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 I am Simon Kilvington and I am currently Director of Microbiology at Advanced Medical Optics, California and also retain an honorary position as senior lecturer in parasitology at the University of Leicester, England. I have been fortunate enough to work on Acanthamoeba for the past 20 years studying the organism’s biology, ecology, pathogenicity and the development of methods for assessing the efficacy of disinfectant and therapeutic agents against the organism.I am Simon Kilvington and I am currently Director of Microbiology at Advanced Medical Optics, California and also retain an honorary position as senior lecturer in parasitology at the University of Leicester, England. I have been fortunate enough to work on Acanthamoeba for the past 20 years studying the organism’s biology, ecology, pathogenicity and the development of methods for assessing the efficacy of disinfectant and therapeutic agents against the organism.

2. Acanthamoeaba is a small free-living amoeba common to virtually all soil and aquatic habitats. It is characterised by a feeding and replicating trophozoite stage which, in response to adversity can transform into a resistant cyst stage. The resistance of the Acanthamoeba cysts to extremes of temperature, desiccation and disinfection accounts the presence of the organism in virtually all natural and manmade aquatic and soil habitats. It is this near ubiquity that presents a constant challenge to the contact lens wearer and the potential risk of acquiring acanthamoeba keratitis.Acanthamoeaba is a small free-living amoeba common to virtually all soil and aquatic habitats. It is characterised by a feeding and replicating trophozoite stage which, in response to adversity can transform into a resistant cyst stage. The resistance of the Acanthamoeba cysts to extremes of temperature, desiccation and disinfection accounts the presence of the organism in virtually all natural and manmade aquatic and soil habitats. It is this near ubiquity that presents a constant challenge to the contact lens wearer and the potential risk of acquiring acanthamoeba keratitis.

3. Need for standardisation of Acanthamoeba efficacy testing Look to ISO 14729:2001 (bacteria and fungi) Test strain Method of culture and storage Assay method Presentation of results All apply equally to Acanthamoeba disinfectant testing The object of my talk is to share some of the years of research undertaken by myself and staff at the University of Leicester, England in attempting to standardise disinfectant efficacy testing against Acanthamoeba. To this end we have looked at the current standards applied to efficacy testing of bacteria and fungi against contact lens disinfectants. Based on this, we have then developed reliable and reproducible methods for assaying the efficacy of contact lens care solutions against Acanthamoeba.The object of my talk is to share some of the years of research undertaken by myself and staff at the University of Leicester, England in attempting to standardise disinfectant efficacy testing against Acanthamoeba. To this end we have looked at the current standards applied to efficacy testing of bacteria and fungi against contact lens disinfectants. Based on this, we have then developed reliable and reproducible methods for assaying the efficacy of contact lens care solutions against Acanthamoeba.

4. 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 The first method we have evolved is a qualitative approach that involves the simple testing of a solution’s ability to kill a challenge inoculum of 50 trophozoites or cysts in a fixed time period (usually 6 hours). Initially we tried 100 organisms (2 log kill) but very few solutions showed kill at this level and have settled on 50 organisms representing a 1.7 log challenge. The use of 10 organisms (1 log) was also tried but discarded as it was not possible to reliable enumerate and adjust cells challenge numbers at this level.The first method we have evolved is a qualitative approach that involves the simple testing of a solution’s ability to kill a challenge inoculum of 50 trophozoites or cysts in a fixed time period (usually 6 hours). Initially we tried 100 organisms (2 log kill) but very few solutions showed kill at this level and have settled on 50 organisms representing a 1.7 log challenge. The use of 10 organisms (1 log) was also tried but discarded as it was not possible to reliable enumerate and adjust cells challenge numbers at this level.

5. Method 1 50 Challenge Inoculum Following inoculation and incubation of the plates you can observed over time the growth of Acanthamoeba in the wells –either from surviving trophozoites or a cyst which has excysted to release the trophozoite which then feeds and replicates on the E. coli lawn. As the assay looks for the ability of a solution to give total kill of the 50 trophozoite or cyst challenge or 1.7 log reduction, if just one trophozoite or cyst survives then the well will be positive. Following inoculation and incubation of the plates you can observed over time the growth of Acanthamoeba in the wells –either from surviving trophozoites or a cyst which has excysted to release the trophozoite which then feeds and replicates on the E. coli lawn. As the assay looks for the ability of a solution to give total kill of the 50 trophozoite or cyst challenge or 1.7 log reduction, if just one trophozoite or cyst survives then the well will be positive.

6. Method 1 Results In this slide we see a summary of the qualitative assay. A positive in red shows survival of the challenge after 6 hours incubation in the test solution and the negative representing total kill. Each solution is assayed in triplicate and we always run a neutraliser control with every solution. Here the test solution is added to the neutralising disinfectant medium and then challenged with Acanthamoeba and this control must always be postive to show that the neutralizer has been affective for the given test solution. The results show variation between the commercial solutions in efficacy against trophozoites and, typically, the cyst form is more resistant. However, a notable exception is MPS-1 which consistently has shown better efficacy against the cyst stage than the trophozoite. In this slide we see a summary of the qualitative assay. A positive in red shows survival of the challenge after 6 hours incubation in the test solution and the negative representing total kill. Each solution is assayed in triplicate and we always run a neutraliser control with every solution. Here the test solution is added to the neutralising disinfectant medium and then challenged with Acanthamoeba and this control must always be postive to show that the neutralizer has been affective for the given test solution. The results show variation between the commercial solutions in efficacy against trophozoites and, typically, the cyst form is more resistant. However, a notable exception is MPS-1 which consistently has shown better efficacy against the cyst stage than the trophozoite.

7. 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 Only qualitative Only qualitative

8. 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 The next method is a preferred quantitative assay that enables the rate of trophozoite or cyst kill to be determined. Here the solution is challenged to give 5 x 104 /ml trophozoites or cysts and the number of surviving organisms determined over time. The approach uses microtitre plate dilutions and most probable number computations to determine the number of survivors at a given time point. The next method is a preferred quantitative assay that enables the rate of trophozoite or cyst kill to be determined. Here the solution is challenged to give 5 x 104 /ml trophozoites or cysts and the number of surviving organisms determined over time. The approach uses microtitre plate dilutions and most probable number computations to determine the number of survivors at a given time point.

9. Acanthamoeba Challenge Testing As per the qualitative assay, the wells of the microtitre plate are examined microscopically for trophozoite replication or excystment and replication on the E. coli feed added to the wells.As per the qualitative assay, the wells of the microtitre plate are examined microscopically for trophozoite replication or excystment and replication on the E. coli feed added to the wells.

10. Acanthamoeba Challenge Testing We then enter and analyse the the plate score results using Excel spreadsheets that calculate the MPN based on Spearman-Karber computations and then plot the findings as the rate of log reduction (Delta Logs) relative to the time zero count.We then enter and analyse the the plate score results using Excel spreadsheets that calculate the MPN based on Spearman-Karber computations and then plot the findings as the rate of log reduction (Delta Logs) relative to the time zero count.

11. A. castellanii (50370) Trophozoites As is then seen in this graph, variation exists in the efficacy of contact lens care solutions against Acanthamoeba trophozoites. Here three MPS failed to produce a 1-log kill after 6 hours incubation and 3 managed a 1.5 – 3.0 log kill. The most efficacious was the 1-step hydrogen peroxide system (in yellow) which gave 3.7-4.0 log kill after 1-2 hours exposure. Importantly, the assay allows the rate of Acanthamoeba kill to be determined and so gain a greater understanding of a solution’s relative efficacy.As is then seen in this graph, variation exists in the efficacy of contact lens care solutions against Acanthamoeba trophozoites. Here three MPS failed to produce a 1-log kill after 6 hours incubation and 3 managed a 1.5 – 3.0 log kill. The most efficacious was the 1-step hydrogen peroxide system (in yellow) which gave 3.7-4.0 log kill after 1-2 hours exposure. Importantly, the assay allows the rate of Acanthamoeba kill to be determined and so gain a greater understanding of a solution’s relative efficacy.

12. Efficacy of Contact Lens Disinfectants Against A. castellanii (50370) Trophozoites (6 hr) This slide summarises the average log kills obtained with a 1-step peroxide (far left) and 17 commercial MPS against A. castellanii after 6 hours contact time. As you will see the peroxide gave over 3 log total kill whist large variation was found among the MPS. Of these sixteen solution, 9 or 56% failed to give 1-log kill, 3 showed kill between the 1-2 log range, 2 were between 2-3 log and 2 were >3 log. Of the 9 solutions which failed to give 1-log kill after 6 hours exposure, increasing the contact time to 24 hours resulted in only 3 of the solutions producing further kill with values between 1-2 log. However, stasis was achieved for all remaining solutions.This slide summarises the average log kills obtained with a 1-step peroxide (far left) and 17 commercial MPS against A. castellanii after 6 hours contact time. As you will see the peroxide gave over 3 log total kill whist large variation was found among the MPS. Of these sixteen solution, 9 or 56% failed to give 1-log kill, 3 showed kill between the 1-2 log range, 2 were between 2-3 log and 2 were >3 log. Of the 9 solutions which failed to give 1-log kill after 6 hours exposure, increasing the contact time to 24 hours resulted in only 3 of the solutions producing further kill with values between 1-2 log. However, stasis was achieved for all remaining solutions.

13. Trophozoite Strain (2000-2008) In this slide we looked at the kill values we obtained for 5 Acanthamoeba keratitis strains tested repeatedly against 3 MPS over the past 8 years. As you will see that, when trophozoites are grown and tested under the same conditions there is little variation between species or strains for a given solution. All strains are of the T4 genotype characteristic of AK isolates but individually are genetically distinct. Therefore, for these strains at least, the trophozoites behave similarly when grown and tested under identical conditions.In this slide we looked at the kill values we obtained for 5 Acanthamoeba keratitis strains tested repeatedly against 3 MPS over the past 8 years. As you will see that, when trophozoites are grown and tested under the same conditions there is little variation between species or strains for a given solution. All strains are of the T4 genotype characteristic of AK isolates but individually are genetically distinct. Therefore, for these strains at least, the trophozoites behave similarly when grown and tested under identical conditions.

14. Effect of culture medium on trophozoite kill (6 hr) Here the effect of the culture conditions used to grow and maintain trophozoites for testing was investigated. Our preferred medium is this AC#6 which is composed of peptones-yeast extract-glucose and vitamin salts. The medium is excellent for adapting clinical and environmental isolates to axenic growth and ensures uniform trophozoite growth to high cell numbers. However, it would appear that the age of the trophozoite culture in this medium (24 hr or 72 hr) results in the latter being slightly more susceptible to disinfection. Also, the strain grown in a less rich medium such as peptone-yeast extract-glucose (PYG) or Trypticase Soy Broth (TSB) are more resistant to disinfection. Here the effect of the culture conditions used to grow and maintain trophozoites for testing was investigated. Our preferred medium is this AC#6 which is composed of peptones-yeast extract-glucose and vitamin salts. The medium is excellent for adapting clinical and environmental isolates to axenic growth and ensures uniform trophozoite growth to high cell numbers. However, it would appear that the age of the trophozoite culture in this medium (24 hr or 72 hr) results in the latter being slightly more susceptible to disinfection. Also, the strain grown in a less rich medium such as peptone-yeast extract-glucose (PYG) or Trypticase Soy Broth (TSB) are more resistant to disinfection.

15. 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) Neff’s constant pH encystment medium (high yield, >99% mature form but increased susceptibility to some MPS?) For efficacy testing against cysts, the same quantitative assay method just described is preferred. However, the method of cyst production can affect significantly the results. This slide summarises some of the methods described for producing Acanthamoeba cysts. I do not have time to describe the methods in detail but the relative merits and weaknesses are summarised in the slide. Of these, we favor the use of cysts produced using Neff’s constant pH encystment medium. This is a high pH defined salts solution that with good aeration and gentle agitation induces synchronised trophozoite encystment during incubation. Large numbers of axenic (free of bacteria) cysts are formed within 7 days incubation and provide sufficient numbers for time-kill disinfection assays. For efficacy testing against cysts, the same quantitative assay method just described is preferred. However, the method of cyst production can affect significantly the results. This slide summarises some of the methods described for producing Acanthamoeba cysts. I do not have time to describe the methods in detail but the relative merits and weaknesses are summarised in the slide. Of these, we favor the use of cysts produced using Neff’s constant pH encystment medium. This is a high pH defined salts solution that with good aeration and gentle agitation induces synchronised trophozoite encystment during incubation. Large numbers of axenic (free of bacteria) cysts are formed within 7 days incubation and provide sufficient numbers for time-kill disinfection assays.

16. Encystment Method It should be noted that cysts produced using the constant pH encystment medium appear more susceptible to certain MPS than their NNA-E. coli or Taurine-NNA counterparts. However, as you will see in the next slides, the cysts produced using Neff’s medium are fully mature and morphologically correct. As such, it would seem that such cysts are inherently more susceptible to certain MPS based on an active ingredient of PHMB (MPS 3, 4, 5). These findings have been consistent on numerous assays with such solutions using at least a dozen different preparations of cysts made with this medium. The results are also consistent when strains of A. polyphaga have been used. It should be noted that cysts produced using the constant pH encystment medium appear more susceptible to certain MPS than their NNA-E. coli or Taurine-NNA counterparts. However, as you will see in the next slides, the cysts produced using Neff’s medium are fully mature and morphologically correct. As such, it would seem that such cysts are inherently more susceptible to certain MPS based on an active ingredient of PHMB (MPS 3, 4, 5). These findings have been consistent on numerous assays with such solutions using at least a dozen different preparations of cysts made with this medium. The results are also consistent when strains of A. polyphaga have been used.

17. A. castellanii (50370) Neff Cysts This slide shows light and electron microscopy of A. castellanii (ATCC 50370) cysts produced using Neff’s constant pH encystment medium. The cysts produced by this method are characteristic of the mature stage with thick double cyst walls and presence of plugged opercula or pores from which the trophozoite emerges. We continue to study Acanthamoeba encystment process and are presently working on improved encystment media that may produce cysts with resistance profiles similar to that observed for NNA-E. coli or NNA-Taurine based methods.This slide shows light and electron microscopy of A. castellanii (ATCC 50370) cysts produced using Neff’s constant pH encystment medium. The cysts produced by this method are characteristic of the mature stage with thick double cyst walls and presence of plugged opercula or pores from which the trophozoite emerges. We continue to study Acanthamoeba encystment process and are presently working on improved encystment media that may produce cysts with resistance profiles similar to that observed for NNA-E. coli or NNA-Taurine based methods.

18. 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

19. Acanthamoeba Regimen Testing Criteria as per ISO 14729:2001 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* We have also developed and evaluated a method for conducting regimen testing with Acanthamoeba trophozoites and cysts in line with ISO 14729 for bacteria and fungi. Here Acanthamoeba are inoculated on to contact lenses and subjected to the solution manufacturers’ recommended regimen for lens cleaning. Quantitative methods have then been developed to enumerate the total number of surviving trophozoites or cysts on the lenses and in the soaking solution. I do not have time to describe this in detail but we do have a manuscript in press addressing regimen efficacy testing against Acanthamoeba.We have also developed and evaluated a method for conducting regimen testing with Acanthamoeba trophozoites and cysts in line with ISO 14729 for bacteria and fungi. Here Acanthamoeba are inoculated on to contact lenses and subjected to the solution manufacturers’ recommended regimen for lens cleaning. Quantitative methods have then been developed to enumerate the total number of surviving trophozoites or cysts on the lenses and in the soaking solution. I do not have time to describe this in detail but we do have a manuscript in press addressing regimen efficacy testing against Acanthamoeba.

20. 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 Ac#6 medium to log phase (75% confluence in flask) Standardise tubes used for testing

21. 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 Neff’s Encystment Medium from late log phase trophozoites grown in Ac#6 medium Need for an improved liquid encystment medium producing cysts with resistance comparable to those from NNA-E. coli or NNA-Taurine methods

22. 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

23. 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@le.ac.uk or James Lonnen jdl3@le.ac.uk) Undertake quality assurance testing (similar to CLI Ring Test undertaken for bacteria and fungi) The work reported here has been conducted at my laboratory at the University of Leicester and we would be pleased to offer support to those interested in learning more about the methods we have developed for conducting Acanthamoeba disinfectant efficacy testing. The work reported here has been conducted at my laboratory at the University of Leicester and we would be pleased to offer support to those interested in learning more about the methods we have developed for conducting Acanthamoeba disinfectant efficacy testing.

24. Acanthamoeba Physiological Response to Contact Lens Solutions

25. 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