First report on the long-term viability of <i>Acanthamoeba</i> species in unpreserved environmental freshwater samples stored at room temperature

<i>Acanthamoeba</i> 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 <i>Acanthamoeba</i> species is needed. This study aimed to define the long-term viability of <i>Acanthamoeba</i> 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 <i>Escherichia coli</i>, 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 <i>Acanthamoeba</i>-specific primers. The study site positivity was 33% (5/15). <i>Acanthamoeba</i> genotype T4 and <i>Acanthamoeba lenticulata</i> were isolated from Luzon; <i>Acanthamoeba divionensis</i> was isolated from Visayas; and <i>Acanthamoeba</i> sp. and genotype T20 were isolated from Mindanao. The long-term viability of <i>Acanthamoeba</i> 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 <i>Acanthamoeba</i> studies can be stored unpreserved at room temperature for several years.