The Hidden Hitchhiker in Water: Unraveling Acanthamoeba's Secrets in Northern Iran

More Than Meets the Eye: Understanding the Acanthamoeba

Acanthamoeba is not your typical parasite. Unlike parasites that require a host to complete their life cycle, this microorganism thrives independently in the environment, particularly in water and soil ¹ ³ .

The Trophozoite

This is the active, feeding, and reproducing form of the amoeba. It moves around its environment, consuming bacteria, yeast, and other microorganisms.

The Cyst

When conditions become unfavorable, the trophozoite transforms into a dormant cyst. This form is incredibly resistant to disinfection, antibiotics, and harsh environmental conditions, allowing it to survive for years ² ⁶ .

Diseases Caused by Acanthamoeba

Keratitis

Acanthamoeba Keratitis (AK)

A painful and sight-threatening infection of the cornea, primarily affecting contact lens wearers. It can be difficult to treat ² .

Risk for contact lens wearers: High

Encephalitis

Granulomatous Amebic Encephalitis (GAE)

A rare but fatal infection of the brain and spinal cord that typically occurs in immunocompromised individuals ¹ ³ .

General population risk: Low

The Acanthamoeba Life Cycle

Understanding the two-stage life cycle is key to comprehending both its survival and pathogenicity.

Trophozoite Stage

The active form that feeds on bacteria, fungi, and other microorganisms in the environment. It reproduces by binary fission and can cause infection if it enters the eye or respiratory system.

Encystment

When conditions become unfavorable (lack of food, extreme temperatures, disinfectants), the trophozoite transforms into a double-walled cyst. This stage is highly resistant to environmental stresses.

Cyst Stage

The dormant form that can survive for years in harsh conditions. Cysts are resistant to chlorine, antibiotics, and extreme temperatures, making eradication difficult.

Excystment

When conditions become favorable again, the cyst transforms back into the active trophozoite stage, resuming feeding and reproduction.

A Scientific Detective Story: The Mazandaran Province Investigation

To understand the risk posed by this parasite, a team of researchers in Northern Iran conducted a study to isolate and identify Acanthamoeba species in the water sources of Mazandaran province ¹ ⁴ .

Map of Mazandaran Province, Iran
Study area for Acanthamoeba research

The Step-by-Step Investigation

The team gathered 77 water samples in duplicate from diverse sources across Sari city and its suburbs. These included lakes, rivers, tap water, swimming pools, rice fields, and the Caspian Sea.

Each sample was filtered to concentrate any microorganisms. The filters were then placed upside down on special non-nutrient agar plates coated with heat-killed E. coli—a food source to encourage the amoebae to grow and multiply.

The plates were sealed and incubated for up to two months. Researchers daily checked them using an inverted microscope, looking for the tell-tale signs of Acanthamoeba cysts and trophozoites.

Once morphological identification suggested the presence of Acanthamoeba, the researchers extracted DNA from the positive samples. They used a Polymerase Chain Reaction (PCR) test, targeting a specific region of the 18S rRNA gene, to confirm the organism's identity at a molecular level.

For a finer-level analysis, they sequenced the PCR fragments from 20 of the positive isolates. Comparing these sequences to a known database allowed them to determine the specific genotype of each Acanthamoeba isolate.

The Revealing Results

The findings were striking and revealed important information about the distribution of Acanthamoeba in Northern Iran's water sources.

Sample Positivity Rate

55.8%

Of the 77 original water samples tested positive for Acanthamoeba based on both morphological and molecular tests ¹ .

Genotype Distribution

The genotyping results were even more revealing. The sequencing of 20 isolates showed a clear dominance of one particular genotype ¹ :

Genotype	Number of Isolates	Percentage	Clinical Relevance
T4	15	83.3%	Most frequently associated with human infections ¹ ²
T2	3	16.7%	Less common in infections, but still potentially pathogenic

Examples of Acanthamoeba Species Identified and Their Sources

Isolate Name	Source of Isolate	Species Identification	Genotype
SI-4-IR	Lake	A. castellanii castellanii	T4
SI-22-IR	Rice field	A. palestinensis	T2
SI-33-IR	Sea sediment	A. castellanii castellanii	T4
SI-15-IR	Dam	A. polyphaga	T4

The Bigger Picture: Why Environmental Monitoring Matters

This study was the first of its kind to report on Acanthamoeba genotypes from water sources in Mazandaran province ¹ .

Pathogenic Dominance

The discovery that the highly pathogenic T4 genotype is the dominant strain in the environment is a crucial piece of the public health puzzle.

Rising Infections

Iran has seen an increase in cases of Acanthamoeba keratitis in recent years, highlighting the need for awareness and prevention ¹ .

Trojan Horse

Acanthamoeba can act as hosts for other harmful bacteria like Legionella pneumophila, protecting them from disinfection ¹ .

The Scientist's Toolkit: Key Tools for Hunting Amoebae

Reagent/Material	Function in the Experiment
Cellulose Nitrate Filter (0.45 µm)	To concentrate and capture amoebae (both trophozoites and cysts) from large volumes of water samples.
Non-Nutrient Agar Plate	A growth medium that does not support bacterial overgrowth, allowing selective cultivation of Acanthamoeba.
Heat-killed E. coli	Serves as a food source to nourish the amoebae and promote their growth and multiplication on the agar plates.
Lysis Buffer & Proteinase K	Chemicals used to break open (lyse) the amoeba cells and digest proteins, freeing the DNA for analysis.
PCR Primers (JDP1 & JDP2)	Short, specific DNA sequences designed to bind to and amplify a unique fragment of the Acanthamoeba 18S rRNA gene.
Polymerase Chain Reaction (PCR) Thermocycler	A machine that performs PCR, rapidly copying the target DNA sequence millions of times.

Public Health Recommendation

For contact lens wearers: Avoid using tap water to rinse lenses or cases, and follow proper disinfection guidelines ² .

A Clearer Vision for the Future

The detective work in Northern Iran's waters reveals an invisible ecosystem where potentially pathogenic organisms like Acanthamoeba are more common than one might think. The dominance of the T4 genotype in the environment is a clear signal that this neglected parasite deserves more attention.

For scientists and public health officials, it highlights the need for continued environmental surveillance, faster diagnostic tools, and more effective treatments to combat the resilient cyst stage of the amoeba ⁹ .

By understanding what lies in the water, we can better protect our health and our vision.