Heterogeneity in cyst morphology within isolates of Acanthamoeba from keratitis patients in Thailand

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1 TMIH563 Tropical Medicine and International Health volume 5 no 5 pp may 2000 Heterogeneity in cyst morphology within isolates of Acanthamoeba from keratitis patients in Thailand Somchai Jongwutiwes 1, Lalida Pariyakanok 2, Malee Charoenkorn 1, Kenji Yagita 3 and Takuro Endo 3 1 Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 2 Department of Ophthalmology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 3 Division of Protozoology, National Institute of Infectious Diseases, Tokyo, Japan Summary We isolated Acanthamoebae from the first two keratitis patients identified in Thailand in 1988 and The patients developed decreased vision, severe photophobia, severe eye pain and foreign body sensation after minor corneal trauma. The lesions included generalized superficial punctate keratitis, stromal corneal ulcer with keratic precipitate and uveitis in one case, and corneal ulcer with abscess in the other. Both cases were diagnosed by isolation of characteristic trophozoites and cysts of Acanthamoeba from corneal tissue by nonnutrient agar culture method. Based on cyst morphology, A. castellanii and A. polyphaga were detected in one case, and A. castellanii and A. triangularis in the other. Restriction fragment length polymorphism analysis of mitochondrial DNA (mtdna-rflp) revealed that each patient harboured a single parasite population. One shared mtdna-rflp with an authentic strain of A. castellanii, and the other gave a new unique pattern. Thus species identification of Acanthamoeba based on cyst morphology per se can be arbitrary, and mtdna-rflp may be more appropriate for accurate species/strain differentiation amongst morphologically heterogeneous populations of Acanthamoebae. keywords Acanthamoeba, keratitis, cyst morphology, Thailand, mitochondrial DNA correspondence Dr Somchai Jongwutiwes, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. fmedsjw@md2.md.chula.ac.th Introduction Acanthamoeba keratitis is a serious corneal infection caused by an amoeba of the Acanthamoebidae family which is abundant in soil, dust, sand and water (Mergeryan 1991). Over the past decade, Acanthamoeba keratitis has been prevalent among contact lens wearers (Moret et al. 1997). Since it manifests as a dendritiform corneal lesion, a paracentral annular infiltrate or an ulcer, clinical diagnosis can be confused with those caused by other pathogens. Consequently, delayed definite diagnosis and early improper management often result in severe outcome (Ficker et al. 1990). Although keratitis due to Acanthamoeba sp. has been detected in many countries, few cases have reportedly been recognized in South-east Asia (Mohammed Kamel & Norazah 1995; Kosrirukvongs et al. 1999). The purposes of this report are to describe the first two diagnosed cases of Acanthamoeba infection of the cornea in Thai patients after minor corneal trauma, and to characterize Acanthamoeba subpopulations in isolates from these patients by the mitochondrial DNA restriction fragment length polymorphism (mtdna-rflp) analysis compared with cyst morphology. Materials and methods Case 1 A 58-year-old Thai woman was struck in her left eye by a straw fragment while she was digging in her garden on the outskirts of Bangkok in May Shortly after she used water kept in a jar near her home to clean some dirt off her face. On the following day, her left eye was mildly painful, had a swollen lid and was producing tears. These symptoms gradually increased in severity over the first week. She was initially given antibacterial eye drops and ointment by a doctor near her home, but as her symptoms could not be alleviated, she was referred to King Chulalongkorn Memorial Hospital on June 15, Blackwell Science Ltd 335

2 On admission her symptoms included decreased vision, severe photophobia, foreign body sensation, redness and severe painful sensation on her left eye. General physical examination revealed that she was otherwise healthy. Visual acuity in the left eye was 20/70, and 20/20 in the right eye. Slit lamp examination revealed mixed injection, generalized superficial punctate keratitis, scleral corneal oedema, keratic precipitate, uveitis and markedly decreased left corneal sensation. The initial clinical impression was a probable herpes simplex infection, and she was treated with 1% trifluorothymidine eye drops and acyclovir eye ointment. Repeated cultures for bacteria and fungi from corneal scrapings gave negative results. Indirect immunofluorescence staining using a monoclonal antibody to herpes simplex virus was also negative. One month later a corneal biopsy for histopathological study revealed several Acanthamoeba cysts. A portion of the corneal button was subjected to culture in 1.5% non-nutrient agar seeded with heat-inactivated Escherichia coli in phosphate buffer saline ph 7.4. Numerous Acanthamoeba trophozoites and cysts were found after 24 and 48 h of incubation at 30 C, respectively. Therapy was changed to 2% ketoconazole eye drops administered alternately at 1-h intervals with neosporin (polymyxin B 5000 units/ml, neomycin 1700 units/ml and gramicidin 25 micrograms/ml), propamidine isethionate eye drops hourly and dibromopropamidine isethionate eye ointment twice daily. Ten days after treatment, a dense peripheral ring infiltration with an area of corneal thinning occurred at the inferonasal rim and a hypopyon developed. One week later a total sclerokeratoplasty with donor size of 13.5 mm diameter was performed due to left corneal perforation. Examination of the removed corneal tissue by serial sections stained with haematoxylin-eosin did not reveal evidence of bacterial, fungal, herpes simplex or Acanthamoeba infection. The remaining tissue was cultured for Acanthamoeba but gave a negative result. On the 37th postoperative day, a large central epithelial defect with white stromal infiltration of the cornea was found, necessitating eviceration on day 60 p.o. due to perforation of the graft with spontaneous lens extraction. Case 2 A 30-year-old Thai man was accidentally struck in his left eye by a tiny piece of bamboo 3 months prior to illness. Thereafter he had mild irritation in his left eye but did not seek medical treatment. One month later, after some contaminated water from a fish pond splashed into his eyes, he developed an itch, severe pain and blurred vision in his left eye. These symptoms became more severe and progressed rapidly over a week despite administration of antibiotic eye drops given by a doctor. The patient was referred to King Chulalongkorn Memorial Hospital on March 12, Physical examination on admission revealed an ulcer with abscess at the central part of his left cornea with a diameter of 1 mm and mild inflammatory reaction in the anterior chamber. The visual acuity of his left eye was 20/200; the right eye was normal. Based on a presumptive diagnosis of fungal corneal abscess, the patient was treated with miconazole and neosporin eye drops for 2 weeks without clinical improvement. No bacteria, fungus or herpes simplex virus could be isolated or detected from corneal scraping. Corneal biopsy and subsequent corneal tissue culture revealed trophozoites and cysts of Acanthamoeba sp. Propamidine isethionate eye drops were administered hourly and dibromopropamidine isethionate eye ointment twice daily. One week after treatment, the diameter of the abscess extended to 3 mm with epithelial defect and left eye vision was limited to counting fingers at a distance of about 7 cm. Penetrating keratoplasty was performed. Culture from the removed corneal tissue did not reveal any cyst or trophozoite of Acanthamoeba, and the rest of the cornea examined by serial sections was also negative. However, one month later, the patient suffered from secondary uncontrolled glaucoma that was corrected by a filtering operation with cyclocryotherapy. After 2.5 years of follow up, the patient s left eye vision has remained at the level of detecting hand motion at a short distance. The patient did not have any other recurrent symptoms. Parasite identification The morphology of cysts from each case was examined by direct smear of cultures under light and phase contrast microscope. Individual cysts were isolated from each sample by micromanipulation and maintained on agar plates seeded with a lawn of Escherichia coli. Each clone was subsequently cultured axenically at 26 C in PYGC medium containing 10 g proteose peptone, 10 g yeast extract, 1 g glucose, 5 g NaCl and 1 g l-cysteine in 1000 ml of 5 mm phosphate buffer ph 7.0 (Wilson et al. 1985). Mitochondrial DNA from each clone was purified as described by Yagita and Endo (1990). Acanthamoeba was typed by digesting 0.15 g of mtdna with Bgl II, Hpa I, Sca I or Eco RI in separate reactions. The reaction was carried out under optimal digestion conditions overnight. The digested products were electrophoresed in 0.3 1% agarose gel with constant voltage of 3 V/cm for 6 h in TBE buffer. After staining with ethidium bromide, the gel was inspected under an ultraviolet transilluminator. Results Histology The corneal section of patient 1, stained with haematoxylin Blackwell Science Ltd

3 and eosin, reveals that the corneal stroma is mostly destroyed. Numerous Acanthamoeba cysts throughout the corneal stroma are mostly surrounded by eosinophils. No trophozoites were found after careful examination of the whole corneal sections (Figure 1). Morphology of Acanthamoeba cysts Acanthamoeba isolates from both patients exhibit heterogeneous cyst morphology (Figure 2). Cloning of Acanthamoeba isolated from patient 1 yields 2 morphologically distinct subpopulations, designated as clones TAC/E1 and TAC/E2 (Figure 2a,b). TAC/E1 is overpopulated with cysts of an average diameter of 15 m, mammilated or wrinkled ectocyst, stellate endocyst with 6 8 rays and compatible with A. castellanii; TAC/E2 is A. polyphaga characterized by smaller cysts with average diameter 12 m, wrinkled ectocyst, oval or roughly polyhedral endocyst. Two distinct clones were isolated from patient 2, designated TAC/E3 and TAC/E4 (Figure 2c,d). Clone TAC/E3 closely resembles TAC/E1 except for its smaller cyst diameter and, thus, is identified as A. castellanii. Clone TAC/E4 contains predominantly triangular endocysts belonging to A. triangularis. Based on cyst morphology, these patients seemed to be coinfected with 2 morphologically different Acanthamoebae. Mitochondrial DNA analysis Figures 3 and 4 show the mtdna-rflp analysis. It is apparent that clones TAC/E1 and TAC/E2 share the same mt-rflp pattern which is identical to that of an authentic strain of A. castellanii, indicating that clones TAC/E1 and TAC/E2 are genetically identical or closely related to each other. Clones TAC/E3 and TAC/E4 exhibit identical RFLP pattern which differs from those of TAC/E1 and TAC/E2. Discussion Although Acanthamoeba keratitis has been recognized as a severe infection associated with contact lens wearers, the disease has been reported less frequently to be associated with primary corneal trauma (Moret et al. 1997). Our report demonstrates widespread Acanthamoeba regardless of association with contact lens use. The source of infection in case 1 may have been soil since Acanthamoeba has been detected together with other protozoa from soil samples collected near her home. Unfortunately, further isolation and characterization attempts were not successful. To date, species differentiation based on morphology of Acanthamoeba has been problematic since there are considerable variations in cyst structure. The number of rays and the size of cysts can change under different culture conditions (Stratford & Griffits 1978). Using mtdna-rflp analysis is useful in characterization of Acanthamoeba strains and clarifying phylogenetic relationships among closely related organisms (Kanno et al. 1998; Yagita et al. 1999). Although morphologically different clones were identified in each patient, these could be typed as a single infection with one strain of Acanthamoeba. Analysis of clones (TAC/E3 and Figure 1 Hematoxylin and eosin stained section of corneal biopsy from patient 1 collected before treatment, showing Acanthamoeba cysts and eosinophilic infiltration in destroyed stroma. Bar 10 microns Blackwell Science Ltd 337

4 Figure 2 Characteristics of morphologically distinct clones of Acanthamoeba cysts isolated from patient 1 (a) TAC/E1 and (b) TAC/E2 and patient 2 (c) TAC/E3 and (d) TAC/E4. Bar 10 microns. TAC/E4) from patient 2 revealed a unique mtdna-rflp pattern comparing with other human isolates from various geographical origins (Yagita et al. 1999). This might be due to the different sources of infections among patients since most isolates studied so far came from contact lens wearers (Kanno et al. 1998; Yagita et al. 1999). The clinical course of Acanthamoeba keratitis usually results in a severe outcome. This is partly due to delayed definite diagnosis since the disease can mimic viral and fungal infections of the cornea, and partly since rapid transformation of the trophozoite to a more resistant cyst stage hampers therapeutic efficacy (Khunkitti et al. 1998). Treatment did not result in clinical improvement in both patients despite antiacanthamoebic drug administration and surgery. Meanwhile, disappearance of Acanthamoeba in the removed corneal tissues suggests the efficacy of anti-amoebic drugs used in both patients. Failure of treatment has been reportedly encountered in advanced keratitis cases, possibly due to massive corneal tissue destruction rather than ineffectiveness of anti-acanthamoebic drugs per se (Ficker et al. 1990; Duguid et al. 1997; Hargrave et al. 1999). Whether different species/strains of Acanthamoebae vary in clinical severity and drug susceptibility remains to be investigated. Little is known about the immune responses against Acanthamoeba infection. An immunohistopathological study of Acanthamoeba keratitis has shown that the corneal stroma contains polymorphonuclear leucocytes, macrophages and few lymphocytes (Mathers et al. 1987). Experimental evidence suggests that lymphokines, released from sensitized T lymphocytes and macrophages, activate neutrophils leading to killing of amoeba while antibody may block invasion (Ferrante 1991). However, corneal infection with Acanth Blackwell Science Ltd

5 Figure 3 Agarose gel electrophoretic patterns for mtdna of clones TAC/E1 digested with Bgl II (lane 1), Eco RI (lane 3), Hpa I (lane 5) and Sca I (lane 7); and clone TAC/E2 with Bgl II (lane 2), Eco RI (lane 4), Hpa I (lane 6) and Sca I (lane 8). Markers are lanes M (mixtures of Sal I-digested DNA and Hae III-digested X174 phage DNA) and N (Hind III-digested DNA). amoeba does not promote systemic cell-mediated or humoral immunity because of the lack of resident antigen-presenting cells in the cornea (Van Klink et al. 1997). On the other hand, histopathology of the cornea of one of our patients revealed predominant eosinophilic infiltration in stroma surrounding Acanthamoeba cysts with few neutrophils and lymphocytes. Although eosinophils are capable of phagocytosis and chemotaxis to various stimuli such as microbial products, their prominent role is dampening immediate hypersensitivity reactions by enzymatic degradation of chemotactic mediators. Nevertheless, there is no apparent evidence in our patient s cornea that cysts surrounded by eosinophils were destroyed. Eosinophilic corneal infiltration is associated with a variety of diseases, including onchocerciasis, recurrent herpes simplex keratitis, fungal keratitis, bacterial ulcers and graft failures (Limberg et al. 1986). The significance of finding eosinophils associated with Acanthamoeba keratitis requires further investigations. In conclusion, delay in diagnosis of Acanthamoeba keratitis often results in a poor outcome despite the effectiveness of amoebicidal agents. Definite identification of Acanthamoeba strains should not rely solely upon parasite mor Blackwell Science Ltd 339

6 Acknowledgements We are grateful to Dr Tuenchai Wongseworaset for support and to Dr Prasert Sitthicharoenchai for encouragement. Figure 4 Agarose gel electrophoretic patterns for mtdna of clones TAC/E3 digested with Eco RI (lane 1) and Bgl II (lane 3); and clone TAC/E4 with Eco RI (lane 2) and Bgl II (lane 4). Markers are lanes M (mixtures of Sal I-digested DNA and Hae III-digested X174 phage DNA) and N (Hind III-digested DNA). phology. Mitochondrial DNA analysis should be fully exploited for precise epidemiological comparison regarding drug susceptibility, pathogenicity and virulence among Acanthamoeba strains from human infections. References Duguid IG, Dart JK, Morlet N et al. (1997) Outcome of Acanthamoeba keratitis treated with polyhexamethyl biguanide and propamidine. Ophthalmology 104, Ferrante A (1991) Immunity to Acanthamoeba. Reviews of Infectious Diseases 13 (Suppl.), S 403 S 409. Ficker L, Seal D, Warhurst D & Wright P (1990) Acanthamoeba keratitis-resistance to medical therapy. Eye 4, Hargrave SL, McCulley JP & Husseini Z (1999) Results of a trial of combined propamidine isethionate and neomycin therapy for Acanthamoeba keratitis. Ophthalmology 106, Kanno M, Yagita K, Endo T, Miyata K, Araie M & Tsuru T (1998) Restriction enzyme analysis of mitochondrial DNA of Acanthamoeba strains isolated from corneal lesions. Japanese Journal of Ophthalmology 42, Khunkitti W, Lloyd D, Furr JR & Russell AD (1998) Acanthamoeba castellanii: growth, encystment, excystation and biocide susceptibility. Journal of Infection 36, Kosrirukvongs P, Wanachiwanawin D & Visvesvara GS (1999) Treatment of Acanthamoeba keratitis with chlorhexidine. Ophthalmology 106, Limberg MB, Margo CE & Lyman GH (1986) Eosinophils in corneas removed by penetrating keratoplasty. British Journal of Ophthalmology 70, Mathers W, Stevens G, Rodrigues M et al. (1987) Immunopathology and electron microscopy of Acanthamoeba keratitis. American Journal of Ophthalmology 103, Mergeryan H (1991) The prevalence of Acanthamoeba in the human environment. Reviews of Infectious Diseases 13 (Suppl.), S 390 S 391. Mohammed Kamel KAG & Norazah A (1995) First case of Acanthamoeba keratitis in Malaysia. Transactions of the Royal Society of Tropical Medicine and Hygiene 89, 652. Moret N, Duguid G, Radford C, Matheson M & Dart J (1997) Incidence of Acanthamoeba keratitis associated with contact lens wear. Lancet 350, 414. Stratford MP & Griffits AJ (1978) Variations in the properties and morphology of cysts of Acanthamoeba castellanii. Journal of General Microbiology 108, Van Klink F, Leher H, Jager ML, Alizadeh H, Taylor W & Niederkorn JY (1997) Systemic immune response to Acanthamoeba keratitis in the Chinese hamster. Ocular Immunology and Inflammation 5, Wilson AG, Cann RL, Carr SM et al. (1985) Mitochondrial DNA and two perspectives on evolutionary genetics. Biological Journal of Linnaeus Society 26, Yagita K & Endo T (1990) Restriction enzyme analysis of mitochondrial DNA of Acanthamoeba strains in Japan. Journal of Protozoology 37, Yagita K, Endo T & Jonckheere JF (1999) Clustering of Acanthamoeba isolates from human eye infections by means of mitochondrial DNA digestion patterns. Parasitology Research 85, Blackwell Science Ltd