Amphizoic Amoebae Research Articles

Prevalence and molecular characterization of Entamoeba moshkovskii in diarrheal patients from Eastern India

Importance of the amphizoic amoeba Entamoeba moshkovskii is increasing in the study of amoebiasis as a common human pathogen in some settings. Limited studies are found on the genetic and phylogenetic characterization of E. moshkovskii from India; hence remain largely unknown. In this study, we determined the prevalence and characterized the E. moshkovskii isolates in eastern India. A three-year systemic surveillance study among a total of 6051 diarrhoeal patients from ID Hospital and BC Roy Hospital, Kolkata was conducted for E. moshkovskii detection via a nested PCR system targeting 18S rRNA locus. The outer primer set detected the genus Entamoeba and the inner primer pair identified the E. moshkovskii species. The 18S rRNA locus of the positive samples was sequenced. Genetic and phylogenetic structures were determined using DnaSP.v5 and MEGA-X. GraphPad Prism (v.8.4.2), CA, USA was used to analyze the statistical data. 4.84% (95%CI = 0.0433-0.0541) samples were positive for Entamoeba spp and 3.12% (95%CI = 0.027-0.036) were infected with E. moshkovskii. E. moshkovskii infection was significantly associated with age groups (X2 = 26.01, P<0.0001) but not with gender (Fisher's exact test = 0.2548, P<0.05). A unique seasonal pattern was found for E. moshkovskii infection. Additionally, 46.56% (95%CI = 0.396-0.537) were sole E. moshkovskii infections and significantly associated with diarrheal incidence (X2 = 335.5,df = 9; P<0.0001). Sequencing revealed that the local E. moshkovskii strains were 99.59%-100% identical to the prototype (GenBank: KP722605.1). The study found certain SNPs that showed a correlation with clinical features, but it is not necessarily indicative of direct control over pathogenicity. However, SNPs in the 18S rRNA gene could impact the biology of the amoeba and serve as a useful phylogenetic marker for identifying pathogenic E. moshkovskii isolates. Neutrality tests of different coinfected subgroups indicated deviations from neutrality and implied population expansion after a bottleneck event or a selective sweep and/or purifying selection in co-infected subgroups. The majority of FST values of different coinfected subgroups were <0.25, indicating low to moderate genetic differentiation within the subgroups of this geographical area. The findings reveal the epidemiological significance of E. moshkovskii infection in Eastern India as the first report in this geographical area and expose this species as a possible emerging enteric pathogen in India. Our findings provide useful knowledge for further research and the development of future control strategies against E. moshkovskii.

An Insight into the Genome of Pathogenic and Non-Pathogenic Acanthamoeba.

Acanthamoeba are amphizoic amoeba majorly responsible for causing Acanthamoeba keratitis (AK) and Granulomatous amoebic encephalitis (GAE). Despite its ubiquitous nature, the frequency of infections is not high, probably due to the existence of non-pathogenic isolates. The whole-genome sequencing and an annotated genome assembly can unravel the biological functions and help in identifying probable genes related to pathogenicity. Illumina and Nanopore sequencing were performed for keratitis, encephalitis, and non-pathogenic environmental isolates. Hybrid assembly was prepared for the AK and GAE isolates, while only the Illumina reads were utilized for a non-pathogenic environmental isolate. Protein coding genes were identified using the GeneMark-ES program and BLASTx module of Diamond used for gene prediction. Additionally, the Kyoto Encyclopedia of Genes and Genomes annotation and cluster of orthologous group's annotation using RPS-blast against the CDD database was performed. The subsequent data analysis and validation will help identify probable pathogenic genes. The genome assemblies of 9.67, 8.34, and 8.89 GBs were reported for GAE, AK, and non-pathogenic isolate, respectively. KEGG reported 22,946 in GAE, 24,231 in keratitis, and 9367 genes in the environmental isolate. The COG annotation revealed 3232 in GAE, 3403 in keratitis, and 1314 genes in the non-pathogenic isolate. The present study has attempted to generate de novo hybrid genome assemblies of Acanthamoeba that would help decode the genome of free-living amoeba and will provide genomic data for a better understanding of virulence-related factors.

Tannic Acid-Modified Silver Nanoparticles in Conjunction with Contact Lens Solutions Are Useful for Progress against the Adhesion of Acanthamoeba spp. to Contact Lenses.

Acanthamoeba spp. are amphizoic amoebae that are widely distributed in the environment and capable of entering the human body. They can cause pathogenic effects in different tissues and organs, including Acanthamoeba keratitis (AK), which may result in a loss of visual acuity and blindness. The diagnostics, treatment, and prevention of AK are still challenging. More than 90% of AK cases are related to the irresponsible wearing of contact lenses. However, even proper lens care does not sufficiently protect against this eye disease, as amoebae have been also found in contact lens solutions and contact lens storage containers. The adhesion of the amoebae to the contact lens surface is the first step in developing this eye infection. To limit the incidence of AK, it is important to enhance the anti-adhesive activity of the most popular contact lens solutions. Currently, silver nanoparticles (AgNPs) are used as modern antimicrobial agents. Their effectiveness against Acanthamoeba spp., especially with the addition of plant metabolites, such as tannic acid, has been confirmed. Here, we present the results of our further studies on the anti-adhesion potential of tannic acid-modified silver nanoparticles (AgTANPs) in combination with selected contact lens solutions against Acanthamoeba spp. on four groups of contact lenses. The obtained results showed an increased anti-adhesion activity of contact lens solutions in conjunction with AgTANPs with a limited cytotoxicity effect compared to contact lens solutions acting alone. This may provide a benefit in improving the prevention of amoebae eye infections. However, there is still a need for further studies on different pathogenic strains of Acanthamoeba in order to assess the adhesion of the cysts to the contact lens surface and to reveal a more comprehensive picture of the activity of AgTANPs and contact lens solutions.

Diseases Caused by Amoebae in Fish: An Overview.

Simple SummaryAmoebae can be found in many different aquatic environments and are also an emerging risk for fish health. Amoebae can display different types of relationships with fish, some of them (amoeba acting as commensals) do not harm fish. However, in many cases they can act as parasites and can be the cause of severe diseases affecting mainly the gills and also causing relevant systemic infections.Parasitic and amphizoic amoebae are ubiquitous and can affect a huge variety of hosts, from invertebrates to humans, and fish are not an exception. Most of the relationships between amoebae and fish are based on four different types: ectocommensals, ectoparasites, endocommensals and endoparasites, although the lines between them are not always clear. As ectocommensals, they are located specially on the gills and particularly the amphizoic Neoparamoeba perurans is the most relevant species, being a real pathogenic parasite in farmed salmon. It causes amoebic gill disease, which causes a progressive hyperplasia of epithelial cells in the gill filaments and lamellae. Nodular gill disease is its analogue in freshwater fish but the causative agent is still not clear, although several amoebae have been identified associated to the lesions. Other species have been described in different fish species, affecting not only gills but also other organs, even internal ones. In some cases, species of the genera Naegleria or Acanthamoeba, which also contain pathogenic species affecting humans, are usually described affecting freshwater fish species. As endocommensals, Entamoebae species have been described in the digestive tract of freshwater and marine fish species, but Endolimax nana can reach other organs and cause systemic infections in farmed Solea senegalensis. Other systemic infections caused by amoebae are usually described in wild fish, although in most cases these are isolated cases without clinical signs or significance.

Molecular characterization of Acanthamoeba isolated from contact lens paraphernalia: An evidence of potentially pathogenic T4 genotype in Malaysia

Background: Amphizoic amoebae generate a serious human health threat due to their pathogenic potential as facultative parasites, causative agents of vision-threatening Acanthamoeba keratitis (AK). Recently, AK incidences have been reported with increasing frequency worldwide, particularly in contact lens wearers. Therefore, this study was designed to identify the genotypes and investigate the pathogenic potential of Acanthamoeba isolates. Methods & Materials: A cross-sectional study was conducted among 180 volunteers who provided their contact lenses, lens storage case and lens solution between 2018 and 2019. All samples were inoculated onto 1.5% non-nutrient agar plates overlaid with Escherichia coli and incubated at 30 °C for 14 days. Polymerase chain reaction was performed on positive isolates and the amplified products were sequenced and compared with GenBank Data. Pathogenic potential of positive isolates were further tested with thermo- and osmo-tolerance assays. Results: Acanthamoeba was successfully isolated from 14 (7.8%) culture-positive samples. Analysis of 18S ribosomal RNA gene indicated that all sequenced positive isolates shared 98–100% homology with Acanthamoeba genotype T4. In vitro assays revealed that 9 (64.2%) Acanthamoeba isolates are able to grow at 42 °C and 1.0 M, indicated a highly pathogenic strain. Conclusion: This is the first report of genotypic identification of Acanthamoeba from contact lens paraphernalia in Malaysia. Potentially pathogenic T4 genotype isolated in this study is the most prevalent genotype in human ocular infection worldwide. Hence, this information would be useful in selecting suitable strain to develop immunological and/or molecular diagnostic assays in the future.

Morphological Description of the Early Events during the Invasion of Acanthamoeba castellanii Trophozoites in a Murine Model of Skin Irradiated under UV-B Light.

Skin infections have been associated with Acanthamoeba, nevertheless the events during skin invasion and UV-B light effects on it are unknown. The early morphological events of Acanthamoeba castellanii skin invasion are shown in SKH-1 mice that were chronically UV-B light irradiated. Mice that developed skin lesions (group 1) were topical and intradermally inoculated with A. castellanii trophozoites and sacrificed 48 h or 18 days later. Mice that showed no skin lesions (group 2) were intradermally inoculated and sacrificed 24, 48 or 72 h later. Mice ventral areas were considered controls with and without trophozoites intradermally inoculated. Skin samples were processed by histological and immunohistochemistry techniques. In group 1, trophozoites were immunolocalized in dermal areas, hair cysts, sebaceous glands, and blood vessels, and collagen degradation was observed. One of these mice shown trophozoites in the spleen, liver, and brain. In group 2, few trophozoites nearby collagenolytic activity zones were observed. In control samples, nor histological damage and no trophozoites were observed. Adherence and collagenolytic activity by A. castellanii were corroborated in vitro. We can infer that UV-B light irradiated skin could favor A. castellanii invasiveness causing damage in sites as far away as the brain, confirming the invasive capacity and pathogenic potential of these amphizoic amoebae.

Changes in distribution, morphology and ultrastructure of chloride cell in Atlantic salmon during an AGD infection.

Amoebic gill disease (AGD) is emerging as one of the most significant health challenges affecting farmed Atlantic salmon in the marine environment. It is caused by the amphizoic amoeba Neoparamoeba perurans, with infestation of gills causing severe hyperplastic lesions, compromising overall gill integrity and function. This study used histology, transmission electron microscopy (TEM), immunohistochemistry and transcript expression to relate AGD-associated pathological changes to changes in the morphology and distribution of chloride cells (CCs) in the gills of Atlantic salmon (Salmo salar L.) showing the progression of an AGD infection. A marked reduction in numbers of immunolabelled CCs was detected, and a changing pattern in distribution and morphology was closely linked with the level of basal epithelial hyperplasia in the gill. In addition, acute degenerative ultrastructural changes to CCs at the lesion site were observed with TEM. These findings were supported by the early-onset downregulation of Na+ /K+ -ATPase transcript expression. This study provides supportive evidence that histological AGD lesion assessment was a good qualitative tool for AGD scoring and corresponded well with qPCR genomic Paramoeba perurans quantification. Ultrastructural changes induced in salmon CCs as a result of AGD are reported here for the first time.

Evaluation of in vitro effect of selected contact lens solutions conjugated with nanoparticles in terms of preventive approach to public health risk generated by Acanthamoeba strains.

Various Acanthamoeba species are free-living organisms widely distributed in the human environment. Amphizoic amoebae as facultative parasites may cause vision-threatening eye disease - Acanthamoeba keratitis, mostly among contact lens wearers. As the number of cases is increasing, and applied therapy often unsuccessful, proper hygienic measures and effective contact lenses disinfection are crucial for the prevention of this disease. Available contact lens solutions are not fully effective against amphizoic amoebae; there is a need to enhance their disinfecting activity to prevent amoebic infections. The use of developing nanotechnology methods already applied with success in the prevention, diagnostic and therapy of other infectious diseases might be helpful regarding amoebic keratitis. This study assesses the in vitro effect of selected contact lens solutions conjugated with nanoparticles against Acanthamoeba trophozoites. Three selected contact lens solutions conjugated with silver and gold nanoparticles in concentration of 0.25-2.5 ppm were used in vitro against the axenically cultured ATCC 30010 type Acanthamoeba castellanii strain. The anti-amoebic efficacy was examined based on the oxido-reduction of AlamarBlue. The cytotoxicity tests based on the measurement of lactate dehydrogenase (LDH) activity were performed using a fibroblast HS-5 cell line. Enhancement of the anti-amoebic activity of contact lens solutions conjugated with selected nanoparticles expressed in the dose dependent amoebic growth inhibition with a low cytotoxicity profile was observed. Results of the study showed that conjugation of selected contact lens solutions with silver nanoparticles might be a promising approach to prevent Acanthamoeba keratitis among contact lens users.

Promising in vitro effects of selected antiseptics against Acanthamoeba Polyphaga strain - the etiological agent of vision-threatening keratitis identified in polish patient

Purpose: In the last decades, Acanthamoeba keratitis, the vision-threatening corneal infection is detected with increasing frequency in Poland. As the disease is difficult to treatdue to extremely high resistance of cysts to chemicals, agents of possible anti-amoebic activity are still tested. In this study, A. polyphagaisolate of T4 genotype from patient with complicated keratitis, a history of swimming in a lake, not using contact lensesand A. castellanii Neff strain were evaluated in terms of their in vitro susceptibility to selected antiseptics. Methods & Materials: Samples of the strains cultured under bacteria-free conditions in BSC calf serum-enriched culture mediumwere exposed to antimicrobial agent Povidone-iodine and Aerodesin disinfectant. Effects of the compounds on Acanthamoeba strains following 48, 96 and 120 hrs exposure compared to the control cultures were monitored: the developmental form dynamics, encystation process, overall number of surviving amoebae, changes in cysts/trophozoites/proportion assessed in the exponential growth phase were determined. Results: The diagnosis was confirmed by confocal microscopy, in vitro culture and by 18S rDNA gene sequences (T4 genotype). Both antiseptics showed some amoebostatic effects on Acanthamoeba strains. Povidone iodine after 120 h from application reduced number of viable amoebae to 17% in comparison to the control samples (100%); Aerodesin indicated expected cysticidal efficacy: no amoebae after 92 h exposure were detected. Conclusion: Risk factors of Acanthamoeba keratitis diagnosed mainly in contact lens wearers include also ocular exposure to water containing cysts and trophozoites of the amphizoic amoebae. Promising in vitro effects of the chemicals against A. polyphaga strain, etiological agent of vision-threatening keratitis justifies further tests with a modified agent application pattern particularly in term of expected cysticidal efficacy.

Thecamoeba quadrilineata (Amoebozoa, Lobosa) as a new member of amphizoic amoebae-first isolation from endozoic conditions.

A free-living soil amoeba Thecamoeba quadrilineata (Carter, 1856) Lepşi, 1960 (Amoebozoa: Thecamoebidae) was isolated from endozoic conditions for the first time. Presence of amoebae was detected after 4days following inoculation of the gut of the earthworm Lumbricus terrestris on agar plate with Escherichia coli. On the basis of our isolation, we consider T. quadrilineata as further amphizoic amoeba species. This study enlarges the range of amphizoic tendency in members of the genus Thecamoeba and stresses the need for further research on the pathogenic potential of Thecamoeba species.

Comparative examination on selected amphizoic amoebae in terms of their in vitro temperature tolerance – a possible indirect marker of potential pathogenicity of Acanthamoeba strains

Acanthamoeba species are ubiquitous in natural and man-made environments worldwide; some strains are able to colonize human eyes as facultative parasites. It has been shown that environmental and clinical isolates/species of Acanthamoeba vary in their pathogenicity. In this study we examine and compare the in vitro effects of the changing temperature on the population dynamics of subsequent amoebic strains. Identification of Acanthamoeba strain by morphological and molecular methods and temperature assays were performed. Monitoring of the corneal and environmental strains showed changes in population densities and a termo-tolerance correlating with pathogenicity of amoebae. Comparative assessment of results indicated differences in viability of amoebic populations in exponential growth phase in vitro cultivation. The increased awareness of the threat is needed for better understanding of impact of factors examined on pathogenesis in human infected with Acanthamoeba strains.

Type 2 diabetes mellitus BALB/c mice are more susceptible to granulomatous amoebic encephalitis: Immunohistochemical study

Granulomatous amoebic encephalitis (GAE) is a chronic, difficult to resolve infection caused by amphizoic amoebae of the genus Acanthamoeba, which in most cases occurs in immunosuppressed persons or with chronic diseases such as diabetes. In this study, we describe the early events of A. culbertsoni infection of GAE in diabetic mice model. Diabetes was induced in male BALB/c mice, with a dose of streptozotocin (130 mg/kg). Healthy and diabetic mice were inoculated via intranasal with 1 × 106 trophozoites of A. culbertsoni. Then were sacrificed and fixed by perfusion at 24, 48, 72 and 96 h post-inoculation, the brains and nasopharyngeal meatus were processed to immunohistochemical analysis. Invasion of trophozoites in diabetic mice was significantly greater with respect to inoculated healthy mice. Trophozoites and scarce cysts were immunolocalized in respiratory epithelial adjacent bone tissue, olfactory nerve packets, Schwann cells and the epineurium base since early 24 h post-inoculation. After 48 h, trophozoites were observed in the respiratory epithelium, white matter of the brain, subcortical central cortex and nasopharyngeal associated lymphoid tissue (NALT). At 72 h, cysts and trophozoites were immunolocalized in the olfactory bulb with the presence of a low inflammatory infiltrate characterized by polymorphonuclear cells. Scarce amoebae were observed in the granular layer of the cerebellum without evidence of inflammation or tissue damage. No amoebas were observed at 96 h after inoculation, suggesting penetration to other tissues at this time. In line with this, no inflammatory infiltrate was observed in the surrounding tissues where the amoebae were immunolocalized, which could contribute to the rapid spread of infection, particularly in diabetic mice.All data suggest that trophozoites invade the tissues by separating the superficial cells, penetrating between the junctions without causing cytolytic effect in the adjacent cells and subsequently reaching the CNS, importantly, diabetes increases the susceptibility to amoebae infection, which could favor the GAE development.

Isolation and molecular characterization of Acanthamoeba from patients with keratitis in Spain

In order to improve our knowledge on the epidemiology of amoebic keratitis, as well as the identification of Acanthamoeba isolates, we have isolated Acanthamoeba spp. from five symptomatic patients in Spain in the present study. All isolates were grown in axenic liquid medium, with only one exception. The morphology of these isolates were characterized by optical and scanning electron microscopy. Their structural features corresponded to those of amphizoic amoebae (namely Acanthamoeba spp.). The molecular characterization of the five Acanthamoeba isolates yielded six sequences. Almost complete 18S rRNA gene sequences (>2000bp) were obtained from three isolates and partial sequences (∼1500bp) from the other two. A robust phylogenetic analysis based on the almost complete 18S rRNA sequence showed that four isolates belonged to the T4 genotype and the other one to the T3 genotype. However, all isolates were identified as T4 genotype using the ASA.S1 fragment. As previously suggested by other researchers, only a robust phylogenetic approach may be helpful in identifying Acanthamoeba genotypes. In addition, new data on the phylogenetic relationships among the Acanthamoeba genotypes is provided and discussed.

Effects of some amphizoic amoeba spread in human environments - new incidences of the emerging threat with the vision-threatening disease of public health significance

Effects of some amphizoic amoeba spread in human environments - new incidences of the emerging threat with the vision-threatening disease of public health significance

Emerging Threats for Human Health in Poland: Pathogenic Isolates from Drug Resistant Acanthamoeba Keratitis Monitored in terms of Their In Vitro Dynamics and Temperature Adaptability.

Amphizoic amoebae generate a serious human health threat due to their pathogenic potential as facultative parasites, causative agents of vision-threatening Acanthamoeba keratitis (AK). Recently, AK incidences have been reported with increasing frequency worldwide, particularly in contact lens wearers. In our study, severe cases of AK in Poland and respective pathogenic isolates were assessed at clinical, morphological, and molecular levels. Misdiagnoses and the unsuccessful treatment in other ophthalmic units delayed suitable therapy, and resistance to applied chemicals resulted in severe courses and treatment difficulties. Molecular assessment indicated that all sequenced pathogenic corneal isolates deriving from Polish patients with AK examined by us showed 98–100% homology with Acanthamoeba genotype T4, the most prevalent genotype in this human ocular infection worldwide. In vitro assays revealed that the pathogenic strains are able to grow at elevated temperature and have a wide adaptive capability. This study is our subsequent in vitro investigation on pathogenic Acanthamoeba strains of AK originating from Polish patients. Further investigations designed to foster a better understanding of the factors leading to an increase of AK observed in the past years in Poland may help to prevent or at least better cope with future cases.

Isolation, identification and biological characterization of Acanthamoeba polyphaga from suspected cases of eye patients and its electron microscopy

Corneal scrapings and tear drop of 90 patients with suspected cases of keratitis and eye infection attending the Govt. Hospital and rural areas patients of Lucknow city were screened for amoebic pathogens including Acanthamoeba. Of these, 6 (6.66percent) were positive for Acanthamoeba, which fulfilled the criteria for suspecting Acanthamoeba keratitis. These were distributed among all ages with the maximum numbers in the 20-45 year age group. None of the patients were using contact lenses and were mostly agricultural labourers and had recent history of swimming in ponds and river and also associated with water sports. The predisposing factor found in this study was trauma of varying degrees. The result of present study confirmed that Amphizoic amoebae colonized almost every conceivable aquatic habitat. This is alarming signal that shows presence of Amphizoic amoebae. Incidences of infection during warm season have been traced in patients suffering from eye infection. Preventive measures include public awareness and maintenance of water body and adequate chlorination.

First report of amphizoic amoebae isolated from edible Oyster mushroom- Pleurotus sajor-caju (Singer, 1949)

The present document serves as the first evidence that describes the presence of amphizoic amoebae in cap, stalk and internal tissues such as gills of the edible Oyster mushrooms, Pleurotus sajor-caju, packaged, and sold for public consumption. Samples tested over a 9 month period from different vegetable markets of Lucknow city revealed that out of 180 mushrooms, 176 (97.77%) yielded amphizoic amoebae, namely, Schizopyrenus, Naegleria, Hartmannella, Acanthamoeba and Vanella sp. Four samples of internal tissues yielded 6 strains of amoebae. A total of 17 Naegleria strains were isolated, out of which 5 were identified as Naegleria fowleri and were tested for their pathogenicity. The strains 2BNf01 and 5BNf04 were mildly pathogenic and strains, 4ANf02i, 4ANf03 and 8ANf05i were highly pathogenic. It was also observed that the most frequently observed species of amoebae belonged to the genus Acanthamoeba and a total 103 Acanthamoeba strains recorded.

A preliminary study on amphizoic amoebae with special reference to their preference for bacterial food

The present study was planned to screen the growth pattern of six different species of pathogenic and non pathogenic amphizoic amoebae viz. Naegleria fowleri, N. gruberi, Acanthamoeba culbertsoni, A. rhysodes, A. polyphaga and A. glebae using six different bacterial species like Escherichia coli (Strain E1 and E2 and E. coli lactose +ve), Proteus, Klebsiella and Pseudomonas as food in their in vitro growth on non-nutrient agar medium. It was observed that out of six amoebae used; the pathogenic N. fowleri and A. culbertsoni were feeding on E. coli (all the strains). Feeding these two species of bacteria, the growth of these two amoebae was luxuriant, but not so good while feeding other strains of bacteria though they fed, survived and formed cysts. The remaining four amoebae were found to feed and survive only on E. coli (all the strain) and formed cysts but showed very poor growth while feeding on other four bacterial strains. It was inferred that E. coli is the most suitable bacterial species for in vitro growth of amphizoic amoebae for various purposes. This also reiterates that there exists a complex inter-relationship between amoebae and bacteria in different habitats.

Acanthamoeba castellanii: In vitro effects of selected biological, physical and chemical factors

Trophozoites and cysts of free-living Acanthamoeba castellanii present a serious risk to human health as causative agents of human diseases such as fatal granulomatous amoebic encephalitis and Acanthamoeba keratitis that is reported from various part of the world, also in Poland, with increasing frequency, particularly in the contact lens wearers. The amphizoic amoebae are generally extremely resistant to different chemical agents, however, several strains/isolates within A. castellanii may differ in virulence. Among the features considered as associated with the amoeba pathogenicity, temperature tolerance and resistance to different environmental conditions are reported. In the present study, A. castellanii strain cultured in 26 °C after several year passages were tested for sensibility/tolerance to instant temperature changes as well as exposition to deuterium oxide, D 2O. Significant decrease of number of viable amoebae during in vitro exposition to D 2O occurred, but no changes in trophozoites/cysts ratio. The ability of the strain examined to develop in higher temperature may indicate a wide adaptation reserve and its pathogenic potential.

Acanthamoeba: Biology and Pathogenesis

The importance of the genus Acanthamoeba has been growing because of the increasing number of human infections and the ability as a reservoir host for pathogenic microbes of this amphizoic amoeba. The most important value of the book is the variety of topics on Acanthamoeba research. The next will be the easy written sentences to follow for the beginners of Acanthamoeba research even for those who live in non-English spoken countries. The author reviewed almost all aspects of Acanthamoeba research to date and composed this book with 9 sections; Biology and Phylogeny, Life Cycle and Genotyping, Acanthamoeba Infections, Pathogenesis, Immune Response, Strategies against Acanthamoeba Infections, Acanthamoeba; Trojan Horse of the Microbial World, Conclusions and Future Studies, and Bibliography. Each section describes central themes and gives the outline of recent research on each topic. One of the excellency of this book is numerous diagrams and illustrations to schematize the concepts and cascades of chemical reactions in metabolism and pathogenesis. Some figures showing the basic principle concepts of Acanthamoeba research especially in pathogenesis are expected to be cited continuously to describe these principles. The most creative section of this book to me is 'Acanthamoeba; Trojan Horses of the Microbial World'. This section describes the role and importance of Acanthamoeba as a vector or a vehicle for pathogenic microbes from environment to humans. The author also introduced the historical event of Legionnaires' disease happened in 1976 in detail. In the 'Conclusions' section, the author listed the future research tasks on Acanthamoeba. Well trained researchers are needed to propel the development of strategies in the prevention, pathogenesis, and therapy for Acanthamoeba infection and this book will be very helpful for this purpose.