Abstract

The present study aims to investigate the occurrence of free living amoeba (FLA) in water resources (rivers and tap water) in Samsun in the Black Sea. The presence of Acanthamoeba spp. was confirmed in 98 of 192 water samples collected from 32 sites of Samsun province (Samsun centre, Terme, Carsamba, Tekkekoy, Bafra) by PCR. Acanthamoeba spp. were found in 15/36 river samples from Samsun, in 58/90 from Terme, in 12/30 from Carsamba, in 7/18 from Tekkekoy and in 6/18 from Bafra. No Acanthamoeba species were detected in tap water samples. The highest rate in river waters contaminated with Acanthamoeba species was in Terme followed by Samsun centre (41.7%), Carsamba (40%), Tekkekoy (38.9%) and Bafra districts (33.3%), respectively. The result of the subsequent sequence analysis showed Haplotype I (A. triangularis) in 5%, Haplotype II (A. polyphaga) in 29.6%, Haplotype III (Acanthamoeba spp.) in 62% and Haplotype IV (A. lenticulata) in 3%. The most common genotype was Acanthamoeba T4 (Acanthamoeba spp., A. polyphaga, A. triangularis) and T5 genotype was also found in 3%. The T4 genotype is the most common genotype associated with Acanthamoeba keratitis (AK) worldwide; therefore, humans and animals living in the area are at risk after contact with such waters.

INTRODUCTION

Acanthamoeba spp. are commonly found in damp and wet soil, freshwater accumulations, sewage, swimming pools, lakes, dam lakes, tap water and air (Khan 2006; Lass et al. 2014) and, in some cases, contact lens holders. More than 80% of people have antibodies against Acanthamoeba (Chappell et al. 2001). This suggests that Acanthamoeba is an organism that is frequently in contact with humans. Acanthamoeba species do not need a host to live; they settle into the tissues causing serious diseases (Lorenzo-Morales et al. 2005; Juárez et al. 2018). The identification of Acanthamoeba species at the genus level is made by distinguishing features of trophozoites and cysts, especially the shape of the double-walled cysts. Acanthamoeba species were initially classified as three different morphological groups (I, II and III) (Pussard & Pons 1977; Page 1991). However, the morphological classification is inadequate to discriminate at the species level of Acanthamoeba (Stothard et al. 1998; Alves et al. 2000). The disadvantage of diagnosis based on morphological characteristics of the cyst is that the morphology differs according to the media used; therefore, it is necessary to use cultures and specialist researchers in this field (Scheid & Balczun 2017).

Acanthamoeba cyst environmental reservoirs have the potential to be transmitted to humans as well as other mammals (Shin & Im 2004; Edagawa et al. 2009; Lass et al. 2014). Since Acanthamoeba species can be found in surface waters, thermal waters, sea water, soil, air, food, drinking water, packaged spring waters, distilled water, chlorinated swimming pool waters and contact lens storage containers they therefore pose the risk of rapid spread. Rapid and reliable identification of Acanthamoeba spp. from both environmental sources and clinical specimens is extremely important for diagnosis and treatment of Acanthamoeba keratitis (AK).

Free-living amoebae (FLA) can serve as tools or hosts for phylogenetically various microorganisms, while some of them amplify intracellularly (Scheid 2014; Scheid et al. 2014; Balczun & Scheid 2017). Taxonomy and classification of these parasites has begun to be revised after the successful application of molecular techniques (Khan et al. 2001; Booton et al. 2002; Kong et al. 2002; Lorenzo-Morales et al. 2005). Classification based on the (SSU) rDNA gene for the detection of Acanthamoeba was established by Gast et al. (1996) and Stothard et al. (1998). The strains in which the differences detected in the (SSU) rDNA gene region were below 5% of all were collected under a single genotype. Analyses of more than 50 strains from three morphological groups were presented as 12 genotypes from T1 to T12 (Stothard et al. 1998). Other genotypes have been identified, T13 to T16, by other researchers (Horn et al. 1999; Hewett et al. 2003; Corsaro & Venditti 2010). Fuerst et al. (2015) showed genotyping T1 to T20 as a result of phylogenetic analysis of Acanthamoeba (SSU) rRNA. The variant among the isolates for the T17 genotype is heterogeneous, while the genotype T18 has only recently been reported as the new nominal A. byersi (Fuerst et al. 2015).

It was determined that 13 Acanthamoeba species found belong to T2, T4 and T6 genotypes. Kao et al. (2012) identified Acanthamoeba genotypes in 211 water samples collected from two water basins in southern Taiwan. Acanthamoeba genotypes reported were T4 (n = 19), T5 (n = 8), T15 (n = 3). Genotype T6, T7/T8, T11 and T12 were detected only once. Genotypes T4, T5, T6, T11 and T15 have been found to be responsible for AK and may cause potential health problems for people in contact with environmental waters (Bouheraoua et al. 2014; Lorenzo-Morales et al. 2015); it has also been recognized as a cause of keratitis in non-CLs wearers too (Juárez et al. 2018). Other pathogenic (T2–T10, T4, T5, T15) or non-pathogenic (T7, T16 and T17) genotypes have been identified for the first time as a result of the molecular characterization of Acanthamoeba genotypes from different waters in the Khyber Pakhtunkhwa region of Pakistan (Tanveer et al. 2013). Maschio et al. (2015) tested marketed mineral water bottles collected from Porto Alegre in the south of Brazil for the determination of Acanthamoeba genotypes. Six of the eight Acanthamoeba genotypes were found to belong to the T5 genotype, one to the T4 genotype and the other to the T11 genotype.

In the present study, we aimed to molecularly identify Acanthamoeba genotypes in river and water samples that were collected from Samsun province within the region of the Black Sea in Turkey based on the (SSU) rDNA target DNA of Acanthamoeba spp.

MATERIALS AND METHODS

Sampling and geography

Samsun is one of the most populated provinces and a major port of the western Black Sea in Turkey. Investigated areas in this study include Terme, Carsamba, Tekkekoy and Bafra districts of Samsun. The most important rivers are the Kizilirmak, Yesilirmak, Terme, Mert, Kurtun, Milic and Akcay. We chose to investigate areas near important rivers, such as part of the Kizilirmak in Bafra (6 samples), part of the Yesilirmak and Irmaksirti (12 samples) in Carsamba, the Terme, Milic, Akcay and Kocaman rivers (58 samples) in Terme, and the Mert and Kurtun rivers (15 samples) in Samsun centre.

Rainfall was seen to be high in a number of days each month of spring and autumn, therefore samples were taken at those periods. Water samples from rivers (as shown in Figure 1 and Tables 1 and 2) were collected from 32 stations in Samsun province during autumn 2016 and spring 2017. A total of 192 samples of river water (i.e., 96 samples twice per year) and 30 tap water samples were collected from five centres of districts, Samsun, Terme, Carsamba, Tekkekoy and Bafra (six tap water samples from each distict) as shown in Figure 1.

Table 1

The presence of Acanthamoeba spp. by PCR in water samples collected from Samsun province

Water typeInvestigated sitesExamined water samplesPCR-positive samples
Tap water All sites 30 
River water Samsun centre 36 15 
Terme 90 58 
Carsamba 30 12 
Tekkekoy 18 
Bafra 18 
Total positive (%)  222 (44%) 
Water typeInvestigated sitesExamined water samplesPCR-positive samples
Tap water All sites 30 
River water Samsun centre 36 15 
Terme 90 58 
Carsamba 30 12 
Tekkekoy 18 
Bafra 18 
Total positive (%)  222 (44%) 
Table 2

Results of the sequencing for Acanthamoeba spp. in river water samples collected from Samsun and its districts

Sequence analysis
Investigated site Samsun centrePCR-positive samples 15Haplotype I A. triangularis (AF316547) T4Haplotype II A. polyphaga (AF019051) T4Haplotype III Acanthamoeba spp. (EU168069) T4Haplotype IV A. lenticulata (U94736) T5
Mert River      
Me1 – – – 
Me2 – – 
Me3 – 
Kurtun River      
Ku1 – – – 
Ku2 – – – 
Ku3 – – 
Terme 58     
Akcay – – – – – 
Milic     – 
Mi1 – – 
Mi2 – – – 
Terme çayı      
T1 – 
T2 – 
T3 – – 
T4 – – 
T5 – 
T6 – – 
Kocaman River      
K1 – 
K2 – 
K3 – – 
K4 – – 
K5 – 
K6 – – – 
Çarsamba 12     
Yesilırmak River      
Y1 – – – 
Y2 – – 
Y3 – – 
Irmaksırtı      
I1 – – 
I2 – – 
Tekkekoy 7     
Gelemen (Te1) – – 
Selyeri (Te2) – –- 
Kirazlık (Te3) – – – 
Bafra 6     
Kızılırmak      
Kı1 – – – – – 
Kı2 – – – 
Kı3 – – 
Total positive (%) 98 5 (%5) 29 (% 29.6) 61 (% 62) 3 (%3) 
Sequence analysis
Investigated site Samsun centrePCR-positive samples 15Haplotype I A. triangularis (AF316547) T4Haplotype II A. polyphaga (AF019051) T4Haplotype III Acanthamoeba spp. (EU168069) T4Haplotype IV A. lenticulata (U94736) T5
Mert River      
Me1 – – – 
Me2 – – 
Me3 – 
Kurtun River      
Ku1 – – – 
Ku2 – – – 
Ku3 – – 
Terme 58     
Akcay – – – – – 
Milic     – 
Mi1 – – 
Mi2 – – – 
Terme çayı      
T1 – 
T2 – 
T3 – – 
T4 – – 
T5 – 
T6 – – 
Kocaman River      
K1 – 
K2 – 
K3 – – 
K4 – – 
K5 – 
K6 – – – 
Çarsamba 12     
Yesilırmak River      
Y1 – – – 
Y2 – – 
Y3 – – 
Irmaksırtı      
I1 – – 
I2 – – 
Tekkekoy 7     
Gelemen (Te1) – – 
Selyeri (Te2) – –- 
Kirazlık (Te3) – – – 
Bafra 6     
Kızılırmak      
Kı1 – – – – – 
Kı2 – – – 
Kı3 – – 
Total positive (%) 98 5 (%5) 29 (% 29.6) 61 (% 62) 3 (%3) 
Figure 1

The map of sampling sites from investigated areas in the Black Sea.

Figure 1

The map of sampling sites from investigated areas in the Black Sea.

Cultivation and isolation of Acanthamoeba species in vitro

For the isolation of Acanthamoeba species, 500 mL of the water samples were used; they were filtered through a cellulose nitrate membrane with pore size 0.45 μm, according to Mahmoudi et al. (2012). Filters were transferred on Ringer agar plates seeded with Gram-negative bacteria (Escherichia coli) as a food source. Plates were incubated at 26 °C and 3 days later, they were microscopically examined for the presence of Acanthamoeba trophozoites. After 3 days' incubation and in the absence of amoebae, plates were monitored at the same conditions for up to 14 days. Acanthamoeba were identified at the genus level, based on morphological characteristics of trophozoites and cysts using microscopy (Figure 2). Amoeba cells were scraped and harvested from culture plates, then they were washed three times with phosphate-buffered saline (PBS 7.2) and the resulting solution was concentrated by centrifugation at 3,000 × rpm for 5 min. PBS was used for resuspension of the pellets and the mixture was counted for the presence of Acanthamoeba in 1 mL by a hemocytometer (Thoma cell counting chamber, Figure 3). The samples were then kept at 4 °C for DNA extraction.

Figure 2

Acanthamoeba cysts cultivated from water samples in inverted microscopy: (a) ×10, (b) ×20, (c) ×40; scale bar 10 μm.

Figure 2

Acanthamoeba cysts cultivated from water samples in inverted microscopy: (a) ×10, (b) ×20, (c) ×40; scale bar 10 μm.

Figure 3

Counting Acanthamoeba cysts collected from plates with hemocytometer (×40).

Figure 3

Counting Acanthamoeba cysts collected from plates with hemocytometer (×40).

DNA extraction and PCR

The pellets resuspended in phosphate buffered saline (PBS) were lysed by treatment with lysozyme (100 mg/mL) and they were frozen and thawed 15 times using liquid nitrogen and heating to 100 °C, as described by Koloren et al. (2011). The samples were then treated with proteinase K (20 mg/mL), and DNA extraction was performed by QIAamp DNA Mini Kit (Qiagen) as described, with minor modifications adapted from Koloren et al. (2011). The presence of Acanthamoeba-specific ASA.S1 region SSU rRNA gene was amplified with primers JDP1 and JDP2 (Schroeder et al. 2001) by standard PCR. A 500-bp fragment of SSU rRNA was amplified by PCR. The PCRs were performed in 25 μL final volumes that included 20 pmol of both primers, 5 × Q solution, 25 mM dNTP, 10 × PCR buffer, 25 mM MgCl2 and HotstarTaq DNA polymerase 5 U/μL. A Veriti thermal cycler was used for amplification that included 35 cycles (94 °C for 60 s, 50 °C for 45 s, 72 °C for 60 s), followed by a final extension at 72 °C for 10 min. PCR products were visualized on a 1.5% agarose gel electrophoresis stained with a solution of ethidium bromide under UV light.

Specificity analysis by PCR

The specificity of the Acanthamoeba for PCR has been demonstrated by comparing Acanthamoeba castellanii (ATCC30010) as a reference strain and the following DNAs were used: Cryptosporidium parvum (Iowa) DNA, Toxoplasma gondii DNA (ATCC50839), Giardia intestinalis (H3) DNA, Babesia bovis (ATCC75575), Blastocystis hominis (ATCC50608D).

Sequence and phylogenetic analysis

Millipore Multiscreen (R) HTSPCR Plates (filter plates) were used for the purification of PCR products according to the manufacturer's specifications. Purified PCR products were sequenced by an ABI PRISM3730 × L Analyzer (96 capillary type) and Genetic Analyzer with Big Dye Terminator V.3.1 cycle sequencing kit (Applied Biosystems). The bi-directional sequences with both primers was arranged by BioEdit software. Clustal W was used to align the reference base sequences from the GenBank and common base sequences from the water samples. Accession numbers of (SSU) rRNA reference sequences for Acanthamoeba were U07400 (T1), DQ992189 (T2), S81337 (T3), AF316547 (T4), U94736 (T5), AY172999 (T6), AF019064 (T7), AF019065 (T8), AF019070 (T10) and AF019068 (T11). Phylogenetic trees were drawn for Acanthamoeba with reference genotypes from GenBank. The neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) were performed using MEGA version 5.05, based on evolutionary distances calculated with the Kimura two-parameter model with 10,000 pseudo replications bootstrap tests. The percentage of the nucleotide similarity and pairwise distances among haplotypes were measured using BioEdit and MEGA (version 5.05), respectively.

RESULTS

Specificity analysis

A. castellanii DNA for target and other parasites such as Cryptosporidium parvum, T. gondii, G. intestinalis, B. bovis and B. hominis DNAs were used for the specificity of PCR. A. castellanii DNA was amplified by PCR, whereas DNAs from the other organisms were not amplified by PCR (Figure 4).

Figure 4

Agarose gel electrophoresis of PCR specificity of Acanthamoeba (SSU) rDNA gene region. M: 100 bp ladder, N: distilled water (negative), P: Acanthamoeba castellanii (ATCC30010), DNA 1: Cryptosporidium parvum DNA, 2: Toxoplasma gondii DNA, 3: Giardia intestinalis DNA, 4: Babesia bovis, 5: Blastocystis hominis.

Figure 4

Agarose gel electrophoresis of PCR specificity of Acanthamoeba (SSU) rDNA gene region. M: 100 bp ladder, N: distilled water (negative), P: Acanthamoeba castellanii (ATCC30010), DNA 1: Cryptosporidium parvum DNA, 2: Toxoplasma gondii DNA, 3: Giardia intestinalis DNA, 4: Babesia bovis, 5: Blastocystis hominis.

Positive water samples by PCR assay and collected from the investigated sites

The PCR results for the occurence of Acanthamoeba spp. in positive water samples taken from the Black Sea are shown in Table 1. Acanthamoeba spp. were not detected in any of the 30 drinking water samples while 98 out of 222 (44%) river water samples were positive for Acanthamoeba. The positive Acanthamoeba water samples collected from sampling sites and detected by PCR (Figure 5), are shown in Table 1. Fifteen of 36 river water samples from Samsun centre were found positive for Acanthamoeba spp. Fifty-eight of 90 river water samples in Terme district, 12/30 in Carsamba district, 7/18 in Tekkekoy district, 6/18 in Bafra district were found to be positive for the presence of Acanthamoeba spp. Ninety-eight of 192 samples from Kizilirmak, Yesilirmak, Terme, Mert, Kurtun, Milic and Akcay rivers yielded a positive result for the detection of Acanthamoeba spp. Five per cent were identified as Haplotype I (A. triangularis, T4), 29.6% as Haplotype II (A. polyphaga, T4), 62% as Haplotype III (Acanthamoeba spp., T4) and 3% as Haplotype IV (A. lenticulata, T5) (Table 2). Haplotype I (A. triangularis) for one water sample, Haplotype II (A. polyphaga) for three samples, Haplotype III (Acanthamoeba spp.) for 11 samples were detected in the 15 water samples positive in the centre of Samsun, but Haplotype IV (A. lenticulata) was not found in the same area. Of the 58 positive water samples taken from the Terme district, four were A. triangularis, 18 were A. polyphaga, 33 were Acanthamoeba spp. and three were identified as A. lenticulata. A. triangularis and A. lenticulata were not observed in the 12 positive water samples taken from Carsamba district. It was determined that five water samples were A. polyphaga and seven samples were Acanthamoeba spp. in Carsamba district. Three of the seven positive water samples in Tekkekoy district were identified as Haplotype II (A. polyphaga) and four as Haplotype III (Acanthamoeba spp.) while Haplotype I and Haplotype IV were not found. One sample was Haplotype II (A. polyphaga) and five were Haplotype III (Acanthamoeba spp.) in six positive water samples collected from Bafra district. Haplotype I and Haplotype IV were not observed (Table 2).

Figure 5

The agarose gel electrophoresis of the (SSU) rDNA gene amplified by the PCR from water samples collected from Samsun province. M: 100 bp ladder, N: distilled water (negative), P: Acanthamoeba castellanii (ATCC30010), 1–22: water samples collected from the investigated areas.

Figure 5

The agarose gel electrophoresis of the (SSU) rDNA gene amplified by the PCR from water samples collected from Samsun province. M: 100 bp ladder, N: distilled water (negative), P: Acanthamoeba castellanii (ATCC30010), 1–22: water samples collected from the investigated areas.

Genotyping and pairwise evolutionary divergence

PCR positive river water samples in Samsun and its districts were clearly arranged and phylogenetic trees with NJ, MP, ML algorithms for Acanthamoeba spp. (SSU) rDNA target sequences were constructed. The tree is represented in Figure 6.

Figure 6

Phylogenetic relationships of Acanthamoeba (SSU) rDNA target sequences in river water samples taken from Samsun and its districts by using NJ, ML, MP analysis. Protacanthamoeba bohemica was selected as an outgroup. Bootstrap values (higher than 50%) from NJ, ML and MP analysis, respectively, are shown in this tree.

Figure 6

Phylogenetic relationships of Acanthamoeba (SSU) rDNA target sequences in river water samples taken from Samsun and its districts by using NJ, ML, MP analysis. Protacanthamoeba bohemica was selected as an outgroup. Bootstrap values (higher than 50%) from NJ, ML and MP analysis, respectively, are shown in this tree.

The NJ phylogeny tree for Acanthamoeba-positive water samples (Haplotype I–IV) and (SSU) rDNA gene region of all Acanthamoeba genotypes taken from GenBank (T1, T2, T3, T4, T5, T6, T7, T8, T10, T11) are given in Figure 6. According to the NJ phylogeny tree, Haplotype I and A. triangularis showed 68%, 70% and 78% homology with sequences of Acanthamoeba T4 which are supported with bootstrap values in NJ, MP, and ML trees, respectively, as shown in Figure 6. Haplotype II and A. polyphaga are represented as sister to Acanthamoeba T4 and supported with 72%, 71% and 74% bootstrap values in the NJ, MP, ML trees, respectively. Haplotype III and Acanthamoeba spp. showed 76%, 78% and 92% homology with sequences of Acanthamoeba T4 which are supported with bootstrap values in NJ, MP and ML trees, respectively. Table 2 shows the sequence analysis results and generated haplotypes. Four haplotypes were found among 98 sequenced river water samples.

Of the 98 positive water samples, five (5%) were identified as Haplotype I, 29 (29.6%) as Haplotype II, 61 (62%) as Haplotype III and three (3%) as Haplotype IV. The nucleotide sequence percentage similarities and pairwise distance between (SSU) rDNA sequences' haplotypes in Samsun and reference sequences for Acanthamoeba spp. from GenBank are illustrated in Table 3.

Table 3

Pairwise evolutionary difference and nucleotide sequence percentage similarities between (SSU) rDNA sequences in water samples from Samsun province and reference sequences of Acanthamoeba spp. from GenBank

Haplotype IAcanthamoeba triangularisHaplotype IIAcanthamoeba polyphagaHaplotype IIIAcanthamoeba sp.Acanthamoeba castellaniiAcanthamoeba sp.Acanthamoeba griffiniAcanthamoeba lenticulataHaplotype IVAcanthamoeba sp.Acanthamoeba astronyxisAcanthamoeba tubiashiAcanthamoeba healyiAcanthamoeba hatchettiProtacanthamoeba bohemica
Haplotype I ID 0.997 0.486 0.486 0.133 0.133 0.354 0.351 0.390 0.333 0.332 0.337 0.291 0.293 0.307 0.387 0.227 
Acanthamoeba triangularis(AF316547) 0.002 ID 0.487 0.487 0.133 0.133 0.354 0.351 0.390 0.333 0.332 0.337 0.290 0.293 0.307 0.387 0.227 
Haplotype II 0.036036036036036 0.036 ID 0.999 0.215 0.215 0.393 0.422 0.410 0.394 0.394 0.427 0.389 0.369 0.379 0.407 0.271 
Acanthamoeba polyphaga(AF019051) 0.036 0.036 0.001 ID 0.215 0.215 0.393 0.422 0.410 0.394 0.394 0.427 0.389 0.369 0.379 0.407 0.271 
Haplotype III 0.045 0.045 0.045 0.045 ID 0.998 0.523 0.512 0.525 0.469 0.469 0.509 0.352 0.385 0.504 0.532 0.482 
Acanthamoebasp. (EU168069) 0.045 0.045 0.045 0.045 0.003 ID 0.523 0.512 0.525 0.469 0.469 0.509 0.352 0.385 0.504 0.532 0.482 
Acanthamoeba castellanii(U07400) 0.054 0.054 0.054 0.054 0.054 0.054 ID 0.844 0.871 0.729 0.729 0.822 0.554 0.621 0.827 0.882 0.628 
Acanthamoebasp. (DQ992189) 0.027 0.027 0.009 0.009 0.036 0.036 0.045 ID 0.883 0.753 0.753 0.926 0.552 0.609 0.792 0.865 0.626 
Acanthamoeba griffini(S81337) 0.009 0.009 0.027 0.027 0.036 0.036 0.045 0.018 ID 0.745 0.743 0.858 0.561 0.620 0.795 0.944 0.635 
Acanthamoeba lenticulata(U94736) 0.054 0.054 0.063 0.063 0.063 0.063 0.045 0.054 0.045 ID 0.998 0.755 0.546 0.590 0.699 0.748 0.604 
Haplotype IV 0.054 0.054 0.063 0.063 0.063 0.063 0.045 0.054 0.045 0.002 ID 0.755 0.546 0.590 0.699 0.748 0.602 
Acanthamoebasp. (AY172999) 0.027 0.027 0.018 0.018 0.036 0.036 0.045 0.018 0.018 0.054 0.054 ID 0.548 0.604 0.781 0.845 0.609 
Acanthamoeba astronyxis(AF019064) 0.090 0.090 0.090 0.090 0.099 0.099 0.072 0.090 0.081 0.063 0.063 0.072 ID 0.825 0.554 0.566 0.458 
Acanthamoeba tubiashi(AF019065) 0.099 0.099 0.090 0.090 0.099 0.099 0.054 0.090 0.090 0.081 0.081 0.072 0.018 ID 0.622 0.625 0.506 
Acanthamoeba healyi(AF019070) 0.054 0.054 0.054 0.054 0.063 0.063 0.018 0.045 0.045 0.063 0.063 0.045 0.090 0.072 ID 0.793 0.587 
Acanthamoeba hatchetti(AF019068) 0.009 0.009 0.027 0.027 0.036 0.036 0.045 0.018 0.000 0.045 0.045 0.018 0.081 0.090 0.045 ID 0.631 
Protacanthamoeba bohemica(AY960120) 0.216216216216216 0.216 0.225 0.225 0.180 0.180 0.216 0.216 0.207 0.171 0.171 0.216 0.234 0.252 0.216 0.207 ID 
Haplotype IAcanthamoeba triangularisHaplotype IIAcanthamoeba polyphagaHaplotype IIIAcanthamoeba sp.Acanthamoeba castellaniiAcanthamoeba sp.Acanthamoeba griffiniAcanthamoeba lenticulataHaplotype IVAcanthamoeba sp.Acanthamoeba astronyxisAcanthamoeba tubiashiAcanthamoeba healyiAcanthamoeba hatchettiProtacanthamoeba bohemica
Haplotype I ID 0.997 0.486 0.486 0.133 0.133 0.354 0.351 0.390 0.333 0.332 0.337 0.291 0.293 0.307 0.387 0.227 
Acanthamoeba triangularis(AF316547) 0.002 ID 0.487 0.487 0.133 0.133 0.354 0.351 0.390 0.333 0.332 0.337 0.290 0.293 0.307 0.387 0.227 
Haplotype II 0.036036036036036 0.036 ID 0.999 0.215 0.215 0.393 0.422 0.410 0.394 0.394 0.427 0.389 0.369 0.379 0.407 0.271 
Acanthamoeba polyphaga(AF019051) 0.036 0.036 0.001 ID 0.215 0.215 0.393 0.422 0.410 0.394 0.394 0.427 0.389 0.369 0.379 0.407 0.271 
Haplotype III 0.045 0.045 0.045 0.045 ID 0.998 0.523 0.512 0.525 0.469 0.469 0.509 0.352 0.385 0.504 0.532 0.482 
Acanthamoebasp. (EU168069) 0.045 0.045 0.045 0.045 0.003 ID 0.523 0.512 0.525 0.469 0.469 0.509 0.352 0.385 0.504 0.532 0.482 
Acanthamoeba castellanii(U07400) 0.054 0.054 0.054 0.054 0.054 0.054 ID 0.844 0.871 0.729 0.729 0.822 0.554 0.621 0.827 0.882 0.628 
Acanthamoebasp. (DQ992189) 0.027 0.027 0.009 0.009 0.036 0.036 0.045 ID 0.883 0.753 0.753 0.926 0.552 0.609 0.792 0.865 0.626 
Acanthamoeba griffini(S81337) 0.009 0.009 0.027 0.027 0.036 0.036 0.045 0.018 ID 0.745 0.743 0.858 0.561 0.620 0.795 0.944 0.635 
Acanthamoeba lenticulata(U94736) 0.054 0.054 0.063 0.063 0.063 0.063 0.045 0.054 0.045 ID 0.998 0.755 0.546 0.590 0.699 0.748 0.604 
Haplotype IV 0.054 0.054 0.063 0.063 0.063 0.063 0.045 0.054 0.045 0.002 ID 0.755 0.546 0.590 0.699 0.748 0.602 
Acanthamoebasp. (AY172999) 0.027 0.027 0.018 0.018 0.036 0.036 0.045 0.018 0.018 0.054 0.054 ID 0.548 0.604 0.781 0.845 0.609 
Acanthamoeba astronyxis(AF019064) 0.090 0.090 0.090 0.090 0.099 0.099 0.072 0.090 0.081 0.063 0.063 0.072 ID 0.825 0.554 0.566 0.458 
Acanthamoeba tubiashi(AF019065) 0.099 0.099 0.090 0.090 0.099 0.099 0.054 0.090 0.090 0.081 0.081 0.072 0.018 ID 0.622 0.625 0.506 
Acanthamoeba healyi(AF019070) 0.054 0.054 0.054 0.054 0.063 0.063 0.018 0.045 0.045 0.063 0.063 0.045 0.090 0.072 ID 0.793 0.587 
Acanthamoeba hatchetti(AF019068) 0.009 0.009 0.027 0.027 0.036 0.036 0.045 0.018 0.000 0.045 0.045 0.018 0.081 0.090 0.045 ID 0.631 
Protacanthamoeba bohemica(AY960120) 0.216216216216216 0.216 0.225 0.225 0.180 0.180 0.216 0.216 0.207 0.171 0.171 0.216 0.234 0.252 0.216 0.207 ID 

DISCUSSION

In the present study, Acanthamoeba spp. and Acanthamoeba genotypes in Samsun province of the Black Sea in Turkey were detected. It was observed that the highest number of Acanthamoeba spp. was in the district of Terme. The rest of the districts (Carsamba, Tekkekoy and Bafra) were less contaminated than Terme orginating from Acanthamoeba spp., respectively. Sequences were successfully taken from 98 samples found positive with Acanthamoeba spp.

The most common genotypes of Acanthamoeba were T4 (96.94%), followed by T5 (3.1%). Also, many cases of Acanthamoeba keratitis (AK) linked to Acanthamoeba infections have been reported in Turkey. The first case of AK in Turkey was reported by Akyol et al. (1996) and the second case by Akisu et al. (1999), but genotyping was not included. Demirci et al. (2006) showed the presence of Acanthamoeba spp. in the eye swabs received from a five-year-old male without genotyping. Another case was reported as A. castellanii (T4) and detected by genotyping from a case with no contact lens (Ertabaklar et al. 2007). Ozkoc et al. (2008) reported A. castellanii T4 genotype in a case without contact lens but with a small trauma history. Ertabaklar et al. (2009) reported A. castellani (T4 genotype) in a 23-year-old woman who was a contact lens wearer, and had red eyes, blurred vision, stinging and burning. Yunlu et al. (2015) investigated the existence of free living amoebas in the case of eyelid swabs taken from 500 people. One example was Acanthamoeba spp. (0.2%) and the other one was Hartmannella spp. (0.2%). Kilic et al. (2004) reported the presence of Acanthamoeba species with genotypes T2, T3, T4, T7 in samples taken from environmental sources in Ankara greater area. All the studies presented in Turkey were contaminations reported from various sources (water, air conditioning, eye swabs, etc.) implicating Acanthamoeba species and in different genotypes. Researchers emphasize the importance of genotyping for the epidemiology of Acanthamoeba species, species separation, determination of pathogenicity and distribution of pathogenic species (Ertabaklar et al. 2009; Yunlu et al. 2015). The present study is the first report of genotyping in the Black Sea region showing the distribution of Acanthamoeba species in environmental waters and it fills a gap in this research area in Turkey.

The high numbers of Acanthamoeba spp. in water, soil and other environmental samples constitutes an important hygiene risk for people with immune deficiency and wearing contact lenses (Lorenzo-Morales et al. 2015). Several studies indicated that the most common Acanthamoeba genotype in the world was T4 (Walochnik et al. 2000a; Schroeder et al. 2001; Ledee et al. 2003; Booton et al. 2005; De Jonckheere 2007). T1, T10, T12 genotypes (Booton et al. 2005), T5 (Barete et al. 2007), T2 (Walochnik et al. 2008), respectively, followed the most dominant T4 genotypes, in other infections without granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). T4 genotype is also the most common genotype in AK (Gast et al. 1996; Walochnik et al. 2008). However, T3 (Ledee et al. 1996; Stothard et al. 1998), T5 (Spanakos et al. 2006; Ledee et al. 2009), T6 (Walochnik et al. 2000b), T11 (Khan et al. 2002), T15 (Di Cave et al. 2009) have also been reported to cause a number of known eye infections (Nagyova et al. 2010).

Regarding environmental samples, 15.9% of the natural water samples from 11 provinces in northeast Thailand were positive for Acanthamoeba and seven samples were T4, one sample was similar to T3, and the other two samples were similar to T5 (Thammaratana et al. 2016). In Iran, a total of 27 surface water samples were investigated, including natural (rivers, lakes, lagoons) and freshwater resources in the Gilan region, a large area of Iran (Mahmoudi et al. (2015). A total of 14 out of 19 (73.7%) water samples were confirmed by the PCR method using primers JDP1 and JDP2. Free living amoebas (Acanthamoeba, Hartmannella and Saccamoeba limax) were identified in 49 surface water samples collected from four provinces in Iran, Guilan, Mazandaran (north of Iran), Alborz and Tehran (capital), as reported by Mahmoudi et al. (2015). Acanthamoeba species were found to be in 18 out of 49 water samples by using primers JDP1 and JDP2 by PCR. Sequence analysis revealed that 16 samples belonged to the T4 genotype and two samples to the T5 genotype. Niyyati et al. (2009) and Rahdar et al. (2013) previously reported T2, T4 and T6 genotypes in environmental samples from rivers, stagnant water and soil in Iran. Acanthamoeba keratitis has been identified in the world as the T4 genotype according to (SSU) rDNA sequencing results (Visvesvara et al. 2007; Booton et al. 2009; Grun et al. 2014).

The nucleotide sequence similarity and evolutionary distance relationship of the (SSU) rDNA gene region of Acanthamoeba genotypes from the GenBank and investigated water samples is shown in Table 3. Briefly, the minimum genetic distance between Haplotype I and A. triangularis was calculated to be 0.002. The highest genetic distance between Haplotype I and A. tubiashi was found to be 0.0991. The nucleotide sequence similarity between Haplotype I and A. triangularis was 99.7%. The nucleotide sequence similarity and genetic divergence between Haplotype II and A. polyphaga was 99.9% and 0.001, respectively (Table 3).

Haplotype III is represented as a sister group to Acanthamoeba spp. with 99.8% nucleotide sequence similarity and the pairwise genetic divergence was 0.003. The minimum genetic distance between Haplotype IV and A. lenticulata was calculated to be 0.002. The highest genetic distance between Haplotype IV and A. tubiashi was found to be 0.081. The nucleotide sequence similarity between Haplotype IV and A. lenticulata was 99.8%.

There was a similarity in just NJ phylogeny trees with 52% bootstrap between Haplotype IV and A. lenticulata (Figure 6). At this point, our research showed similarity to studies (Walochnik et al. 2000a; Schroeder et al. 2001; Ledee et al. 2003; Booton et al. 2005) which reported that the most common Acanthamoeba genotype in the world was T4. At the same time, the identification of the Acanthamoeba T4 and T5 genotypes in our study resembled the study previously reported by Mahmoudi et al. (2015). In addition, there were seven out of nine Acanthamoeba positive samples for T4 and two samples for T15 genotype in 20 public pools in Hungary (Kiss et al. 2014); in Pakistan, T4 and T15 genotypes in water and soil samples (Tanveer et al. 2015) and in Taiwan, T4 and T2 genotypes were found to be dominant genotypes (Kao et al. 2015). In the following years, also reported were: T4 in seven, T3 in one, T5 in two out of samples taken from natural water resources in North East Thailand (Thammaratana et al. 2016); T1, T2, T4, T5, T6 and T11 in the samples collected from environmental and tap water in Uganda (Sente et al. 2016); T4, T3 in the samples taken from environmental waters in Egypt (Tawfeek et al. 2016); T4, T10 and T11 in water taken from a hospital (surgical services, intensive care unit, operating room and water storage tanks) in Tunisia (Trabelsi et al. 2016); and T4, T5 and T16 in tap water samples and soil samples collected from China's Yanji area (Xuan et al. 2017).

Worldwide, Acanthamoeba keratitis is the most common pathology caused by the T4 genotype. The present study suggests the possibility of AK as a risk for people and animals living in the region due to human and animal contact with such waters and due to the most common occurrence of the T4 genotype in the investigated waters, as found in our study.

Genotyping studies are answering questions such as the difference in the specificity of Acanthamoeba spp., which causes infection in different organs such as the brain and eye, and whether there are differences in the pathogenicity of the species. Genotyping of Acanthamoeba species isolated from environmental sources may lead to an important pathway for AK diagnosis in the investigated region as both the T4 and T5 are genotypes are responsible for infections in the eye. The presence of both genotypes in the region suggests that individuals with a compromised immune system or others, such as contact lens users, should be very careful using these waters as they are a risk to public health. The spread of Acanthamoeba disease may increase as contact lenses are sold by non-experts which may place emphasis on the risk to users. Health authorities and employees need to raise awareness of the public, especially young people, who use lenses for cosmetic purposes, highlighting the pathogenesis of Acanthamoeba and the ocular risk source from this parasite.

The present study emphasizes the importance of the Acanthamoeba species in the investigated area and that these waters are also found to be used in agriculture and animal husbandry, increasing the risk of contamination. Since such surface, recreational waters and spring waters are used by many people, the public should be alerted to the risk of potential waterborne pathogens and water quality should be monitored periodically in these areas. The results suggest that Acanthamoeba strains belonging to T4 genotype, which are related to AK cases, are present in surface water samples examined in Samsun province. The diversity of Acanthamoeba strains, their prevalence, genotyping and pathogenic potential studies should be performed for larger-scale water samples and in different sites that have not been studied previously in the Black Sea region. Acanthamoeba diagnosis is under-reported, and only available in a few hospitals of large cities but not in rural areas where knowledge about personal hygiene and periodic checks of the water quality are rather limited. There is an urgent need to alert the public health authorities and draw the attention of Acanthamoeba to clinicians and public healthcare providers in rural and urbanized areas in Turkey.

ACKNOWLEDGEMENTS

We thank Ordu University Scientific Research Projects Coordination Unit (BAP- BY1719) for supporting part of this work.

REFERENCES

Akısu
C.
Baka
M.
Durak
I.
Orhan
V.
1999
A case of Acanthamoeba keratitis; light and electron microscope findings
.
Acta Parasitologica Turcica
23
,
340
342
.
Akyol
N.
Ascı
Z.
Kukner
S.
1996
Acanthamoeba keratitis: the first reported case from Turkey
.
Ophthalmic Practice
2
,
46
48
.
Alves
J. M.
Gusmao
C. X.
Teixeira
M. M.
Freitas
D.
Foronda
A.
Affonso
H. T.
2000
Random amplified polymorphic DNA profiles as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba
.
Brazilian Journal of Medical and Biological Research
33
,
19
26
.
Barete
S.
Combes
A.
De Jonckheere
J. F.
Datry
A.
Varnous
S.
Martinez
V.
Ptacek
S. G.
Caumes
E.
Capron
F.
Francès
C.
Gibert
C.
Chosidow
O.
2007
Fatal disseminated Acanthamoeba lenticulata infection in a heart transplant patient
.
Emerging Infectious Diseases Journal
13
,
736
738
.
Booton
G. C.
Visvesvara
G. S.
Byers
T. J.
Kelly
D. J.
Fuerst
P. A.
2005
Identification and distribution of Acanthamoeba species genotypes associated with non-keratitis infections
.
Journal of Clinical Microbiology
43
,
1689
1693
.
Bouheraoua
N.
Labbé
A.
Chaumeil
C.
Liang
Q.
Laroche
L.
Borderie
V.
2014
Acanthamoeba keratitis
.
Journal Français D'Ophtalmologie
37
,
640
652
.
Chappell
C. L.
Wright
J. A.
Coletta
M.
Newsome
A. L.
2001
Standardized method of measuring Acanthamoeba in sera from healthy human subjects
.
Clinical and Diagnostic Laboratory Immunology
8
,
724
730
.
Demirci
G.
Ay
G. M.
Karabas
L. V.
Altintas
O.
Tamer
G. S.
Cağlar
Y.
2006
Acanthamoeba keratitis in a 5-year-old boy without a history of contact lens usage
.
Cornea
25
(
3
),
356
368
.
Di Cave
D.
Monno
R.
Bottalico
P.
Guerriero
S.
D'Amelio
S.
D'Orazi
C.
Berrilli
F.
2009
Acanthamoeba T4 and T15 genotypes associated with keratitis infections in Italy
.
European Journal of Clinical Microbiology
28
,
607
612
.
Edagawa
A.
Kimura
A.
Kawabuchi-Kurata
T.
Kusuhara
Y.
Karanis
P.
2009
Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan
.
Parasitology Research
105
,
1109
1117
.
Ertabaklar
H.
Türk
M.
Dayanır
V.
Ertug
S.
Walochnik
J.
2007
Acanthamoeba keratitis due to Acanthamoeba genotype T4 in a non-contact-lens wearer in Turkey
.
Parasitology Research
100
,
241
246
.
Ertabaklar
H.
Dayanır
V.
Apaydın
P.
Ertuğ
S.
Walochnik
J.
2009
Acanthamoeba keratitis
.
Turkish Journal of Parasitology
33
,
283
285
.
Fuerst
P. A.
Booton
G. C.
Crary
M.
2015
Phylogenetic analysis and the evolution of the 18S rRNA gene typing system of Acanthamoeba
.
Journal of Eukaryotic Microbiology
62
,
69
84
.
Gast
R. J.
Ledee
D. R.
Fuerst
P. A.
Byers
T. J.
1996
Subgenus systematics of Acanthamoeba: four nuclear 18S rDNA sequence types
.
Journal of Eukaryotic Microbiology
43
,
498
504
.
Hewett
M. K.
Robinson
B. S.
Monis
P. T.
Saint
C. P.
2003
Identification of a new Acanthamoeba 18S rRNA gene sequence type, corresponding to the species Acanthamoeba jacobsi Sawyer, Nerad and Visvesvara, (Lobosea: Acanthamoebidae)
.
Acta Protozoologica
42
,
325
329
.
Horn
M.
Fritsche
T. R.
Gautom
R. K.
Schleifer
K. H.
Wagner
M.
1999
Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus
.
Environmental Microbiology
1
,
357
367
.
Juárez
M. M.
Tártara
L. I.
Cid
A. G.
Real
J. P.
Bermúdez
J. M.
Rajal
V. B.
Palma
S. D.
2018
Acanthamoeba in the eye, can the parasite hide even more? Latest developments on the disease
.
Contact Lens & Anterior Eye
41
,
245
251
.
Kao
P. M.
Hsu
B. M.
Chen
N. H.
Huang
K. H.
Huang
C. C.
Ji
D. D.
Chen
J. S.
Lin
W. C.
Huang
S. W.
Chiu
Y. C.
2012
Molecular detection and comparison of Acanthamoeba genotypes in different functions of watersheds in Taiwan
.
Environmental Monitoring and Assessment
184
,
4335
4344
.
Kao
P. M.
Hsu
B. M.
Hsu
T. K.
Liu
J. H.
Chang
H. Y.
Ji
W. T.
Tzeng
K. J.
Huang
S. W.
Huang
Y. L.
2015
Seasonal distribution of potentially pathogenic Acanthamoeba species from drinking water reservoirs in Taiwan
.
Environmental Science and Pollution Research
22
,
3766
3773
.
Khan
N. A.
2006
Acanthamoeba: biology and increasing importance in human health
.
FEMS Microbiological Reviews
30
,
564
595
.
Khan
N. A.
Naveed
A.
Timothy
A. P.
2002
Molecular tools for speciation and epidemiological studies of Acanthamoeba
.
Current Microbiology
44
,
444
449
.
Kılıc
A.
Tanyuksel
M.
Sissons
J.
2004
Isolation of Acanthamoeba isolates belonging to T2, T3, T4, and T7 genotypes from environmental samples in Ankara, Turkey
.
Acta Parasitologica
49
,
246
252
.
Ledee
D. R.
Hay
J.
Byers
T. J.
Seal
D. V.
Kirkness
C. M.
1996
Acanthamoeba griffini molecular characterization of a new corneal pathogen
.
Investigative Ophthalmology & Visual Science
37
,
544
550
.
Ledee
D. R.
Vooton
G. C.
Awwad
M. H.
2003
Advantages of using mitochondrial 16S rDNA sequences to classify clinical isolates of Acanthamoeba
.
Ophthalmology & Visual Science
44
,
1142
1149
.
Ledee
D. R.
Iovieno
A.
Miller
D.
Mandal
N.
Diaz
M.
Fell
J.
2009
Molecular identification of T4 and T5 genotypes in Acanthamoeba keratitis patients
.
Journal of Clinical Microbiology
47
,
1485
1462
.
Lorenzo-Morales
J.
Lindo
J. F.
Martinez
E.
Calder
D.
Figueruelo
E.
Valladares
B.
Ortega-Rivas
A.
2005
Pathogenic Acanthamoeba strains from water sources in Jamaica, West Indies
.
Annals of Tropical Medicine and Parasitology
99
,
751
758
.
Mahmoudi
M. R.
Taghipour
N.
Eftekhar
M.
Haghighi
A.
Karanis
P.
2012
Isolation of Acanthamoeba species in surface waters of Gilan province-north of Iran
.
Parasitology Research
110
,
473
477
.
Maschio
V. J.
Chies
F.
Carlesso
A. M.
Carvalho
A.
Rosa
S. P.
Van Der Sand
S. T.
Rott
M. B.
2015
Acanthamoeba T4, T5 and T11 isolated from mineral water bottles in southern Brazil
.
Current Microbiology
70
,
6
9
.
Niyyati
M.
Lorenzo-Morales
J.
Rezaie
S.
Rahimi
F.
Mohebali
M.
Maghsood
A. H.
Motevalli-Haghi
A.
Martín-Navarro
C. M.
Farnia
S.
Valladares
B.
Rezaeian
M.
2009
Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran
.
Experimental Parasitology
121
,
242
245
.
Ozkoc
S.
Tuncay
S.
Delibas
S. B.
Akisu
C.
Ozbek
Z.
Durak
I.
Walochnik
J.
2008
Identification of Acanthamoeba genotype T4 and Paravahlkampfia spp. from two clinical samples
.
Journal of Medical Microbiology
57
,
392
396
.
Page
F. C.
1991
Nackte Rhizopoda
. In:
Protozoenfauna
(
Matthes
D.
, ed.).
Band 2. G. Fischer
,
Stuttgart, New York
, pp.
3
145
.
Pussard
M.
Pons
R.
1977
Morphologie de la paroi kystique et taxonomie du genre Acanthamoeba. (Morphology of the cystic wall and taxonomy of the genus Acanthamoeba)
.
Protistologica
13
,
557
598
.
Rahdar
M.
Niyyati
M.
Salehi
M.
Feghhi
M.
Makvandi
M.
Pourmehdi
M.
Farnia
S.
2013
Isolation and genotyping of Acanthamoeba strains from environmental sources in Ahvaz City, Khuzestan Province, Southern Iran
.
Iranian Journal of Parasitology
7
,
22
26
.
Schroeder
J. M.
Booton
G. C.
Hay
J.
Niszl
I. A.
Seal
D. V.
Markus
M. B.
Fuerst
P. A.
Byers
T. J.
2001
Use of sub-genic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of Acanthamoeba from humans with keratitis and from sewage sludge
.
Journal of Clinical Microbiology
39
,
1903
1911
.
Sente
C.
Erume
J.
Naigaga
I.
Magambo
P. K.
Ochwo
S.
Mulindwa
J.
Namara
B. G.
Charles
D. K.
Sebyatika
G.
Muwonge
K.
Ocaido
M.
2016
Occurrence and genetic characterisation of Acanthamoeba spp. from environmental and domestic water sources in Queen Elizabeth Protected Area, Uganda
.
Parasites and Vectors
9
,
127
.
Shin
H. J.
Im
K. I.
2004
Pathogenic free-living amoebae in Korea
.
Korean Journal of Parasitology
42
,
93
119
.
Stothard
D. R.
Schroeder-Diedrich
J. M.
Awwad
M. H.
Gast
R. J.
Ledee
D. R.
Rodriguez-Zaragoza
S.
Dean
C. L.
Fuerst
P. A.
Byers
T. J.
1998
The evolutionary history of the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types
.
Journal of Eukaryotic Microbiology
45
,
45
54
.
Tawfeek
G. M.
Bishara
S. A.
Sarhan
R. M.
ElShabrawi Taher
E.
El-Saady Khayyal
A.
2016
Genotypic, physiological, and biochemical characterization of potentially pathogenic Acanthamoeba isolated from the environment in Cairo, Egypt
.
Parasitology Research
115
,
1871
1881
.
Trabelsi
H.
Dendana
F.
Neji
S.
Sellami
H.
Cheikhrouhou
F.
Makni
F.
Ayadi
A.
2016
Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia
.
Parasitology Research
115
,
431
435
.
Walochnik
J.
Haller-Schober
E. M.
Kölli
H.
Picher
O.
Obwaller
A.
Aspöck
H.
2000a
Discrimination between clinically relevant and nonrelevant Acanthamoeba strains isolated from contact lens-wearing keratitis patients in Austria
.
Journal of Clinical Microbiology
38
,
3932
3936
.
Walochnik
J.
Aichelburg
A.
Assadian
O.
Steuer
A.
Visvesvara
G.
Vetter
N.
2008
Granulomatous amoebic encephalitis caused by Acanthamoeba amoebae of genotype T2 in a human immunodeficiency virus negative patient
.
Journal of Clinical Microbiology
46
,
338
340
.
Xuan
Y.
Shen
Y.
Ge
Y.
Yan
G.
Zheng
S.
2017
Isolation and identification of Acanthamoeba strains from soil and tap water in Yanji, China
.
Environmental Health and Preventive Medicine
22
,
58
.