**1. Introduction**

*Acanthamoeba* keratitis (AK), the vision-threatening corneal disease that was first time recog‐ nized in 1973 in the United States in a Texas rancher [1], is reported with increasing prevalence in different regions and countries year after year [1- 7]. This corneal, usually acute and progressive infection is becoming increasingly significant for human health worldwide.

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Eye diseases affecting the cornea are a major cause of blindness worldwide. Among different infectious agents, bacteria, fungi, viruses and protozoans may be causes of keratitis in contact lens users. At present, the epidemiology of microbial keratitis is complicated, diverse, and even controversial; the use of contact lenses is considered as the most important risk factor of corneal infections in humans [4,8]. It was emphasized in several reviews [8- 10] that the incidence rates of particular organisms causing keratitis associated with contact lens wear differ between parts of the world, from country to country and even from one population to another. Economic factors, various frequencies of contact lens wear in particular geographical locations, different availabilities and standards of eye care, and different methods of etiological agent isolation and culture in particular surveys were mentioned as factors influencing the differences and criteria taken into consideration in the studies.

The frequency of microbial keratitis cases caused by Gram-negative bacteria from genus *Pseudomonas* (as percent of total isolates from given location) varied in Europe: from 6.6% in Turkey to 72.2% in Italy and from 12% to 28.5% in various surveys from the United Kingdom [8]. Climate also influenced the incidence of bacterial keratitis: e.g.in Australia, the frequency of *P. aeruginosa* contact lens keratitis was increased in tropical zones than temperate zones.

The highest proportion of fungal corneal infections (*Aspergillus* sp., *Candida* sp.), 67% was found in India [9]; in Europe, the highest frequency of 22.2% was found in Turkey.

Correlations between the contact lens wear and percentages of bacterial and fungal keratitis were statistically significant.

The viruses from *Herpes* spp. are other agents of keratitis. There are relatively few studies on the epidemiology of *Herpes* keratitis in humans. According to review by Farooq and Shukla [11], in developed countries, *Herpes* keratitis is believed to be an important cause of infectious blindness mainly resulting from stromal opacification, e.g. it was reported from France that the incidence was 25.8 (21.2–30.4) per 100,000 person-years; 95% of the cases occurred in contact lens users. These authors found that the incidence of this keratitis is about 1.5 million, includ‐ ing 40,000 new cases of severe visual impairment each year, however, it is difficult to ascer‐ tain the frequency of the viral keratitis because of a lack of surveillance-based epidemiologic studies [11].

Protozoan eye disease, *Acanthamoeba* keratitis (AK), is rare in the general population (estimated incidence: 1.4 per million person- year) but much frequent in contact lens wearers. [4,12,13]. In the United States, an estimated 85% of cases occur in contact lens users. The incidence of the disease in developed countries is approximately 1-33 cases per million contact lens wearers, e.g. in England 17-21 cases per million. Currently, it is emphasized that an awareness and knowledge about AK -the serious, vision-threatening eye disease are still insufficient [12,18].

Previous and recent studies continued in many centers with a participation of practicing researchers and other scientists are crucial for a better understanding of *Acanthamoeba* keratitis [4,14-18]. Advances in the field are expected by both laboratory and clinical practitioners; particularly, an improvement in duration from first symptoms until suitable diagnosis as well as in efficacy of the therapeutic management and prophylaxis is the urgent need. Here, we present selected aspects of this multi-factorial human disease, including the results of our studies and own experiences in the topic.

#### **1.1. Possible environmental sources of** *Acanthamoeba* **spp.**

Eye diseases affecting the cornea are a major cause of blindness worldwide. Among different infectious agents, bacteria, fungi, viruses and protozoans may be causes of keratitis in contact lens users. At present, the epidemiology of microbial keratitis is complicated, diverse, and even controversial; the use of contact lenses is considered as the most important risk factor of corneal infections in humans [4,8]. It was emphasized in several reviews [8- 10] that the incidence rates of particular organisms causing keratitis associated with contact lens wear differ between parts of the world, from country to country and even from one population to another. Economic factors, various frequencies of contact lens wear in particular geographical locations, different availabilities and standards of eye care, and different methods of etiological agent isolation and culture in particular surveys were mentioned as factors influencing the differences and

The frequency of microbial keratitis cases caused by Gram-negative bacteria from genus *Pseudomonas* (as percent of total isolates from given location) varied in Europe: from 6.6% in Turkey to 72.2% in Italy and from 12% to 28.5% in various surveys from the United Kingdom [8]. Climate also influenced the incidence of bacterial keratitis: e.g.in Australia, the frequency of *P. aeruginosa* contact lens keratitis was increased in tropical zones than temperate zones.

The highest proportion of fungal corneal infections (*Aspergillus* sp., *Candida* sp.), 67% was

Correlations between the contact lens wear and percentages of bacterial and fungal keratitis

The viruses from *Herpes* spp. are other agents of keratitis. There are relatively few studies on the epidemiology of *Herpes* keratitis in humans. According to review by Farooq and Shukla [11], in developed countries, *Herpes* keratitis is believed to be an important cause of infectious blindness mainly resulting from stromal opacification, e.g. it was reported from France that the incidence was 25.8 (21.2–30.4) per 100,000 person-years; 95% of the cases occurred in contact lens users. These authors found that the incidence of this keratitis is about 1.5 million, includ‐ ing 40,000 new cases of severe visual impairment each year, however, it is difficult to ascer‐ tain the frequency of the viral keratitis because of a lack of surveillance-based epidemiologic

Protozoan eye disease, *Acanthamoeba* keratitis (AK), is rare in the general population (estimated incidence: 1.4 per million person- year) but much frequent in contact lens wearers. [4,12,13]. In the United States, an estimated 85% of cases occur in contact lens users. The incidence of the disease in developed countries is approximately 1-33 cases per million contact lens wearers, e.g. in England 17-21 cases per million. Currently, it is emphasized that an awareness and knowledge about AK -the serious, vision-threatening eye disease are still insufficient [12,18].

Previous and recent studies continued in many centers with a participation of practicing researchers and other scientists are crucial for a better understanding of *Acanthamoeba* keratitis [4,14-18]. Advances in the field are expected by both laboratory and clinical practitioners; particularly, an improvement in duration from first symptoms until suitable diagnosis as well as in efficacy of the therapeutic management and prophylaxis is the urgent need. Here, we

found in India [9]; in Europe, the highest frequency of 22.2% was found in Turkey.

criteria taken into consideration in the studies.

were statistically significant.

100 Advances in Common Eye Infections

studies [11].

Free-living amoebae belonging to *Acanthamoeba* genus are ubiquitous and widely distributed in natural and man-made environments of many parts of the world [4,19-25]. The amoebae have been isolated from a wide range of soil and aquatic habitats; they occur in sea, freshchlorinated- and tap- water, drinking water systems, bottled mineral water, thermal recrea‐ tional waters, swimming pools, air, air-conditioning systems including humidifiers, soil and dust, and sewage. The amoeboid protists have been found in fruits, vegetables, and also healthy, diseased or dead animals [12,18]. The amoebae have been isolated also from clinical settings and the hospital environment: on surfaces of different equipment and accessories, in water and air-conditioning systems, on surgical instruments, in dental irrigation units, in contact lenses and their cases, and in dialyzers [7,26,27].

#### **1.2. The developmental forms and classification of** *Acanthamoeba* **spp.**

The amoeboid, mitochondria-bearing protist is known as free-living organism that exists in two morphologic forms: trophozoite and cyst [4,12,18]. The life cycle of *Acanthamoeba* is asexual; the reproduction of trophozoite is by binary fission. This active vegetative stage contains one nucleus with large, central nucleolus (endosome), ectoplasm and granular endoplasm, with a large contractile vacuole as well as numerous mitochondria and digestive vacuoles. The trophozoite is changing in shape, 15–45µm in size and moving by cytoplasmictransparent pseudopodia that create characteristic protrusions: spine-like acanthopodia. The dormant stage, cyst is smaller, 8-24µm in size, rounded or polygonal in shape, and double walled, with wrinkled or rippled outer layer; the form indicates minimal metabolic activity. The outer wall of the cyst, the so-called ectocyst, contains lipids and proteins; the inner cyst wall, endocyst contains cellulose that is not present in trophozoite stage. The two cyst walls are separated by a space, except of points in that both walls meet; in these points - pores, i.e. ostioles occur that are covered by plugs, so-called opercula. The plugs are removed when trophozoites emerge from cysts during excystation [4,12]. Trophozoites transform into cyst stages after the growth developmental phase of amoeba population, in which high cell density occurs as well as under harmful environmental conditions (e.g. extremes in temperature and pH, increased or decreased osmolarity, lack of nutrients). The therapeutic experience and many *in vitro* studies showed that the cysts are highly resistant to antimicrobial drugs and a variety of chemical and physical agents [reviewed in 4,12]; they can remain viable under prolonged desiccation, starvation, heat and cold. Also, they can survive *in vitro* in distilled water as long as 25 years [28] and maintain their viability and virulence.

Following the recognition of the amoebae and increasing number of isolates belonging to the genus *Acanthamoeba*, for years they were classified using morphological criteria. In this classification, a size of cysts and the number of characteristic arm -like structures visible within a single cyst in light microscope were mainly taken into consideration [4,12,18,29,30]. At the time, 18 species have been determined and placed in three morphological groups I, II and III. **Figure 1.** *Acanthamoeba* sp. - wet-mount slides from corneal scrapings; note characteristic spine-like acanthopodia in trophozoites and the double-walled cysts; light micrographs; scale bar = 10µm

The classification of *Acanthamoeba* species /isolates changed with the advances in molecular methods. Recently, it is based on genotype associations - the 18S rRNA gene sequence [5,20,31-33]. The modern approach differs from morphology solely: the species identification is based on combination of the morphological and molecular characterization [3,5,15,20]. At present, 18 or19 genotypes are distinguished for diagnostics and for the characterization of clinical and environmental *Acanthamoeba* isolates [12,15,18,31-33].
