Free-living amoebas, or FLAs, are unicellular protozoa that are widely distributed throughout the world and are distinguished by their ubiquity and amphizoid ability (1). Air, soil, swimming pools, sea, drinking water, cleaning solutions, lenses, household animals, and hot water have all been kept apart from them (1).

Its morphological stages include the trophozoite, a vegetative form that harms the host, and the resistant cyst, which may survive in the environment for extended periods of time. As a result, water transfer is crucial to the disease's epidemiology (2). Furthermore, considering that they have also been detected in dental offices and dialysis units, the epidemiological risk within healthcare facilities needs to be considered (3). Their typical food consists of bacteria, fungus, and minute organic particles. Despite their potential to infect humans quickly and with a high death rate, they have been categorized as emerging pathogens (3).

The illness known as Acanthamoeba Keratitis (AK) causes serious corneal infections, mostly because it is difficult to diagnose and has no effective therapy. AK is a rare but potentially blinding corneal infection that is brought on by the Acanthamoeba protozoan's trophozoite stage. Contact lens wearers may get severe keratitis due to the organism species Acanthamoeba hatchetti, Acanthamoeba polyphaga, and Acanthamoeba castellanii (1).

During the 1980s, there was a marked rise in the incidence of AK in correlation with the growing clinical use of soft contact lenses. This was mainly because to swimming while wearing lenses, the use of nonsterile lens solutions, and inadequate disinfection efforts (1)(2). Research has indicated that more than 90% of instances were associated with contact lens wearers, while non-wearers may also be affected (4). An infection with Acanthamoeba starts on the epithelium and gradually spreads into the stroma. While most occurrences of AK are unilateral, researchers think it can also damage both eyes (5).

Granulomatous Amoebic Encephalitis (GAE), a condition known to cause immune system weakness, is one of the recognized infections that the Acanthamoeba can cause in the Central Nervous System (CNS). Furthermore, reports of skin infections and Acanthamoeba pneumonia have been made. Besides its carcinogenic properties. As a "Trojan Horse," Acanthamoeba can aid in the introduction of organisms that carry a host inside higher, which can result in bacterial or mixed infections that are a secondary cause of protozoan disease (6). Currently, it is thought to be unreliable to identify various strains of Acanthamoeba keratitis based solely on physical characteristics like size. Initially, a monophylogenetic categorization based on ultrastructural traits and biochemical data was attempted (1).

Free-living amoebas (FLA)

Free-living amoeba (FLA) are unicellular protozoa organisms that are known for their ubiquity and amphizoica ability. They are known to live in the environment, withstand harsh circumstances, and be kept apart from both natural and artificial reservoirs, including sewage, household sediments, swimming pool water, aquariums, irrigation channels, sewers, hot springs, lakes, rivers, and puddles (7). They are also kept apart from living fish, amphibians, insects, reptiles, and even deceased creatures (1). FLAs like as Naegleria fowleri, Sappinia, Acanthamoeba spp., and Balamuthia mandrillaris are believed to be harmful to humans (10). Water transmission plays a role in the survival of both trophozoites, which are nutritional forms that harm the host, and cysts, which are resistant forms that may live for extended periods of time in the environment.

Historical Background

In 1912, the unicellular protozoa Acanthamoeba were assigned to the Hartmannellidae family (10). The first recorded instance of Acanthamoeba was in 1913, when Puschkarew reported finding an Amoeba polyphagus in the dust. Page would subsequently identify this organism as Acanthamoeba polyphaga (11). A second description of Acanthamoeba was provided in 1930 by Sir Aldo Castellani, who noted that amoeba could be found in Cryptococcus pararoseus cells. The amoeba belonging to the genus Hartmannella was given the name Hartmannella castellanii by Douglas (1930). A year later, Volkonsky (1931) split the genus Hartmannella into three separate genera on the basis of their morphological features: Acanthamoeba, which has double-walled cysts with pointed chromatic spindles during mitosis, Gleseria, which has nuclear divisions in cysts, and Hartmannella, which has smooth-walled cysts. In the first part of the 20th century, the free-living amoeba was recognized as a ground amoeba and was thought to be a non-pathogenic protozoon.Since then, no reports of amoeba have surfaced. In the late 1950s, it was discovered that Acanthamoeba contaminated tissue cultures and was harmful since it could infect mice and monkeys fatally (12).

In (1967) regarded the spine's form as a dubious criterion for species identification. He thinks that the cyst's position and the existence of Acanthopodia might be enough to differentiate Hartmannella from Acanthamoeba. In 1975, Griffin and Sawyer proposed the Acanthamoebidae as a new family (9). The initial diagnosis of Acanthamoeba as the cause of brain abscesses in Hodgkin's disease patients was made in 1972 (13). In 1974, the first incidence of Acanthamoeba keratitis was documented in the scientific literature (14).

The identification of endosymbiotic bacteria (15) and its capacity to serve as a reservoir for mycobacteria (16) and Legionella pneumophila (17) is another fundamental aspect that has contributed to the "popularity" of this protozoan.

The amoebas were initially identified as Acanthamoeba spp. strain, but in honor of Dr. Culbertson (18), Later on, they were renamed Acanthamoeba castellanii. As of right now, it is known that people and animals can contract Acanthamoeba castellani, A. culbertsoni, A. polyphaga, and probably other species. Over 150 instances of Granulomatous Amoebic Encephalitis (GAE) and approximately 10,000 cases of keratitis are caused by Acanthamoeba spp. (19). Numerous bacteria, yeasts, and viruses, including Escherichia coli,Legionella pneumophila, Helicobacter pylori, Vibrio cholerae, Mycobacterium spp., and Listeria monocytogenes, can react with Acanthamoeba (20).

Taxonomy and classification

Numerous revisions to the FLA classification have been made, and new information from genome sequencing research has been added (21). The International Society of Protozoology recently eschewed the old taxonomy in favor of a contemporary paradigm that emphasizes molecular phylogeny, metabolic pathways, and ultrastructural aspects of morphological alterations (5).

The classical classification placed Naegleria and other Vahlkampfiid amebae under the order Schizopyrenida; Family Vahlkampfiidae, while Acanthamoeba, Hartmannella, and Balamuthia were placed under the Phylum Protozoa, sub phylum Sarcodina, Superclass Rhizopodea, Class Lobosea, Order Amoebida (Figure 1) (22).

Figure 1: The classification scheme of free-living amoebae based on

morphological characteristics (22).

Based on morphological criteria, 25 species were recently identified and documented in the genus Acanthamoeba. They were divided into three groups (Figure 2) according to the morphological characteristics and cyst size (1).

The four species in Group I are Astrium tubiashi, Astronyxis, Echinulata, and Comandoni. With an average diameter of less than 18 μm, these species have the largest cysts and trophozoites in the genus.Group II: Found in both natural and man-made habitats, they are the most prevalent and widely dispersed. The cysts are medium-sized, with an average diameter of less than 18 μm. The endocyst is typically ovoid, triangle-shaped, or polygonal, but the exocyst is typically rough and wavy. There are variations in the area between the inner and outer walls. Eleven species were classified in this category (1).

Third Group. Small cysts are the least common cause of them. less than 18 μm in diameter.

The endocyst is ovoid or globose, but it is never polygonal or stellate. The exocyst is extremely fluffy and frequently undetectable when it has creases. Five species were grouped together (1).

Figure 2: Classification of Acanthamoeba cysts based on morphological characteristics (23).

1.2.5 Life Cycle of Acanthamoeba

The active vegetative stage, also known as the trophozoite, and the cyst are the two life stages of the Acanthamoeba species. The compound organism Acanthamoeba morphs morphologically from a trophozoite to a cyst in order to demonstrate its tolerance to extreme environmental conditions (24).

During the trophozoite stage, mitotic division and binary fission are the methods used for amoeba reproduction. The four stages of binary fission are prophase, metaphase, anaphase, and telophase. Both types of Acanthamoeba have the ability to infect humans when the right circumstances are met (1). Different illnesses can be caused by the same route of entry: inhaled through the lungs and nasal passages generates pneumonia, or through the eye causes amoebic keratitis. Skin lesions can also induce pneumonia. Granulomatous encephalitis can be caused by hematogenous spread from a source site in the skin or lungs, or by direct entry via the mucosa of the nose (25) into the Central Nervous System (CNS).

Corneal anatomy:

With a diameter of 12 mm and a thickness of around 0.5 mm, the cornea is the primary refractive surface of the eye and accounts for 79.0 percent of the total refractive force of the eye, making it the most important part of the optical system. occupying one-sixth of the outer surface of the eye and having an extremely durable structure (26). The cornea has five histological layers and is a multilayered organ (27).

1. The epithelium is the topmost layer; it acts as a physical barrier to fluids and microbes and has an optical function.

2. Bowman's membrane is a fiber-based, transparent tissue.

3. Collagen, keratocytes, and matrix comprise the stroma.

4. The basement membrane of the endothelium is known as Descemet's membrane.

5. The endothelium Blindness can result from an illness called infection keratitis anywhere in the world.In individuals with infectious keratitis, contact lens wear, ocular surface illness, and trauma are the most often encountered risk factors (28). Care must be initiated as soon as possible because the condition can have disastrous effects, including corneal scarring, ocular perforation, endophthalmitis, and, in the most severe cases, failure of the eyeball with permanent vision loss.

Pathogenesis of Acanthamoeba

Although it can infect a number of host species under the right circumstances, Acanthamoeba does not require a host to complete its life cycle (1). Patients with Acanthamoeba keratitis are often in good health. At any age, infections can happen. There are almost equal numbers of male and female patients. Most individuals suffer from unilateral infections (1).

The two main diseases caused by Acanthamoeba pathogens are granulomatous amoebic encephalitis (GAE), an infection of the central nervous system (CNS) that affects both people and animals, and Acanthamoebic keratitis, an infection of the cornea. Moreover, skin infections frequently associated with GAE are caused by Acanthamoeba (29). Additionally, from a drug-exposed bone graft in a patient with mandibular osteomyelitis, cannabinoid cystamoeba has been isolated (30).

Acanthamoeba keratitis (AK)

The symptoms of Acanthamoeba keratitis include severe corneal inflammation, which results in redness, photophobia, and epiphora—the lack of tears. Physical examination findings could include infiltrates, erosions, or epithelial opacities. There are a number of ways that amoebic keratitis can occur, but the most common ones include the entry of Acanthamoeba spp. into the body by ocular damage, which is typically brought on by contact lenses exposed to contaminated water or by improper hygiene when using disinfectant solutions (31).

The infection started after Acanthamoeba trophozoite reaches cornea

1-The acanthamoeba trophozoite attaches itself to polysaccharide or mannosylated glycoproteins on the corneal epithelium. It then secretes cytolytic substances (neuraminidase, cysteine proteinases, serine proteases, plasminogen, and phospholipases activators) that collectively produce a potent cytopathic effect that causes the extracellular matrix to degrade, the extracellular matrix to break down, and ultimately, the death of epithelial cells.

2- Deep cracks and exfoliation of the epithelium are caused by the parasite's invasion of the underlying collagenous stroma, which attacks and lyses keratocytes, causing significant damage to the collagen matrix. Simultaneously, optic nerve inflammation occurs, resulting in partial or total loss of vision. All of these factors contribute to the progressive development of necrotizing keratitis in the cornea (32). Due to the immune response's neutrophil and macrophage multiplication, this injury causes a severe inflammatory response that manifests in patients as a ring-shaped epithelial infiltrate in the corneal stroma (33)

Among the various factors influencing parasite adherence is temperature. At 25 °C, Acanthamoeba's adhesive attraction to the rabbit corneal epithelium is relatively active. As the temperature rises to 35 °C, the attraction becomes more constant. On the other hand, adhesion does not occur at 4 °C. 34. Pathogenicity is significantly influenced by genotypes. After isolating Acanthamoeba strains from patients with keratitis in China, the 18S rRNA gene sequencing revealed that most pathogenic bacteria causing keratitis in China have the T4 genotype, with a small number of cases having the T3 genotype (35).

Diagnosis of Acanthamoeba

It is challenging to diagnose AK since the symptoms are vague and resemble keratitis from a different origin. Most of the time, it is misdiagnosed as fungal keratitis, which can cause significant delays in diagnosis and weeks of insufficient antimicrobial treatment for the patients. While AK often affects one eye, it can affect both in contact lens wearers (36), in certain cases the disease presents as a mixed infection, complicating diagnosis (32, 33).

Even though non-contact lens wearers account for between 3% and 15% of AK cases in the US and the UK, the diagnosis of AK is more difficult and takes longer in this population due to the lower suspicion for infection. Non-contact lens wearers get worse visual outcomes than those with contact lens-associated AK because of diagnostic delays (36). The symptoms of Acanthamoeba keratitis can be characteristic and last for several days to weeks. People who wear contact lenses or experience severe ocular pain, redness, photophobia, and stromal penetration are more likely to have this condition, which can be dangerous for their eyesight and is frequently misdiagnosed as bacterial, fungal, or Herpes simplex keratitis (37). Ulceration, ring infiltrates, and secondary sterile anterior uveitis with hypopyon are typical as the condition worsens. Although perineural infiltrates eventually go away, they can harm the iris and result in a permanent dilation of the pupil, glaucoma, cataracts, corneal melt, and perforations. a cataract, a symptom with poor prognosis. Bacterial superinfection should always be taken into account, especially in cases when 

Acanthamoeba studies in Iraqi:

The examination and identification of Acanthamoeba spp. parasites in Iraq have been the subject of very few investigations. In Iraq, a 25-year-old lady with nodules and an ulcer on her left foot was the first patient to be diagnosed with cutaneous Acanthamoebiasis. Following four days of cultivation, the fluid from the nodules on the non-nutritious agar was used to determine the morphological features of the cyst and trophozoite of Acanthamoeba spp. (39). In the southern Iraqi province of Basra, Acanthamoeba polyphaga was isolated from soils and rivers. Acanthamoeba was found in 18 out of 33 samples, and 2 of those samples tested positive for Acanthamoeba polyphaga based on the physical characteristics of a cyst formed with non-nutrient media (40). A total of fifty-eight drinking water samples were gathered from family water tanks in various Baghdad reigns, Al-Wahdaa, and the Al-Rasheed Drinking Project. Direct wet examination was used to find FLA cysts following cultivation and concentration techniques. There were findings of both Acanthomoeba and Naegleria cysts (41). In a different work by (42) Acanthamoeba genotype T3 was isolated using a molecular approach from various Iraqi water sources, including the Tigris, Euphrates, Dukan, and Najaf Seas. Acanthamoeba genotype T3 has been assigned the accession number MN462973 in the GenBank.

Acanthamoeba-bacteria interaction:

Depending on how they recognize each other and how bacteria internalize, Acanthamoeba species and bacteria have a wide range of intricate interactions (29). The main food source for Acanthamoeba is bacteria. Although it can eat both gram-negative and gram-positive bacteria, it prefers the former, which is why isolating this amoeba is currently so common. At low concentrations, gram-negative bacteria were expected to promote Acanthamoeba proliferation. Acanthamoeba may readily digest bacteria such as Salmonella spp., Escherichia coli, and Pseudomonas aeruginosa; but, at higher concentrations (over 10:1), these bacteria would infest Acanthamoeba growth and cause lysis (29). Certain bacteria are deemed indigestible by Acanthamoeba, potentially because of the presence of toxins, poisonous pigments, or other features like bacterial cell walls that prevent them from being digested (43).Pathogenic bacteria, including E. Coli O157:H7, Chlamydia pneumonia, and Mycobacterium avium, have been demonstrated to be able to survive intracellularly, grow and multiply within amoeba cells, evade other microbes' predation, and withstand antibacterial treatments through in vitro studies using amoeba-bacterium microcosms (43).Acanthamoeba has a great potential to operate as bacterial reservoirs for human infection, according to the data gathered (29). Additionally, from the eye wash solution, Acanthamoeba, Pseudomonas spp., and Legionella were recovered (43).

Additionally, 90% of instances of infectious keratitis can be caused by fungi, viruses, parasites, and bacteria, the most often discovered pathogens being Staphylococcus aureus and Pseudomonas aeruginosa (28).

Escherichia coli- Acanthamoeba interaction

A rod-shaped, Gram-negative bacterium known as Escherichia coli (E. coli) can cause diseases of the intestines as well as the extraintestines (44). Acanthamoeba is greatly impacted by the presence of E. coli because it promotes the organism's growth, helps it maintain its trophozoite stage, and slows down the encystment process (45). For the purpose of identifying, isolating, and maintaining Acanthamoeba spp., this bacterium has been used a lot (64). The way that E. Coli and Acanthamoeba interact is influenced by their respective virulence traits.Research has shown that virulent strains have a higher probability of infiltrating and surviving intracellularly within Acanthamoeba cells. While the non-pathogenic strain E. Coli was found to be fully digested, the pathogenic strain E. coli K1 was found to be surviving intracellularly within Acanthamoeba cells under harsh conditions, such as a lack of nutrients. When conditions improved, the bacteria managed to escape Acanthamoeba by lysing and killing the cell and growing exponentially (47). Above, with sufficient nutrition, this bacterium's nonmucoid phenotypic features would postpone amoebal phagocytosis, leading to an overabundance of bacteria in the amoebal population and preventing them from reproducing (46).

Moreover, interactions and multiplication of the dangerous strain of Escherichia coli within Acanthamoeba have been observed. This strain is responsible for bloody diarrhea and renal failure. It was discovered that this strain could endure inside Acanthamoeba for three weeks, and that the amoeba's presence enhanced the strain's growth (48). Additionally, a study discovered that because of the bacteria's intracellular survival within amoebal cells, a portion of the bacterial genome is produced differently when E. coli interacts with Acanthamoeba (48).

Pseudomonas aeruginosa- Acanthamoeba interaction

A rod-shaped, Gram-negative bacterium known as Pseudomonas aeruginosa (P. aeruginosa) is starting to be identified as an opportunistic illness. Many epidemiological studies tracking the incidence of this bacteria as a nosocomial infection show that antibiotic resistance is emerging in clinical isolates. This bacteria is typically seen in cystic fibrosis patients and is the main contributor to elevated morbidity and death (43).Furthermore, P. aeruginosa is present in a wide range of habitats in natural ecosystems, including soil, water, plant and animal surfaces, and degraded organic waste; nevertheless, the precise ecological niche occupied by the microbe is yet unknown (49).Given their coexistence in both soil and aquatic environments, P. aeruginosa and Acanthamoeba are probably going to interact in the wild. Gram-negative bacteria, such P. aeruginosa, are grazed by Acanthamoeba. As a result, these bacteria can attach to and phagocytose Acanthamoeba trophozoites, preserving their viability within the amoebal host. Studies have indicated that because pigmented bacteria, including P. aeruginosa, can produce amoebicidal exoenzymes such phospholipase C, proteases, and the blue pigment pyocyanin, they decrease Acanthamoeba.

Additionally, P. aeruginosa can create alginate, an exopolysaccharide that acts as a barrier against adverse environments, preventing amoebal grazing and ultimately leading to the extinction of amoebal populations (43). The persistence of P. aeruginosa has been linked to its affiliation with Acanthamoeba (29). Nonetheless, some researchers have determined that P. aeruginosa is not shielded from hydrogen peroxide-based contact lens cleaning solutions by Acanthamoeba (50).

Legionella pneumophila- Acanthamoeba interaction

Although strains have occasionally been observed to remain stationary, Legionella pneumophila is an aerobic, gram-negative, non-capsulated, motile bacillus with one or more polar or lateral flagella. Aerosol inhalation or microsuction of tainted water can cause infection (17). Acanthamoeba contaminated with bacteria resembling Legionella has been isolated from soil samples (11). Legionella pneuomophila, the etiological agent of legionellosis, is one of the extensively researched endosymbionts (11). Rowbotham (1980) An amoeba carrying a Legionella organism may be a human-infectious particle rather than the free form of bacteria.Additionally, every vesicle in A. polyphaga and A. castellanii includes hundreds of L. pneumophila (28). According to the majority of research, amoebae's capacity to increase the quantity of L. pneumophila plays a significant part in the epidemiology of legionellosis (28). Together with Acanthamoeba, which is created by membrane transporters, the endoplasmic reticulum, and fusion with other membrane-bound vesicles, L. pneumophila creates a membrane-enclosed vesicle (53).

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