Genotyping determination of Acanthamoeba strains: an original study and a systematic review in Iran

Acanthamoeba is a cosmopolitan free-living amoeba (FLA) and an opportunistic protozoan parasite which can be found in a variety of environments, including saltwater and freshwater, soil, and decaying plants as well as the skin, cornea, lung, and brain of infected humans (Tsvetkova et al. 2004; Khan 2006; Rezaeian et al. 2008; Bagheri et al. 2010; Magnet et al. 2012). Two forms of the parasite, representing distinct stages of its life cycle, have been identified: a vegetative trophozoite and a cyst with a double-layer coat, which is resistant to disinfectants and dryness and can survive for many years in the environment (Schuster & Visvesvara 2004). Contaminated environments are considered as major resources for transmission of Acanthamoeba spp. to humans (Zolfaghari Emameh et al. 2016).

Acanthamoeba is very important due to serious and sometimes fatal infections in humans and animals (Marciano-Cabral & Cabral 2003; Schuster & Visvesvara 2004; Visvesvara et al. 2007). Some Acanthamoeba spp. cause two major diseases: amoebic keratitis (AK) of the eye in contact lens wearers and granulomatous amoebic encephalitis in immune-deficient subjects (Prasher et al. 2004; Huang & Hsu 2010; Lass et al. 2014). This amoeba has been classified based on the 18S rRNA (ribosomal RNA) sequence of the trophozoite into 20 different genotypes (T1–T20) (Di Filippo et al. 2015). Among these, T3, T4, and T5 are the most common pathogenic genotypes. Members belonging to the T4 genotype are the most common causative agents of infections (Maghsood et al. 2005; Khan 2006), and more than 90% of AK cases are due to T3 and T4 Acanthamoeba (Schuster & Visvesvara 2004). In addition, T2 and T4 genotypes have been reported in Iran's water (Maghsood et al. 2005; Niyyati et al. 2009b).

Unfortunately, in recent years, there has been an upward trend in the number of immunocompromised individuals (Shafiei et al. 2011; Ahmadpour et al. 2014; Rahimi et al. 2015; Abdollahian et al. 2017). In addition, the high potential for resistance and the ability of the parasite to adapt to a variety of environmental conditions presents optimal conditions for the increased incidence of infection and the emergence of this opportunistic parasite as a major health concern. Considering all the abovementioned threats and their potential impacts on human health, early detection of pathogenic Acanthamoeba in aquatic environments plays a crucial role in the control and prevention of the disease (Zolfaghari Emameh et al. 2018).

Zahedan is located in Sistan and Baluchistan province, in the southeast of Iran, and literature review indicates that, so far, no study has been performed to clarify the situation of Acanthamoeba in this area. Therefore, the objective of this study was to investigate and determine the genotype of potentially pathogenic Acanthamoeba spp. from different water resources in Zahedan. In addition, we systematically reviewed genotyping studies of Acanthamoeba in Iran during 2005–2018.

In the present descriptive cross-sectional study, a total of 50 water samples were randomly collected from February to December of 2016 from different resources in Zahedan, where human activity and colonization were identified. Sampling was carried out from tap water, stagnant water of public squares, parks, swimming pools, agricultural channels (each of them had nine samples), and hospitals (five samples). Each sample contains 1,000 mL of water that was collected inside a sterile and contamination-free container and transferred to Parasitology and Mycology Laboratory in the Zahedan University of Medical Sciences, Zahedan, Iran. Samples were filtered using a vacuum pump through a sterile multi-pore nitrocellulose Durapore Membrane filter (pore size, 0.45 μm). The sediments on the membranes were conveyed in the top-down direction in 1.5% non-nutrient agar medium containing amoeba page saline (pH 7.2–7.4) that was overlaid by heat-killed Escherichia coli (Niyyati et al. 2009b). Plates were incubated (Cole-Parmer Digital Incubator) at 28–30 °C for 7 days. Then, incubating plates were observed daily for outgrowth evaluation of Acanthamoeba cysts and trophozoites using an inverted microscope (Zeiss), for up to 1 month. The plates which showed growth of amoeba were transferred to new plates and subcultures were made to receive a plate without any microorganism (fungal or bacteria) contamination. Finally, the plates were subjected to the following procedures (Niyyati et al. 2016).

Positive Acanthamoeba plates were gently scraped and washed with 5 mL of phosphate-buffered saline (Shokri et al. 2016). The solution was transferred to sterile tubes and after centrifugation 5,000 g for 2 min, the sediments having amoebas were collected. The Dyna Bio kit (Takapou Zist, Iran) was used for DNA extraction to extract the total genomic DNA from the collected debris according to the manufacturer's instructions.

A pair of primers, including JDP1: 5′-GGCCCAGATCGTTTACCGTGAA-3′ as forward and JDP2: 5′-TCTCACAAGCTGCTAGGGGAGTCA-3′ as the reverse, were used to amplify a fragment of approximately 500 bp in Acanthamoeba genus-specific 18S rRNA gene (Schroeder et al. 2001). The polymerase chain reaction (PCR) mixtures (25 μL) contained 3 μL of extracted DNA solution, 1.25 units of Taq DNA polymerase (Cinnagen, Iran), 2.5 μL of 10× PCR buffer, 2 mM of MgCl₂, 50 pmol/25 mL reaction mixtures of both forward and reverse primers, 0.4 mM of dNTPs, and 15.5 mL of distilled water. PCR amplification was run on the thermocycler (Eppendorf Mastercycler Gradient) according to the following details: 94°C (3 min), [94°C (35 s), 57°C (45 s), 72°C (1 min): 33 cycles], 72°C (5 min), each of the PCR cycles were followed by cooling at 4°C. PCR products of positive isolates were purified from the agarose gel using a PCR purification kit (Bioneer, Korea) and sequenced in both forward and reverse directions (Applied Biosystems, DNA Analyzers Sequencing, Bioneer, Korea, Sanger method) using the same primers that were used in the PCR (Huang & Hsu 2010).

A total of 20 (18S rRNA) sequences were aligned using Clustal Omega (Sievers et al. 2011) and the obtained alignment was trimmed on each end to the location where nucleotides were present for at least 75% of entries. With the subsequent edited aligned sequences, a maximum likelihood-based phylogenetic analysis was performed using the IQTree package (Minh et al. 2013; Nguyen et al. 2015). The best model for analysis was predicted to be TVMe + G4 with Model Finder (Kalyaanamoorthy et al. 2017), and the analysis was run with 100,000 bootstrap replicates of the SH-like approximate likelihood ratio test and 100,000 ultrafast bootstrap replicates (parameters set to ‘-alrt 100,000 -bb 100,000 -nt AUTO’ and all other options run as default). The resulting consensus tree had a final log-likelihood value of −2,571.798, and the internal nodes represent the percentage of replicates supporting the configuration at the node. The final figure was visualized using the Python-based ETE3 toolkit library (Huerta-Cepas et al. 2016), with representations of sequence data accompanying each leaf of the phylogenetic tree. In this analysis, Balamuthia mandrillaris (a single-celled FLA) 18S rRNA sequence was used as the out group.

Along with genotyping of the samples as a major goal of the current study, we systematically reviewed studies of Acanthamoeba spp. genotyping from 2005 to 2018 in Iran. A literature search of the current systematic review was carried out through various methods including database search, data collection, and data assessment. The database search was performed using the Medical Subject Heading (MeSH) of specified search terms, alone or in combination, within the following databases: Science Direct, ISI Web of Science, MEDLINE, EBSCO, Scopus, and Google Scholar. The MeSH terms used ‘Acanthamoeba’, ‘Free-living amoeba’, ‘Immunosuppressed individuals’, ‘Keratitis’, ‘Environment’, ‘Water’, ‘Isolation’, and ‘Genotyping’. References of identified articles were also similarly checked. From those identified studies conducted during 2005–2018 (14 year period), the collected information consists of data such as the year of publication, first author, study areas, sample type, and genotype. Data collection was limited to the study cases that were published in English and/or Persian. It is worth noting that repetitive studies were excluded.

A total of 17 (34%) out of 50 water samples were identified as positive for Acanthamoeba spp., through both culture and PCR methods (Figure 1). All 17 positive samples without any contamination were successfully sequenced after two times gel purification and then well typed. The prevalence and frequency of the detected Acanthamoeba were summarized in Table 1. In addition, the results of nucleotide sequencing indicated that 15 samples (88.23%) belonged to the T4 genotype and two samples (11.74%) belonged to the T5 genotype.

The process of study design for systematic review was depicted in a flowchart in Figure 2. A list of 39 published studies (2005–2018), identifying the genotypes of Acanthamoeba spp. determined in what sample type and in which defined areas of Iran, is presented in Table 2. The different genotypes detected in the descending order of number of studies were identified: T4 (35 studies), T5 (19 studies) and T3 (11 studies), T11 (8 studies), T2 (6 studies), T15 (2 studies), and T1, T6, T7, T9 and T13 (1 study) isolates were the different genotypes detected from different areas of Iran. Phylogenetic analysis results are presented in Figure 3, which depicts a phylogenetic tree of 17 Acanthamoeba 18S rRNA sequences from water samples of different areas of Iran including Zahedan.

Phylogenetic analysis was performed in 18S rRNA sequences. A. castellanii and A. griffini predominantly colocalize with T4 genotypes, while A. lenticulata groups with the only two T5 genotypes. Due to the likely incomplete nature of a number of the sequences used in this analysis, it would be premature to make any final conclusions on the evolutionary relationships between these organisms. Specifically, the single T3 sequence (KT985968.1) appears to be significantly truncated in the 3′-end, as is the sequence for A. lenticulata, though to a lesser extent. As a result, the overall tree topology is weakly supported at some nodes, yet leaf pairing is strongly supported in some cases and can point to likely associations between known and unknown genotypes. Regardless, this provides a preliminary glance at the evolutionary relationships of these medically important organisms using the most current sequence data.

In the present study, the prevalence and genotype of Acanthamoeba spp. was determined in different water resources of Zahedan in the Sistan and Baluchistan province of southeast Iran. Among the water samples, 34% were positive in culture and based on 18S rRNA sequencing, all of Acanthamoeba isolates were categorized as T4 and T5, with potentially pathogenic amoebae which are the predominant environmental genotype. Since Acanthamoeba spp. are resistant to extreme environmental physical conditions such as temperature, pH, and exposure to various chemicals can increase both their pathogenicity as well as endosymbiont virulence bacteria (Khan 2006). The isolates which belong to the T4 genotype have shown more affinity for host cells and subsequently greater cytotoxicity in comparison with T2, T3, and T11 genotypes (Memari et al. 2017).

Based on 18S rRNA sequencing results, most of Acanthamoeba which were isolated from keratitis in the world have been categorized as the T4 genotype (Visvesvara et al. 2007). Several genotypes of Acanthamoeba including T2, T3, T4, and T11 cause AK. T4 is the most predominant genotype in Iran, which leads to AK (Maghsood et al. 2005; Niyyati et al. 2009b; Hajialilo et al. 2015, 2016).

Several studies have reviewed the presence of different genotypes of Acanthamoeba spp. in a variety of water resources in different parts of Iran and have reported eight pathogenic and nonpathogenic genotypes of Acanthamoeba spp., including T2–T5, T7, T11, T13, and T15.

In some studies including Mazandaran (Dodangeh et al. 2018), West Azerbaijan (Haniloo et al. 2017), and Ardabil provinces (Badirzadeh et al. 2011), T4 (100%) was found in water sources as the predominant genotype of Acanthamoeba. Also in Semnan province on drinking water (Javanmard et al. 2017) and in East Azerbaijan province on surface resting waters (Fallah et al. 2017), T5 (100%) was the only found genotype, while T4 and T5 genotypes were detected in some studies by Niyyati et al. and Mahmoudi et al. in Guilan, Mazandaran, Alborz, West Azerbaijan, and Tehran provinces on different water sources (Nazar et al. 2011; Mahmoudi et al. 2015; Niyyati et al. 2015a, 2015b; Khezri et al. 2016). Based on our results, the prevalence of T4 as A. castellanii was higher than T5 as A. griffini which is in accordance with previous findings that suggested T4 as the most prevalent genotype not only in Iran but also in the world.

Moreover, in other studies, other genotypes of Acanthamoeba were detected in Iran. T2, T7, and T11 genotypes were reported from Isfahan province (Golestani et al. 2018), and T3 was found from stagnant water of Zabol in Sistan and Baluchistan province (Aghajani et al. 2016). Considering the fact that both Zabol and Zahedan are located in Sistan and Baluchistan province, T3 genotype was not detected in our samples.

T2 as A. palestinensis was reported from Qazvin, Mazandaran, and Ilam provinces (Hooshyar et al. 2013; Niyyati et al. 2016; Shokri et al. 2016). The T2 genotype has a pathogenic potential and it can grow at high temperature and osmolality and has been reported as a causative agent of keratitis in Iran and worldwide (Maghsood et al. 2005; Niyyati et al. 2016; Golestani et al. 2018).

The T15 genotype is frequently isolated from warm water such as hot springs (Huang & Hsu 2010), but the T15 genotype was detected in two occasions from cold rivers of Tehran (Niyyati et al. 2012) and surface and tap water of Fars province (Armand et al. 2016).

In our study, the highest and lowest prevalence rate of the amoeba in the examined samples was observed in public square (55.5%) and pipe (11.1%) water samples, respectively. But 40% of the hospital drinking water samples were positive for Acanthamoeba. A similar investigation on tap water at hospitals in 13 cities of Iran detected 48% Acanthamoeba spp. in the examined samples that our findings are in agreement with this study (Bagheri et al. 2010). In fact, the isolation of potentially pathogenic Acanthamoeba spp. from either hospital tap water or hospital wards can put patients particularly immunodeficient ones in danger of acquiring the infection.

In the current study, Acanthamoeba was detected in 33.3% of swimming pool samples. In a study in Zabol from Sistan and Baluchistan province in the southeast of Iran, 93 samples of pools and stagnant water were evaluated for Acanthamoeba. Overall, 40.8% of the samples were positive that the following genotypes were detected: T4 (89.47%), T5 (7.9%), and T3 (2.63%) (Aghajani et al. 2016). Another study by Maghsood et al. (2005) on 12 pool and waterfall samples showed Acanthamoeba T4 (33.3%) and T2 (58.3%) genotypes. Although the result of our study was almost similar to the findings of the two abovementioned surveys, T2 and T3 genotypes were not observed in our swimming pool specimens. An acceptable justification for the contamination of swimming pool waters might be associated with cyst form which has a double-layer coat that is resistant to disinfectants despite normal chlorination/disinfection procedures. Therefore, we could consider this fact as an alarm and precaution for soft contact lens wearers. They should not wear lenses while they swim in pools due to the probable danger of acquiring Acanthamoebiasis corneal infection.

The T4 genotype is the most common genotype detected in water samples and patients in Iran. Moreover, many studies have been undertaken to identify Acanthamoeba in natural resources and patients in other countries. The results show that the T4 genotype is the dominant genotype in the examined specimens (Zhao et al. 2010; Coskun et al. 2013; Lass et al. 2014). Even though in many studies, T4 is the most common genotype, other genotypes including T3, T5, T11, and T13 are common genotypes in drinking water (tank and tap water) (Behniafar et al. 2015), T2, T3, T5, and T15 in surface and groundwater (Mahmoudi et al. 2012; Niyyati et al. 2012; Rahdar et al. 2012; Behniafar et al. 2015), T3 in recreational water (pools, springs, and the sea) (Badirzadeh et al. 2011; Solgi et al. 2012; Behniafar et al. 2015), and T2, T3, T5, and T15 in stagnant water (ponds, fountains, and streams) (Hooshyar et al. 2013; Armand et al. 2016).

Overall, the occurrence of T4 and T5 genotypes of Acanthamoeba as potentially pathogenic agents in different water resources in Zahedan of Iran reflects an urgent need to improve water treatment procedures in the city to prevent Acanthamoeba-related infections, especially for immunodeficient patients.

Authors from the current study draw the conclusion that Acanthamoeba spp. is present in the water sources of Zahedan and the predominant genotype is T4, which is pathogenic particularly for high-risk individuals. Furthermore, the current systematic review elucidates that the most common genotypes isolated from clinical and environmental samples from Iran were T4 and T5. Phylogenetic analysis on 18S rRNA sequences indicates that A. castellanii and A. griffini predominantly colocalize with the T4 genotype.

This study was a part of the MSc thesis of Maryam Mansouri Nia and supported financially by the Cellular and Molecular Research Center of the Zahedan University of Medical Sciences, Zahedan, Iran (Grant No. 7139).

The authors declare that they have no conflicts of interest.

This manuscript does not report on animal or human data.

This manuscript does not contain individual-level data or identifying information for any person.