Genotyping and phylogenetic analysis of free-living amoeba (Acanthamoeba and Naegleria) in treated and untreated water in the northeastern provinces of Iran

Different genera of free-living amoebae (FLA), including Balamuthia, Acanthamoeba, Naegleria, Sappinia and Vermamoeba (Hartmannella), can cause disease in humans (Qvarnstrom et al. 2009).

FLA is widely distributed in the environment and can be transmitted to humans (Teixeira et al. 2009; Bagheri et al. 2010).

FLA is also known as amphizoic amoebae because of their ability to live in a host and in different sources, namely soil, air, and water (i.e. sea, lakes, ponds, pools, dams, hospital drinking water sources, and treated water) (Ahmadi et al. 2021). Amoebic cysts are very resistant to temperature variation, pH, chlorine, detergents and common disinfectants. Several species of FLA can cause accidental, opportunistic or non-opportunistic infections (Niyyati et al. 2015). These parasites can be found in hemodialysis units, hospitals, and contact eye lens and cleaning solutions along with bacteria or yeast (Greub & Raoult 2003; Niyyati et al. 2017).

FLA belonging to the genus Acanthamoeba are the causative agents of keratitis in healthy individuals, often among people who wear contact lenses. In opportunistic infections, Acanthamoeba species can cause skin infections, fatal granulomatous amebic encephalitis (GAE), and pneumonitis (Marciano-Cabral et al. 2000). The phylogenetic analyses based on the nuclear 18S ribosomal RNA (rRNA) gene have recently distinguished at least 22 genotypes (T1–T22) Acanthamoeb (Fuerst & Booton 2020). Clinical isolates and environmental samples in Iran and other parts of the world are mainly infected with the T4 genotype (Niyyati et al. 2015).

Acanthamoeba parasites are increasing significantly in Iran (Pazoki et al. 2020). Despite this fact, a number of regional researchers have focused their molecular discoveries on the discrimination of Acanthamoeba sp. (Karamati et al. 2016; Spotin et al. 2017). In different parts of Iran, due to the clinical importance of Acanthambiasis, genetic analysis of the parasite can be useful to target endangered populations and explain how the parasite spreads in different geographical areas (Spotin et al. 2017).

Meanwhile, more than 40 species of Naegleria have been identified (Spotin et al. 2017); only Naegleria fowleri, commonly found in freshwater lakes, rivers and hot springs, is responsible for a rare and fatal brain infection called primary amebic meningoencephalitis (PAM). Naegleria australiensis and Naegleria italica cause disease in laboratory animals (Di Filippo et al. 2017).

Acanthamoeba keratitis and PAM have been documented in Iran (Esboei et al. 2020; Ahmadi et al. 2021). Although the number of reported cases is low, FLA must not be overlooked as a possible cause of disease. The fact that reported cases are infrequent could be due to the lack of familiarity of physicians and laboratory personnel incorrectly identifying FLA infections (Movahedi et al. 2012; Mohammadpour et al. 2018).

Limited studies are available about FLA prevalence in the various sources of water samples in northern and northeastern Iran. Therefore, this study aimed to investigate the prevalence and genotype distribution of Acanthamoeba and Naegleria in drinking water and surface water samples by microscopic identification, staining, and molecular methods.

This information would be helpful for early detection of possible pathogenic FLA contamination in various water sources and further assess the risks of human contact with FLA. Humans can also be exposed to FLA through contact with contaminated water or through open wounds; hence, this study is crucial to fill the gaps of knowledge.

First, a review of sampling areas and diagnostic methods used in this study is provided (Figure 1).

This cross-sectional review study was conducted in the three north and northeast Iran provinces (Golestan, North Khorasan and Semnan). It was approved by the Mashhad University of Medical Sciences ethics committee (Ethics number: IR.MUMS.MEDICAL.REC.1398.865).

Golestan province with coordinate's 36.8393°N 54.4444°E is located along the Caspian Sea. Moreover, it has a temperate climate. The annual rainfall is similar to other Mediterranean climates. Thousands of domestic and foreign tourists visited the province due to the rivers, hot springs and beautiful beaches. In contrast, North Khorasan province with coordinates of 37.4761°N 57.3317°E and Semnan in the geographical position of 35.5769°N 53.3953°E are semi-desert areas with lower average rainfall (Figure 2).

A total of 60 samples of treated or untreated water were collected at various locations in North Khorasan (20), Golestan (20), and Semnan (20). Each sample was collected in two 200 mL sterile tubes to detect FLA by filtration and centrifugation methods. All samples were transferred to the Parasitology Research Laboratory, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, and processed the soonest time possible.

Firstly, one of the 200 mL tubes of water sample was passed through the cellulose nitrate membrane with 0.22 μm pore size. The filter was then placed on a non-nutrient agar plate coated with Escherichia coli (NNA-E. coli). In order to compare the filtration with the centrifuge method, all samples were also centrifuged in a volume of 200 mL at 2,000 rpm for 15 min. A few drops of resuspended samples were spread on non-nutrient agar plates coated with a layer of E. coli. Then, the media were incubated at room temperature (24–28 °C) for four weeks.

All the media were checked daily using an inverted microscope (Olympus, CKX53) maximum of 30 days. Giemsa and methylene blue-stained used a suspension of 30 μL from the plates containing the amoebae, and observed with a light microscope (Olympus BX51, Japan). Morphological features identified the differences between amoeba cysts and trophozoites (Page 1967; Page 1988).

Acanthamoeba spp. cysts and trophozoites were removed from the culture plates by adding PBS and scraping of the media. The eluate was transferred into 1.5 mL sterile tubes. According to the manufacturer's instructions, DNA was then extracted using a commercial DNA kit (Blood Mini Kit, Bioneer, Korea). A nanodrop device (Thermo ND-ONE, USA) was used to determine the quality of DNA and finally stored at −20 °C until further analysis.

All PCR amplifications were carried out in 20 μL reactions containing 1 μL of genomic DNA template, 10 μL of Amplicon Taq DNA Polymerase Master Mix RED, 0.25 μL each primer (25 μM stock), and 8.5 μL of de-ionized water (ddH20).

A 450 bp fragment of 18S rRNA region of Acanthamoeba species was amplified using genus-specific forward primers JDP1 (5′-GGCCCAGATCGTTACCGTGAA-3′) and reverse JDP2 (5′-TCTCACAAGCTGCTAGGGAGTCA) (Booton et al. 2002). With the following parameters: the initial denaturation at 94 °C for 3 min, 35 cycles of 94 °C for 35 seconds, 56 °C for 45 seconds, 72 °C for 1 min, followed by a final expansion at 72 °C for 7 min.

Naegleria species was detected by region-specific amplification ITS (ITS1, 5.8S, and ITS2) using species-specific forward primers: ITS1 forward primer (5-GAA CCT GCG TAG GGA TCA TTT-3) and ITS2 reverse primer (5-TTT CTT TTC CTC CCC TTA TTA-3) (Pélandakis et al. 2000). The amplification was performed with an initial denaturation at 94 °C for 2 min, 35 cycles of 94 °C for 35 seconds, 56 °C for 60 seconds and 72 °C for 1 min followed by a final extension at 72 °C for 7 min.

PCR products with positive and negative control of Acanthamoeba and Naegleria were evaluated by electrophoresis on 1.5% agarose gel at 100 voltage for 45 min. Also, a 100 bp DNA ladder (Yekta Tajhiz Azma, YT8503, Iran) was used to compare the expected amplitude of the gel. Subsequently, it was detected under ultraviolet (UV) light and images were taken (PhotoDoc-It gel imaging system).

Favourable PCR products with good quality were sequenced with an ABI 310 automated fluorescent sequencing system using the primers above. The similarity between FLA sequences was found with other FLA species by the Basic Local Alignment Search Tool (BLAST) software provided by the National Center for Biotechnology Information (NCBI) in GenBank.

The sequences were analyzed by CLC Genomics Workbench 12 software and edited manually. Maximum likelihood reconstructions were performed using the phylogenetic program MEGA software version X. The trees were generated using the neighbour-joining method with the Kimura 2-parameter model. Bootstrap values estimated with 1,000 replicate data sets were indicated at each node. In addition, the samples in our study were compared with similar isolates in Iran and other parts of the world (Tamura et al. 2011).

Thirty-five percent (21/60) of water samples, including a sample from North Khorasan (7), Golestan (9), and Semnan (5), were positive for Acanthamoeba by culture (as shown in Tables 1 and 2). Similar results were also obtained using molecular analysis.

Meanwhile, 26% (16/60) of samples, including samples from North Khorasan (5), Golestan (7) and Semnan (4), were positive for Naegleria by culture and PCR analysis (as described in Tables 1 and 2). Only seven Acanthamoeba and five Naegleria favourable PCR products were sent for sequencing.

The filtration method and centrifugation method showed the same results to detect FLA in media.

In culture, trophozoites and cyst stages were typically produced after 2–3 and 5–6 days of cultivation, respectively. The morphology of all isolates is distinguishable based on the motile stage of trophozoites/flagella.

Acanthamoeba cysts were identified by their star shape and irregular trophozoites with a superior design of acanthapodia in multidirectional motion.

In Figure 3(d), Naegleria trophozoites are cylindrical, and the cyst is circular with a relatively large central nucleus (Figure 3(c), 3(g) and 3(h)).

The figure also shows Acanthamoeba trophozoites with Acanthapodia legs (Figure 3(b)), and a star-shaped cyst (Figure 3(a), 3(e) and 3(f)).

In order to identify the morphology of pathogenic amoebae, Giemsa and methylene blue staining methods were compared to non-stained slides. In both staining methods, two separate layers of cysts (ectocyst and endocyst) were observed for Acanthamoeba isolates. Naegleria cysts were also observed in the form of a circle with a central nucleus in both stainings (Figure 3).

Using Giemsa and methylene blue staining, the parasite cysts were observed well-contrasted against the background and stained purple and dark blue, respectively. However, there is no distinct demarcation between the nucleus and the cytoplasm (Figure 3(e) and 3(f)).

Meanwhile, for the Naegleria cyst stage, trophozoites and flagellation were observed.

Amplification of the 18S rRNA region of Acanthamoeba produced a 450 bp PCR product. All isolates of Acanthamoeba belonged to the T4 genotype. Five isolates (MW040579, MW040577, MW040576, MW040580, and MW040582) were identified as A. castellanii, and two isolates (MW040581, MW040579) were identified as A. polyphaga (Table 3, Figure 4).

A PCR product length of about 500 bp was observed for Naegleria after amplification of the ITS region. The sequencing analysis showed that Naegleria isolates (MW042257 and MW042256) were 100% similar to N. americana, and two isolates (MW042259 and MW042258) detected in Semnan province have 96% and 93% similarity to N. dobsoni and N. pagei, respectively; another isolate (MW042260) was similar to N. australiensis (Table 3, Figure 5).

The ability of free-living amoebae (FLA) to cause severe infections in humans and their essential role in ecosystems has led to more researches on FLA (Abdul Majid et al. 2017). Previously, important studies have been reported on Acanthamoeba genotypes isolated from water in Iran (Rezaeian et al. 2008; Niyyati et al. 2015; Ahmadi et al. 2021). However, there is little information about this parasite in northeastern Iran. This study aimed to determine Naegleria and Acanthamoeba species’ existence and molecular identity in treated water and untreated water samples from three northeastern provinces of Iran, especially North Khorasan, Semnan and Gorgan.

With an increasing number of FLA cases, several studies have been conducted on various water sources as a habitat to gather epidemiological data that would help reduce levels of FLA transmission worldwide (Behniafar et al. 2015).

Accordingly, a high percentage of Acanthamoeba and Naegleria species in water resources have been reported in recent Iranian studies, showing a significant increase for FLA in water resources in Iran, similar to the work done by Rezaian et al. In Tehran, 46.25% of the samples contained Acanthamoeba (Rezaeian et al. 2008). In a study in Shiraz, out of 82 water samples collected from different parts of the city, 48 (58.53%) samples were positive for FLA. Acanthamoeba was observed in 39 cases (47.56%). The results (62.96%) showed the T4 genotype (Armand et al. 2016b).

In another study, Niyyati et al. found that 38.2 per cent of 55 tap water samples collected from Kish Island in Iran were contaminated with FLA (Niyyati et al. 2015).

For the first time in Iran, Movahedi et al. reported (PAM) caused by N. fowleri. (Movahedi et al. 2012).

Najafpour et al. found several species of Naegleria in the water of parks’ pools in Mashhad, Iran, including N. Americana, N. clarki, N. andersoni, N. fultoni, N. carteri, and N. pagei, but no N. fowleri (Najafpoor et al. 2018).

Previous studies in Iran showed that the T4 genotype of Acanthamoeba is the dominant type in patients with Acanthamoeba keratitis (Maghsood et al. 2005; Niyyati et al. 2009).

The results showed that the pathogenic Acanthamoeba and non-pathogenic Naegleria species by both microscopy and PCR techniques in North Khorasan, Semnan and Golestan for the first time in Iran.

Due to the recent advances in molecular detection, conventional PCR has been developed as a reliable method for routine screening of FLA in environmental samples. This is sufficient to distinguish between species of different organisms (Abdul Majid et al. 2017).

The classification status of amoebae isolated from water sources is shown in (Table 3) using BLAST software. In this classification, five samples were identified as A. castellanii (MW040577, MW040579, MW040580, MW040582, and MW040576), while two were confirmed as A. polyphaga (MW040578, MW040581).

Only one family of amoebae pathogenic for humans belonging to the genera of Acanthamoeba (Acanthamoebidae) was found following PCR and sequencing.

The pathogenic strain of Acanthamoeba was observed in 35% of water resources in the study area. The highest incidence of FLA was observed in Golestan province (45% Acanthamoeba and 35% Naegleria) then in north Khorasan FLA (35% Acanthamoeba and 25% Naegleria) and Semnan (25% Acanthamoeba and 20% Naegleria).

Based on the 18S rRNA sequence, most Acanthamoeba isolated in this study has been identified as T4 genotype, known as the most important causative agent of amoebic keratitis and GAE in the world (Visvesvara et al. 2007; Booton et al. 2009; Grün et al. 2014).

Since the provinces under study have touristic attractions, this isolation in many drinking water sources indicates a severe public health threat for indigenous peoples and tourists. Moreover, the risk of infection with FLA in individuals with immunocompromised states should be considered in these provinces. GAE is frequently related to human immunodeficiency virus and organ transplant. Although, it has also been reported from immunocompetent people (Basher et al. 2018).

The phylogenetic analysis also confirms the correct placement of the samples in the T4 genotype (Figure 4). It clearly shows the difference between different genotypes of Acanthamoeba. Many previous studies have similarly reported that T4 is the most common genotype among clinical and environmental samples in Iran and other parts of the world (Izumiyama et al. 2003; Niyyati et al. 2009; Lorenzo-Morales et al. 2015; Armand et al. 2016b).

Based on one step PCR assay with specific primers targeting the ITS region and 18S rRNA, there was no positive sample for N. fowleri. This may result in the proliferation activity of N. fowleri at high temperatures (up to 45 °C) (Izumiyama et al. 2003).

Although some cases of primary amoebic meningoencephalitis (PAM) caused by N. fowleri has been reported in Iran, the diagnosis of PAM should never be ignored because of its severity and fatality. The lack of skills and knowledge of physicians and laboratory personnel regarding the correct diagnosis of this amoeba can cause rare reported cases (Movahedi et al. 2012; Mohammadpour et al. 2018). Naegleria sequence analyses were related to N. americana (MW042256, MW042257), N. australiensis (MW042260), N. dobsoni (MW042259), and N. pagei (MW042258) species (Table 3). The phylogenetic analysis also confirms the correct placement of Naegleria specimens (Figure 5). It clearly shows the difference between the various species of Naegleria. However, these species of Naegleria are non-pathogenic to humans, which indicates the importance of poor quality of drinking water and surface water as a severe risk to human health.

Based on the centrifuge methods’ findings, in the absence of facilities such as filtration for researchers, the centrifuge method is a promising tool to detect FLA.

In this study, various staining techniques were used in comparison with non-staining samples (Figure 3). We found that Giemsa staining, commonly used for blood parasites, provided a good contrast for identifying Acanthamoeba and Naegleria cysts. In addition, methylene blue staining had good quality in detecting Acanthamoeba and Naegleria cysts and distinguishing the outer and the inner layer of the cysts.

A previous study by Abdul Majid et al. (2017) stated that all stages (cysts and trophozoites) of FLA were defined better in cellular features and organs using Giemsa staining (Abdul Majid et al. 2017).

The findings showed that methylene blue and Giemsa staining could identify and diagnose FLA for clinical epidemiology and public health purposes.

However, the classification of FLA based on morphological criteria is insufficient and requires more precise identification, such as molecular PCR methods. Also, we can claim that this is probably the first time that methylene blue staining has been used to identify Acanthamoeba.

Despite conventional water treatment methods (filtration and disinfection) being used throughout Iran, treated waters were contaminated with FLA in all three provinces. It seems the FLA cyst stage is resistant to biocides and chlorination.

This finding increased knowledge about the distribution of FLA isolates in North Khorasan, Golestan and Semnan. Local authorities should not overlook the high prevalence of amoebae in the provinces. Appropriate precautions must be taken to ensure high water quality. It is suggested to add some other disinfectants such as chlorinated cyanuric acid, Baquacil and chlorine dioxide to water to control the growth of FLA (Marciano-Cabral 1988; Abd Ghani et al. 2010). Water used for recreational purposes (i.e. river, sea, dam) should also be closely monitored because untreated water may contain large amounts of FLA to transmit infections to humans (Ma et al. 1990; Szénási et al. 1998; Niyyati et al. 2015; Esboei et al. 2020).

Therefore, our results, in line with the results of other Iranian researchers who have studied in other cities of Iran (Rezaeian et al. 2008; Niyyati et al. 2015; Armand et al. 2016a; Esboei et al. 2020; Ahmadi et al. 2021) show the abundance of FLA in various water sources in Iran.

The results show the significance of an urgent need to seriously consider FLA where human activity has been observed in understudy water resources. Polluted surface waters investigated can act as potential sources for the distribution and transmission of Acanthamoeba, especially recreational waters and hot springs. They need periodic monitoring, particularly during the summer when millions of people use this surface water. Future research on FLA occurrence in treated and untreated water with larger sample sizes should be taken.

According to the results of this study and the pathogenic role of these protozoa in immunocompromised patients and the ability of these microorganisms to carry other pathogens such as Legionella, further studies are needed. Importantly, to provide a safe drinking water system in hospitals, you must be extremely careful to prevent nosocomial infections in immunocompromised patients, such as HIV patients.

As a result, the present study indicates the high presence of Acanthamoeba and Naegleria in water resources, including drinking water in northeastern and northern Iran.

This paper was extracted from a student thesis. It was approved by the Research Committee of the Faculty of Medicine and financially supported by the Research Deputy (No. 980212) of Mashhad University of Medical Sciences, Mashhad, Iran.

Conceptualization: MZ HM.

Data curation: YS OA.

Formal analysis: YS OA BRHF NK.

Funding acquisition: MZ.

Investigation: YS OA.

Methodology: YS EM AP FB AB.

Project administration: MZ HM.

Resources: MZ HM.

Software: RFA YS.

Supervision: MZ HM.

Validation: MZ HM.

Writing ± original draft: YS OA.

Writing ± review & editing: YS OA BRHF MZ HM.

All relevant data are included in the paper or its Supplementary Information.

The Ethics Committee of Mashhad University of Medical Sciences in Iran approved this study.

This work with (Grant No: 980212) was supported by Mashhad University of Medical Sciences.