Acanthamoeba are free-living protozoa that are ubiquitous and inhabit various ecosystems, such as soil, water, and air. They have been isolated from swimming pools, water supply networks, cooling and dental equipment, hemodialysis machines, medical equipment (lens solution), etc.
Among free-living amoebae, only four groups include representatives that are opportunistically pathogenic to humans and animals: Acanthamoeba, Balamuthia, Naegleria, and Sappinia. Their taxonomic position has been revised several times in accordance with the criteria of modern systematics. Based on the classification proposed by the International Society of Protozoologists, these microorganisms have been assigned to so-called ‘supergroups’. Acanthamoeba and Balamuthia were classified as belonging to the supergroup Amoebozoa: Acanthamoevidae; Naegleria fowleri to Excavata: Heterolobosia: Vahlkampfiidae; and Sappinia to Amoebozoa: Flabellinea: Thecamoevidae. Free-living amoebae can cause diseases in humans such as amoebic keratitis, granulomatous encephalitis, meningitis and meningoencephalitis, sinusitis, skin lesions, etc [2].
Human exposure to Acanthamoeba is common, by means of inhalation of airborne cysts. Organisms can be recovered from the nasopharynx6 and circulating IgG antibodies to Acanthamoeba antigens are present in up to 80% of healthy individuals. Similarly, tears in healthy individuals also contain specific IgA antibodies to Acanthamoeba antigens.
Acanthamoeba is resilient to many forms of disinfection and decontamination. It exists in two forms, the active trophozoite that moves by contractions of finger-like projections, and the dormant cyst that is encased in a double layer cellulose wall. The cyst is able to withstand environmental conditions including extended periods of extreme temperature, pH and desiccation. Transfer to the cyst form may occur in environments of nutrient limitation, accumulation of metabolic products and toxin in culture media and in adverse environmental conditions. This differentiation process is also likely to be important in the pathogenesis of Acanthamoeba keratitis.
There is also evidence for a symbiotic relationship between Acanthamoeba and the organisms it ingests. Pathogenic organisms may be retained within the cytoplasm, conferring protection to the ingested organism from disinfection or host defences and allowing growth and replication.
Acanthamoeba most often causes keratitis in human, however some strains (different from those that cause keratitis) can also very rarely infect the brain (granulomatous amoebic encephalitis) in immunocompromised individuals, and the skin. The genus Acanthamoeba has been classified into 18 different sub-types, denoted T1-T18. Genotyping has been increasingly used to characterize strains causing human disease. Both contact-lens related and non-contact-lens related corneal isolates are most commonly associated with the T4 genotype which includes the species A. castellanii, A. polyphaga and A. culbertsonii.
Acanthamoeba keratitis is usually a unilateral disease, but it can occur bilaterally. Early symptoms are typically pain, photophobia and watering, sometimes to a greater extent than suggested by the clinical picture. However, Acanthamoeba keratitis can also occur without pain.
In the early stages, Acanthamoeba keratitis is confined to the epithelium with punctate keratopathy and often pseudodendrites. In later disease, the condition progresses to the stroma and is characterised by epithelial breakdown with a recurrent course and stromal inflammation. Immune ring ulcers and scleritis, thought to be sterile inflammatory reactions, tend to occur later in 15–20% of cases. If scleritis develops, often, very severe, persistent pain is reported. In the later stages of disease, stromal thinning, corneal opacification, hypopyon, corneal perforation, cataract, glaucoma and posterior segment inflammation may also be present. Limbitis is present in 90% of cases in both the early and late stages of the disease and an anterior chamber response is commonly seen.
Diagnosis
Corneal culture. The gold standard in the diagnosis of Acanthamoeba keratitis remains corneal culture. While confocal microscopy remains a useful adjunct, particularly in identifying cysts in the later stage of disease and for monitoring therapy, diagnostic sensitivity and specificity remains observer dependent.
Histopathology. Frequently, histopathology is also performed at diagnosis or later in the disease course from a corneal biopsy sample. If a sample for histology is also collected, it is usually mounted and fixed on a glass slide for immunohistochemistry. The sample is stained for organisms or their specific antigens for visualisation under light or fluorescent microscopy.
Polymerase chain reaction. Polymerase chain reaction (PCR) is a molecular technique which allows genotypic identification of a microbe based on sequence analysis of ribosomal DNA present in a tissue or fluid sample. This technique using a swab or epithelial sample is sensitive in up to 85% of cases for Acanthamoeba and has utility in culture negative cases [1].
The diagnosis of Acanthamoeba infections by microscopic examination of clinical samples is challenging and requires skilled laboratory personnel. In addition, prolonged incubation of cultures may be necessary, particularly when patients have been treated already. However, Acanthamoeba diagnosis has been facilitated and improved by the establishment of several PCR assays targeting various regions of the nuclear small subunit 18S rRNA gene (Rns) [3].
That’s why early and rapid diagnosis is important for successful and proper treatment. A new product from the Austrian manufacturer Ingenetix GmbH is the ParoReal® Kit Acanthamoeba T4 (CE-IVD) reagent kit, which allows to obtain results in less than 2 hours after sample collection.
(Source reference —https://ingenetix.com/en/product/human/Kit+Acanthamoeba+T4/)
ParoReal® Kit Acanthamoeba T4 is a non-automated IVD real-time PCR test for the qualitative detection of DNA (18S rRNA gene) of Acanthamoeba species of genotype T4 (A. castellani, A. lugdunensis, A. mauritaniensis, A. polyphaga, A. rhysodes, A. royreba). As already mentioned, T4 genotype is the most prevalent (approx. 86%) Acanthamoeba genotype causing keratitis worldwide. Proper specimens are DNA extracts isolated from human clinical specimens associated with keratitis (ocular swabs, corneal biopsies, ocular punctates, corneal scrapings) as well as contact lenses and contact lens solution. A probe-specific amplification-curve in the FAM channel indicates the amplification of Acanthamoeba specific DNA. An internal DNA positive control (IPC) is detected in Cy5 and serves as a control for DNA extraction and possible real-time PCR inhibition. This kit is compatible with a wide range of real-time PCR systems [5].
Conclusions
In summary, the global increase in incidence is prompting ophthalmologists to consider Acanthamoeba keratitis when very similar symptoms occur. 80% of AK cases are misdiagnosed! – 48% of these are mistakenly treated with steroids for Herpes simplex virus (HSV), which has been shown to worsen the course of AK [4].
Consequently, rapid and high-quality PCR diagnostics will facilitate correct medication prescriptions and the patient's quickest possible recovery.
References
1. Carnt N, Stapleton F. Strategies for the prevention of contact lens-related Acanthamoeba keratitis: a review Ophthalmic Physiol Opt 2016; 36: 77–92. doi: 10.1111/opo.12271.
2. Chobotar, A. (2019). Акантамеби як резервуар патогенних бактерій та вірусів. Інфекційні хвороби. 66–74. doi.org/10.11603/1681-2727.2019.2.10328.
3. Lamien-Meda, A.; Köhsler, M.; Walochnik, J. Real-Time PCR for the Diagnosis of Acanthamoeba Genotype T4. Microorganisms 2022, 10, 1307. https://doi.org/10.3390/ microorganisms10071307.
4. Acanthamoeba Keratitis Eye Foundation. Information about Acanthamoeba keratitis: a rare but serious infection of the cornea caused by a free-living amoeba. Available at: akeyefoundation.com/acanthamoebakeratitis/.
5. Ingenetix / ParoReal Kit Acanthamoeba T4. Manufacturer and diagnostic kit for detecting amoeba DNA (genotype T4) using PCR/real-time methods, used to confirm the diagnosis of Acanthamoeba keratitis. Available at: ingenetix.com/en/product/human/Kit+Acanthamoeba+T4/.
