Acanthamoeba Genotype Research Articles

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

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.

Non-Contact-Lens-Related Acanthamoeba Keratitis Caused by Acanthamoeba sp. Group T4D/T4e

Acanthamoeba keratitis (AK) is a rare but serious infection of the cornea, typically associated with contact lens wear. Here, we present a case of AK caused by the Acanthamoeba genotype T4D/T4e in a patient without identifiable risk factors: a 34-year-old woman who initially presented with signs and symptoms suggestive of herpetic keratitis, and who did not respond to conventional treatment. Corneal culture and targeted metagenomic analysis (18S rRNA, 16S-like rRNA) revealed the presence of an Acanthamoeba species closely related to the ‘Nagington’ strain. Despite intensive anti-Acanthamoeba therapy, complications arose necessitating penetrating keratoplasty. In conclusion, this case underscores the importance of considering Acanthamoeba as a causal agent of keratitis in non-contact-lens wearers. The identification of Acanthamoeba genotype T4D/T4e challenges the previous understanding of its pathogenic potential. Furthermore, it emphasizes the need for ongoing research into the pathogenicity of different Acanthamoeba subtypes. Early diagnosis and treatment are essential for preventing vision-threatening complications associated with AK.

First report of Acanthamoeba spp. isolation in a recreational hot spring in the Philippines.

Natural hot springs are ideal places and environmental matrices that offer relaxation to people and microorganisms of different types. A total of 40 surface water samples were collected from the five identified collection sites, eight water samples for each site. Collection sites are designated 200 m apart to cover the entire study site. Surface water samples were collected approximately 10-20 cm from the surface. Water samples were filtered, cultured, and microscopically observed for 14 days. After 14 days of cultivation, eight (20%) water samples revealed cystic and trophozoite stages. Polymerase chain reaction using JDP1 and JDP2 specific primers confirmed the presence of Acanthamoeba spp. from two of our isolates in the hot spring, isolates 1.1 and 5.1. Further sequencing revealed that the isolates are Acanthamoeba T20 and Acanthamoeba genotype T7. Sequences were deposited to GenBank and were assigned accession numbers PP741726 and PP741727, respectively. The isolation of Acanthamoeba spp. in hot springs has significant health implications, especially for those who use it for recreational activity. Private resort owners are highly encouraged to regularly monitor and maintain hot spring resorts to avoid future infections.

Isolates of Acanthamoeba species in the marine environment in the Philippines.

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acanthamoeba sp., Acanthamoeba culbertsoni, Acanthamoeba castellani, and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs.

First Report of Acanthamoeba Genotype T4 from the Newly Formed Tajogaite Volcano Tephra (La Palma, Canary Islands).

The Tajogaite Volcano erupted on the western slope of the Cumbre Vieja mountain range on La Palma Island in the Canary Islands, Spain, in 2021. As one of the multiple consequences of this eruption, a layer of tephra was deposited, to a variable extent, over a large part of the island. Tephra deposits affect all aspects of vegetation recovery, the water cycle, and the long-term availability of volcanic nutrients. Protozoa, including free-living amoeba (FLA), are known to be among the first microorganisms capable of colonizing harsh environments. In the present study, the presence of FLA has been evaluated in the Tajogaite Volcano deposits. Samples of the tephra were collected and incubated at 26 °C on 2% non-nutrient agar plates with a layer of heat-killed E. coli. Morphological features, as well as the DF3 region sequence of the 18S rDNA, confirmed the presence of a T4 genotype strain of Acanthamoeba. Thermotolerance and osmotolerance assays were used to evaluate the strain's pathogenic potential. This strain was considered thermotolerant but poorly osmotolerant. To the best of our knowledge, this is the first report of Acanthamoeba being isolated from a recently erupted volcano.

Commercial reverse osmosis point-of-use systems in Egypt failed to purify tap water.

This study addresses the heightened global reliance on point-of-use (PoU) systems driven by water quality concerns, ageing infrastructure, and urbanization. While widely used in Egypt, there is a lack of comprehensive evaluation of these systems. We assessed 10 reverse osmosis point-of-use systems, examining physicochemical, bacteriological, and protozoological aspects of tap water (inlets) and filtered water (outlets), adhering to standard methods for the examination of water and wastewater. Results showed significant reductions in total dissolved solids across most systems, with a decrease from 210 ± 23.6 mg/L in tap water to 21 ± 2.8 mg/L in filtered water for PoU-10. Ammonia nitrogen levels in tap water decreased from 0.05 ± 0.04 to 2.28 ± 1.47 mg/L to 0.02 ± 0.04 to 0.69 ± 0.64 mg/L in filtered water. Despite this, bacterial indicators showed no significant changes, with some systems even increasing coliform levels. Protozoological analysis identified prevalent Acanthamoeba (42.5%), less frequent Naegleria (2.5%), Vermamoeba vermiformis (5%), and potentially pathogenic Acanthamoeba genotypes. Elevated bacterial indicators in filtered water of point-of-use systems, combined with essential mineral removal, indicate non-compliance with water quality standards, posing a public health concern. Further research on the long-term health implications of these filtration systems is essential.

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

Abstract Acanthamoeba, a widely distributed free-living amoeba with 20 genotypes identified through rRNA gene sequencing, exhibits varying degrees of pathogenicity influenced by its genotype. This study focuses on assessing the prevalence of Acanthamoeba species in the surface waters of Ilam, located in western Iran, utilizing morphological analysis and sequencing of the 18S rRNA gene through the PCR method. A total of 50 water samples were collected from various regions within Ilam city, situated in the southwest of Iran. To isolate Acanthamoeba parasites from the samples, a culture method was used, and all utilized culture media were scrutinized through microscopic and molecular techniques. The parasite's genotype was determined by sequencing a 500-bp fragment of the 18S rRNA gene. Using microscopic and molecular methods, 19 and 16 water samples tested positive, respectively. The 18S rRNA sequences revealed that the isolates belonged to the T4, T2, and T11 genotypes. This study emphasizes the presence and inclination for close contact with highly pathogenic genotypes of Acanthamoeba in the surface waters of Ilam City.

Molecular identification, phylogenetic analysis and histopathological study of pathogenic free-living amoebae isolated from discus fish (Symphysodon aequifasciatus) in Iran: 2020–2022

Free-living amoebae (FLA) are capable of inhabiting diverse reservoirs independently, without relying on a host organism, hence their designation as “free-living”. The majority of amoebae that infect freshwater or marine fish are amphizoic, or free-living forms that may colonize fish under particular circumstances. Symphysodon aequifasciatus, commonly referred to as the discus, is widely recognized as a popular ornamental fish species. The primary objective of the present study was to determine the presence of pathogenic free-living amoebae (FLA) in samples of discus fish. Fish exhibiting clinical signs, sourced from various fish farms, were transferred to the ornamental fish clinic. The skin, gills, and intestinal mucosa of the fish were collected and subjected to culturing on plates containing a 1% non-nutrient agar medium. The detection of FLA was conducted through morphological, histopathological and molecular methods. The construction of the phylogenetic tree for Acanthamoeba genotypes was achieved using the maximum likelihood approach. The molecular sequence analysis revealed that all cultures that tested positive for FLA were T4 genotype of Acanthamoeba and Acanthamoeba sp. The examination of gill samples using histopathological methods demonstrated the presence of lamellar epithelial hyperplasia, significant fusion of secondary lamellae, and infiltration of inflammatory cells. A multitude of cysts, varying in shape from circular to elliptical, were observed within the gills. The occurrence of interlamellar vesicles and amoeboid organisms could be observed within the epithelial tissue of the gills. In the current study, presence of the Acanthamoeba T4 genotype on the skin and gills of discus fish exhibiting signs of illness in freshwater ornamental fish farms was identified. This observation suggests the potential of a transmission of amoebic infection from ornamental fish to humans, thereby highlighting the need for further investigation into this infection among ornamental fish maintained as pets, as well as individuals who interact with them and their environment.

Acanthamoeba Keratitis in China: Genotypic and Clinical Correlations.

To investigate the relationship between Acanthamoeba genotypes, clinical manifestations, and outcomes in Acanthamoeba keratitis (AK) patients. This retrospective study included 159 culture-confirmed AK patients. Patients' data were collected, including demographics, initial diagnosis, treatments, and clinical features. The genotype of Acanthamoeba was identified through sequencing the Diagnostic Fragment 3 (DF3) region in the small ribosomal subunit RNA genes. The phylogenetic tree was constructed using the ClustalW model and maximum likelihood method. Cases with "poor outcome" were defined based on specific clinical criteria, including corneal perforation, keratoplasty, other eye surgery, duration of anti-amoebic therapy ≥8.0months, and final visual acuity ≤20/80. "Better outcome" cases were the remainder. The correlation between T4 subtypes, clinical phenotypes, and clinical prognosis were further analyzed. In this study, AK was primarily attributed to the T4A genotype, with a positive correlation between geographical and genetic distances. The primary clinical associated with T4 subtypes was deep stromal infiltration. Results was also showed a significant association between T4 subtypes and clinical outcomes (P = 0.021). Further analysis revealed that T4C was closely associated with a better prognosis (P = 0.040) and T4D with worse outcomes (P = 0.013). In China, AK was predominantly caused by the T4A subtype. Geographical distance positively correlated with genetic distance. Clinical prognosis varied among different subtypes, notably in T4C and T4D. This study demonstrated the association between T4 subtypes and clinical phenotypes, as well as the effects of T4 subtypes on clinical prognosis.

Phylogenetic analysis of Acanthamoeba isolated from soil samples and nasal cavities of patients with malignancy: a public health concern in the northwest of Iran.

The genus Acanthamoeba is reported from various environmental sources and can cause multiple complications, including chronic amoebic aeratitis and amoebic granulomatous encephalitis. This study investigated the presence and genotyping of Acanthamoeba in the soil of parks and patients with malignancies referred to health centers in Zanjan city, Iran. In this cross-sectional study, 200 soil samples were collected from amusement parks in Zanjan city from September 2017 to May 2018. Samples were cultured on 1.5% non-nutrient agar, and the Acanthamoeba genus was identified using the morphological method. PCR was performed on all positive environmental samples, and six microscopically positive clinical samples belonged to our previous study. DNA sequencing of 18S rRNA was performed to analyze the genetic pattern of some PCR-positive isolates. Microscopic results showed that 96 (48%) soil samples were positive. PCR confirmed all positive cases of clinical samples and 84 soil samples. Out of the PCR-positive samples, 20 soil samples and five clinical samples were sequenced successfully. All soil isolates belonged to the T4 genotype, and three and two clinical samples belonged to T4 and T5 genotypes, respectively. : The presence of Acanthamoeba in both the environment and clinical samples of Zanjan city suggests paying greater attention to the infections caused by it.

The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model.

Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba. This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. Household water and corneal swabs were collected from AK patients. Acanthamoeba strains and genotypes were confirmed by sequencing. Acanthamoeba isolates were assessed for the presence of intracellular bacteria using sequencing, fluorescence in situ hybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne-functionalized D-alanine (alkDala). Primary human macrophages were used to compare the intracellular survival and replication of the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats were challenged intrastromally with Acanthamoeba containing or devoid of P. aeruginosa and evaluated for the clinical response. Domestic water and corneal swabs were positive for Acanthamoeba. Both strains belonged to genotype T4F. One of the Acanthamoeba isolates harboured P. aeruginosa which was seen throughout the Acanthamoeba's cytoplasm. It was metabolically active and could be seen undergoing binary fission. This motile strain was able to replicate in macrophage to a greater degree than strain PAO1 (p<0.05). Inoculation of Acanthamoeba containing the intracellular P. aeruginosa in rats eyes resulted in a severe keratitis with increased neutrophil response. Acanthamoeba alone induced milder keratitis. Our findings indicate the presence of live intracellular bacteria in Acanthamoeba can increase the severity of acute keratitis in vivo. As P. aeruginosa is a common cause of keratitis, this may indicate the potential for these intracellular bacteria in Acanthamoeba to lead to severe polymicrobial keratitis.

Saline-Tolerant Pathogenic Acanthamoeba spp. Isolated from a Geothermal Power Plant.

Few studies have been conducted in the cooling systems of power plants; they have focused on Naegleria fowleri, leaving a gap in the knowledge of other pathogenic free-living amoebae in this environment. The objective of this study was to determine the occurrence of saline-tolerant pathogenic Acanthamoeba in a geothermal power plant. The identification of isolated amoebae at genus level was carried out, observing their morphological characteristics; the determination of genotype and species of Acanthamoeba was performed via molecular biology (PCR). Water temperature ranged from 18 to 43 °C and conductivity from 4.0 × 104 to 8.7 × 104 μS/cm; this last value was greater than the seawater value. Only five amoeba genera were found. Acanthamoeba was in all the sampling sites, showing high saline tolerance. The high temperature, but mainly high conductivity, were the environmental conditions that determined the presence of pathogenic free-living amoebae in the hot water. All the strains of Acanthamoeba culbertsoni killed the mice, having a mortality of 40 to 100%. Acanthamoeba genotypes T10 and T5 were identified, T10 is rarely isolated from the environment, while T5 is more frequent. This is the first time that genotypes T5 and T10 have been reported in the environment in Mexico.

Isolation and genotyping of Acanthamoeba species and Vahlkampfiidae in the harsh environmental conditions in the centre of Iran.

Different species of free-living amoeba (FLA) have been abundantly isolated in harsh environmental conditions such as hot springs and brackish water. The present study aimed to isolate, genotype, and evaluate the pathogenicity of FLAs in Qom Roud, a large river, in the centre of Iran. About 500 mL of water samples (n = 30) were collected from each sampling site and were investigated for the presence of FLAs using morphological and molecular characters. Genotype identification was performed using DNA sequencing and a phylogenetic tree was constructed with the MEGA X software. The pathogenic potential of all positive isolates was evaluated using the tolerance ability test. Morphological and molecular analysis indicated that 14 (46.66%) and two (6.66%) water samples were positive for Acanthamoeba species and Vahlkampfiidae, respectively. According to sequence analysis, Acanthamoeba isolates related to the T4 genotype and Vahlkampfiidae sequences were similar to Naegleria philippinensis. In the next step, thermo- and osmotolerance tests indicated four Acanthamoeba strains are extremely pathogenic. Our data showed the presence of potentially pathogenic Acanthamoeba T4 genotype and N. philippinensis in the super harsh Qom Roud. Contamination of water with virulent T4 genotype of Acanthamoeba may pose risk factors for contact lens users, children, and immunocompromised people.

Molecular characterisation and potential pathogenicity analysis of Acanthamoeba isolated from recreational lakes in Peninsular Malaysia.

The present study aims to identify the Acanthamoeba genotypes and their pathogenic potential in three recreational lakes in Malaysia. Thirty water samples were collected by purposive sampling between June and July 2022. Physical parameters of water quality were measured in situ while chemical and microbiological analyses were performed in the laboratory. The samples were vacuum filtered through nitrate filter, cultured onto non-nutrient agar and observed microscopically for amoebic growth. DNAs from positive samples were extracted and made to react with polymerase chain reaction using specific primers. Physiological tolerance tests were performed for all Acanthamoeba-positive samples. The presence of Acanthamoeba was found in 26 of 30 water samples by PCR. The highest rate in lake waters contaminated with amoeba was in Biru Lake (100%), followed by Titiwangsa Lake (80%) and Shah Alam Lake (80%). ORP, water temperature, pH and DO were found to be significantly correlated with the presence of Acanthamoeba. The most common genotype was T4. Temperature- and osmo-tolerance tests showed that 8 (30.8%) of the genotypes T4, T9 and T11 were highly pathogenic. The presence of genotype T4 in habitats related to human activities supports the relevance of this amoeba as a potential public health concern.

Identification and genotyping of Acanthamoeba spp. in the water resources of western Iran

BackgroundAcanthamoeba spp. is opportunistic amoeba that resides in water, soil, and air. Some pathogenic genotypes of the genus of Acanthamoeba can cause granulomatous amoebic encephalitis (GAE) in people with a defective immune system. The parasite can also cause Acanthamoeba keratitis (AK) among contact lens users. This study was conducted to isolate and identify the Acanthamoeba genotypes in water resources in Lorestan province, western Iran. MethodsCollected 72 water samples from surface and groundwater (springs and aqueducts) in Lorestan province. Samples were filtered and cultured in non-nutrient 1.5% agar medium covered with Escherichia coli (E. coli) at 25 °C. DNA extraction was done and the PCR reaction was performed to detect the Acanthamoeba spp. The positive PCR products were sequenced to determine the genotypes of Acanthamoeba. ResultsOut of 72 examined water samples, 23.61% were positive for Acanthamoeba sp. by PCR. From PCR-positive samples, 8 (47.05%) samples were T4 genotypes and others were other Acanthamoeba genotypes (T1-T23). Therefore, approximately half of the genotypes belong to the pathogenic T4 genotype. ConclusionsThe water examined samples in western provinces of Iran have the potential risk factor for public health. Therefore, the efforts of healthcare providers are needed to identify, train, and prevention from human infections.

Genotyping of Environmental Isolates of Acanthamoeba in Hamadan, West of Iran.

Different genotypes of Acanthamoeba have been abundantly isolated in environmental samples such as water, soil, and dust, as well as in different hospital departments and eyewash stations. This protozoan is a potential hazard for immunocompromised patients and contact lens wearers. The aim of the present study was isolation and genotyping of environmental and corneal isolates of Acanthamoeba in Hamadan, west of Iran. During 2018-2020, a total of 104 environmental samples including, water, soil, and dust and 16 corneal scraping samples were collected and investigated for the presence of Acanthamoeba using morphological and molecular identification tools. Genotypes were determined using sequence analysis of the diagnostic fragment 3 (DF3) from Acanthamoeba-specific amplimer S1 (ASA.S1) gene. Phylogenetic tree was constructed with the MEGA7 software using Neighbor-Joining method. The presence of Acanthamoeba spp. was determined in 87.5% of water, 53.1% of soil, and 25% of dust samples. From 30 dust samples collected from eight wards of three hospitals, 7 (23.3%) were contaminated with Acanthamoeba. Sequencing analysis of environmental samples revealed that the T4 genotype was the most prevalent (92.6%) one. Genotypes T2 (1.9%), T2/T6 (1.9%), and mixed T4 and T2/T6 (3.7%) were also identified in environmental samples. Acanthamoeba was seen in none of the examined corneal scraping samples from patients with suspected keratitis. The widespread occurrence of this potentially pathogenic amoeba in most hospital wards and environmental resources and areas of the region highlights a strong need to increase awareness regarding this ubiquitous amoeba among susceptible individuals, such as immunocompromised patients and contact lens wearers.

Genotyping of Acanthamoeba Isolated from Hospital Environments and Thermal Water of Recreational Baths in Markazi Province, Iran.

Due to the opportunism character of Acanthamoeba, the presence of this parasite in the thermal water of recreational baths and hospital environments can be a risk to the health of staff, patients and others. The aim of this study was to investigate the distribution of potentially pathogenic Acanthamoeba genotypes isolated from the hospital environment and the thermal water of recreational baths in Markazi Province, central Iran. Overall, 180 samples including thermal water from recreational baths in Mahallat City and dust, soil and water from different hospitals of Arak, Farahan and Komijan cities, central Iran were collected. The presence of Acanthamoeba was investigated using microscopic examination and molecular methods. The PCR and sequencing was performed based on a specific 18S fragment of ribosomal DNA. Based on the microscopic survey, totally 134 positive samples were detected including 35% in thermal water samples and 44.7% in hospital samples. In molecular analysis, 53.5% of the samples were identified as Acanthamoeba and 46.7% as Protacanthamoeba bohemica. The genotypes were detected as T4 (33.3%), T2 (10%), T11 (6.7%), and T5 (3.3%). The T4 was the most common genotype found in hospitals sampling sites while the T2 genotype and P. bohemica were detected in thermal water sampling sites.

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

Research on free-living amoebae (FLA) and its public health implication as an etiologic agent of parasitic infection in humans has recently gained traction in the Philippines. This study aimed to identify potential FLAs in collected groundwater samples from Masinloc, Zambales, Philippines. Fifty-four (54) water samples were collected in 250-mL sterile polyethylene containers by purposive sampling from selected groundwater sources in six (6) barangays of Masinloc. The samples were vacuum filtered through a 1.2-μm pore glass microfiber filter, cultured onto non-nutrient agar (NNA) lawned with Escherichia coli, and observed microscopically for amoebic growth for 14 days using light microscopy. Amoebic growth was observed in 11.1% (6/54) of water samples. DNAs from positive samples were extracted and were made to react with polymerase chain reaction using Acanthamoeba-specific JDP1 (5'-GGCCCAGATCGTTTACCGTGAA-3') and JDP2 (5'-TCTCACAAGCTGCTAGGGAGTCA-3') primers, and universal primer Euk A (5'-AACCTGGTTGATCCTGCCAGT-3') and Euk B (5'-TGATCCTTCTGCAGGTTCACCTAC-3'). The presence of Acanthamoeba genotypes T4, T7, and T11 was confirmed using molecular and phylogenetic analysis. Our results confirmed that groundwater sources from two of six sampling sites (33.3%) in Masinloc, Zambales, were contaminated with potentially pathogenic FLAs. Proper identification of risk factors that may cause contamination consequently leads to the implementation of programs that will prevent future infections.

Occurrence and molecular characterization of Acanthamoeba, Naegleria fowleri and Blastocystis in water samples from various sources in Egypt

PCR-testing coupled to isolate sequencing was conducted to detect prevalence and various genotypes/subtypes of 3 neglected waterborne protists (Acanthamoeba, Naegleria fowleri and Blastocystis) in water samples from various sources in Dakahlia governorate, Egypt. Out of 62 protozoan-suspected samples by microscopy, Acanthamoeba was molecularly confirmed in 24 (38.7%) samples from various sources including tap water. Twenty Acanthamoeba isolates were successfully-sequenced; 18 were designated as the genotype T3 and 2 as T4. Naegleria spp. were detected in 6 (9.6%) samples from the Nile, of them 2 (3.2%) were identified as N. fowleri. Blastocystis spp. were found in 4 (6.4%) samples from waste water and ground water. Blastocystis subtype 2 was found in a sample from waste water, which may reflect human infection with this subtype and constitutes a public health hazard because waste water is occasionally discharged in the Nile with minimal treatments. Findings of the present study were analyzed in combination with those of earlier surveys from the other Egyptian governorates to evaluate the whole situation of the 3 protists in water from Egypt. Results of this analysis showed that Acanthamoeba had a high mean prevalence (43.03%) throughout Egypt, with insignificant variations among various water sources. Various Acanthamoeba genotypes were detected, and the highly pathogenic T4 was the most significantly identified type. A common T4 haplotype circulated in water from Egypt and 3 other countries (Tanzania, Rwanda, Uganda) located on the Nile basin, and included isolates from keratitis-infected patients, which confirms the potential role of water in the epidemiology of Acanthamoeba keratitis infecting humans in these countries. The estimated mean prevalence for Naegleria spp. was 23.79%, being the highest in the Nile water. In the present study, occurrence of 3 potentially pathogenic protists has been confirmed in water from Egypt, which should alert the authorities to revise the procedures for controlling these pathogens in water.

Distribution and Current State of Molecular Genetic Characterization in Pathogenic Free-Living Amoebae.

Free-living amoebae (FLA) are protozoa widely distributed in the environment, found in a great diversity of terrestrial biomes. Some genera of FLA are linked to human infections. The genus Acanthamoeba is currently classified into 23 genotypes (T1-T23), and of these some (T1, T2, T4, T5, T10, T12, and T18) are known to be capable of causing granulomatous amoebic encephalitis (GAE) mainly in immunocompromised patients while other genotypes (T2, T3, T4, T5, T6, T10, T11, T12, and T15) cause Acanthamoeba keratitis mainly in otherwise healthy patients. Meanwhile, Naegleria fowleri is the causative agent of an acute infection called primary amoebic meningoencephalitis (PAM), while Balamuthia mandrillaris, like some Acanthamoeba genotypes, causes GAE, differing from the latter in the description of numerous cases in patients immunocompetent. Finally, other FLA related to the pathologies mentioned above have been reported; Sappinia sp. is responsible for one case of amoebic encephalitis; Vermamoeba vermiformis has been found in cases of ocular damage, and its extraordinary capacity as endocytobiont for microorganisms of public health importance such as Legionella pneumophila, Bacillus anthracis, and Pseudomonas aeruginosa, among others. This review addressed issues related to epidemiology, updating their geographic distribution and cases reported in recent years for pathogenic FLA.