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Editorial | Previous Editorials
February 2009


Acanthamoeba testing for multipurpose disinfecting solutions

Professor Mark Willcox, BSc, PhD

Mark Willcox is a microbiologist who has specialised in the areas or ocular and oral microbiology. Mark’s research has examined particularly the interactions of the normal microbiota of the eye with contact lenses. In addition, the interactions of the normal and pathogenic microbiota with host defences has been of interest. Mark has recently begun developing novel antimicrobials and other therapies that will help treat ocular infections. Mark has published extensively in the international literature with over 110 refereed journal articles, 5 patents, 7 reviews, 6 book chapters, 30 conference papers, and over 300 conference abstracts. Mark is a member of several national and international scientific societies including ARVO, International Society for Contact Lens Research, and the Tear Film and Ocular Surface Society. Mark has refereed many papers for several scientific journals and is on the editorial board of Eye & Contact Lens and Current Eye Research.


In May 2007 Advance Medical Optics recalled their multipurpose disinfecting solution (MDPS) named Complete MoisturePlus from sale after it was found that use of this solution resulted in a 7-17% increased risk of developing Acanthamoeba keratitis (Joslin et al., 2007; Bryant et al., 2007). During the licensing of MPDS for sale in most countries, companies are required to test the MPDS by using a variety of techniques to ensure there is a minimum level of disinfecting activity. The disinfection criteria can be either the so-called Stand Alone test which is published as ISO standard 14729 (reviewed by Rosenthal et al. 2002) where solutions are required to reduce the load of specific strains of microbes by specific amounts (1 log reduction for Fusarium and Candida; 3 log reduction for Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus), or the MPDS can meet the  Regimen Criteria where the solutions must not leave >10 colony forming units of any of the tested microbes (see above) adhered to a contact lens after rubbing and rinsing the lenses ( Rosenthal et al., 2002). As will be obvious from these testing requirements, there has been no requirement on manufactures to test their solutions for efficacy against Acanthamoeba species.

In Jan 2009, the Federal Drug Administration organised a workshop to discuss the issue of incorporating Acanthamoeba testing into the requirement for licensing MPDS . The workshop participants also discussed the use of more “real world” testing of MPDS.  At the workshop, presentations were given by experts covering areas including epidemiology of Acanthamoeba keratitis (AK), the biology and immunology of AK and proposals for MPDS testing methods. The agenda can be found at the FDAs web site and handouts, slides and discussion items can be found at here.

The FDA requirements from the workshop were to form a consensus around a number of issues relating to Acanthamoeba testing. These issues included (1) what should be the challenge size for testing MPDS in the absence of a contact lens?; (2) which strains of Acanthamoeba should be tested?; (3) how should the cells be grown in order to obtain cells in the trophozoite stage of growth?; (4) how should cells in the cyst stage be produced?; (5) how should the number of survivors of trophozoites or cysts be measured?; (6) what should be the protocol to use when testing for MPDS efficacy in the presence of a contact lens?; (7) what should the overall performance criteria be for the different possible test scenarios?; (8) should the MPDS ability to cause the Acanthamoeba cells to encyst be measured?

Below is a general overview of the consensus that was emerging from the meeting, although it should be kept in mind that these are merely proposals at this stage and can be changed. One of the interesting points to emerge from the discussions was the fact that any new criteria applied for Acanthamoeba testing must not be so strict that currently sold MPDS might fail this. This seems appropriate given that epidemiological studies have not identified risks associated with the use of other MPDS, apart from Complete MoisturePlus which is no longer available for sale world-wide.

What should be the challenge size for testing MPDS in the absence of a contact lens? The initial thoughts were that the challenge level should be less than that for bacteria and fungi used in current FDA/ISO standard testing, and that perhaps an inoculum of no greater than 103 - 104 cysts or trophozoites would be appropriate. The outcome measure from this might be killing of at least 1 log unit of this inoculum.

Which strains of Acanthamoeba should be tested? The general feeling was that a strain of Acanthamoeba castellanii  should be used, perhaps with addition of one other species. The use of clinical isolates was discussed, but it was felt that these should not be incorporated into the standard, but manufactures may need to test against a range and provide this data to FDA during their submission process. Representatives from many of the companies selling MPDS were present at the workshop, and pointed out that  they already test their solutions in a range of tests outside of the FDA process against a range of microbes and under non-standard conditions.

How should the cells be grown in order to obtain cells in the trophozoite stage of growth? Acanthamoeba usually grow by eating bacteria as a food source, and they can be grown in the laboratory this way. However, it was felt that this method of growth might add an extra level of complexity to the standardisation process. Axenic growth, that is growth in synthetic media in absence of bacteria, should be more able to be standardised.

How should cells in the cyst stage be produced? Acanthamoeba generally encyst under environmental stress, such as during starvation. A method that placed trophozoites into a non-nutrient solution (perhaps Neff’s constant pH buffer) was preferred by many as the method to produce large levels of cysts.

How should the number of survivors of trophozoites or cysts be measured? In general a method that measured the number of viable cells was preferred over direct microscopic count methods.  Methods for enumerating survivors could then use the numbers of track-forming units, or the most probable number, on lawns of bacteria on agar plates.

What should be the protocol to use when testing for MPDS efficacy in the presence of a contact lens? There are essentially two methods that could be used. One is essentially using the current FDA/ISO Regimen testing, perhaps adding slightly fewer cells of Acanthamoeba compared to fungal cells used in current standard assay. Another method that is being investigated in several laboratories for use with the standard bacterial and fungal strains, that could be modified for use with Acanthamoeba, is inoculating microbial cells onto a contact lens, placing the lens into a contact lens case, adding MPDS, and measuring survival of microbial cells over periods of time (from the manufactures minimum recommended disinfection time [usually 4-6h depending on solution/manufacturer], up to 30 days).

Should the MPDS ability to cause the Acanthamoeba cells to encyst be measured?
The concensus was that this test was important. It has been shown by Kilvington et al. (2008) and Borazjani et al. (2008) that components of Complete MoisturePlus MPDS, especially propylene glycol and potassium ions, can induce Acanthamoeba trophozoites to encyst. This does not appear to happen with other commercially available MPDS tested in these studies, and so provides the first possibility as to why this particular MPDS was associated with AK.

What should the overall performance criteria be for the different possible test scenarios? This question was deferred until testing has occurred with the proposed methods, once they have been agreed to. It was felt that in the absence of knowledge of the performance of currently marketed MPDS, performance criteria could not be set. Thus, there is a need for all of the currently marketed (in the USA at least) MPDS to be tested in a variety of laboratories.
In conclusion, the FDAs hope at this workshop was to reach consensus of experts on many issues, and I think we succeeded in achieving this. There is still a way to go – but it is clear that regulators, manufacturers and researchers are all eager for standards to be set. This may only be a first step on the way to reducing this disease, but it is an important one.
One final note. Whilst Acanthamoeba keratitis is a distressing disease, and can lead to the need for surgery to improve visual outcome, or in rare cases can lead to blindness, we must not forget that this disease is still relatively uncommon. Table 1 shows the crude incidence rates per 10,000 contact lens wearers for Acanthamoeba keratitis in various geographical regions. The rates in UK have been higher than rates elsewhere for many years, in part due to differences in domestic water supply. Wherever AK is occurring, it is always at a rate much below the general rate for microbial keratitis, which has remained largely static over the past 20 year at around 20 per 10,000 if people wear lenses on an extended wear schedule, to 4 per 10,000 if people wear lenses on a daily wear schedule (Stapleton et al., 2007).

Table 1. Incidence data for Acanthamoeba keratitis

Study type/Lenses






Seal et al., 1999

Hong Kong



Lam et al., 2002




Radford et al., 2002




Schaumberg et al., 1998




Stapleton 2009, personnel communication


  • Joslin CE, Tu EY, Shoff ME, Booton GC, Fuerst P, McMahon TT, Anderson RJ,  Dworkin MS, Sugar J, Davis FG, Stayner LT.  2007. The Association of Contact Lens Solution Use and Acanthamoeba Keratitis. Am J Ophthalmol. 144: 169 –180.
  • Bryant K, Chang T, Chen S, et al. Morbidity and Mortality Weekly Report. 2007. June 1. Acanthamoeba keratitis Multiple States 2005-2007. 56: 532-534.
  • Kilvington S, Heaselgrave W, Lally JM, Ambrus K, Powell H. 2008. Encystment of Acanthamoeba during incubation in multipurpose contact lens disinfectant solutions and experimental formulations. Eye & Contact Lens: Science & Clinical Practice. 34:133-139
  • Borazjani RN, Kilvington S, Meadows DL. 2008. A method to study the rate of encystment for Acanthamoeba spp. and the effect of multi purpose solutions. ARVO general meeting, Fort Lauderdale, May 2008.
  • Seal DV, Kirkness CM, Bennett HGB, Peterson M and Keratitis Study Group, 1999. Acanthamoeba keratitis in Scotland: risk factors for contact lens wearers, Contact Lens Ant Eye 23 (1999), pp. 56–68.
  • Lam DS, Houang E, Fan DS, Lyon D, Seal D, Wong E, and Hong Kong Microbial Keratitis Study Group. 2002. Incidence and risk factors for microbial keratitis in Hong Kong: comparison with Europe and North America. Eye. 16:608-618.
  • Radford CF, Minassian DC, Dart JK. 2002. Acanthamoeba keratitis in England and Wales: incidence, outcome, and risk factors. British Journal of Ophthalmology. 86:536-542.
    Schaumberg DA, Snow KK, Dana MR. 1998. The epidemic of Acanthamoeba keratitis: where do we stand? Cornea.17:3-10.
  • Stapleton F, Keay L, Jalbert I, Cole N. 2007. The epidemiology of contact lens related infiltrates. Optometry & Vision Science. 84:257-272.

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