Year : 1990 | Volume
: 38 | Issue : 2 | Page : 50--56
Diagnosis of acanthamoeba keratitis-A report of four cases and review of literature
Savitri Sharma, M Srinivasan, Celine George
Aravind Eye Hospital & Post-graduate Institute of Ophthalmology, Madurai-625 020, India
Aravind Eye Hospital & Post-graduate Institute of Ophthalmology, Madurai-625 020
Acanthamoeba keratitis is not reported often in India. We reported the first case diagnosed in this country a year back. In this communication, four more cases of Acanthamoeba keratitis diagnosed since then are being reported along with a brief review of literature. Diagnosis in all these patients was based on observation of acanthamoeba cysts in 10% KOH wet mount of corneal ulcer scrapings and subsequent culture. The characteristic ring infiltration of cornea was seen in all of them. All cases were treated medically with the available antiamoebic drugs (Miconazole, Neosporin, Ketoconazole and Metronidazole) in different combinations. Only one out of four, responded with complete healing of the ulcer. Acanthamoeba keratitis is probably not as uncommon in India as it is thought to be. With increased awareness and performance of minimal laboratory tests the condition may be diagnosed more often.
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Sharma S, Srinivasan M, George C. Diagnosis of acanthamoeba keratitis-A report of four cases and review of literature.Indian J Ophthalmol 1990;38:50-56
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Sharma S, Srinivasan M, George C. Diagnosis of acanthamoeba keratitis-A report of four cases and review of literature. Indian J Ophthalmol [serial online] 1990 [cited 2020 Apr 1 ];38:50-56
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Reports of scattered cases of Acanthamoeba keratitis have appeared in the European and American literature ,,,, Diagnosis by clinical or laboratory methods is said to be difficult, delaying accurate identification for months . It is possible that both clinicians and microbiologists are now more aware of the condition. However, reports of Acanthamoeba keratitis are lacking in Indian literature. Inadequate facilities for microbiological investigations in ophthalmic institutions probably is responsible for this lacuna. We reported the first case diagnosed in India . Corneal scraping examined in 10% KOH wet mount demonstrated cysts of Acanthamoeba which provided the clue and lead to a successful culture on non-nutrient agar with Escherichia coli overlay. A high level of clinical suspicion  and wet mount examination of specimens from infected tissue  are essential to aid in rapid diagnosis of Acanthamoeba keratitis. However, a majority of cases described have been diagnosed following histopathology of the corneal button or corneal biopsy or only after culture 
Subsequent to the first experience  we were able to detect Acanthamoeba keratitis on four more occasions from May 1988 through September 1988, all by meticulous examination of 10% KOH wet mount preparations of corneal ulcer scrapings. The fact of utmost importance appears to be the familiarity of the investigator with the appearance of the organism. the purpose of this communication, on the basis of observations in our five patients, including the previously reported one, is to point out that the entity probably is much more frequent than is generally believed. In addition, simple readily available microbiologic studies deserve more emphasis than they have received in earlier publications. Four cases are considered here in detail with a brief review of literature.
Case 1 : A 26-year-old female patient, a house wife, consulted us in May 1988 with history of pain and lacrimation from the left eye of 40 days duration. She was unaware of any episode of ocular trauma. On examination, the visual acuity recorded in the affected eye was counting fingers close to the face. There was a central corneal lesion of 6x6 mm. ring infiltration with 2 mm hypopyon [Figure 1] with no associated chemosis or lid oedema. The ulcer extended upto the anterior stroma. The right eye was normal.
Corneal ulcer scrapings were examined in 10% KOH wet mount which showed stellate, polygonal, double walled cysts suggestive of Acanthamoeba. Cysts were also demonstrated in corneal ulcer scrapings stained by Gram stain and Giemsa stain. Deep stormal scrapings of the corneal ring inoculated on non-nutrient agar with Escherichia coli overlay grew Acanthamoeba in 48 hours. No bacteria or fungi was isolated on culture. The isolate was confirmed to be Acanthamoeba castellanii by the Centre for Disease Control, Atlanta, U.S.A The species identification was done by indirect immunofluorescence method using rabbit antisera against A.castellanii, A.polyphaga, and A. culbertsoni.
With topical application of Miconazole (10 mg/m) at hourly intervals and Neosporin TM drops (Neomycin, Polymyxin-B, Bacitracin) six hourly there was a slight resolution in stromal infiltration by the 13th day. Another evaluation of corneal scrapings at this stage revealed the persistence of cysts in 100/8 KOH wet mount and was positive in culture for Acanthamoeba. Thereafter Neosporin was continued and Miconazole was replaced with hourly drops of Metronidazole (5 mg/ml) which was continued for nine days. A daily clinical evaluation showed decreasing infiltration and receding hypopyon. On the 22nd day the patient was discharged with instructions to use Neosporin four times a day and cycloplegics once a day. Subsequent reviews on the 45th and 75th day showed remarkable progress towards healing of the ulcer [Figure 2]. When seen four and half months later the eye was quiet with minimal scar and the visual acuity with dilatation had improved to 6/60. Keratoplasty has been deferred so far.
Case 2 : A 40-year male labourer presented to us with history of mud falling in his left eye. After four weeks he reported to us in July 1988 with pain, photophobia and defective vision of the left eye. Prior to that he was treated elsewhere with 0.3% Gentamicin eye drops and ointment. A central, well demarcated disc shaped corneal ulcer with extensive corneal oedema and 6 mm hypopyon was noted [Figure 3]. Anterior chamber details were not visible and the nasolacrimal passage was free. The patient complained of severe pain. The visual acuity was PL. There was no ocular disease in the fellow eye.
Examination of scrapings from the corneal ulcer in 10% KOH mount revealed cysts of Acanthamoeba which was confirmed by culture. The Acanthamoeba isolate was subsequently identified as A.polyphaga at CDC, U.S.A. There was no evidence of fungus either in the direct examination or culture, however, staphylococcus aureus was cultured.
The patient was started on hourl6topical Metronidazole drops and six hourly Neosporin TM drops with topical 1 atropine thrice a day. After seven days of this treatment regimen, no change in the ocular condition was discernible except that the corneal oedema had cleared. The corneal scrapings were again positive for Acanthamoeba cysts in direct microscopy and Acanthamoeba was grown in culture. Culture was negative for Staphylococcus aureus at this stage. Metronidazole drops were substituted by Miconazole and Neosporin was continued as before. With this regimen, which continued for 16 days, the ulcer responded very slowly. On the 23rd post admission day Metronidazole was again added to Miconazole and Neosporin in the same dosage mentioned before. With this combination the ulcer appeared to heal faster. By 40th day the ulcer had healed leaving a residual 4 mm central scar with anterior synechiae. The vision remained at perception of light (PL). No material could be obtained on corneal scraping for laboratory tests at this stage. He was advised keratoplasty for which he was not willing. When he reported to us two months later the cornea was vascularized, there was 2 mm hyphaema with rubeosis iridis and anterior synechiae.
Case 3 : A 47-year-old male patient, who had been treated elsewhere (details not known) for corneal ulcer reported to us in August 1988. Ten days prior to consulting us he had an injury in the eye with vegetable matter which was followed by pain, photophobia and defective vision. On examination there was a paracentral disc shaped corneal ulcer in the right eye with ring infiltration. There was no lid oedema, chemosis or hypopyon. However, the right duct was found partially blocked and an emergency dacryocystectomy was done on the day of admission. The visual acuity recorded in that eye was counting fingers close to the face. The fellow eye was normal. The evaluation of corneal scrapings was positive for Acanthamoeba cysts in KOH mount and culture. No fungus or bacteria was grown on culture.
Treatment with hourly topical drops of Miconazole and six hourly Neosporin was started. Patient wanted to leave the hospital on the 7th post-admission day. He was discharged on request, with the advice to use Neosporin six hourly. Subsequently the patient was seen on the 17th and 28th day. Each time the culture was positive for Acanthamoeba species. The ulcer did not respond to treatment and ultimately the patient was lost to follow up.
Case 4 : A 30-year-old fisherman developed pain and photophobia of the left eye after dirty sea water fell into the eye. He was treated elsewhere for a bacterial corneal ulcer and referred to us after one month. Visual acuity was HM in that eye. On slit-lamp examination, a 6x6 mm, round, paracentral corneal ulcer with raised surface, indistinct borders [Figure 4], infiltration upto the anterior stroma; no folds in the Descemet's membrane was noted. There was no hypopyon, no chemosis or lid oedema but the patient complained of severe pain. The other eye was normal.
Acanthamoeba cysts were observed in 10% KOH wet mount preparation of the corneal scrapings. The corneal scrapings also yielded Acanthamoeba in culture. direct examination as well as culture were negative for bacteria and fungus.
Metronidazole drops were started and given hourly for 7 days. But on the seventh day the ulcer size was remaining the same, the corneal oedema had increased and a ring infiltrate had developed [Figure 5]. In addition, a hypopyon of 2 mm had developed. However, there was no lid oedema or chemosis. Intraocular pressure recorded digitally was high. Metronidazole was withdrawn and replaced with hourly topical administration of Ketoconazole (1% drops), six hourly topical administration of Neosporin drops, and twice a day 1% Atropine. Oral Acetazolamide and nonsteroidal anti-inflammatory drugs were given. Seven days later (fourteenth post admission day) the progress of the ulcer seemed to have been arrested. On the 18th post-admission day the patient was discharged on request with advice to use topical Ketoconazole 1 % drops 4 hourly, Neosporin eye drops 6 hourly and 1% Atropine drops twice daily. He has not yet come back for follow-up.
DISCUSSION AND REVIEW OF LITERATURE
Acanthamoeba and Naegleria are two genera of the order Amoebida of free-living amoebae which normally feed on bacteria and are ubiquitous. They are found in soil, fresh water, well water, sea water, brackish water, sewage, soil and air , In trophozoite form Naegleria measures 10 to 35 um and has a flagellate stage. Cysts are spherical and measure 7 to 15 µm. Although slightly larger (15-45 um), the Acanthamoeba trophozoite is a honflagellate and produces fine, tapered, cytoplasmic projections called acanthopodia (Gr. Acantha: thorn). Its cyst is double-walled and stellate, measuring 10-25 µm. Both are uninucleate and contain a large, dense, centrally located nucleolus.
At least 22 species of Acanthamoeba have been distinguished by cyst morphology, immunofluorescence antibody tests, or isoenzyme studies. Several species including A.culbertsoni, A:castellanil, A.polyphaga and A.astronyxis are considered pathogenic to humans . The ability of the trophozoite to encyst in adverse conditions renders Acanthamoeba highly resistant to freezing, dessication, standard chlorination of water supplies, and a variety of antimicrobial agents. Nevertheless, routine sterilization techniques of autoclaving and gassterlization using ethylene oxide are effective against Acanthamoeba trophozoites and Cysts 
The factors responsible for tissue invasion and virulence have not been determined though Acanthamoeba has been shown to contain Phospholipase A and to activate the alternate pathway of complement  . Serological studies have shown that most immunocompetent adults have been exposed to the organism and have antibodies to Acanthamoeba spp. There is experimental evidence that both humoral and cell mediated immunity play a role in natural immunity to Acanthamoeba .
Both Naeglaria and Acanthamoeba can cause granulomatous meningoencephalitis in humans by invading the nasopharynx and becoming blood borne through the cribriform plate of ethmoid . These infections are believed to be acquired by inhaling the cysts or by direct contact with contaminated water. Apart from meningoencephalitis Acanthamoeba has been implicated in the causation of skin infections, pneumonitis, external otitis and osteomyelitis ,.
Ocular infection caused by Acanthamoeba was first reported in June 1973  in Washington. The organisms had probably infected the eye by blood-borne dissemination and did not affect the cornea.
Corneal infection with Acanthamoeba is believed to result from direct corneal contact with contaminated material or water. It is a serious infection of the cornea though relatively rare. Less than 100 persons have been diagnosed as having Acanthamoeba keratitis since the disease was first described . The disease probably remained undiagnosed prior to 1970 as is indicated by Cohen et al  in a retrospective examination of eight cases of corneal transplant performed at the Wills Eye Hospital. Historically, the infection has been associated with penetrating corneal trauma ,,, More recently,' an association - with contact-lens wear has become apparent . However, a few patients have had neither a history of trauma nor of contact lens wear , Three out of four cases reported in this communication had definite history of antecedent trauma but none were contact lens wearers. One case reported by us earlier  had also resulted following splashing of dirty water into the eye.
Of the reported cases of Acanthamoeba keratitis there was no specific occupational prerequisite necessary for the infection. Cases have been reported in farmers, office workers, electricians, teachers, engineers and students . All cases seen by us (including the one reported earlier) came from economically backward groups, three of them were labourers and one a fisherman. One patient, however was a housewife. In most cases the infection has been unilateral, although a transient idiopathic contralateral keratouveitis after penetrating keratoplasty of the infected eye has been described .A recent report describes chorioretinitis of the right eye of a patient with contact lens associated Acanthamoeba keratitis in the left eye during an acute exacerbation of keratitis . A haematogenous dissemination from the corneal infection has been suggested. The contralateral ocular condition of all our patients was normal with no signs suggestive of uveitis.
Of the several cases diagnosed in the United States there was one which originated in India . This was in a 28-year-old female patient with low myopia (-4.00 D) who developed ulcer cornea in both eyes a month after switching over from hard contact lenses to soft ones. She was initially diagnosed and treated as a case of herpetic keratitis. Subsequently she underwent penetrating keratoplasty of both eyes in the United States. Prior to keratoplasty the corneal scrapings were negative for virus, bacteria and fungus. It was only after histopathological study of the corneal button that the diagnosis of Acanthamoeba keratitis was made. The first case that was diagnosed in this country was seen in July 1987 by us  and had features very much contrary to the one just described. It was an unilateral keratitis following splashing of dirty water into the eye. The case was treated as a bacterial corneal ulcer with subconjunctival Gentamicin for over a week and then referred to us. Routine examination of the corneal scraping in 10% KOH wet mount provided the clue to the presence of Acanthamoeba cysts and it was confirmed by successful culture of A. castellanii on non-nutrient agar with Escherichia coli overlay.
Acquiring familiarity with the morphology of Acanthamoeba in direct examination of corneal scrapings as well as in culture, probably, was the impetus which lead us to quick diagnosis of four more cases within a short period between May 1988 and September 1988. Three out of four had been treated earlier as bacterial ulcers without any response. A ring infiltrate of the central or paracentral cornea was the presenting feature in three cases (case 1,2,3) whereas in the fourth case a ring infiltrate developed during the course of treatment. This conformed to clinical features described in over two-thirds of earlier reported cases . We, however, subjected the cases to microbiological investigations as a routine procedure followed by us, without any clinical bias. Accurate diagnosis could be made on observation of cysts of Acanthamoeba in the corneal ulcer scrapings in all the cases. Nevertheless, a clinical suspicion on the finding of central or paracentral stromal infiltration within an outer annular infiltrate must include Acanthamoeba keratitis in the differential diagnosis. Majority of cases reviewed by Auran et al [1 0] had characteristic ring infiltrates which were segmental or circumferential, progressive and often involved thinning or furrowing. In many cases, however, especially the early ones, Acanthamoeba infection was considered only after failure of treatment for suspected bacterial, viral and fungal infections .
In most reported cases, a primary diagnosis of Acanthamoeba keratitis was not made and a delay in correct diagnosis ranged from 7 weeks to 12 months. In three cases the diagnosis was made after enucleation of the eye sub . Diagnosis was based on histopathology of the corneal button or corneal biopsy in 56% of cases, after corneal scrapings for smear and culture were negative for Acanthamoeba  .Examination of the corneal scrapings by various methods, for the observation of cysts or trophozoites of Acanthamoeba, has had still lesser success in the early diagnosis of Acanthamoeba keratitis. In two cases the diagnosis was made only after "wavelike tracks" were observed on the surface of initial blood agar culture plates, leading the investigators to perform a diagnostic wet mount examination ,. In another case a Giemsa-stained specimen taken after 6 weeks into the course of the keratitis revealed a single cyst . In contrast, every single case of ours was first suspected on the observation of large number of cysts in 10% KOH wet mount examination of corneal ulcer scrapings. Potassium-hydroxide serves to weaken the corneal lamellar structure and epithelial cell outlines rendering the background almost homogenous and is useful in the diagnosis of keratomycosis . We could identify the double walled cysts of Acanthamoeba, having outer wrinkled wall (ectocyst) and inner polygonal, stellate wall (endocyst), in corneal scraping KOH preparations. The clarity of the cysts in this preparation is truly remarkable [Figure 6]. Except on the first occasion , in the presently described four cases we were certain of the identity of the cysts which was substantiated by successful culture. Attempts were made to locate trophozoites/cysts in normal saline wet mount preparations of the corneal scrapings in all cases. No trophozoites could be discerned in any of the four cases but cysts were visible in two. However, the intact outlines of corneal stroma, epithelial cells and pus cells hindered a clear view. In addition. motile macrophages may be mistaken for trophozoites. The cysts could also be seen by Gram stain and Giemsa stain of corneal scrapings in one case (not attempted in other cases) but the structure of cysts was not as remarkable as KOH wet mount examination and these techniques did not seem to offer any additional advantage. However, in KOH preparation slight disintegration was noted in the cyst morphology after one hour had passed and a permanent preparation cannot obviously be made. We recommend the simple method of 10% KOH wet mount examination of corneal scrapings for rapid identification of Acanthamoeba cysts and Gram stain or Giemsa stain if preservation of the slide is desired. A large number of stains have been described The organisms stain well with haematoxylin-eosin  sub periodic acid-schiff(PAS) . Giemsa , Wright's . Trichrome , Heidenhain's iron haematoxylin-eosin , hemacolour . gomori-methenamine-silver , Wilder's reticulum , Bauer chromic acid , calcofluor white with Evan's blue . and indirect fluorescent antibody staininq ,
The yield of Acanthamoeba in culture greatly increases with increased suspicion. Inclusion of special growth media consisting of non-nutrient agar (1.5% agar in normal saline) with an overlay of Escherichia coli  along with standard bacteriological and mycological media might lead to an increased detection of Acanthamoeba keratitis. Culture on NNA and detection of the trophozoites and cysts in culture are simpler than what is generally believed. Trophozoites can be detected within 48-72 hours on the surface of the medium [Figure 7], directly examined under the microscope, by their slow directional movement. They begin to change into cysts after this period and can be easily recognized by their double walled structure [Figure 8]. The observation of both cysts and trophozoites can be facilitated by transferring it onto a slide in a drop of saline. Identification to generic level is based on their morphology and failure to form flagellate forms in enflagellation experiments . For species identification, however, the cultures should be sent to reference laboratories.
Acanthamoeba can grow on blood agar, chocolate agar or nutrient agar but the observation of these media under the microscope may be difficult owing to their opacity. Moreover, NNA minimizes the growth of toxic, inedible bacteria and only the appropriate bacterial lawn is made available to the amoebae as food. Corneal scrapings, corneal button or corneal biopsy specimens may all be used for successful culture of Acanthamoeba . Cultures of conjunctival scrapings. though done in several cases, has a low yield of Acanthamoeba and may even result in erroneous diagnosis of bacterial keratitis.
Acanthamoeba castellanii is the commonest species reported in most well described cases . Other species of Acanthamoeba that have been recovered from keratitis cases are A.polyphaga ,, A.culbertsoni ,, A.rhysodes , and A.hatchetti . The most reliable method of speciation is immunofluorescence method using monoclonal or polyclonal antibodies . Immunoperoxidase methods may also be used for the identification of species 
Current medical therapy for Acanthamoeba keratitis includes the empiric use of topical 0.1% Propamidine isethionate, 0.15% Dibromopropamidine isethionate,1% Miconazole, Neosporin, Corticosteroids and systemic Ketoconazole. Medical therapy with specific drugs used alone or in combination has had variable results. There have been promising reports of successful prolonged medical therapy ,,. Early diagnosis may be a prerequisite for successful medical therapy sub . All cases that were seen by us were in the advanced stages with large ulcers. In absence of Propamidine we used a combination of Miconazole and Neosporin in cases 1,2 and 3. Metronidazole topical drops were given empirically on a trial basis to case 1 when no improvement was visible after thirteen days of treatment with a combination of Miconazole and Neosporin.
Surprisingly, corneal infiltration and hypopyon was reduced after nine days therapy with a combination of Metronidazole and Neosporin. However, Acanthamoeba spp isolated from this patient was not found to be sensitive to Metronidazole (MIC25iig/ml) invitro. Consequently the patient was discharged with only Neosporin and was found to have a completely healed ulcer after one and half months. The impression that Metronidazole may have had some action on the amoebae invivo made us try this drug in case 2 also in conjunction with Miconazole and Neosporin. There was an apparent transitory clinical improvement but ultimately the ulcer progressed leading to perforation. The lack of favourable action of Metronidazole became evident in case 4 when it was given as a single drug for 7 days and the ulcer worsened rapidly. Thus, with the available drugs. medical therapy succeeded in only one out of four cases of Acanthamoeba keratitis in this series.
Various investigators have performed sensitivity tests against the Acanthamoeba strains they have isolated, with varying results ,. Recent sensitivity studies  suggest that Ketoconazole and Miconazole may be effective. Wright et a1  have shown Propamidine and Dibromopropamidine to be highly effective both invivo and invitro. Dibromopropamidine ointment (0.15%) and Propamidine isethionate (0.1 %)eye drops (Brolene TM) are sold freely in Great Britain for antibacterial and antifungal therapy, but are not available in India. Systemic Ketoconazole along with other topical antiamoebic drugs and corticosterids has been found effective by Cohen et al .Even though patients may have an initial favourable response to medical therapy, in certain cases the clinical course may become progressive after a transient improvement, as was seen in our cases 2 and 3.
Penetrating keratoplasty has been successful in treating Acanthamoeba infection in a majority of cases . The indications for penetrating keratoplasty have been specified as descemetocele formation, impending or actual perforation of cornea, thinning of the cornea with extension of the infiltrates and failure to improve on medical therapy. At first, most agree, a trial of specific antiamoebic therapy is reasonable in the management of Acanthamoeba keratitis before corneal transplantation. The greatest constraint in India is, however, the non-availability of specific drugs which under the circumstances may hinder adequate therapy even in the face of accurate diagnosis.
The cases of Acanthamoeba keratitis reported here and earlier by us  are probably the tip of a large iceberg of unreported and unrecognized cases. There may have been several instances of amoebic keratitis, missed because of unfamiliarity with the entity on the part of all involved clinicians, pathologists and microbiologists.
Noting certain characteristic features of Acanthamoeba corneal infection, such as the slow relapsing course with development of ring abscess, recurrent epithelial breakdown, hypopyon which waxes and wanes, disproportionately severe pain and secondary glaucoma, may be worthwhile. A corneal ulcer scraping examination in 10% KOH wet mount and culture will readily confirm the diagnosis if performed knowledgeably.
The authors thank Dr. G.S. Visvesvara, Ph.D, Research Microbiologist, Parasitic Diseases branch, Centres for Disease Control, Atlanta, U.S.A. for helping-with speciation of Acanthamoeba isolates and providing important literature. Technical assistance of Miss. K. NagaJyothi and Mr. M.Murugesan is gratefully acknowledged.
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