Opportunistic Free-living Amoebae Research Articles

Nanoparticle-Terpene Fusion: A Game-Changer in Combating Primary Amoebic Meningoencephalitis Caused by Naegleria fowleri.

Pathogenic Naegleria fowleri (N. fowleri) are opportunistic free-living amoebae and are the causative agents of a very rare but severe brain infection called primary amoebic meningoencephalitis (PAM). The fatality rate of PAM in reported cases is more than 95%. Most of the drugs used againstN. fowleri infections are repurposed drugs. Therefore, a large number of compounds have been tested againstN. fowleri in vitro, but most of the tested compounds showed high toxicity and an inability to cross the blood-brain barrier. Andrographolide, forskolin, and borneol are important natural compounds that have shown various valuable biological properties. In the present study, the nanoconjugates (AND-AgNPs, BOR-AgNPs, and FOR-AgNPs) of these compounds were synthesized and assessed against both stages (trophozoite and cyst) ofN. fowleri for their antiamoebic and cysticidal potential in vitro. In addition, cytotoxicity and host cell pathogenicity were also evaluated in vitro. FOR-AgNPs were the most potent nanoconjugate and showed potent antiamoebic activity againstN. fowleriwith an IC50 of 26.35 μM. Nanoconjugates FOR-AgNPs, BOR-AgNPs, and AND-AgNPs also significantly inhibit the viability of N. fowleri cysts. Cytotoxicity assessment showed that these nanoconjugates caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 μg/mL, while also effectively reducing the cytopathogenicity of N. fowleri trophozoites to the HaCaT cells. The outcomes of our experiments have unveiled substantial potential for AND-AgNPs, BOR-AgNPs, and FOR-AgNPs in the realm of developing innovative alternative therapeutic agents to combat infections caused by N. fowleri. This study represents a significant step forward in the pursuit of advanced strategies for managing such amoebic infections, laying the foundation for the development of novel and more effective therapeutic modalities in the fight against free-living amoebae.

Malabaricones from the fruit of Myristica cinnamomea King as potential agents against Acanthamoeba castellanii

Acanthamoeba castellanii is an opportunistic free-living amoeba (FLA) pathogen which can cause fatal central nervous system (CNS) infection, granulomatous amoebic encephalitis (GAE) and potentially blinding ocular infection, Acanthamoeba keratitis (AK). Acanthamoeba species remain a challenging protist to treat due to the unavailability of safe and effective therapeutic drugs and their ability to protect themselves in the cyst stage. Natural products and their secondary metabolites play a pivotal role in drug discovery against various pathogenic microorganisms. In the present study, the ethyl acetate extract of Myristica cinnamomea King fruit was evaluated against A. castellanii (ATCC 50492), showing an IC50 of 45.102 ± 4.62 µg/mL. Previously, the bio-guided fractionation of the extract resulted in the identification of three active compounds, namely Malabaricones (A-C). The isolated and thoroughly characterized acylphenols were evaluated for their anti-amoebic activity against A. castellanii for the first time. Among tested compounds, Malabaricone B (IC50 of 101.31 ± 17.41 µM) and Malabaricone C (IC50 of 49.95 ± 6.33 µM) showed potent anti-amoebic activity against A. castellanii trophozoites and reduced their viability up-to 75 and 80 %, respectively. Moreover, both extract and Malabaricones also significantly (p < 0.05) inhibit the encystation and excystation of A. castellanii, while showed minimal toxicity against human keratinocyte cells (HaCaT cells) at lower tested concentrations. Following that, the explanation of the possible mechanism of action of purified compounds were assessed by detection of the state of chromatin. Hoechst/PI 33342 double staining showed that necrotic cell death occurred in A. castellanii trophozoites after 8 h treatment of Malabaricones (A-C). These findings demonstrate that Malabaricones B and C could serve as promising therapeutic options against A. castellanii infections.

Anti-amoebic effects of synthetic acridine-9(10H)-one against brain-eating amoebae

Pathogenic A. castellanii and N. fowleri are opportunistic free-living amoebae. Acanthamoeba spp. are the causative agents of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK), whereas Naegleria fowleri causes a very rare but severe brain infection called primary amebic meningoencephalitis (PAM). Acridinone is an important heterocyclic scaffold and both synthetic and naturally occurring derivatives have shown various valuable biological properties. In the present study, ten synthetic Acridinone derivatives (I-X) were synthesized and assessed against both amoebae for anti-amoebic and cysticidal activities in vitro. In addition, excystation, encystation, cytotoxicity, host cell pathogenicity was also performed in-vitro. Furthermore, molecular docking studies of these compounds with three cathepsin B paralogous enzymes of N. fowleri were performed in order to predict the possible docking mode with pathogen. Compound VII showed potent anti-amoebic activity against A. castellanii with IC50 53.46 µg/mL, while compound IX showed strong activity against N. fowleri in vitro with IC50 72.41 µg/mL. Compounds II and VII showed a significant inhibition of phenotypic alteration of A. castellanii, while compound VIII significantly inhibited N. fowleri cysts. Cytotoxicity assessment showed that these compounds caused minimum damage to human keratinocyte cells (HaCaT cells) at 100 µg/mL, while also effectively reduced the cytopathogenicity of Acanthamoeba to HaCaT cells. Moreover, Cathepsin B protease was investigated in-silico as a new molecular therapeutic target for these compounds. All compounds showed potential interactions with the catalytic residues. These results showed that acridine-9(10H)-one derivatives, in particular compounds II, VII, VIII and IX hold promise in the development of therapeutic agents against these free-living amoebae.

Real-Time PCR for the Diagnosis of Acanthamoeba Genotype T4.

Acanthamoeba spp. are ubiquitous and opportunistic free-living amoebae (FLA) that can cause Acanthamoeba keratitis and other infections in the human host. A quick and efficient diagnosis is often challenging. Our study aimed to establish a qPCR assay to detect and, at the same time, quantify the predominant Acanthamoeba genotype T4. DNA from clinical corneal scrapings and Acanthamoeba reference strains, including genotypes T3, T4, T5, T6, T10, T11, and T12, were used to develop the new T4 assay and it was compared to published protocols and one commercial kit for evaluation. The T4 assay showed no amplification with Acanthamoeba genotypes T3, T5, T6, T10, T11, and T12. The efficiencies ranged from 92.01 to 97.59% (R2 of 0.9768 to 0.9951). The calculated LOD range was 3.63 to 33.27 cells/µL. The protocol published by Qvarnstrom and colleagues was more sensitive compared to the other assays, and an overall good agreement was observed between the new T4 and the Qvarnstrom assays. We successfully developed and validated a genotype T4 assay that could be run in duplex with the Qvarnstrom assay to reliably and simultaneously diagnose Acanthamoeba genotype T4 and other genotypes from clinical samples.

Discovery of Amoebicidal Compounds by Combining Computational and Experimental Approaches.

Pathogenic and opportunistic free-living amoebae such as Acanthamoeba spp. can cause keratitis (Acanthamoeba keratitis [AK]), which may ultimately lead to permanent visual impairment or blindness. Acanthamoeba can also cause rare but usually fatal granulomatous amoebic encephalitis (GAE). Current therapeutic options for AK require a lengthy treatment with nonspecific drugs that are often associated with adverse effects. Recent developments in the field led us to target cAMP pathways, specifically phosphodiesterase. Guided by computational tools, we targeted the Acanthamoeba phosphodiesterase RegA. Computational studies led to the construction and validation of a homology model followed by a virtual screening protocol guided by induced-fit docking and chemical scaffold analysis using our medicinal and biological chemistry (MBC) chemical library. Subsequently, 18 virtual screening hits were prioritized for further testing in vitro against Acanthamoeba castellanii, identifying amoebicidal hits containing piperidine and urea imidazole cores. Promising activities were confirmed in the resistant cyst form of the amoeba and in additional clinical Acanthamoeba strains, increasing their therapeutic potential. Mechanism-of-action studies revealed that these compounds produce apoptosis through reactive oxygen species (ROS)-mediated mitochondrial damage. These chemical families show promise for further optimization to produce effective antiacanthamoebal drugs.

Ac-HSP20 Is Associated With the Infectivity and Encystation of Acanthamoeba castellanii.

Acanthamoeba castellanii is a pathogenic and opportunistic free-living amoeba that causes Acanthamoeba keratitis (AK) and granulomatous amebic encephalitis (GAE) in immunocompromised individuals. The biological and pathogenic characterizations behind this opportunistic protozoan is not fully understood. This study aimed to determine the biological functions of heat shock protein (HSP)-20 of A. castellanii (Ac-HSP20) involved in the maintenance of life cycle and the infectivity of A. castellanii. Immunoscreening A. castellanii cDNA library with A. castellanii infected rabbit sera identified three positive clones, one of them was a putative heat shock protein (Ac-HSP20). The recombinant 23 kDa Ac-HSP20 protein (rAc-HSP20) was successfully expressed in Escherichia coli BL21 (DE3) and purified using metal affinity chromatography. The rabbits immunized with rAc-HSP20 produced high titer antibody (1:25,600). Immunolocalization with the antibody identified the expression of native Ac-HSP20 on the surface of both A. castellanii trophozoites and cysts. Further, Western blot with antibody identified that the expression of native Ac-HSP20 was 7.5 times higher in cysts than in trophozoites. Blocking Ac-HSP20 on the membrane of trophozoites with specific antibody or silencing Ac-hsp20 gene transcription by siRNA inhibited their transformation into cysts at the early stage but returned to normal at the late stage by stimulating the transcription of Ac-hsp20. Incubation of trophozoites with anti-Ac-HSP20 IgG increased macrophage-involved phagocytosis to the protozoa and inhibited trophozoite infectivity on the cornea of rabbits compared with that without antibody. Our study provides that Ac-HSP20 is a surface antigen involved in the encystation and infectivity of A. castellanii and thus an important target for vaccine and drug development.

Reviewing the Effects of Miltefosine and Suggesting It for the Treatment of Coronavirus Disease (COVID-19).

Objective:Miltefosine is an anti-cancer drug used to treat leishmaniasis and deadly opportunistic free-living amoeba and other deadly pathogenic microorganisms. Several studies have demonstrated its antiviral effect. In this study, we discuss the effectiveness of this drug on pathogenic microorganisms, and according to the functional system of the medicine, we present this drug as a therapeutic proposal to treat Coronavirus disease (COVID-19)Methods:A literature search was conducted in electronic databases, including Pubmed, Science Direct, Elsevier, and Google Scholar, and articles published from 2006 to 2020 (the last decade) were selected. The search keywords included Miltefosine, microorganism, pathogen, and treatment.Results:The studies indicated that Miltefosine had therapeutic effects on leishmaniasis and deadly opportunistic free-living amoeba and other deadly pathogenic microorganisms. Several studies have proven its antiviral effect.Conclusion:Owing to the beneficial effects of this drug on pathogenic and deadly microorganisms and antiviral effects, and due to the epidemic of Coronavirus and the lack of effective treatment and vaccine, this drug is recommended as one of the treatment options for this disease.

Molecular Identification and Genotyping of Acanthamoeba Isolated from Environmental Sources in Kashan, Central Iran

Background: Acanthamoeba is one of the most common opportunistic free-living amoebae, with ubiquitous presence in various en- vironmental sources. Pathogenic strains of Acanthamoeba are the causative agents of amoebic keratitis and granulomatous amoebic encephalitis. Objectives: The aim of the present study was to identify Acanthamoeba genotypes in soil, hospital dust, and stagnant water samples from Kashan, Central Iran. Methods: In this cross sectional study, a total of 122 samples from soil (n, 32), hospital dust (n, 40), and stagnant water (n, 50) were collected and examined for the presence of free-living amoebae and Acanthamoeba species. All the samples were cultured onto non-nutrient agar plates for detection of free-living amoebae. Acanthamoeba species was identified by polymerase chain reaction (PCR) assay, using specific primers. A total of 29 Acanthamoeba isolates were sequenced, and different genotypes were detected via sequence analysis. Results: The results showed that 82.8% (101/122) of samples were positive for free-living amoebae. The PCR assay revealed that 62.5%, 52.5%, and 50% of soil, hospital dust, and stagnant water samples were positive for Acanthamoeba species, respectively. Moreover, T4, T5, T2, T7, and T11 genotypes were identified. The most common genotype was T4 (76%), isolated from stagnant water. Conclusions: Acanthamoeba is a prevalent species in the soil, hospital dust, and stagnant water of Kashan. As this protozoon can cause severe infections, health education and improvement of sanitation services are recommended for prevention of infection. Keywords: Acanthamoeba, Soil, Hospital Dust, Stagnant Water, Genotype, Iran

Human infections caused by free-living amoebae.

[b]Abstract Introduction[/b]. Among free-living amoebae that are widely distributed in nature only four genera/species are known as agents of human infections:[i] Acanthamoeba spp., Naegleriafowleri, Balamuthia mandrillaris[/i] and[i] Sappiniapedata[/i]. These amoebae are not well adapted to parasitism, and could exist in the human environment without the need for a host. Infections due to these amoebae, despite low morbidity, are characterized by relatively high mortality rate and pose serious clinical problems. [b]Objectve[/b]. This review study presents and summarizes current knowledge about infections due to pathogenic and opportunistic free-living amoebae focused on epidemiology, clinical manifestations, diagnosis and treatment based on global literature. [b]State of knowledge[/b]. All four genera have been recognized as etiologic factors of fatal central nervous system infections and other serious diseases in humans. [i]N. fowleri[/i] causes an acute fulminating meningoencephalitis in children and young adults. [i]Acanthamoeba spp[/i]. and [i]B.mandrillaris[/i] are opportunistic pathogens causing granulomatous amoebic encephalitis and disseminated or localized infections which could affect the skin, sinuses, lungs, adrenals and/or bones. [i]Acanthamoeba spp[/i]. is also the main agent of acute eye infection -[i] Acanthamoeba keratitis, [/i]mostly in contact lens wearers. However, there is only one recognized case of encephalitis caused by [i]S. pedata. [/i] [b]Conclusions[/b]. Amoebic diseases are difficult to diagnose which leads to delayed treatment, and result in a high mortality rate. Considering those issues, there is an urgent need to draw more attention to this type of diseases.

Presence of Balamuthia mandrillaris in hot springs from Mazandaran province, northern Iran.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause cutaneous lesions and Balamuthia amoebic encephalitis. The biology and environmental distribution of B. mandrillaris is still poorly understood and isolation of this pathogen from the environment is a rare event. Previous studies have reported that the presence of B. mandrillaris in the environment in Iran may be common. However, no clinical cases have been reported so far in this country. In the present study, a survey was conducted in order to evaluate the presence of B. mandrillaris in hot-spring samples of northern Iran. A total of 66 water samples were analysed using morphological and molecular tools. Positive samples by microscopy were confirmed by performing PCR amplification of the 16S rRNA gene of B. mandrillaris. Sequencing of the positive amplicons was also performed to confirm morphological data. Two of the 66 collected water samples were positive for B. mandrillaris after morphological and molecular identification. Interestingly, both positive hot springs had low pH values and temperatures ranging from 32°C to 42°C. Many locals and tourists use both hot springs due to their medicinal properties and thus contact with water bodies containing the organism increases the likelihood of infection. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from hot-spring sources related to human activity. Therefore, B. mandrillaris should be considered as a possible causative agent if cases of encephalitis are suspected following immersion in hot springs in addition to Acanthamoeba and Naegleria.

Isolation of Balamuthia mandrillaris from soil samples in North-Western Iran

Balamuthia mandrillaris is an opportunistic free-living amoebae (FLA) which has been reported as the causative agent of the fatal Balamuthia amoebic encephalitis (BAE). Currently, the transmission dynamics of this pathogen remain poorly understood although the organism has been associated with soils, and thus, soil exposure has been identified as a risk factor for this pathogenic amoeba. Nevertheless, environmental isolation of B. mandrillaris is a rare event and strains of this amoebic species have been isolated from soil and dust sources only in seven previous reports (Iran, Mexico, USA, Peru, Costa Rica, Guinea Bissau, and Jamaica). In Iran, a previous study reported the isolation of B. mandrillaris from dust collected in the city of Tehran and free of known infectious involvement. Therefore, in this work, 55 soil samples collected from public and recreational areas of East Azerbaijan, North-Western Iran, were investigated for the presence of this pathogen. Samples were cultured in 2% non-nutrient agar plates and were monitored for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S ribosomal DNA (rDNA) of B. mandrillaris. The obtained results revealed the presence of this emerging pathogen in 5 of 55 samples included in the study. Homology analysis of the obtained DNA sequences revealed high similarity with previously isolated strains from clinical and environmental sources. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from soil sources in Iran related to human activity and the second time that this pathogen is isolated from the environment in this country.

Cellulose degradation: a therapeutic strategy in the improved treatment of Acanthamoeba infections.

Acanthamoeba is an opportunistic free-living amoeba that can cause blinding keratitis and fatal brain infection. Early diagnosis, followed by aggressive treatment is a pre-requisite in the successful treatment but even then the prognosis remains poor. A major drawback during the course of treatment is the ability of the amoeba to enclose itself within a shell (a process known as encystment), making it resistant to chemotherapeutic agents. As the cyst wall is partly made of cellulose, thus cellulose degradation offers a potential therapeutic strategy in the effective targeting of trophozoite encased within the cyst walls. Here, we present a comprehensive report on the structure of cellulose and cellulases, as well as known cellulose degradation mechanisms with an eye to target the Acanthamoeba cyst wall. The disruption of the cyst wall will make amoeba (concealed within) susceptible to chemotherapeutic agents, and at the very least inhibition of the excystment process will impede infection recurrence, as we bring these promising drug targets into focus so that they can be explored to their fullest.

The isolation of Balamuthia mandrillaris from environmental sources from Peru

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding.

Free living amoebic infections causing CNS disease - diagnostic techniques

Free-living amoebae belonging to the genera Acanthamoeba, Balamuthia and Naegleria are important causes of disease in humans. Naegleria fowleri produces an acute, and usually lethal, central nervous system (CNS) disease called primary amoebic meningoencephalitis (PAM). Acanthamoeba spp. and Balamuthia mandrillaris are opportunistic free-living amoebae capable of causing a more chronic granulomatous amoebic encephalitis (GAE), often in immunocompromised individuals. In Naegleria infections, the diagnosis can be made by microscopic examination of cerebrospinal fluid (CSF) demonstrating motile trophozoites, and a Giemsa-stained smear will show trophozoites with typical morphology. In Acanthamoeba and Balamuthia infections, organisms are usually not present in CSF and the diagnosis can be made from microscopic examination of stained smears of biopsy specimens (brain tissue, skin, cornea) containing both trophozoites and cysts. Identification by direct immunofluorescent antibody may also prove useful. PCR-based techniques have been described for free-living amoebic infections in clinical samples and a multiplex real-time PCR has been adopted 1 at Sullivan Nicolaides Pathology which has proved useful both for detection and identification of the species in two locally acquired cases due to Balamuthia mandrillaris and Naeglaria fowleri .

Isolation and genotyping of potentially pathogenic Acanthamoeba strains from dust sources in Iran

Opportunistic free-living amoebae of the Acanthamoeba genus present genotypes/strains that are causative agents of Acanthamoeba keratitis (AK), with genotype T4 related to most AK cases worldwide. In order to prove that exposure to dirt and dust could be an important risk factor for contact lens wearers in Iran, 13 strains isolated from dust samples in this area were classified at the genotype level. Results revealed the presence of T4, T5 and T11 genotypes within these samples. To our knowledge, this is the first study presenting the identification of pathogenic genotypes of Acanthamoeba in dust samples in Iran and around the world.