Australian and New Zealand Journal of Ophthalmology (1998) 26, 231-236

Original Article

Acanthamoeba keratitis in New Zealand, including two cases with in vivo resistance to polyhexamethylene biguanide David Murdoch, FRACO, I Trevor B Gray, FRAC0,I Ray Cursons, PhD3 and Dinah Parr, BSc2 'Eye Department and 2Mycology Unit, Auckland Hospital, Auckland and 3Department of Biological Sciences, University of Waikato, Hamilton, New Zealand

ABSTRACT Background: Acanthamoeba keratitis is an uncommon

corneal infection that can run a protracted course with, at times, serious visual results. Eightyfive per cent of cases occur in soft contact lens wearers.The first New Zealand case occurred in 1990 and only seven cases have been identified in New Zealand to the end of 1996.

Methods: We surveyed the ophthalmologists looking after these seven cases of acanthamoeba keratitis as t o time t o diagnosis, treatment and outcome.

Results: New Zealand has a low incidence of this disease. All cases were soft contact lens wearers with defective care in every instance. After an initial two late-diagnosed cases, the time t o diagnosis for four of the five other cases has been within 2 weeks. Medical treatment has varied over this series, but since the introduction of the cationic antiseptics polyhexamethylene biguanide (PHMB) and chlorhexidine in 1992, the last five cases were all treated with PHMB. One case diagnosed within 2 weeks ran a devastating course, despite intensive PHMB, and a second case remained culture positive after I year of PHMB and the late addition of chlorhexidine. Debridement, 0. I % PHMB and hexami- dine eventually settled this eye.

Conclusions: For treatment PHMB, hexamidine rather than propamidine and surgical debridement are favoured. While all Acanthamoeba isolates show good in vitro sensitivity t o PHMB, the in vivo response is not always proportionate. A bacterial endosymbiont may have been a factor in the favourable outcome o f one protracted case.

Key words: acanthamoeba keratitis, bacterial endosymbionts, polyhexamethylene biguanide resistance.

INTRODUCTION Since 1990, seven cases of acanthamoeba keratitis have been diagnosed and treated in New Zealand. New Zealand has a population of 3.52 million ( 1995), of whom 30 000-50 000 are thought to be soft contact lens wearers, the group most at risk for this condition. The reported cases have been scattered throughout the country and only Auckland, with two cases and sharing in the care of a third case, has had to manage more than one case. Australia, with a population of 17.84 million (1994), has had possibly I30 cases since the first case in 1984' (P Badenoch, pers. comm., 1997). Six cases were described from Western Australia (population 1.7 million) over an 18 month period prior to 1992.2 The Australian and New Zealand experience is very limited compared with Moorfields Eye Hospital (London, UK), where 67 new cases were treated in 1995 (TB Gray, unpubl. obs., 1996). Following are summaries of our seven cases.

CASE

Case 1 1990

A 27-year-old female daily wear soft contact lens wearer presented with a left stromal keratitis that was treated with topical steroids and not diagnosed until 4 months after presentation by culture from an epithelial scrape. Her contact lens care was deficient, with her saline coming from an i.v. bag and no sterilization used. She was unres- ponsive to oral itraconazole (200 mg daily) and topical miconazole ( 1 % hourly) and finally underwent penetrating keratoplasty with a good result. Post-graft she had raised intra-ocular pressure (IOP) for 4 months and mild anterior uveitis that eventually settled and all topical therapy could be stopped 1 year after grafting. She maintains a clear graft

Correspondence. David Murdoch, 197 Hurstmere Road, Takapuna, Auckland 9, New Zealand.

Murdoch et al . 232

and achieves 6/6 with -6/-2.50,,,.,, which is a very similar refraction to her unaffected right.

Case 2 1991

A 29-year-old female daily wear soft contact lens wearer presented with a left superficial epithelial keratitis that was thought to be adenoviral and, 1 month later when stromal involvement began, herpetic. Topical steroids were used throughout and, because of poor response, scrapings taken at

Gray et a1.3 as the first successful Australasian case treated with PHMB. During the course of the disease the patient developed a cataract and an unreactive pupil. After cataract surgery, he was found to have a pale disc and a best vision of 6/60. The optic atrophy was felt to be due to neurotoxicity from intensive NeosporinB. Both neomycin and polymyxin B (ingredients of Neosporin@) are known neurotoxins possessing retinotoxic potential.4 There had been no evidence of glaucoma during the course of his disease.

3 months were suspicious for Acantbamoeba cysts, but there was no growth on culture. The cornea seemed to be improving on 1 % prednisolone drops q.i.d., so the condition was thought to be metaherpetic. By 6 months, with a long history of unremit- ting pain, a full-thickness keratitis with a ring infiltrate plus hypopyon and a central epithelial defect (Fig. I ) , it was real- ized this was classical acanthamoeba keratitis and the patient was commenced on 0.1 % propamidine hourly, NeosporinB (neomycin-polymyxin B-gramicidin, Claxo Wellcome, Auck- land, New Zealand) hourly, oral 200 mg itraconazole daily and topical 1% miconazole 2 hourly with little effect at this late stage. One month later, a central corneal perforation occurred requiring a penetrating keratoplasty. The corneal specimen yielded Acantbamoeba both on histology and culture. Post- operative 1 80° angle closure glaucoma responded to synechio- lysis using Healon (Pharmacia & Upjohn, Auckland, New Zealand). Two years after her graft, the patient required a trabeculectomy to control persistent glaucoma and, later, a cataract extraction and intra-ocular lens (IOL). She sees 616 unaided through a clear graft, but has had two rejection episodes and continues to use 0.12% prednisolone drops 0.d. to prevent rejection. She had been scrupulous in her lens care using an Allergan (Auckland, New Zealand) hydrogen per- oxide system, but had been supplied by a chemist with some saline made with tapwater.

A 41 -year-old male disposable contact lens wearer presented with an epithelial irregularity treated with topical steroids and chloramphenicol and, when stromal involvement began 14 days after presentation, corneal scrapings that were taken were culture positive for Acantbamoeba. He discarded his lenses every 2 weeks, but had done nothing more with regard to hygiene than soak them overnight in non- preserved saline. No sterilization or case cleaning had ever been performed. His case was heavily contaminated with bacteria and Acantbamoeba. Propamidine 0.1 % hourly, Neosporinm hourly, 0.1 % miconazole hourly, oral 200 mg itraconazole daily plus 0.1 % prednisolone failed to control his disease. Polyhexamethylene biguanide (PHMB) 0.02% hourly plus 0.12% prednisolone t.i.d. cured the keratitis within 3 months and this case was reported in this journal by

Case 4 1994

A 30-year-old male daily wear soft contact lens wearer pre- sented with an 8 day history of an irritable eye. Epithelial changes affected 10% of the cornea and, after no change after 2 days observation and treatment with topical dexamethasone with neomycin, they were scraped and Acantbamoeba spp. were grown. The patient was started on 0.1% propamidine and 0.02% PHMB hourly. The propamidine was stopped after 1 month and the PHMB was reduced to 2 hourly and con- tinued for a total of 4 months, by which time the keratitis was totally resolved. His corrected vision was 6/6. His lens care had been poor with no sterilization used.

Case 5 1994

A 35-year-old female daily wear soft contact lens wearer pre- sented with a 4 day history of photophobia from an unusual 4 mm central area of epithelial change. She had had several previous episodes of sterile infiltrates from her Hema soft lens wear. After 4 weeks of slow spread while being treated with dexamethasone with neomycin drops, Acantbamoeba was suspected, but scrapings were delayed when a second opinion suggested this was a toxicity reaction. Two months after presentation, scrapings showed cysts and culture grew Acantbamoeba. Polyhexamethylene biguanide 0.02% and 0.1 % propamidine, both six times a day, were commenced and steroids were continued as 1 % prednisolone t.i.d. After 4 months, the acuity dropped to 6/24 from 611 2, with a wide- spread greyish microcystic epithelial change that was inter- preted as propamidine toxicity and the propamidine was stopped. The cornea went back to its previous state of patchy epithelial involvement and, because of a lack of progress at 5 months, 0.1 % chlorhexidine was added to the PHMB, both four times a day. The disease remained superficial, with epithelial and minor anterior stromal involvement, so, after 7 months of treatment, a limited epithelial scraping was taken from which amoebae still grew in culture.

Efforts to obtain a minimum inhibitoty concentration (MIC) against this Acantbamoeba strain were frustrated by the pres- ence of a bacterial contaminant that prevented an axenic culture being obtained both in Adelaide and in Auckland. Within 24 h of culture, the medium was turbid with bacterial

Acanthamoeba keratitis 233

Figure I . tation showing full-thickness keratitis.

Case 2. A late diagnosed case 6 months after presen-

Figure 2. Case 5 . Persistent epithelial haze 16 months after presentation and 2 months after total epithelial debridement.

overgrowth. This was felt to be due to either a bacterial contaminant or an endosymbiont. An axenic culture was fin- ally obtained by one of the authors (RC) and showed excel- lent i M oitro sensitivity to PHMB with an MIC of 0.0015% (1 5 pg/cm-3). Polyhexamethylene biguanide was fortified to 0.1 % six times a day and, 1 1 months after commencing treat- ment, the patient undewent a total epithelial debridement (Fig. 2). After this, chlorhexidine was stopped and 0.1% PHMB was continued six times a day and 0.1 % hexamidine was added in at the same rate. The cornea then slowly settled to heal fully after 20 months of treatment. The patient had had persistent photophobia for this period. Her central 5 mm of cornea was clear but was surrounded by an irregular ring of anterior stromal scarring. This had steepened her central cornea by 1.25 D, measured by keratometer and her spectacle prescription similarly. Her lens is clear and she still corrects to 6/6. Her contact lens care had been exemplary, but she had

used AOsept (hydrogen peroxide with metal catalyst neutrali- zation; ClBA Vision, Auckland, New Zealand) for sterilization and routinely wore the lenses in some hot natural thermal pools, which contain numerous varied amoeba species.

Case 6 1995

A 26-year-old female soft contact lens wearer presented with an irritable eye due to epithelial corneal changes accompanied by a radial keratoneuritis. She used AOsept for sterilization, regularly boiled her case, but wore her lenses to aquarobics in a chlorinated public pool. Acanthamoeba keratitis was sus- pected at 5 days after presentation and a positive culture was returned from an epithelial scrape by 16 days. Poly- hexamethylene biguanide 0.02% and propamidine were used 2 hourly, with some infrequent topical steroid. The treatment showed no apparent effect on the disease, which progressed inexorably with widespread stromal involvement, leading to a perforated cornea after 1 1 months. The organisms MIC to PHMB was 0.0015% ( 1 5 &cm-3). A penetrating keratoplasty was performed and the graft remains clear. The patient has subsequently had an intracapsular cataract extraction followed by three retinal detachment operations. She still requires topical steroid and timolol plus acetazolamide for secondary glaucoma from complete angle closure. The eye is now stable with a best corrected vision of 6/18.

Case 7 1995

A 50-year-old male soft toric lens wearer presented with a 1 week history of discomfort in his left eye. He used AOsept for sterilization and had two abnormal areas of corneal epithelium, the larger 2 x 3 mm in size. After no change on chloramphenicol drops for 3 days, epithelial scrapings were taken and four double walled cysts were seen in them. Culture was unsuccessful because the plates were overrun by an Aspergillus contaminant. There was some delay in the report and, 3 weeks after scraping, the patient had minor anterior stromal haze at the site of the lesions and he was put on 0.02% PHMB six times a day. This may have been unnecessary as debridement may have been curative. Six weeks later, the cornea was clear and he corrected to 616 and treatment stopped. No amoebae were cultured from either his case or on his lens.

DISCUSSION Since the first New Zealand case presented in 1990, we have seen one case each year with no sign of any increase in this rare condition. Since the first case was described in 1975,s America and England have seen increasing numbers of cases. Between 1984 and 1992, Moorfields Eye Hospital saw 72 cases,* but numbers have increased ever since with 67 new

234 Murdoch et al.

cases in 1995 (TB Gray, unpubl. obs., 1996). Our cases have been scattered throughout the country. With the chronic course of this condition, there is plenty of time to consult with experts in Australia and the UK, which we have done freely. Diagnosis was late (over 2 months) in our first two cases because of unfamiliarity with the disease, but has been early in four of the last 5 cases (10 days to 2 months; mean 22 days), although it is still not yet down to the 9.3 days mean diagnos- tic delay reported from Moorfields in 1992.6 Early diagnosis is known to improve the outcome.6 New Zealand is thought to have between 30 000 and 50 000 soft contact lens wearers, giving an annual incidence of approximately one case in 40 000. Our incidence is very low compared with Australia and the UK.

All our patients have been soft contact lens wearers, but only one of seven patients wore disposable lenses. We have yet to see the British experience’ of increasing numbers of disposable lens wearers affected through failure to disinfect or through the use of inefficient chlorine release disinfection systems. The latter systems are not used here. There was non-existent disinfection in three of our cases and another three cases who showed good care used AOsept. This hydrogen peroxide method with neutralization by a metal catalyst, which is complete at 15 min, is known to be inef- fective against Acantbamoeba, which require 1-2 h to kill.8 Two of the AOsept users used their- lenses with swimming, one in natural thermal pools. The remaining case had been given saline made with tap water by a chemist.

Acanthamoeba keratitis is a preventable disease and all of our seven cases used inadvisable contact lens practices.

All of our seven cases showed cysts on epithelial scrap- ings after digestion with KOH and staining with ink. Six of the seven scrapings grew Acanthamoeba on non-nutrient agar with an Escbericbia coli overlay. In a series of 72 patients from Moorfields, 64% were culture positive.6 They found culture- negative eyes more common in cases with an early presen- tation. Case seven in the present study, whose culture was over-run by contaminant fungi, may well have been negative with his light infective load, only four cysts being found in a careful inspection of all his abnormal epithelium. Biopsy may be necessary in suspicious culture-negative cases, as reported by Crawford from Perth2 in six late-presenting cases referred to him over 18 months to the end of 199 1 .

All the New Zealand isolates have had their MIC deter- mined by one of us (RC). It was difficult to obtain an MIC in case 5 for some months because of a contaminant bac- teria, which prevented an axenic culture. It was finally eliminated by continuing dilution. Whether this was a con- taminant or an endosymbiont is uncertain. Acantbamoeba are host to a variety of non-culturable endosymbionts in up to 2 1 % of isolates.9 They are usually Gram-negative rods and occasionally Gram-negative cocci, which are chlamydia-like

in nature. They are not possible to culture and their relation- ship to the host amoeba’s physiological economy and viru- lence potential are unknown. One cannot determine an MIC until an axenic culture is obtained. Bacterial overgrowth, such as we experienced, would cause an inaccurate estimate of Acanthamoeba sensitivity because the bacteria could preferentially absorb the therapeutic agent tested. A recent reportlo is of an Acantharnoeba strain that showed increased virulence at 37OC when separated from its endosymbiont. This may explain why case 5 in the present study remained superficially affected despite a long duration of the disease.

Many different drugs have been used against Acanthamoeba, many with limited success.

Aromatic diamidines: Propamidine and hexamidine. Neomycin

Wright et ~ 1 . ~ ~ reported the first successful medical treat- ment of acanthamoeba keratitis in 1985, 10 years after the disease was first described. It consisted of topical prop- amadine isethionate (Brolenea; RhBne-Poulenc Rorer, Auck- land, New Zealand) plus Neosporina (neomycin-polymyxin B-gramicidin). This combination had a 50% cure rate. Neomycin has surface toxicity and poor in uitro sensitivity and should no longer be used. Moorfields Eye Hospital no longer uses propamidine as it is associated with chronic con- junctival infection and discomfort when used for prolonged periods6 We used it in the treatment of case 5 as it was sug- gested at the time that combination therapy was an advan- tage.12 We stopped using propamidine after 4 months when a widespread epitheliopathy commenced, which resolved on stopping propamidine. This was first described by O’Day et ~ 1 . 1 3 in two cases where a line of epithelial microcystic change occurred in the central cornea along the line of the lacrimal lake. It was asymptomatic and reversed on stopping the drug. Like many anti-amoeba1 drugs, propamidine has a poor cysticidal effect and can induce encystment in the trophozoite, making it resistant to any therapy.

Propamidine isethionate (BroleneB) is an aromatic diami- dine. Another more promising diamidine is hexamidine, which is sold in pharmacies in France as a biocide, much as BroleneB is in Australasia. It is manufactured by Laboratoire Chauvin (Perpignan, France) and is marketed as Desomedin@; Laboratoire Chauvin sent supplies to us for the treatment of case 5. At the 1995 American Academy of Ophthalmology meeting, Colin et all4 reported four cases of superficial acan- thamoeba keratitis successfully treated within 1-2 months on 0.1 % hexamidine drops alone. The strong amoebicidal activ- i ty against cysts and trophozoites in uitro also occurred in uiuo. Brasseur et a l l 5 reported the successful treatment of two cases of acanthamoeba keratitis with 0.1 % hexamidine diisethion- ate, which was effective both in uitro and in uiuo. They also used

Acanthamoeba keratitis 235

neomycin in both cases and fluconazole in one case. Hexamidine was well tolerated and halved the time required to inactivate both trophozoites and cysts in oitro that was achieved by propamidine.

In a recent study, Brasseur et al. 16 did not find a difference of efficacy between 0.1 % hexamidine and 0.1 % propami- dine. The difference between the two agents was the poten- tial toxicity of propamadine against epithelial cells and keratocytes both in oioo and in oitro. Case five in the present study began to improve after an extensive debridement and the addition of hexamidine to her PHMB regimen.

Cationic antiseptics: Chlorhexidine and PHMB In 1992, Larkin et all7 reported the successful treatment of five of six cases with 0.02% PHMB, which is present in a 20% concentration in the ICI (Auckland, New Zealand) pool disinfectant BaquaciP. In 1983, Dawson et al. at Massey University (New Zealand) demonstrated the efficacy of Baquacil against free-living amoebae.'* ICI NZ kindly supply the pure chemical to hospital pharmacies to make up as 0.02% eye drops in 0.3% methyl cellulose and all New Zealand cases have been treated with PHMB since 1992. This concentration was selected17 to exceed, by approxi- mately 2OO-fold, the minimal cysticidal concentration of one of their isolates. It is well tolerated and is usually commenced 1 hourly until improvement occurs, when it is reduced to six times a day. Moorfields Eye Hospital uses 0.02% PHMB alone on new cases. They have occasionally used 0.1% PHMB in resistant cases who were repeatedly culture posi- tive after intensive PHMB 0.02%. 0.1 % PHMB is used with care, as experience is limited and accurate ideas of potential toxicity are uncertain. There is no evidence of intra-ocular toxicity and while reversible surface toxicity is suspected, it is hard to separate from surface effects of the active disease. At least one case from Moorfields Eye Hospital was con- verted from culture positive to negative by increasing the strength of PHMB from 0.02 to 0.1 %. Case 5 in the present study had no toxicity after 5 weeks of intensive 0.1 % PHMB, but remained culture positive. Paradoxically, this same isolate exhibited good in oitro sensitivity to PHMB with an MIC of 0.0015% (15 ~ g / c m - ~ ) .

No isolates from the more than 200 cases of acantha- moeba keratitis seen at Moorfields Eye Hospital have been resistant to 0.02% PHMB o n in uitro testing, but paradoxical in oioo resistance to PHMB has been observed in some cases.19 Case 5 in the present study, who was still culture positive after treatment for 1 year of the superficial disease with PHMB plus 6 months treatment with additional chlorhexidine, seems such a case. Case six in the present study showed no clinical response to 0.02% PHMB, despite early diagnosis, and demonstrated sensitivity to PHMB.

Moorfields Eye Hospital reports a success rate of 88% with medical treatment6 based on PHMB. Cases of failure with PHMB do not seem to be due to the development of drug resistance. 19

Chlorhexidine was shown by Kirkness et a1.12 to be an equally effective treatment for acanthamoeba keratitis and the authors also demonstrated that propamidine gave an additive effect. Chlorhexidine is a bis-biguanide while PHMB is a polymeric biguanide; both are effective acan- thamoebacides with good effect against both trophozoites and cysts. Moorfields Eye Hospital favours PHMB but, in the North of England and Scotland, the preference of Kirkness et al. for chlorhexidine in combination with Brolenea is largely followed. Desperate to cure case 5 , we used both PHMB and chlorhexidine without initial success.

Other treatments

The aminoglycoside paromomycin (2.5%) has been thought to be helpful, but exhibits almost no in oitro efficacy against Acantbamoeba spp.20 Ketoconazole and itraconazole have been used, but also have poor in oitro efficacy. In 1990, lshibashi et al.20 described three cases of acanthamoeba kera- titis that resolved with up to 200 mg oral itraconazole, topical 0.1 % miconazole hourly and multiple debridement. One case was debrided six times and no sensitivity data were offered. We suspect debridement may have been an impor- tant part of this treatment. Total epithelial debridement in isolated epithelial acanthamoebal infection has been docu- mented to result in a cure without the use of anti-amoebic agents.22 Coster et al.23 resolved two superficially involved cases with wide epithelial debridement alone. Case 7 in the present study was almost certainly cured by debridement alone, although the patient was given prophylactic PHMB for 6 weeks afterwards. Case five in the present study would probably have been helped, in retrospect, by earlier wide- spread debridement to reduce her infective load, as her involvement remained superficial throughout.

The numbers in the present study are too few to comment on surgical management of the condition. The four cases requiring surgery have had 12 major procedures between them. The three corneal grafts are all surviving, despite the known poor outlook for keratoplasties in actively inflamed eyes,6 and all three have had varying prob- lems with glaucoma postoperatively.

Acanthamoeba keratitis is a rare condition in New Zealand, with no evidence of an increasing incidence. Factors that have been implicated with the increasing inci- dence seen in the UK are the greater use of disposable lenses with inadequate sterilizing systems and, since 1990, the use of non-preserved saline due to thiomersal toxicity. Thiomersal did kill 90% of Acantbamoeba within 24 h and was

236 Murdoch et al.

more effective than any of the currently used disinfectants, including chlorhexidine a n d PHMB.8 Unfortunately, Acantbamoeba infections are too uncommon to interest phar- maceutical companies in developing new drugs against the organism and the advances have continued t o come from academic units in the UK. Early diagnosis and the institution of anti-amoeba1 therapy remain the mainstay of successful management. After two initial late diagnosed cases, which were fully reported to colleagues, we seem to be achieving early recognition. The last five cases have all been recog- nized as atypical keratitis a t presentation and Acantbamoeba has been high o n the list of differential diagnosis. We favour the use of 0.02% PHMB as the primary treatment and 0. i % hexamidine for combined therapy. Debridement of superficial cases is very valuable.

ACKNOWLEDGEMENTS Thanks t o Drs Heather Mackintosh (Taranaki Base Hospital), Thiers Halliwell and Paul Herrick (Wellington Hospital), Ken Tam (Christchurch Hospital) and Brett Rogers (Invercargill Eye Clinic) for clinical details of the cases under their care and Bret Robinson (Australian Centre for Water Quality Research, Salisbury, SA, Australia) for his advice.

REFERENCES 1.

2.

3.

4.

5

6

Jackson TN, Heinze JB, Tuxen J, Weiner JM. Successful medical treatment of a corneal ulcer due to Acantbamoeba polypbaga. Aust. N.Z. J . Opbtbalmol. 1986; 14: 139-42. Tay-Kearney M-L, McGhee CNJ, Crawford GC, Trown K. Acanthamoeba keratitis: A masquerade of presentation in six cases. Aust. N.Z. J. Opbtbalmol. 1993; 21: 237-45. Gray TB, Gross KA, Cursons RTM, Shewan JF. Acanthamoeba keratitis: A sobering case and a promising new treatment. Aust. N.Z. J. Opbtbalmol. 1994; 22: 73-6. D'Amico DJ, Caspers-Velu L, Libert J et al. Comparative toxi- city of intravitreal aminoglycoside antibiotics. Am. J. Opbtbalmol.

Jones DB, Visvesvara GS, Robinson NM. Acantbamoeba polypbaga keratitis and acanthamoeba uveitis associated with fatal meningoencephalitis. Trans. Opbtbalmol. SOC. UK 1975;

Bacon AS, Frazer DG, Dart JKG, Matheson M, Ficker LA, Wright P. A review of 72 consecutive cases of acanthamoeba keratitis, 1984-1992. Eye 1993; 7: 719-25.

1985; 100: 264-75.

95: 221-32.

7.

8.

9.

10.

11

12

13

14

15.

16.

17.

18.

19

20

21

22. Holland GN, Donzis PB. Rapid resolution of early acantha- moeba keratitis after epithelial debridement. Am. J. Opbtbalmol.

23. Brooks JG, Coster DJ, Badenoch PR. Acanthamoeba kera- titis: Resolution after epithelial debridement. Cornea 1994;

1987; 104: 87-9.

13: 186-9.

bdford CF, Bacon AS, Dart JKG, Minassian DC. Risk factors lor acanthamoeba keratitis in contact lens users: A case- zontrol study. B M J 1995; 3 10: 1567-70. Levey SB, Cohen EJ. Methods of disinfecting contact lenses to avoid corneal disorders. Surv. Opbtbalmol. 1996;

Gautom RK, Fritsche TR. Characterisation of bacterial endo- symbionts of Acantbamoeba spp. In: 7th International Conference on Small Freeliving Amoebae. 1 996, Adelaide, SA, Australia. (Abstract). Macsai MS, Hall JE. The effect of bacterial endosymbionts on the temperature dependent growth and cytopathogenicity of Acanthamoeba. Invest. Optbalmol. Vis. Sci. 1996; Suppl.: s420. Wright P, Warhurst D, Jones BR. Acanthamoeba keratitis successfully treated medically. Br. J. Opbtba lmol . 1985;

Seal D, Hay J, Kirkness C et al. Successful medical therapy of acanthamoeba keratitis with topical chlorhexidine and propamidine. Eye 1996; 10: 4 1 3-2 1. Johns KJ, Head WS, ODay DM. Corneal toxicity of prop- amidine. Arch. Opbtbalmol. 1988, 106: 68-9. Colin J, Nalet F, Richard C. Successful treatment of superficial acanthamoeba keratitis with 0.1 % hexamidine ophthalmic solution. Opbtbalmology 1995; 102-9A: 129. Brasseur G, Favenec L, Perrine D, Chenu JP, Brasseur P. Successful treatment of acanthamoeba keratitis by hexami- dine. Cornea 1994; 13: 459-62. Olle-dela Haye P, Zafirakis E, Akpek E, Brasseur G, Eupherte L, Foster CS. Acanthamoeba keratitis: Efficiency of hexami- dine us propamidine in vim and in vitro. Invest. Opbtbalmol. Vis. sci. 1997; suppl.: s230. Larkin DFP, Kilvington S, Dart JKG. Treatment of acan- thamoeba keratitis with polyhexamethylene biguanide. Opbtbalmology 1992; 99: 185-9 1 . Dawson MW, Brown TJ, Till DG. The effect of Baquacil on free-living amoebae. N.Z. J . Mar. Freshwat. Res. 1983; 17:

Elder MJ, Kilvington S, Dart JKG. A clinicopathological study of in vitro sensitivity testing and acanthamoeba keratitis. Invest. Optbalmol. Vis. Sci. 1994, 35: 1059-64. Hay J, Kirkness CM, Seal DV, Wright P. Drug resistance and acanthamoeba keratitis: The quest for alternative antiproto- zoal chemotherapy. Eye 1994; 8: 555-63. lshibashi Y, Matsumoto Y, Kabata T et al. Oral itraconazole and topical miconazole with debridement for acanthamoeba keratitis. Am. J . Obbtbalmol. 1990; 109: 121-6.

41: 245-51.

69: 778-82.

305-30.