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 Table of Contents  
CASE SERIES
Year : 2022  |  Volume : 12  |  Issue : 2  |  Page : 119-123  

Acanthamoeba keratitis: Experience from a tertiary eye care center in North India


1 Department of Ocular Microbiology, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
2 Department of Ophthalmology, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India

Date of Submission28-Feb-2022
Date of Decision29-May-2022
Date of Acceptance29-Jun-2022
Date of Web Publication24-Nov-2022

Correspondence Address:
Nishat Hussain Ahmed
Ocular Microbiology Section, Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tp.tp_16_22

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   Abstract 


The free-living amebae of genus Acanthamoeba are an important cause of microbial keratitis. The clinical appearance of Acanthamoeba keratitis (AK) usually mimics viral or fungal keratitis. Thus, microbiological workup plays a significant role in the diagnosis and timely treatment of such cases. We report a retrospective case series of seven culture-confirmed AK cases from a tertiary eye care center in North India. Various risk factors and triggers of infection, clinical presentations, microbiological findings, and management of AK are elucidated.

Keywords: Acanthamoeba keratitis, coexisting bacterial infections, nonnutrient agar, polyhexamethylene biguanide, ring infiltrates


How to cite this article:
Ahmed NH, Rathod PG, Satpathy G, Tandon R, Sharma N, Titiyal JS. Acanthamoeba keratitis: Experience from a tertiary eye care center in North India. Trop Parasitol 2022;12:119-23

How to cite this URL:
Ahmed NH, Rathod PG, Satpathy G, Tandon R, Sharma N, Titiyal JS. Acanthamoeba keratitis: Experience from a tertiary eye care center in North India. Trop Parasitol [serial online] 2022 [cited 2022 Dec 4];12:119-23. Available from: https://www.tropicalparasitology.org/text.asp?2022/12/2/119/361951




   Introduction Top


Acanthamoeba species are free-living amebae that are widely distributed in the natural and man-made environment and are known to cause serious central nervous system and ophthalmic infections. Ocular infections by Acanthamoeba species include keratitis, uveitis, and endophthalmitis.[1],[2]

The clinical manifestations of Acanthamoeba keratitis (AK) overlap with those of viral and fungal keratitis. This leads to misdiagnoses and delay in the administration of appropriate therapy. Furthermore, AK has a prolonged course of disease which may be complicated by corneal scarring and visual morbidity.[3] Thus, microbiological diagnosis plays a significant role in the management of AK.

We report a retrospective case series of culture-proven AK from a tertiary eye care center in North India. The approval of the institutional ethics committee was obtained before commencing the study. Medical records of microbiologically confirmed AK patients from 2016 to 2020 were reviewed and demographic, clinical, and microbiological details of the cases were noted.

Investigations to diagnose AK involved the collection of corneal specimens by trained ophthalmologists using a sterile surgical blade (no. 15). The microbiological workup included primary microscopy using wet mounts and Giemsa staining; and culture on Nonnutrient agar (NNA) with  Escherichia More Details coli overlay. In Giemsa staining, the Acanthamoeba trophozoites appear as blue spherical or oval structures (size 12–45 μ) with needle-like fine projections on pseudopodia (acanthopodia) against the purple background. The cysts of Acanthamoeba are of the same size as trophozoites, they are double-walled of which the inner wall is retracted to give a polygonal- or star-shaped appearance [Figure 1]. The NNA plates were incubated for 2 weeks at 37°C and were observed daily under a low-power (×10) microscope to look for the presence of trails of Acanthamoeba growth along with bacterial clearing [Figure 2]. The trails were scraped and wet mounts and Giemsa stained smears were observed under high power (×40) of a light microscope to confirm the presence of Acanthamoeba trophozoites and cysts.
Figure 1: Giemsa stained smear showing oval, blue trophozoite of Acanthamoeba (a), and double-walled cyst (the inner wall is retracted to give a polygonal- or star-shaped appearance) (b)

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Figure 2: Nonnutrient agar culture plate showing the presence of trails of Acanthamoeba growth along with bacterial clearing

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   Case Series Top


From January 2016 to December 2020, 142 cases were investigated for AK, out of which seven were positive for Acanthamoeba. A summary of the epidemiological and clinical details of the cases is given in [Table 1].
Table 1: Epidemiological and clinical details of seven Acanthamoeba keratitis cases

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Case 1

A 31-year-old male patient developed severe pain, redness, and diminution of vision after a trivial injury to the left eye while traveling. He was referred to our center due to nonresponse to medications given by a private practitioner (details of treatment not available). On examination, the visual acuity recorded perception of light and a large (9 mm × 9.5 mm) central corneal ulcer was seen along with corneal opacity and scleritis. A provisional diagnosis of fungal and AK was made and corneal scrapings were sent to the ocular microbiology section. The direct microscopy by Gram stain, Giemsa stain, and potassium hydroxide mount was negative for any pathogen. NNA culture showed trails of Acanthamoeba growth in 2 days. The patient was started on 0.02% topical polyhexamethylene biguanide (PHMB) two hourly, 5% topical Natamycin two hourly and oral Voriconazole 200 mg/day. However, the symptoms did not improve and a therapeutic deep anterior lamellar keratoplasty was performed following which the visual acuity improved to hand movement close to face (HMCF), and the patient was discharged with advice for weekly visits. The patient was lost to follow-up.

Case 2

A 35-year-old male patient gave a history of trauma in the left eye by caustic soda while cleaning his vehicle, followed by washing the injured eye with tap water. After an asymptomatic period of 2 months, he developed severe pain, watery discharge, redness, and diminution of vision. On examination, the visual acuity was HMCF and a central corneal ulcer (8.5 mm × 8.5 mm) along with ring infiltrates was seen. Corneal scrapings sent for microbiological culture grew Acanthamoeba species on the NNA plate. Topical PHMB 0.02%, moxifloxacin 0.5%, and oral voriconazole 400 mg were started. The patient improved symptomatically after continuous treatment for 3 months.

Case 3

A 55-year-old male patient complained of severe pain, redness, watering, and diminished vision in the left eye for 2 months duration, following an episode of rubbing the eye while bathing. He was diagnosed with viral keratitis at a private center and was being managed by bandage contact lenses and topical steroids. However, his symptoms did not improve and he was referred to our center. On examination, the visual acuity in the affected eye was HMCF and a 3.5 mm × 5 mm ulcer along with 5 mm ring infiltrates was seen in the central part of the cornea. Confocal microscopy showed evidence of Acanthamoeba cysts. Corneal scrapings were sent for microbiological diagnosis. Growth of Staphylococcus aureus and Pseudomonas aeruginosa was obtained in bacterial culture; and that of Acanthamoeba on NNA plates. Topical PHMB 0.02%, propamidine isethionate 0.1%, and moxifloxacin 0.5% were given 1 hourly for the initial 3 days, followed by 2 hourly for 2 weeks after which the symptoms resolved. The antiamebic treatment was continued for two more weeks after the resolution of symptoms.

Case 4

A 20-year-old male farmer developed pain, redness, watering, and gradual diminution of vision in the right eye following a fall in muddy water on the road 45 days ago. Visual acuity was restricted to HMCF. The cornea was edematous and a central 5.5 mm × 7 mm ulcer with ring infiltrates and hypopyon was seen. An empirical treatment with vancomycin 5% eye drops and oral doxycycline was started. Corneal scraping sent for microbiological evaluation grew Staphylococcus aureus. Thus, the same treatment was continued. However, the symptoms worsened and a therapeutic penetrating keratoplasty (TPK) was performed. The excised corneal tissue grew Acanthamoeba on NNA along with S. aureus on blood agar. PHMB 0.02% and chlorhexidine 0.02% were added to the patient's treatment. The visual acuity improved to 2/60 and symptoms resolved after continuous treatment for 3 months.

Case 5

A 35-year-old male patient presented with pain, redness, burning sensation, watering, and diminution of vision in the left eye for 3 months duration. He gave a history of trauma by a foreign body while traveling by motorcycle. He was diagnosed with necrotizing stromal keratitis in a private setup and referred to our center due to nonresponse to the treatment. The visual acuity was HMCF and a large 9 mm × 9 mm, central corneal ulcer with deep stromal infiltrates was seen. Empirical treatment with tobramycin 1.3%, cefazolin 5%, natamycin 5% eye drops, and oral voriconazole 200 mg was started and corneal scrapings were sent for microbiological diagnosis. Acanthamoeba trails were seen in the NNA culture plate after 3 days of incubation. Topical PHMB 0.02%, propamidine isethionate 0.1%, and moxifloxacin 0.5% eye drops were started. However, after 2 weeks the ulcer perforated; thus a TPK was performed and the patient improved symptomatically. Antiamebic treatment was continued for 2 weeks after the resolution of symptoms.

Case 6

A 38-year-old male patient presented with severe pain, irritation, watering, and diminution in vision in the left eye after around 1 month of an episode of swimming while wearing contact lenses. On examination, visual acuity of HMCF was noted and an ulcer of 4.5 mm × 5 mm was observed in the central cornea along with reduced corneal sensations. The corneal scrapings as well as contact lenses and lens solution were sent for microbiological evaluation. The growth of Acanthamoeba was obtained after 2 days of incubation in NNA culture. The patient was started on topical PHMB 0.02%, propamidine 0.1%, and moxifloxacin 0.5%. The patient was lost to follow-up after discharge.

Case 7

A 31-year-old male patient presented with severe pain and diminution of vision in the right eye. He recalled an episode of fall of a dust particle in the eye in the recent past. The ophthalmic examination revealed congested conjunctiva and 7 mm × 8 mm central corneal ulcer along with hypopyon. Visual acuity was HMCF. Confocal microscopy showed the presence of fungal elements. Topical natamycin 5%, voriconazole 1% eye drops, and oral voriconazole 200 mg were started empirically, and corneal scrapings were sent for microbiological evaluation. Direct microscopy by Gram stain, potassium hydroxide, and Giemsa stain did not reveal any organism. Bacterial and fungal cultures were sterile. However, NNA culture showed trails of Acanthamoeba growth after 4 days. Thus, PHMB 0.02% and propamidine isethionate 0.1% were added to the treatment and continued for 5 weeks. The patient has not improved clinically and surgical management with TPK is planned. The patient is still being followed.


   Discussion Top


Worldwide, AK is increasingly being reported as a sight-threatening infection. Epidemiologically, its incidence is known to be high in middle-aged males as this group of individuals is mainly involved in outdoor activities and exposed to various habitats of the free-living amebae.[3],[4] All the cases in the present series are males with five of them in the age range of 31–38 years.

The knowledge of AK is expanding both in developed and developing countries. In our country, the prevalence of AK ranges from 1.04% in South India to 3.6% in North India.[3],[5] In India, the most common triggers for AK are corneal trauma and exposure to contaminated water or soil.[3],[5],[6],[7],[8],[9] In developed countries, AK is usually reported from contact lens wearers.[10],[11] In Indian urban setup, various studies have reported AK associated with contact lens wear in the range of 0.9%–27% of corneal ulcer cases.[3],[4],[8] Trauma to the cornea, especially with vegetable matter is also the most common trigger for fungal corneal ulcers in developing countries. As some cases of AK can clinically mimic fungal keratitis – in patients having a history of trauma, if there is nonresponse to antifungal treatment, AK should be ruled out at the earliest. In the present case series, the triggers for Acanthamoeba infection included trauma to the cornea, exposure to mud, exposure to tap water, washing of chemically injured eye with unsterile water, and swimming with contact lenses on.

The classical clinical features of AK include out of proportion pain, radial keratoneuritis, and ring infiltrates.[4] However, typical clinical presentation may not be seen in all cases. All the cases of our series complained of severe pain and three cases had ring infiltrates. A study from South India has reported excruciating pain and ring infiltrates in 23% and 33% of AK cases, respectively.[4] A previous study from the same institute has reported radial keratoneuritis and severe pain as nonsignificant clinical features of AK.[12] Such variation in the clinical presentation often leads to misdiagnoses and delay in initiation of appropriate therapy. In a large retrospective study of 21 years duration from Austria, 95.3% of patients had severe pain; the most frequent ophthalmological finding was stromal infiltration, including ring infiltrates in 68.2% of cases.[13] The variation in clinical presentation and low index of suspicion often causes delay in diagnosis of AK which might lead to adverse clinical outcomes.

The diagnosis of AK endures several challenges. The sensitivity of primary microscopy is low. We use wet mounts and Giemsa stained smears for primary microscopy of corneal specimens. Several staining techniques have been evaluated for the identification of the trophozoite and cyst forms of Acanthamoeba species. These include temporary wet mounts by iodine, eosin, methylene blue, lactophenol cotton blue, potassium hydroxide, and calcofluor white stain; and permanent staining of smears using Gram, Gimenez, Giemsa, chlorazol black, H and E, iron hematoxylin, periodic acid–Schiff, Masson's trichrome, Field's, Gomori's methenamine silver, and acridine orange stains.[14],[15] The temporary wet mounts are simple, single-step, rapid, and inexpensive methods that have an added advantage of demonstrating the motility of the trophozoite forms. However, they are transient preparations; and therefore, may not be available for further review and evaluation.[14],[16] In Egypt, two large multi-attribute ranking analyses of different staining methods for the identification of morphological forms of Acanthamoeba were done in 2015 and 2019. Among the temporary mounts evaluated, it was found that the iodine wet mount was better than others; among permanent stains, Gimenez stain and Giemsa stain were found to be more effective in demonstrating morphological details.[14],[16]

The culture of Acanthamoeba is the gold standard diagnostic modality for AK. However, it has low sensitivity particularly when the patient is treated with antibiotics having amebicidal activity; and often the culture results are obtained after prolonged incubation. Recently, many nucleic acid amplification tests have been developed that claim improved the diagnosis of AK.[17],[18],[19] Molecular methods have an advantage of a shorter turnaround time compared to culture, and the theoretical detection threshold is one ameba per sample. However, the expertise and infrastructure for doing molecular tests are not available at all centers. In a previous study done at our center, a polymerase chain reaction (PCR) assay based on 464-bp region within 18S rRNA gene was compared with culture on NNA. Acanthamoeba spp. could be detected by culture from 15.84% of cases, and by PCR from 17.48% of cases of suspected amebic keratitis patients.[19] In a study from Canada, clinical specimens from suspected AK cases were subjected to microscopy, culture, two gel-based PCR assays, and two real-time quantitative PCR assays. The best sensitivity (89.3%) was found to be of one of the real-time PCR assays. Furthermore, 13 culture- and microscopy-negative specimens were found to be positive by real-time PCR.[20] A study from Europe compared the performance of five PCR-based assays on corneal samples for the diagnosis of AK. The authors reported that the sensitivity of these PCR assays ranged from 73.3% to 86.7%. However, these sensitivities did not differ significantly from that of culture (66.7%).[21] Our series endures a big limitation that molecular tests were not done in suspected cases of Acanthamoeba keratitis.

Treatment of AK is also challenging owing to the resistant cystic stage of the ameba and nonavailability of a single effective drug therapy. The current therapeutic approaches for AK include both medical and surgical management. Topical biguanides and diamidines are reported to have in vitro efficacy against cysts, thus forming the mainstay of medical treatment.[4] In our study, all seven cases were given PHMB. Three patients who worsened with corneal thinning and perforation underwent surgical intervention. The antiamebic treatment is continued for at least 2 weeks after the resolution of symptoms to clear the cysts from deeper layers of the cornea.[9]

To conclude, despite the low prevalence, AK is of concern due to the visual morbidity associated with it. High clinical suspicion and timely application of microbiological tests are necessary to guide an effective management for better ophthalmic outcomes.

Ethical clearance

Permission of Institute Ethics Committee was obtained prior to beginning the study, reference no. IEC-751/12.11.2021, dated 15-11-2021.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

The study was supported by internal funding from Dr. R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Jones DB, Visvesvara GS, Robinson NM. Acanthamoeba polyphaga keratitis and Acenthamoeba uveitis associated with fatal meningoencephalitis. Trans Ophthalmol Soc U K (1962) 1975;95:221-32.  Back to cited text no. 1
    
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Rammohan R, Hajib Naraharirao M, Veerappan S, Vijayaraghavan P, Rajaraman R, Manayath GJ, et al. Cluster of post-operative endophthalmitis caused by Acanthamoeba T10 genotype – A first report. Cornea 2021;40:232-41.  Back to cited text no. 2
    
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Megha K, Thakur A, Khurana S, Sehgal R, Gupta A. Acanthamoeba keratitis: A 4-year review from a tertiary care hospital in North India. Nepal J Ophthalmol 2020;12:83-90.  Back to cited text no. 3
    
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Garg P, Kalra P, Joseph J. Non-contact lens related Acanthamoeba keratitis. Indian J Ophthalmol 2017;65:1079-86.  Back to cited text no. 4
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Chidambaram JD, Venkatesh Prajna N, Srikanthi P, Lanjewar S, Shah M, Elakkiya S, et al. Epidemiology, risk factors, and clinical outcomes in severe microbial keratitis in South India. Ophthalmic Epidemiol 2018;25:297-305.  Back to cited text no. 7
    
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Moe CA, Lalitha P, Prajna NV, Mascarenhas J, Srinivasan M, Das M, et al. Outcomes of amoebic, fungal, and bacterial keratitis: A retrospective cohort study. PLoS One 2022;17:e0264021.  Back to cited text no. 9
    
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Randag AC, van Rooij J, van Goor AT, Verkerk S, Wisse RP, Saelens IE, et al. The rising incidence of Acanthamoeba keratitis: A 7-year nationwide survey and clinical assessment of risk factors and functional outcomes. PLoS One 2019;14:e0222092.  Back to cited text no. 10
    
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Stehr-Green JK, Bailey TM, Visvesvara GS. The epidemiology of Acanthamoeba keratitis in the United States. Am J Ophthalmol 1989;107:331-6.  Back to cited text no. 11
    
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Sharma S, Garg P, Rao GN. Patient characteristics, diagnosis, and treatment of non-contact lens related Acanthamoeba keratitis. Br J Ophthalmol 2000;84:1103-8.  Back to cited text no. 12
    
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List W, Glatz W, Riedl R, Mossboeck G, Steinwender G, Wedrich A. Evaluation of Acanthamoeba keratitis cases in a tertiary medical care centre over 21 years. Sci Rep 2021;11:1036.  Back to cited text no. 13
    
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Eldeek H, Attia R, Nageeb M, Sakla A. Comparative evaluation of multiple staining techniques for identification of different developmental stages of Acanthamoeba and Naegleria. J Egypt Soc Parasitol 2019;49:409-22.  Back to cited text no. 14
    
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El-Sayed NM, Hikal WM. Several staining techniques to enhance the visibility of Acanthamoeba cysts. Parasitol Res 2015;114:823-30.  Back to cited text no. 16
    
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Maubon D, Richarme C, Post L, Robert MG, Bernheim D, Garnaud C. Development, optimization, and validation of a multiplex real-time PCR assay on the BD MAX platform for routine diagnosis of Acanthamoeba keratitis. J Mol Diagn 2020;22:1400-7.  Back to cited text no. 17
    
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Holmgaard DB, Barnadas C, Mirbarati SH, O'Brien Andersen L, Nielsen HV, Stensvold CR. Detection and identification of Acanthamoeba and other nonviral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis. J Clin Microbiol 2021;59:e02224-20.  Back to cited text no. 18
    
19.
Behera HS, Satpathy G, Panda A, Pooja B, Manjari T. Evaluation of PCR assay over culture for the detection of Acanthamoeba Spp. in amoebic keratitis and amoebic meningitis/meningoencephalitis patients in India. J Ophthalmol 2019;4:000184.  Back to cited text no. 19
    
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Khairnar K, Tamber GS, Ralevski F, Pillai DR. Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis. Diagn Microbiol Infect Dis 2011;70:499-506.  Back to cited text no. 20
    
21.
Yera H, Ok V, Lee Koy Kuet F, Dahane N, Ariey F, Hasseine L, et al. PCR and culture for diagnosis of Acanthamoeba keratitis. Br J Ophthalmol 2021;105:1302-6.  Back to cited text no. 21
    


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