Acanthamoeba trophozoites with scanty inflammatory cells in a corneal stroma seen, stained with H&E stain. Trophozoites resembles the macrophages, with cytoplasmic vacuolation (red arrow), retraction cleft and distinct nucleus in tissue section. The epithelium is not seen. (100x view)
Acanthamoeba trophozoites with scanty inflammatory cells in a corneal stroma seen, stained with H&E stain. Trophozoites resembles the macrophages, with retraction cleft in few and distinct nucleus. The epithelium is not seen. (100x view)
Acanthamoeba cyst form shows thick double walled cyst with cytoplasmic retraction in tissue sections with H&E stain (Green circle). Trophozoite form resembles the macrophages, with cytoplasmic retraction and distinct nucleus (blue box) (100x view).
Acanthamoeba cyst form shows cytoplasmic retraction in tissue sections with H&E stain (Green circle) (100x view). Many other trophozoite resembles macrophages (red arrow) (100x view).
Practical Points of Pathoclinics:
- Acanthamoeba keratitis is an uncommon but a serious eye-sight threatening infection of contact lenses users, caused by acanthamoeba. Nowadays, about 5% of contact-lens-associated keratitis is caused by acanthamoeba and the rates have been steadily increasing in recent years.
- Acanthamoebas are small, free-living ubiquitous opportunistic pathogens, found in soil and water. Acanthamoeba exists in an active trophozoite and a dormant cyst forms, later is responsible for severity of infection. Antibiotics, low temperatures, high doses of UV-light, and gama-radiation have no effect on cyst wall, and cyst form may survive over 24 years. So, the cyst form of acanthamoeba is difficult to eradicate.
- Use of long standing contaminated lens wash saline solutions and usage of lenses during swimming or bathing in hot tubs are responsible for acanthamoeba infection in contact lens users. Acanthamoeba become adherent to the surface of old wear and tear contact lenses and proliferate in association with other fungi and bacteria, as they feed on bacteria, algae, and yeasts. Use of disposable contact lenses can decrease the incidence of acanthamoeba keratitis. Only hydrogen-peroxide-containing contact lens cleaners are effective against all acanthamoeba strains. Non-contact lens users, those use tap water to wash their eyes are also at risk of acanthamoeba infection. Among immunocompromised person, severe life threatening granulomatous amoebic encephalitis infection of the central nervous system can develop with very high mortality of 95-95%.
- In early stages of the infection, about 75-90% of all patients are misdiagnosed, as initial clinical signs and symptoms resemble other corneal conditions, and so management get delayed. A high index of clinical suspicion is necessary for early diagnosis and management, to prevent vision loss. Clinically, corneal infection is extremely painful, due to cornea involvement and perineuritis. A ring or annular infiltrate is a characteristic feature, but usually develops in the late in course of disease and in approximately 50% of cases only. Ring infiltrates also seen among other corneal ulcers and fungal keratitis, so it is not specific clinical findings. Keratoconjunctivitis with cloudy corneal opacities and photophobia may be seen. Clinical course is relapse and remitting type, as cyst forms remain in corneal tissue for a prolonged period of time and inability to eradicate the cyst form of acanthamoeba, once they penetrate deeper corneal stroma. Cyst forms are responsible for persistent corneal and scleral inflammation.
- Acanthamoeba keratitis can be diagnosed using in vivo confocal microscopy, corneal Scraping, polymerase chain reaction (PCR), corneal biopsy and imprint cytology and culture.
- Initial early tentative diagnosis can be made on in vivo confocal microscopy, which shows hyper-reflective, well defined spherical structures due to double cyst wall. Vivo confocal microscopy has high sensitivity and specificity in experienced person; however, only acanthamoeba cysts are well recognized using confocal microscopy.
- Acanthamoeba keratitis can be diagnosed using scrapping and smears and tissue biopsy, with a sensitivity of 31-65%. Number of amoeba are related with severity of infection. Biopsy usually reveals variable degree of corneal epithelium detachment and ulceration along with necrosis and chronic inflammation. A useful early histopathological diagnostic clue is widespread disappearance of stromal keratocytes, which presumably have been killed and phagocytosed. In an initial phase of acanthamoeba keratitis, infiltration of multiple cysts and/or trophozoites of acanthamoeba are limited to the corneal epithelium only with minimal chronic inflammation, later parasite invades the underlying stroma with extensive damage to collagen matrix and initiate intense inflammation, in second phase. Chronic inflammatory reaction in a corneal stroma may mask the amoebic cysts. Amoebic cysts are readily found in routine hematoxylin and eosin stained sections or smears. The chitinous walls of the Acanthamoeba cysts are round or oval and stain with methenamine-silver, periodic acid-Schiff, Masson’s trichrome, and iron-haematoxylin- eosin stains. The cytoplasm typically retracts from the cyst wall in tissue sections. The cysts contain a small round nucleus with a distinct nucleolus. Trophozoites form of Acanthamoeba are usually difficult to identify because they resemble macrophages. The fluorochrome – calcofluor white can stain the trophozoites (bright red-orange) and cyst cell walls (fluoresce bright apple-green with red orange cytoplasm), gives rapid and reliable results on corneal scrapping cytology, under ultraviolet fluorescent microscope. Stromal neovascularization is usually not found despite long-standing corneal inflammation. A granulomatous reaction with many multinucleated giant cells, some with engulfed cysts of Acanthamoeba, can be present in the posterior corneal stroma and anterior chamber along Descemet’s membrane. Severe keratitis with neutrophilic abscess formation can be seen in the late stages of the disease.
- Acanthamoebas can be identified by immunohistochemistry using anti-acanthamoeba antibodies in tissue sections.
- Genotyping of acanthamoebas show twenty-two different genotypes (T1–T22) based on 18s rRNA gene typing. Different genotypes show variation in clinical presentation and response to medical therapy. At least eight of the genotypic classes (T2, T3, T4, T5, T6, T10, T11, and T15) have been shown to cause Acanthamoeba keratitis, with the most common causative genotype being T4. The most common infectious species are Acanthamoeba castellani and Acanthamoeba polyphaga, both from the T4 genotype.
- Polymerase chain reaction (PCR) of corneal scrapings for acanthamoeba DNA has highest sensitivity of 84-100%, with rapid results in 60 minutes. However, non-living acanthamoeba may give a positive result.
- Acanthamoebas are cultured as a line on agar plates with a layer of E. coli. But culture takes 3 weeks for results. Serological techniques have no diagnostic value as specific antibodies are also detected in apparently healthy people due to the ubiquity of Acanthamoeba.
- In some of acanthamoeba keratitis, a mixed infection with virus, bacteria, or fungi can be present. Initial stage of acanthamoeba keratitis often confused with HSV keratitis, with anterior stromal infiltrates, disciform keratitis, keratoneuritis, and ring infiltrate. Anterior chamber reaction is rare. In contrast to bacterial or fungal keratitis, the anterior stroma of acanthamoeba keratitis, in many cases contains only a few sparse infiltrate of inflammatory cells, mainly neutrophils and macrophages. Stromal necrosis is usually inconspicuous, and stromal neovascularization is not observed.
- Many cases of acanthamoeba keratitis are resolve with early diagnosis and aggressive medical therapy with use of biguanide and aromatic diadine eye drops for several months. Early epithelial debridement is helpful for success of medical therapy, by removing the majority of organisms. Effective treatment is aimed at the eradication of viable cysts (which are more resistant to treatment) and trophozoites. Some cases of medical management failure are due to infection with coexistent herpes simplex and other amoeba-like protozoa (e.g., Vahlkampfi a , Hartmannella and Naegleria ). In a medically refractory cases, penetrating keratoplasty (corneal transplantation) may be necessary. Intractable cases can develop endophthalmitis.
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