**6. Differential diagnosis of AK**

#### **6.1. A tentative diagnosis of AK**

the amoebae as well as in the proportion of trophozoites and cysts dependent on the environ‐ mental conditions are also listed as the amoebic pathogenesis determinants. The temperature tolerance, osmotolerance and growth at different pH allow the amphizoic *Acanthamoeba* to exist in different environments and simultaneously easy adapt to different human organ and tissues, including the human cornea. Moreover, it has been shown that both, clinical and environmental *Acanthamoeba* strains/isolates vary in their among the oral cavity microbiota pathogenicity. The thermal tolerance and ability to grow at high temperature are considered

In several studies it is underlined that the initiating of AK is a multi-factorial process, in which both host and environmental determinants are likely involved, apart connected with *Acantha‐*

It is considered that *Acanthamoeba* keratitis is mainly related to contact lens wear, although, *Acanthamoeba* corneal infections are also detected in persons not using contact lenses [7,12-14, 46]. After the first case of AK associated with contact lenses in Central Europe was reported from Germany, more than 85% of all incidences of the disease have been recognized in different

The estimation of AK findings in several countries showed various, generally relatively low, but constantly increasing number of the cornel disease incidents during the last few decades i.e. 1.36 cases per million contact lens wears in the United States, 17 to 21 cases per million in England, 1 per 30,000 contact lens wears in France, 0.05 per 10,000 in Holland [4,12,13]. However, "it is noteworthy", as Khan [4] concluded "that variations in the incidence rate of *Acanthamoeba* keratitis do not reflect the geographical distribution of *Acanthamoeba*, and are most likely due to variations in the extended wear of soft contact lenses, varied awareness of the potential risk associated with the contact lens wear, enhanced detection, and/or local conditions that promote growth of pathogenic *Acanthamoeba* only e.g.. water hardness or salinity, or conditions that suppress growth of non-pathogenic *Acanthamoeba*." Interestingly, in Austria, women and men were affected almost equally; the highest AK incidences occurred in the 21-30-years-old patients; simultaneously, poor contact lens hygiene is indicated as the

Some micro-traumas occurring earlier or appearing in connection with the use of the lenses predispose to contract AK; a human organism's susceptibility, tissue specificity, tear factors, and secretory immunoglobulin A (sIgA), important in the specific immune defense mecha‐ nism, are among other host factors influencing development of this corneal disease. Environ‐ mental conditions such as temperature, osmolarity, and pH may be important in initiating AK. Simultaneously, the amoebae were found in contact lens and in storage cases that may be potential sources and reservoirs of the facultative parasites [12,18,47-53]. In spite of this, the incidence rate of AK in wearers of contact lenses is remarkably low in comparison with the

to be indirect markers of the pathogenicity of *Acanthamoeba* strains [12,17,18,43-45].

**4. Predisposing/ risk factors sufficient to contract AK**

countries in wearers of contact lenses [12,17,18,47-50].

most important risk factor of AK in this country [46].

contact lens storage cases contaminated with *Acanthamoeba*.

*moeba* pathogenic strains [4,12].

104 Advances in Common Eye Infections

The non-invasive methods are useful for the tentative diagnosis of AK, in which the slit-lamp that provides magnification from 10 to 25 times and *in vivo* confocal microscopy (Figures 2 and 3) are applied [4,12,44,50,56].

The use of a slit lamp is indicated in any acute situation that requires magnification to inspect the anterior segment of the eye. Active epithelial inflammations and hyper reflective tissues in the affected eye may be visualized by the slit lamp; a corneal ulcer and characteristic, ringlike corneal infiltration may occur in some patients. However it should be underlined that the characteristic ring infiltrate is seen in approximately 50% of AK cases.

**Figure 2.** Slit-lamp photograph of corneal ulceration of severe *Acanthamoeba* keratitis case.

**Figure 3.** Hyper reflective objects - *Acanthamoeba* cysts in the affected eye with the late diagnosed severe keratitis; *in vivo* confocal microscopy; scale bar = 50µm

Different clinical presentations may occur in various causes of keratitis. However, in clinical AK practice there are nonspecific signs variable in their intensity starting with photophobia, redness, and excessive tearing that present with similar symptoms as viral, bacterial and fungal keratitis (4,7, 9-12].

In the slit lamp, corneal epithelial disease caused by *Herpes simplex* virus may be seen as dendritic keratitis or the geographic ulcer. The edges of these lesions with swollen epithelial cell stain with rose bengal while the central part stains with fluorescein [11,12,18].

Bacterial corneal infection appears typically as a one gray-white stromal infiltrate with welldemarked borders. Critical sings for fungal keratitis, e.g. *Fusarium* keratitis, are stromal graywhite or yellow-white infiltrate with a feathery border, satellite lesions surrounding the primary lesion; co-infections with fungi and bacteria can complicate the fungal keratitis [9,10,12].

In the early epithelial stage of *Acanthamoeba* keratitis, this infection can be misdiagnosed as a *Herpes simplex* keratitis because of irregular grayish lesions and pseudodendrites that are frequently observed in the epithelium. In advanced stage, AK can be mistaken as a fungal keratitis [9,11,12].

in the affected eye may be visualized by the slit lamp; a corneal ulcer and characteristic, ringlike corneal infiltration may occur in some patients. However it should be underlined that the

**Figure 3.** Hyper reflective objects - *Acanthamoeba* cysts in the affected eye with the late diagnosed severe keratitis; *in*

Different clinical presentations may occur in various causes of keratitis. However, in clinical AK practice there are nonspecific signs variable in their intensity starting with photophobia, redness, and excessive tearing that present with similar symptoms as viral, bacterial and fungal

In the slit lamp, corneal epithelial disease caused by *Herpes simplex* virus may be seen as dendritic keratitis or the geographic ulcer. The edges of these lesions with swollen epithelial

Bacterial corneal infection appears typically as a one gray-white stromal infiltrate with welldemarked borders. Critical sings for fungal keratitis, e.g. *Fusarium* keratitis, are stromal graywhite or yellow-white infiltrate with a feathery border, satellite lesions surrounding the primary lesion; co-infections with fungi and bacteria can complicate the fungal keratitis [9,10,12].

In the early epithelial stage of *Acanthamoeba* keratitis, this infection can be misdiagnosed as a *Herpes simplex* keratitis because of irregular grayish lesions and pseudodendrites that are

cell stain with rose bengal while the central part stains with fluorescein [11,12,18].

characteristic ring infiltrate is seen in approximately 50% of AK cases.

**Figure 2.** Slit-lamp photograph of corneal ulceration of severe *Acanthamoeba* keratitis case.

*vivo* confocal microscopy; scale bar = 50µm

106 Advances in Common Eye Infections

keratitis (4,7, 9-12].

Active epithelial inflammations usually progresses from the outermost layer of the cornea -a superficial keratitis -to deeper stroma -the stromal or interstitial keratitis.

In the initial epithelial phase, typical signs of AK include epithelial or sub-epithelial infiltrates, pseudodendrites resembling these observed in *Herpes* keratitis, radial keratoneuritis (infil‐ trates along corneal nerves) and recurrent puncture staining of the corneal epithelium. Perineural infiltrates -a radial keratoneuritis are described as pathognomonic for the diagnosis of *Acanthamoeba* keratitis [12,18]; they are evoked by tropism of the amoebic organism for corneal nerves. Radial keratoneuritis is the reason for the extreme pain and is usually seen during the first one to four weeks of disease. Anterior stromal infiltrates are another common sing of AK. They gradually enlarge and coalesce to form a ring abscess, commonly located in the center of the cornea. Less specific signs of AK are satellite stromal infiltrates, diffuse stromal infiltrates and endothelial plaques observed in many of the patients.

Later signs of AK develop in 3-8 weeks and include a deep inflammation of the cornea consisting of a central stromal thinning and melting, anterior chamber cells and flare, hypo‐ pyon and extension of inflammation into sclera. The latter is generally reactive reaction rather than extension of infection; later in the disease course, the slowly progressive stromal opaci‐ fications and neovascularization may occur.

Etiological agents of infectious keratitis can be differing using *in vivo* confocal microscopy that is confirmed as useful tool for rapid diagnosis with high sensitivity [4,12,18,56,57].

Common findings in viral keratitis are: highly reflective, desquamating epithelial cells in superficial epithelial layer, multiple dendritic cells in basal epithelial layer, the absence of subepithelial nerve plexus, and hyperreflective keratocytes in the anterior stroma.

In bacterial keratitis, confocal micrographs typically reveal leucocytes infiltrating the corneal stroma and adherent to vessel walls. In some cases, the dendritic cells are present intrastro‐ mally; the bacteria themselves cannot be detected with the confocal microscopy.

Filamentous fungi and bacteria (e.g. *Nocardia*) can form filamentous structures that are large enough to be distinguished by confocal microscopy [57]. Another characteristics sing of filamentous fungal infection is hyphae branching, in a case of *Aspergillus* at 45o and in a case of *Fusarium* at 90o .

The examination of affected eyes by *in vivo* confocal microscopy make possible to distinguish AK from the aforementioned infectious keratitis. Lately, confocal scan features of cysts and trophozoites as well as associated corneal epithelial and stromal findings were described as criteria to specify AK in clinical diagnosis [57]. Presumable *Acanthamoeba* cysts can be visible as numerous hyper reflective, double-walled ovoid or spherical objects, 10-25µm in diameter, localized typically in deeper parts of epithelium and in anterior layers of the corneal stroma [4,18,57]. These findings should be distinguished from the well-delineated individual epithe‐ lial cell nuclei or leucocytes; the latter are larger and more regular than *Acanthamoeba* cysts. The outer wall of the cyst is more reflective than internal wall; with time, some cysts are not seen and the others become calcified. Trophozoites are also described as visible in confocal scan images, however, false results can occur because the forms are difficult to distinguish from nuclei of leukocytes and keratocytes [4,12,18]. Although confocal microscopy, if available, is non-invasive, high- sensitive tool for rapid *in vivo* diagnosis, examiners have to be familiar with morphology of *Acanthamoeba* forms. Also, differences in strain pathogenicity and viability can be taken into consideration.

It was evident also in our studies on monitoring of *in vitro* dynamics of *Acanthamoeba* strains isolated from infected eyes [17,50]; the presence of hyper reflective objects/cysts was revealed by this non-invasive *in vivo* confocal microscopy mainly for severe, late diagnosed infections with strains of which strong viability was indicated by intensive multiplying of trophozoites *in vitro* and their long survival time (42 months) in culture medium. Contrary to this, no cysts were detected by the confocal technique in material from corneal scraping if infections with weak viability strains occurred; a low amoeba number in the exponential growth phase and short (10 days) survival time of such amoeba strains were manifested *in vitro* in the culture medium. Also, no cyst was found in confocal microscopy images when mixed infections occurred, although finally the infection with *Acanthamoeba* was confirmed by laboratory methods.

Negative results of the *in vivo* confocal microscopy were reported if patients have already been pre-treated, thus the amoeba density was very low [12,50].

#### **6.2. Why clinical manifestations are not sufficient to indicate a causative agent of keratitis**

Knowledge and awareness of threat are necessary as the most important step in proper AK diagnosis as it is underlined by J. Lorenzo-Morales et al. [12].

The careful anamnesis is very important and helpful. Most of the clinical symptoms of *Acanthamoeba* keratitis are nonspecific and frequently a variability in their intensity is observed and reported from different world regions. AK is often misdiagnosed as viral infection with *Herpes simplex,* bacterial with *Pseudomonas aeruginosa* or keratitis caused by fungi of genera *Fusarium* or *Candida*; moreover, bacterial, viral, and fungal co-infections with *Acanthamoeba* may occur [2,4,46]. This is why the clinical symptoms alone, as non- pathognomonic, are not sufficient to indicate an etiological agent of human keratitis.

Undoubtedly, the non-invasive *in vivo* confocal microscopy is a valuable technique, however usefulness of it is limited if bacterial or viral keratitis occurs, thus it should be applied for the tentative diagnosis.

Also, in our several studies we analyzed serious keratitis cases regarding patients who were under suspicion of *Acanthamoeba* etiology of the infections. Although, in our hospital, finally AK was determined, there were mistakes in results of earlier diagnostics. The mixed amoebic, bacterial (*P. aeruginosa, E. faecalis*), and fungal (*Candida* sp.) infections have been revealed by us in more than 50% cases regarding persons previously unsuccessfully treated only with antibacterial and antifungal medications in other ophthalmic units [17,50].

It has been reported that *Acanthamoeba* protozoans may carry more than 20 species pathogenic for humans, among others bacteria belonging to genera *Legionella, Pseudomonas, Mycobacterium,* *Listeria* and *Escherichia*, protozoa *Cryptosporidium* sp., and fungi *Cyrptococcus neoformans*. The microorganisms are able not only to survive within cells but even proliferate inside the amoebae; thus, secondary infections can occur and influence diagnostic difficulties [4,12, 18].

#### **6.3. Laboratory evaluation**

seen and the others become calcified. Trophozoites are also described as visible in confocal scan images, however, false results can occur because the forms are difficult to distinguish from nuclei of leukocytes and keratocytes [4,12,18]. Although confocal microscopy, if available, is non-invasive, high- sensitive tool for rapid *in vivo* diagnosis, examiners have to be familiar with morphology of *Acanthamoeba* forms. Also, differences in strain pathogenicity and viability

It was evident also in our studies on monitoring of *in vitro* dynamics of *Acanthamoeba* strains isolated from infected eyes [17,50]; the presence of hyper reflective objects/cysts was revealed by this non-invasive *in vivo* confocal microscopy mainly for severe, late diagnosed infections with strains of which strong viability was indicated by intensive multiplying of trophozoites *in vitro* and their long survival time (42 months) in culture medium. Contrary to this, no cysts were detected by the confocal technique in material from corneal scraping if infections with weak viability strains occurred; a low amoeba number in the exponential growth phase and short (10 days) survival time of such amoeba strains were manifested *in vitro* in the culture medium. Also, no cyst was found in confocal microscopy images when mixed infections occurred, although finally the infection with *Acanthamoeba* was confirmed by laboratory

Negative results of the *in vivo* confocal microscopy were reported if patients have already been

**6.2. Why clinical manifestations are not sufficient to indicate a causative agent of keratitis** Knowledge and awareness of threat are necessary as the most important step in proper AK

The careful anamnesis is very important and helpful. Most of the clinical symptoms of *Acanthamoeba* keratitis are nonspecific and frequently a variability in their intensity is observed and reported from different world regions. AK is often misdiagnosed as viral infection with *Herpes simplex,* bacterial with *Pseudomonas aeruginosa* or keratitis caused by fungi of genera *Fusarium* or *Candida*; moreover, bacterial, viral, and fungal co-infections with *Acanthamoeba* may occur [2,4,46]. This is why the clinical symptoms alone, as non- pathognomonic, are not

Undoubtedly, the non-invasive *in vivo* confocal microscopy is a valuable technique, however usefulness of it is limited if bacterial or viral keratitis occurs, thus it should be applied for the

Also, in our several studies we analyzed serious keratitis cases regarding patients who were under suspicion of *Acanthamoeba* etiology of the infections. Although, in our hospital, finally AK was determined, there were mistakes in results of earlier diagnostics. The mixed amoebic, bacterial (*P. aeruginosa, E. faecalis*), and fungal (*Candida* sp.) infections have been revealed by us in more than 50% cases regarding persons previously unsuccessfully treated only with

It has been reported that *Acanthamoeba* protozoans may carry more than 20 species pathogenic for humans, among others bacteria belonging to genera *Legionella, Pseudomonas, Mycobacterium,*

antibacterial and antifungal medications in other ophthalmic units [17,50].

pre-treated, thus the amoeba density was very low [12,50].

diagnosis as it is underlined by J. Lorenzo-Morales et al. [12].

sufficient to indicate an etiological agent of human keratitis.

can be taken into consideration.

108 Advances in Common Eye Infections

methods.

tentative diagnosis.

Literature data as well as results of our studies indicated that microscopic visualization of amoebae in unstained or stained slides prepared directly from corneal scraping is usefulness for AK diagnostics. Also, laboratory examinations of specimens from *in vitro* cultivated corneal isolates allow to identify directly the facultative pathogens and to verify previous misdiagno‐ ses [4,12,46,50].

**Figure 4.** Live *Acanthamoeba* trophozoites and cysts in unstained preparations from corneal isolates cultured *in vitro*; light micrographs. Scale bars = 10µm

Moreover, culture methods are considered as the gold standard of diagnosis, which needs, however, collaboration between clinicians and laboratory staff and, also, the familiarization with a morphological characteristic of *Acanthamoeba* stages [12,14,18,55,58-60]. Non-nutrient (NN) agar plates seeded with Gram- negative, non-mucous bacteria: *Escherichia coli* or *Enterobacter aerogenes* are applied for isolation/growth of *Acanthamoeba* trophozoites both from environmental and clinical samples (corneal scrapings, biopsies, swabs). The incubation of the plates at 30ºC promotes a transformation of amoeba trophozoites into cysts within approxi‐ mately 1 week. Also, the cultivation of amoebae in bacteria-free (axenic) conditions in a modified enriched culture medium containing antibiotics (penicillin, streptomycin) is useful for classification cysts to the morphological level.

Simultaneously, molecular methods of classification of *Acanthamoeba* isolates, with the use of sensitive PCR techniques, basing on genotype associations are distinguishing for diagnostics and for the characterization of clinical and environmental *Acanthamoeba* isolate [12,18,27,32,33,59]. In this modern approach, the species identification is based on the combi‐ nation of morphological and molecular characteristics of amoebae. Additionally, our experi‐ ences gave convincing evidences of an importance of a clinical examination of the affected eyes and laboratory differentiations/identification of amoebic forms in material deriving from infected corneas.
