Successive Acanthamoeba Corneal Isolates Identified in Poland Monitored in Terms of In Vitro Dynamics

Background: Amoebae of the genus Acanthamoeba cause a sight-threatening infection called Acanthamoeba keratitis. It is considered a rare disease in humans but poses an increasing threat to public health worldwide, including in Poland. We present successive isolates from serious keratitis preliminary examined in terms of the identification and monitoring of, among others, the in vitro dynamics of the detected strains. Methods: Clinical and combined laboratory methods were applied; causative agents of the keratitis were identified at the cellular and molecular levels; isolates were cultivated in an axenic liquid medium and regularly monitored. Results: In a phase-contrast microscope, Acanthamoeba sp. cysts and live trophozoites from corneal samples and in vitro cultures were assessed on the cellular level. Some isolates that were tested at the molecular level were found to correspond to A. mauritanensis, A. culbertsoni, A. castellanii, genotype T4. There was variability in the amoebic strain dynamics; high viability was expressed as trofozoites’ long duration ability to intense multiply. Conclusions: Some strains from keratitis under diagnosis verification and dynamics assessment showed enough adaptive capability to grow in an axenic medium, allowing them to exhibit significant thermal tolerance. In vitro monitoring that was suitable for verifying in vivo examinations, in particular, was useful to detect the strong viability and pathogenic potential of successive Acanthamoeba strains with a long duration of high dynamics.


Introduction
The primary free-living acanthamoebae (FLA) distributed in natural and man-made environments exist in two forms: as the active vegetative trophozoites with characteristic protrusions-acanthopodia-and as double-walled dormant cysts developing after the growth phase and in harsh conditions. The amoebae occur in soil, air, dust, on/in vegetables, in animals, and in different aquatic habitats; they survive in domestic tap water, municipal sewage systems, in chlorinated swimming pools, and in air conditioning units [1][2][3][4][5][6][7]. They have also been detected in hospital environments on equipment surfaces, on accessories, in dialyzers, on surgical instruments, in dental irrigation units, and in contact lenses and their boxes. As Acanthamoeba strains can enter human tissues and exist as endozoic forms, they are believed to be amphizoic organisms able to exist as free-living forms and as facultative parasites. The amoebae were detected on skin, in paranasal sinuses and lungs, and among oral cavity microbiota associated with gingivitis and periodontitis [8][9][10]. They are known as causative agents of granulomatous amoebic encephalitis (GAE), a rare, usually fatal disease developing in immunocompromised persons, patients under immunosuppressive therapy, and HIV/AIDS patients [2,5,11].
The treatment of AK is often unsuccessful due to extremely high resistance of the cysts of Acanthamoeba to chemical agents, disinfectants, and drugs [20,38,51].
In this interdisciplinary study, we present successive isolates originating from complicated keratitis incidents previously recognized incorrectly and unsuccessfully treated in various units. We examined and evaluated the material to identify etiological agents of this keratitis, assess the usefulness of particular methods for a comparison of the detected strains that are pathogenic for humans, and monitor of their population dynamics.

Materials and Methods
The study pertains to material originating from incidents of serious eye infections in 12 women aged 22-48 years and 8 men aged 18-70 years who were ineffectively treated in various ophthalmic units. Because of previous unsuccessful treatment of keratitis, there was a need for diagnostic verification. The study was performed in accordance with the tenets of the Declaration of Helsinki. In the search for pathogenic agents of keratitis, clinical symptoms that were visible in slit-lamp and in vivo confocal microscopy, appearing in the early and advanced infection stages, were taken into account.
Laboratory methods of species identification were based on combination of morphological and molecular characterization.
Samples originating from keratitis material that were initially examined using the phasecontrast microscope were assessed in wet-mount slides based on the amoebic morphology.
Ten samples of the material were investigated for specific identification using molecular techniques. DNA extraction from the material was performed using the commercial Sherlock AX Kit (A&ABiotechnology, Gdynia, Poland). The ASA.S1 fragment of Acanthamoeba 18S ribosomal RNA gene was amplified using JDP1 (5 GGCCCAGATCGTTTACCGTGAA3 ) and JDP2 (5 TCTCACAAGCTGCTAGGGAGTCA3 ) primers targeting~450 bp fragment of the gene [57]. The reaction mixture consisted of 12.5 µL PCR Master MixPlus High GC (ready-to-use PCR mixture containing Taq DNA polymerase, PCR buffer, MgCl 2 , and dNTPs; A&A Biotechnology), 1 µL each primer (concentration 10 µm), and 2.5 µL DNA template, supplemented with deionized water up to 25 µL. Amplifications were performed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Waltham, MA, USA) according to following protocol: 3 min at 95 • C (initial denaturation) followed by 35 cycles of denaturation at 95 • C for 60 s, annealing of primers at 60 • C for 60 s, strand elongation at 72 • C for 3 min, and a final extension step of 10 min at 72 • C. PCR products were analyzed using the GelDoc-It Imaging Systems (UVP, Upland, CA, USA) after electrophoresis on agarose gel (Sigma, St. Louis, MO, USA), stained with Midori Green DNA Stain (Nippon Genetics Europe, Duren, Germany). Direct sequencing was performed using standard procedures and amplification primers; obtained sequences were analyzed using GeneStu-dio™ Professional (Thermo Fisher Scientific, Waltham, MA, USA) and compared with the sequences available in the GenBank using NCBI BLAST (identification of Acanthamoeba isolates to species level) (http://www.ncbi.nlm.nih.gov/BLAST (accessed on 1 September 2021-30 January 2023).
Simultaneously, to assess in vitro dynamics of particular strain, the cultivation of the samples was performed under axenic conditions in the absence of external live food organisms, as in our earlier studies [31,39,50,58,59]. Cultures were grown in vitro in sterile 15 mL tubes with the liquid medium composed of Bacto Casitone, Difco (BSC) dissolved in water enriched with 10% calf serum, with addition of aqueous solution of antibiotics, namely streptomycin and penicilin, at 24 • C and sub-cultured into this medium twice a month. The dynamics of Acanthamoeba strains parallel cultivated were in vitro monitored and compared to one another. Changes in overall numbers of amoebae, the ability of trophozoites to multiply, and trophozoite and cyst proportions were directly counted with aid of the Bürker hemocytometer. Ranges of three counts of amoebae calculated for 1 mL of the medium were compared for particular strains and assays. Samples of several amoeba strains were also exposed to 37 • C (near human eye temperature) to test a tolerance to changes in temperature on 5th day after a sub-culturing and monitored from 7th day 3-5 times a week in the exponential growth phase during in vitro cultivation. Dynamics of amoebic strains were monitored during each sub-culturing for 7 days in the logarithmic growth phase. Results were analyzed statistically (ANOVA, Student-Newman-Keuls method; the level of statistical significance was set at p < 0.05).
During laboratory differential diagnosis, samples obtained from corneal material were also routinely tested with microbiological techniques. Preliminary identification of Gram-positive and Gram-negative bacteria strains and conventional in vitro techniques were used for detection of fungi and bacteria.

Results
The material assessed in our study originated from 20 keratitis cases in which one of the two eyes was affected. In 14 of the incidents (70%), antibacterial or/and antifungal medications were unsuccessfully applied in other units; thus, misdiagnoses were taken into account by us. Investigations were performed to identify the etiological factors of keratitis to verify diagnoses.
Wearing contact lenses (CL), which is a predisposing risk factor for amoebic eye infections, was documented in 17 cases (85%). Among these cases, several were associated with washing CL in tap water and showering and/or swimming in a pool with CL on. In the patients who were not wearing contact lenses, swimming in lakes and swimming in pools were probable risk factors.
There were different durations as well as intensities of clinical symptoms in 18 keratitis cases-ranging from 5 to 38 days-until proper diagnosis and various times when the anti-Acanthamoeba therapy was started. In two other misdiagnosed keratitis cases, the affected eyes were treated unsuccessfully over six months. Drug-combined therapy involved mainly chlorhexidine digluconate, polyhexamethylene biguanide PHMB, and propamidine isethionate Brolene with an addition of antibiotics; steroids were also used. In some cases of AK, surgical interventions, deep anterior lamellar keratoplasty (DALK) or penetrating keratoplasty, amniotic membrane transplantations, or cataract surgery were needed.
In the clinical picture of the keratitis incidents included in our study, photophobia, reduced visual acuity, lid edema, and redness appeared in all cases with different intensities. Symptoms that were visible in slit-lamp and in vivo confocal microscopy, appearing in the early and advanced infection stages, were taken into account. The hyperreflective tissues, epithelial inflammation, size and depth of stromal infiltration or ulcer, and presence or absence of hypopyon were assessed by non-invasive in vivo tests in the slit-lamp biomicroscopy. The occurrence of unbearable pain, epithelial diffuse edema progress to dendriform ulcer, and characteristic ring-shaped infiltration successively involving deep stromal layers, which were considered as factors indicating Acanthamoeba keratitis, were found in 12 incidents during the monitoring of clinical pictures.
In vivo confocal microscopy was applied in 14 cases for the assessment/verification of the diagnosis. The presence of hyper-reflective objects scattered or arranged in characteristic chains-double-walled, polygonal, or round Acanthamoeba cysts, with their outer wall more reflective than the internal walls-was detected by this technique. The visualization of hyper-reflective objects was positive in 16 incidents (80%). However, there were various effects of monitoring related to the previous lack of progress of therapy; in the majority of incidents, hyper-reflective objects, namely amoeba cysts, were detected in the confocal microscopy three weeks after the first keratitis symptoms appeared. The duration of the severe keratitis symptoms was longer, and proper diagnosis was delayed, ranging from 21 to 38 days. Representative confocal microscopy images of corneal layers showed the presence of the hyper-reflective objects; the double-walled Acanthamoeba cysts are presented in Figure 1. anti-Acanthamoeba therapy was started. In two other misdiagnosed keratitis cases, the affected eyes were treated unsuccessfully over six months. Drug-combined therapy involved mainly chlorhexidine digluconate, polyhexamethylene biguanide PHMB, and propamidine isethionate Brolene with an addition of antibiotics; steroids were also used. In some cases of AK, surgical interventions, deep anterior lamellar keratoplasty (DALK) or penetrating keratoplasty, amniotic membrane transplantations, or cataract surgery were needed.
In the clinical picture of the keratitis incidents included in our study, photophobia, reduced visual acuity, lid edema, and redness appeared in all cases with different intensities. Symptoms that were visible in slit-lamp and in vivo confocal microscopy, appearing in the early and advanced infection stages, were taken into account. The hyperreflective tissues, epithelial inflammation, size and depth of stromal infiltration or ulcer, and presence or absence of hypopyon were assessed by non-invasive in vivo tests in the slit-lamp biomicroscopy. The occurrence of unbearable pain, epithelial diffuse edema progress to dendriform ulcer, and characteristic ring-shaped infiltration successively involving deep stromal layers, which were considered as factors indicating Acanthamoeba keratitis, were found in 12 incidents during the monitoring of clinical pictures.
In vivo confocal microscopy was applied in 14 cases for the assessment/verification of the diagnosis. The presence of hyper-reflective objects scattered or arranged in characteristic chains-double-walled, polygonal, or round Acanthamoeba cysts, with their outer wall more reflective than the internal walls-was detected by this technique. The visualization of hyper-reflective objects was positive in 16 incidents (80%). However, there were various effects of monitoring related to the previous lack of progress of therapy; in the majority of incidents, hyper-reflective objects, namely amoeba cysts, were detected in the confocal microscopy three weeks after the first keratitis symptoms appeared. The duration of the severe keratitis symptoms was longer, and proper diagnosis was delayed, ranging from 21 to 38 days. Representative confocal microscopy images of corneal layers showed the presence of the hyper-reflective objects; the double-walled Acanthamoeba cysts are presented in Figure 1.

Laboratory Differential Diagnosis
During the differential diagnosis, the material obtained from affected eyes was tested in the parasitological laboratory for confirmation of Acanthamoeba keratitis. Initially, during the light microscopy examinations, samples of scrapings were directly examined in wet-mount slides using a phase-contrast light microscope (100× and 400×) to visualize cysts or/and trophozoites and assess amoebic strains at the cellular level. Isolated amoebae were identified at the group level (Acanthamoeba spp., group I-III), according to Pussard and Pons (1977) [60], mainly based on size, cyst morphology, and the number of opercula. The most commonly detected amoebae corneal isolates were identified and classified as belonging to Acanthamoeba sp., group II: live trophozoites (14-38 µm), with pseudopodia and characteristic protrusions, acanthopodia with a nucleus and a prominent centrally placed nucleolus, cysts (7-24 µm) with their two cyst walls wrinkled ectocyst, and a polygonal or round-to-ovoid endocyst.
The results of 10 corneal isolates examined at the molecular level revealed 99.1-100% homology in the obtained sequences, with those available in the GenBank identifying particular isolates as belonging to the T4 genotypes. The material was derived from AK cases: eight contact lens wearers and two non-contact-lens wearers. The obtained sequences are deposited in GenBank under accession numbers MZ401143-MZ401152; the cultured subsequent isolates were in line with A. mauritanensis, A. culbertsoni, and A. castellanii, genotype T4. These results were in line with those from the corneal isolates that were identified on the cellular level as species belonging to the Acanthamoeba genus.
A compilation of data on the material investigated for the confirmation of Acanthamoeba keratitis (AK) is presented in Table 1.

Assessment of In Vitro Cultivation
The cultures were maintained as long as Acanthamoeba corneal strains survived. In vitro cultured corneal samples were examined with a phase-contrast light microscope as the direct scraping material. Regular monitoring confirmed AK for 12 cases by the detection of live amoebae. The Acanthamoeba infections were confirmed based on the morpho-physiology of the developmental stages. The trophozoites (~18-40 µm) moving by acanthopodia with spine-like protrusions and the double-walled cysts of Acanthamoeba (~10-20 µm) were revealed. Light micrographs of live Acanthamoeba trophozoites and cysts in the wet-mount slides from cultured corneal isolates are presented in Figure 2.

Assessment of In Vitro Cultivation
The cultures were maintained as long as Acanthamoeba corneal strains survived. In vitro cultured corneal samples were examined with a phase-contrast light microscope as the direct scraping material. Regular monitoring confirmed AK for 12 cases by the detection of live amoebae. The Acanthamoeba infections were confirmed based on the morphophysiology of the developmental stages. The trophozoites (~18-40 μm) moving by acanthopodia with spine-like protrusions and the double-walled cysts of Acanthamoeba (~10-20 μm) were revealed. Light micrographs of live Acanthamoeba trophozoites and cysts in the wet-mount slides from cultured corneal isolates are presented in Figure 2.  A comparative assessment of the cultures showed changes in protozoan density that appeared with varying intensity in particular amoebic populations. A low overall number of amoebae was detected in the cultures of some strains with short symptom duration before proper diagnosis.
Acanthamoeba corneal isolate dynamics were evaluated in regard to in vitro viability of the particular strains and were expressed as, among others, the trophozoites' ability to intensely multiply, the overall amoeba number, and the duration of the survival time of the cultivated amoebae. For example, the lowest number of amoebae, 12.0-28.0 × 10 3 (in the range of three counts), detected in the log growth phase was revealed for the weak corneal strain that indicated a short survival time of about one cycle of sub-culturing. For comparison, a distinctly higher number of amoebae was revealed in the corneal strains, which indicated high in vitro activity during 6 or 24 cycles of sub-culturing, in the range of amoeba numbers 24.0-60.0 × 10 3 , 54.3-80.0 × 10 3 , respectively (the level of statistical significance was set at p < 0.05).
A comparison of the in vitro dynamics of particular Acanthamoeba strains cultured in BSC medium is presented in Table 2. The population density of subsequent Acanthamoeba isolates with the long-time cultivation (3.5 months and more) transferred to 37 • C and monitored during many cycles of sub-culturing was compared with the effect obtained for samples of the strain that were surviving in a culture medium during only one cycle of sub-culturing; there was a statistically significantly higher overall number of amoebae cells revealed in strains after exposure to 37 • C in comparison with their density in 24 • C. Simultaneously, some reduction in Acanthamoeba population density was found in the strain, with short survival times in cultures exposed to changed, higher temperatures.
The representative effects of Acanthamoeba T4 corneal strains to changed temperatures in the exponential growth phase of the cultivation are presented in Table 3.
The microbiological examinations of scrapings from corneas performed for the verification of initial diagnoses revealed concomitant infections in 10 of 20 AK incidents. The Gram-positive strain Enterococcus faecalis, the Gram-negative Pantoea agglomerans, Enterobacter cloacae, Pseudomonas aeruginosa, and fungal co-infections with Fusarium sp. and Candida spp. were detected. In this interdisciplinary study, the data on infections with Acanthamoeba spp. alone as well as the etiologically mixed keratitis, namely Acanthamoeba spp. and concomitant fungal and/or bacterial infections, have been included. Table 3. Effect of in vitro exposure of Acanthamoeba corneal strains from exponential growth phase of the cultivation to changed temperature.

No.
Acanthamoeba Strain The range of three counts calculated for 1 mL of culture medium is compared for five strains. The level of statistical significance was set at p < 0.05; statistically significant differences in relation to data of 24 • C have been bolded.
The cases included in the study presented challenges in terms of AK. There was a wide range of initial non-specific, confusing clinical symptoms, the incorrect identification of pathogenic factors, and ambiguous initial diagnoses or misdiagnoses influencing the prolonged and severe course of this eye disease.
Although a standardized protocol is missing in AK management, it is emphasized that the clinical picture alone is not sufficient to identify the etiological agent of amoebic keratitis in humans [38,39,48]. Clinical manifestations of AK are similar to those appearing in viral, bacterial, or fungal keratitis. Moreover, the amoebae are known as vehicles/reservoirs/sources of various bacteria, fungi, viruses, and protists, which can survive and multiply within the amphizoic amoebae [32][33][34][35][36][37]. These microorganisms may be causative agents of concomitant/secondary/mixed corneal infections.
In the present study, mistakes in the initial identification of the infectious causative agents of keratitis were found in most cases. It is noteworthy that while AK was finally shown in all 20 incidents, concomitant infections with bacteria and/or fungi that are pathogenic for humans were revealed in 50% of the analyzed cases. It was not possible to assess if the microorganisms were endogenous or exogenous in origin; however, their influence on the difficulties of the correct assessment of corneal isolates under suspicion with AK cannot be excluded.
Diagnostic approaches for AK include methods with different sensitivities: in vivo confocal microscopy, direct microscopic examinations of corneal material, in vitro cultures useful for the detection of causative factors of keratitis, and PCR-based techniques. It is considered that the direct detection of a causative agent in a cornel scrape specimen is the only reliable diagnostic method for AK; culture remains the gold standard of the Acanthamoeba laboratory diagnosis, and several PCR-based techniques have been wellestablished and have increased sensitivity significantly [38,52,57,64].
In this study, the direct detection of the causative agents-amoeba developmental forms-was very important for the proper diagnosis as well as for the verification of common misdiagnoses. A comparison of characteristics of the successively detected isolates identified on the cellular level as Acanthamoeba sp. showed the suitability of contrast phase microscopy for the direct visualization of trophozoites and cysts derived from both corneal isolate material and from in vitro cultures, particularly for cases with a long duration of symptoms until proper diagnosis.
Molecular examinations of cultured samples deriving from ten corneal isolates revealed 99.1-100% homology obtained sequences with those available in the GenBankidentifying the individual isolates to species level, e.g., A. mauritanensis, A. culbertsoni, and A. castellanii belonging to the T4 genotype. The results of molecular tests were in line with the effect of a cellular-level direct microscopic examination of Acanthamoeba based on the morpho-physiology of trophozoites and cysts.
It is known that Acanthamoeba spp. are able to tolerate various conditions in natural and man-made environments and growth in different culture media, e.g., non-nutrient agar plates seeded with Escherichia coli, the most commonly used medium peptone-yeast extract-glucose medium PYG, Bacto Casitone, Difco (Bactocasitone, BSC), and the hardly used PYG-Bactocasitone [38,50,63,64].
In our comparative investigations, we used mainly BSC [31,53,59,62]. We considered the axenic medium with the absence of external live food organisms suitable in this study for in vitro cultivation and for monitoring the subsequent Acanthamoeba strains.
In the present study, the evaluation of in vitro cultivation of the Acanthamoeba corneal strain populations performed under axenic conditions showed different durations of high in vitro activity of amoebic strains from one cycle of sub-culturing to many months and high viability. The thermo-tolerance detected in the subsequent Acanthamoeba isolates cultured in vitro expressed that clear strong population dynamics, higher amoeba density, and the ability to intense multiply at high temperatures corresponded to strong viability and long survival time in the axenic culture medium. The ability shown in this study for subsequent Acanthamoeba isolates is considered the indicator of the pathogenic potential of a given isolate [31,38,53].

Conclusions
Our work, based on data from the verification process of Acanthamoeba corneal isolates from complicated AK cases, provides information about the differences in amoeba population density and in the duration and the in vitro ability to intensely multiply. To our best knowledge, this is the first Polish study evaluating the amoeba strains A. mauritanensis and A. culbertsoni in terms of their possibility to grow in vitro that resulted in the confirmation of long survival time of the amoebae isolates in this cultivation conditions. Furthermore, we demonstrated that some of the Acanthamoeba strains under dynamic assessments indicated enough adaptive capability to grow in an axenic culture medium, allowing them to exhibit significant thermal tolerance that is considered an indirect marker of pathogenic strains. The in vitro monitoring was especially useful to reveal variability in the dynamics of these Acanthamoeba strains that were obtained from keratitis cases with a long duration of symptoms until proper diagnosis.
The results of the presented study, our experience, and the literature data indicate the various levels of usefulness/value of particular methods of keratitis assessment that depend on, among others, the amoebic population viability and density. Therefore, in complicated incidents that are suspected to be AK, a combined approach involving different methods is highly recommended.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki. It has been taken note of Bioethic Commission of Medical University of Warsaw (11.2022,01.2023,AKBE/4) and did not require ethical approval.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data are not publicly available due to patient's privacy and ethical reasons.

Conflicts of Interest:
The authors declare no conflict of interest.