DEPARTMENT OF PATHOLOGY
The Johns Hopkins Medical Institutions

Vol 16, No. 29

THE JOHNS HOPKINS MICROBIOLOGY NEWSLETTER

Monday, August 4, 1997

A. Provided by Carmela Groves, R.N., M.S., Chief, Division of Outbreak Investigation, Maryland Departent of Health and Mental Hygiene

B. The Johns Hopkins Hospital: Information provided by David M. Steinberg, M.D., Chief Resident, Pathology

History: A 73 year old white female from New York with no significant past medical history was on a cruise ship in the Mediterranean. She ran out of sterile cleansing solution for her contact lenses and started to rinse her lenses with tap water. Within a few days she complained of increasing eye irritation, intense pain, and blurred vision. She was flown off the ship to Italy where she was clinically diagnosed with herpes keratitis. Subsequently, she went back to NYC where the clinical diagnosis of herpes keratitis was again made. Eye exam revealed diffuse corneal swelling. All cultures and biopsies remained negative. Despite 6 weeks of antiviral therapy, her symptoms worsened. She went to the Wilmer Institute for a second opinion. As part of the work-up, a special culture of the corneal scrapings was sent to microbiology. This culture was positive for Acanthamoeba. Appropriate anti-amoebicidal treatment with polyhexamethylene biguanide, miconazole, and neomycin was instituted immediately.

Acanthamoeba Keratitis

Introduction: Acanthamoeba are small, free-living protozoa that have been isolated from many environmental sources including fresh, marine and chlorinated water, arctic ice, soil, vegetable matter, dust, and air. Like other protozoa, they are unicellular and can exist in two forms, trophozoite and cyst. The trophozoite form (15-45 m long) contains granular and abundant cytoplasm, mitochondria and a single nucleus with a prominent central round nucleolus. The trophozoite is motile, proliferates, and feeds on bacteria, fungi, and other unicellular organisms. In adverse conditions, the trophozoites encyst, forming a double wall containing cellulose. The cyst form (10-25 m in diameter) is much more resistant to extreme environments, as well as to chlorine and antimicrobial agents.

Epidemiology: Acanthamoeba keratitis is a relatively uncommon but potentially devastating corneal infection. Minor trauma to the corneal epithelium appears to be an important factor in the pathogenesis of the infection. The majority of recent cases are unilateral and have been associated with contact lens wearers who use contaminated cleaning solutions, including tap water, well water, homemade saline solutions, and saliva. In these cases, Acanthamoeba is usually isolated from one or more of the aforementioned sources, along with bacteria and fungi, which may serve as a food source for the Acanthamoeba. The infection is most common in young healthy adults.

Clinical Features: The clinical features are classified into three stages: initial, transient, and completed. Severe pain is characteristic during all three stages. The predilection of the amoebae for neural tissue may explain the initial severe pain, which is disproportionate to the stromal keratitis. In the initial stage, signs and symptoms include redness, irritation, foreign body sensation, tearing, blepharospasm, conjunctivitis, blurred vision, decreased corneal sensation, and photophobia. A dendriform corneal epithelial pattern may be seen before stromal involvement. The corneal epithelium may either be intact or have a localized punctate staining pattern. In the transient stage, stromal infiltrates form partial or complete rings. The ring infiltrates are almost pathognomonic for Acanthamoeba in a patient whose cultures and scrapings are repeatedly negative for any other organism. In the completed stage, opacification, corneal abscesses and central stromal edema are often observed. Double rings have been noted in some cases of established infection. In advanced cases, the ulcer may perforate.

Diagnosis: Many patients present with intense ocular pain that is responsive to topical corticosteroids, narcotics, or modified retrobulbar alcohol injections. The infection is frequently misdiagnosed initially as a herpes simplex virus or bacterial or even fungal keratitis. False-positive cultures for herpesvirus have been reported as a result of the cytopathic effect of Acanthamoeba in tissue culture. A definitive diagnosis is made by confirmation of Acanthamoeba in corneal lesions with direct examination or culture of corneal scrapings. For deeper infections, a corneal biopsy specimen can be obtained. A wet mount is prepared by scraping a corneal specimen or other material in a drop of saline solution on a glass slide under a coverslip. Acridine orange, Gram's, Giemsa, lactophenol cotton blue and Parker ink-KOH stains can reveal the Acanthamoeba cysts and motile trophozoites. Histopathologic sections can be stained with methenamine silver, periodic acid-Schiff (PAS), trichrome, and hematoxylin-eosin stains to demonstrate the organisms. Acute and chronic inflammation may or may not be present. Material for culture isolation is preferably inoculated directly onto culture medium. Acanthamoeba grow best on horse blood agar and nonnutrient agar seeded with an overlay of dead or living gram-negative rods, like Escherichia coli. Plates are incubated at room temperature and 37 °C and examined daily with a dissecting microscope to show the depressions or trails that motile trophozoites leave on the agar surface. If amoebae are observed, they are checked for the presence of a contractile vacuole, spiny pseudopods known as acanthapodia, and a well-defined ectoplasm and endoplasm. After most of the food source on the plate is exhausted, the double walled cysts are seen.

Treatment: In general, treatment for Acanthamoeba keratitis has been disappointing, partially because the infection is frequently well advanced before diagnosis and partially because the available treatment is suboptimal. Successful treatment requires early diagnosis and aggressive surgical and medical management. Evaluation of drug therapy has been restricted by the small number of cases treated, the varying stages at which treatment was initiated, the natural waxing and waning of the disease, and the differences among different Acanthamoeba strains. Several weeks to months of pharmacotherapy is necessary. Drugs to use include topical aromatic diamidines (e.g. propamidine isethionate and broline), topical and oral imidazoles (e.g. miconazole), topical aminoglycosides (e.g. neomycin), and topical antiseptics (e.g. polyhexamethylene biguanide). Biguanides, used as swimming pool biocides at higher concentrations, interfere with cytoplasmic membrane integrity and inhibit essential respiratory enzymes of many microbes. Treatment failures may be due to acquired resistance, poor penetration, and induced encystment from subcysticidal drug levels. When the infection has spread to the sclera, enucleation may be necessary. Some authors have had good success using triple procedures for Acanthamoeba keratitis; this method combines systemic antifungal drugs, topical anti-amoebic eyedrops, and multiple surgical debridements of the corneal lesion.

Prevention: Studies estimate that if all contact lens wearers used proper techniques to avoid contamination of the contact lens, 80% of Acanthamoeba keratitis would be prevented. Prevention must be aimed at educating eye care practitioners and contact lens wearers. Contact lens wearers must be informed of the importance of not wearing their lenses while swimming in fresh water and of always using commercially prepared sterile solutions preserved with benzalkonium chloride. Nonpreserved commercially prepared solutions in larger quantities and home-brew cleaning solutions can become contaminated during storage.

References

1. Perry HD, Donnenfeld ED, Foulks GN, Moadel K, Kanellopoulos AJ. Decreased corneal sensation as an initial feature of Acanthamoeba keratitis. Ophthalmology. 102(10):1565-8, 1995 Oct.

2. Chynn EW, Lopez MA, Pavan-Langston D, Talamo JH. Acanthamoeba keratitis. Contact lens and noncontact lens characteristics. Ophthalmology. 102(9):1369-73, 1995 Sep.

3. D'Aversa G, Stern GA, Driebe WT Jr. Diagnosis and successful medical treatment of Acanthamoeba keratitis. Archives of Ophthalmology. 113(9):1120-3, 1995 Sep.

4. Mathers WD, Sutphin JE, Folberg R, Meier PA, Wenzel RP, Elgin RG. Outbreak of keratitis presumed to be caused by Acanthamoeba. American Journal of Ophthalmology

HHV6 Announcement

Human Herpes Virus Type 6 (HHV-6) PCR testing is being offered through the Microbiology Laboratories. This is a send-out test, performed by Viro Med Labs in Minnesota. Acceptable sources include CSF, pleural fluid, whole blood in EDTA, bone marrow in EDTA and tissue. Turn around time is between 48 and 72 hours of receipt by Viro Med. The analytical sensitivity is 10 copies. Specificity is greater than 99%. The cost of the assay is $250.00 Use the Pathology requisition number 5. Check off source and write "HHV-6 PCR" in the comment box.


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