Detection of Acanthamoeba spp. in groundwater sources in a rural area in the Philippines

Free-living amoebas (FLAs) are a group of ubiquitous protozoans that thrive in several environmental matrices. According to the World Health Organization (2003), four genera of FLA, namely Acanthamoeba spp., Naegleria spp., Balamuthia mandrillaris, and Sappinia spp. are considered medically important due to FLA-related infections (Visvesvara et al. 2007; Angelici et al. 2021). Among the FLAs, Acanthamoeba spp. is known to be widely distributed in the environment. These protozoans can cause both non-fatal and fatal health conditions (Balczun & Scheid 2017; Milanez et al. 2020). Several genotypes are known to cause granulomatous amoebic encephalitis (GAE), Acanthamoeba keratitis (AK), and disseminated cutaneous infections in both immunocompetent and immunocompromised hosts (Khan 2006). Acanthamoeba spp. has been reported to be isolated from a variety of natural and man-made environments such as soil (Cruz & Rivera 2014; Xuan et al. 2017; Meighani et al. 2018), marine water (Hussein et al. 2021), lakes (Ballares et al. 2020; Milanez et al. 2022), and swimming pools (Esboei et al. 2020). Due to their ability to thrive in a plethora of environments, the isolation of these protozoans in major aquatic sources is important to mitigate potential infections due to anthropogenic activities (Masangkay et al. 2022). Groundwater is a significant source of potable drinking water worldwide (Carrard et al. 2019). The Centers for Disease Control and Prevention (CDC) reported that groundwater, such as those coming from wells, is naturally filtered to some extent (CDC 2022). The lack of knowledge on the maintenance of groundwater sources and their potential biological contaminants may pose a public health threat due to its possible exploitation and contamination of soil-thriving microorganisms that may cause morbid to mortal health conditions (Giordano 2009). This has been validated by recent reports on the simultaneous contamination of protozoan pathogens in well water sources in the Southeast Asian region (Masangkay et al. 2022), and of tap water sources as well (Coşkun et al. 2013; Üstüntürk-Onan & Walochnik 2018).

In a study conducted in 10 Southeast Asian countries, groundwater is preferred over surface water for drinking and domestic use (Carrard et al. 2019). In the Philippines, especially in rural areas, groundwater is the main water source for domestic and irrigation purposes (Inson et al. 2021). Groundwater is the most extracted raw material globally and is prone to pollution/contamination (Alsalme et al. 2021). Due to the vast availability and accessibility of groundwater, it is overexploited in many parts of the world (Molle et al. 2018). Although there are monitoring guidelines currently being implemented to check the quality of the groundwater sources in terms of pH, temperature, electrical conductivity, total dissolved solids, salinity, and static water level (Oppus et al. 2020), there appears to be no established protocol for protozoans such as Acanthamoeba species. This study aimed to isolate and identify Acanthamoeba spp. from groundwater sources in the province of Masinloc, Zambales, Philippines.

A total of 54 (three samples from each water pump source) groundwater samples from six selected barangays were collected in 250-mL sterile polyethylene containers (Table 1). The samples were transported to the laboratory at the Department of Medical Technology, Far Eastern University Manila, Philippines for processing. Samples were vacuum filtered through a 1.2-μm pore size glass microfiber filter. Filters containing sediments were placed sediment-side down on non-nutrient agar (NNA) lawned with live Escherichia coli and incubated aerobically at 33 °C for 14 days. NNA plates were examined microscopically for amoebic growth using a compound microscope (Nikon Eclipse E100) under 400× magnification. The agar surface was scanned for the presence of cysts and trophozoites. Positive plates were subcultured to obtain homogenous growth following previously established protocols (Milanez et al. 2020). Briefly, approximately 1 cm × 1 cm of agar block from an identified area with abundant growth was cut using a sterile scalpel blade and then placed culture-side down onto a fresh NNA plate lawned with live E. coli and was incubated and examined under similar conditions as before. These steps were repeated until a homogenous culture was obtained.

Suspected Acanthamoeba spp. cysts and trophozoites were harvested from culture plates by flooding the agar surface with cold phosphate-buffered saline solution (pH 7) and gently scraped with a sterile scalpel blade (Milanez et al. 2019). The fluid was then aspirated and transferred to microcentrifuge tubes, and DNA was extracted using QIAamp^® DNA mini stool kit, following the manufacturer's instructions. Primer pairs JDP1 5′-GGCCCAGATCGTTTACCGTGAA-3′ and JDP2 5′-TCT CAAGCTGCTAGGGGAGTCA-3′ were used to amplify the ASA1.S1 region of the Acanthamoeba partial sequence (Booton et al. 2005). Thermal cycling conditions were set at initial denaturation of 95 °C for 7 min, 40 cycles at 95 °C for 1 min, annealing temperature at 55 °C for 1 min, extension at 72 °C for 2 min, and a final extension of 72 °C for 15 min (Booton et al. 2005). PCR products were visualized by performing 1.5% agarose gel electrophoresis stained with ethidium bromide. To further detect other FLAs in our samples, gel electrophoresis-negative samples were subjected to another round of PCR using universal eukaryotic primers Euk A (5′-AACCTGGTTGATCCTGCCAGT-3′) and Euk B (5′-TGATCCTTCTGCAGGTTCACCTAC-3′). Thermal cycling conditions for this primer set were set at 94 °C for 5 min initial denaturation, 30 cycles of denaturation at 94 °C for 45 s, annealing temperature of 52 °C for 1 min, extension at 72 °C for 2 min, and a final extension of 72 °C for 7 min (Medlin et al. 1988) and visualized through gel electrophoresis as before. Acanthamoeba spp. genotypes were identified through DNA sequencing and phylogenetic analysis. PCR amplicons were sent to a commercial sequencing company (Macrogen, Seoul, South Korea). Sequences were aligned using ClustalW of BioEdit with careful visual consideration of gaps and ambiguous sequences and were deposited in GenBank. Phylogenetic analysis of isolate sequences along with reference strains from GenBank was performed using the maximum likelihood (ML) tree constructed with 1,000 replicates using MEGA 11.

Thermo-tolerance assay on sequenced isolates was performed following previously established protocols (Walochnik et al. 2000). Briefly, 1 μl of cystic stage suspension of isolates was cultured at the center of freshly prepared NNA plates lawned with live E. coli and incubated in varying temperatures, i.e. 30, 37, and 40 °C. Amoebic growth was observed for 48 h with emphasis on the outward migration of trophozoites from the point of inoculation.

Acanthamoeba spp. causes two important health conditions: non-fatal but morbid ocular infection (AK) and fatal encephalitis (GAE). AK is commonly observed in contact lens wearers (CLWs), with the usual cause of infection being unhygienic cleaning and storage of the lenses (Shimmura-Tomita et al. 2018). Nevertheless, non-contact lens wearers (NCLWs) may also contract AK through the exposure of the eye to contaminated water, or trauma with a contaminated object (Singh et al. 2020). This was the suspected cause of the AK case of a non-contact lens wearer in the Philippines who used tap water to regularly cleanse the face (Buerano et al. 2014). This case provided evidence of the potential health hazards of using water contaminated with Acanthamoeba spp. for domestic purposes.

Although there have been no known reports of contracting fatal Acanthamoeba infection through groundwater ingestion, there is an increasing number of AK cases reported recently in different parts of the world (Alver et al. 2020; Bagga et al. 2020; Satitpitakul et al. 2021). Also, there was a peculiar demonstration of Acanthamoeba spp. in urine samples of critically ill patients (Santos et al. 2009). The capacity of Acanthamoeba spp. to trigger an immune response in the gastrointestinal tract (GIT) is an argument that is yet to be resolved. Although this is the case, it is important to consider that FLAs have been isolated from the animal intestines and bat guano (Mulec et al. 2016; Milanez et al. 2017). More importantly, Acanthamoeba spp. is known to harbor a plethora of microorganisms and safely host them within the cytoplasm of cystic stages (Scheid 2018). This mechanism enables the latter to evade the immune responses of the host, allowing the process of disease causation. Thus, the capacity of FLAs as Trojan horses to introduce human pathogenic endocytobionts in the GIT and other anatomic sites requires consideration.

In our study, we have identified two potentially pathogenic Acanthamoeba spp. isolates, namely IN2.1 (OP411026) and IN2.2 (OP411028) confirmed through an in vitro thermo-tolerance assay. This suggests that groundwater contaminated with Acanthamoeba spp. may cause FLA-related infections in humans. This is the first report of A. astronyxis in the Philippines. However, its pathogenic potential remains elusive to the present date (Callicott 1968; Martinez 1980).

The findings of several Acanthamoeba spp. in groundwater sources in the country necessitate the revisiting of the local national water standards to include the screening of pathogenic protozoans from freshwater sources for human use. Also, the Philippines, at present, has no case definition for FLA-related infections (Milanez et al. 2022). This hints at the lack of reports of FLA-related infections in the country as such cases may either be misdiagnosed, underreported, or neglected.

This is the first report of A. astronyxis genotype T7 in the Philippines. The thermo-tolerance characteristics exhibited by local Acanthamoeba spp. from groundwater sources is evidence of a pathogenic potential for humans. To date, the detection of pathogenic protozoans in freshwater sources for human use and a case definition for FLA-related infections is non-existent in the country. It is necessary to craft country policies that address FLA-related health concerns. The role of Acanthamoeba to trigger an immune response in the gastrointestinal tract and other anatomic sites is still unclear but requires consideration as it can introduce pathogenic endocytobionts.

The authors would like to thank the Department of Medical Technology, Far Eastern University-Manila for facilities assistance. Gratitude is extended to Mr Joseph Dionisio and Ms Dess Policarpio for providing technical support.

All relevant data are included in the paper.

The authors declare there is no conflict of interest.