Surface water contamination with Acanthamoeba spp. in Ilam city, Iran

Free-living amoebae are opportunistic protozoans distributed globally in a wide range of environmental sources including water, soil, air, hot springs, and urban water supply systems (Siddiqui & Khan 2012; Padzik et al. 2019). Acanthamoeba belongs to the Acanthamoebidae family, exhibiting both trophozoite and cystic forms throughout its life cycle (Visvesvara 1991; Panjwani 2010; Eslamirad et al. 2020). Characterized by their high resistance to various environmental factors, they maintain their pathogenicity even after prolonged exposure to temperature changes, pH variations, and chlorination (Mosayebi et al. 2014; Behniafar et al. 2015). Granulomatous encephalitis, Acanthamoeba keratitis, and cutaneous Acanthamoeba represent the most significant clinical manifestations resulting from infestation by the pathogenic genotypes of the Acanthamoeba parasite in humans (Nazar et al. 2011). In addition, complications such as sinus infections, dermatitis, and the development of severe skin ulcers are associated with this protozoan, particularly in individuals with compromised immune systems (Niyyati et al. 2016). Morphological taxonomic studies have identified 24 species of Acanthamoeba. However, the rRNA gene sequence confirms only 20 genotypes (T1–T20) (Hosseinbigi et al. 2012), with the T3 and T4 genotypes exhibiting the highest pathogenicity in their hosts (Booton et al. 2005). Genotyping of clinical isolates of Acanthamoeba has revealed that the T4 genotype is accountable for 90% of amoebic keratitis. The varying pathogenicity observed among different Acanthamoeba genotypes and their roles in disease prognosis, particularly in cases of amoebic keratitis, underscores the significance of Acanthamoeba genotyping in the diagnostic and treatment processes (Khan et al. 2002). In light of the amoeba's close association with human habitation, its ecological characteristics, widespread presence in the environment, and the rising frequency of immune diseases, as well as common recreational activities such as swimming in seasonal water, utilizing surface water in the region for drinking, washing, and bathing, it becomes crucial to consider the multitude of surface water sources. These sources may potentially be contaminated by resilient cysts of the parasite, carried through sand and dust storms originating from neighboring countries (Iraq, Kuwait, and Saudi Arabia). The assessment of the Acanthamoeba fauna in the environment holds significant implications for public health. This study, utilizing morphological analysis and sequencing of the 18S rRNA gene through the PCR method, aims to investigate the prevalence of Acanthamoeba species in the surface waters of Ilam City, located in the western part of Iran.

All containers were shaken for 3 min to ensure homogeneity of the contents, and water filtration was performed using a nitrocellulose filter with a pore diameter of 45 μm, coupled with a vacuum pump.

After filtration, the nitrocellulose filters were placed upside down on the surface of a sterile non-nutrient agar medium coated with Escherichia coli bacteria. To mitigate culture media evaporation, the plates were sealed with parafilm and incubated at 25 °C for 24 h. Subsequently, the filters were removed from the culture medium to prevent the proliferation of fungal saprophytes. Microscopic examination for the presence of Acanthamoeba trophozoites, particularly along the culture medium margins, was conducted 4 days after parasite culture initiation. Given the slow growth of certain Acanthamoeba species, the cultured media were incubated for one month, with microscopic assessments performed at various intervals.

To eliminate potential contamination by bacteria, fungi, and free worm larvae, amoeba cloning was executed. Under a light microscope, Acanthamoeba cysts devoid of bacterial or fungal colonies in their vicinity were carefully excised using a sterile scalpel and subsequently transferred to a fresh culture medium. Daily observations were made on the new plates to monitor amoeba growth and detect the emergence of contaminants. Through several successive passages in culture media, amoeba plates obtained were ultimately free from any contamination.

The isolation of Acanthamoeba from the culture medium involved the following steps: 5 mL of phosphate-buffered saline (PBS) buffer was added to the plates, and the medium was sectioned using a sterile scalpel blade. The plate surface was washed 10 times with a sterile Pasteur pipette until cysts and trophozoites floated in the PBS. The resulting mixture was transferred to 1.5 mL microtubes and centrifuged at 200g for 10 min. Subsequently, the supernatant was discarded, and the sediments were washed three times with PBS to eliminate any agar residue. The isolated parasites were then collected and stored at −20 °C for morphological and molecular identification.

A volume of Giemsa stain and 40 volumes of the isolated sample were mixed and stained for 12 min. Following this, wet smear slides were prepared and examined under a light microscope ×10 and ×40 lenses.

Genomic DNA was extracted using the Favor Prep DNA Isolation Mini Kit according to the manufacturer's instruction. Primers (18S rRNA) were designed by Schroeder and produced a 500-bp fragment (Table 2). PCR was performed in a final reaction volume of 25 μl using 12.5 μl of Taq Master Mix (Yekta Tajhiz Azma Company, Iran; which contained 0.2 mM dNTPs, 0.1units/μl Ampliqon Taq DNA polymerase), 1.5 μl of each primer (10 pmol), and 4 μl of DNA template, bringing the final volume with the sterile distilled water (5.5 μl) to 25 μl. The Bio RAD (New York, USA) thermocycler was used for the PCR reactions. The temperature profile was an initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 60 s, annealing at 57 °C for 60 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 5 min. The PCR product was electrophoresed on 1.5% agarose gel stained with DNA-safe stain dye (Pishgam Biotech Co., Tehran, Iran) for 45 min at 100 V and visualized using a UV transilluminator.

The PCR products were sequenced in one direction using a forward primer (Takapozist Co., Tehran, Iran). Subsequently, the generated sequences were manually organized and compared to analogous Acanthamoeba sequences in the Gene Bank utilizing the Chromas software version 2.5.0. Alignments were performed with similar sequences derived from Acanthamoeba species isolated from various regions. Distances between the sequences were calculated, and a phylogenetic tree was constructed using MEGA version 7 software, and the maximum likelihood method with a generation of 1,000 bootstrapped replicates.

All culture plates were meticulously examined daily under a light microscope. Clear spots detected on the culture medium were interpreted as the presence of trophozoites. Employing the Giemsa stain method, Acanthamoeba endocysts appeared stained blue, while ectocysts exhibited a very pale to colorless appearance. On the fifth day after culture initiation, 17 out of the 50 plates (34%) tested positive. All cysts displayed a distinct wall with spherical, polyhedral, and star-shaped endocysts (Figure 2). Due to the slow growth rate of certain Acanthamoeba species, two plates required an additional 3 weeks to yield positive results. A total of 19 samples (38%) tested positive within a 4-week culture period on non-nutrient agar media (Table 1).

The findings of this investigation provide evidence for the presence of Acanthamoeba in surface water within Ilam, western Iran. These results align with prior studies that have successfully isolated this amoeba from various regions across Iran (Nazar et al. 2011). Additional studies have reported higher frequencies of Acanthamoeba contamination in the hot springs of Ilam and stagnant surface waters of Qazvin compared to the outcomes observed in our study (Hosseinbigi et al. 2012; Niyyati et al. 2016). The variation in Acanthamoeba prevalence among the studied areas can likely be attributed to distinctions in the sources of water samples and the environmental conditions specific to each region. When comparing molecular and microscopic examination methods, molecular techniques demonstrated greater sensitivity and specificity in identifying Acanthamoeba genotypes (Khan et al. 2002; Booton et al. 2005), and the current study reinforces these data, as alignment of the 18S rRNA gene sequences obtained herein with those registered in the GenBank reveals that the T4 genotype of Acanthamoeba predominates in the water resources of Ilam. Consistent with previous research, it is well established that the T4 genotype serves as the most prevalent causative agent of both amoebic keratitis and granulomatous Acanthamoeba encephalitis (Zhang et al. 2004; Sheng et al. 2009; Liang et al. 2010), and this matter might be related to the higher prevalence (Shoff et al. 2008), virulence, and potential transmissibility associated with the T4 genotype in the environment (Khan 2006). The T4 genotype has a higher tendency to bind to the host cells compared to the T2, T3, and T11 genotypes (Alsam et al. 2003). The urban pools yielded the most pathogenic genotypes in this study, specifically T4 and T2. This underscores the significance of determining genotypes in both clinical and environmental samples. In the context of Iran, T4 emerges as the dominant genotype, alongside reports of T2, T3, T5, T6, T9, T11, T13, and T15 genotypes (Badirzadeh et al. 2011; Spotin et al. 2017). Previous investigations have highlighted a close relationship between the T11 and T4 genotypes and supported and validated this established association (Walochnik et al. 2000). The prevalence of the free-living amoebae in the treated water network is observed to be lower compared to untreated surface water. The chlorination system is effective in destroying amoebic trophozoites. However, it is noteworthy that certain studies have reported instances of contamination in treated and chlorinated water with this protozoan (Latifi et al. 2015). Hence, the proximity of surface water to potential pollution sources likely constitutes a major factor contributing to the higher contamination levels with the Acanthamoeba parasite, distinguishing it from other water sources. Considering that the Acanthamoeba parasite is highly pathogenic in all climatic regions and can cause many pathogenic lesions including eye, skin, lung, mucosal lesions, etc., in all people of the society, the results of this study can be a serious warning for health officials and clinicians to design a plan to prevent and treat this disease and be used as a reliable document by healthcare decision-makers.

In conclusion, this study revealed a widespread prevalence of Acanthamoeba in various water resources. The analysis of the 18S rRNA gene sequence further confirmed the presence of the highly pathogenic T4 genotype, demonstrating its highest frequency in the surface water of Ilam. These findings underscore the importance of continued monitoring and awareness of Acanthamoeba contamination in water sources for effective public health management.

The authors sincerely thank all professors and students of the Department of Parasitology, School of Allied Medical Sciences, Ilam University of Medical Sciences, Iran.

This study was funded by Ilam University of Medical Sciences, Ilam, Iran, under grant number 982004/41.

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

The authors declare there is no conflict.