ABSTRACT
Acanthamoeba is an opportunistic, free-living amoeba ubiquitous in the environment. Despite reports of its wide distribution in the Philippines’ freshwater resources, more information on the long-term viability of the Acanthamoeba species is needed. This study aimed to define the long-term viability of Acanthamoeba species in unpreserved environmental freshwater samples after 3 years of storage at room temperature. Stored water samples from 15 study sites were filtered through a 1.2-μm pore size glass microfiber filter, cultured in non-nutrient agar (NNA) lawned with Escherichia coli, and observed for amoebic growth for 14 days using light microscopy. Isolates from positive NNA culture were subjected to polymerase chain reaction (PCR) using JDP1 and JDP2 Acanthamoeba-specific primers. The study site positivity was 33% (5/15). Acanthamoeba genotype T4 and Acanthamoeba lenticulata were isolated from Luzon; Acanthamoeba divionensis was isolated from Visayas; and Acanthamoeba sp. and genotype T20 were isolated from Mindanao. The long-term viability of Acanthamoeba species is an added risk factor for the sustained contamination of aquatic resources and other sample matrices. This heightens the risk of transmission to humans and animals. This study demonstrated that water samples fated for Acanthamoeba studies can be stored unpreserved at room temperature for several years.
HIGHLIGHTS
Acanthamoeba spp. remain viable for 3 years in freshwater samples.
Pathogenic species and genotypes exhibit long-term viability.
Long-term viability is an added risk factor.
Storage conditions are uncomplicated.
INTRODUCTION
Acanthamoeba species is an opportunistic, free-living amoeba (FLA) broadly distributed in the environment and contaminates contact lens fluids, tap water, pool water, hospital room dust, environmental fresh and brackish water resources, and wastewater (Milanez et al. 2022; Mirabedini et al. 2022). The life cycle of Acanthamoeba includes an inactive cyst stage and an active and feeding trophozoite stage (Siddiqui & Khan 2012). Acanthamoeba spp. trophozoites can host endosymbionts composed of various pathogenic bacteria, fungi, and viruses that potentially enhance their virulence (Hamilton et al. 2016; Mungroo et al. 2021).
Acanthamoeba primarily affects older people, children, and immunocompromised individuals and rarely affects immunocompetent individuals (Hamilton et al. 2016). Acanthamoeba may enter through the eyes, nose, or broken skin and cause Acanthamoeba keratitis (AK), a painful eye inflammation affecting the corneal tissue and commonly associated with persons wearing contact lenses and granulomatous amoebic encephalitis (GAE), a neurological infection with a high incidence of mortality (Lorenzo-Morales et al. 2015; Parija et al. 2015; Rayamajhee et al. 2021; Centers for Disease Control and Prevention (CDC) 2022).
Although there have been numerous reports regarding Acanthamoeba spp. and studies on agents against Acanthamoeba in ASEAN countries, the available information regarding Acanthamoeba infections remains notably limited (Bunsuwansakul et al. 2019). Acanthamoeba infections, particularly AK cases, have been reported to increase worldwide, but their global incidence is yet to be resolved (Zhang et al. 2023).
Meanwhile, research on FLA in the Philippines is still in its early phase due to the lack of established case definitions for FLA-related infections within the country's surveillance programs (Masangkay et al. 2022). Freshwater contamination has emerged as one of the country's growing environmental concerns. This issue negatively impacts local economies and their suitability for human use and consumption, contributing to the transmission of waterborne parasitic protozoans (Andrews 2018; Milanez et al. 2019). A nationwide epidemiological survey in the Philippines reported the predominant Acanthamoeba genotypes, specifically T3, T4, T5, T7, T11, and T15, in significant freshwater resources utilized by the populace (Milanez et al. 2022).
Existing literature showcases Acanthamoeba viability in controlled environments (Mazur et al. 1995). However, literature on its potential long-term viability is unavailable when left unpreserved in its source matrix. This study aimed to explore Acanthamoeba viability in unpreserved water samples stored for 3 years at room temperature.
MATERIALS AND METHODS
Study sites and samples
Taal Lake, the third-largest lake in the Philippines, has seen significant growth in its aquaculture sector over the years. What was once a thriving fishery has now expanded into a major aquaculture hub. This evolution, coupled with its roles as a recreational destination, a tourist attraction, a navigational aid, and a food source for ducks, underscores the lake's multifaceted importance. Moreover, it is a vital water source for Tagaytay and the nearby agricultural fields (Mutia et al. 2018).
Pantabangan Dam is an earth fill dam in Pantabangan, Nueva Ecija. The multipurpose embankment dam is a water source for irrigation and hydroelectric power. In addition, its reservoir, Pantabangan Lake, provides flood control (Masangkay et al. 2020a).
Laguna de Bay, the country's largest lake, is significant in the economic, political, and socio-cultural landscape. Its importance is underscored by its role as a major source of freshwater fish and its use for irrigation, livestock, and waste management. This understanding fosters a deep respect for its significance (Iizuka et al. 2017; Masangkay et al. 2020a).
Lake Buhi is a lake in Buhi, Camarines Sur in Southern Luzon. The lake and its surroundings are habitats for many land, water, and flying animals. Lake Buhi is home to the Sinarapan (Mistichthys luzonensis), an endemic edible fish deemed the smallest commercially harvested fish in the world (Masangkay et al. 2020a).
The San Roque Multipurpose Project is an embankment dam on the Agno River of Pangasinan in Luzon Island. Considered the largest dam in the country, it provides power benefits, flood control, irrigation, and enhanced water quality to its surrounding areas in Luzon (Masangkay et al. 2020a).
Ambuklao Dam is in Brgy. Ambuclao, Bokod, Benguet province in Northern Luzon. It is part of a hydroelectric facility capable of producing 105 MW of electricity for the Luzon grid. The Agno River springs from Mount Data and is the dam's primary water source (Masangkay et al. 2020a).
The northern part of Pacijan Island, one of the minor islands in Cebu Province's Camotes Islands chain, is home to Lake Danao Sakanao and is considered one of the island's most visited tourist destinations (Masangkay et al. 2020a).
Lake Danao, with its unique guitar or violin-like shape, is a significant water source in Ormoc, Leyte. It was initially named Lake Imelda and was renamed Lake Danao National Park under R.A. No. 7586 in 1998. This lake is not just a tourist attraction but also a source of potable water for the seven towns of Leyte, underscoring its vital role in the region (Masangkay et al. 2020a).
Lake Bito, nestled in Leyte and surrounded by picturesque rice fields and mountains, is a potential ecotourism hotspot. The operation of tilapia fish pens and freshwater collection is a significant source of income for its residents. The local government's vision for Lake Bito as a thriving ecotourism destination is a promising prospect that can enhance the local economy (Masangkay et al. 2020a).
Lake Sebu is one of the most important watersheds in the nation, as South Cotabato and Sultan Kudarat significantly rely on it for irrigation. Additionally, it is situated in the ancestral lands of the T'boli and Ubo tribes. The Department of Tourism now promotes the lake as one of Mindanao's top ecotourism destinations (Masangkay et al. 2020a).
Pulangi River, a key headwater of the Rio Grande de Mindanao, is a lifeline for Mindanao. As the second-largest river system in the Philippines, it provides essential services such as hydropower, irrigation, and fisheries. Originating in Barangay Kalabugao, Impasugong, and Bukidnon, it traverses most of Bukidnon's towns and cities, demonstrating its crucial role in the region's development (Masangkay et al. 2020a).
Lake Lanao, the Philippines' second-largest lake and Mindanao's largest lake, is a natural wonder. As one of the world's 15 most ancient lakes, it is a testament to the region's rich natural heritage. Its unique ecosystem comprises five watersheds with significant creeks and rivers totaling 431 km. The hydroelectric plant installed along Lanao Lake and Agus River generates 70% of the electricity the people of Mindanao use, further highlighting its importance (Masangkay et al. 2020a).
Lake Mainit is the Philippines' fourth largest and deepest lake. It borders Surigao del Norte and Agusan del Sur provinces. It supports an important fishery, a major livelihood source for the communities surrounding it (Masangkay et al. 2020a).
The Tagunay River, Anibongan River, and Ising River comprise most of the Tagunay River systems. The principal river draining this basin is the 101 km-long Tagunay River. The Tagum Agricultural Development Company, Incorporated (TADECO) watershed, which provides fresh water to Davao, is connected to this river (Masangkay et al. 2020a).
Comparison of Acanthamoeba isolates between initial and 3-year-old water samples
Study sites (N = 15) . | Initial results . | Reference . | Three-year-old results . | |||
---|---|---|---|---|---|---|
Vol (mL) . | Molecular . | Vol (mL) . | Culture . | Molecular . | ||
Luzon (n = 7) | ||||||
Ambuklao Dam | NT | NA | Masangkay et al. (2020a) | 3,400 | − | NA |
San Roque Dam | NT | NA | Masangkay et al. (2020a) | 900 | − | NA |
Lake Pantabangan | 250 | − | Milanez et al. (2020a) | 3,000 | − | NA |
Laguna De Bay | NT | NA | Masangkay et al. (2020a) | 3,000 | − | NA |
Lake Taal-Crater Lake | NT | NA | Masangkay et al. (2020a) | 3,700 | − | NA |
Lake Taal-San Nicolas | NT | NA | Masangkay et al. (2020a) | 5,000 | + | Acanthamoeba genotype T4 (PP564835) A. lenticulata (PP564837) |
Lake Buhi | 50 | PCR + | Hagosojos et al. (2020) | 4,550 | − | NA |
Culture positive | 14% (1/7) | |||||
PCR positive (from cultures positive) | 100% (2/2) | |||||
New positive study sites | 1 | |||||
Visayas (n = 3) | ||||||
Lake Danao | 250 | A. lenticulata (MN685223) | Milanez et al. (2020a) | 2,200 | + | PCR +1 |
Lake Bito | 250 | A. lenticulata (MN685221) | Milanez et al. (2020a) | 3,800 | − | NA |
Lake Sakanaw | NT | NA | Masangkay et al. (2020a) | 3,750 | − | NA |
Culture positive | 33% (1/3) | |||||
PCR positive (from cultures) | 100% (1/1) | |||||
New positive study sites | 0 | |||||
Mindanao | ||||||
Lake Mainit | 250 | A. castellanii (MN685226) A. lenticulata (MN685268) A. jacobsi (MN685227) A. jacobsi (MN685242) A. jacobsi (MN685244) | Milanez et al. (2020a) | 3,400 | − | NA |
Pulangi River | 250 | − | Milanez et al. (2020a) | 2,600 | + | Acanthamoeba sp. (PP564836) |
Lake Lanao | 250 | − | Milanez et al. (2020a) | 2,850 | − | NA |
Tagunay River | 250 | − | Milanez et al. (2020a) | 850 | + | PCR +2 |
Lake Sebu | 250 | − | Milanez et al. (2020a) | 2,810 | + | Acanthamoeba sp. (PP564846) |
Culture positive | 60% (3/5) | |||||
PCR positive | 100% (3/3) | |||||
New positive study sites | 3 | |||||
Total culture positive | 33% (5/15) | |||||
Total PCR positive | 100% (6/6) | |||||
Total new isolates (deposited in GenBank) | 4/6 | |||||
Total new positive study sites | 4 |
Study sites (N = 15) . | Initial results . | Reference . | Three-year-old results . | |||
---|---|---|---|---|---|---|
Vol (mL) . | Molecular . | Vol (mL) . | Culture . | Molecular . | ||
Luzon (n = 7) | ||||||
Ambuklao Dam | NT | NA | Masangkay et al. (2020a) | 3,400 | − | NA |
San Roque Dam | NT | NA | Masangkay et al. (2020a) | 900 | − | NA |
Lake Pantabangan | 250 | − | Milanez et al. (2020a) | 3,000 | − | NA |
Laguna De Bay | NT | NA | Masangkay et al. (2020a) | 3,000 | − | NA |
Lake Taal-Crater Lake | NT | NA | Masangkay et al. (2020a) | 3,700 | − | NA |
Lake Taal-San Nicolas | NT | NA | Masangkay et al. (2020a) | 5,000 | + | Acanthamoeba genotype T4 (PP564835) A. lenticulata (PP564837) |
Lake Buhi | 50 | PCR + | Hagosojos et al. (2020) | 4,550 | − | NA |
Culture positive | 14% (1/7) | |||||
PCR positive (from cultures positive) | 100% (2/2) | |||||
New positive study sites | 1 | |||||
Visayas (n = 3) | ||||||
Lake Danao | 250 | A. lenticulata (MN685223) | Milanez et al. (2020a) | 2,200 | + | PCR +1 |
Lake Bito | 250 | A. lenticulata (MN685221) | Milanez et al. (2020a) | 3,800 | − | NA |
Lake Sakanaw | NT | NA | Masangkay et al. (2020a) | 3,750 | − | NA |
Culture positive | 33% (1/3) | |||||
PCR positive (from cultures) | 100% (1/1) | |||||
New positive study sites | 0 | |||||
Mindanao | ||||||
Lake Mainit | 250 | A. castellanii (MN685226) A. lenticulata (MN685268) A. jacobsi (MN685227) A. jacobsi (MN685242) A. jacobsi (MN685244) | Milanez et al. (2020a) | 3,400 | − | NA |
Pulangi River | 250 | − | Milanez et al. (2020a) | 2,600 | + | Acanthamoeba sp. (PP564836) |
Lake Lanao | 250 | − | Milanez et al. (2020a) | 2,850 | − | NA |
Tagunay River | 250 | − | Milanez et al. (2020a) | 850 | + | PCR +2 |
Lake Sebu | 250 | − | Milanez et al. (2020a) | 2,810 | + | Acanthamoeba sp. (PP564846) |
Culture positive | 60% (3/5) | |||||
PCR positive | 100% (3/3) | |||||
New positive study sites | 3 | |||||
Total culture positive | 33% (5/15) | |||||
Total PCR positive | 100% (6/6) | |||||
Total new isolates (deposited in GenBank) | 4/6 | |||||
Total new positive study sites | 4 |
N, total; n, subtotal; Vol (mL), volume of water sample processed; NT, not tested; PCR +, polymerase chain reaction positive but sequencing was not performed; −, negative; +, positive; MN and PP, GenBank accession numbers; NA, not applicable. 1Acanthamoeba divionensis (100% similarity; 14% Query cover); 2Acanthamoeba genotype T20 (96.77% similarity; 49% Query cover). Note: It is possible that the Acanthamoeba cysts from the water samples are more than 3 years old, supposing they were in the water sources before sampling.
Mapping of study sites of unpreserved 3-year-old environmental freshwater samples stored at room temperature in the Philippines. Mapping shows the results for Acanthamoeba culture in 0 and 3 years of stored samples. Previous culture result/present culture result: NT/ − , not tested/negative; NT/ + , not tested/positive; −/ − , negative/negative; −/ + , negative/positive; +/ − , positive/negative; +/ + , positive/positive.
Mapping of study sites of unpreserved 3-year-old environmental freshwater samples stored at room temperature in the Philippines. Mapping shows the results for Acanthamoeba culture in 0 and 3 years of stored samples. Previous culture result/present culture result: NT/ − , not tested/negative; NT/ + , not tested/positive; −/ − , negative/negative; −/ + , negative/positive; +/ − , positive/negative; +/ + , positive/positive.
Sample processing
Stored 3-year-old unpreserved environmental freshwater samples from previous studies (Table 1) were subjected to vacuum filtration with a Buchner funnel setup using a 1.2-μm pore size glass microfiber filter (47 mm diameter). Glass microfiber filters were recovered and replaced upon clogging. This is expected when processing high-volume environmental water samples. While still moist, the glass microfiber filters were scraped with a sterile scalpel blade and washed with 2 mL of sterile distilled water to harvest the sediments. The sediment suspensions were placed in 2 mL microtubes and processed to detect viable Acanthamoeba cells within 24 h.
Acanthamoeba culture and molecular analysis
Culture and PCR results of unpreserved 3-year-old environmental freshwater samples stored at room temperature demonstrating long-term viability of Acanthamoeba species. (a) Four-quadrant inoculation of filtered sediments in an NNA plate lawned with live Escherichia coli (Lake Taal-San Nicholas); (b) Acanthamoeba spp. cysts (Lake Taal-San Nicholas); (c) Acanthamoeba spp. cysts (Pulangi River); (d) Acanthamoeba spp. trophozoites with extended acanthopodia (Lake Sebu); 400× magnification; Scale bars at 10 μm. (e) PCR results using JDP1 and JDP2 Acanthamoeba-specific primer amplifying the ASA1.S1 region of the Acanthamoeba genome revealed bands along the 500 bp region of the ladder (L) on five study sites: Lanes 3 and 4 (Lake Taal-San Nicolas), 5 (Lake Danao), 9 (Pulangi River), 10 (Tagunay River), and 11 (Lake Sebu).
Culture and PCR results of unpreserved 3-year-old environmental freshwater samples stored at room temperature demonstrating long-term viability of Acanthamoeba species. (a) Four-quadrant inoculation of filtered sediments in an NNA plate lawned with live Escherichia coli (Lake Taal-San Nicholas); (b) Acanthamoeba spp. cysts (Lake Taal-San Nicholas); (c) Acanthamoeba spp. cysts (Pulangi River); (d) Acanthamoeba spp. trophozoites with extended acanthopodia (Lake Sebu); 400× magnification; Scale bars at 10 μm. (e) PCR results using JDP1 and JDP2 Acanthamoeba-specific primer amplifying the ASA1.S1 region of the Acanthamoeba genome revealed bands along the 500 bp region of the ladder (L) on five study sites: Lanes 3 and 4 (Lake Taal-San Nicolas), 5 (Lake Danao), 9 (Pulangi River), 10 (Tagunay River), and 11 (Lake Sebu).
RESULTS
Viability of Acanthamoeba spp. cysts and molecular results
The results of the present study provided evidence of the long-term viability of Acanthamoeba spp. from environmental freshwater samples despite being stored without any preservatives/additives at room temperature for 3 years. Overall, culture and PCR positivity were at 33% (5/15) and 100% (6/6), respectively, with two isolates simultaneously identified from Lake Taal-San Nicolas (Table 1). No trend was observed between culture positive samples and sample volume. After 14 days of incubation, primary culture in NNA plates demonstrated cysts (Figure 2(b) and 2(c)) and trophozoites morphologically consistent with Acanthamoeba spp. Double-layered hexagonal or polygonal cysts with prominent endocysts and ectocysts measuring 20 μm in diameter were observed (Figure 2(c)). Cyst size and morphology were consistent with Page's morphological criteria (Page 1967). After 7 days of subculture, motile and feeding trophozoites were abundant, with some exhibiting prominent acanthopodia (Figure 2(d)). Isolates from subcultures subjected to PCR using JDP1 and JDP2 Acanthamoeba-specific primers demonstrated DNA amplification (bands) along the 500 bp region of the 100 kb reference ladder. This confirms that the FLAs from the subcultures (pure cultures) belong to the genus of Acanthamoeba. From the four new study sites that demonstrated viable Acanthamoeba species through culture and PCR (Lake Taal-San Nicolas, Pulangi River, Tagunay River, and Lake Sebu), only three study sites had isolates that were successfully sequenced, i.e., Acanthamoeba genotype T4 and A. lenticulata (Lake Taal-San Nicolas), Acanthamoeba sp. (Pulangi River), and Acanthamoeba sp. (Lake Sebu). The sequences were deposited in GenBank under the accession numbers PP564835, PP564837, PP564836, and PP564846. The two sequences from the Acanthamoeba spp. isolates from Lake Danao (Acanthamoeba divionensis) and Tagunay River (Acanthamoeba genotype T20) could not be deposited in GenBank as although BLAST results showed 100 and 96.77% similarity, respectively, the Query Cover was low at 14 and 49% only after trimming, cleaning and consensus of forward and reverse sequences.
Among the study sites (Figure 1 and Table 1), Mindanao Island demonstrated the most samples with viable Acanthamoeba spp. cysts at 60% (3/5), followed by Visayas Island with 33% (1/3) and Luzon Island with 14% (1/7). Aside from demonstrating the long-term viability of Acanthamoeba species, the study's results also provided the opportunity to expand the mapping of the geographic distribution of Acanthamoeba spp. in the Philippines' environmental freshwater resources (Figure 1). This study demonstrated four new study sites as habitats for Acanthamoeba spp.: Lake Taal-San Nicolas in Luzon and Pulangi River, Tagunay River, and Lake Sebu in Mindanao. It is also important to note that although long-term viability was demonstrated in the 3-year-old stored water samples, the three study sites that previously tested positive for Acanthamoeba spp., namely Lake Buhi, Lake Bito, and Lake Mainit, may have organic or inorganic components that inhibited the FLA growth in NNA culture in the present study, aside from other factors like changes in pH and osmolarity.
DISCUSSION
Growth conditions for Acanthamoeba spp.
Acanthamoeba spp. are highly adaptable to many environmental conditions, enabling them to thrive in various habitats. Its trophozoite stage primarily feeds on microorganisms such as bacteria, algae, or yeasts. An abundance of food supply, neutrality in pH, appropriate temperature, and an osmolarity between 50 and 80 mOsm can maintain the trophozoite stage of Acanthamoeba (Khan 2006). Its cystic stage can withstand harsh environmental conditions that might harm trophozoites, providing a protective mechanism for the amoeba. Cysts resist ecological stressors, including temperature fluctuations, nutrient scarcity, and exposure to disinfectants or chemicals, and help maintain the viability of Acanthamoeba spp. over extended periods (Mazur et al. 1995). The present study's culture results from 3-year-old unpreserved environmental freshwater samples stored at room temperature demonstrated amoebic growth of Acanthamoeba species in previously culture-negative study sites (Table 1 and Figure 1). This may be attributed to the increased volume of water sample processed, which ranged from 850 to 5,000 mL, compared with the lower sample volume of 250 mL processed in previous studies (Table 1), except for the 2020 study that demonstrated PCR positive results for Acanthamoeba spp. in low-volume (50 mL) water sample (Hagosojos et al. 2020). Also, introducing the four-quadrant method of NNA plate inoculation (Figure 2(a)) maximized the culture plate surfaces for amoebic growth, eased microscopic monitoring for the cystic and trophic stages, and increased test analysis from a single test to quadruplicate tests in a single NNA plate for each sample. Acanthamoeba spp., A. lenticulata, A. castellanii, and A. jacobsi were previously reported in Buhi Lake (Hagosojos et al. 2020), Lake Bito, and Lake Mainit (Milanez et al. 2020a) but showed no amoebic growth in the present study. Cyst dormancy could have contributed to this lack of amoebic growth. Also, variations in the culture methods may have influenced the detection of Acanthamoeba spp. and the presence of unknown organic and inorganic matter that may have been harmful to Acanthamoeba species cysts in the stored water samples. While cysts are resistant structures, their long-term viability is not indefinite. Over time, even cysts may lose viability, leading to a decline in amoebic growth potential (Coulon et al. 2010).
Long-term viability of Acanthamoeba spp. cysts
The long-term viability of Acanthamoeba species cysts is attributed to several adaptive features that allow these organisms to endure adverse conditions. Acanthamoeba can transform into a cyst to protect itself against environmental stressors like nutrient depletion, desiccation, and temperature fluctuations (Niederkorn 2021). The protozoan cyst is a protective structure that encases the amoeba, shielding it from harsh external conditions. Its robust outer layer provides resistance to physical and chemical stress. Inside the cyst, Acanthamoeba undergoes a state of dormancy with a significant reduction in metabolic activity (Siddiqui & Khan 2012). Metabolic dormancy allows the amoeba to conserve energy and withstand prolonged periods without actively feeding or reproducing. Acanthamoeba spp. cysts can be associated with biofilms, with their communities of microorganisms providing a protected environment that aids in cyst survival (Masangkay et al. 2022). It has been demonstrated that several Acanthamoeba species can remain viable in vitro in dried-up NNA plates for up to 20 years (Sriram et al. 2008). Also, a recent study noted the viability of A. lenticulata and A. hatchetti in 2-year-old groundwater samples (Masangkay et al. 2022). Despite 3 years of water sample storage, unpreserved, at room temperature, this supports the isolation of A. lenticulata and other Acanthamoeba species/genotypes in the present study. The combination of cyst formation, resistance to environmental stressors, metabolic dormancy, and adaptability to different habitats contributes to the long-term viability of Acanthamoeba cysts. This resilience allows them to persist in the unpreserved environmental freshwater samples in the present study. It is important to note that although it was defined that the environmental freshwater samples were stored at room temperature for 3 years, it is possible that the Acanthamoeba spp. cysts captured were present in the source water well before the sample collection 3 years prior and have already been subjected to numerous changes in environmental conditions, such as UV exposure, fluctuating water temperatures, and precipitation levels. Also, as the stored water samples had no preservatives/additives, the bacterial population present may have served as a food source for Acanthamoeba spp. trophozoites prior to encystation.
From perspectives of how the changes in pH and osmolarity in the stored water samples may have affected the viability of Acanthamoeba spp. cysts, the decomposition of organic material generally decreases pH. Also, bacterial growth may have influenced changes in pH depending on the concentration of acidic or alkaline metabolites they produce. This also influences the fate of the cycle of bacterial populations in the stored sample (Ratzke & Gore 2018). In terms of changes in osmolarity due to water volume, no significant change in water volume due to dehydration was observed as the polyethylene containers were tightly sealed with their cover and parafilm. Sustaining bacterial population becomes challenging as osmolarity increases and becomes more complex regarding mutations due to growth trade-offs across varying osmolarities (Cesar et al. 2020). In this regard, monitoring these parameters and their effects on the long-term viability of Acanthamoeba species and other FLAs would be interesting.
This cost-effective method of storing environmental freshwater samples destined for Acanthamoeba studies hints at the potential of other FLAs with cystic stages to remain viable in other sample matrices for a long time. It is then a curiosity to observe Acanthamoeba spp. and other FLA cysts' long-term viability in estuarine and marine/saltwater samples. The long-term viability of Acanthamoeba spp. should be considered in the following contexts. Firstly, it is an added risk factor for the sustained contamination of aquatic resources and possibly other sample matrices. Secondly, this potentially increases the risk of transmission to humans and animals; thirdly, as a means for the cost-effective storage of water samples fated for Acanthamoeba studies; and lastly, the applicability of the same to other FLAs.
Updated geographic distribution of Acanthamoeba spp. in the Philippines
Acanthamoeba spp. inhabit diverse ecosystems due to their adaptability to different environmental conditions. This study presented data from 15 significant environmental freshwater resources throughout the Philippines, 33% (5/15) of which demonstrated viable Acanthamoeba spp. after 3 years of unpreserved storage at room temperature. Acanthamoeba spp. previously documented in Lake Danao from Visayas Island remained favorable for amoebic growth. New study sites identified to be inhabited by Acanthamoeba species were Lake Taal (San Nicolas) from Luzon Island, along with Pulangi River, Tagunay River, and Lake Sebu from Mindanao Island.
Acanthamoeba and the threat of increasing global temperature
Increasing global temperature and climate change contribute to the degradation of water resources through extreme precipitation events that carry possible pathogens and contaminants into waterways via runoff and flooding (DeNicola et al. 2015). Also, Acanthamoeba spp., previously isolated from soil and dust (Cruz & Rivera 2014), may contaminate surface and groundwater sources due to soil runoff (Masangkay et al. 2020b; Milanez et al. 2020a). Further, increasing water temperatures due to global warming promotes disease and may significantly affect the proliferation of waterborne pathogens such as Acanthamoeba spp. (Karvonen et al. 2010). The scarcity of clean water sources may induce the populace to utilize water contaminated with waterborne protozoan pathogens (Masangkay et al. 2020a). The genotype T4 of Acanthamoeba, the most prevalent genotype in human ocular infections worldwide, was reported to survive at elevated temperatures of up to 42 °C and have a broad adaptive capability (Chomicz et al. 2015). With this considered, it is essential to investigate the limits of the thermo-adaptive capacity of Acanthamoeba spp. and how we can safely inactivate viable cysts in water resources for human and animal use or use barriers that may mitigate transmission.
Health implications
Acanthamoeba spp. are generally waterborne, opportunistic pathogens detected in various water resources, including fresh and brackish water. These organisms also survive in artificial habitats, such as cooling towers, water tanks, tap water systems, wastewater treatment plants, sewage, and recreational pools (Scheid 2018). Exposure of contact lens fluids to these contaminated waters is the leading cause of AK, a sight-threatening infection affecting the corneas. Although rare, trauma and water contamination are the primary risk factors for non-contact lens users for GAE (Garg et al. 2017). Since the majority of Acanthamoeba spp. infections cause amoebic keratitis, it is paramount to practice adequate disinfection and good contact lens hygiene by avoiding exposure of contact lenses to tap water (Carnt et al. 2018). Also, Acanthamoeba is an underdiagnosed but potentially common infection in animals. These organisms have been reported in a variety of animal species, including cats, dogs, and even fishes, amphibians, and reptiles (Sesma & Ramos 1989; Dyková et al. 1999; Walochnik et al. 1999). Like humans, immunocompromised animals are more likely to be infected with Acanthamoeba. Due to the proximity between humans and animals in many settings (Cooper et al. 2021), knowing the epidemiology, pathogenesis, transmission, diagnosis, and treatment options for Acanthamoeba infection is essential.
Acanthamoeba spp. can act as a ‘Trojan horse’ for other microbes by helping transmit pathogenic endosymbionts to susceptible hosts. This process was demonstrated in 2016 when Fukumoto et al. detected the presence of Acanthamoeba, which carried an endosymbiotic chlamydial pathogen, Protochlamydia W-9, from hospital floors, sinks, and drainages (Fukumoto et al. 2016). Other examples of endosymbiotic bacteria related to Acanthamoeba are Escherichia coli, Methicillin-resistant Staphylococcus aureus, Mycobacterium spp., and Shigella spp., to name a few. Additionally, it was also reported to harbor a variety of viruses such as enterovirus, adenoviruses, or coxsackieviruses and yeast cells, i.e., Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum (Siddiqui & Khan 2012).
Significant human activity in these freshwater resources and their human utility may heighten the risk of human infections, indicating a need for water treatment and purification before human consumption or other uses, as well as a warning to the public that freshwater resources, in general, may harbor pathogenic FLAs. From a tourism perspective, it may be helpful for both the local economy and the tourists to explore the production, sales, and advocacy of using locally made nose clips and goggles when engaging in water recreational activities.
The present study had certain limitations. Firstly, osmo-tolerance and thermotolerance assays were not performed. Secondly, the data on the water samples' pH, temperature, and osmolarity during collection were not obtained. Thirdly, the 3-year-old samples' pH and osmolarity data were not noted. Further studies on the long-term viability of Acanthamoeba species and other FLAs are recommended to assess the viability of cysts, along with patterns of osmo-thermotolerance demonstrated by isolates from fresh and stored samples. Also, data collection of the sample pH, temperature during collection, and osmolarity may assist in explaining the FLAs' behavior relative to long-term viability.
This groundbreaking research provides the first evidence of the long-term viability of Acanthamoeba species in unpreserved environmental freshwater samples stored at room temperature for 3 years. The potential impact of this finding on our understanding of Acanthamoeba spp. and its survival in environmental freshwater samples cannot be overstated, as it is crucial for future decision-making on water treatment and reuse, wastewater treatment, environmental water quality, and public health safety.
CONCLUSIONS
This is the first report on the long-term viability of Acanthamoeba species in unpreserved environmental freshwater samples stored at room temperature for 3 years. The introduction of the four-quadrant method of NNA inoculation seems an acceptable alternative to the traditional FLA culture method as it maximizes a single NNA plate to accommodate quadruplicate testing for each sample. The three central islands of the Philippines demonstrated the presence of Acanthamoeba genotype T4 and A. lenticulata in Luzon, Acanthamoeba divionensis in Visayas, and Acanthamoeba sp. and Acanthamoeba genotype T20 in Mindanao. The long-term viability of Acanthamoeba species is an added risk factor for the sustained contamination of aquatic resources and other sample matrices. This can increase the risk of transmission to humans and animals. This study also demonstrated that water samples fated for Acanthamoeba studies can be stored unpreserved at room temperature for several years. The findings of this study are crucial for future decision-making on water treatment and reuse, wastewater treatment, environmental water quality, and public health safety.
ACKNOWLEDGEMENTS
We sincerely thank Mr Carlo Honesto B. Botor, Mr Emmanuel D. Zambrano, Mr Robert Li, and Mr Romie Cultura of the University of Santo Tomas, Department of Medical Technology for their invaluable assistance with laboratory logistics. We also thank the Department of Medical Technology, Faculty of Pharmacy, University of Santo Tomas, for their support and guidance.
CREDIT AUTHORSHIP CONTRIBUTION STATEMENT
F.R.M.: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Validation, Taxonomy of parasites, Writing – original draft; Writing – review and editing, Supervision; F.C.R.: Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review and editing; A.J.G.P.: Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review and editing; R.L.M.P.: Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review and editing; M.S.S.R.: Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review and editing; J.K.M.R.: Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review and editing; M.J.E.S.: Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review and editing; M.C.I.S.: Data curation, Formal analysis, Investigation, Resources, Visualization, Writing – original draft, Writing – review and editing; M.F.F.E.P.: Methodology, Visualization; M.K.: Validation, Writing – review and editing; J.T.: Validation, Writing – review and editing; G.D.M.: Formal analysis, Methodology, Visualization, Validation, Taxonomy of parasites; P.K.: Validation, Taxonomy of parasites, Writing – review and editing.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
CONFLICT OF INTEREST
The authors declare there is no conflict.