Megan Shoff, MS, Phd

1
Anti-Acanthamoeba Methods

• Megan Shoff, MS, Phd

2
Previous Studies Variables

• Organism Strain
• 1-5 strains
• Newly isolated, lab strains (30 years)

3
Previous Studies Variables

• Organism Strain
• 1-5 strains
• Newly isolated, lab strains (30 years)
• Trophs, cysts, cysts trophs
• Cyst production
• Axenic, bacterized

4
Previous Studies Variables

• Organism Strain
• 1-5 strains
• Newly isolated, lab strains (30 years)
• Trophs, cysts, cysts trophs
• Cyst production
• Axenic, bacterized
• Innoculum preparation

5
Previous Studies Variables

• Organism Strain
• 1-5 strains
• Newly isolated, lab strains (30 years)
• Trophs, cysts, cysts trophs
• Cyst production
• Axenic, bacterized
• Innoculum preparation
• Innoculum size
• 250-1 million cells/ml

6
Previous Studies Variables

• Organism Strain
• 1-5 strains
• Newly isolated, lab strains (30 years)
• Trophs, cysts, cysts trophs
• Cyst production
• Axenic, bacterized
• Innoculum preparation
• Innoculum size
• 250-1 million cells/ml
• Assay
• Plate, microtiter, centrifuged and resuspended

7
Previous Studies Variables

• Organism Strain
• 1-5 strains
• Newly isolated, lab strains (30 years)
• Trophs, cysts, cysts trophs
• Cyst production
• Axenic, bacterized
• Innoculum preparation
• Innoculum size
• 250-1 million cells/ml
• Assay
• Plate, microtiter, centrifuged and resuspended
• Quantification and viability
• Log reduction, Most Probable Number (MPN),
  positive/negative growth

8
Methodologies

• One or two strains tested
• Strains vary greatly in response to biocides (and
  virulence)
• Species names often are misleading

9
Methodologies

• One or two strains tested
• Strains vary greatly in response to biocides
• Species names often are misleading
• Strains grown axenically
• Amoebae lose ability to encyst

10
Methodologies

• One or two strains tested
• Strains vary greatly in response to biocides
• Species names often are misleading
• Strains grown axenically
• Amoebae lose ability to encyst
• Suspended cells
• Surface attachment is more natural
• Biofilms provide protection

11
Methodologies

• One or two strains tested
• Strains vary greatly in response to biocides
• Species names often are misleading
• Strains grown axenically
• Amoebae lose ability to encyst
• Suspended cells
• Surface attachment is more natural
• Biofilms provide protection
• Old stock strains
• Amoebae lose virulence over time in culture

12
Methodologies

• One or two strains tested
• Strains vary greatly in response to biocides
• Species names often are misleading
• Strains grown axenically
• Amoebae lose ability to encyst
• Suspended cells
• Surface attachment is more natural
• Biofilms provide protection
• Old stock strains
• Amoebae lose virulence over time in culture
• Varying concentrations
• High enough to adequately measure kill

13
Methodologies, cont.

• Inactivation of media
• A neutralization step needs to be included

14
Methodologies, cont.

• Inactivation of media
• Quantification
• Recovery and quantification of organisms
• Viability check of survivors
• Infective dose unknown

15
Methodologies, cont.

• Inactivation of media
• Quantification
• Recovery and quantitation of organisms
• Viability check of survivors
• Infective dose unknown
• Trophs vs Cysts
• Trophs are more susceptible
• Cysts are able to attach to surfaces

16
Proposed Method Improvements

• Strains tested (Shoff, M., et al., Optom Vis
  Sci, 2007)
• Multiple strains should be used
• Minimum of 3-5?
• Strains should be chosen by genotype
• Both recent clinical and environmental isolates
  should be used (Shoff, M., et al., Cornea, 2008)

TW
TW
AK
AK
AK
17
Proposed Method Improvements

• Strains tested (Shoff, M., et al., Optom Vis
  Sci, 2007)
• Multiple strains should be used
• Minimum of 3-5?
• Strains should be chosen by genotype
• Both recent clinical and environmental isolates
  should be used (Shoff, M., et al., Cornea, 2008.
  )
• Growth media (Brandt, F.H., D.A. Ware, and G.S.
  Visvesvara, Applied and Environmental
  Microbiology, 1989.)
• NNAS bacteria
• Rinse and enumerate

18
Proposed Method Improvements

• Strains tested (Shoff, M., et al., Optom Vis
  Sci, 2007)
• Multiple strains should be used
• Minimum of 3-5?
• Strains should be chosen by genotype
• Both recent clinical and environmental isolates
  should be used (Shoff, M., et al., Cornea, 2008.
  )
• Growth media (Brandt, F.H., D.A. Ware, and G.S.
  Visvesvara, Applied and Environmental
  Microbiology, 1989.)
• NNAS bacteria
• Rinse and enumerate
• Suspended cells
• Allow cells to attach before treatment
• Time?

19
Proposed Method Improvements

• Old stock strains
• Do not use 30 year old ATCC strains
• Isolate recent clinical and environmental
  (tapwater) strains

20
Proposed Method Improvements

• Time?
• Old stock strains
• Do not use 30 year old ATCC strains
• Isolate recent clinical and environmental
  (tapwater) strains
• Concentrations
• High enough to adequately measure kill

21
Proposed Method Improvements, cont.

• Inactivation of media
• A neutralization step needs to be included

22
Proposed Method Improvements, cont.

• Inactivation of media
• A neutralization step needs to be included
• Quantification
• Log reduction not adequate
• Growth of cells on NNAS bacteria for
  verification

23
Proposed Method Improvements, cont.

• Inactivation of media
• A neutralization step needs to be included
• Quantification
• Log reduction not adequate
• Growth of cells on NNAS bacteria for
  verification
• Tests with lenses
• Incubate with amoebae before testing

24
Proposed Method Improvements, cont.

• Inactivation of media
• A neutralization step needs to be included
• Quantification
• Log reduction not adequate
• Growth of cells on NNAS bacteria for
  verification
• Tests with lenses
• Incubate with amoebae before testing
• Trophs vs Cysts
• Both trophs and cysts should be tested
• Encystment method matters! (Kilvington, S. and
  C. Anger, British Journal of Ophthalmology, 2001)

25
Conclusions

• Methodology changes are needed to
• obtain real-world results
• Obtain more robust conclusions
• Protect industry and public

26
Papers

• Pickup, Z.L., R. Pickup, and J.D. Parry, A
  comparison of the growth and starvation responses
  of Acanthamoeba castellanii and Hartmannella
  vermiformis in the presence of suspended and
  attached Escherichia coli K12. FEMS Microbiology
  Ecology, 2007. 59(3) p. 556-563.
• Perkovich, B.T., et al., Adherence of
  Acanthamoeba to soft contact lenses, ed. K.R.
  Wilhelmus and D.B. Jones. 1991. S421-S422.
• Shoff, M., et al., Variable responses of
  Acanthamoeba strains to three multipurpose lens
  cleaning solutions. Optom Vis Sci, 2007. 84(3)
  p. 202-7.
• Sankaridurg, P.R., et al., Bacterial Colonization
  of Disposable Soft Contact Lenses is Greater
  during Corneal Infiltrative Events than during
  Asymptomatic Extended Lens Wear. Journal of
  Clinical Microbiology, 2000. 38(12) p.
  4420-4424.
• Kohsler, M., et al., Acanthamoeba strains lose
  their abilities to encyst synchronously upon
  prolonged axenic culture. Parasitol Res, 2008.
  102(5) p. 1069-72.
• Kilvington, S., et al., Encystment of
  Acanthamoeba during incubation in multipurpose
  contact lens disinfectant solutions and
  experimental formulations. Eye Contact Lens,
  2008. 34(3) p. 133-9
• Thomas, V., et al., Biodiversity of amoebae and
  amoebae-resisting bacteria in a drinking water
  treatment plant. Environ Microbiol, 2008.

27
Papers

• Storey, M., et al., The efficacy of heat and
  chlorine treatment against thermotolerant
  Acanthamoebae and Legionellae. Scand J Infect
  Dis, 2004. 36(9) p. 656-62.
• Connor, C.G., S.L. Hopkins, and R.D. Salisbury,
  Effectivity of contact lens disinfection systems
  against Acanthamoeba culbertsoni. Optometry and
  Vision Science, 1991. 68 p. 138-41.
• Cancrini, G., A. Iori, and R. Mancino,
  Acanthamoeba adherence to contact lenses, removal
  by rinsing procedures, and survival to some
  ophthalmic products. Parassitologia, 1998. 40(3)
  p. 275-278.
• Beattie, T.K., et al., Enhanced attachment of
  Acanthamoeba to extended-wear silicone hydrogel
  contact lenses a new risk factor for infection?
  Ophthalmology, 2003. 110 p. 765-771.
• Beattie, T., et al., Determination of Amoebicidal
  Activities of Multipurpose Contact Lens Solutions
  by Using a Most Probable Number Enumeration
  Technique. Journal of Clinical Microbiology,
  2003. 41(7) p. 2992-3000.
• Buck, S.L., R.A. Rosenthal, and B.A. Schlech,
  Methods used to evaluate the effectiveness of
  contact lens solutions and other compounds
  against Acanthamoeba a review of the literature.
  Contact Lens Association of Ophthalmologists
  Journal, 2000. 26 p. 72-84.
• Brandt, F.H., D.A. Ware, and G.S. Visvesvara,
  Viability of Acanthamoeba cysts in ophthalmic
  solutions. Applied and Environmental
  Microbiology, 1989. 55(5) p. 1144-1146.
• Hughes, R. and S. Kilvington, Comparison of
  Hydrogen Peroxide Contact Lens Disinfection
  Systems and Solutions against Acanthamoeba
  polyphaga. Antimicrobial Agents Chemotherapy,
  2001. 45(7) p. 2038-2043.

28
Papers

• Hugo, E.R., et al., Quantitative enumeration of
  Acanthamoeba for evaluation of cyst inactivation
  in contact lens solutions. Investigative
  Ophthalmology Visual Science, 1991. 32 p.
  655-57.
• Hiti, K., et al., One- and two-step hydrogen
  peroxide contact lens disinfection solutions
  against Acanthamoeba How effective are they?
  Eye, 2005. 19 p. 1301-1305.
• Kilvington, S. and C. Anger, A comparison of cyst
  age and assay method of the efficacy of contact
  lens disinfectants against Acanthamoeba. British
  Journal of Ophthalmology, 2001. 85 p. 336-340.
• Kilvington, S., et al., Effect of contact lens
  disinfectants against Acanthamoeba cysts, ed.
  K.R. Wilhelmus and D.B. Jones. 1991. S414-S415.
• Liedel, K.K. and G.C. Begley, The effectiveness
  of soft contact lens disinfection systems against
  Acanthamoeba on the lens surface. Journal of the
  American Optometric Association, 1996. 67 p.
  135-142.
• Ludwig, I.H., et al., Susceptibility of
  Acanthamoeba to soft contact lens disinfection
  systems. Invest Ophthalmol Vis Sci, 1986. 27(4)
  p. 626-8.
• Behlau, I. and M.S. Gilmore, Microbial biofilms
  in ophthalmology and infectious disease. Arch
  Ophthalmol, 2008. 126(11) p. 1572-81.