• Meier, et al., 1998. An epidemic of presumed
  Acanthamoeba keratitis that followed regional
  flooding. Results of a case-control
  investigation. Arch Ophthalmol 1161090-4.
• USA 138 patients identified by CDC 2005-2007
• 46 case interviews, 39 were soft CLW (85) and
  21/36 (58) used Complete MoisturePlus
• MMWR May 2007 Acanthamoeba keratitis Multiple
  States, 2005 - 2007

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Trophozoite Encystment Following Exposure to MPS
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Complete MoisturePlus Induced Encystment
A, castellanii trophozoites following 24 hour
incubation in Complete Moisture Plus a) phase
contrast and b) fluorescence with calcofluor
white staining (x100)
TEM of A, castellanii trophozoites following 24
hour incubation in a) Complete MoisturePlus and
b) mature cysts formed in Neffs constant pH
encystment medium
Kilvington, S., W. Heaselgrave, J. M. Lally, et
al., (2008). Encystment of Acanthamoeba during
incubation in multipurpose contact lens
disinfectant solutions and experimental
formulations. Eye Contact Lens 34133-9.
8
Trophozoite Aggregation
MPS-J 2 hr exposure (x10)
MPS-J 2 hr exposure (x20)
MPS-D 6 hr exposure (x4)
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MPS-J A. castellanii Trophozoite Efficacy
10
Trophozoite Aggregation
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Encystment and Aggregation Method

• Add 3 ml of test solution to 24 well microtitre
  plate
• Add 30 µl of log phase trophozoites to give 3-5
  x 105 /ml (make actual haemocytometer count in
  well)
• Observe microscopically over 24 hours and score
  aggregation
• Add sarkosyl (15) to 0.25 (50 µl) and pipette
  vigorously
• Make haemocytometer counts and calculate
  encystment prepare wet films for microscopy
  (e.g. phase contrast and calcafluor white)

12
Conclusions

• Some MPS can induce trophozoite encystment which
  may favour protection from disinfection and
  survival
• Some MPS induce trophozoite aggregation which may
  afford protection from disinfection and aid
  encystment
• The physiological affects of MPS on Acanthamoeba
  (and other organisms) need to be considered when
  assessing antimicrobial efficacy

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Recommendations

• Need to develop standardised testing methods for
  Acanthamoeba
• Biocidal efficacy
• Regimen efficacy
• Need to assess the physiological response of
  Acanthamoeba to contact lens solutions
• Encystment
• Aggregation
• Need to develop multipurpose contact lens
  disinfectants with efficacy against Acanthamoeba

14
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 centre 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)
• Need to take these recommendations forward to new
  ISO and ANSII standards

15
Acknowledgements

• Wayne Heaselgrave and James Lonnen, 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